Design, synthesis, and qualitative structure-activity evaluations of novel β-secretase inhibitors as potential Alzheimer's drug leads

Article abstract of DOI:10.1021/jm201181f

We have identified highly selective imidazopyr-idines armed with benzimidazol and/or arylimidazole as potent β-secretase inhibitors. The most effective and selective analogues demonstrated low nanomolar potency for the BACE1 enzyme as measured by FRET and cell-based (ELISA) assays and exhibited comparable affinity (K_I) and high ligand efficiency (LE). In addition, these motifs were highly selective (>200) against the structurally related aspartyl protease BACE2. Our design strategy followed a traditional SAR approach and was supported by molecular modeling studies based on the previously reported hydroxyethylene transition state inhibitor derived from iso-phthalic acid I. Of the most potent compounds, 34 displayed an IC₅₀ for BACE1 of 18 nM and exhibited cellular activity with an EC₅₀ of 37 nM in the cell-based ELISA assay, as well as high affinity (K_I = 17 nM) and ligand efficiency (LE = 1.7 kJ/mol). Compound 34 was found to be 204-fold more selective for BACE1 compared to the closely related aspartyl protease BACE2. (Figure presented)

Full text of DOI:10.1021/jm201181f

Article
pubs.acs.org/jmc
Design, Synthesis, and Qualitative Structure−Activity Evaluations of
Novel β-Secretase Inhibitors as Potential Alzheimer’s Drug Leads
Taleb H. Al-Tel,*,† Mohammad H. Semreen,† Raed A. Al-Qawasmeh,§ Marco F. Schmidt,∥
Raafat El-Awadi,† Mustafa Ardah,⊥ Rania Zaarour,‡ Shashidhar N. Rao,# and Omar El-Agnaf⊥
‡
†College of Pharmacy and Sharjah Institute for Medical Research, University of Sharjah, P.O. Box 27272, Sharjah, UAE
§Chemistry Department, University of Jordan, 11942, Amman, Jordan
∥University Chemical Laboratory, University of Cambridge, Lensfield Road, Cambridge CB2 EW1, U.K.
⊥Faculty of Medicine & Health Sciences, United Arab Emirates University, P.O. Box 17666 Al-Ain, UAE
#Tripos International, Inc., 1699 South Hanley Road, St. Louis, Missouri 63144, United States
S
* Supporting Information
ABSTRACT: We have identified highly selective imidazopyr-
idines armed with benzimidazol and/or arylimidazole as potent
β-secretase inhibitors. The most effective and selective analogues
demonstrated low nanomolar potency for the BACE1 enzyme as
measured by FRET and cell-based (ELISA) assays and exhibited
comparable affinity (KI) and high ligand efficiency (LE). In
addition, these motifs were highly selective (>200) against the
structurally related aspartyl protease BACE2. Our design
strategy followed a traditional SAR approach and was supported
by molecular modeling studies based on the previously reported
hydroxyethylene transition state inhibitor derived from iso-
phthalic acid I. Of the most potent compounds, 34 displayed an
IC50 for BACE1 of 18 nM and exhibited cellular activity with an
EC50 of 37 nM in the cell-based ELISA assay, as well as high
affinity (KI = 17 nM) and ligand efficiency (LE = 1.7 kJ/mol). Compound 34 was found to be 204-fold more selective for
BACE1 compared to the closely related aspartyl protease BACE2.
in the brain is a hallmark of AD.5 Thus, the most direct
approach in AD therapy is the reduction of Aβ42 production.
Aβ is generated proteolytically from a large precursor molecule,
APP, by the sequential action of two proteases: β-secretase
INTRODUCTION
■
Alzheimer’s disease (AD) is the most common form of
irreversible dementia.1 More than 25 million people are
suffering from dementia, and the annual socioeconomic
worldwide costs have been estimated to exceed U.S. $200
(also known as β-site APP cleaving enzyme 1, BACE1) and
γ-secretase. A third protease, α-secretase, competes with
billion.1 According to the Alzheimer’s Association, in 2009 an
β-secretase for the APP substrate and can prevent the production
estimated 5.3 million Americans had Alzheimer’s disease (AD),
making it the sixth leading cause of death in the United States.
As increased age is the major risk factor of the disease, AD
frequency is expected to increase to an estimated 7.7 million
cases in 2030 and 11−16 million cases in 2050 in the United
States alone. These numbers do not encompass the large
of Aβ by cleaving the peptide into two fragments. These
molecular pathways evoke at least three strategies to reduce Aβ:
inhibition of γ-secretase, inhibition of β-secretase, and
stimulation of α-secretase. The main approach for targeting Aβ
production involves targeting β-secretase.6
Tissue culture and animal studies have shown that β-secretase
is expressed in all tissues, with higher expression levels in the
number of people with mild cognitive impairment, a
considerable proportion of whom will progress to AD.2 Genetic
brain. Enhanced β-secretase activity has been detected in the
and pathological evidence strongly supports the amyloid
cascade hypothesis for AD. This states that Aβ, a proteolytic
derivative of the large transmembrane amyloid precursor
protein (APP), in particular the least soluble 42 amino acid
brains of patients with sporadic AD.7 Furthermore, BACE1
knockout mice are scarce in Aβ production, demonstrating that
there are no redundant molecular mechanisms for β-secretase
cleavage in mice. More interestingly, the knockout mice did not
manifest severe health defects as a result of the lack of β-secretase;
fragment Aβ42 isoform, has an early and imperative role in all
forms of AD.3
To date, treatment of AD only partly reduces the symptoms
and does not affect the underlying progression of the disease.4
Received: June 17, 2011
The accumulation and aggregation of extracellular Aβ plaques
Published: November 1, 2011
© 2011 American Chemical Society
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Figure 1. De novo design strategies utilized to find P1−P1′ and P3 fragments based on isophthalic acid motif I.
they had no aberrant phenotype and were fertile, and clinical
chemistry parameters were normal in both young and old
animals. Both the absence of Aβ production and the lack of
distinct pathology of the BACE1 knockout mice emphasized the
potential effectiveness of drugs targeting β-secretase. However,
the development of specific inhibitors for β-secretase has proven
to be highly challenging.8
To date, one company has reported clinical data with
γ-secretase inhibitor, and over the past 2 decades many groups
and Biotech Pharma have investigated a diversity of approaches
to the design of BACE1 inhibitors.9 These efforts have
produced low molecular weight BACE1 inhibitors with little
or no peptidic character.10 In addition, recent reports have
revealed the application of fragment-based lead generation
approaches as an alternative route to the design of potent small-
molecule BACE1 inhibitors.11 The discovery of low molecular
weight BACE1 inhibitors has also led to the improvement of
the physicochemical properties of the compounds as verified by
their high-permeability in cell-based assays.12
replace the hydroxyethylene (HE) amide portion of compound
I with benzimidazole and/or arylimidazole derivatives, leading
to compounds of type III. We hypothesized that this might
improve the affinity of such motifs against BACE1. Accordingly,
the IC50 of such motifs (6−8 and 12−14) ranged from 1.08 to
0.187 μM (Tables 1 and 2). The results were promising.
However, such series displayed high polar surface area (PSA),
an indication of low oral bioavailability,15 and a high peptidic
nature; hence, we decided to design more rigid motifs to lower
the peptidic portions and the PSA. We therefore carried out
detailed SAR studies around compounds II and III that
ultimately led us to contract the diazepam ring, leading to
imidazopyridine derivative of type IV. This resulted in some
cases of up to 60-fold improvement of the ligand affinity (34 vs 6)
toward BACE1 enzyme (34, IC50 = 18 nM; Table 5) and com-
parable activity in the cell-based ELISA assay (EC50 = 37 nM).
In addition, compound 34 demonstrated about 204-fold
selectivity against the closely related aspartyl protease,
BACE2, and its solubility in water was 10 mg/mL. Further-
more, we found that linear terephthalic acid like scaffolds (e.g.,
19−21) on the targeted ligand at the vicinity of this aberration
generated steric conflict between the ligand and this protein,
thus impacting the ligand’s affinity and efficiency. These results
were in contrast to the isophathalic acid like motifs (e.g., 24−
26 and 31−34), which lacked such steric compression and
exhibited high protein affinity.
Synthesis. The preparation of the compounds needed to
delineate the SAR for this study was done according to
synthetic Schemes 1 and 2. Two types of scaffolds were synthe-
sized. In the first, sulfone amide carboxylic acid 2, synthesized
using well established procedures,16 was coupled with the
desired diaminobenzene, 3−5 (Scheme 1). Thus, treatment of 2
with TBTU and DIPEA in DMF at 0 °C followed by the addi-
tion of the desired diaminobenzene (3, 4, or 5) and subsequent
reaction with NH4OAc followed by silica gel chromatography
furnished compounds 6−8 in fairly good yields. In the second
approach, compound 2 (Scheme 2) was coupled with the
desired α-bromoketone 9−11 using DIPEA in DMF at 0 °C.
Subsequently, the product from this step was subjected to
cyclization reaction with the amidic carbonyl group in refluxing
acetic acid to deliver compounds 12−14.
BACE2 is a closely related aspartyl protease. The
physiological functions of BACE2 have to be fully charac-
terized, although BACE1/BACE2 double knockout mice have
been reported to be viable.13
RESULTS AND DISCUSSION
■
We have previously reported on the design and synthesis of
benzodiazepine motifs of type II (Figure 1) as potential lead
molecules for BACE1 inhibition.14 As a continuation of this
work, we report herein on the design and synthesis of novel
motifs derived from isophthalic and terephthalic acid as
potential BACE1 inhibitors. As could be concluded from
Figure 1, we followed a traditional medicinal chemistry strategy,
namely, extension/contraction, rigidification/cyclization, posi-
tional ring variations, and bioisostere replacement, to arrive at
our intended motifs. In this respect, we considered the
extensive structure−activity relationship (SAR) investigations
presented in our previous article.14 We learned that the trunca-
tion of the benzimidazole segment (benzodiazepine derivatives
of type II, Figure 1) as an isostere of the amide handle of
compound I led to more rigid and potent motifs that possessed
an IC50 of about 200 nM against BACE1.14 Thus, we decided to
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a
Table 1. SAR of BACE1 Inhibitors
BACE1
BACE2
ELISA
EC50 (nM)
compd
IC50 (nM)
KI (nM)
LE (kJ/mol)
tPSA (Å2)
IC50 (nM)
KI (nM)
6
7
8
1080 ± 303
545 ± 63
786 ± 262
1041 ± 342
525 ± 82
758 ± 254
NA
1.07
1.05
1.06
90.9
100.1
90.9
2130
2425
2127
2067
2354
2065
NA
230 ± 54
a
IC50 and EC50 values are the mean values of at least three experiments ± SD. NA means not applicable.
a
Table 2. SAR of BACE1 Inhibitors
BACE1
BACE2
ELISA
EC50 (nM)
compd
IC50 (nM)
KI (nM)
LE (kJ/mol)
tPSA (Å2)
IC50 (nM)
KI (nM)
12
13
14
315 ± 112
77 ± 11
187 ± 21
304 ± 98
62 ± 13
180 ± 27
450 ± 87
179 ± 36
NA
1.09
1.25
1.16
90.9
100.1
90.9
1786
2350
1873
1733
2281
1818
a
IC50 and EC50 values are the mean values of at least three experiments ± SD. NA means not applicable.
Scheme 1. Synthesis of the First Series of BACE1 Inhibitors
6−8
Scheme 2. Synthesis of the Second Series of BACE1
Inhibitors 12−14
a
a
a
Reagents and conditions: (a) DMF, DIPEA, 0 °C to room temp, 6 h
a
Reagents and conditions: (a) TBTU, DIPEA, DMF, 0 °C, 6 h, then
(95%), then AcOH, AcONH4, reflux, 8 h.
AcOH, reflux, 6 h.
The fourth series of compounds (Scheme 4) were synthe-
sized using the Greobock−Blackburn protocol as described
before. Alternatively, isophthalaldehyde 22 was used in this
reaction instead of terephthalic acid derivative 16. With
compound 23 in hand, it was subjected to coupling reaction
with the desired diaminobenzene followed by reaction with
NH4OAc furnishing imidazopyridine motifs 24−26. To
enhance SAR diversity, 2-aminopyridine derivatives 15 and
17 (Scheme 5) were subjected to tandem coupling and
cyclization reaction with α-bromoketone 28 to deliver adducts
For the synthesis of the third class of compounds, namely,
imidazopyridine analogues of type 19 (Scheme 3), Greobock−
Blackburn [4 + 1] cycladdition protocol of aminopyridine (15)
with phthalaldehyde 16 and isonitrile 17 was followed to afford
imidazopyridine 18 in 75% yield. Without further purification,
compound 18 was subjected to coupling with the desired di-
aminobenzene derivatives followed by acid catalyzed ring closure
leading to benzimidazole derivatives 19−21.
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flash column chromatography, employing variable concen-
trations from EtOAc/DCM mixtures.
Scheme 3. Synthesis of the Third Series of BACE1 Inhibitors
19−21
a
Biological Activities and SAR. Our goal in this study was
to develop selective and potent BACE1 inhibitors. Toward this
objective, we have first replaced the P1−P1′ portions in
compound I by a benzimidazole arm to project directly toward
the S1−S1′ subpockets of the BACE1 active site. This was
initiated based on our earlier extensive SAR analysis of
compound II (Figure 1). To prove the concept that the HE
transition state mimic in compound I could be replaced by
benzimidazole group, we have subjected compound I to
traditional medicinal chemistry strategies leading to scaffolds
such as III and IV (Figure 1). With the necessary tools needed
to fully investigate the SAR in hand, the diverse array of
compounds were profiled for their potency at the target
enzyme BACE1, and their selectivity toward the closely related
aspartyl protease BACE2 was investigated. The BACE1/
BACE2 primary screening assay utilized for this program was
the fluorescence resonance energy transfer (FRET) protocol.
We used an APP-based peptide substrate (rhodamine-
EVNLDAEFK-quencher, KM of 20 μM) carrying the Swedish
mutation and containing rhodamine as a fluorescence donor
and a quencher acceptor at each end. IC50 values were
calculated by plotting the obtained relative fluorescence unit
per hour (RFU/h) against the logarithmic of inhibitor
concentration. The measured inhibition data were analyzed in
GraphPad Prism 4 for Windows (GraphPad Software Inc., La
Jolla, CA, U.S.) by nonlinear regression (curve fitting).
The observed inhibition constant in the presence of
substrate, KI (app), is calculated according to the following
equation:17
a
Reagents and conditions: (a) Sc(OTf)3, DCM−MeOH, 12 h; (b)
TBTU, DIPEA, DMF, 0 °C, 6 h, then AcOH, reflux, 8 h.
Scheme 4. Synthesis of the Fourth Series of BACE1
Inhibitors 24−26
a
where vI is the steady state rate of substrate hydrolysis in
the presence of inhibitor concentration [I] and where v0 is
the uninhibited rate. The overall equilibrium constant is
obtained taking into account the substrate concentration
[S] according to the following equation:17
a
Reagents and conditions: (a) Sc(OTf)3, DCM−MeOH, 12 h; (b)
TBTU, DIPEA, DMF, 0 °C, 6 h, then AcOH, reflux, 8 h.
Scheme 5. Synthesis of the Fifth Series of BACE1 Inhibitors
31−34
a
As an indication of the efficiency of the compounds toward binding
BACE1 compared to known BACE1 inhibitors, the ligand efficiency
was calculated for each compound based on the free energy
equation,
and the equation of binding energy per atom (ligand
efficiency, LE),
Cellular potency of advancing compounds was done via cell-
based Aβ inhibition (Aβ40 or Aβ42) in an enzyme-linked
immune sandwich assay (ELISA) in H4 cells (human
neuroglioma cell line) expressing the double Swedish mutation
(K595N/M596L) of human APP (APPsw). The concentration
at which the cellular production of Aβ40 or Aβ42 was reduced
by 50% (EC50) was determined and reported in Tables 1−5.17
Compared to compounds I and II (Figure 1), the initial
unsubstituted baseline benzimidazole analogue 6 (Table 1)
a
Reagents and conditions: (a) EtOH, reflux 2 h, then NaHCO3, reflux
12 h; (b) TBTU, DIPEA, DMF, 0 °C, 6 h, then AcOH, reflux, 8 h.
29 and 30. These acids were coupled with the desired
diaminobenzene to deliver the corresponding benzimidazole
derivatives 31−34. The final products were then purified using
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showed about 108-fold less in potency when compared to
compound I (Figure 1) and a 5-fold decrease in potency when
compared to the compounds of type II.14 As we were not
satisfied with this result, we decided to include a fluorine atom
on the benzimidazole appendage. By doing so, we thought that
the fluorine might be engaged in a network of hydrogen
bonding in S1−S1′ subpockets leading to higher affinity.
Thus, compound 8 was synthesized according to Scheme 1
and was found to possess about 1.5-fold improvement in
potency compared to compound 6. By replacement of the
fluorine atom in 8 with a methoxy group (compound 7), the
activity was enhanced by 2-fold compared to that of 6.
Compounds 7 and 8 exhibited fair selectivity (5-fold) against
BACE2 and possessed modest activity against BACE 1
compared to compound I. Compound 8 possessed an EC50
of 230 nM in the cell-based ELISA assay. To understand the
biological sensitivity to which compounds 6−8 were subject
and to find a plausible explanation for the difference in the
activity profiles between 6−8 and compound I, one needed to
consider their docking profile in the enzyme active site in an
attempt. Thus, we analyzed the X-ray cocrystal structures of
BACE1 protein available in Protein Data Bank reported for
compound I (2B8L) and used this structure to calculate the
molecular modeling (Figure 2) data for our compounds using
Figure 3. Top-ranked docked pose of compound 7 (ball and stick
rendering, carbon atoms in gray) into the BACE1 binding site based
on 2B8L X-ray structure from the Protein Data Bank. The protein
atoms are shown in tube rendering. The four subsites S1′, S1, S2, and
S3 are also marked for clarity with blue arcs.
Accordingly, we envisioned that an extension strategy around
the imidazole ring would allow for direct projection toward
the unoccupied S1−S1′ subpockets. To this end, we have
introduced a phenyl group at the 4-position of the imidazole
moiety leading to compounds 12−14 (Scheme 2). These
compounds were tested for their activity profile against BACE1
and BACE2 (Table 1). As expected, compound 13 was found
to be 7-fold more potent against BACE1 when compared to
compound 7. Comparable activities for compounds 12 and 13
were found in the cellular assay with an EC50 of 450 and 179 nM
(Table 2), respectively. The selectivity index for BACE1/
BACE2 was calculated to be 30. When docked using Sybyl-X
1.3 under induced-fit mode, it was found that ligand 13,
containing the more flexible methoxyphenylimidazole arm,
projected directly toward S1−S1′ subpockets (Figure 4) and the
Figure 2. Top-ranked docked pose of compound I (ball and stick
rendering, carbon atoms in red) into the BACE1 binding site based on
2B8L X-ray structure from the Protein Data Bank. The pose has a
rmsd (root mean square deviation) of 0.5 Å of the pose reported for a
very similar ligand in 2B8L. The protein atoms are shown in tube
rendering. The four subsites S1′, S1, S2, and S3 are also marked for
clarity with blue arcs.
Sybyl-X program, version 1.3 (for details, see Experimental
Section).18 Two important observations could be concluded
from Figure 3. First, the methoxybenzimidazole group at the
P1−P1′ position appeared not to be perfectly projected toward
the S1−S1′ subpockets. Second, the NH of the imidazole ring
was not properly oriented to be engaged with hydrogen
bonding interaction with Asp32. At the outset of this analysis, it
could be more than idle speculation at this level to suggest that
such factors were responsible for lowering the affinity between
these ligands and BACE1. With these results in hand, we next
turned our attention toward finding proper groups that fit in
the S1−S1′ subpockets. The first task in finding such potent
BACE1 inhibitors was centered on proper functionalization of
the imidazole ring in III (Figure 1).
Figure 4. Top-ranked docked pose of compound 13 (green) into the
BACE1 binding site.
imidazole ring and methoxy phenyl groups were properly
oriented to form van der Waals (vdW) contacts with the enzyme
wall as well as hydrogen bonding with Asp32 (Figure 4).
This analysis could explain the enhanced potency of
compounds 12−14 compared to the previous series. On the
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a
Table 3. SAR of BACE1 Inhibitors
BACE1
BACE2
ELISA
EC50 (nM)
compd
IC50 (nM)
KI (nM)
LE (kJ/mol)
tPSA (Å2)
IC50 (nM)
KI (nM)
19
20
21
5511 ± 248
2479 ± 313
2241 ± 424
5312 ± 447
2389 ± 402
2160 ± 505
NA
1.04
1.03
1.08
52
5675
3725
2895
5509
3617
2810
NA
61.2
52.1
2405 ± 521
a
IC50 and EC50 values are the mean values of at least three experiments ± SD. NA means not applicable.
other hand, the high selectivity index found for this series could
be due to the difference in both the dynamic motion and the
protein amino acid sequences of the “flap” loops of the BACE1
and BACE2 upon ligand engagement. This apparently may
have influenced the ligand/protein contacts and impeded the
ligand’s affinity for the BACE2 site, thus resulting in increased
selectivity.
With these promising results in hand, efforts were shifted
toward further rigidification/cyclization of the prime side P3, as
it was felt that this strategy was more likely to lead to the
identification of druglike inhibitors of BACE1 with the
potential for good oral bioavailability and CNS penetration.
In this regard we calculated the PSA of compounds 6−8 and
12−14, and these were found to be high >90 (Tables 1
and 2).15a It has been noted that molecules possessing a large
PSA may encounter difficulty in crossing biological membranes.
This inability to cross membranes may result in poor
absorption or lack of blood−brain barrier (BBB) penetration.
Some of the first work correlating PSA and oral absorption was
published by Palm15b who found that drugs that were highly
absorbed (>90%) had a polar surface area less than 60 Å2 while
drugs that were poorly absorbed (<10%) had a polar surface
area greater than 140 Å2.15c
To improve the PSA values, we next synthesized a series of
fused-ring compounds, 19−21 (Scheme 3), which were
subsequently tested for their activities against BACE1 and
BACE2 (Table 3). The intended goals for the synthesis of such
scaffolds were threefold: to eliminate the peptidic nature, to
rigidify the system, and to decrease the PSA (Table 3).
Unfortunately, this brought about a 72-fold decrease in potency
toward BACE1 and a sharp decrease in the selectivity toward
BACE2 when compared to the previous series. As shown in the
docking pose of compound 20 (Figure 5), its functional groups
were not properly oriented between S3−S1′ subpockets,
resulting in low affinity toward the protein.
The decreased selectivity of these ligands could be attributed
to the increased linearity of the two handles on the central
phenyl ring (terephthalic acid derivative). Despite these
discouraging results, it was decided to synthesize the
regioisomers of compounds 19−21 (Scheme 4) so that
the intended derivatives possessed isophthalic acid backbone
similar to that present in compound I. The FRET assay results
of these motifs against BACE1 were promising (Table 4).
Compound 26 was about 12-fold more potent than its
regioisomer 21 and was found to be 23-fold more selective
toward BACE1 when compared to BACE2. Furthermore, the
activity in the cell based assay was importantly sustained during
this investigation. Compound 26 tracked well with its increased
Figure 5. Top-ranked docked pose of compound 20 into the BACE1
binding site. The four subsites S1′, S1, S2, and S3 are marked for clarity
with blue arcs.
molecular binding with an EC50 of 310 nM in the cell based
ELISA assay (Table 4).
Docking analysis of compound 26 (Figure 6) indicated
extensive vdW and H-bonding interactions with the protein.
These included hydrogen bonding with Thr72, Gly34, Asp 32,
Asp228, and Tyr14 and vdW interactions with the active site
amino acid sequence. At this junction, we envisioned that the
proximity of the tert-butyl group in 26 toward the polar parts in
the active site, as could be concluded from Figure 6, may result
in mutual steric clashing leading to lower affinity between this
class of ligands and the enzyme active site.
Therefore, we turned our attention toward the synthesis of
similar compounds devoid of such bulky arm. Thus, we carried
out the synthesis of compounds 31−34 (Scheme 5). Their
activities against BACE1 and BACE2 are shown in Table 5.
Compound 34 was found to be a strongly potent inhibitor with
an IC50 of 18 nM, which is comparable to that of compound I
(Figure 1). Furthermore, compound 34 was found to be 204-
fold more selective toward BACE1 when compared to BACE2
(IC50 = 3675 nM). The ligand efficiency of this compound was
found to be highly promising (LE = 1.7 kJ/mol) and the PSA
was 40 with Mwt of 346 Da.15 Furthermore, compound 34
exhibited potent activity in the cell-based ELISA assay (EC50
=
37 nM). The enhanced potency of compound 34 could be
rationalized from its docking pose (Figure 7), which is
characterized by two sets of interactions: (a) hydrophobic
dispersion interactions of the aromatic rings in the ligand with
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a
Table 4. SAR of BACE1 Inhibitors
BACE1
BACE2
ELISA
EC50 (nM)
compd
IC50 (nM)
KI (nM)
LE (kJ/mol)
tPSA (Å2)
IC50 (nM)
KI (nM)
24
25
26
514 ± 110
312 ± 34
178 ± 37
495 ± 121
301 ± 44
253 ± 56
NA
1.24
0.74
1.21
52
3785
4234
4123
3674
4110
4003
930 ± 89
310 ± 29
52
61.3
a
IC50 and EC50 values are the mean values of at least three experiments ± SD. NA means not applicable.
comparable cellular potency. Moreover, since the peptidic
nature and the number of rotatable bonds of these motifs are
low, they represent a novel class of druglike leads. On the basis
of the extensive SAR analysis of the transition state inhibitors of
BACE1, a pharmacophoric model (Figure 8) is proposed. In
this model, a central aryl ring, disubstituted on the meta-
positions with heteroaryl groups, is needed to properly orient
the ligands in the active site of the protein. A hydrogen bond
acceptor on the aromatic portion, resident on the heteroaryl
group located on the right side of the model, enhances the
affinity to BACE1 by projecting directly toward S1−S1′
subpockets. Furthermore, a fused heteroaromatic ring append-
age on the left side of the proposed model projects toward S3
subpocket of the protein and is needed for improved activity.
To further support this proposed model, an overlay of the most
potent compounds described in this article, in the enzyme
active site is depicted in Figure 9. The results described here
merit further investigations and developments in our
laboratories.
Figure 6. Top-ranked docked pose of compound 26 into the BACE1
binding site. The four subsites S1′, S1, S2, and S3 are also marked for
clarity with blue arcs.
EXPERIMENTAL SECTION
the neighboring hydrophobic side chains of amino acid residues
and (b) water mediated interactions of the polar atoms in the
ligand with the side chains of Asp32, Thr72, Gln328 as well as
direct electrostatic interactions with the side chain of Asp228.
Removal of the tert-butylamine group and introduction of a
fluoro- and/or methoxybenzimidazol group at the meta-
position of the central phenyl nucleus (e.g., compounds 33
and 34, Scheme 5) sharply increased the ligand potency (10-
fold increase) for BACE1 and highly improved the ligand’s
selectivity (about 300-fold) against BACE2. Interestingly,
replacement of the imidazopyridine nucleus with a fluoroimi-
dazopyridine moiety, 32−34, resulted in an additional
enhancement (40-fold) of the ligand’s affinity (34 vs 31).
Furthermore, the 34·HCl salt had appreciable solubility in
distilled water (10 mg/mL; for details see Experimental
Section).
■
Chemistry. Materials and General Methods. Reagents and
solvents were purchased from commercial sources and were used as
received. Reaction progress was monitored by thin-layer chromatog-
raphy on Merck silica gel 60 F-254 with detection by UV. Silica gel 60
1
(Merck 40−63 μm) was used for flash column chromatography. H
NMR and 13C NMR spectra were recorded with Bruker 300 and
Bruker AMX-400 spectrometers using DMSO-d6 or CDCl3. Data are
presented as follows: chemical shift (parts per million, ppm),
multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet;
br, broad; bs, broad singlet; bd, broad doublet), coupling constant
J (in hertz), and integration. Carbon magnetic resonance (13C NMR)
spectra were recorded at 75 or 125 MHz. Data for 13C NMR are
reported in terms of chemical shifts (ppm). High resolution mass
spectra were recorded in positive ion mode by electrospray ionization
(ESI) on a Bruker instrument. The samples were dissolved in
acetonitrile, diluted in spray solution (methanol/water (1:1 v/v)−
0.1% formic acid), and infused using a syringe pump with a flow rate of
2 mL/min. External calibration was conducted using arginine cluster in
a mass range m/z 175−871. For all HRMS data, mass error was 0.00−
0.50 ppm.
CONCLUSION
■
In this work we have optimized the synthesis of novel, low
molecular weight, and potent transition state inhibitors of
BACE1. Furthermore, we have developed peptidomimetic
groups to replace P1−P1′ and P3 moieties resident on
compound I. The synthetic methodologies followed were
simple, efficient, and economic. Several of the developed low
molecular weight scaffolds possessed high affinity, promising
ligand efficiency, and low PSA. Many of these motifs exhibited
All compounds were determined to be >95% pure by high-
performance liquid chromatography (HPLC). Purity of compounds
was determined on a Phenomenex Luna C18 (2) 3 mm column, 4.6
mm i.d. × 30 mm length, with 30−75% acetonitrile/water/0.1%
trifluoroacetic acid and 1.0 mL/min elution at room temperature using
210, 254, or 280 nm wavelength.
General Procedure A for the Synthesis of Compounds 6−8,
19−21, 24−26, and 31−34. To a solution of the desired carboxylic
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a
Table 5. SAR of BACE1 Inhibitors
BACE1
BACE2
ELISA
EC50 (nM)
compd
IC50 (nM)
KI (nM)
LE (kJ/mol)
tPSA (Å2)
IC50 (nM)
KI (nM)
31
32
33
34
961 ± 66
35 ± 8
26 ± 7
18 ± 2
926 ± 100
34 ± 9
25 ± 8
1550 ± 231
120 ± 21
30 ± 9
0.72
1.38
1.4
40
40
40
40
3890
3173
3321
3675
3777
3081
3224
3568
17 ± 3
37 ± 11
1.7
a
IC50 and EC50 values are the mean values of at least three experiments ± SD.
by dropwise addition of the appropriate α-bromoketone (1.1 mmol,
1.1 equiv) in DMF. The resulting mixture was stirred at 0 °C for 6 h,
then quenched with ice−water. The precipitated solid was filtered,
washed with water, and dissolved in EtOAc. The organic phase was
washed with a 1 N HCl aqueous solution, saturated NaHCO3 aqueous
solution, H2O, dried over MgSO4, and concentrated in vacuo, which
was used in the next stage without further purification. To the
appropriate α-keto ester were added AcOH (25 mL) and AcONH4
(924 mg, 12 mmol, 12 equiv), and the resulting suspension was
refluxed for 8 h, cooled to room temperature, concentrated in vacuo,
and diluted with crushed ice. The brown solid was filtered, washed
thoroughly with water. The crude cake was dissolved in EtOAc,
washed with a saturated NaHCO3 aqueous solution, H2O, dried over
MgSO4, and concentrated in vacuo. The crude product was purified by
flash chromatography on silica gel (DCM/AcOEt, 8/2 to 7/3) to give
compounds 12 (65%), 13 (69%), and 14 (73%).
General Procedure C for the Synthesis of Compounds 18
and 23. A mixture of the desired 2-aminopyridine (15 or 27)
(5.1 mmol) in MeOH−DCM (2:3, 15.0 mL) and phthalaldehyde
derivative (16 or 22, 5.0 mmol) containing 5 mol % of Sc(OTf)3 was
stirred for 1 h at room temperature, followed by the addition of 5.1
mmol of the tert-butyl isocyanide, and the mixture was stirred for
another 12 h at room temperature. Then 2 mL of hexane was added,
and the resulting yellowish solid was filtered, washed three times with
hexane−ethyl acetate mixture (5:1, 20 mL), and triturated with ethyl
acetate−hexane. The crude product was used in the next step without
further purification.
General Procedure D for the Synthesis of Compounds 29
and 30. In a two-necked round-bottom flask connected with a
condenser and under N2 were introduced 2-aminopyridin derivative
(15 or 27, 1.15 mmol), 2 methyl 3-(2-bromoacetyl)benzoate (28)
(334 mg, 1.3 mmol), and absolute EtOH (12 mL). The reaction
mixture was stirred at reflux for 2 h. After the reaction mixture had
cooled down, NaHCO3 (0.15 g, 1.75 mmol) was added and the
mixture refluxed for another 6 h. Solvent was removed and the residue
dissolved in EtOAc. The organic phase was washed thoroughly with
water (three times), dried over MgSO4, and filtered. The solvent was
removed, and crude products were purified by washing with 5 mL of
hexane. The resulting yellowish solid was filtered, washed three times
with hexane−ethyl acetate mixture (5:1, 10 mL), and triturated with
ethyl acetate−hexane to give crudes 29 and 30 in 67% and 61% yield,
respectively. The crude products were used in the next step without
further purification.
Figure 7. Top-ranked docked pose of compound 34 (ball and stick
rendering, carbon atoms in magenta) into the BACE1 binding site
based on 2B8L X-ray structure from the Protein Data Bank. The
protein atoms are shown in tube rendering. The four subsites S1′, S1,
S2, and S3 are also marked for clarity with blue arcs.
acids (2, 18, 23, 29, or 30) (1.0 mmol, 1 equiv) in DMF (8 mL) at
0 °C was added DIPEA (0.21 mL, 1.2 mmol, 1.2 equiv). After 10 min,
TBTU (551 mg, 1.2 mmol, 1.2 equiv) was added and the resulting
mixture stirred at the same temperature for 30 min. Then the desired
diaminobenzene derivative (1.1 mmol, 1.1 equiv) was added. The
resulting mixture was stirred at 0 °C for 6 h and then quenched with
ice−water. The precipitated solid was filtered, washed with water, and
dissolved in EtOAc. The organic phase was washed with a 1 N HCl
aqueous solution, then with a saturated NaHCO3 aqueous solution
and finally H2O, dried over MgSO4, and concentrated in vacuo, which
was used in the next stage without further purification. To the
appropriate amide was added AcOH (30 mL), and the resulting
suspension was refluxed for 6 h, cooled to room temperature,
concentrated in vacuo, and diluted with crushed ice. The brown solid
was filtered and washed thoroughly with water. The crude was
dissolved in EtOAc, washed with a saturated NaHCO3 aqueous
solution and with H2O, dried over MgSO4, and concentrated in vacuo.
The crude product was purified by flash chromatography on silica gel
(DCM/AcOEt, 8/2 to 7/3) to give the desired benzimidazole
derivative.
BACE1/BACE2 Enzymatic Assay. BACE1/BACE2 assays were
carried out according to the manufacturer described protocol available
from Invitrogen, U.S. (http://tools.invitrogen.com/content/sfs/
manuals/L0724.pdf). Briefly, BACE1/BACE2 in vitro assays were
carried out using fluorescence resonance energy transfer (FRET). An
APP-based peptide substrate (rhodamine-EVNLDAEFK-quencher, KM
General Procedure B for the Synthesis of Compounds 12−
14. To a solution of the carboxylic acids 2 (1.0 mmol, 1 equiv) in
DMF (10 mL) at 0 °C was added DIPEA (0.21 mL, 1.2 mmol, 1.2
equiv). The resulting mixture was stirred at 0 °C for 30 min followed
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Figure 8. Proposed pharmacophoric model based on isophthalic acid BACE1 inhibitors.
where vI is the steady state rate of substrate hydrolysis in the presence
of inhibitor concentration [I] and where v0 is the uninhibited
rate. From there KI(app) can be obtained. In the case of v0/vI = 2,
KI(app) = IC50. An IC50 is relative because it depends on substrate
concentration. Therefore, an absolute KI value can be calculated by
taking into account the substrate concentration [S] and the affinity of
the substrate to the enzyme, indicated by the KM, according to the
following equation:
As an indication of the efficiency of the compounds toward binding
BACE1 compared to known BACE1 inhibitors, the ligand efficiency
was calculated for each compound based on the free energy equation,
Figure 9. Overlap of top-ranked docked poses of compounds 7 (gray
carbons), 13 (yellow carbons), 20 (magenta carbons), 26 (orange
carbons), and 34 (red carbons) into the BACE1 binding site based on
2B8L X-ray structure from the Protein Data Bank. The overlap defines
the pharmacophore of their interactions in the four subsites S1′, S1, S2,
and S3 marked for clarity with blue arcs. The protein atoms are shown
in tube rendering.
and the equation of binding energy per atom (ligand efficiency, LE),
Cell Based Assay. Aβ42 and Aβ40 were measured in culture
medium of H4 cells (human neuroglioma cell line) expressing the
double Swedish mutation (K595N/M596L) of human APP (APPsw).
Cells were seeded onto 24-well plates (2 × 105 cells well−1) and
allowed to grow for 24 h in 5% CO2/95% air in a humidified
atmosphere. Increasing concentrations of compounds were added to
the cells for overnight in a final volume of 0.5 mL. R-Flurbiprofen was
used as positive control. DMSO-d6 (1%) was used as negative control.
At the end of the incubation, an amount of 100 μL of supernatant was
removed and treated with a biotinylated mouse monoclonal antibody
(4G8, Signet Laboratories Inc., Dedham, MA, U.S.), specifically
recognizing the 17−24 amino acid region of Aβ and two rabbit
polyclonal antibodies (C-term 42 and C-term 40, BioSource
International, Camarillo, CA, U.S.), specifically recognizing the
C-terminus of Aβ42and Aβ40, respectively. Antigen−antibodies 5
complexes were recognized by TAG-donkey anti-rabbit IgG (Jackson
Immuno Research Laboratories, Soham, U.K.). Streptavidin coated
magnetic beads captured the complexes, and the signals were read by
an electrochemiluminescence instrument (Origen M8 analyzer,
BioVeris Corporation, Gaithersburg, MD, U.S.). The cytotoxicity
potential of test compound was assessed in the same cells of the Aβ
assay using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide (MTT) assay. After medium removal for Aβ42 and Aβ40
determination, cells were incubated for 3 h with 500 μL of culture
medium containing 0.5 mg mL−1 MTT at 37 °C, 5% CO2, and
saturated humidity. After removal of the medium, an amount of 25−
500 μL of 100% DMSO-d6 was added to each well. The amount of
formed formazan was determined by reading the samples at 570 nm
of 20 μM) carrying the Swedish mutation and containing a rhodamine
as a fluorescence donor and a quencher acceptor at each end was used.
The intact substrate is weakly fluorescent and becomes highly
fluorescent upon enzymatic cleavage. The assays were conducted for
both enzymes in 50 mM sodium acetate buffer, pH 4.5, in a final
enzyme concentration (1 U/mL). Inhibitor (first line screening, 30,
10, 3, 1, and 0.3 μM) compounds that showed high activity at 0.3 μM
were validated at low concentration, and both substrates were used at
750 nM. Inhibitor compounds were diluted from stock solutions to
result in 3.3% DMSO final concentration. The mixture was incubated
for 60 min at 25 °C under dark conditions and then stopped with
2.5 M sodium acetate. Fluorescence was measured with a Victor3 1420
(Wallac) microplate reader at 545 nm excitation and 585 nm emission.
The assay kit was validated by manufacturer. The obtained values are
the mean values of three different experiments. IC50 values were
calculated by plotting the obtained relative fluorescence unit per hour
(RFU/h) against the logarithmic of inhibitor concentration. The
measured inhibition data were analyzed in GraphPad Prism 4 for
Windows (GraphPad Software Inc., La Jolla, CA, U.S.) by nonlinear
regression (curve fitting). The observed inhibition constant in the
presence of substrate, KI (app), is calculated according to the following
equation:
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(background, 630 nm) using a microplater reader (model 450, Bio-
Rad, Hercules, CA, U.S.).
cooled to 0 °C. To this solution was added dropwise 1 equiv of cold
solution of concentrated HCl in 2 mL of DCM. The mixture was
stirred at 0 °C for 45 min and then evaporated to dryness under high
vacuum.
Molecular Modeling. All molecular modeling studies were
carried out using the Sybyl-X program, version 1.3. The X-ray
structures of the proteins in 2B8L and 3FKT were “prepared” in the
presence of their respective bound ligands. These two protein−ligand
complexes were chosen in light of the fact that their active sites, while
being similar in size and shape, bind to their respective ligands in
somewhat different modes. Interestingly, the overall three-dimensional
topologies of the protein structures are very similar with minor
conformational changes seen in the residues near the active site. The
bound ligands were modeled to reflect their correct chemical states
relevant to the binding interactions in the active site. Since the original
X-ray structures do not have explicit bond orders, it is important to
represent the aromaticity of rings, planarity of amides, and
hybridization states of heteroatoms appropriately. The ligand
preparation in Sybyl-X accounts for these states in an automated
fashion. Hydrogen atoms were added on the protein atoms and on the
water oxygen atoms. The orientations of water hydrogen atoms were
randomly chosen. The states and orientations of the imidazole rings in
histidine side chains close to the active site were adjusted to optimize
interactions with the ligand and neighboring residues. In addition, the
side chain amide moieties in Asn and Gln residues were also optimized
for their interactions with neighboring functional groups in the bound
ligand and protein residues. In each of the two cases, the protein−
ligand−water complex thus obtained was subjected to a staged energy
minimization which consisted of six stages: (1) minimization of
hydrogen positions, (2) minimization of waters, (3) minimization of
side chain atoms, (4) minimization of the entire protein structure
minus the Cα atoms, (5) minimization of the ligand, and (6)
minimization of all the atoms. In each stage, 100 cycles of energy
minimizations were carried out using the Tripos force field. The main
goal of this minimization is to clean up the original X-ray structure
after the addition of hydrogen atoms and to orient the waters with
optimal interactions toward protein residues and the bound ligand.
Thus, the above procedure maintains the integrity of the experimental
structures of 2B8L and 3FKT. The prepared protein−ligand−water
complexes were used to generate respective protomols in Surflex-
Dock18 with the option of “Ligand”. The protomols essentially
represent the signatures of the active sites in docking studies with
Surflex-dock. The X-ray crystallographic water molecules were retained
as a segment of the protein structures during the protomol generation.
Such water molecules are treated flexibly in the docking process in
Surflex-Dock, with the hydrogen atoms being allowed to move while
the oxygen atoms are held in place. The ligands (7, 13, 20, 26, and 34)
were prepared using CONCORD (in the ligand preparation module of
Sybyl-X) with the generation of all possible tautomeric and ionization
states, where applicable. They were energy minimized using the
MMFF94s force field in Sybyl-X. The prepared ligands were docked
into the prepared 2B8L and 3FKT using the above generated
protomols in Surflex-Dock with the GEOMX docking mode. Protein
flexibility including heavy atoms and hydrogen atoms within 12 Å of
the ligand poses was incorporated in the postdocking treatment of the
docked poses.18 The docked poses were examined for their hydrogen
bonding interaction active site residues using the Surflex-Dock results
browser in the Sybyl-X interface.
Calibration Curve. The calibration curve was plotted with five
different concentrations of 34·HCl salt. Five standard solutions (Table 6)
Table 6. Calibration Curve of Compound 34 in Acetonitrile/
Methanol
concn (mg/mL)
area
slope
intercept
21.18
r2
1.44
2.5
1739
3100
11854
0.9995
5.0
5992
10.0
18.0
11557
21505
were prepared in a mixture of acetonitrile/methanol, 50:50 v/v. The final
concentrations of the standard solutions were 1.44, 2.50, 5.0, 10.0, and
18.0 mg/mL. The linearity was evaluated by linear regression analysis,
which was calculated by the least-squares regression method. Before the
solutions were injected, the column was equilibrated for at least 45 min
with the mobile phase flowing through the system. Three determinations
were carried out for each solution. Peak areas were recorded for all the
solutions. The correlation graph was constructed by plotting peak areas
obtained at the optimum wavelength of detection versus the injected
amounts. A linear response was observed over the examined
concentration range. Table 7 summarizes the correlation coefficient,
slope, and intercept.
Table 7. Compound 34·HCl Salt in Distilled Water
concn (mg/mL)
area
recovery (%)
99.7
10 (HCl in distilled water)
12073.27
Solubility Test. An accurately weighed sample of 34·HCl salt was
dissolved in distilled water to obtain a final concentration equivalent to
10 mg/mL. The solution was sonicated in a water bath for 48 h,
filtered through a 0.45 μm Millipore filter, then injected into the
HPLC system after running a calibration curve (Figure 10) of 34·HCl
Compound 34·HCl Solubility Test. Acetonitrile (HPLC grade)
was purchased from Sigma-Aldrich. Water used in the mobile phase
was deionized, distilled, and filtered through a 0.45 μm Millipore filter
(Sartorius, Germany) under vacuum before use. Methanol (Fluka) was
HPLC grade. An Agilent HPLC system (U.S.) was used to perform
the solubility test. A reversed phase high-performance liquid
chromatographic method was used to test the solubility of the
34·HCl salt in distilled water. The mobile phase was a mixture of
acetonitrile and water (30:70, v/v). The flow rate was 1.0 mg/mL. The
UV detector wavelength was set at 280 nm. A Waters reversed-phase
C18 column (25 cm × 4.6 mm i.d., particle size 5 μm) was utilized as
stationary phase.
Figure 10. Calibration graph of standard solutions of 34·HCl.
salt dissolved into a mixture of acetonitrile/methanol, 50:50 v/v. The
recovery of 34·HCl salt was found to be 99.7% (Table 7). We found
that increasing the concentration of 34 above 10 mg/mL decreased the
recovery proportionally to the increasing concentration of 34·HCl.
Therefore, we concluded that the solubility is around 10 mg/mL.
Preparation of Compound 34·HCl Salt. An amount of 1 mmol
of compound 34 was dissolved in 10 mL of DCM, and the mixture was
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(S)-3-(Methylsulfonamido)-5-(1-phenylethylcarbamoyl)-
benzoic Acid (2). 1H NMR (DMSO-d6): δ 9.14 (1H, d, J = 8.1Hs),
8.39 (1H, d, J = 1.5Hs), 8.11 (1H, dd, J = 1.8, 3.6 Hz), 8.06 (1H, dd,
J = 1.8, 3.3 Hz), 7.41−7.22 (5H, m), 5.18 (1H, t, J = 7.2 Hz), 3.00
(3H, s), 2.49 (3H,s), 1.49 (3H, d, J = 7.2 Hz). HRMS: m/z [M + Na]
calcd for C17H18N2O5SNa, 385.3922 g/mol; found, 385.3795.
(S)-3-(1H-Benzo[d]imidazol-2-yl)-5-(N-methylmethylsulfona-
mido)-N-(1-phenylethyl)benzamide (6). The synthetic procedure
was the same as general procedure A. Yield 68%. 1H NMR (CDCl3): δ
8.29 (1H, t, J = 1.8 Hz), 7.60 (2H, m), 7.45 (1H, bs), 7.36 (1H, bs),
7.21 (2H, dd, J = 3.0, 6.6 Hz), 7.17−6.97 (6H, m), 6.38 (1H, bs), 5.33
(1H, dd, J = 8.3, 13.9 Hz), 3.14 (3H, S), 2.76 (3H, s), 1.51 (3H, d, J =
6.8). 13C NMR (CDCl3): δ 167.1, 155.9, 143.0, 139.7, 139.2, 136.9,
132.5, 129.1, 129.0, 128.5, 123.6, 122.1, 120.5, 118.1, 111.4, 52.8, 41.1,
34.4, 14.2. HRMS: m/z [M + Na] calcd for C24H24N4O3SNa,
471.5299 g/mol; found, 471.1449.
(S)-3-(5-Methoxy-1H-benzo[d]imidazol-2-yl)-5-(N-methyl-
methylsulfonamido)-N-(1-phenylethyl)benzamide (7). The
synthetic procedure was the same as general procedure A. Yield
72%. 1H NMR (CDCl3): δ 8.30 (1H, bs), 8.1 (1H, bs), 7.74 (1H, bs),
7.41 (1H, bd, J = 8.7 Hz), 7.27−7.15 (6H, m), (1H, bs), 6.85 (1H, dd,
J = 2.1, 9.0 Hz), 5.19 (1H, t, J = 7.2 Hz), 3.77 (3H, s), 3.15 (3H, s),
2.70 (3H,s), 1.48 (3H, d, J = 6.9 Hz). 13C NMR (CDCl3): δ 169.7,
160.1, 158.3, 146.1, 145.1, 141.2, 138.5, 133.4, 131.2, 129.7, 128.8,
128.2, 123.4, 121.1, 120.3, 114.2, 111.3, 99.1, 55.3, 48.2, 38.2, 32.3,
14.3. HRMS: m/z [M + Na] calcd for C25H26N4O4SNa, 501.5560
g/mol; found, 501.1554.
(S)-3-(5-Fluoro-1H-benzo[d]imidazol-2-yl)-5-(N-methylme-
thylsulfonamido)-N-(1-phenylethyl)benzamide (8). The syn-
thetic procedure was the same as general procedure A. Yield 62%.
1H NMR (CDCl3): δ 8.28, (1H, dd, J = 1.8, 4.2 Hz), 8.25 (1H, d, J =
8.7 Hz), 7.50 (1H, dd, J = 2.7, 5.4 Hz), 7.47 (1H, d, J = 4.5 Hz), 7.37
(1H, d, J = 4.8 Hz), 7.14 (3H, m), 6.97 (3H, m), 6.37 (1H, bs), 5.34
(1H, dd, J = 8.4, 14.4 Hz), 3.15 (3H, s), 2.72 (3H, s), 1.46 (3H, d, J =
6.9 Hz). 13C NMR (CDCl3): δ 167.1, 156.8, 155.9, 153.3, 143.8, 143.7,
143.1, 139.7, 136.7, 131.4, 128.4, 128.1, 127.6, 121.2, 119.6, 116.2,
116.1, 114.9, 114.6, 112.2, 111.8, 110.2, 49.7, 38.4, 31.9, 14.3. HRMS:
m/z [M + Na] calcd for C24H23FN4O3SNa, 489.5203 g/mol; found,
489.1360.
4-(3-(tert-Butylamino)imidazo[1,2-a]pyridin-2-yl)benzoic
Acid (18). The synthetic procedure was the same as general
procedure C. Yield 73%. The synthetic procedure was the same as
general procedure A. Yield 67%. 1H NMR (DMSO-d6): δ 8.39 (1H, d,
J = 6.9 Hz), 8.26 (2H, m), 7.99 (2H, bs), 7.44 (1H, bs), 7.20 (1H, d,
J = 6.9 Hz), 6.88 (1H, t, J = 6.9 Hz), 4.65 (1H, bs), 0.98 (9 H, s).
HRMS: m/z [M + Na] calcd for C18H19N3O2Na, 332.3538 g/mol;
found, 332.3419.
2-(4-(1H-Benzo[d]imidazol-2-yl)phenyl)-N-tert-butylimidazo-
[1,2-a]pyridin-3-amine (19). The synthetic procedure was the same
1
as general procedure A. Yield 73%. H NMR (CDCl3): δ 6.04 (1H, d,
J = 6.9 Hz), 5.94 (2H, d, J = 8.4 Hz), 5.82 (2H, d, J = 8.4 Hz), 5.41
(1H, dd, J = 3.3, 6.0 Hz), 5.28 (1H, d, J = 9.0 Hz), 5.01 (4H, m), 4.61
(1H, t, J = 6.6 Hz), 1.20 (9H, s). 13C NMR (CDCl3): δ 152.1, 147.8,
144.8, 139.9, 137.9, 133.9, 132.2, 132.1, 124.7, 124.4, 122.5, 121.0,
120.2, 119.6, 118.5, 112.3, 110.7, 107.7, 51.8, 26.0. HRMS: m/z [M +
H] calcd for C24H24N5, 382.4830 g/mol; found, 382.2047.
N-tert-Butyl-2-(4-(6-fluoro-1H-benzo[d]imidazol-2-yl)-
phenyl)imidazo[1,2-a]pyridin-3-amine (20). The synthetic pro-
1
cedure was the same as general procedure A. Yield 65%. H NMR
(CDCl3): δ 8.24 (1H, d, J = 6.9 Hz), 8.0 (2H, d, J = 8.4 Hz), 7.81 (2H,
d, J = 8.4 Hz), 7.5 (1H, d, J = 9.3 Hz), 7.47 (1H, m), 7.16 (2H, m),
6.91 (1H, t, J = 1.8 Hz), 6.82 (1H, t, J = 6.9 Hz), 0.97 (9H, s). 13C
NMR (CDCl3): δ 161.0, 157.8, 153.2, 144.8, 144.4, 141.8, 138.4,
136.0, 128.9, 128.5, 126.8, 125.0, 124.1, 123.6, 116.1, 111.4, 110.8,
110.4, 56.4, 30.1. HRMS: m/z [M + Na] calcd for C24H22FN5Na,
422.4553 g/mol; found, 422.1749.
N-tert-Butyl-2-(4-(6-methoxy-1H-benzo[d]imidazol-2-yl)-
phenyl)imidazo[1,2-a]pyridin-3-amine (21). The synthetic pro-
1
cedure was the same as general procedure A. Yield 69%. H NMR
(CDCl3): δ 8.77 (1H, d, J = 7.8 Hz), 8.14 (1H, d, J = 8.4 Hz), 7.87
(3H, m), 7.48 (1H, d, J = 8.4 Hz), 7.16 93H, m), 6.85 (1H, t, J = 6.6
Hz), 3.86 (3H, s), 1.20 (9H, s). 13C NMR (CDCl3): δ 155.2, 152.1,
144.8, 142.3, 133.3, 131.5, 128.6, 127.9, 124.2, 123.7, 118.7, 117.1,
113.2, 112.0, 105.3, 98.3, 55.3, 54.6, 31.1. HRMS: m/z [M + Na] calcd
for C25H25N5ONa, 434.4910 g/mol; found, 434.1947.
3-(3-(tert-Butylamino)imidazo[1,2-a]pyridin-2-yl)benzoic
Acid (23). The synthetic procedure was the same as general
procedure C. Yield 71%. 1H NMR (DMSO-d6): δ 8.84 (1H, bs),
8.44 (1H, bs), 8.42 (1H, bs), 7.84 (1H, bs), 7.51 (1H, bs), 7.48 (1H,
bs), 7.24 (1H, d, J = 8.1 Hz), 6.93 (1H, t, J = 6.9 H), 4.71 (1H, bs),
1.01 (9H, s). HRMS: m/z [M + Na] calcd for C18H19N3O2Na,
332.3538 g/mol; found, 332.3431.
(S)-3-(N-Methylmethylsulfonamido)-5-(5-phenyl-1H-imida-
zol-2-yl)-N-(1-phenylethyl)benzamide (12). The synthetic proce-
dure was the same as general procedure B. Yield 65%. 1H NMR
(CDCl3): δ 8.0 (1H, bs), 7.84 (1H, bs), 7.68 (2H, bd, J = 7.2H), 7.50
(2H, bs), 7.35−7.19 (10H, m), 5.13 (1H, t, J = 7.2 Hz), 3.04 (3H, s),
2.60 (3H, s), 1.48 (3H, d, J = 6.9 Hz). 13C NMR (CDCl3): δ 171.1,
166.0, 145.4, 142.9, 141.8, 135.9, 131.5, 128.7, 128.5, 127.3, 127.1,
126.2, 126.1, 125.7, 124.9, 124.2, 122.3, 60.3, 49.9, 37.5, 35.4, 21.6,
20.9, 14.1. HRMS: m/z [M + Na] calcd for C26H26N4O3SNa,
497.5673 g/mol; found, 497.1621.
2-(3-(1H-Benzo[d]imidazol-2-yl)phenyl)-N-tert-butylimidazo-
[1,2-a]pyridin-3-amine (24). The synthetic procedure was the same
1
as general procedure A. Yield 65%. H NMR (CDCl3): δ 8.77 (1H, d,
J = 7.8 Hz), 8.51 (1H, bs), 8.35 (1H, t, J = 8.1 Hz), 8.20 (1H. d, J = 9.0
Hz), 7.85 (2H, m), 7.59 (2H, m), 7.23 (3H, m), 6.84 (1H, t, J = 8.1
Hz), 1.2 (9H, s). 13C NMR (CDCl3): δ 153.2, 144.8, 138.8, 137.1,
132.0, 130.0, 129.1, 128.7, 127.9, 125.0, 124.0, 123.7, 123.5, 118.7,
116.1, 112.0, 105.2, 54.6, 31.1. HRMS: m/z [M + Na] calcd for
C24H23N5Na, 404.4649 g/mol; found, 404.1840.
(S)-3-(5-(3-Methoxyphenyl)-1H-imidazol-2-yl)-5-(N-methyl-
methylsulfonamido)-N-(1-phenylethyl)benzamide (13). The
synthetic procedure was the same as general procedure B. Yield
69%. 1H NMR (CDCl3): δ 8.37 (1H, d, J = 2.1 Hz), 8.00 (1H, d, J =
9.97), 7.52 (1H, dd, J = 8.4 Hz), 7.45 (2H, d, J = 4.8 Hz), 7.43 (1H, s),
7.15 (2H, dd, J = 2.7, 4.5 Hz), 7.05−6.92 (5H, m), 5.34 (1H, t, J = 8.4
Hz), 3.91 (3H, s), 2.71 (3H,s), 1.50 (3H, d, J = 6.9 Hz). 13C NMR
(CDCl3): δ 170.0, 162.1, 151.2, 145.1, 141.3, 139.7, 138.2, 133.6,
130.5, 129.6, 129.6, 129.1, 128.5, 125.4, 123.3, 123.1, 121.9, 120.2,
119.8, 112.1, 110.5, 53.4, 50.1, 39.3, 32.2, 14.1. HRMS: m/z [M + Na]
calcd for C27H28N4O4SNa, 527.5934 g/mol; found, 527.5832.
(S)-3-(5-(3-Fluorophenyl)-1H-imidazol-2-yl)-5-(N-methylme-
thylsulfonamido)-N-(1-phenylethyl)benzamide (14). The syn-
N-tert-Butyl-2-(3-(6-fluoro-1H-benzo[d]imidazol-2-yl)-
phenyl)imidazo[1,2-a]pyridin-3-amine (25). The synthetic pro-
1
cedure was the same as general procedure A. Yield 68%. H NMR
(CDCl3): δ 6.61 (1H, bs), 6.49 (1H, d, J = 6.7 Hz), 6.24 (1H, d, J =
7.8 Hz), 6.10 (1H, d, J = 7.8 Hz), 5.69 (3H, m), 5.53 (1H,d, J = 6.9
Hz), 5.41 (1H, t = 7.8 Hz), 5.20 (1H, dt, J = 2.4, 9.0, 11.4 Hz), 5.03 (1
H, t, J = 6.6 Hz), 1.00 (9H, s). 13C NMR (CDCl3): δ 156.8, 153.3,
153.2, 144, 143.4,143.3, 137.1, 133.0, 132.9, 132.1, 130.1, 129.5, 129.0,
128.2, 125.1, 124.1, 123.9, 119.1, 117.0, 116.4, 115.3, 114.9, 113.1,
111.6. HRMS: m/z [M + Na] calcd for C24H22FN5Na, 422.4553
g/mol; found, 422.1741.
1
thetic procedure was the same as general procedure B. Yield 73%. H
N-tert-Butyl-2-(3-(6-methoxy-1H-benzo[d]imidazol-2-yl)-
NMR (CDCl3): δ 8.13, (1H,bs), 7.94 (1H,bs), 7.63 (3H, m), 7.32−
7.23 (6H, m), 7.09 (1H, bs), 7.00 (2H, t, J = 8.7 Hz), 5.18 (1H, t, J =
7.21 Hz), 3.16 (3H, s), 2.73 (3H, s), 1.51 (3H, d, J = 6.9 Hz). 13C
NMR (CDCl3): δ 171.1, 166.0, 163.7, 160.3, 145.4, 142.8, 141.9,
135.9, 131.6, 128.6, 127.3, 126.5, 126.4, 126.0, 124.2, 123.4, 115.6,
115.1, 60.3, 49.9, 37.7, 35.5, 14.1. HRMS: m/z [M + Na] calcd for
C26H25FN4O3SNa, 515.5578 g/mol; found, 515.1522.
phenyl)imidazo[1,2-a]pyridin-3-amine (26). The synthetic pro-
1
cedure was the same as general procedure A. Yield 58%. H NMR
(CDCl3): δ 8.11 (1H, bs), 8.00 (1H, d, J = 6.9 Hs), 7.68 (1H, d, J =
7.8 Hz), 7.59 (1H, d, J = 7.8 Hz), 7.23−7.09 (3H, m), 6.85 (1H, t, J =
8.1 Hz), 6.76 (1H, d, J = 1.8 Hz), 6.56 (1H, dd, J = 2.1, 8.7 Hz), 6.5
(1H, d, J = 6.6 Hz), 3.52 (3H,s), 1.12 (9H, s). 13C NMR (CDCl3): δ
156.3, 151.31, 141.6, 137.9, 135.1, 129.4, 129.3, 128.8, 125.8, 125.1,
8383
dx.doi.org/10.1021/jm201181f|J. Med. Chem. 2011, 54, 8373−8385

Journal of Medicinal Chemistry
Article
124.9, 124.3, 124.0, 115.8, 113.1, 111.5, 55.9, 55.3, 29.5. HRMS: m/z
[M + Na] calcd for C25H25N5ONa, 434.4910 g/mol; found, 434.1954.
2-(3-(Imidazo[1,2-a]pyridin-2-yl)phenyl)-1H-benzo[d]-
imidazole (31). The synthetic procedure was the same as general
activity relationship; PSA, polar surface area; LC−MS, liquid
chromatography−mass spectrometry; DCM, dichloromethane;
DMF, N,N-dimethylformamide; DIPEA, N,N-diisopropylethyl-
amine; HE, hydroxyethylene; FRET, fluorescence resonance
energy transfer; FRU/h, relative fluorence unit per hour; LE,
ligand efficiency; ELISA, enzyme-linked immunosorbent assay;
HRMS, high resolution mass spectrometry
1
procedure A. Yield 66%. H NMR (CDCl3): δ 8.59 (1H, d, J = 8.1
Hz), 8.38 (2H, m), 7.89 (1H, bs), 7.83 (1H, m), 7.59 (6H, m), 7.34
(1H, m), 7.23 (2H, dd, J = 3.3, 6.3 H), 7.00 (2H, t, J = 1.2 Hz). 13C
NMR (CDCl3): δ 153.2, 151.0, 147.5, 138.8, 133.9, 130.6, 128.7,
128.4, 126.6, 126.2, 125.7, 123.9, 123.5, 116.1, 115.0, 112.4, 103.0.
HRMS: m/z [M + Na] calcd for C20H14N4Na, 333.3435 g/mol; found,
333.2437.
REFERENCES
■
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6-Fluoro-2-(3-(imidazo[1,2-a]pyridin-2-yl)phenyl)-1H-benzo-
[d]imidazole (32). The synthetic procedure was the same as general
1
procedure A. Yield 61%. H NMR (CDCl3): δ 9.06 (1H, bs), 8.59
(1H, d, J = 8.1 Hz), 8.37 (2H, m), 8.25 (1H, d, J = 9.0 Hz), 7.93 (1H,
bs), 7.82 (1H, dd, J = 1.2, 3.0 Hz), 7.64 (1H, bd, J = 8.4 Hz), 7.51
(1H, dd, J = 2.7, 5.4 Hz), 7.35 (1H, t, J = 6.6 Hz), 7.14 (1H, t, J = 3.9
Hz), 6.97 (1H,t, J = 5.4 Hz). 13C NMR (CDCl3): δ 156.8,153.4, 153.2,
151.0, 147.5, 143.5, 143.1, 133.9, 133.0, 132.7, 129.8, 129.0, 128.6,
127.0, 126.8, 126.1, 123.8, 117.1, 116.7, 115.3, 115.1, 114.7, 113.0,
129.6, 111.8, 103.1. HRMS: m/z [M + Na] calcd for C20H13FN4Na,
351.3340 g/mol; found, 351.3253.
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2-(3-(Imidazo[1,2-a]pyridin-2-yl)phenyl)-6-methoxy-1H-
benzo[d]imidazole (33). The synthetic procedure was the same as
1
general procedure A. Yield 69%. H NMR (CDCl3): δ 9.06 (1H, bs),
8.6 (1H,d, J = 8.1 Hz), 8.40 (2H,m), 7.93 (1H, bs), 7.81 (1H, dd, J =
1.5, 4.7 Hz), 7.75 (1H, bd, J = 8.3 Hz), 7.48 (1H, bd, J = 8.4 Hz), 7.34
(1H, t, J = 8.7 Hz), 7.15 (1H, dd, J = 2.7, 8.4 Hz), 7.10 (1H, bd, J = 3.0
Hz), 7.0 (1H, t, J = 6.6 Hz), 3.86 (3H,s). 13C NMR (CDCl3): δ 155.2,
153.2, 151.0, 147.5, 141.1, 133.8, 130.5, 130.3, 128.7, 128.4, 126.6,
126.2, 125.7, 123.9, 117.1, 115.0, 113.2, 112.4, 103.0, 98.3, 55.3.
HRMS: m/z [M + Na] calcd for C21H16N4ONa, 363.3696 g/mol;
found, 363.3479.
6-Fluoro-2-(3-(7-fluoroimidazo[1,2-a]pyridin-2-yl)phenyl)-
1H-benzo[d]imidazole (34). The synthetic procedure was the same
1
as general procedure A. Yield 63%. H NMR (CDCl3): δ 9.14 (1H,s),
8.29 (2H,m), 8.24 (2H,m), 7.93 (1H,bs), 7.82 (2H,m), 7.51 (1H, dd,
J = 2.7,5.4 Hz), 7.26 (1H, bd, J = 1.0 Hz), 7.13 (1H, dd, J = 2.4,5.1
Hz), 7.10 (1H, dd, J = 2.4, 5.1 Hz). 13C NMR (CDCl3): δ 156.8,
153.4, 153.2, 151.0, 147.6, 147.5, 143.4, 143.3, 133.9, 133.1, 133.0,
129.6, 128.7, 128.4, 126.5, 126.4, 125.7, 123.9, 116.2, 116.1, 114.9,
114.6, 112.2, 111.8, 105.0, 104.6, 103.0, 98.1, 97.9. HRMS: m/z [M +
Na] calcd for C20H12F2N4Na, 369.3244 g/mol; found, 369.3122.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures for the synthesis of 6−34, in vitro
assays and protocols for the determination of KI, and dose
response curves. This material is available free of charge via the
Internet at http://pubs.acs.org.
(9) CoMentis. Press Release Jul 28, 2008. CoMentis and Astellas To
Present Alzheimer’s Disease Research at International Conference on
Alzheimer’s Disease (ICAD). http://www.athenagen.com/index.
php?/athenagen/press_releases/52/ (2008).
AUTHOR INFORMATION
Corresponding Author
*Phone: (971) 50 1732950. Fax: +971 6 5585812. E-mail: taltal@
sharjah.ac.ae.
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■
ACKNOWLEDGMENTS
■
The authors are grateful to the College of Graduate Studies and
Research (University of Sharjah, UAE) for partially funding
this research project. Dr. Thomas Nittoli and Dr. Sana Enayeh
are greatly acknowledged for their valuable discussions and
proofreading.
ABBREVIATIONS USED
■
AD, Alzheimer’s disease; APP, amyloid precursor protein;
BACE1, β-site amyloid precursor protein cleaving enzyme 1;
HPLC, high performance liquid chromatography; SAR, structure−
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