Design and synthesis of 1,4-dihydropyridine derivatives as BACE-1 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2010.02.046

BACE-1 has been shown to be an attractive therapeutic target in Alzheimer's disease (AD). Using a 1,4-dihydropyridine (DHP) scaffold, we synthesized new inhibitors of BACE-1 by modifying the known BACE inhibitor 2 containing a hydroxyethylamine (HEA) motif. Using structure-based drug design based on computer-aided molecular docking, the isophthalamide ring of 2 was replaced with a 1,4-dihydropyridine ring as a brain-targeting strategy. Several of the new dihydropyridine derivatives were synthesized and their BACE-1-inhibitory activities were evaluated using a cell-based, reporter gene assay system that measures the cleavage of alkaline phosphatase (AP)-APP fusion protein by BACE-1. Most of the 1,4-DHP analogs showed BACE-1-inhibitory activities with IC₅₀ values in the range 8-30?μM, suggesting that the 1,4-DHP skeleton may be utilized to develop brain-targeting BACE-1 inhibitors.

Full text of DOI:10.1016/j.ejmech.2010.02.046

European Journal of Medicinal Chemistry 45 (2010) 2578e2590
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design and synthesis of 1,4-dihydropyridine derivatives as BACE-1 inhibitors
Soo-Jeong Choi a, Joong-Heui Cho a, Isak Im a, So-Deok Lee a, Ji-Yeon Jang b, Yu-Min Oh b,
,
Yong-Keun Jung b, Eun-Seok Jeon c, Yong-Chul Kim a
*
a Department of Life Science, Gwangju Institute of Science and Technology, 1 Oryong-dong, Gwangju 500-712, Republic of Korea
b School of Biological Science, Seoul National University, Seoul 151-742, Republic of Korea
c Department of Medicine, Sungkyunkwan University School of Medicine, Cardiac and Vascular Center, Samsung Medical Center, Seoul 135-710, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
BACE-1 has been shown to be an attractive therapeutic target in Alzheimer's disease (AD). Using a 1,4-
dihydropyridine (DHP) scaffold, we synthesized new inhibitors of BACE-1 by modifying the known BACE
inhibitor 2 containing a hydroxyethylamine (HEA) motif. Using structure-based drug design based on
computer-aided molecular docking, the isophthalamide ring of 2 was replaced with a 1,4-dihydropyr-
idine ring as a brain-targeting strategy. Several of the new dihydropyridine derivatives were synthesized
and their BACE-1-inhibitory activities were evaluated using a cell-based, reporter gene assay system that
measures the cleavage of alkaline phosphatase (AP)-APP fusion protein by BACE-1. Most of the 1,4-DHP
Received 20 October 2009
Received in revised form
17 February 2010
Accepted 19 February 2010
Available online 23 February 2010
Keywords:
analogs showed BACE-1-inhibitory activities with IC50 values in the range 8e30 mM, suggesting that the
1,4-DHP skeleton may be utilized to develop brain-targeting BACE-1 inhibitors.
BACE-1 inhibitors
Hydroxyethylamine
1,4-Dihydropyridine
Alzheimer's disease
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
A
b
production, has been
a
major therapeutic target in AD
-site APP cleaving enzyme or
treatment. Particularly, -secretase (
b
b
Alzheimer's disease (AD) is the most common form of dementia
and no disease modification therapy is currently available [1]. Much
research on AD is based on the amyloid cascade hypothesis, which
BACE-1), a type _ membrane-associated aspartyl protease [5], has
been considered to be attractive therapeutic target in AD, because
the enzyme catalyzes the first step in Ab production [4].
suggests that accumulation of amyloid-
the formation of plaques in the brain early in the course of the
disease are significant in most patients with AD. These Ab40 and
b
-42 (Ab42) peptides and
A strategy frequently employed in designing BACE-1 inhibitors is
replacement of the scissile amide bond of APP with noncleavable
transitionstateisosteressuchasstatine,hydroxyethylene, norstatine,
or hydroxyethylamine (HEA), which can interact with aspartates in
the active site of BACE-1 [6e9]. Among known motif of the aspartyl
protease inhibitors, hydroxyethylamine (HEA) dipeptide isoster has
been extensively applied to HIV protease and renin inhibitors [10] as
well as several BACE-1 inhibitors (Fig.1) [9,11e13]. For example, Elan
Corporation reported an isophthalamide-HEA based derivative, 1, as
a potent and cell permeable BACE-1 inhibitor [11]. Introduction of
a pyrrolidine scaffold led to development of a conformationally
constrained HEA inhibitor, compound 3 (by Schering) [12], and the
5-substituted isophthalamide analog, 2 (by Merck), inhibited BACE-1
enzymatic activity (IC50 ¼ 15 nM) and in a cell-based assay
(IC50 ¼ 29 nM), with impressive selectivity against other biologically
relevant aspartyl proteases [9]. Because of impermeability through
the bloodebrain barrier and P-glycoprotein (Pgp)-mediated efflux,
however, compound 2 was poorly distributed within the CNS.
Recently GSK reported a modified isophthalamide inhibitor, 4, which
was the first orally bioavailable compound but had low brain pene-
tration because of Pgp-efflux [13].
A
b42 peptides are secreted as a result of proteolytic processing of
a large transmembrane protein, amyloid precursor protein (APP),
by two enzymes, the - and -secretases [2,3]. Therefore, the
development of specific inhibitors of these key proteases, to block
b
g
Abbreviations: 1,4-DHP,1,4-Dihydropyridine; Ab40, 40-Residue
b42, 42-Residue -amyloid peptide; AD, Alzheimer's diseases; AP, Alkaline phospha-
tase; APP, -Amyloid precursor protein; APPs , Soluble APP product from -secretase
cleavage; BACE-1, -Site APP cleaving enzyme; Bn, Benzyl; BSA, Bovine serum albumin;
b-amyloid peptide;
A
b
b
b
b
b
CNS, Central nervous system; DCM, Dichloromethane; DIPA, Diisopropylamine; DIPEA,
Diisopropylethylamine; DMAP, Dimethylaminopyridine; DMEM, Dulbecco's modified
eagle medium; DMF, Dimethylformamide; EDAC, 1-Ethyl-3-(3-dimethylaminopropyl)
carbodiimide; FBS, Fetal bovine serum; HATU, 2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate methanaminium; HEA, Hydroxyethyl-
amine; HEK, Human embryonic kidney; pNPP, p-Nitrophenylphosphate; PyBOP,
(Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate; SAR, Struc-
ture-activity relationship; TEA, Triethylamine; THF, Tetrahydrofuran.
* Corresponding author. Tel.: þ82 62 970 2502; fax: þ82 62 970 2484.
E-mail address: yongchul@gist.ac.kr (Y.-C. Kim).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.02.046

S.-J. Choi et al. / European Journal of Medicinal Chemistry 45 (2010) 2578e2590
2579
Fig. 1. Hydroxyethylamine (HEA) BACE-1 inhibitors.
As a BACE-1 inhibitor must act in the brain, the material must be
able to cross the bloodebrain barrier and achieve sustainable drug
levels in the brain [14]. To introduce brain-targeting properties for
BACE-1 inhibitors, we attempted to replace the isophthalamide
ring of 2 with a 1,4-dihydropyridine (DHP) scaffold, which has been
utilized to target the brain, because of an ability to be metabolized
into charged quaternary amine molecules, resulting in the material
becoming locked into the brain [15]. Moreover, 1,4-DHP scaffolds
can be used to design diverse analogs through derivatization at
various positions (Fig. 2). In this perspective, 1,4-DHP scaffold was
investigated as a new skeleton of BACE-1 inhibitor by incorporation
of structural information from known BACE-1 inhibitors such as
compound 2, estimation of binding through molecular docking, and
evaluation of the inhibitory activity in a cell-based assay system.
(Fig. 3a). Hydrophobic interactions of the
a
-methylbenzyl group,
hydrogen bonding of the sulfonamide group, and interactions of
the cyclopropyl amine moieties in the S3, S2, and S10 pockets,
respectively, were the same for the two compounds. A detailed
analysis of the hydrogen bonding network of 9a is shown in Fig. 3b.
The sulfonamide group of 9a was hydrogen-bonded to the carbonyl
oxygen of Gln73 and the amine nitrogen to Gly230, and the HEA
isostere of 9a was hydrogen-bonded to both catalytic aspartates,
Asp32 and Asp228, findings consistent with those observed for
compound 2.
Based on molecular docking results, we designed 1,4-DHP
derivatives as BACE-1 inhibitors using five strategies. These were
(1) replacement of the bulky a-methylbenzamide group in 2 with
a benzyl ester or smaller acetyl group for binding in the S3 pocket;
(2) modification of the sulfonamide group in the aromatic scaffold
of 2 with alkyl ester or amide groups, maintaining the important
hydrogen bonding with Asn233 in the S2 binding pocket; (3)
incorporation of additional hydrophobic interactions into the S1
binding pocket by introduction of alkyl or aryl groups, including
methyl, ethyl, propyl, isopropyl, and phenyl groups; (4) alteration of
the cyclopropyl group at the R4 position to other aromatic groups,
thus changing hydrophobic interactions in the S20 binding pocket
by extension toward the prime-side of the enzyme; and, (5) alter-
ations at the 2 and 6 positions of the 1,4-DHP scaffold by synthesis
of 2-monomethyl and 2,6-unsubstituted analogs.
2. Results and discussion
2.1. Inhibitor design and molecular docking study
To estimate the binding of 1,4-DHP-based BACE-1 inhibitors, we
used a molecular modeling program, Discovery Studio (Accelrys),
to dock 9a into the active site of BACE-1. Fig. 3a and b show the
interaction models of docked compounds 2 and 9a, respectively.
Overlay of docking structures showed that the two compounds
occupied the same binding pocket in the active site of BACE-1
Fig. 2. 1,4-dihydropyridine (DHP) BACE-1 inhibitors.

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S.-J. Choi et al. / European Journal of Medicinal Chemistry 45 (2010) 2578e2590
Fig. 3. a. Overlay of 2 (green) and 9a in the BACE-1 active site, b. Interactions of 9a in the active site of BACE-1. Hydrogen bonds are shown with dotted lines (For interpretation of
the references to colour in this figure legend, the reader is referred to the web version of this article).
2.2. Chemistry
or a phenyl group, at the R1 position, and a benzyl ester or acetyl
group instead of the (R)-methylbenzylamine at the R2 position. We
also replaced the sulfonamide moiety at the R3 position with
several alkyl ester groups (methyl, ethyl, or isopropyl) for extension
of carbon chains. The aromatic building blocks (m-fluorophenyl,
m-methoxyphenyl, or m-methoxybenzyl) at the R4 position were
introduced in place of the cyclopropyl group (10j, 10len).
2.2.1. 2,6-Dimethyl-1,4-dihydropyridine derivatives
As molecular docking suggested that the 1,4-DHP analog, 9a,
and the 5-amino isophthalate analog, 2, had similar binding
properties, we synthesized various 1,4-DHP derivatives by
Hantzsch condensation using three components; a
b-ketoester, an
aldehyde, and a -enaminoester, as shown in Schemes 1e6 [16].
b
Compound 13 was synthesized to assess the effects of an alkyl
chain linker on the R3 group (Scheme 3). Compound 11 was
hydroborated using borane-methylsulfide; subsequent oxidation
with H2O2 resulted in an alcohol compound [18,19], which was
converted to 12 by acetylation. Monohydrolysis of one of the benzyl
ester groups followed by a coupling reaction with 5a yielded the
carbon chain-extended analog, 13.
Compounds 16aec, containing amides at the N-1 position
instead of esters, were prepared as outlined in Scheme 4. To
introduce an amide moiety into the secondary amine group of
1,4-DHP, the tert-butyl acetate group of 14a was selectively
hydrolyzed and coupled with several secondary amine building
blocks to yield 15aec. Monohydrolysis of the benzyl ester group
The initial 2,6-dimethyl-1,4-DHP analogs, 9aeb, corresponding to
2, were synthesized as shown in Scheme 1. Briefly, the N-1 posi-
tions of the 1,4-DHP dibenzyl esters, 6aeb, were mesylated,
followed by monohydrolysis at 0 ꢀC with aluminum chloride as
a Lewis-acid, resulting in compounds 7aeb [17]. PyBOP-mediated
amide coupling of 7aeb with (R)-methylbenzylamine, hydrolysis
of the resulting esters, 8aeb, and coupling with compound 5a, an
HEA isoster moiety [9], yielded 9aeb. The HEA isoster moieties,
5aed, were prepared in two steps as described [9].
A second series of 2,6-substituted 1,4-DHP-based inhibitors,
10aen, was prepared as depicted in Scheme 2. Compounds 10aen
contained various alkyl chains (methyl, ethyl, propyl, or isopropyl),

S.-J. Choi et al. / European Journal of Medicinal Chemistry 45 (2010) 2578e2590
2581
Scheme 1. Synthesis of 1-methylsulfonamide-2,6-dimethyl-1,4-dihydropyridine derivatives. Reagent: (a) NH4OAc, Ethanol, 90 ꢀC, 24 h, 98%; (b) Methane sulfonylchloride, NaH,
DMF, 0e60 ꢀC, 4 h, 35%; (c) AlCl3, Anisole, DCM, ꢁ50 ꢀC to RT, 2 h, 21%; (d) R-methylbenzylamine, PyBOP, DIPA, DCM, 1 h, 70%; (e) AlCl3, Anisole, DCM, ꢁ50 ꢀC to RT, 2 h, 30%;
(f) Compound 5a, PyBOP, DIPEA, DCM, 15 min, 65%.
Scheme 2. Synthesis of 1-alkyl acetate-2,6-dimethyl-1,4-dihydropyridine derivatives. Reagent: (a) NH4OAc, Ethanol, 90 ꢀC, 24 h, 98%; (b) R3 building blocks, NaH, DMF, 0e60 ꢀC, 5 h,
30%; (c) AlCl3, Anisole, DCM, ꢁ50 ꢀC to RT, 1 h, 20%; (d) Compounds 5aed, PyBOP, DIPEA, DCM, 15 min, 49%.

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S.-J. Choi et al. / European Journal of Medicinal Chemistry 45 (2010) 2578e2590
Scheme 3. Synthesis of 1-(3-acetoxypropyl)-2,6-dimethyl-1,4-dihydropyridine derivatives. Reagent: (a) Ally bromide, NaH, DMF, 1 h, 45%; (b) i. Borane-methylsulfide, THF, 2 h ii. 3N
NaOH, 0 ꢀC, iii. 30% H2O2 in water, 0 ꢀC, 35%; (c) AcCl, TEA, DCM, 2 h, 50%; (d) AlCl3, Anisole, DCM, ꢁ50 ꢀC to RT, 2 h, 21%; (e) Compound 5a, PyBop, DIPEA, DCM, 15 min, 45%.
and PyBOP-mediated amide coupling with 5a afforded compounds
16aec.
dimethyldihydropyridines in the binding site of BACE-1 (Scheme 5).
Benzyl coumalate, 18, was subjected to a condensation reaction with
benzylacetoacetate or acetylacetone in the presence of ammonium
acetate, yielding 19aeb [20]. Subsequent reactions, including N-
alkylation, hydrolysis, and coupling with 5a and 5bed were the same
as described in the reaction schemes above. Compounds 21aec were
2.2.2. 2-Monomethyl 1,4-dihydropyridine derivatives
The 2-monomethyl-1,4-dihydropyridines, 21aec, were synthe-
sized to assess the possible steric effects of methyl groups of
Scheme 4. Synthesis of 1-acetamide-2,6-dimethyl-1,4-dihydropyridine derivatives. Reagent: (a) t-butyl bromoacetate, NaH, DMF, 0e60 ꢀC, 4 h, 33%; (b) TFA 50% in DCM, 2 h, 47%;
(c) PyBop, R3 building blocks, DCM, 2 h, 49%; (d) AlCl3, Anisole, DCM, ꢁ40 ꢀC to RT, 2 h, 29%; (e) Compound 5a, PyBop, DIPEA, DCM, 15 min, 40%.

S.-J. Choi et al. / European Journal of Medicinal Chemistry 45 (2010) 2578e2590
2583
Scheme 5. Synthesis of 2-monomethyl-1,4-dihydropyridine derivatives. Reagent: (a) Benzyl alcohol, DMAP, EDC, DCM, 5 h, 70%; (b) Benzylacetoacetate or acetylacetone, acetic acid,
NH4OAc, 24 h, 33%; (c) Isopropylbromoacetate, NaH, 0e60 ꢀC, 4 h, DMF, 69%; (d) AlCl3, Anisole, DCM, ꢁ50 ꢀC to RT, 2 h, 20%; (e) Compounds 5a or 5b or 5d, HATU, DIPEA, DCM,1 h, 42%.
Scheme 6. Synthesis of 2,6-unsubstituted-1,4-dihydropyridine derivatives. Reagent: (a) NH4OAc, Ethanol, 90 ꢀC, 24 h, 15%; (b) Isopropylbromoacetate, NaH, DMF, 0e60 ꢀC, 4 h, 58%;
(c) 96% formic acid, 0 ꢀC, 20 min, 45%; (d) Compounds 5a,c, HATU, DIPEA, DCM, 1 h, 55%; (e) 96% formic acid, 0 ꢀC, 20 min, 49%; (f) R-methylbenzylamine, PyBop, DIPA, DCM, 1 h,
59%; (g) 96% formic acid, 0 ꢀC, 30 min, 60%; (h) Compounds 5a,c, HATU, DIPEA, DCM, 1 h, 48%.

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S.-J. Choi et al. / European Journal of Medicinal Chemistry 45 (2010) 2578e2590
also prepared as a mixture of diastereomers and confirmed by NMR
2.3. BACE-1-inhibitory activity
spectrum of R1-group.
We utilized an alkaline phosphatase (AP)-APP-cell-based assay
system to evaluate the BACE-1-inhibitory activity of the synthe-
sized 1,4-DHP derivatives. A plasmid encoding a fusion protein of
APP with AP, and containing the BACE-1 cleavage site, was stably
transfected into HEK293 cells, allowing BACE-1-inhibitory activity
to be measured indirectly by quantification of secreted AP after
2.2.3. 2,6-Unsubstituted 1,4-dihydropyridine derivatives
We synthesized the 2,6-unsubstituted-1,4-dihydropyridine
analogs 24aec and 26aec using 22aeb, which had beenprepared by
reaction with tert-butylpropiolate and acetaldehyde or butylalde-
hyde (Scheme 6) [21,22]. N-alkylation with isopropylbromoacetate
followed by monohydrolysis of a t-butyl ester with 96% formic acid
[23], and the HATU-mediated coupling of a carboxylic acid group
with derivatives of 5a, c yielded 24aec. Compounds 26aec were
synthesized by monohydrolysis of 23aeb, followed by amide
coupling with (R)-methylbenzylamine, hydrolysis of the remaining
ester, and final amide coupling, as shown for 24.
cleavage of the
b-site of APP by BACE-1. The BACE-1-inhibitory
activities of the 2,6-dimethyl-1,4-DHP derivatives are summarized
in Table 1. The known BACE-1 inhibitor, 2 (reported IC50 ¼ 29 nM),
was utilized as a positive control. We found that the IC50 value of 2
was 0.25
mM in our indirect cell-based assay system, which
exhibited less than 8-fold loss of BACE-1-inhibitory activity
compared to the direct measurement of BACE-1 activity in cells [9].
Initially, to test the effect on BACE-1 enzyme activity of the
replacement of the isophthalate ring of 2 by a 1,4-DHP scaffold,
In the case of compound 9a and 9b, each single diastereomer
could be separated by silica gel column chromatography. However,
all other final compounds were obtained and tested as diastereo-
meric mixtures.
we determined the activities of 9a (R1
¼
methyl) and 9b
Table 1
BACE-1-inhibitory activities of 2,6-dimethyl-1,4-DHP analogs.
Compound
R1
R2
R3
R4
IC50 (m
M)a
2b
0.25 ꢂ 0.18
16.1 ꢂ 2.1
16.2 ꢂ 0.6
14.9 ꢂ 0.6
9.5 ꢂ 0.4
9ac
Methyl
Ethyl
Methyl
Ethyl
eNH-(
eNH-(
eOBn
eOBn
eOBn
eOBn
eOBn
eOBn
eOBn
eOBn
eCH3
eCH3
eCH3
eCH3
eCH3
eCH3
eOBn
a
a
)methylBn
)methylBn
eSO2CH3
eSO2CH3
eSO2CH3
eSO2CH3
eSO2CH3
eSO2CH3
eSO2CH3
eCH2COOCH3
eCH2COOCH2CH3
CH2COOCH(CH3)2
CH2COOCH(CH3)2
CH2COOCH(CH3)2
CH2COOCH(CH3)2
CH2COOCH(CH3)2
CH2COOCH(CH3)2
CH2COOCH(CH3)2
e(CH2)3OCOCH3
Cyclopropyl
Cyclopropyl
Cyclopropyl
Cyclopropyl
Cyclopropyl
Cyclopropyl
Cyclopropyl
Cyclopropyl
Cyclopropyl
Cyclopropyl
Cyclopropyl
m-Fluorophe
Cyclopropyl
m-Fluorophe
m-Methoxyphe
m-MethoxyBn
Cyclopropyl
9bc
10ad
10bd
10cd
10dd
10ed
10fd
10gd
10hd
10id
10jd
10kd
10ld
10md
10nd
13d
Propyl
Isopropyl
Phenyl
Methyl
Methyl
Methyl
Methyl
Methyl
Propyl
Propyl
Propyl
Propyl
Methyl
8.1 ꢂ 0.8
11.0 ꢂ 3.0
21.4 ꢂ 3.5
15.2 ꢂ 3.1
15.5 ꢂ 0.3
9.0 ꢂ 0.6
29.3 ꢂ 2.3
26.4 ꢂ 0.4
33.1 ꢂ 3.1
27.1 ꢂ 1.3
26.6 ꢂ 2.1
23.2 ꢂ 0.4
26.9 ꢂ 2.8
16ad
Methyl
eOBn
Cyclopropyl
39.1 ꢂ 2.5
16bd
Methyl
eOBn
Cyclopropyl
28.4 ꢂ 3.0
16cd
Methyl
eOBn
Cyclopropyl
28.2 ꢂ 3.1
a
Calculated as the average from four experiments.
The known BACE-1 inhibitor 2 was synthesized as described [9] and used as a positive control in the BACE-1 cell-based assay.
Although each single diastereomer of 9aeb was purified and tested, there was no difference of the inhibitory activity between each single diastereomer of 9a and 9b.
Compounds 10aen, 13, and 16aec were obtained and tested as diastereomeric mixtures.
b
c
d

S.-J. Choi et al. / European Journal of Medicinal Chemistry 45 (2010) 2578e2590
2585
Table 2
(R1 ¼ ethyl), which had the same side chains as 2. We found that
these compounds showed a 60-fold decrease in inhibitory activity
compared with 2. There was no difference of the inhibitory activity
between each single diastereomer of 9a and 9b. In the course of an
effort to reduce the peptidic nature, benzyl ester for R2 group
instead of (R)-methylbenzylamine of 9a,b was found to be tolerated
as shown in the activity of 10a,b. Replacement of side chains at the
R1 position of 2,6-dimethyl-1,4-DHP (10aee), such as methyl, ethyl,
propyl, isopropyl, and phenyl, showed that longer or bulkier alkyl
groups slightly increased BACE-1-inhibitory activity (10aec),
whereas a phenyl group (10e) at the R1 position diminished activity
more than two-fold. Therefore, following the first round of opti-
mization of 1,4-DHP-based BACE-1 inhibitors, we found that 10c,
with a propyl group at the R1 position and a benzyl ester group at
the R2 position, showed a two-fold enhancement of inhibitory
activity compared with the initial compound 9.
The sulfonamide group in the isophthalate scaffold of 2 has been
shown to contribute to important hydrogen bonding interactions
with the backbone residues of Asn233 and Arg235 in the active site
of BACE-1 [9]. We therefore performed SAR at the R3 position of
1,4-DHP derivatives by assessing the effects of ester groups (10feh)
or amide groups (16aec) instead of the sulfonamide group of
10aee. Compound 10f, with a methylacetate group at the R3 posi-
tion, showed an inhibitory activity similar to that of 10a, suggesting
that the oxygen molecules of the ester could provide a degree of
hydrogen bonding similar to that of the sulfonamide group of 10a.
Among the ester groups of 10feh, the isopropyl ester group of 10h
BACE inhibitory activities of 2-methyl-1,4-DHP analogs and 2,6-unsubstituted-1,4-
DHP analogs.
Compounda
R1
R2
R3
IC50 (mM)
21a
21b
21c
24a
24b
24c
26a
26b
26c
2
Methyl
Methyl
Methyl
Methyl
Propyl
Propyl
Methyl
Propyl
Propyl
eOBn
Cyclopropyl
16.1 ꢂ 1.3
29.3 ꢂ 2.1
26.1 ꢂ 0.9
31.9 ꢂ 1.7
24.6 ꢂ 0.9
30.4 ꢂ 2.3
32.8 ꢂ 0.8
29.6 ꢂ 0.3
31.2 ꢂ 2.8
0.3 ꢂ 0.11
Methyl
Methyl
eOtBu
eOtBu
eOtBu
eNH-(
eNH-(
eNH-(
m-Fluorophenyl
m-MethoxyBn
Cyclopropyl
Cyclopropyl
m-Methoxyphe
Cyclopropyl
a
a
a
)methylBn
)methylBn
)methylBn
Cyclopropyl
m-Methoxyphe
a
All compounds were obtained and tested as diastereomeric mixtures.
Whereas molecular modeling predicted that 1,4-DHP derivatives
could have similar docking fits, the synthesized compounds were
experimentally less potent than 2.
resulted in increased inhibitory activity, with an IC50 value of 9 mM.
To determine the effect of the carbon chain spacer of the R3 group,
we synthesized 13, with three carbon chains. However, 13 showed
an approximately two-fold loss of BACE-1-inhibitory activity
compared with 10f, suggesting that space in the S2 binding pocket
was limited. Amide substitutions (dimethyl, morpholine, and
N-isopropyl-N-methyl amide) at the R3 position (16aec) showed an
average three-to-four-fold loss of BACE-1-inhibitory activity
compared with compounds containing ester group substitutions
(10feh), suggesting that two hydrogen bond donors could enhance
binding.
We also attempted to incorporate different hydrophobic inter-
actions at the R2 and R4 positions [11,24] by reducing the size of the
R2 group (e.g., to an acetyl group) and by introducing aromatic
building blocks at the R4 position for extensions toward the
prime-side of BACE-1 (i.e., an m-fluorophenyl, m-methoxyphenyl,
3. Conclusion
We have described a series of BACE-1 inhibitors, synthesized
using a 1,4-dihydropyridine scaffold to replace the isophthalate
ring of the known BACE-1 inhibitor 2. The designed analogs
showed BACE-1-inhibitory activities with IC50 values of 8e30 mM,
suggesting that a chemical probe may block APP processing
induced by BACE-1. Further study of chemical delivery systems for
BACE-1 inhibitors with the 1,4-DHP scaffold are currently under
investigation.
4. Experimental protocols
or m-methoxybenzyl group instead of
a cyclopropyl group
[10ien]). However, replacement of the benzyl group at the R2
position (10h) by an acetyl group (10i) resulted in a three-fold
decrease in BACE-1-inhibitory activity. The remaining compounds
designed according to this concept also showed significant
decreases in potency.
4.1. Chemistry
Proton nuclear magnetic resonance spectroscopy was per-
formed on a JEOL JNM-LA 300WB spectrometer, and spectra were
taken in CDCl3 or DMSO-d6. Unless otherwise noted, chemical shifts
are expressed as ppm downfield from internal tetramethylsilane, or
relative ppm from DMSO (2.5 ppm). Data reported include chemical
shift, multiplicity (s, singlet; d, doublet; t, triplet; m, multiplet;
b, broad), coupling constants, and integration. Mass spectroscopy
was performed on ESI. Chromatographic purifications were
achieved using kieselgel 60 (Merck) 0.040e0.063 mm column
chromatography. TLC was performed on fluorescent silica gel plates
(60 F254 from Merck).
As 2,6-dimethyl groups may increase steric hindrance in the
maintenance of hydrogen bonding interactions between the two
oxygens at the R3 position and the S2 binding site, we tested the
effects of 2,6-dimethyl groups in DHP derivatives on BACE-1-
inhibitory activity by evaluating 2-monomethyl and 2,6-unsub-
stituted derivatives (Table 2). However, we found that 21a, with
a monomethyl group, showed a two-fold reduction in inhibitory
potency compared with 10h. In addition, an acetyl group at the R2
position (e.g., 21bec) did not enhance inhibitory activity, compared
with the corresponding benzyl group of 21a. 2,6-Unsubstituted 1,4-
DHPs (24aec and 26aec), containing combinations of various
active groups around the DHP skeleton, also showed decreased
BACE-1-inhibitory activities, without a clear trend in SAR. These
results indicated that methyl groups at the 2 and 6 positions could
contribute to inhibitory activity without interfering with the
hydrogen bonding of the ester group at the N-1 position of DHP.
4.1.1. General procedure for synthesis of derivatives of 2,6-
substituted 1,4-DHP (6aeg)
For compounds 6aee: 2 equimolar amounts of benzylacetoa-
cetate, R1-aldehyde, and ammonium acetate (1.5 eq) were dissolved
in anhydrous ethanol [16]. The mixture was refluxed for 24 h. After
cooling to room temperature, the solvent was evaporated and the
residue was purified by silica gel column chromatography.

2586
S.-J. Choi et al. / European Journal of Medicinal Chemistry 45 (2010) 2578e2590
For compounds 6feg: Benzylacetoacetate (1 eq), acetylacetone
(1 eq), and ammonium acetate (1.5 eq) were dissolved in anhydrous
ethanol. The reaction condition and purification was same as 6aee.
4.1.4. General procedure for the coupling reaction with HEA
moieties (9aeb, 10aen, 13, 16aec, 21aec, 24aec, 26aec)
A solution containing a monoacid compound such as 7 in DCM
was treated sequentially with an HEA moiety (5aed) (1.19 eq), DIPEA
(2.2 eq), and PyBop (1 eq) or HATU (1 eq). The reaction mixture was
stirred at ambient temperature for 15 min. After evaporation, the
product was purified by silica gel column chromatography.
In the case of compound 9aeb, two diastereomers could be
separated by silica gel column chromatography. However,
compound 10aen, 13, 16aec, 21aec were only obtained as
a mixture of diastereomers.
4.1.1.1. Dibenzyl 2,4,6-trimethyl-1,4-dihydropyridine-3,5-dicarbox-
ylate (6a). Yield: 65%, 1H NMR (300 MHz, CDCl3)
d (ppm) 7.36
(m, 10H), 5.59 (s, 1H, NeH), 5.19 (s, 1H), 5.17 (s, 1H), 3.97
(q, J ¼ 6.3 Hz, 1H), 2.27 (s, 6H), 1.01 (d, J ¼ 6.6 Hz, 3H)
4.1.1.2. Benzyl 5-acetyl-2,6-dimethyl-4-propyl-1,4-dihydropyridine-
3-carboxylate (6g). Yield: 31%, 1H NMR (300 MHz, CDCl3)
d (ppm)
7.35 (m, 5H), 5.6 (s, 1H, NeH), 5.21 (d, J ¼ 2.7 Hz, 2H), 3.94
(t, J ¼ 5.7 Hz, 1H), 2.30 (s, 3H), 2.26 (s, 3H), 2.25 (s, 3H), 1.25 (m, 4H),
0.80 (t, J ¼ 6.9 Hz, 3H)
4.1.4.1. N3-((2S,3R)-4-(cyclopropylamino)-3-hydroxy-1-phenyl-
butan-2-yl)-2,4,6-trimethyl-1-(methylsulfonyl)-N5-((R)-1-phenyl-
ethyl)-1,4-dihydropyridine-3,5-dicarboxamide (9a). (9ae1: single
4.1.2. General procedure for the Lewis-acid mediated hydrolysis
reaction (7aej)
diastereomer) Yield: 65%, 1H NMR (300 MHz, CDCl3)
d (ppm) 7.4
(m, 4H), 7.25e7.15 (m, 6H), 7.08 (s, 1H, NeH), 6.97 (s, 1H, NeH), 5.17
(q, J ¼ 7 Hz, 1H), 4.28 (m, 1H), 3.69 (m, 1H), 3.18 (s, 3H), 3.10 (m, 1H),
3.08 (q, J ¼ 6.9 Hz, 1H), 2.85 (m, 3H), 2.09 (s, 3H), 1.85 (s, 3H), 1.52
(d, J ¼ 7.2 Hz, 3H), 0.99 (d, J ¼ 6.9 Hz, 3H), 0.49e0.39 (m, 4H). ESI
[M þ H] ¼ 595.5
To a solution of 2,6-substituted 1,4-DHP (100 mg, 0.25 mmol)
and NaH (15 mg, 0.3 mmol) dissolved in DMF (5 mL) was added
methane sulfonylchloride or bromoacetate (0.025 mL, 0.3 mmol)
dropwise at 0 ꢀC. The reaction mixture was heated to 60 ꢀC and
stirred for 4 h. After cooling, the reaction mixture was parti-
tioned between a saturated ammonium chloride solution and
ethylacetate. The organic layer was dried over sodium sulfate,
filtered and evaporated under vacuum. The resulting product was
purified by silica gel column chromatography. To a solution of
0.13 mmol of each product in DCM was added anisole
(0.13 mmol) dropwise. The reaction mixture was stirred at
ꢁ50 ꢀC for 30 min and AlCl3 (51 mg, 0.39 mmol) was added at the
same temperature. The reaction mixture was stirred for 1 h with
warming to room temperature, quenched with water, and
partitioned between DCM and water. The resulting inorganic
precipitates were removed by filtration and the filtrate was
dried over sodium sulfate, filtered, and evaporated. The product
was purified by silica gel column chromatography (CHCl3:
CH3OH ¼ 20:1).
(9ae2: single diastereomer) Yield: 60%, 1H NMR (300 MHz,
CDCl3)
d (ppm) 7.39e7.30 (m, 5H), 7.26e7.13 (m, 5H), 6.16 (s, 1H,
NeH), 6.01 (s, 1H, NeH), 5.18 (q, J ¼ 6.9 Hz, 1H), 4.28 (m, 1H), 4.13
(q, J ¼ 6.9 Hz, 1H), 3.60 (m, 1H), 3.21 (s, 3H), 3.12 (m, 2H), 2.86e2.73
(m, 2H), 2.13 (s, 3H), 2.10 (m,1H),1.84 (s, 3H),1.53 (d, J ¼ 6.9 Hz, 3H),
0.99 (d, J ¼ 6.9 Hz, 3H), 0.47e0.35 (m, 4H).
4.1.4.2. N3-((2S,3R)-4-(cyclopropylamino)-3-hydroxy-1-phenyl-
butan-2-yl)-4-ethyl-2,6-dimethyl-1-(methylsulfonyl)-N5-((R)-1-
phenylethyl)-1,4-dihydropyridine-3,5-dicarboxamide (9b). (9be1:
single diastereomer) Yield: 68%, 1H NMR (300 MHz, CDCl3)
d
(ppm) 7.36e7.29 (m, 5H), 7.22e7.14 (m, 5H), 6.33 (s, 1H, NeH),
6.04 (s, 1H, NeH), 5.18 (q, J ¼ 7.2 Hz, 1H), 4.29 (m, 1H), 3.70 (m, 1H),
3.18 (s, 3H), 3.10 (t, J ¼ 4.8 Hz, 1H), 3.02e2.78 (m, 4H), 2.21 (m, 1H),
2.10 (s, 3H), 1.97 (s, 3H), 1.51 (d, J ¼ 7.2 Hz, 3H), 1.31 (m, 2H), 0.76
(t, J ¼ 7.5 Hz, 3H), 0.55e0.48 (m, 4H). ESI [M þ H] ¼ 609.5
(9be2: single diastereomer) Yield: 51%, 1H NMR (300 MHz,
4.1.2.1. 5-(benzyloxycarbonyl)-2,4,6-trimethyl-1-(methylsulfonyl)-
1,4-dihydropyridine-3-carboxylic acid (7a). Yield: 21%, 1H NMR
CDCl3) d (ppm) 7.34e7.28 (m, 5H), 7.26e7.13 (m, 5H), 6.76 (s, 1H,
(300 M Hz, CDCl3)
d
(ppm) 7.37 (m, 5H), 5.25 (s, 2H), 3.84
NeH), 6.38 (s, 1H, NeH), 5.18 (q, J ¼ 6.2 Hz, 1H), 4.25 (m, 1H), 3.83
(m, 1H), 3.23 (s, 3H), 3.11 (t, J ¼ 6.5 Hz, 1H), 3.10e2.94 (m, 3H), 2.80
(m, 1H), 2.24 (m, 1H), 2.14 (s, 3H), 1.93 (s, 3H), 1.56 (d, J ¼ 6 Hz, 3H),
1.27 (m, 2H), 0.72e0.64 (m, 7H). ESI [M þ H] ¼ 609.5
(q, J ¼ 5.1 Hz, 1H), 3.38 (s, 3H), 2.50 (s, 3H), 2.48 (s, 3H), 1.21
(d, J ¼ 6.6 Hz, 3H)
4.1.2.2. 5-(Benzyloxycarbonyl)-1-(2-methoxy-2-oxoethyl)-2,4,6-tri-
methyl-1,4-dihydropy ridine-3-carboxylic acid (7f). Yield: 16%, 1H
4.1.4.3. Benzyl 5-((2S,3R)-4-(cyclopropylamino)-3-hydroxy-1-phenyl-
butan-2-ylcarbamoyl)-2,4,6-trimethyl-1-(methylsulfonyl)-1,4-dihy-
dropyridine-3-carboxylate (10a). Yield: 59.1%, 1H NMR (300 MHz,
NMR (300 MHz, CDCl3)
d
(ppm) 7.38 (m, 5H), 5.20 (q, J ¼ 12.6 Hz,
2H), 4.35 (s, 2H), 3.92 (q, J ¼ 6.3 Hz, 1H), 3.78 (s, 3H), 2.37(s, 3H),
2.34 (s, 3H), 1.01 (d, J ¼ 6.6 Hz, 3H)
CDCl3)
d (ppm) 7.35 (m, 5H), 7.28e7.14 (m, 5H), 6.11 (s, 1H, NeH),
4.34 (m, 1H), 3.71 (m, 1H), 3.45 (q, J ¼ 6.6 Hz, 1H), 3.26 (s, 3H),
3.18e3.12 (m, 1H), 2.99e2.95 (m, 1H), 2.89e2.83 (m, 2H), 2.43
(m, 3H), 2.19 (m, 1H), 1.78 (s, 3H), 1.08 and 0.96 (2d, J ¼ 6.6 Hz, 3H,
mixture of two diastereomers), 0.53 (m, 4H). ESI [M þ H] ¼ 582.6
4.1.2.3. 5-(Benzyloxycarbonyl)-1-(2-isopropoxy-2-oxoethyl)-2,4,6-
trimethyl-1,4-dihydro pyridine-3-carboxylic acid (7h). Yield: 25%, 1H
NMR (300 MHz, CDCl3)
d (ppm) 7.34 (m, 5H), 5.18 (s, 2H), 5.01
(m, 1H), 4.28 (s, 2H), 3.99 (q, J ¼ 6.3 Hz, 1H), 2.37(s, 3H), 2.34 (s, 3H),
1.25 (d, J ¼ 3.9 Hz, 6H), 1.01 (d, J ¼ 6.6 Hz, 3H)
4.1.4.4. Benzyl 5-((2S,3R)-4-(cyclopropylamino)-3-hydroxy-1-phenyl-
butan-2-ylcarbamoyl)-4-ethyl-2,6-dimethyl-1-(methylsulfonyl)-1,4-
dihydropyridine-3-carboxylate (10b). Yield: 51%, 1H NMR (300 MHz,
4.1.3. (R)-Benzyl 2,4,6-trimethyl-1-(methylsulfonyl)-5-(1-phenyl-
ethylcarbamoyl)-1,4-dihydropyridine-3-carboxylate (8a)
CDCl3) d (ppm) 7.39e7.31 (m, 5H), 7.25e7.13 (m, 5H), 6.2 (s, 1H, NeH),
A solution containing 7a (135 mg, 0.36 mmol) in 5 mL DCM,
PyBOP (222 mg, 0.42 mmol), (R)-methylbenzylamine (0.055 mL,
0.42 mmol), and DIPA (0.151 mL, 1.07 mmol) was stirred at ambient
temperature for 1 h. The solvent was evaporated and the product
was purified by silica gel column chromatography. Yield: 70%, 1H
5.22 (m, 2H), 4.33 (m, 1H), 3.81 (m, 1H), 3.48 (t, J ¼ 7.2 Hz, 1H), 3.26 (s,
3H), 3.17e2.76 (m, 4H), 2.45 (s, 3H), 2.09 (s, 1H), 1.76 (s, 2H), 1.48e1.42
(m, 2H), 0.78 (t, J ¼ 7.5 Hz, 3H), 0.71e0.64 (m, 4H). ESI [M þ H] ¼ 596.5
4.1.4.5. Benzyl 5-((2S,3R)-4-(cyclopropylamino)-3-hydroxy-1-phenyl-
butan-2-ylcarbamoyl)-2,6-dimethyl-1-(methylsulfonyl)-4-propyl-1,4-
dihydropyridine-3-carboxylate (10c). Yield: 65%, 1H NMR (300 MHz,
NMR (300 MHz, CDCl3) d (ppm) 7.35 (m, 10H), 5.87 (s, 1H), 5.20 (m,
3H), 3.58 (q, J ¼ 6.3 Hz, 1H), 3.29(s, 3H), 2.46 (s, 3H), 2.30 (s, 1H),
1.54 (d, J ¼ 6.9 Hz, 3H), 1.67 (d, J ¼ 6.9 Hz, 3H)
CDCl3)
d (ppm) 7.36e7.30 (m, 5H), 7.21e7.16 (m, 5H), 6.11 (s, 1H,

S.-J. Choi et al. / European Journal of Medicinal Chemistry 45 (2010) 2578e2590
2587
NeH), 5.27e5.10 (m, 2H), 4.41e4.23 (m, 1H), 3.68 (m, 1H), 3.56 (t,
J ¼ 7.2 Hz, 1H), 3.28 (s, 3H), 3.11e2.81 (m, 4H), 2.42 (s, 3H), 2.16 (s,
1H), 2.08 (s, 1H), 2.04 (m, 1H), 1.77 (s, 2H), 1.39e1.05 (m, 4H),
0.80e0.69 (m, 3H), 0.48e0.40 (m, 4H). ESI [M þ H] ¼ 610.9
4.47 (m, 1H), 4.36 (m, 1H), 4.19 (s, 2H), 3.94 (q, J ¼ 6.6 Hz, 1H), 3.34
(m, 1H), 3.22e3.02 (m, 3H), 2.29 (s, 3H), 2.12 (s, 3H), 1.93 (s, 2H),
1.87 (s, 1H), 1.25 (dd, J ¼ 4.5, 1.5 Hz, 6H), 0.92 and 0.86
(2d,
J
¼
6.6 Hz, 3H, mixture of two diastereomers). ESI
[M þ H] ¼ 566.7
4.1.4.6. Benzyl 5-((2S,3R)-4-(cyclopropylamino)-3-hydroxy-1-phenyl
butan-2-ylcarbamoyl)-4-isopropyl-2,6-dimethyl-1-(methylsulfonyl)-
1,4-dihydropyridine-3-carboxylate (10d). Yield: 67%, 1H NMR
4.1.4.13. Isopropyl 2-(3-acetyl-5-((2S,3R)-4-(cyclopropylamino)-3-
hydroxy-1-phenylbutan-2-ylcarbamoyl)-2,6-dimethyl-4-propylpyr-
idin-1(4H)-yl)acetate (10k). Yield: 35%, 1H NMR (300 MHz, CDCl3)
(300 MHz, CDCl3)
d (ppm) 7.39e7.35 (m, 5H), 7.26e7.16 (m, 5H),
6.25 (s, 1H, NeH), 5.18 (s, 2H), 3.68 (m, 1H), 3.41 (d, J ¼ 3 Hz, 1H),
3.39 (m, 1H), 3.27 (s, 3H), 2.96e2.7 (m, 4H), 2.41 (s, 3H), 2.17
(m, 1H), 1.95 (m, 1H), 1.82 (s, 3H), 1.58 (m, 1H), 0.75 (d, J ¼ 6.6 Hz,
3H), 0.66 (d, J ¼ 7.2 Hz, 3H), 0.51e0.39 (m, 4H). ESI [M þ H] ¼ 610.8
d (ppm) 7.33e7.19 (m, 5H), 5.73 (s, 1H, NeH), 5.09 (m, 1H), 4.29
(m, 1H), 4.17 (s, 2H), 3.62 (m, 1H), 3.26e2.79 (m, 5H), 2.22 (s, 3H),
2.19 (s, 3H), 2.12 (m, 1H), 1.86 (s, 3H), 1.26 (dd, J ¼ 6.4, 1.5 Hz, 6H),
1.19e1.06 (m, 4H), 0.76 (t, J ¼ 6.3 Hz, 3H), 0.49e0.35 (m, 4H).
ESI [M ꢁ H] ¼ 540.2
4.1.4.7. Benzyl 5-((2S,3R)-4-(cyclopropylamino)-3-hydroxy-1-phenyl
butan-2-ylcarbamoyl)-2,6-dimethyl-1-(methylsulfonyl)-4-phenyl-
1,4-dihydropyridine-3-carboxylate (10e). Yield: 62.6%, 1H NMR
4.1.4.14. Isopropyl 2-(3-acetyl-5-((2S,3R)-4-(3-fluorophenylamino)-
3-hydroxy-1-phenylbutan-2-ylcarbamoyl)-2,6-dimethyl-4-propyl-
pyridin-1(4H)-yl)acetate (10l). Yield: 28.1%, 1H NMR (300 MHz,
(300 MHz, CDCl3)
d (ppm) 7.30e7.28 (m, 5H), 7.24e7.10 (m, 10H),
5.84 (s, 1H, NeH), 5.11 (s, 2H), 4.80 (s, 1H), 4.39e4.21 (m.1H), 3.42
(m, 1H), 3.19 (s, 3H), 3.0 (m, 1H), 2.79 (m, 1H), 2.73e2.62 (m, 2H),
2.51 (s, 3H), 2.04 (m, 1H), 1.79 (s, 3H), 0.44e0.26 (m, 4H). ESI
[M þ H] ¼ 644.8
CDCl3) d (ppm) 7.40e7.28 (m, 5H), 7.12 (m, 1H), 6.47 (m, 1H), 6.41
(m, 1H), 6.36 (m, 1H), 5.65 (s, 1H, NeH), 5.07 (m, 1H), 4.38 (m, 1H),
4.17 (s, 2H), 3.85 (m, 1H), 3.32 (dd, J ¼ 12.3, 3.6 Hz, 1H), 3.17e3.02
(m, 3H), 2.80 (m, 1H), 2.19 (d, J ¼ 6.3 Hz, 6H), 1.89 (s, 3H), 1.25
(d, J ¼ 6.3 Hz, 6H), 1.05 (m, 2H), 0.81 (m, 2H), 0.70 (t, J ¼ 6.6 Hz, 3H).
ESI [M ꢁ H] ¼ 592.2
4.1.4.8. Benzyl 5-((2S,3R)-4-(cyclopropylamino)-3-hydroxy-1-phenyl
butan-2-ylcarbamoyl)-1-(2-methoxy-2-oxoethyl)-2,4,6-trimethyl-
1,4-dihydropyridine-3-carboxylate (10f). Yield: 21%, 1H NMR
4.1.4.15. Isopropyl 2-(3-acetyl-5-((2S,3R)-3-hydroxy-4-(3-methoxy
phenylamino)-1-phenylbutan-2-ylcarbamoyl)-2,6-dimethyl-4-pro-
pylpyridin-1(4H)-yl)acetate (10m). Yield: 21%, 1H NMR (300 MHz,
(300 MHz, CDCl3)
d (ppm) 7.36 (m, 5H), 7.26e7.15 (m, 5H), 7.35
(s, 1H, NeH), 5.25e5.15 (m, 2H), 4.24 (s, 2H), 3.76 (s, 3H), 3.58
(m, 1H), 3.38 (q, J ¼ 4.2 Hz, 1H), 3.17 (m, 1H), 3.16e3.08 (m, 2H),
2.90e2.78 (m, 2H), 2.35 (s, 3H), 1.98 (s, 2H), 1.80 (s, 1H), 0.94 and
0.78 (2d, J ¼ 6.5 Hz, 3H, mixture of two diastereomers), 0.48e0.41
(m, 4H). ESI [M þ H] ¼ 576.4
CDCl3)
d
(ppm) 7.32e7.28 (m, 3H), 7.22 (m, 2H), 7.09 (t, J ¼ 8.4 Hz,
1H), 6.33e6.24 (m, 3H), 5.71 (m, 1H), 4.39 (m, 1H), 4.16 (s, 2H),
3.9 (m, 1H), 3.77 (s, 3H), 3.33 (m, 1H), 3.18 (m, 1H), 3.14e2.85
(m, 3H), 2.20 (s, 3H), 2.19 (s, 1H), 2.13 (s, 2H), 1.89 (s, 3H), 1.25
(dd, J ¼ 4.5, 2.7 Hz, 6H), 1.19e1.11 (m, 4H), 0.72 (t, J ¼ 6.9 Hz, 3H).
ESI [M ꢁ H] ¼ 604.2
4.1.4.9. Benzyl 5-((2S,3R)-4-(cyclopropylamino)-3-hydroxy-1-phenyl-
butan-2-ylcarbamoyl)-1-(2-ethoxy-2-oxoethyl)-2,4,6-trimethyl-1,4-
dihydropyridine-3-carboxylate (10g). Yield: 20%, 1H NMR (300 MHz,
4.1.4.16. Isopropyl 2-(3-acetyl-5-((2S,3R)-3-hydroxy-4-(3-methoxy
benzylamino)-1-phenyl butan-2-ylcarbamoyl)-2,6-dimethyl-4-propyl
pyridin-1(4H)-yl)acetate (10n). Yield: 12%, 1H NMR (300 MHz,
CDCl3)
d (ppm) 7.36e7.27 (m, 5H), 7.22e7.16 (m, 5H), 5.81 (s, 1H,
NeH), 5.24e5.15 (m, 2H), 4.25e4.18 (m, 4H), 3.79 (m, 1H), 3.63
(t, J ¼ 8.1 Hz, 1H), 3.35e2.89 (m, 5H), 2.32 (s, 3H), 2.27 (s, 3H), 1.65 (m,
1H), 1.24 (t, J ¼ 7.8 Hz, 3H), 0.87 and 0.79 (2d, J ¼ 8.1 Hz, 3H, mixture of
two diastereomers), 0.59e0.41 (m, 4H). ESI [M þ H] ¼ 590.9
CDCl3)
d
(ppm) 7.29e7.23 (m, 5H), 7.16 (dd, J ¼ 6.9, 1.5 Hz, 1H), 6.92
(m, 1H), 6.85(m, 2H), 5.09 (m, 1H), 4.25 (m, 1H), 4.16 (s, 2H), 3.82
(s, 3H), 3.74 (s, 2H), 3.65 (t, J ¼ 3.9 Hz, 1H), 3.16 (m, 1H), 2.97e2.73
(m, 4H), 2.19 (s, 6H), 1.86 (s, 3H), 1.27 (d, J ¼ 6.3 Hz, 6H), 1.18e0.85
(m, 4H), 0.76 (t, J ¼ 6.6 Hz, 3H). ESI [M þ H] ¼ 620.2
4.1.4.10. Benzyl 5-((2S,3R)-4-(cyclopropylamino)-3-hydroxy-1-phenyl-
butan-2-ylcarbamoyl)-1-(2-isopropoxy-2-oxoethyl)-2,4,6-trimethyl-
1,4-dihydropyridine-3-carboxylate (10h). Yield: 37%, 1H NMR
4.1.5. Dibenzyl 1-allyl-2,4,6-trimethyl-1,4-dihydropyridine-
3,5-dicarboxylate (11)
(300 MHz, CDCl3)
d (ppm) 7.37e7.34 (m, 5H), 7.21e7.15 (m, 5H),
5.77 (s, 1H, NeH), 5.19 (m, 2H), 5.09 (m, 1H), 4.23 (m, 1H), 4.18 (s,
2H), 3.56 (m, 1H), 3.37 (q, J ¼ 6.6 Hz, 1H), 3.07 (m, 1H), 2.87e2.78
(m, 3H), 2.31 (s, 3H), 2.16 (m, 1H), 1.94 (s, 2H), 1.80 (s, 1H), 1.25 (d,
J ¼ 15.9 Hz, 3H), 0.94 and 0.78 (2d, J ¼ 6.6 Hz, 3H, mixture of two
diastereomers), 0.46 (m, 4H). ESI [M þ H] ¼ 604.9
To a solution of compound 6a (100 mg, 0.25 mmol) in DMF
(5 mL) and NaH (15 mg, 0.3 mmol) was added dropwise allyl
bromide (0.05 mL, 0.4 mmol) at 0 ꢀC. The solution was stirred at
ambient temperature for 1 h and partitioned between a saturated
ammonium chloride solution and ethylacetate. The organic layer
was dried over sodium sulfate, filtered and evaporated under
vacuum. The product was purified by silica gel column chroma-
tography, using hexane:ethylacetate 7:1 (v/v).
4.1.4.11. Isopropyl
hydroxy-1-phenylbutan-2-ylcarbamoyl)-2,4,6-trimethylpyridin-1(4H)-
yl)acetate (10i). Yield: 10%, 1H NMR (300 MHz, CDCl3)
(ppm)
2-(3-acetyl-5-((2S,3R)-4-(cyclopropylamino)-3-
d
Yield: 45%, 1H NMR (300 MHz, CDCl3)
d (ppm) 7.36e7.28
7.41e7.19 (m, 5H), 5.75 (s, 1H, NeH), 5.07 (q, J ¼ 6.6 Hz, 1H), 4.32
(m, 1H), 4.17 (s, 2H), 3.63 (m, 1H), 3.2e3.0 (m, 2H), 2.83 (m, 3H),
2.23 (s, 3H), 2.19 (s, 3H), 2.14 (m, 1H), 1.83 (s, 3H), 1.26 (d, J ¼ 6 Hz,
6H), 0.91 and 0.85 (2d, J ¼ 6.6 Hz, 3H, mixture of two diastereo-
mers), 0.47 (m, 4H). ESI [M þ H] ¼ 512.2
(m, 10H), 5.82e5.78 (m, 1H), 5.26e5.21 (m, 2H), 5.19 (s, 2H), 4.22
(m, 2H), 3.97 (q, J ¼ 6.6 Hz, 1H), 2.40 (s, 6H), 0.95 (d, J ¼ 6.6 Hz, 3H).
4.1.6. Dibenzyl 1-(3-acetoxypropyl)-2,4,6-trimethyl-1,4-
dihydropyridine-3,5-dicarboxylate (12)
0.12 mL of 2 M borane-methylsulfide complex solution was
slowly added dropwise to compound 11 (100 mg, 0.232 mmol) in
THF and under nitrogen gas. The reaction mixture was stirred for
2 h, cooled in an ice-bath, and quenched with one drop of NaOH
solution. The flow of nitrogen was stopped when effervescence
4.1.4.12. Isopropyl 2-(3-acetyl-5-((2S,3R)-4-(3-fluorophenylamino)-
3-hydroxy-1-phenylbutan-2-ylcarbamoyl)-2,4,6-trimethylpyridin-1
(4H)-yl)acetate (10j). Yield: 11%, 1H NMR (300 MHz, CDCl3)
d
(ppm)
7.39e7.21 (m, 5H), 7.12 (q, J ¼ 6.6 Hz,1H), 6.42 (m, 3H), 5.64 (m, 1H),

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S.-J. Choi et al. / European Journal of Medicinal Chemistry 45 (2010) 2578e2590
ceased. After the addition of 3 M aqueous NaOH (1.5 mL) and 30%
aqueous H2O2 (0.69 mL), the reaction mixture was stirred at room
temperature for 2e3 h. The solution was saturated with solid NaCl
and products were extracted with diethyl ether. The ether extract
was washed with brine, dried over sodium sulfate, filtered and
evaporated under vacuum. The residue was purified by silica gel
column chromatography using chloroform: methanol 20:1. To
a solution of the purified product in 6 mL of DCM was added
dropwise acetic anhydride (0.02 mL) and TEA (0.019 mL). The
reaction mixture was stirred for 2 h and partitioned between
saturated sodium bicarbonate solution and DCM. The organic layer
was dried over sodium sulfate, filtered and evaporated under
vacuum. The product was purified by silica gel column chroma-
tography using hexane:ethylacetate 4.5:1.
4.1.10. Benzyl 5-((2S,3R)-4-(cyclopropylamino)-3-hydroxy-
1-phenylbutan-2-ylcarbamoyl)-2,4,6-trimethyl-1-(2-morpholino-
2-oxoethyl)-1,4-dihydropyridine-3-carboxylate (16b)
Yield: 61%, 1H NMR (300 MHz, CDCl3)
d (ppm) 7.37e7.29 (m, 5H),
7.21e7.13 (m, 5H), 5.86 (s, 1H, NeH), 5.14 (m, 2H), 4.27 (s, 2H), 4.20
(m, 1H), 3.71e3.68 (m, 4H), 3.62e3.54 (m, 4H), 3.45 (m, 1H), 3.38
(q, J ¼ 6.6 Hz, 1H), 3.06 (m, 1H), 2.83e2.75 (m, 3H), 2.29 (s, 3H), 1.89
(s, 2H), 1.79 (s, 1H), 0.97 and 0.81 (2d, J ¼ 6.6 Hz, 3H, mixture of two
diastereomers), 0.46e0.35 (m, 4H). ESI [M þ H] ¼ 631.9
4.1.11. Benzyl 5-((2S,3R)-4-(cyclopropylamino)-3-hydroxy-
1-phenylbutan-2-ylcarbamoyl)-1-(2-(isopropyl(methyl)amino)-2-
oxoethyl)-2,4,6-trimethyl-1,4-dihydropyridine-3-carboxylate (16c)
Yield: 54%, 1H NMR (300 MHz, CDCl3)
d (ppm) 7.39e7.28
Yield: 50%, 1H NMR (300 MHz, CDCl3)
d
(ppm) 7.31 (m, 10H), 5.19
(m, 5H), 7.21e7.13 (m, 5H), 5.79 (s, 1H, NeH), 5.24e5.10 (m, 2H),
4.83 (q, J ¼ 6.6 Hz, 1H), 4.30 (s, 2H), 3.53 (m, 1H), 3.37 (q, J ¼ 6 Hz,
1H), 3.19e3.05 (m, 2H), 2.85 (s, 3H), 2.84e2.75 (m, 2H), 2.29 (s, 3H),
2.09 (s, 1H), 1.83 (s, 3H), 1.25 and 0.82 (2d, J ¼ 6 Hz, 3H, mixture of
two diastereomers), 1.11 (dd, J ¼ 6.6, 2.4 Hz, 6H), 0.45e0.41 (m, 4H).
ESI [M þ H] ¼ 617.9
(s, 4H), 4.09 (t, J ¼ 6.9 Hz, 2H), 3.97 (q, J ¼ 6.9 Hz, 1H), 3.75
(t, J ¼ 7.8 Hz, 2H), 2.41 (s, 6H), 2.06 (s, 3H), 1.83 (m, 2H), 0.93
(d, J ¼ 6.9 Hz, 3H).
4.1.7. Benzyl 1-(3-acetoxypropyl)-5-((2S,3R)-4-(cyclopropylamino)-
3-hydroxy-1-phenylbutan-2-ylcarbamoyl)-2,4,6-trimethyl-1,4-
dihydropyridine-3-carboxylate (13)
4.1.12. General procedure for synthesis of derivatives of
2,6-monosubstituted 1,4-DHP (19aeb)
The methods for Lewis-acid mediated hydrolysis and the
coupling reaction with the HEA moiety are described above.
4-DMAP (348 mg, 0.2 mmol) was added to a stirred suspension
of coumalic acid (2 g, 14.27 mmol) in DCM/THF solution maintained
at room temperature. After stirring for 15 min, benzyl alcohol
(1.5 mL) and EDC (2.73 g, 14.27 mmol) were added. The reaction
mixture was stirred for 5 h and partitioned between water and
DCM. The organic layer was dried over sodium sulfate, filtered and
evaporated under vacuum. The product (18) was purified by silica
gel column chromatography using hexane:ethylacetate 4.5:1
(yield: 70%)
To a solution of compound 18 (100 mg, 0.434 mmol), and
acetylacetone (0.045 mL, 0.434 mmol) or benzylacetoacetate
(0.075 mL, 0.434 mmol) in ethanol (1 mL)/acetic acid (0.1 mL) [20],
was added ammonium acetate (50.2 mg, 0.651 mmol) and the
reaction mixture was stirred for 24 h. Ethanol was removed by
evaporation and the products (19aeb) were extracted with DCM,
washed with saturated sodium bicarbonate solution, and purified
by silica gel column chromatography using hexane:ethylacetate 3:1
(v/v) (yield: 33%)
Yield: 45%, 1H NMR (300 MHz, CDCl3)
d (ppm) 7.36e7.29
(m, 5H), 7.22e7.15 (m, 5H), 5.16 (m, 2H), 4.24 (m, 1H), 4.07 (m, 2H),
3.67 (m, 1H), 3.57 (m, 2H), 3.34 (q, J ¼ 6.6 Hz, 3H), 3.11 (m, 1H),
2.88e2.82 (m, 3H), 2.36 (s, 3H), 2.17 (m, 1H), 2.07 (s, 3H), 1.94
(s, 2H), 1.89 (s, 1H), 1.81 (m, 2H), 0.85 and 0.72 (2d, J ¼ 6.6 Hz, 3H,
mixture of two diastereomers). ESI [M þ H] ¼ 604.8
4.1.8. Dibenzyl 1-(2-(dimethylamino)-2-oxoethyl)-2,4,6-trimethyl-
1,4-dihydropyridine-3,5-dicarboxylate (15a)
Synthesis of 15a was identical to that of 11 except for using
t-butyl bromoacetate. Briefly, N-alkylated 14a (50 mg, 0.099 mmol)
was hydrolyzed with TFA (50%, v/v) in DCM for 2 h at room
temperature. After removing the solvent by evaporation, the acid
product was extracted with DCM and washed with sodium
bicarbonate. The organic layer was dried over sodium sulfate,
filtered and evaporated under vacuum. After purification by
silica gel column chromatography using chloroform: methanol 10:1
(v/v), the acid compound was reacted PyBop (60 mg) and R3 amine
building blocks in DCM (6 mL). The reaction mixture was stirred for
2 h and washed with saturated ammonium chloride solution. The
organic layer was extracted and dried over sodium sulfate. The
product was purified by silica gel column chromatography using
hexane:ethylacetate 1: (v/v).
4.1.12.1. Benzyl 5-acetyl-4,6-dimethyl-1,4-dihydropyridine-3-carboxy-
late (19b). 1H NMR (300 MHz, CDCl3)
d (ppm) 7.38e7.32 (m, 5H),
7.19 (s, 1H), 6.07 (s, 1H, NeH), 5.21 (s, 2H), 3.83 (q, J ¼ 6.6 Hz, 1H),
2.28 (s, 3H), 2.21 (s, 3H), 1.03 (d, J ¼ 6.6 Hz, 3H). ESI [M ꢁ H] ¼ 284.0
Yield 49%, 1H NMR (300 MHz, CDCl3)
d
(ppm) 7.35e7.29
4.1.13. Benzyl 5-acetyl-1-(2-isopropoxy-2-oxoethyl)-4,6-dimethyl-
1,4-dihydropyridine-3-carboxylate (20b)
(m, 10H), 5.18 (s, 4H), 4.36 (s, 2H), 4.02 (q, J ¼ 6.6 Hz, 1H), 3.06
(s, 3H), 3.02 (s, 3H), 2.33 (s, 6H), 1.04 (d, J ¼ 6.6 Hz, 3H).
ESI [M þ H] ¼ 477.9
The synthetic method was the same as that for compound 11,
except for using isopropylbromoacetate.
Yield: 69%, 1H NMR (300 MHz, CDCl3)
d (ppm) 7.37e7.31 (m, 5H),
4.1.9. Benzyl 5-((2S,3R)-4-(cyclopropylamino)-3-hydroxy-1-phenyl-
butan-2-ylcarbamoyl)-1-(2-(dimethylamino)-2-oxoethyl)-2,4,6-
trimethyl-1,4-dihydropyridine-3-carboxylate (16a)
The methods of Lewis-acid mediated hydrolysis and the
coupling reaction with the HEA moiety have been described
above.
7.04 (s, 1H), 5.22 (s, 2H), 5.08 (m, 1H), 4.14 (s, 2H), 3.80 (q, J ¼ 6.6 Hz,
1H), 2.31 (s, 3H), 2.17 (s, 3H), 1.26 (d, J ¼ 5.7 Hz, 6H), 1.06
(d, J ¼ 6.6 Hz, 3H).). ESI [M ꢁ H] ¼ 384.2
4.1.14. Benzyl 5-((2S,3R)-4-(cyclopropylamino)-3-hydroxy-
1-phenylbutan-2-ylcarbamoyl)-1-(2-isopropoxy-2-oxoethyl)-
2,4-dimethyl-1,4-dihydropyridine-3-carboxylate (21a)
Yield: 40%, 1H NMR (300 MHz, CDCl3)
d (ppm) 7.36e7.30
(m, 5H), 7.21e7.14 (m, 5H), 5.81 (s, 1H, NeH), 5.24e5.09 (m, 2H),
4.23 (s, 2H), 4.20 (m, 1H), 3.55 (m, 1H), 3.40 (q, J ¼ 6.3 Hz, 1H), 3.07
(m, 1H), 3.03 (s, 3H), 3.00 (s, 3H), 2.81e2.70 (m, 3H), 2.30 (s, 3H),
2.09e2.08 (m, 1H), 1.91 (s, 2H), 1.78 (s, 1H), 1.0 and 0.81
(2d, J ¼ 6.3 Hz, 3H, mixture of two diastereomers), 0.41e0.30
(m, 4H). ESI [M þ H] ¼ 589.7
The methods of Lewis-acid mediated hydrolysis and the
coupling reaction with the HEA moiety have been described above.
Yield: 37%, 1H NMR (300 MHz, CDCl3)
d (ppm) 7.39e7.27 (m, 5H),
7.20e7.15 (m, 5H), 6.98 (s, 1H), 5.81 (s, 1H, NeH), 5.19 (m, 2H), 5.03
(m,1H), 4.28 (m,1H), 4.03 (s, 2H), 3.58 (q, J ¼ 6 Hz,1H), 3.38 (m,1H),
3.17e2.79 (m, 4H), 2.18 (m, 1H), 1.81 (s, 3H), 1.25 (d, J ¼ 6.3 Hz, 6H),

S.-J. Choi et al. / European Journal of Medicinal Chemistry 45 (2010) 2578e2590
2589
1.12 and 0.82 (2d, J ¼ 6 Hz, 3H, mixture of two diastereomers),
0.52e0.35 (m, 4H). ESI [M þ H] ¼ 590.2
4.1.19.2. Tert-butyl 5-((2S,3R)-4-(cyclopropylamino)-3-hydroxy-1-
phenylbutan-2-ylcarbamoyl)-1-(2-isopropoxy-2-oxoethyl)-4-propyl-
1,4-dihydropyridine-3-carboxylate (24b). Yield: 34.2%, 1H NMR
4.1.15. Isopropyl 2-(3-acetyl-5-((2S,3R)-4-(3-fluorophenylamino)-
3-hydroxy-1-phenylbutan-2-ylcarbamoyl)-2,4-dimethylpyridin-1
(4H)-yl)acetate (21b)
(300 MHz, CDCl3) d (ppm) 7.35e7.22 (m, 5H), 6.94 (s, 1H), 6.91
(s, 1H), 5.97 (s, 1H, NeH), 5.08 (m, 1H), 4.25 (m, 1H), 3.91
(d, J ¼ 11.4 Hz, 2H), 3.78 (q, J ¼ 6.6 Hz,1H), 3.65 (m,1H), 3.17 (m, 2H),
2.86 (m, 2H), 2.2 (m, 1H), 1.49 (s, 9H), 1.25 (d, J ¼ 2.1 Hz, 6H), 1.15
(m, 4H), 0.80 (t, J ¼ 6.6 Hz, 3H), 0.49 (m, 4H). ESI [M ꢁ H] ¼ 568.3
Yield: 45%, 1H NMR (300 MHz, CDCl3)
d (ppm) 7.36e7.34
(m, 2H), 7.24e7.20 (m, 3H), 7.11 (q, J ¼ 7.8 Hz, 1H), 6.78 (s, 1H),
6.46e6.33 (m, 3H), 5.07 (m, 1H), 4.27 (m, 1H), 4.27e4.0 (m, 2H),
3.64 (q, J ¼ 6.6 Hz, 1H), 3.29 (m, 1H), 3.17e2.99 (m, 3H), 2.31
(s, 3H), 2.28 (s, 3H), 1.26 (d, J ¼ 5.7 Hz, 6H), 1.06 and 0.71
4.1.19.3. Tert-butyl 5-((2S,3R)-3-hydroxy-4-(3-methoxyphenylamino)-
1-phenylbutan-2-ylcarbamoyl)-1-(2-isopropoxy-2-oxoethyl)-4-propyl-
1,4-dihydropyridine-3-carboxylate (24c). Yield: 42.1%, 1H NMR
(2d,
J
¼
6.6 Hz, 3H, mixture of two diastereomers).
ESI [M þ H] ¼ 552.2
(300 MHz, CDCl3)
d
(ppm) 7.38e7.24 (m, 5H), 7.05 (t, J ¼ 7.5 Hz, 1H),
6.94 (s,1H), 6.93 (s,1H), 6.31e6.25 (m, 3H), 5.65 (s,1H, NeH), 5.06 (m,
1H), 3.93 (d, J ¼ 6.9 Hz, 2H), 3.86 (m,1H), 3.81 (d, J ¼ 2.4 Hz, 3H), 3.48
(m,1H), 3.29(m, 2H), 3.15e3.08(m, 2H),1.44 (s, 9H),1.27 (d, J ¼ 4.8 Hz,
6H), 1.25e1.01 (m, 2H), 0.68 (t, J ¼ 2.4 Hz, 3H). ESI [M ꢁ H] ¼ 634.2
4.1.16. Isopropyl 2-(3-acetyl-5-((2S,3R)-3-hydroxy-4-(3-methoxy-
benzylamino)-1-phenylbutan-2-ylcarbamoyl)-2,4-dimethylpyridin-1
(4H)-yl)acetate (21c)
Yield: 59%, 1H NMR (300 MHz, CDCl3)
d (ppm) 7.30e7.24
(m, 5H), 7.21 (m, 1H), 6.99e6.82 (m, 2H), 6.73 (s, 1H), 6.20 (s, 1H,
NeH), 6.14 (s, 1H, NeH), 5.05 (m, 1H), 4.24 (m, 1H), 4.1 (s, 2H), 3.96
(m, 1H), 3.82 (s, 3H), 3.80 (s, 2H), 3.39 (q, J ¼ 6 Hz, 1H), 3.24e3.09
(m, 2H), 2.96e2.79 (m, 2H), 2.28 (s, 3H), 2.13 (s, 3H), 1.26 (m, 6H),
0.81 and 0.78 (2d, J ¼ 6 Hz, 3H, mixture of two diastereomers).
ESI [M þ H] ¼ 578.2
4.1.20. Tert-butyl 1-(2-isopropoxy-2-oxoethyl)-4-methyl-5-((R)-1-
phenylethylcarbamoyl)-1,4-dihydropyridine-3-carboxylate (25a)
Yield ¼ 45.9%, 1H NMR (300 MHz, CDCl3)
d (ppm) 7.35 (s, 1H),
7.21 (m, 5H), 7.04 (s, 1H), 6.00 (s, 1H, NeH), 5.22 (m, 1H), 5.13
(m, 1H), 4.49 (d, J ¼ 6.3 Hz, 2H), 3.63 (t, J ¼ 3.9 Hz, 1H), 1.50 (s, 3H),
1.47 (s, 9H), 1.25 (d, J ¼ 3.6 Hz, 6H), 1.08 (d, J ¼ 6.3 Hz, 3H).
4.1.17. General procedure for synthesis of derivatives of
2,6-unsubstituted 1,4-DHP (22a,b)
4.1.21. Isopropyl 2-(3-((2S,3R)-4-(cyclopropylamino)-3-hydroxy-
1-phenylbutan-2-ylcarbamoyl)-4-methyl-5-((R)-1-phenyl-
ethylcarbamoyl)pyridin-1(4H)-yl) acetate(26a)
A solution of ammonium acetate (1.5 eq), tert-butylpropiolate
(2 eq) and butylaldehyde (1 eq) (acetaldehyde or butylaldehyde)
was refluxed at 90 ꢀC, and stirred for 24 h [21,22]. After cooling to
room temperature, the solvent was removed by evaporation and
the residue purified by silica gel column chromatography.
Yield ¼ 28.4%, 1H NMR (300 MHz, CDCl3)
d (ppm) 7.34e7.22
(m, 10H), 6.79 (s, 1H), 6.72 (s, 1H), 5.23 (m, 1H), 5.06 (m, 1H), 4.23
(m, 1H), 3.94 (d, J ¼ 6.0 Hz, 2H), 3.79e3.68 (m, 2H), 2.98e2.86
(m, 4H), 2.08 (m, 1H), 1.56 (t, J ¼ 4.2 Hz, 3H), 1.24 (d, J ¼ 2.1 Hz, 6H),
0.98 and 0.91 (2d, J ¼ 6.3 Hz, 3H, mixture of two diastereomers),
0.55e0.53 (m, 4H). ESI [M þ H] ¼ 589.3
4.1.17.1. Di-tert-butyl 4-methyl-1,4-dihydropyridine-3,5-dicarboxylate
(22a). Yield: 12.8%, 1H NMR (300 MHz, CDCl3)
d (ppm) 7.10 (s, 1H),
7.08 (s, 1H), 5.82 (s, 1H, NeH), 3.75 (q, J ¼ 6.6 Hz, 1H), 1.46 (s, 18H),
4.1.22. Isopropyl 2-(3-((2S,3R)-4-(cyclopropylamino)-3-hydroxy-
1-phenylbutan-2-ylcarbamoyl)-5-((R)-1-phenylethylcarbamoyl)-
4-propylpyridin-1(4H)-yl)acetate (26b)
1.08 (d, J ¼ 6.6 Hz, 3H)
4.1.17.2. Di-tert-butyl 4-propyl-1,4-dihydropyridine-3,5-dicarboxylate
Yield: 48.2%, 1H NMR (300 MHz, CDCl3)
d (ppm) 7.32 (m, 5H),
(22b). Yield: 17%, 1H NMR (300 MHz, CDCl3)
d
(ppm) 7.17 (s, 1H), 7.16
7.21 (m, 5H), 6.94 (s, 1H), 6.85 (s, 1H), 5.97 (s, 1H, NeH), 5.32
(m, 1H), 5.04 (m, 1H), 4.25 (m, 1H), 3.79 (d, J ¼ 7.8 Hz, 2H), 3.68
(m, 2H), 2.93e2.82 (m, 4H), 2.14 (m, 1H), 1.51 (m, 3H), 1.24
(q, J ¼ 3.9 Hz, 9H), 1.18e1.05 (m, 4H), 0.73 (t, J ¼ 3.2 Hz, 3H), 0.48
(m, 4H). ESI [M þ H] ¼ 617.2
(s, 1H), 5.92 (s, 1H, NeH), 3.79 (t, J ¼ 5.1 Hz, 1H), 1.43 (s, 18H),
1.37e1.32 (m, 4H), 0.82(t, J ¼ 7.2 Hz, 3H)
4.1.18. Di-tert-butyl 1-(2-isopropoxy-2-oxoethyl)-4-methyl-
1,4-dihydropyridine-3,5-dicarboxylate (23a)
Yield: 37.8%, 1H NMR (300 MHz, CDCl3)
d
(ppm) 7.1 (s, 1H), 6.87
4.1.23. Isopropyl 2-(3-((2S,3R)-3-hydroxy-4-(3-methoxyphenyl-
amino)-1-phenylbutan-2-ylcarbamoyl)-5-((R)-
(s, 1H), 5.13 (m, 1H), 4.62 (m, 1H), 4.19 (s, 2H), 1.43 (s, 18H), 1.28
(d, J ¼ 2.1 Hz, 9H)
1-phenylethylcarbamoyl)-4-propylpyridin-1(4H)-yl)acetate (26c)
Yield: 12.7%, 1H NMR (300 MHz, CDCl3)
d (ppm) 7.31e7.19
4.1.19. General procedure for the hydrolysis reaction and the
coupling reaction with the HEA moiety (24aec)
(m, 10H), 7.08 (t, J ¼ 7.8 Hz, 1H), 6.89 (s, 1H), 6.79 (s, 1H), 6.28
(m, 1H), 5.23 (m, 1H), 5.05 (m, 1H), 4.19 (m, 1H), 3.89 (d, J ¼ 5.4 Hz,
2H), 3.76 (s, 3H), 3.42 (t, J ¼ 3.9 Hz, 1H), 3.28 (m, 1H), 3.05 (m, 4H),
1.52 (s, 3H), 1.21 (q, J ¼ 6.6 Hz, 6H), 1.12 (m, 4H), 0.74 (t, J ¼ 6.9 Hz,
3H). ESI [M þ H] ¼ 683.2
N-alkylated compounds (23a,b) were hydrolyzed with 96%
formic acid for 20 min at 0 ꢀC to room temperature. The solvent was
removed by evaporation and the residue was purified by silica gel
column chromatography using hexane:ethylacetate 3:1. The
coupling reaction with the HEA moiety was as described above.
4.2. BACE-1-inhibitory activities
4.1.19.1. Tert-butyl 5-((2S,3R)-4-(cyclopropylamino)-3-hydroxy-1-phe-
nylbutan-2-ylcarbamoyl)-1-(2-isopropoxy-2-oxoethyl)-4-methyl-1,4-
dihydropyridine-3-carboxylate(24a). Yield ¼ 55%, 1H NMR (300 MHz,
4.2.1. Cell-based assay
To evaluate the BACE-1-inhibitory activity, an alkaline phos-
phatase (AP)-APP-cell-based assay was carried out in HEK293 cells
with pCDNA-neo-SEAP-APP plasmid. The plasmid encoding
a fusion protein of APP and AP containing the BACE-1 cleavage site,
was stably transfected into HEK293 cells. BACE-1-inhibitory activity
of test compounds was measured indirectly by quantification of
CDCl3) d (ppm) 7.31e7.24 (m, 5H), 6.84 (s, 1H), 6.81 (s, 1H), 6.13 (s, 1H,
NeH), 5.06 (m, 1H), 4.21 (m, 1H), 3.98 (d, J ¼ 3.9 Hz, 2H), 3.65 (m, 1H),
3.42 (q, J ¼ 1.2 Hz, 1H), 2.98e2.82 (m, 4H), 2.03 (m, 1H), 1.47 (s, 9H),
1.26 (dd, J ¼ 5.4 Hz, 1.8 Hz, 6H), 0.95 and 0.92 (2d, J ¼ 6.3 Hz, 3H,
mixture of two diastereomers), 0.61 (m, 4H). ESI [M þ H] ¼ 542.2
secreted AP after cleavage of the b-site of APP by BACE-1. Cells in

2590
S.-J. Choi et al. / European Journal of Medicinal Chemistry 45 (2010) 2578e2590
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DMEM containing 10% FBS and antibiotics were plated at a density
of 2 ꢃ 104 cells per well of a 96-well plate and allowed to adhere for
24 h. The culture medium was aspirated and DMEM containing
1 mg/mL BSA was added to each well. A BACE-1 inhibitor, diluted in
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DMEM containing 1 mg/mL BSA and 5
no F6886), was added, and the cells were incubated for 6 h. A 50
aliquot of conditioned medium was transferred to each well of
a 96-well fluorescence assay plate and 50 L of a yellow liquid
mM forskolin (Sigma; catalog
mL
m
alkaline phosphatate substrate was added. Absorbance was
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ꢀ
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bysimulated annealingat700 K. Finalenergy minimizationwasset as
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determined on the basis of energy.
Acknowledgment
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This study was supported by a grant of the Korea Healthcare
technology R&D Project, Ministry for Health, Welfare & Family
Affairs, Republic of Korea (A090125) and by a grant from the
Institute of Medical System Engineering (iMSE) in the GIST, Korea.
References
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