Thiophene substituted acylguanidines as BACE1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.08.010

A series of thiophene-substituted acylguanidines were designed from a pyrrole substituted acylguanidine HTS lead. This template allowed a greater flexibility, through differential Suzuki couplings, to explore the binding site of BACE1 and to enhance the i

Full text of DOI:10.1016/j.bmcl.2007.08.010

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5353–5356
Thiophene substituted acylguanidines as BACE1 inhibitors
William F. Fobare,a,* William R. Solvibile,a Albert J. Robichaud,a Michael S. Malamas,a
Eric Manas,a Jim Turner,b Yun Hu,b Erik Wagner,b Rajiv Chopra,c Rebecca Cowling,d
Guixan Jind and Jonathan Bardb
aChemical and Screening Sciences, Wyeth Research, CN8000, Princeton, NJ 08543-8000, USA
bNeurosciences Discovery Research, Wyeth Research, CN8000, Princeton, NJ 08543-8000, USA
cChemical and Screening Sciences, Wyeth Research, 200 Cambridge Park Drive, Cambridge, MA 02140, USA
dChemical and Screening Sciences, Wyeth Research, 401 N. Middletown Road, Pearl River, NY 10965, USA
Received 22 June 2007; revised 3 August 2007; accepted 6 August 2007
Available online 11 August 2007
Abstract—A series of thiophene-substituted acylguanidines were designed from a pyrrole substituted acylguanidine HTS lead. This
template allowed a greater flexibility, through differential Suzuki couplings, to explore the binding site of BACE1 and to enhance the
inhibitory potencies. This exploration provided a 25-fold enhancement in potency to yield compound 10a, which was 150 nM in a
BACE1 FRET assay.
Ó 2007 Elsevier Ltd. All rights reserved.
Alzheimer’s disease (AD) is an age-related and irrevers-
ible brain disorder that occurs gradually and results in
memory loss, behavior, and personality changes, a de-
cline in cognition and eventually death.1 These losses
are related to the breakdown of the connections between
nerve cells in the brain and the eventual death of many
of these cells. The course of this disease varies from per-
son to person, as does the rate of decline. On average,
patients with AD live for 8–10 years after they are diag-
nosed, though the disease can last for up to 20 years.2
One primary theory on the etiology of AD is that in
AD patients, amyloid precursor protein (APP) is
processed in the brain and converted to beta-amyloid
protein, a precursor to amyloid plaques.3 b-Secretase
(b-site APP cleavage enzyme or BACE1) and c-secretase
are two important enzymes involved in the amyloid syn-
thetic cascade. BACE1 is an aspartyl protease that initi-
ates APP proteolytic cleavage generating the membrane
bound C-terminal APP fragment (bCTF/C99). Further
processing of this fragment by c-secretase liberates the
pathogenic Ab40ꢀ42 peptide.4 Aggregation of Ab40–42
peptide eventually leads to oligomeric amyloid plaques,
a hallmark of AD. It is believed that inhibition of
BACE1 or c-secretase will inhibit this amyloidogenic
APP cleavage thus preventing the formation of
b-amyloid.
Our goal was to produce a potent and selective non-pep-
tidic small molecule inhibitor of BACE1. While there
have been numerous reported substrate–based peptide
inhibitors, there have been only a few examples of
non-peptide based inhibitors.5 In the course of running
an HTS, the tri-substituted pyrrole 16 was identified,
which had an IC50 of 3.7 lM for BACE1 in a FRET as-
say and an ED50 of 8.9 lM in our cellular ELISA. Our
initial efforts at examining the scope and breadth of the
SAR of the pyrrole lead 1 were limited due to the diffi-
culty in the substitution of a wide variety of aryl and
heteroaryl moieties on the pyrrole ring. It became obvi-
ous to us very early that the sometimes unstable and
NH2
NH2
O
N
O
Y
O
X
N
S
2
1
difficult to prepare a-substituted halopyrrole precursors
would prevent a rapid entry to divergent substitution
patterns. Instead, we proposed that substitution of a thi-
Keywords: Beta-secretase; BACE1; Aspartyl protease; Acylguanidine.
*
Corresponding author. Tel.: +1 732 274 4477; fax: +1 732 274
4505; e-mail: fobarew@wyeth.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.08.010

5354
W. F. Fobare et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5353–5356
ophene ring for the core pyrrole would allow for a facile
examination of the pendant aryl ring SAR while main-
taining the disposition of these key moieties and comple-
ment the developing SAR around the pyrrole series.
Thus, intermediate 2 with the aforementioned thiophene
ring (X, Y = Br) would be a viable surrogate for the pyr-
role. Previous experience with 2,4-dihalo-substituted thi-
ophenes had shown that it was possible to perform
Suzuki7 couplings on the thiophene ring with either dib-
romo- or diiodothiophenes in a regiospecific manner.
This proposed sequence would allow us to append the
aryl substituents at a later stage in the synthesis, making
the strategy more amenable to library generation.
Toward that end, the synthesis of key intermediate 2 (X,
Y = Br) was initiated by lithium halogen exchange of the
readily available 2,3,5-tribromo-4-methylthiophene 3 in
ether at ꢀ78 °C and quenching with water to yield the
2,4-dibromo-3-methylthiophene (Scheme 1).8 Radical
induced bromination of the methyl group followed by
displacement with cyanide yielded the (2,4-dibromo-
thiophen-3-yl)-acetonitrile 4. Acid catalyzed methanoly-
sis of the nitrile and regioselective Suzuki coupling with
a suitably substituted arylboronic acid at the 2-position
of the thiophene yield a variety of intermediates 5 in 75–
91% yield. This selective coupling is best achieved when
only 1.1 equiv of boronic acid is used at a temperature
of 45 °C. The second Suzuki coupling, at the 4-position
of the thiophene ring, is significantly slower and occurs
at elevated reaction temperatures (70 °C) and longer
reaction times. While the yields are quite good (66–
84%) the slower reaction and increased temperatures
give rise to small amounts (3–6%) of dehalogenated ad-
ducts, which are easily separable by chromatography on
silica gel. Basic hydrolysis of esters 5 with NaOH in eth-
anol, followed by carbonyl diimidazole (CDI) activation
under basic conditions and displacement with guani-
dine, affords the desired final products 6.
Figure 1. Depiction of an X-ray crystal structure of Compound 1
bound in the active site of BACE1. The relative locations of the
different binding areas of the active site are labeled along with key
amino acid residues. RCSB ID code: rcsb044055; PDB code: 2QU2.
of compound 1 co-crystallized with the BACE1 enzyme
shows that the acylguanidine group binds to the key
aspartates Asp32 and Asp228 as predicted. The surface
area around the S1–S3 pocket is fairly large and has pre-
dominately hydrophobic residues while that of S20 has
more polar or charged residues. This led us to believe that
it should be straightforward to discern between the two
pockets. Our initial adduct, 6a (Table 1), was meant to
mimic the binding of the lead pyrrole, 1, and to confirm
the favorable substitution of the core heterocycle. Com-
parable binding affinity shows that this is indeed the case.
A thorough examination of the molecular modeling of 6a
had suggested that the substitution pattern on the aryl
group extending into the S1–S3 pocket would be better
served by placing substituents on the para-position. Sub-
stitution of larger functionalities on the para-position
that extend into the S3 pocket containing more polar
functions had somewhat mixed results (e.g., 6b and 6c).
While there is a 10-fold enhancement in binding for 6b,
attempts to further enhance affinity by extension of a pyr-
azole ring into S3, that is 6c, were deleterious. Additional
observations from the modeling predicted that a para
propoxy group on the aryl targeted for the S1–S3 pocket
would possibly enhance binding affinity. Furthermore,
an examination of the S20 pocket suggested that an ortho
chlorine on the aryl group projecting into S20 would
increase binding through interactions with Trp76. Com-
pounds 6d–6g were prepared to challenge this hypothesis
on the thiophene core nucleus. There are two alternate
substitution patterns on the thiophene ring for these par-
ticular placements. The first has the proposed S1–S3
binding substituents on C-2 of the thiophene and the
S20 substituents on the C-4 (6d and 6f), and the second
has the exact opposite (6e and 6g). The presence of the
ortho chloro group on the S20 aryl group of 6g shows en-
hanced affinity for BACE1 in comparison to 6e, demon-
strating the positive effects of both substitutions.
However, when the substituents are not favorably dis-
placed, thus positioning the molecule with the proper aryl
At the outset of our SAR exploration, our first goal was
to differentiate the two pendant aryl rings from each
other. That is, to define which aryl ring, Ar1 or Ar2, pro-
jects into the S1–S3 pocket and which occupies the S20
pocket (see Fig. 1). An X-ray crystal structure analysis
CN
a,b,c
Br
d,e
Br
Br
Br
S
S
Br
3
4
NH2
NH2
O
N
O
O
Ar2
f,g,h
Ar1
Ar1
Br
S
S
5
6
Scheme 1. Reagents: (a) n-BuLi, Et2O, ꢀ78 °C, H2O (quench), 82%;
(b) NBS, AIBN, hm, CCl4, 91%; (c) KCN, EtOH, 86%; (d) H2SO4,
MeOH, 88%; (e) Ar1B(OH)2, PdCl2(dppf)2, aq K2CO3, dioxane, 45 °C,
75–91% ; (f) Ar2B(OH)2, PdCl2(dppf)2, aq K2CO3, dioxane, 70 °C, 66–
84%; (g) NaOH, EtOH, 95%; (h) CDI, DMF, guanidine hydrochlo-
ride, DIEA, 60–83%.

W. F. Fobare et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5353–5356
5355
Table 1. SAR of substitution patterns for Ar1 and Ar2 on thiophene
CO2CH3
CO2CH3
Br
a,b Ar1
c,d
Br
S
S
Cl
8
7
NH2
NH2
N
N
H
OH
N
N
N
O
O
e
Ar1
Ar1
Compound Ar1
Ar2
IC50
(lM)
S
S
Cl
Cl
10
9
6a
8.3
Scheme 2. Reagents: (a) Ar1B(OH)2 (1.1 equiv), aq K2CO3 (2.0 equiv),
PdCl2(dppf)2 (3 mol%), dioxane 45 °C; (b) Ar1B(OH)2 (2.0 equiv), aq
K2CO3 (4.0 equiv), PdCl2(dppf)2 (3 mol%), dioxane 70 °C; (c) NaOH,
EtOH; (d) CDI, CH2Cl2, pyrazole carboxamidine hydrochloride,
DIEA; (e) CH2Cl2, aminopropanol, DIEA.
6b
6c
6d
6e
6f
0.75
6.9
moieties projecting into the proposed pockets, this added
chloro group has no effect on the affinity (compare 6d and
6f). Molecular modeling suggests that the ortho chloro
substituent on the C-4 aryl group on 6f skews the aryl
group because of interactions with the C-3 substituent
and C-5 hydrogen to enhance p-stacking with Pro70.
The absence of this substituent, as in 6e, onto C2 of the
thiophene nucleus is not optimized for this p-stacking
and this may be the cause of decreased binding affinity.
0.68
1.89
0.63
0.59
The original hypothesis that the propyloxyphenyl sub-
stituent would enhance affinity and project into the
S1–S3 pocket of the BACE1 site was confirmed when
an X-ray co-crystal structure of analog 6g with the
BACE1 enzyme was attained (Fig. 2).
In an attempt to optimize the potency and to identify
opportunities to exploit additional space within the
enzyme active site, the S10 area was explored. Struc-
ture–activity relationships (SAR) being developed
concurrently with the pyrrole series (Ref. 6) of acylgua-
nidines, focused on exactly that goal, revealed this
opportunity. On those derivatives it had been shown
that substitution of a functionalized alkyl group, in par-
ticular 3-hydroxypropyl, on the terminus of the guani-
dine portion of the molecule projecting into the S10
pocket of BACE1 enhanced binding. Synthesis of the
analogous derivatives from this series was undertaken
and is described in Scheme 2.
6g
Routine Suzuki coupling of an aryl boronic acid with (2,4-
dibromo-thiophen-3-yl)-acetic acid methyl ester 7 (pre-
pared by methanolysis of compound 4) at the 2-position
followed by a second Suzuki coupling at the 4-position
with 2-chlorophenyl boronic acid gave excellent yields
of the diaryl substituted thiophenyl esters 8. Hydrolysis
of the ester followed by activation with carbonyl diimi-
dazole, as before, then coupling with pyrazole carboxami-
dine yielded the pyrazole substituted acylguanidines 9.9
Displacement of the pyrazole with 3-aminopropanol
readily occurs and affords the desired final products.
Figure 2. Molcad surface colored for the electrostatic potential of an
X-ray of compound 6g bound to BACE1. The green and light green
surfaces indicate hydrophobic regions and the blue region represent
negatively charged surfaces. RCSB ID code: rcsb044056; PDB ID
code: 2QU3.
It can be seen from the data in Table 2 that substitution
of the hydroxyl containing 3-carbon fragment that
reaches into the S10 pocket enhances the BACE1 FRET

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W. F. Fobare et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5353–5356
Table 2. SAR of substitution patterns for Ar1 on thiophene–propanol
substituted acylguanidines
23-fold selective versus cathepsin D, and displayed only
16% inhibition at 100 lM for pepsin.
In conclusion, we have investigated the substitution of a
thiophene ring for the key pyrrole nucleus in a series of
substituted acylguanidines. This replacement allowed us
to more easily probe the size constraints of the S1 and S3
pockets of the BACE1 enzyme. Furthermore, utilizing
results from the thiophene SAR allowed for optimized
derivatives possessing S10 moieties to be prepared which
showed potencies below 200 nM. These potent inhibi-
tors of the enzyme supplied us with molecules to co-crys-
tallize with the protein and with X-ray analysis further
expand our understanding of the requirements for small
molecule inhibition of BACE1.
Compound
R1
R2
H
R2
H
IC50 (lM)
10a
0.15
10b
10c
10d
10e
10f
10g
10h
CH3
H
H
1.54
1.55
0.20
0.26
0.14
0.28
0.93
CH3
H
Acknowledgments
The authors thank Dr. Kristi Fan for her assistance in
computational chemistry and the Chemical Technolo-
gies group for performing spectral analysis.
H
H
H
References and notes
H
H
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H
H
activity by a factor of 4-fold (10a, compare to 6f) and
afforded one of the most potent compounds in the
series. Utilizing this optimized binding of S10 and S20,
further examination of small substituents on the S1 aryl
group showed that this pocket is fairly narrow and
intolerant of even small changes. Substitution of a sim-
ple methyl group at either the ortho- or meta-position of
the phenyl ring decreases activity by 10-fold (10b and
10c). Reaching out further to the S3 pocket, with the
synthesis of several substituted amides (10d–10h), affor-
ded results that were supported by molecular modeling.
Not surprisingly, each pocket has tolerances and addi-
tion of a branched alkyl chain reaching into the S3
pocket reduced binding by 6-fold (10h). Furthermore,
it was shown that substitution on the 4-position of the
phenyl group could tolerate five to six atoms before
binding is adversely affected. Of the amides synthesized
the cyclopropylmethyl substituted compound 10f was
the most closely related to 10a in activity in the
in vitro assay.
Compound 6a, our initial thiophene compound, had an
IC50 of 8.3 lM for BACE1 in a FRET assay, and was
similar in activity to the pyrrole substituted compound
1 found through HTS, which had an IC50 of 3.7 lM.
In terms of selectivity, compound 10a, which had an
IC50 of 0.15 lM, was 7-fold selective versus BACE2,
7. Suzuki, A. Chem. Commun. 2005, 38, 4759, and references
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