Diamide amino-imidazoles: A novel series of γ-secretase inhibitors for the treatment of Alzheimers disease

Article abstract of DOI:10.1016/j.bmcl.2010.12.117

The synthesis and structure-activity relationship (SAR) of a novel series of di-substituted imidazoles, derived from modification of DAPT, are described. Subsequent optimization led to identification of a highly potent series of inhibitors that contain a β-amine in the imidazole side-chain resulting in a robust in vivo reduction of plasma and brain Aβ in guinea pigs. The therapeutic index between Aβ reductions and changes in B-cell populations were studied for compound 10h.

Full text of DOI:10.1016/j.bmcl.2010.12.117

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2631–2636
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Diamide amino-imidazoles: A novel series of
for the treatment of Alzheimer’s disease
c-secretase inhibitors
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 a, Barbara Tate b, Jeffrey W. Van Deusen c, Douglas E. Wood b,
Kathleen M. Wood b
⇑
a Neuroscience Medicinal Chemistry, Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, USA
b Neuroscience Research Unit, Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, USA
c Pharmacokinetics, Dynamics, and Metabolism, Pfizer Global 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:
The synthesis and structure–activity relationship (SAR) of a novel series of di-substituted imidazoles,
derived from modification of DAPT, are described. Subsequent optimization led to identification of a
highly potent series of inhibitors that contain a b-amine in the imidazole side-chain resulting in a robust
in vivo reduction of plasma and brain Ab in guinea pigs. The therapeutic index between Ab reductions and
changes in B-cell populations were studied for compound 10h.
Received 1 November 2010
Revised 22 December 2010
Accepted 23 December 2010
Available online 30 December 2010
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
c
-Secretase inhibitors
Amino-imidazoles
Ab reduction
Dementia results from a wide variety of distinctive pathological
processes. Alzheimer’s disease (AD) is the most common cause of
dementia, affecting nearly half of all people past the age of 85,
the most rapidly growing portion of the population. The number
of AD patients in the United States is expected to increase from 5
million to about 15 million by the middle of the next century.1
Two hallmark pathologies of the AD brain are the accumulation
of extracellular insoluble deposits known as amyloid plaques and
abnormal lesions within neurons called neurofibrillary tangles.2
Definitive diagnosis of AD is currently dependent upon observation
of both of these pathological events in post-mortem brain tissue.
The primary component of amyloid plaques are aggregated Ab
(such as Ab1–40 and Ab1–42) which result from sequential enzymatic
processing of the APP (amyloid protein precursor) by b-secretase
(GSI) such as LY-4501391, BMS-299897, GSI-953, and BMS-708163
have advanced into human clinical trials for the treatment of AD
(Fig. 1).5
F
CO2H
O
H
N
O
S
O
HO
N
N
H
F
N
F
O
O
Cl
LY-450139 (Semagacestat)
BMS-299897
NH2
O
F3C
CF3
OH
CF3
(BACE) and
c
-secretase.3 The causes of sporadic AD are unclear,
however several familial forms of AD stem either from APP variants
HN
S
N
O
F
or PSI mutations. Furthermore, both soluble and insoluble Ab
S
O
O
S
assemblies demonstrate neurotoxicity.4 As such,
c
-secretase is a
O
compelling target for the treatment of AD by inhibiting the produc-
tion of toxic Ab-peptides. To date, numerous c-secretase inhibitors
Cl
Cl
GSI-953 (Begacestat)
N
N
O
BMS-708163
⇑
Corresponding author. Tel.: +1 860 715 6168.
E-mail address: michael.a.brodney@pfizer.com (M.A. Brodney).
Figure 1. c-Secretase inhibitors that have advanced to human clinical trials.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.117

2632
M. A. Brodney et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2631–2636
H
N
O
H
N
O
R
F
F
O
N
H
N
H
O
O
O
1
F
F
DAPT
Figure 2. N-Terminal replacements of DAPT.
In 2001, coworkers at Eli Lilly and Elan reported on DAPT, the
first report of a centrally active -secretase inhibitor with an
MED = 30 mg/kg in the PDAPP mouse (Fig. 2).6 While whole cell po-
tency (0.02 M) and low molecular weight (MW = 432.5) made
this a compelling lead, further pharmacokinetic (PK) profiling re-
vealed potential acid instability of the t-butyl ester group. In light
of this result, our medicinal chemistry efforts focused on develop-
ing novel C-terminal heterocycles (R) as replacements for the ester
functionality in DAPT. Initial efforts in this area led to a potent ser-
ies of amino-thiazoles7 and this manuscript describes the in vitro
and in vivo characterization of a novel series of diamide amino imi-
dazoles (1).
c
A general synthetic strategy for the preparation of 4-substituted
amino imidazoles is illustrated in Scheme 1.8 Starting from 4-nitro-
imidazole 2, regioselective N-alkylation with an appropriate alkyl
halide provides 3-substituted imidazoles 3a–b. Subsequent reduc-
tion of the ester 3b to the alcohol 3c was accomplished with stan-
dard conditions. Alternatively, reduction of ester 3b with DIBAL
afforded the intermediate aldehyde that underwent reductive
amination to furnish nitro imidazoles 3d. Reduction of the
l
Table 1
In vitro potency of representative GSI
O
N
H
N
R1
N
F
O
F
N
H
R2
6a-q
Compound
R1
R2
CFA
IC50 (nM)
WCA
IC50 (nM)
Total Ab in hd
(brain @ 3 h)
a,b
b,c
CO2Me
OH
6a
6b
6c
6d
6e
6f
57.7
263
82.8
52.7
0.39
2.7
306
114
2.4
N
<10%
32%
CO2Me
5.1
CO2Me
1.2
1.2
OH
6g
6h
6i
2.5
14.6
20.3
226
0.8
OH
3.6
N
9.1
N
6j
0.9
43%
a
b
c
IC50 values in the cell free assay (CFA) were obtained from human HeLa cells by measuring Ab1–40 by DELFIA-based immunoassay.10
Values are geometric mean of at least two experiments; compounds were typically dosed at log intervals from 0.1 nM to 10 lM.
10
IC50 values in the whole cell assay (WCA) were obtained from H4 APPSw cells by measuring Ab1ÀX
.
d
In vivo activity was determined by measuring Ab1ÀX, Ab1À40, and Ab1À42 in guinea pig brain and plasma by Delfia ELISA. Extracts were analyzed for
changes in Ab1ÀX using an IGEN assay.11 A significant difference between groups was 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.

M. A. Brodney et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2631–2636
2633
N
N
N
a
R1
R1
b or c, d
N
N
NH
O2N
O2N
O2N
e, f
3a; R1 = alkyl
1
3c;
R = C(CH3)2CH2OH
3d; R1 = C(CH3)2CH2NR4R5
2
1
3b;
R = C(CH3)2CO2Me
O
O
N
O
N
H
N
R1
N
R1
N
g
H2N
Boc
N
H
N
H
5
R2
R2
4
O
H
N
N
F
H
N
N R1 3a-d,
OH
e, f
R3
h
N
H
O
O
R2
7
F
6a-q
Scheme 1. Reagents and conditions: (a) R1-Br, K2CO3, Et3BnNCl, CH3CN, 80 °C, 30–65%; (b) LiAlH4, THF, reflux; (c) DIBAL, CH2Cl2, À30 °C; (d) R4R5-NH2, 4AoMS, CH2Cl2, then
Na(OAc)3BH, 60% over 2 steps; (e) Pd/C (10%), H2 (40 psi), MeOH, rt; (f) BocNCHR2CO2H, TPTU, iPr2EtN, DMF, 40–80% for 2 steps; (g) TFA, CH2Cl2; (h) R3CO2H, EDC, HOBt,
iPr2EtN, rt, 70–95%.
nitroimidazole was accomplished via hydrogenation with Pd/C.9
Due to the instability of the resultant aminoimidazole, coupling
with an N-Boc protected amino acid was immediately carried for-
ward to amide 4. Removal of the Boc protecting group provided
amine 5, which was acylated to provide 6 bearing structural varia-
tions in the R1, R2, and R3 positions. Alternatively, amino-acid 7 is
coupled to the intermediate amino-imidazoles derived from nitro-
imidazoles 3a–d to provide the desired analogs.
free assay (CFA) and whole cell assay (WCA) potencies.10 The syn-
thesis and evaluation of a wide range of straight chain and
branched alkyls off the imidazole ring demonstrated the impor-
tance of
a-branching for whole cell potency. From this early SAR,
the 3-pentyl analog 6d provided excellent in vitro potency in
cell-free and whole assays. Analogous to the alkyl side chains,
incorporation of a-branching on the R1 side chain resulted in a sig-
nificant improvement in whole cell potency for esters (6a vs 6e),
alcohols (6b vs 6g) and b-amines (6c vs 6j). The tether length off
the imidazole side chain was investigated to understand the im-
pact of spacing on potency. For esters 6e/6f and alcohols 6g/6h, a
modest preference based upon whole cell potency was identified
for the longer tethered substituents. Interestingly, the b-amine 6j
Preliminary SAR at the R1 position of compounds 6a–j (Table 1)
revealed a range of substituents (esters, alcohols, and b-amines)
were well tolerated on the imidazole side chain with respect to cell
and the c-amine 6i gave comparable cell free potency but 6j was
Table 2
SAR of central amino amide R2 substituent
ꢀ100-fold more potent than 6i in the whole cell assay. To assess
the efficacy of inhibitors 6d, 6f, 6j, guinea pigs were dosed acutely
with subcutaneous (sc) injection and Ab1ÀX was measured in brain
and plasma by Delfia ELISA at the 3 h time point. While 6d failed to
reduce total brain Ab1ÀX after a 10 mg/kg, sc screening dose due to
poor central exposure, inhibitors 6f and 6j significantly reduced
brain Ab1ÀX (32% and 43%, respectively) after a 10 mg/kg, sc screen-
ing dose in guinea pig.11
With a desirable R1 substituent, the central amino acid (R2) of
compound 6j was varied (Table 2). A range of alkyl (6k–m), cyclo-
alkyl (6n), and aryl (6o) groups were tolerated at this position but
the propyl analog (6j) derived from (S)-norvaline proved superior
with respect to in vitro potency. Attempts to incorporate polarity
into the R2 group such as ether 6p resulted in a 10-fold lose in po-
tency while the dimethylamide analog 6q was inactive. Additional
in vivo characterization of 6k and 6l in the guinea pig after an
acute dose of 10 mg/kg, sc failed to show a reduction of total brain
Ab1ÀX at a 3 h time point. We speculated that the lack of robust
efficacy was due to poor central exposure.
In orderto furtherinvestigatecellularpotencyinthe b-amineSAR
on the imidazole side chain, a more versatile route was utilized
to prepare a set of secondary and tertiary amines (Scheme 2).
Amide coupling of an amino imidazole derived from nitroimidazole
8 with acid 7 utilizing the standard coupling procedure afforded
diamide 9. Subsequent reduction of the ester and reductive
amination afforded the desired analogs 10a–i.
In comparison to the pyrrolidine analog 6j (Table 1), morpho-
line 10a showed comparable whole cell potency while smaller
alkyl amines such as monomethyl amine 10b and dimethyl
amine 10c resulted in a ꢀ90-fold and ꢀ5-fold lose in potency,
Compound
R1
R2
CFA
IC50 (nM)
WCA
IC50 (nM)
a,b
b,c
Me
6k
6l
12.6
7.8
N
N
7.0
4.3
2.5
N
N
6m
6n
11.9
55.0
21.6
N
6o
6p
6q
51.1
9.4
N
N
39.7
8.2
O
O
>10,000
>10,000
NMe2
a
IC50 values obtained from human HeLa cells and were used to test compounds
in a cell free assay, and Ab1À40 was detected using a DELFIA-based immunoassay.10
b
Values are geometric mean of at least two experiments; compounds were
typically dosed at log intervals from 0.1 nM to 10 lM.
c
IC50 values obtained from H4 APPSw cells were used to test compounds in a
whole cell assay, and Ab1ÀX was measured.10

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M. A. Brodney et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2631–2636
Table 3
Cell free potency (CFA), whole cell potency (WCA), and notch effects (FTOC) of GSI’s with b-amino side chains
d
Compound
R1
CFA
IC50 (nM)
WCA
IC50 (nM)
FTOC EC50
(lM)
a,b
b,c
O
10a
0.6
0.4
0.1
N
10b
10c
45.7
3.8
89.5
4.8
NT
0.7
NH
N
10e
10f
29.0
64.6
7.9
>10.0
>10.0
NH
H
N
72.7
OH
10g
10h
1.7
1.1
3.1
0.4
4.3
1.2
NH
NH
O
10i
0.5
0.4
4.0
NH
NT = not tested.
a
IC50 values obtained from human HeLa cells and were used to test compounds in a cell free assay, and Ab1–40 was detected using a
DELFIA-based immunoassay.10
b
Values are geometric mean of at least two experiments; compounds were typically dosed at log intervals from 0.1 nM to 10
IC50 values obtained from H4 APPSw cells were used to test compounds in a whole cell assay, and Ab1ÀX was measured.10
Fetal thymic organ cultures (FTOC) were prepared for assessment of compound effects on notch processing.13
lM.
c
d
respectively (Table 3). A range of amines were tolerated at this
position but secondary amines 10g–i resulted in robust whole cell
potency. To evaluate the potential effects on notch processing,
compounds 10a–i were evaluated in fetal thymic organ culture
(FTOC) B- and T-cell populations. Interestingly, a range of selectiv-
ity was observed for reductions in Ab as measured in the whole cell
assay versus reductions in B- and T-cell populations. This selectiv-
ity in vitro suggested the potential for a therapeutic index (TI) in
vivo.
3 h post treatment (Fig. 3A).11,12 The reduction of brain Ab was dose
dependent from 1 to 32 mg/kg, sc resulting in a brain ED50
=
7.3 mpk. The reductions in plasma Ab were sustained at ꢀ60%
and appeared to be a maximal reduction for this analog despite
the increasing drug concentration in plasma. The corresponding
drug exposures measured at 3.2 mg/kg, sc (3 h post dose) showed
good coverage of the in vitro IC50 in brain (12 ng/g, 24 nM). To ob-
tain an understanding of the Ab changes over time in brain, CSF
and plasma, guinea pigs were dosed acutely with 10h and tissues
were collected at 2 h intervals from 1 to 7 h post dose (Fig. 3B).
At 1 h after dose, a significant reduction in plasma and brain Ab
could be detected while CSF was not significantly different than
To further characterize the in vivo performance of this series of
compounds, guinea pigs were treated with compound 10h from 1
to 32 mg/kg and total Ab1ÀX was evaluated in brain and plasma at
CO2Me
O
N
H
N
N
F
N
CO2Me
a, b
N
H
N
O
O2N
8
F
9
O
N
NR4R5
c, d
H
N
N
F
N
H
O
F
10a-i
Scheme 2. Reagents and conditions: (a) Pd/C (10%), H2 (40 psi), MeOH, rt; (b) compound 7, TPTU, iPr2EtN, DMF, 75% for 2 steps; (c) DIBAL, CH2Cl2, À30 °C; (d) R4R5-NH2,
4AoMS, CH2Cl2, then Na(OAc)3BH, 60% over 2 steps.

M. A. Brodney et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2631–2636
2635
preparations after multiple doses in
a
24 h time period
100
80
60
40
20
0
800
600
400
200
0
A
(Fig. 3C).13,14 Guinea pigs were treated with 5 and 25 mg/kg, sc
of compound 10h with three doses over a 24 h period (time 0,
12, and 24 h) and tissues were collected at 3 h after the final dose.
A dose responsive relationship was observed for brain and plasma
Ab with compound 10h and the 5 mg/kg, sc dose closely estimated
a 50% reduction of brain Ab(1ÀX). Despite the significant separation
between APP processing in the whole cell assay and Notch inhibi-
tion in the FTOC assay, analysis of blood B cell and marginal zone
B-cells (MZB) from the spleen suggested no therapeutic index
(TI) was achieved for this GSI (Fig. 3C).
In conclusion, a series of amino imidazoles were synthesized
based on DAPT. Efforts varying the N-terminal substituent on the
imidazole ring lead to compound 10h with robust cellular potency
1
3.2
10
32
in the
c-secretase inhibition assay and significant reductions of
Brain Aβ1-X
Plasma Aβ1-X
Dose (mg/kg)
Brain exposure
Plasma exposure
brain, plasma, and CSF Ab in guinea pigs after an acute dose.
Despite in vitro selectivity between APP processing in the whole
cell assay and B-cell reductions in FTOC, in vivo studies examining
Ab reductions and B cell changes within the same animals sug-
gested that further optimization of the selectivity profile or
distribution properties of these inhibitors will be required to
obtain a therapeutic index. Future publications will focus on more
detailed in vivo characterization and Notch related safety of this
100
B
Brain Aβ1-X
CSF Aβ1-X
Plasma Aβ1-X
80
60
40
20
0
series of c-secretase inhibitors.
References and notes
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Time (h)
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C
100
80
60
40
20
0
B Cell
MZB
Plasma Aβ
Brain Aβ
vehicle
5 mg/kg
25 mg/kg
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Treatment
Figure 3. In vivo characterization of GSI 10h: (A) dose responsive of brain and
plasma Ab; (D) time course of brain, plasma, CSF Ab; (C) reduction of marginal and
blood B-cell populations. (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.11,13
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.11,13
8. Brodney, M.A.; Coffman, K.C. US2005215610.
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controls (Fig. 3B). From 3 to 7 h, Ab was significantly reduced in all
compartments. A potential liability associated with inhibiting the
c
-secretase complex is substrate specificity.15 Cleavage of Notch
by c-secretase is necessary for differentiation of certain cell types
12. Representative synthesis of compound 10h: To
a solution of (S)-2-(2-(3,5-
within the intestine and blood cell populations. To evaluate the po-
tential for Notch related toxicity in relationship to efficacy over
time for this class of GSI, B-cell populations were analyzed by fluo-
rescence activated cell sorting (FACS) of whole blood and spleen
difluorophenyl)acetamido)-N-(1-(2-methyl-1-oxopropan-2-yl)-1H-imidazol-
4-yl)pentanamide in DCM is added 2,2-dimethylpropan-1-amine (4.4 equiv),
pTSOH (0.2 equiv), 4AoMS, and the solution is stirred for 4 h and then
Na(OAc)3BH (3.3 equiv) is added. The reaction is stirred for 12 h, quenched

2636
M. A. Brodney et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2631–2636
with sodium bicarbonate, extracted with DCM, concentrated and purified by
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.
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Wolfe, M. S.; Schroeter, E. H.; Kopan, R. Proc. Natl Acad. Sci. USA 2001,
98, 7487.
flash chromatography to provide 10h; 1H NMR (400 MHz, chloroform-d) d ppm
0.81 (t, J = 7.32 Hz, 3H) 1.20–1.34 (m, 2H) 1.51 (s, 6H) 1.56–1.70 (m, 5H) 1.71–
1.88 (m, 1H) 2.25–2.40 (m, 4H) 2.67 (s, 2H) 3.49 (s, 2H) 4.76 (q, J = 7.35 Hz, 1H)
6.62–6.70 (m, 1H) 6.73–6.85 (m, 2 H) 7.41 (s, 1H) 7.59 (s, 1 H) 11.26 (s, 1H).
13. 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.;
15. (a) McCarthy, J. V.; Twomey, C.; Wujek, P. Chem. Mol. Life Sci. 2009, 66, 1534; (b)
Lleo, A. Curr. Top. Med. Chem. 2008, 8, 9.