Conformational restriction approach to β-Secretase (BACE1) inhibitors: Effect of a cyclopropane ring to induce an alternative binding mode

Article abstract of DOI:10.1021/jm3011405

Improvement of a drugs binding activity using the conformational restriction approach with sp³ hybridized carbon is becoming a key strategy in drug discovery. We applied this approach to BACE1 inhibitors and designed four stereoisomeric cyclopropane compounds in which the ethylene linker of a known amidine-type inhibitor 2 was replaced with chiral cyclopropane rings. The synthesis and biologic evaluation of these compounds revealed that the cis-(1S,2R) isomer 6 exhibited the most potent BACE1 inhibitory activity among them. X-ray structure analysis of the complex of 6 and BACE1 revealed that its unique binding mode is due to the apparent CH-π interaction between the rigid cyclopropane ring and the Tyr71 side chain. A derivatization study using 6 as a lead molecule led to the development of highly potent inhibitors in which the structure-activity relationship as well as the binding mode of the compounds clearly differ from those of known amidine-type inhibitors.

Full text of DOI:10.1021/jm3011405

Article
pubs.acs.org/jmc
Conformational Restriction Approach to β‑Secretase (BACE1)
Inhibitors: Effect of a Cyclopropane Ring To Induce an Alternative
Binding Mode
Shuji Yonezawa,*,†,⊥ Takahiko Yamamoto,‡ Hidekuni Yamakawa,‡ Chie Muto,‡ Motoko Hosono,‡
Kazunari Hattori,‡ Kenichi Higashino,† Takashi Yutsudo,‡ Hideo Iwamoto,§ Yutaka Kondo,§
Masahiro Sakagami,‡ Hiroko Togame,∥ Yoshikazu Tanaka,† Toru Nakano,† Hiroshi Takemoto,‡
Mitsuhiro Arisawa,⊥ and Satoshi Shuto*,⊥
†Shionogi Innovation Center for Drug Discovery, Shionogi & Co., Ltd., Kita-21 Nishi-11 Kita-ku, Sapporo 001-0021, Japan
‡Pharmaceutical Research Division, Medicinal Research Laboratories, and Innovative Drug Discovery Research Laboratories, Shionogi
& Co., Ltd., 1-1, Futaba-cho 3-chome, Toyonaka, Osaka 561-0825, Japan
§Shionogi Techno Advance Research Co., Ltd., 1-1, Futaba-cho 3-chome, Toyonaka, Osaka 561-0825, Japan
∥Quality, Safety and Regulatory Affairs Management Division, Shionogi & Co., Ltd., 1-8, Doshomachi 3-chome, Chuo-ku, Osaka
541-0045, Japan
⊥Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan
S
* Supporting Information
ABSTRACT: Improvement of a drug’s binding activity using the
conformational restriction approach with sp3 hybridized carbon is
becoming a key strategy in drug discovery. We applied this approach to
BACE1 inhibitors and designed four stereoisomeric cyclopropane
compounds in which the ethylene linker of a known amidine-type
inhibitor 2 was replaced with chiral cyclopropane rings. The synthesis
and biologic evaluation of these compounds revealed that the cis-(1S,2R) isomer 6 exhibited the most potent BACE1 inhibitory
activity among them. X-ray structure analysis of the complex of 6 and BACE1 revealed that its unique binding mode is due to the
apparent CH−π interaction between the rigid cyclopropane ring and the Tyr71 side chain. A derivatization study using 6 as a
lead molecule led to the development of highly potent inhibitors in which the structure−activity relationship as well as the
binding mode of the compounds clearly differ from those of known amidine-type inhibitors.
signs of significant dysfunction.3 Therefore, BACE1 inhibitors
INTRODUCTION
■
are considered to be an attractive target for a causative
Alzheimer’s disease (AD) is a progressive neurologic disorder
treatment of AD.4 Many pharmaceutical companies are now
of the brain, now considered to be the main form of dementia.
competing to develop these inhibitors, and clinical trials with
AD causes a gradual and progressive loss of memory and
cognitive ability, impaired orientation to surroundings, and
impaired language ability, and it eventually leads to death. In
some compounds have recently begun.5
The cyclic amidine unit is the main common component of
many targeted BACE1 inhibitors. The first report of a cyclic
amidine-type inhibitor using a fragment-based drug design
the amyloid cascade hypothesis, overproduction of amyloid-
beta (Aβ) is considered to trigger the onset of AD.1 Aβ
technique was made by AstraZeneca/Astex reseachers in 2007
gradually accumulates in the brain, forming soluble oligomers
and senile plaques. Neurofibrillary tangles caused by hyper-
phosphorylation of the τ protein are also observed and lead to
neuronal death. Two key proteases, γ-secretase and β-secretase
(BACE1), are considered to have an important role in Aβ
production. BACE1 first cleaves amyloid precursor protein
(APP) to generate soluble amyloid precursor protein β
(Figure 1).6 They used nuclear magnetic resonance screening
to identify the first small fragment hit 1 containing an
aminopyrimidone ring system. They then studied the
structure/activity relationship (SAR) of the hit 1 using a
fragment-growing approach based on the X-ray structure of the
complex with BACE1, and they developed an effective inhibitor
with submicromolar order inhibitory activity by extending the
hydrophobic aryl substituent with an ethylene linker. They also
reported the X-ray structures of some other amidine-based
(sAPPβ) and C99 peptide, which remains bound to a
membrane.2 The γ-secretase cuts the C99 peptide to produce
mature Aβ peptides of various lengths, including Aβ42, which is
thought to be the most pathogenic among the Aβs produced.
inhibitors complexed with BACE1, in which a common binding
Inhibition of BACE1 activity decreases the production of all
forms of Aβ, and knockdown of BACE1 expression in mice
stops the production of Aβ40 and Aβ42 in vivo, without any
Received: August 3, 2012
Published: September 21, 2012
© 2012 American Chemical Society
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Figure 1. Basic concept of the compound design.
mode was disclosed.6,7f,g Two nitrogen atoms in the cyclic
amidine ring make tight hydrogen bonds with the side chains of
catalytic residues Asp32 and Asp228. The hydrophobic aryl part
of the inhibitors is accommodated in the S1 and S3 pocket of
the active sites, allowing for an effective hydrophobic
interaction with BACE1. This binding mode is analogous in
complexes of other amidine-type inhibitors.7
histamine H3 agonists,14a,b and histamine H3/H4 antago-
nists,14c,d whose conformation is effectively controlled by the
characteristic stereochemical features of cyclopropane. Its low
molecular weight and rigid framework may also be suitable for
fragment-based drug design, especially in a fragment growing
approach, as well as a component in a fragment compound
library.
To convert the fragments into more effective lead
compounds, saturated alkyl chains or sp2 hybridized carbon-
including linkers, such as carbon−carbon double bonds or
amide bonds, are commonly utilized for fragment-growing.8
Saturated alkyl chains are probably the most commonly utilized
linkers because of their structural simplicity and facile synthesis,
but their high degree of free rotation may impede their binding
affinity. sp2 linkers are also widely used for their availability as
building blocks due to the recent development of coupling
reactions. They are also advantageous as a facile method for
introducing conformational fixation to molecules, due to their
low degree of free rotation. On the other hand, the
incorporation of sp2 carbon-based components sometimes
causes undesirable physical properties, e.g., low solubility,
probably due to intermolecular static interactions caused by
the planar sp2 carbon.9 Further, identification of a structurally
new drug candidate is often difficult because of the shortage of
sp2-carbon based chemical entities in a novel structure and its
limited three-dimensional structural diversity. On the other
hand, the use of rigid structures based on sp3 carbons is now
becoming a key topic in drug design in terms of improved
selectivity.10 This approach may enable the development of
novel, structurally diverse compounds and potential binders
with improved activity by compensating for the entropic loss
generated by binding to the target compound.11
Cyclopropane is widely used in drug design and is
incorporated in many drugs that are currently on the market.
It is the smallest, rigid sp3-carbon based ring system and has
potential application in a conformational restriction approach,
but the number of successful applications to date is small,
probably due to the limited number of examples of its
stereoselective and industrially feasible synthetic methods.12
Our laboratory has been studying the synthesis of optically
active cyclopropanes from commercially available chiral
epichlorohydrin and their utilization in drug design. We
successfully produced novel, selective NMDA antagonists,13
Therefore, here we investigated the feasibility of replacing the
ethylene part of a known BACE1 inhibitor 2 with a
cyclopropane ring, and we designed the conformationally
restricted analogs 6 and 7 and their enantiomers ent-6 and ent-
7 to improve their BACE1 inhibitory activity by placing the
substituents at a position suitable for effective binding and by
reducing the active entropic energy loss upon binding BACE1
due to the conformational restriction (Figure 1). Using the
most active compound 6 among them as a lead, we obtained
SAR results that were clearly different from those of the
unrestricted inhibitors to identify potent BACE1 inhibitors
based on the characteristic binding mode of the compounds
due to the conformational restriction. In this report, we
describe the results in detail.
RESULTS AND DISCUSSION
■
Docking Simulations of the Conformationally Re-
stricted Analogs. First, the conformations of these four
stereoisomers with the lowest energy were calculated in the
MMFF94x force field15 using the Molecular Operating
Environment (MOE).16 The structures obtained by the
calculations were superimposed on the X-ray crystallographic
structure of the ethylene linker compound 3 complexed with
human BACE1 (PDB code no. 2VA5), as shown in Figure 2. In
the BACE1 complex, compound 3 adopts a folded con-
formation that is not likely to be the most stable in its unbound
state. While the two cis isomers 6 and ent-6 assume
conformations similar to that of compound 3, the benzene
rings of the trans isomers 7 and ent-7 located at positions
clearly different from that of the indole ring in 3. On the basis
of this observation, the inhibitory activities of the trans isomers
were predicted to be weaker than those of the cis isomers.
Comparison of the two cis isomers suggested that ent-6 was
superimposed on compound 3 more precisely than 6.
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was deprotected with TBAF to isolate the cis alcohol 12. After
oxidation of 12 into the carboxylic acid 14, the β-ketoester 15
was successfully obtained from 14 using the Masamune
conditions.18 The minor trans isomer was completely removed
by SiO2 column chromatography at this stage. According to the
aminopyrimidone synthesis method reported previously,6b,c 15
was treated with guanidine carbonate in the presence of
MeONa. Epimerization at the 1-position of cyclopropane
occurred concomitantly during cyclization to give a mixture of
diastereomers 16 in a ratio of cis/trans = 1.3:1. Highly basic
guanidine may have caused the isomerization via the
enolization of the keto-cyclopropyl moiety in 15. Selective
methylation of 16 with MeI occurred at the lactam nitrogen,
and finally the desired 6 was obtained after the reversed phase
preparative HPLC purification, although the yield was poor. Its
enantiomer, ent-6, was synthesized using the same reaction
sequence starting from (R)-(−)-epichlorohydrin.
The trans-(1R,2R) isomer 7 and its enantiomer trans-(1S,2S)
ent-7 were prepared according to a procedure similar to those
for the cis-isomers 6 and ent-6, from the known chiral
phenylcyclopropane carboxylic acid 17 and ent-17 (Scheme
2).19 Neither epimerization nor a decrease in enantiomeric
Figure 2. Superimpositions of the X-ray structure of compound 3 are
in gray (PDB 2VA5) and the predicted conformations of each
stereoisomer are in magenta: (a) 6 [cis-(1S,2R)]; (b) ent-6 [cis-
(1R,2S)]; (c) 7 [trans-(1R,2R)]; and (d) ent-7 [trans-(1S,2S)].
a
Scheme 2. Synthesis of 7
We planned to synthesize compounds 6, ent-6, 7, and ent-7,
to examine their BACE1 inhibitory activities to confirm the
above predictions and to identify the most active stereoisomer.
Synthesis of Compounds. First generation synthesis of
the cis-cyclopropane derivative 6 was performed based on our
previous report of the stereoselective synthesis of phenyl-
cyclopropane carboxylic acid 14 (Scheme 1).17 Lactone 9a was
obtained by the condensation of optically pure (S)-(+)-epi-
chlorohydrin 8 and phenylacetonitrile under basic conditions.
Subsequent alkaline hydrolysis of the lactone followed by
silylation gave the disilylated product on both the carboxyl and
hydroxyl moieties, and then the silyl ester, which was selectively
hydrolyzed under aqueous basic conditions to yield the
carboxylic acid 10. 10 was subjected to Barton decarboxylation,
giving the cis-product selectively, which, without purification,
a
Reagents and conditions: (a) (i) 1.5 equiv of CDI, MeCN, rt, 1 h, (ii)
2.1 equiv of potassium ethyl malonate, 2.5 equiv of MgCl2, 3.2 eq
Et3N, MeCN, r.t., then 80 °C, 2 h, 82%; (b) 2 equiv of guanidine
carbonate, 2 equiv of EtONa, EtOH, 80 °C, 5 h, 57%; (c) 1.1 equiv of
MeI, 1.1 equiv of K2CO3, DMF, rt, 18 h, 67%.
a
Scheme 1. First-Generation Synthesis of 6
a
Reagents and conditions: (a) (i) 1.1 equiv of PhCH2CN, 2.5 equiv of NaNH2, benzene, rt, 1 h, (ii) 10 M KOH aq, EtOH 20 h, reflux, 56%; (b) (i)
10 M KOH aq, EtOH, rt, 1 h, (ii) 4.5 equiv of TBDPS-Cl, 5 equiv of imidazole, DMF, rt, overnight, (iii) 10 M KOH aq, EtOH, 1 h, rt, 48%; (c) (i)
1.2 equiv of 11, 1.5 equiv of n-Bu3P, 3 equiv of (TMS)3SiH, 0.2 equiv of AIBN, benzene, rt, 1 h, then 80 °C, 6.5 h, (ii) 1.5 equiv of TBAF, THF,
45%, cis/trans = 10.7: 1; (d) 0.1 equiv of TPAP, 3 equiv of NMO, MS4A, CH2Cl2, rt, 1 h, 75%, cis/trans = 10.6:1; (e) 1.5 equiv of NaClO2, 1.5 equiv
of H2NSO3H, MeOH/H2O, 0 °C, 1 h, 90%, cis/trans = 7.6: 1; (f) (i) 1.5 equiv of CDI, MeCN, rt, 1 h, (ii) 2.1 equiv of potassium ethyl malonate, 2.5
equiv of MgCl2, 3.2 equiv of Et3N, MeCN, rt, then 80 °C, 2 h, 77%; (g) 2 equiv of guanidine carbonate, 2 equiv of EtONa, EtOH, 80 °C, 5 h, 80%,
cis/trans = 1.3:1; (h) 1.3 equiv of MeI, 1.3 equiv of K2CO3, DMF, rt, 22 h, 47%; (i) preparative HPLC separation, 6 23%, 7 56%.
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a
Scheme 3. Improved Synthesis of cis-Cyclopropane Derivatives
a
Reagents and conditions: (a) (i) ArCH2CN, NaNH2, benzene or toluene, rt, (ii) 10 M KOH aq, EtOH, reflux, 58% (9a), 61% (9b), 63% (ent-9b),
62% (9c); (b) (i) 10 M KOH aq, EtOH, H2O, rt, (ii) 1 equiv of MeONa, Et2O, 80% (20a), 87% (20b), 94% (ent-20b), 95% (20c); (c) cat. RuCl3,
NaIO4, MeCN, H2O, rt, 75% (21a), 87% (21b), 80% (ent-21b), 69% (21c); (d) (i) CDI, (ii) potassium ethyl malonate, MgCl2, Et3N, MeCN, rt,
then 80 °C; (e) (i) EtONa, (ii) guanidine carbonate, EtOH, 90 °C, 14 h; (f) MeI, K2CO3, DMF, rt, 32% (24a), 34% (24b), 29% (ent-24b), 33%
(24c) for 3 steps; (g) Boc2O, DMAP, CH2Cl2, rt, 85% (25a), 89% (25b), 82% (ent-25b), 92% (25c); (h) 10 M KOH aq, EtOH, H2O, 60 °C; (i) (i)
N,O-bistrimethylsilylacetamide, (ii) Boc2O, DMAP, CH2Cl2, rt, 78% (27a), 80% (27b), 78% (ent-27b), 72% (27c) for 2 steps.
purity was observed during the synthesis of these trans isomers,
indicating that intermediate ketoester 18 and ent-18 were likely
to be thermodynamically far more stable than the cis-oriented
ketoester 15 under basic conditions.
As described below, the cis-(1S,2R) isomer 6 was the most
potent BACE1 inhibitor among the four conformationally
restricted stereoisomers. The first generation synthetic route
shown in Scheme 1 involved an undesired epimerization at the
1-position in the cyclopropane ring to the trans-isomer at the
cyclization step, which was a big problem for scale-up synthesis
and efficient exploration of the SAR, so we developed an
alternative stereoelective synthetic route for the cis-cyclo-
propane derivatives (Scheme 3).
We planned to prepare various derivatives of the cis-(1S,2R)
isomer 6 that have a substituent at the 3- or 4-position of the
benzene ring using the Suzuki−Miyaura coupling reaction, and
therefore, 3- or 4-bromo substituted derivatives of 6 were also
required. Thus, the 3- or 4-bromo substituted lactones 9b, ent-
9b, and 9c were prepared according to the procedure for the
synthesis of 9a described above, the enantiomeric excesses of
which were confirmed to be around 95%. Compounds 9 were
then hydrolyzed under aqueous basic conditions to convert
them to a stable and fine crystalline carboxylic acid salt 20,
subsequent ruthenium tetroxide oxidation of which gave the
bis-carboxylic acids 21 in good yields. Next, we introduced the
β-ketoester moiety into 21. β-Ketoesterification using Masa-
mune conditions18 proceeded selectively at the sterically less
hindered carboxy group to yield the desired 22 as a sole
product. Treatment of 22 with sodium ethoxide and
subsequent heating with guanidine carbonate produced 23;
then methylation of 23 with MeI proceeded selectively at the
carboxyl group and the lactam nitrogen in the amino-
pyrimidone ring to give 24 with ∼30% yield from 21. After
protection of the amino moiety of 24 with two Boc groups,
alkaline hydrolyses of the resulting methyl ester 25 were carried
out, where one of the two Boc groups was simultaneously
removed to furnish 26. In situ silylation of the carboxy group
with N,O-bistrimethylsilylacetamide (BSA), and then a trial of
Boc protection yielded 27, the substrate for stereoselective
Barton decarboxylation (Scheme 4).17,20
Optimization studies for the radical decarboxylative isomer-
ization reaction were performed using ent-27b as a substrate
(Table 1). First, according to the best conditions determined in
our previous report,17 ent-27b was heated with 11 (1.2 equiv),
Bu3P (3 equiv), and TMS3SiH (3 equiv) in the presence of
AIBN as a radical initiator to give ent-28b in 32% yield, whereas
none of the trans isomer was produced. The thio-pyridine
containing compound 29 was also isolated in 42% yield as a
byproduct (entry 1).21 We deduced that the side-reaction might
proceed due to the low reactivity of (TMS)3SiH. When 6 equiv
of (TMS)3SiH was used to promote trapping of the radical
intermediate by the reagent, the yield of ent-28b was slightly
improved, but there was still significant production of 29 (entry
2 in Table 1). Thus, using a more reactive reductant, Bu3SnH,
the desired product ent-28b was successfully obtained in 57%
yield (entry 3) without producing the trans isomer under the
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Scheme 4. Barton Decarboxylation and Cross Coupling
Reactions
Table 2. BACE1 Inhibitory Activity of 2 and Its
Conformationally Restricted Analogs
a
BACE1 IC50
ligand efficiency (LE) (kcal/mol per heavy
b
compd
(μM)
atom)
a
2
171
157
0.30
0.29
0.24
6
ent-6
7
619
>2000
>2000
ent-7
a
Compound 2 was prepared according to the reported procedures.6b,c
IC50 in ELISA. Values are means of at least two experiments.
b
exhibited significant inhibitory activity [IC50 = 157 μM, ligand
efficiency (LE) = 0.29], while the activity of its enantiomer ent-
6 was approximately four times weaker (IC50 = 619 μM, LE =
0.24). The two trans isomers did not show any inhibitory
activity (>2000 μM). Although the IC50 value and LE of 6 were
not definitely improved, it was equally active with the
nonrestricted ethylene linker compound 2 (IC50 = 171 μM,
LE = 0.30). Thus, the stereochemistry of the cyclopropane ring
was closely related to the inhibitory activity of BACE1, and the
cis-(1S,2R) isomer 6 was the most suitable for further design of
compounds with improved activity.
a
Reagents and conditions: (a) 11 (1.2 equiv), n-Bu3P (3 equiv), n-
Bu3SnH (3 equiv), AIBN (0.2 equiv), toluene, rt, then 80 °C, 53%
(28a), 48% (28b), 52% (ent-28b), 38% (28c); (b) TFA, CH2Cl2, rt,
75% (6), 81% (31), 80% (ent-31); (c) R-B(OH)2 or R-Bpin, K2CO3,
PdCl2(PPh3)2, dioxane, H2O, 100 °C (Method A), 150 °C under
microwave, 30 min (Method B).
X-ray Crystallographic Structure Analysis of 6
Complexed with BACE1. To clarify the bioactive con-
formation of 6 and to gain more detailed insight into the
structure modification of 6, we prepared the crystalline complex
of human BACE1 and 6 and analyzed its X-ray crystallographic
structure (Figure 3). As expected from the structures of other
previously reported cyclic amidine-based inhibitors complexed
with BACE1, the two nitrogen atoms of the amidine moiety
formed hydrogen-bond contacts with the side chain of Asp32
and Asp228 of BACE1. The benzene ring of 6 was
accommodated in the S1 pocket with favorable hydrophobic
interactions. Superimposition of this structure on the previously
reported structure of the ethylene linker compound 3−BACE1
complex (PDB code no. 2VA5)6b (Figure 3) revealed some
interesting differences. In the structure of 6, the side chain of
Tyr71 in the BACE1 enzyme flipped, which made space for the
compound to be incorporated.22 Furthermore, to our surprise,
6 was located at a position remarkably closer to the flap region
of BACE1 compared with that of the ethylene linker compound
3, making it possible for the cyclopropane ring to interact with
same conditions. Although another radical initiator, 2,2′-
azobisvaleronitrile, which initiates radical chain reactions at a
lower temperature, was also tested, the reaction did not
proceed (entry 4).
Using the conditions described for entry 3, Barton
decarboxylation of other substrates was performed, and the
desired cis-substituted cyclopropane compounds 28a−c were
obtained in 38−53% yield. Deprotection of 28a gave the cis-
(1S,2R) isomer 6, whose enantiomeric excess was determined
to be 94.8% by chiral column analysis (see Experimental
Section). Compounds 28b and ent-28b were also deprotected
to give 31 and ent-31, respectively. Using 28b, ent-28b, and 29c
as substrates for the Suzuki−Miyaura couplings, we obtained a
variety of target compounds.
BACE1 Inhibitory Activities of Conformationally
Restricted Analogs. The BACE1 inhibitory activities of the
four isomers 6, ent-6, 7, and ent-7 against human BACE1 are
shown in Table 2. Among the four cyclopropane-based
conformationally restricted analogs, the cis-(1S,2R) isomer 6
Table 1. Optimization of Barton Decarboxylation of ent-27b
yields (%)
entry
reductant (eq)
initiator
temp (°C)
ent-28b
29
42
1
2
3
4
TMS3SiH (3)
TMS3SiH (6)
n-Bu3SnH (3)
n-Bu3SnH (3)
AIBN
AIBN
AIBN
80
80
80
65
32
38
57
35
10
a
b
b
AVN
N.D.
N.D.
a
b
2,2′-Azobisvaleronitrile. Not detected.
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component that interacts with the two catalytic Asp side chains,
and they also contain a biaryl-based structure, which fits
hydrophobic S1 and S3 pockets. In these biaryl cyclic amidine
BACE1 inhibitors,4 substituting the amidine moiety at the meta
position on the aromatic ring usually leads to improved
inhibitory activity by efficiently occupying the bent-shaped
active site of BACE1.
We designed several compounds by taking these reported
results and the structure of 6 complexed with BACE1 into
account. First, we intended to introduce another benzene ring
at the meta position of the benzene ring on 6. A docking study
was performed using the X-ray data of Figure 3; energy
minimization of a newly designed ligand 32, which bears a
benzene ring at the meta position, was performed with MOE
and then merged with the protein structure shown in Figure 3.
An unfavorable steric crash between the binding site of BACE1
and the terminal benzene ring, however, was clearly observed
(Figure 5), probably due to the position of this compound in
Figure 3. X-ray crystallographic structure of the BACE1 (in cyan)
complexed with 6 (in green), superimposed with ligand 3 in PDB
2VA5 (3 in gray and Tyr71 side chain in white). Key interactions
between 6 and the side chains of Asp32 and Asp228 are depicted as
yellow dashed lines.
the side chain moiety of Tyr71. The distance of the aromatic
ring in Tyr71 and a carbon or hydrogen in the cyclopropane
ring was around 3.4 Å or 2.7 Å, respectively (Figure 4),
Figure 5. Docking study result for compound 32, in which a phenyl
substituent was introduced at the meta position of compound 6.
the active site being more proximal to the flap region compared
with that of the ethylene linker compound 3. Thus, it was
anticipated that the introduction of an additional aromatic ring
into the meta position of the benzene ring in 6 might lead to
weaker activity. We observed a new space around the para
position of the benzene ring in 6, however, which was not
present in the complex structures of the known biaryl cyclic
amidine BACE1 inhibitors (Figure 6). Therefore, we expected
that introducing another substituent into this position might
make it possible to develop a new type of potent inhibitor, and
Figure 4. Distances between the π plane of Tyr71 and the
cyclopropane carbon atom or hydrogen atom of compound 6 in the
complex.
suggesting a CH−π interaction between those moieties.23
A
tight conformational restriction by the cyclopropane ring might
have caused the unexpected induced-fit on the binding of 6
with BACE1, in which the effective CH−π interaction of the
cyclopropane ring and the Tyr71 side chain was likely to
stabilize the complex structure. The cyclopropane ring is more
polarizable than the ethylene, which might result in effective
dispersion interactions, leading to this interesting CH−π
interaction mode.24 Moreover, the sp2-like hybrid orbital nature
of cyclopropane might also make favorable contact with the
Tyr71 side chain through electrostatic interactions.25
Thus, an alternative inhibitor-binding mode of BACE1
induced by the rigid cyclopropane ring was identified. We
expected that the SAR, different from those reported for the
derivatives of ethylene linker compounds 2, might be
determined in derivatization studies of 6 as a lead.
Compound Design Based on the Structure of 6
Complexed with BACE1. To explore more active compounds
against BACE1, we performed docking studies using the 6 and
BACE1 complex (Figure 3). Many potent BACE1 inhibitors
reported to date bear a cyclic amidine moiety as the key
Figure 6. Possible substitutions at the para position of 6.
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Table 3. BACE1 Inhibitory Activity of the meta-Substituted Derivatives
a
b
c
IC50 in ELISA. Values are means of at least two experiments. Ligand efficiency. Compound 30 was prepared according to the reported
procedures.6b,c
we planned to synthesize the following two types of new
compounds and examine their BACE1 inhibitory activity to
verify our hypotheses: (a) meta-substituted biaryl derivatives to
examine whether the SAR of our compounds differed from
those of the previous biaryl cyclic amidine BACE1 inhibitors,
and (b) para-substituted biaryl derivatives to improve the
activity.
Structure−Activity Relationship of the meta-Substi-
tuted Biaryl Derivatives. We synthesized several meta-aryl
substituted cis-(1S,2R) cyclopropane derivatives 31−33 and the
corresponding ethylene linker compounds 30, 4, and 5, and we
compared their BACE1 inhibitory activity. We also prepared
their enantiomers, ent-31−33, to reaffirm the suitable stereo-
configuration of the cyclopropane ring. The structures and
activities of these compounds are shown in Table 3. When a
bromo or a phenyl group was introduced into the meta-position
of these three series of compounds, the IC50 values against
BACE1 significantly decreased over 500 μM in the cis-(1S,2R)-
type compounds (31, 32) and over 1 mM in the cis-(1R,2S)-
type compounds (ent-31, 32), while the corresponding
ethylene linker-type compounds had remarkable inhibitory
activity (30, 4; IC50 = 19.8−6.5 μM). The declining activity of
the cyclopropane derivatives was just as predicted from the
docking study shown in Figure 5. To our surprise, however,
substitution of the m-methoxyphenyl group on the cis-(1S,2R)
cyclopropane compound dramatically improved the activity,
with an IC50 value (33; 4.6 μM) comparable to that of the
ethylene linker compound 5 (4.2 μM). The identical
modification on the cis-(1R,2S) cyclopropane compound was
also effective, but its IC50 value (ent-33; 103 μM) was still ∼20
times weaker than those of 33 and 5. As a result, compound 6
with the cis-(1S,2R)-cyclopropane structure was the most
effective lead compared with its enantiomer ent-6 having a
(1R,2S) cyclopropane structure, in these meta-substituted biaryl
derivatives.
Figure 7. (a) X-ray crystallographic structure of 33 (in yellow)
complexed with BACE1 (in beige), superimposed with BACE1 (in
cyan) complexed with 6 (in green). The hydrogen binding network of
the methoxy group of 33, a water molecule, and the main chain of
Ser229 is drawn in green dashed lines. (b) Seen from the bottom side
of part a. Some residues were omitted for clarity.
X-ray Structure of 33 Complexed with BACE1. We then
performed the X-ray structure analysis of the compound 33 and
BACE1 complex to elucidate why only 33 among the meta-
substituted cyclopropane derivatives exhibited strong inhibitory
activity against BACE1. The aminopyrimidine moiety of 33
occupied a position almost identical to that of 6, and its
nitrogens interacted with the aspartic acid catalytic centers in
the same manner (Figure 7a). The central benzene ring was
placed in the S1 pocket, creating an effective hydrophobic
interaction with the enzyme pocket. As predicted from the SAR
study summarized in Table 3, the methoxy group on the
terminal benzene ring played a key role in the interaction with
BACE1. The methoxy oxygen formed a hydrogen bond with
the water molecule, placed in the deeper position within the S3
pocket, which makes a hydrogen bond with the main chain of
Ser229.26 Interestingly, the side chain of Leu30 of the S1
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pocket eventually rotated toward the indole ring of Trp115 to
make a space accommodating the terminal aromatic ring
(Figure 7b). Such an induced fit initiated by the interaction
between the amidine-based BACE1 inhibitor and the Leu30
side chain is the first example, to our knowledge, among the
many X-ray structures of the BACE1 and its inhibitor complex
reported so far.27 The hydrogen bond network mediated by the
water molecule placed in the S3 pocket should be indispensable
for the remarkable inhibitory activity of 33. The unexpected
binding mode of 33 to BACE1 would originate from the
induced fit due to the rigid cyclopropane ring with the cis-
(1S,2R) stereochemistry.
The X-ray structure of compound 36 complexed with
BACE1 was then analyzed to obtain information for further
structural modification (Figure 8). The biaryl moiety was in an
SAR of the para-Substituted Biaryl Derivatives. We
examined the BACE1 inhibitory activities of the para-
substituted biaryl derivatives 34−37 with the (1S,2R)-cyclo-
propane structure and the corresponding ethylene linker-type
compound 38 (Table 4). As expected from the X-ray structural
Figure 8. X-ray crystallographic structure of 36 (in orange) complexed
with BACE1.
Table 4. Primary SAR of the para-Substituted Biaryl
Derivatives
almost planar conformation, and the terminal methyl group was
directed toward the unoccupied S3 pocket. On the basis of this
observation, we further examined the SAR using two strategies:
(a) replacing the terminal benzene ring of 36 with a smaller
heteroaromatic ring or a pyridine ring to more precisely fit the
compound to the active site of BACE1 and to reduce the steric
repulsion between the two benzene rings in 36 in the planar
conformation; and (b) replacing the substituent at the meta
position of the terminal benzene ring to effectively occupy the
vacant space of the S3 pocket.
The SAR results based on strategy a are summarized in Table
5. Replacing the benzene ring with a sterically less-demanding
2-thienyl group (compound 39) improved the activity (14.1
μM) and LE (0.29), but replacing the benzene ring with a 3-
thienyl group or a 2-furanyl group was not effective. Further,
introduction of a methyl group onto the thiophene ring of 39,
i.e., compounds 42 and 43, was not effective, probably due to
the different orientation of the methyl group from that in
compound 36. Replacement with a substituted pyridine ring
was also carried out (compounds 44, 45), but this resulted in
largely decreased activity. On the other hand, replacement with
a pyrazine ring was fairly effective (compound 46), with an IC50
value of 12.6 μM, but the LE was not significantly improved.
Replacing the terminal substituent based on strategy b was
also investigated (Table 6). Various alkoxy chains were
introduced in which the ethoxy-substituted compound 48 had
excellent activity (IC50 = 10.2 μM). Introduction of shorter
(47), longer (49), or branched (50, 51) alkoxy chains led to
dropped activity. Simple linear alkyl chains or alkylsulfanyl
chains were fairly effective (52−55), with IC50 values of 10−20
μM. Ethylsulfanyl-substituted compound 55 exhibited the best
activity among the para-substituted compounds synthesized
(IC50 = 9.1 μM). The corresponding ethylene linker-type
congener 61, having the same ethylsulfanyl substituent as 55,
had no inhibitory activity against BACE1. Replacement with
other substituents, such as MeOCH2, NCCH2, HOCH2CH2,
AcHN, or H2NOC, was less effective (compounds 56−60).
These data support the fact that the remaining space in the S3
pocket is a narrow “pipe”-like shape of approximately three
single bonds in length with a hydrophobic nature.
a
IC50 in homogeneous time-resolved fluorescence (HTRF) assay.
b
Values are means of at least two experiments. Ligand efficiency.
analysis of the parent compound 6, substitution of a benzene
ring in the para position of 6 successfully improved the activity;
compound 34 had an IC50 value of 25.6 μM, which is about 5-
fold more active than 6, although the LE decreased slightly to
0.26. The corresponding ethylene linker compound 38 was
completely inactive. These results suggest that the novel
structural modification could be spread out by restricting the
conformation of the ethylene chain using the (1S,2R)-
cyclopropane structure.
Compounds 35−37 methylated at the ortho-, meta-, or para-
position on the terminal benzene ring of 34 were examined to
investigate the feasibility of additional structural modification.
Among those compounds, the meta-methylated 36 had
improved activity with an IC50 of 17.1 μM, of which the LE
(0.26) was equal to that of compound 34. Thus, substitution at
the meta position of the terminal benzene ring improved the
inhibitory activity of these derivatives.
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Table 5. SAR of the para-Substituted Biaryl Derivatives:
Introduction of Heteroaromatics
Table 6. SAR of the para-Substituted Biaryl Derivatives:
Modification of the Terminal Substituent
a
: IC50 in HTRF assay. Values are means of at least two experiments.
Ligand efficiency.
b
CONCLUSION
■
a
IC50 in HTRF assay. Values are means of at least two experiments.
Ligand efficiency.
We designed and synthesized cyclopropane-based conforma-
tionally restricted analogs of the amidine-type BACE1 inhibitor
2, and we identified the active cis-(1S,2R) isomer 6. X-ray
crystallographic structure analysis of 6 complexed with BACE1
revealed an unexpected binding mode that was clearly different
from those of the known amidine-type inhibitors, which was
induced by a CH−π interaction between the rigid cyclopropane
ring and the Tyr71 side chain. On the basis of this unique
binding mode, we performed a derivatization study using 6 as
the lead compound to clarify the novel SAR, and we
successfully developed potent BACE1 inhibitors. This study
revealed a new feature of the conformational restriction
approach: replacement of a flexible ethylene chain with a
rigid cyclopropane ring triggered an induced fit of the target
protein, leading to a unique space arrangement of the binding
site and thus an alternative SAR, different from that for the
conformationally unrestricted parent compound.
b
raphy was performed on a Yamazen Hi-Flash Column (Yamazen
Corporation) using an automated flash chromatography system W-
prep 2XY (Yamazen Corporation). Biotage Initiator was used for
microwave irradiation reactions. The purity of the final products was
≥95% as determined by LCMS analysis. LC conditions are as follows:
column, intakt Unison U-18 4.6 mm × 75 mm (3 μm); temperature,
50 °C; eluent, A H2O (0.1% HCO2H), B MeCN (0.1% HCO2H);
gradient condition, Eluent B 10%−95% 6 min, 95% 2 min; flow rate, 2
mL/min. For detailed data of each compounds, see Supporting
Information.
2-Amino-3-methyl-6-((1S,2R)-2-phenylcyclopropyl)pyrimidin-
4(3H)-one (6). To a solution of 16 (105 mg, 0.460 mmol) in DMF
(3.1 mL) was added potassium carbonate (63.6 mg, 0.460 mmol) and
iodomethane (28.7 μL, 0.460 mmol) under N2 atmosphere, and the
mixture was stirred for 25 h at room temperature. Then iodomethane
(8.6 μL, 0.138 mmol) was added, and the mixture was stirred for
another 15 h. Another portion of potassium carbonate (63.6 mg, 0.460
mmol) and iodomethane (28.7 μL, 0.460 mmol) were added, and the
reaction mixture was stirred for 3 h and then partitioned between
AcOEt and water. The organic layer was washed with water and brine,
dried with Na2SO4, and evaporated. The residue was purified by silica
gel chromatography (NH silica gel, CHCl3/MeOH = 30:1) to obtain
EXPERIMENTAL SECTION
■
General Experimental Methods for the Syntheses of
1
Compounds. H- and 13C-NMR chemical shifts were reported in
parts per million (ppm) relative to tetramethylsilane (0.00 ppm).
Coupling constants (J) were reported in hertz. Silica-gel chromatog-
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the mixture of 6 and 7 (52.6 mg, 0.218 mmol, 47%) as a colorless
foam.
(+)-epichlorohydrin (8) (10.0 g, 108 mmol) in benzene (30 mL) for
25 min, maintaining the temperature below 20 °C in an ice-bath, and
the mixture was stirred for 30 min at room temperature. The solvent
was evaporated, and to the residue was added EtOH (150 mL) and 10
M KOH solution (50 mL). The reaction mixture was stirred for 8 h
under reflux, and the solvent was concentrated to approximately half
amount. The residue was added to ice-cooled concentrated HCl (90
mL) and water (90 mL) with stirring. The mixture was extracted with
AcOEt, and the organic layer was washed with saturated NaHCO3,
H2O, and brine and dried with Na2SO4. The solvent was evaporated,
and the residue was purified by silica gel chromatography (Hex/AcOEt
= 2:1) to obtain 9b (16.74 g, 66.1 mmol, 61%) as a light brown oil.
[α]D +58.3° (c = 1.015, CHCl3); H NMR (CDCl3, 400 MHz) δ
1.38−1.42 (1H, m), 1.62−1.67 (1H, m), 2.56−2.62 (1H, m), 4.31
(1H, d, J = 9.2 Hz), 4.47 (1H, dd, J = 9.2, 4.2 Hz), 7.23 (1H, t, J = 7.8
Hz), 7.40 (1H, d, J = 7.8 Hz), 7.44 (1H, d, J = 7.8 Hz), 7.56 (1H, s);
13C NMR (CDCl3, 125 MHz) δ 20.48, 25.20, 31.25, 67.99, 61.22,
The mixture of 6 and 7 (94.2 mg, 0.390 mmol) was dissolved in
MeCN (6 mL) and water (2 mL), and separated by preparative
reversed-phase HPLC (column: GL Science Inertsil-ODS-3, 10 mm ×
250 mm; eluent: A 0.1% TFA in H2O, B 0.1% TFA in MeCN; gradient
condition: B 10%−60% 40 min; flow rate: 3 mL/min). All fractions
were collected and concentrated to approximately half volume. After
alkalinizing with 5% potassium carbonate in water, the product was
extracted with AcOEt. The organic layer was washed with water and
brine, dried with Na2SO4, and evaporated. The residue was purified
again via the same method to give 6 (21.2 mg, 0.0879 mmol, 23%) and
7 (52.7 mg, 0.218 mmol, 56%) as a colorless solid, respectively. 6: mp
22
1
22
1
148−150 °C; [α]D −34.1° (c = 0.511, CHCl3); H NMR (CDCl3,
400 MHz) δ 1.34−1.41 (1H, m), 1.57−1.65 (1H, m), 2.15−2.23 (1H,
m), 2.52−2.62 (1H, m), 4.79 (2H, br s), 5.64 (1H, s), 7.07−7.20 (5H,
m); 13C NMR (CDCl3, 150 MHz) δ 10.67, 25.14, 25.92, 27.45, 101.83,
126.05, 127.69, 129.30, 137.30, 153.68, 162.24, 164.29; HRMS (ESI)
calcd for C14H16N3O: 242.1288 [(M + H)+], found 242.1288; Anal.
Calcd for C14H15N3O·0.2H2O: C, 68.66; H, 6.34; N, 17.16. Found: C,
68.83; H, 6.38; N, 16.91. Optical purity: 96.9% ee (column: Daicel
CHIRALPAK AY-H; eluent: MeCN (0.1% diethylamine), 1.0 mL/
min, 40 °C, 259 nm; retention time: 4.2 min).
122.61, 127.01, 130.15, 130.86, 131.23, 136.39, 175.29; HRMS (ESI)
calcd for C11H10BrO2: 252.9859 [(M + H)+], found 252.9861; Anal.
Calcd for C11H9BrO2: C, 52.20; H, 3.58; Br, 31.57. Found: C, 51.93;
H, 3.54; Br, 31.29; Optical purity: 96.4% ee (column: Daicel
CHIRALCEL OJ-H 4.6 mm × 150 mm; eluent: hexane/2-propanol
98:2, 0.6 mL/min, 25 °C, 200 nm; retention time: 70.2 min).
(1S,5R)-1-(3-Bromophenyl)-3-oxabicyclo[3.1.0]hexan-2-one (ent-
9b). ent-9b (17.19 g, 67.9 mmol, 63%) was obtained as a brown oil
from (R)-(−)-epichlorohydrin (10.0 g, 108 mmol) and m-
bromophenylacetonitrile (23.29 g, 119 mmol) by the same procedure
2-Amino-3-methyl-6-((1R,2S)-2-phenylcyclopropyl)pyrimidin-
4(3H)-one (ent-6). According to the procedure used to prepare 6, a
mixture of ent-6 and ent-7 (35.0 mg, 0.145 mmol, 42%) was obtained
as a colorless powder from ent-16 (77.8 mg, 0.342 mmol). Then a
mixture of ent-6 and ent-7 (69.1 mg, 0.286 mmol) was purified to give
ent-6 (17.9 mg, 0.0742 mmol, 26%) and ent-7 (35.7 mg, 0.148 mmol,
52%). ent-6: [α]D22 +32.7° (c = 0.513, CHCl3); HRMS (ESI) calcd for
C14H16N3O: 242.1288 [(M + H)+], found 242.1288. Optical purity:
93.0% ee (column: Daicel CHIRALPAK AY-H 4.6 mm × 250 mm,
eluent: MeCN (0.1% diethylamine), 1.0 mL/min, 40 °C, 259 nm;
retention time: 5.0 min).
2-Amino-3-methyl-6-((1R,2R)-2-phenylcyclopropyl)pyrimidin-
4(3H)-one (7). To a solution of 19 (80.9 mg, 0.356 mmol) in DMF
(2.4 mL) was added potassium carbonate (49.2 mg, 0.356 mmol) and
iodomethane (22.2 μL, 0.356 mmol) under N2 atmosphere, and the
mixture was stirred for 18 h at room temperature. Then the reaction
mixture was partitioned between AcOEt and water. The organic layer
was washed with water and brine, dried with Na2SO4, and evaporated.
The residue was purified by silica gel chromatography (NH silica gel,
CHCl3/MeOH = 30:1) to obtain 7 (57.8 mg, 0.240 mmol, 67%) as a
colorless foam. mp 185−187 °C; [α]D22 −399.2° (c = 0.512, CHCl3);
1H NMR (DMSO-d6, 400 MHz) δ 1.29−1.35 (1H, m), 1.51−1.58
22
used to prepare 9b. [α]D −58.9° (c = 1.002, CHCl3); HRMS (ESI)
calcd for C11H10BrO2: 252.9859 [(M + H)+], found 252.9859; Anal.
Calcd for C11H9BrO2: C, 52.20; H, 3.58; Br, 31.57. Found: C, 52.06;
H, 3.60; Br, 31.21; Optical purity: 94.7% ee (column: Daicel
CHIRALCEL OJ-H 4.6 mm × 150 mm, eluent: hexane/2-propanol
98:2, 0.6 mL/min, 25 °C, 200 nm; retention time: 64.0 min).
(1S,5R)-1-(4-Bromophenyl)-3-oxabicyclo[3.1.0]hexan-2-one (9c).
9c (17.07 g, 67.4 mmol, 62%) was obtained as a pale brown solid from
(S)-(+)-epichlorohydrin (10.0 g, 108 mmol) and p-bromophenylace-
tonitrile (23.29 g, 119 mmol) by the same procedure used to prepare
9b. [α]D22 +59.5° (c = 1.010, CHCl3); 1H NMR (CDCl3, 600 MHz) δ
1.37−1.40 (1H, m), 1.59−1.62 (1H, m), 2.54−2.58 (1H, m), 4.29
(1H, d, J = 9.6 Hz), 4.46 (1H, dd, J = 9.6, 4.5 Hz), 7.31 (2H, d, J = 8.7
Hz), 7.48 (2H, d, J = 8.7 Hz); 13C NMR (CDCl3, 125 MHz) δ 20.46,
25.13, 31.21, 68.01, 61.22, 121.78, 129.91, 131.76, 133.19, 175.47;
HRMS (ESI) calcd for C11H10BrO2: 252.9859 [(M + H)+], found
252.9863; Anal. Calcd for C11H9BrO2: C, 52.20; H, 3.58; Br, 31.57.
Found: C, 52.01; H, 3.55; Br, 31.21; Optical purity: 94.8% ee (column:
Daicel CHIRALCEL OJ-H 4.6 mm × 150 mm; eluent: hexane/2-
propanol 90:10, 0.6 mL/min, 25 °C, 200 nm; retention time: 38.3
min).
(1H, m), 1.92−1.98 (1H, m), 2.32−2.39 (1H, m), 3.21 (3H, s), 5.65
(1H, s), 7.05 (2H, br s), 7.11−7.19 (3H, m), 7.24−7.29 (2H, m); 13C
NMR (CDCl3, 150 MHz) δ 17.16, 26.47, 27.55, 28.63, 100.08, 126.00,
126.05, 128.45, 141.65, 154.87, 162.26, 166.70; HRMS (ESI) calcd for
C14H16N3O: 242.1288 [(M + H)+], found 242.1285; Anal. Calcd for
C14H15N3O·0.2H2O: C, 68.66; H, 6.34; N, 17.16. Found: C, 68.80; H,
6.45; N, 16.94. Optical purity: 95.3% ee (column: Daicel
CHIRALPAK AY-H 4.6 mm × 250 mm; eluent: MeCN (0.1%
diethylamine), 1.0 mL/min, 40 °C, 259 nm; retention time: 4.9 min).
2-Amino-3-methyl-6-((1S,2S)-2-phenylcyclopropyl)pyrimidin-
4(3H)-one (ent-7). ent-7 (78.8 mg, 0.327 mmol, 53%) was obtained as
a colorless foam from ent-19 (141 mg, 0.620 mmol) by the same
(1R,2S)-2-((tert-Butyldiphenylsilyloxy)methyl)-1-phenylcyclopro-
panecarboxylic Acid (10). To a solution of 9a13a (7.34 g, 42.1 mmol)
in EtOH (59 mL) was added a 10 M KOH solution (6.3 mL), and the
mixture was stirred for 1 h at room temperature. The reaction mixture
was added to chilled AcOEt (50 mL), concentrated HCl (8 mL), and
water (30 mL) with vigorous stirring, and the resulting mixture was
extracted with AcOEt. The organic layer was washed with water and
brine and dried with Na2SO4. DMF (59 mL) was added to the
solution, Na2SO4 was filtered off, and the mother liquid was
evaporated. Under argon atmosphere, imidazole (14.3 g, 211 mmol)
and tert-butylchlorodiphenylsilane (32.9 mL, 126 mmol) were added
to the solution at 0 °C, and the reaction mixture was stirred at rt for 30
min. Then tert-butylchlorodiphenylsilane (11.0 mL, 42.1 mmol) and
DMF (15 mL) were added and stirred at rt for 30 min. Then another
batch of tert-butylchlorodiphenylsilane (5.48 mL, 21.1 mmol) and
DMF (15 mL) was added, and the reaction mixture was stirred at rt
for 30 min. AcOEt and water were added, and the reaction mixture
was extracted with AcOEt. The organic layer was washed with water
and brine and dried with Na2SO4 and evaporated. The residue was
dissolved in EtOH (80 mL), 10 M KOH solution (25 mL) was added,
and the mixture was stirred for 1 h at rt. The solvent was evaporated to
approximately half volume, and the residue was partitioned between
22
procedure used to prepare 7. [α]D +435.9° (c = 0.515, CHCl3);
HRMS (ESI) calcd for C14H16N3O: 242.1288 [(M + H)+], found
242.1288; Anal. Calcd for C14H15N3O·0.1H2O: C, 69.17; H, 6.30; N,
17.29. Found: C, 69.23; H, 6.15; N, 17.31. Optical purity: 98.4% ee
(column: Daicel CHIRALPAK AY-H 4.6 mm × 250 mm; eluent:
MeCN (0.1% diethylamine), 1.0 mL/min, 40 °C, 259 nm; retention
time: 6.7 min).
(1R,5S)-1-(3-Bromophenyl)-3-oxabicyclo[3.1.0]hexan-2-one (9b).
Sodium amide (10.5 g, 270 mmol) was suspended in benzene (60
mL), and m-bromophenylacetonitrile (23.29 g, 119 mmol) in benzene
(30 mL) was added for 30 min, maintaining the temperature below 20
°C in an ice-bath. The reaction mixture was stirred at room
temperature for 30 min. Then to the mixture was added (S)-
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22
AcOEt and water. The water layer was acidified with HCl to pH = 1
and extracted with AcOEt. The organic layer was washed with water
and brine, dried with Na2SO4, and evaporated. The residue was
purified by silica gel chromatography (hexane/AcOEt = 5:1) to give
10 (8.69 g, 20.2 mmol, 48%) as a pale brown oil. The spectrum data
were identical with that reported previously.17 Compound ent-10
(7.49 g, 43.0 mmol, 56%) was obtained in the same manner from ent-
9a (10.20 g, 58.6 mmol).
18. [α]D +424.0° (c = 0.50, CHCl3); HRMS (ESI) calcd for
C14H17O3: 233.1172 [(M + H)+], found 233.1175.
2-Amino-6-((1R,2R)-2-phenylcyclopropyl)pyrimidin-4(3H)-one
(19). 19 (105 mg, 0.462 mmol, 57%) was obtained as a colorless solid
from 18 (189 mg, 0.814 mmol) by the same procedure used to
prepare 16. [α]D25 −501.8° (c = 0.51, DMSO); 1H NMR (DMSO-d6,
400 MHz): δ 1.28−1.34 (1H, m), 1.50−1.57 (1H, m), 1.93−1.99 (1H,
m), 2.33−2.39 (1H, m), 5.56 (1H, s), 6.46 (2H, br s), 7.11−7.19 (2H,
m), 7.24−7.29 (3H, m), 10.54 (1H, br s); 13C NMR (DMSO-d6, 150
MHz) δ 17.79, 26.41, 29.70, 99.60, 126.65, 129.26, 142.61, 156.75,
163.19, 169.35; HRMS (ESI) calcd for C13H14N3O: 228.1131 [(M +
H)+], found 228.1130.
Compounds 11−14 and ent-11−14 were prepared according to the
reported procedure.17
Ethyl 3-Oxo-3-((1S,2R)-2-phenylcyclopropyl)propanoate (15). To
a solution of 14 (201 mg, 1.24 mmol) in MeCN (2 mL) was added
carbonyldiimidazole (221 mg, 1.36 mmol) under N2 atmosphere at
room temperature, and the mixture was stirred at the same
temperature for 45 min to prepare the imidazolide solution of 14.
Using another independent reaction vessel, magnesium chloride (294
mg, 3.09 mmol), potassium ethyl malonate (442 mg, 2.60 mmol), and
triethylamine (552 μL, 3.96 mmol) were suspended in MeCN (6 mL)
under N2 atmosphere, and the reaction mixture was stirred for 40 min.
Then the prepared imidazolide solution of 14 was added, and the
resulting suspension was stirred for 2 h at 80 °C. The insoluble solid
was filtered off and washed with MeCN, and the mother liquid was
concentrated. The residue was partitioned between AcOEt and 1 M
HCl. The organic layer was washed with water and brine, dried with
Na2SO4, and evaporated. The residue was purified by silica gel
chromatography (hexane/AcOEt = 5:1) to give 15 (220 mg, 0.947
2-Amino-6-((1S, 2S)-2-phenylcyclopropyl)pyrimidin-4(3H)-one
(ent-19). ent-19 (176 mg, 0.776 mmol, 78%) was obtained as a
colorless solid from ent-18 (232 mg, 0.995 mmol) by the same
25
procedure used to prepare 19. [α]D +504.5° (c = 0.51, DMSO);
HRMS (ESI) calcd for C13H14N3O: 228.1131 [(M + H)+], found
228.1129.
Sodium (1R,2S)-2-(Hydroxymethyl)-1-phenylcyclopropanecar-
boxylate (20a). To a suspension of 9a13a (10.6 g, 61.0 mmol) in
EtOH (53 mL) and water (9.2 mL) was added 10 N KOH solution
(9.2 mL, 92 mmol), and the solution was stirred for 1 h at room
temperature. The reaction mixture was poured into the chilled Et2O
(120 mL) and 2 M HCl (61 mL) with vigorous stirring. The mixture
was extracted with Et2O. The organic layer was washed with water and
brine and dried with MgSO4. MgSO4 was filtered off, and to the
mother liquid was added 28% sodium methoxide solution MeOH
(12.5 mL). Precipitate was separated by filtration and washed with
Et2O to give 20a (10.4 g, 48.6 mmol, 80%) as a pale brown solid.
[α]D +76.2° (c = 0.50, MeOH); H NMR (DMSO-d6, 600 MHz) δ
0.86−0.89 (1H, m), 1.62−1.67 (1H, m), 1.09−1.14 (1H, m), 1.22−
1.28 (1H, m), 3.26−3.31 (1H, m), 3.79−3.84 (1H, m), 6.18 (1H, br
s), 7.05−7.09 (1H, m), 7.16−7.20 (2H, m), 7.24−7.27 (2H, m); 13C
NMR (CD3OD, 125 MHz) δ 18.63, 28.88, 39.65, 64.50, 127.01,
128.96, 129.78, 145.15, 179.85; HRMS (ESI) calcd for C11H13O3:
193.0859 [(M − Na + 2H)+], found 193.0860.
Sodium (1R,2S)-1-(3-Bromophenyl)-2-(hydroxymethyl)-
cyclopropanecarboxylate (20b). 20b (16.26 g, 55.5 mmol, 87%)
was obtained as a colorless solid from 9b (16.13 g, 63.7 mmol) by the
22
1
mmol, 77%) as a colorless oil: [α]D −5.1° (c = 0.51, CHCl3); H
NMR (CDCl3, 400 MHz) δ 1.22 (3H, t, J = 7.3 Hz), 1.39−1.43 (1H,
m), 1.88−1.94 (1H, m), 2.55−2.61 (1H, m), 2.74−2.83 (1H, m), 3.31,
3.36 (2H, ABq, J = 15.1 Hz), 4.05−4.19 (2H, m), 7.17−7.30 (5H, m);
13C NMR (CDCl3, 150 MHz) δ 12.73, 14.05, 14.07, 29.69, 50.76,
23
1
61.22, 126.90, 127.99, 129.19, 135.38, 167.08, 197.93; HRMS (ESI)
calcd for C14H17O3: 233.1172 [(M + H)+], found 233.1174.
Ethyl 3-Oxo-3-((1R,2S)-2-phenylcyclopropyl)propanoate (ent-
15). ent-15 (195 mg, 0.840 mmol, 56%) was obtained from ent-14
(10.20 g, 58.6 mmol) as a colorless oil by the same procedure used to
22
prepare 15. [α]D +4.3° (c = 0.51, CHCl3); HRMS (ESI) calcd for
C14H17O3: 233.1172 [(M + H)+], found 233.1175.
2-Amino-6-((1S,2R)-2-phenylcyclopropyl)pyrimidin-4(3H)-one
and the 1R Trans Isomer (16). To a solution of 15 (149 mg, 0.642
mmol) in EtOH (3.0 mL) was added guanidine carbonate (116 mg,
0.642 mmol), 20% sodium ethoxide solution in EtOH (503 μL, 1.284
mmol), and the mixture was stirred for 5 h at 80 °C under N2
atmosphere. The reaction mixture was partitioned between AcOEt and
water, and neutralized with diluted HCl to pH = 6. The organic layer
was washed with water and brine, dried with Na2SO4, and evaporated.
The residue was purified by silica gel chromatography (NH silica gel,
CHCl3/MeOH = 5:1) to give 16 (116 mg, 0.511 mmol, 80%) as a
22
same procedure used to prepare 20a. [α]D +64.0° (c = 0.50,
MeOH); 1H NMR (DMSO-d6, 600 MHz) δ 0.90−0.93 (1H, m),
1.17−1.20 (1H, m), 1.23−1.30 (1H, m), 3.34−3.36 (1H, m), 3.77−
3.82 (1H, m), 6.01 (1H, br s), 7.15 (1H, t, J = 7.8 Hz), 7.20 (1H, d, J =
7.8 Hz), 7.27 (1H, d, J = 7.8 Hz), 7.46 (1H, s); 13C NMR (DMSO-d6,
125 MHz) δ 16.85, 28.50, 37.43, 61.58, 120.58, 127.08, 127.68, 129.42,
131.57, 148.43, 174.18; HRMS (ESI) calcd for C11H12BrO3: 270.9964
[(M − Na + 2H)+], found 270.9967.
Sodium (1S,2R)-1-(3-Bromophenyl)-2-(hydroxymethyl)-
cyclopropanecarboxylate (ent-20b). ent-20b (13.20 g, 45.0 mmol,
94%) was obtained as a colorless solid from ent-9b (12.15 g, 48.0
mmol) by the same procedure used to prepare 20a. [α]D22 −65.6° (c =
0.50, MeOH); HRMS (ESI) calcd for C11H12BrO3: 270.9964 [(M −
Na + 2H)+], found 270.9969.
Sodium (1S,2R)-1-(4-Bromophenyl)-2-(hydroxymethyl)-
cyclopropanecarboxylate (20c). 20c (18.40 g, 62.8 mmol, 95%)
was obtained as a colorless solid from 9c (16.69 g, 65.9 mmol) by the
1
colorless foam: H NMR (DMSO-d6, 400 MHz): cis-isomer δ 1.23−
1.29 (1H, m), 1.61−1.67 (1H, m), 2.11−2.19 (1H, m), 2.44−2.50
(1H, m), 5.20 (1H, s), 6.26 (2H, br s), 7.05−7.29 (5H, m), 10.4 (1H,
1
br s). cis:trans ratio was 1.3:1 from H NMR.
2-Amino-6-((1R,2S)-2-phenylcyclopropyl)pyrimidin-4(3H)-one
and the 1S Trans Isomer (ent-16). ent-16 (113 mg, 0.498 mmol,
79%) was obtained as a colorless foam from ent-15 (147 mg, 0.632
mmol) by the same procedure used to prepare 16. The cis:trans ratio
22
1
same procedure used to prepare 20a. [α]D +64.0° (c = 0.50,
was 1.5:1 from H NMR.
MeOH); 1H NMR (DMSO-d6, 600 MHz) δ 0.86−0.90 (1H, m),
1.17−1.20 (1H, m), 1.21−1.27 (1H, m), 3.31−3.35 (1H, m), 3.77−
3.81 (1H, m), 7.21 (2H, t, J = 8.7 Hz), 7.35 (2H, d, J = 8.7 Hz); 13C
NMR (DMSO-d6, 125 MHz) δ 16.77, 28.41, 37.08, 61.56, 117.77,
130.00, 130.77, 145.08, 174.44; HRMS (ESI) calcd for C11H12BrO3:
270.9964 [(M − Na + 2H)+], found 270.9969.
Ethyl 3-Oxo-3-((1R,2R)-2-phenylcyclopropyl)propanoate (18). 18
(306 mg, 1.32 mmol, 82%) was obtained from 1719 (261 mg, 1.61
mmol) by the same procedure used to prepare 15. [α]D22 −422.4° (c =
1
0.50, CHCl3); H NMR (CDCl3, 400 MHz): δ 1.25 (3H, t, J = 7.3
Hz), 1.44−1.50 (1H, m), 1.74−1.79 (1H, m), 2.27−2.34 (1H, m),
2.56−2.64 (1H, m), 3.60 (2H, s), 4.18 (2H, q, J = 7.3 Hz), 7.09−7.13
(2H, m), 7.20−7.32 (3H, m); 13C NMR (CDCl3, 150 MHz) δ 14.08,
19.50, 30.12, 32.35, 50.33, 61.41, 126.19, 126.74, 128.55, 139.80,
167.08, 200.86; HRMS (ESI) calcd for C14H17O3: 233.1172 [(M +
H)+], found 233.1174.
Ethyl 3-Oxo-3-((1S,2S)-2-phenylcyclopropyl)propanoate (ent-18).
ent-18 (333 mg, 1.41 mmol, 81%) was obtained as a colorless oil from
ent-1719 (288 mg, 1.78 mmol) by the same procedure used to prepare
(1R,2S)-1-Phenylcyclopropane-1,2-dicarboxylic Acid (21a). To a
solution of 20a (1.75 g, 8.17 mmol) in MeCN (17.5 mL) and water
(8.8 mL) was added sodium periodate (3.50 g, 16.3 mmol) and
ruthenium(III) chloride (51 mg, 0.245 mmol), and the mixture was
stirred for 25 h at room temperature. The reaction mixture was poured
into the chilled AcOEt (60 mL), 2 M HCl (20 mL), and water (60
mL) with vigorous stirring, and the resulting mixture was extracted
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with AcOEt. Then the product was extracted with saturated NaHCO3
two times. The water layer was acidified with HCl, and the mixture was
extracted with AcOEt. The organic layer was washed with water and
brine, dried with Na2SO4, and evaporated. The residual solid was
washed with hexane to give 21a (1.27 g, 6.16 mmol, 75%) as a pale
gray solid. [α]D +166.3° (c = 0.51, MeOH); H NMR (CDCl3, 500
MHz) δ 1.61−1.65 (1H, m), 2.19−2.23 (1H, m), 2.35−2.40 (1H, m),
7.30−7.36 (3H, m), 7.42−7.45 (2H, m); 13C NMR (CDCl3, 125
MHz) δ 19.32, 29.61, 38.49, 128.34, 128.69, 129.75, 137.18, 176.78,
176.86; HRMS (ESI) calcd for C11H11O4: 207.0652 [(M + H)+],
found 207.0654.
(1R,2S)-1-(3-Bromophenyl)cyclopropane-1,2-dicarboxylic Acid
(21b). 21b (16.20 g, 56.8 mmol, 87%) was obtained as a pale gray
solid from 20b (19.25 g, 65.7 mmol) by the same procedure used to
prepare 21a. [α]D22 +155.0° (c = 0.50, CHCl3); 1H NMR (CDCl3, 500
MHz) δ 1.62−1.65 (1H, m), 2.20−2.24 (1H, m), 2.35−2.39 (1H, m),
7.22 (1H, t, J = 7.8 Hz), 7.37 (1H, dt, J = 7.8, 1.8 Hz), 7.46 (1H, dt, J
= 7.8, 1.8 Hz), 7.58 (1H, t, J = 1.8 Hz); 13C NMR (CDCl3, 125 MHz)
δ 19.23, 29.73, 37.87, 122.49, 128.49, 130.19, 131.61, 132.89, 139.17,
176.47; HRMS (ESI) calcd for C11H10BrO4: 284.9757 [(M + H)+],
found 284.9763.
was added, and the suspension was stirred for 25 h at 95 °C. Then the
reaction mixture was partitioned between AcOEt and water. The
organic layer was washed water, brine, dried with Na2SO4, and
evaporated. The residue was purified by silica gel chromatography
[CHCl3/(CHCl3/MeOH/28% NH3 = 32:6:0.5) = 3:1] to obtain 24a
22
1
23
(676 mg, 2.26 mmol, 32% from 21a) as a colorless foam. [α]D
+178.0° (c = 0.50, CHCl3); 1H NMR (CDCl3, 600 MHz) δ 1.51−1.55
(1H, m), 2.19−2.23 (1H, m), 2.43−2.47 (1H, m), 3.36 (3H, s), 3.53
(3H, s), 5.24 (2H, s), 5.98 (1H, s), 7.27−7.36 (3H, m), 7.44−7.47
(2H, m); 13C NMR (CDCl3, 125 MHz) δ 18.83, 27.49, 32.40, 38.34,
52.24, 102.30, 127.60, 128.51, 129.44, 139.39, 154.79, 162.43, 162.78,
171.16; HRMS (ESI) calcd for C16H18N3O3: 300.1343 [(M + H)+],
found 300.1346.
(1R,2S)-Methyl 2-(2-amino-1-methyl-6-oxo-1,6-dihydropyrimi-
din-4-yl)-1-(3-bromophenyl)cyclopropanecarboxylate (24b). 24b
(2.94 g, 7.77 mmol, 34% from 21b) was obtained as a colorless
foam from the mixture of 22b and ethyl malonate (6.63 g) by the same
procedure used to prepare 24a. [α]D22 +165.4° (c = 0.50, CHCl3); 1H
NMR (CDCl3, 600 MHz) δ 1.51−1.55 (1H, m), 2.19−2.22 (1H, m),
2.40−2.44 (1H, m), 3.39 (3H, s), 3.54 (3H, s), 5.16 (2H, s), 5.98 (1H,
s), 7.21 (1H, t, J = 7.8 Hz), 7.38 (1H, d, J = 7.8 Hz), 7.42 (1H, d, J =
7.8 Hz), 7.61 (1H, s); 13C NMR (CDCl3, 125 MHz) δ 18.69, 27.59,
32.64, 37.80, 52.39, 102.52, 122.33, 128.29, 130.02, 130.85, 132.75,
141.63, 154.78, 162.28, 162.39, 170.58; HRMS (ESI) calcd for
C16H17BrN3O3: 378.0448 [(M + H)+], found 378.0453.
(1S,2R)-1-(3-Bromophenyl)cyclopropane-1,2-dicarboxylic Acid
(ent-21b). ent-21b (168.6 mg, 0.591 mmol, 80%) was obtained as a
pale gray solid from ent-20b (218 mg, 0.743 mmol) by the same
22
procedure used to prepare 21a. [α]D −143.2° (c = 0.50, CHCl3);
HRMS (ESI) calcd for C11H10BrO4: 284.9757 [(M + H)+], found
284.9760.
(1S,2R)-Methyl 2-(2-amino-1-methyl-6-oxo-1,6-dihydropyrimi-
din-4-yl)-1-(3-bromophenyl)cyclopropanecarboxylate (ent-24b).
ent-24b (2.08 g, 5.50 mmol, 29% from ent-21b) was obtained as a
colorless foam from the mixture of ent-22b and ethyl malonate (6.80
(1R,2S)-1-(4-Bromophenyl)cyclopropane-1,2-dicarboxylic Acid
(21c). 21c (13.43 g, 47.1 mmol, 69%) was obtained as a grayish
brown solid from 20c (20.13 g, 68.7 mmol) by the same procedure
22
g) by the same procedure used to prepare 24a. [α]D −164.6° (c =
22
1
used to prepare 21a. [α]D +174.0° (c = 0.50, CHCl3); H NMR
(CDCl3, 500 MHz) δ 1.59−1.63 (1H, m), 2.18−2.23 (1H, m), 2.32−
2.36 (1H, m), 7.30 (2H, d, J = 8.6 Hz), 7.48 (2H, d, J = 8.6 Hz); 13C
NMR (CDCl3, 125 MHz) δ 19.27, 29.69, 37.84, 122.59, 131.46,
131.85, 136.11, 176.43; HRMS (ESI) calcd for C11H10BrO4: 284.9757
[(M + H)+], found 284.9765.
0.50, CHCl3); HRMS (ESI) calcd for C16H17BrN3O3: 378.0448 [(M +
H)+], found 378.0455.
(1R,2S)-Methyl 2-(2-Amino-1-methyl-6-oxo-1,6-dihydropyrimi-
din-4-yl)-1-(4-bromophenyl)cyclopropanecarboxylate (24c). 24c
(3.22 g, 8.51 mmol, 33% from 21c) was obtained as a colorless
foam from the mixture of 20c and ethyl malonate (10.02 g) by the
(1R,2S)-2-(3-Ethoxy-3-oxopropanoyl)-1-phenylcyclopropanecar-
boxylic Acid (22a). The mixture of 22a and ethyl malonate (2.33 g)
was obtained as a light brown oil from 21a (1.52 g, 7.37 mmol) by the
same procedure used to prepare 15, except for silica gel
chromatography eluent (CHCl3/MeOH = 30:1). It was used in the
next step without further purification. 1H NMR (CDCl3, 600 MHz) δ
1.24−1.30 (1H, m), 1.31 (3H, t, J = 6.9 Hz), 1.58−1.61 (1H, m),
2.58−2.62 (1H, m), 2.98 (2H, br s), 4.23−4.28 (2H, m), 7.29−7.45
(5H, m).
22
same procedure used to prepare 24a. [α]D +198.0° (c = 0.50,
1
CHCl3); H NMR (CDCl3, 600 MHz) δ 1.49−1.52 (1H, m), 2.19−
2.22 (1H, m), 2.36−2.40 (1H, m), 3.39 (3H, s), 3.53 (3H, s), 5.07
(2H, s), 5.97 (1H, s), 7.33 (2H, d, J = 8.1 Hz), 7.46 (2H, d, J = 8.1
Hz); 13C NMR (CDCl3, 125 MHz) δ 18.69, 27.60, 32.62, 37.77, 52.34,
102.53, 121.71, 131.34, 131.60, 138.47, 154.68, 162.36, 162.48, 170.65;
HRMS (ESI) calcd for C16H17BrN3O3: 378.0448 [(M + H)+], found
378.0453.
(1R,2S)-Methyl 2-(2-(Bis(tert-butoxycarbonyl)amino)-1-methyl-6-
oxo-1,6-dihydropyrimidin-4-yl)-1-phenylcyclopropanecarboxylate
(25a). To a solution of 24a (612 mg, 2.04 mmol) in CH2Cl2 (6.1 mL)
was added N,N-dimethylaminopyridine (74.9 mg, 0.613 mmol) and di-
tert-butyl carbonate (1.19 mL, 5.11 mmol) under N2 atmosphere, and
the mixture was stirred for 1 h at room temperature. The solvent was
evaporated, and the residue was purified by silica gel chromatography
(hexane/AcOEt = 1:1) to obtain 25a (863 mg, 1.73 mmol, 85%) as a
colorless foam. [α]D +102.0° (c = 0.51, CHCl3); H NMR (CDCl3,
600 MHz) δ 1.47 (9H, s), 1.49 (9H, s), 1.65−1.68 (1H, m), 2.16−2.19
(1H, m), 2.48−2.52 (1H, m), 3.43 (3H, s), 3.52 (3H, s), 6.43 (1H, s),
7.26−7.36 (3H, m), 7.44−7.47 (2H, m); 13C NMR (CDCl3, 125
MHz) δ 19.11, 27.82, 27.85, 30.13, 32.28, 39.18, 52.27, 84.76, 85.13,
111.55, 127.70, 128.49, 129.50, 139.22, 148.51, 148.53, 148.55, 161.85,
162.62, 170.05; HRMS (ESI) calcd for C26H34N3O7: 500.2391 [(M +
H)+], found 500.2394.
(1R,2S)-1-(3-Bromophenyl)-2-(3-ethoxy-3-oxopropanoyl)-
cyclopropanecarboxylic Acid (22b). The mixture of 22b and ethyl
malonate (6.63 g) was obtained from 21b (6.56 g, 23.0 mmol) by the
same procedure used to prepare 22a, which was used in the next step
without further purification. 1H NMR (CDCl3, 600 MHz) δ 1.18−1.30
(1H, m), 1.32 (3H, t, J = 7.3 Hz), 1.56−1.58 (1H, m), 2.59−2.63 (1H,
m), 2.95 (2H, br s), 4.24−4.30 (2H, m), 7.23 (1H, t, J = 7.8 Hz), 7.39
(1H, d, J = 7.8 Hz), 7.45 (1H, d, J = 7.8 Hz), 7.57 (1H, s).
(1S,2R)-1-(3-Bromophenyl)-2-(3-ethoxy-3-oxopropanoyl)-
cyclopropanecarboxylic Acid (ent-22b). The mixture of ent-22b and
ethyl malonate (6.90 g) was obtained from ent-21b (5.51 g, 19.3
mmol) by the same procedure used to prepare 22a, which was used in
the next step without further purification.
(1R,2S)-1-(4-Bromophenyl)-2-(3-ethoxy-3-oxopropanoyl)-
cyclopropanecarboxylic Acid (22c). The mixture of 22c and ethyl
malonate (10.15 g) was obtained from 21c (7.51 g, 26.3 mmol) by the
same procedure used to prepare 22a, which was used in the next step
without further purification. 1H NMR (CDCl3, 600 MHz) δ 1.17−1.29
(1H, m), 1.31 (3H, t, J = 6.9 Hz), 1.53−1.56 (1H, m), 2.57−2.60 (1H,
m), 2.93 (2H, br s), 4.23−4.29 (2H, m), 7.31 (2H, d, J = 8.7 Hz), 7.49
(2H, d, J = 8.7 Hz).
22
1
(1R,2S)-Methyl 2-(2-(Bis(tert-butoxycarbonyl)amino)-1-methyl-6-
oxo-1,6-dihydropyrimidin-4-yl)-1-(3-bromophenyl)-
cyclopropanecarboxylate (25b). 25b (3.92 g, 6.78 mmol, 89%) was
obtained as a colorless foam from 24b (2.87 g, 7.59 mmol) by the
22
same procedure used to prepare 25a. [α]D +105.4° (c = 0.50,
1
CHCl3); H NMR (CDCl3, 600 MHz) δ 1.47 (9H, s), 1.50 (9H, s),
(1R,2S)-Methyl 2-(2-Amino-1-methyl-6-oxo-1,6-dihydropyrimi-
din-4-yl)-1-phenylcyclopropanecarboxylate (24a). To a solution of
the mixture of 22a and ethyl malonate (2.23 g) in EtOH (33 mL) was
added 20% sodium ethoxide EtOH solution (2.76 mL, 7.05 mmol).
After stirring for 10 min, guanidine carbonate (635 mg, 3.53 mmol)
1.63−1.67 (1H, m), 2.17−2.20 (1H, m), 2.47−2.50 (1H, m), 3.43
(3H, s), 3.53 (3H, s), 6.42 (1H, s), 7.21 (1H, t, J = 7.8 Hz), 7.39 (1H,
d, J = 7.8 Hz), 7.42 (1H, d, J = 7.8 Hz), 7.60 (1H, s); 13C NMR
(CDCl3, 125 MHz) δ 19.03, 27.83, 27.86, 30.16, 32.40, 38.50, 52.40,
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22
84.83, 85.18, 111.74, 111.76, 122.34, 128.33, 130.02, 130.91, 132.68,
141.39, 148.53, 148.54, 148.67, 161.28, 162.57, 169.54; HRMS (ESI)
calcd for C26H34BrN3O7: 578.1496 [(M + H)+], found 578.1501.
(1S,2R)-Methyl 2-(2-(Bis(tert-butoxycarbonyl)amino)-1-methyl-6-
oxo-1,6-dihydropyrimidin-4-yl)-1-(3-bromophenyl)-
cyclopropanecarboxylate (ent-25b). ent-25b (1.32 g, 2.28 mmol,
82%) was obtained as a colorless foam from ent-24b (1.05 g, 2.78
mmol) by the same procedure used to prepare 25a. [α]D22 −101.0° (c
= 0.50, CHCl3); HRMS (ESI) calcd for C26H34BrN3O7: 578.1496 [(M
+ H)+], found 578.1503.
ent-26b (729 mg) by the same procedure used to prepare 27a. [α]D
−112.7° (c = 0.30, CHCl3); HRMS (ESI) calcd for C25H31BrN3O7:
564.1340 [(M + H)+], found 564.1345.
(1R,2S)-2-(2-(Bis(tert-butoxycarbonyl)amino)-1-methyl-6-oxo-
1 , 6 - d i h y d r o p y r i m i d i n - 4 - y l ) - 1 - ( 4 - b r o m o p h e n y l ) -
cyclopropanecarboxylic Acid (27c). 26c (2.90 g) was obtained as a
colorless solid from 25c (3.89 g, 6.72 mmol) by the same procedure
used to prepare 26a. Then 27c (2.71 g, 4.80 mmol, 72% from 25c)
was obtained as an orange brown foam from 26c (2.88 g) by the same
procedure used to prepare 27a. [α]D22 +122.0° (c = 0.50, CHCl3); 1H
NMR (CDCl3, 600 MHz) δ 1.44 (9H, s), 1.49 (9H, s), 1.71−1.74
(1H, m), 2.24−2.27 (1H, m), 2.60−2.64 (1H, m), 3.44 (3H, s), 6.48
(1H, s), 7.37 (2H, d, J = 8.7 Hz), 7.50 (2H, d, J = 8.7 Hz); 13C NMR
(CDCl3, 125 MHz) δ 19.34, 27.78, 27.84, 30.22, 33.79, 38.17, 84.99,
85.22, 111.58, 121.93, 131.58, 131.74, 138.02, 148.36, 148.52, 148.61,
160.80, 162.94, 172.64; HRMS (ESI) calcd for C25H31BrN3O7:
564.1340 [(M + H)+], found 564.1343.
2-Bis(tert-butoxycarbonyl)amino-3-methyl-6-((1S,2R)-2-
phenylcyclopropyl)pyrimidin-4(3H)-one (28a). To a suspension of
27a (74.0 mg, 0.152 mmol) and 11 (46.1 mg, 0.183 mmol) in toluene
(11 mL) was added tri-n-butyltin hydride (0.122 mL, 0.457 mmol),
azoisobutyronitrile (5.0 mg, 0.030 mmol), and tri-n-butylphosphine
(0.114 mL, 0.457 mmol) under N2 atmosphere and shading by
wrapping the vessel with aluminum foil, and the mixture was stirred for
20 min at room temperature and then for 4.5 h at 80 °C. The solvent
was evaporated, and the residue was purified by silica gel
chromatography (hexane/AcOEt = 1:1) to obtain 28a (35.5 mg,
0.084 mmol, 53%) as a colorless solid. [α]D22 +4.0° (c = 0.30, CHCl3);
1H NMR (CDCl3, 600 MHz) δ 1.41 (9H, s), 1.53 (9H, s), 1.72−1.75
(1H, m), 2.21−2.24 (1H, m), 2.62−2.66 (1H, m), 3.42 (3H, s), 6.46
(1H, s), 7.28−7.36 (3H, m), 7.45−7.48 (2H, m); 13C NMR (CDCl3,
125 MHz) δ 11.03, 25.21, 27.02, 27.79, 27.81, 29.92, 84.54, 84.75,
110.09, 126.48, 128.03, 129.33, 136.27, 147.70, 148.15, 148.36, 162.51,
163.04; HRMS (ESI) calcd for C24H32N3O5: 442.2336 [(M + H)+],
found 442.2343.
2-Bis(tert-butoxycarbonyl)amino-3-methyl-6-[(1S,2R)-2-(3-
bromophenyl)cyclopropyl]pyrimidin-4(3H)-one (28b). 28b (442 mg,
0.850 mmol, 48%) was obtained as a colorless solid from 27b (1.00 g,
1.77 mmol) by the same procedure used to prepare 28a. [α]D22 +1.6°
(c = 0.50, CHCl3); 1H NMR (CDCl3, 500 MHz) δ 1.38 (9H, s), 1.40
(9H, s), 1.45−1.51 (1H, m), 1.60−1.65 (1H, m), 2.32−2.38 (1H, m),
2.60−2.66 (1H, m), 3.31 (3H, s), 6.06 (1H, s), 7.00−7.06 (2H, m),
7.23 (1H, d, J = 7.9 Hz), 7.36 (1H, s); 13C NMR (CDCl3, 125 MHz) δ
11.04, 25.31, 26.41, 27.81, 27.81, 29.98, 84.61, 84.80, 110.52, 122.07,
127.59, 129.45, 129.62, 132.71, 138.85, 147.96, 148.20, 148.34, 162.46,
162.56; HRMS (ESI) calcd for C24H31BrN3O5: 520.1442 [(M + H)+],
found 520.1445.
(1R,2S)-Methyl 2-(2-(Bis(tert-butoxycarbonyl)amino)-1-methyl-6-
oxo-1,6-dihydropyrimidin-4-yl)-1-(4-bromophenyl)-
cyclopropanecarboxylate (25c). 25c (4.43 g, 7.66 mmol, 92%) was
obtained as a colorless foam from 24c (3.15 g, 8.33 mmol) by the
22
same procedure used to prepare 25a. [α]D +118.8° (c = 0.50,
1
CHCl3); H NMR (CDCl3, 600 MHz) δ 1.47 (9H, s), 1.49 (9H, s),
1.61−1.65 (1H, m), 2.17−2.20 (1H, m), 2.40−2.48 (1H, m), 3.43
(3H, s), 3.52 (3H, s), 6.41 (1H, s), 7.33 (2H, d, J = 8.1 Hz), 7.46 (2H,
d, J = 8.1 Hz); 13C NMR (CDCl3, 125 MHz) δ 19.00, 27.82, 27.85,
30.15, 32.49, 38.36, 52.36, 84.81, 85.16, 111.73, 121.80, 131.34,
131.62, 138.26, 148.52, 148.54, 148.64, 161.33, 162.57, 169.64; HRMS
(ESI) calcd for C26H34BrN3O7: 578.1496 [(M + H)+], found
578.1502.
(1R,2S)-2-(2-(Bis(tert-butoxycarbonyl)amino)-1-methyl-6-oxo-
1,6-dihydropyrimidin-4-yl)-1-phenylcyclopropanecarboxylic Acid
(27a). To a solution of 25a (825 mg, 1.65 mmol) in EtOH (8.2
mL) was added water (0.8 mL) and 10 M KOH solution, and the
mixture was stirred for 4 h at 60 °C. The reaction mixture was poured
into the chilled AcOEt (20 mL), 2 M HCl (6 mL), and water (10 mL)
with vigorous stirring, and the insoluble solid was filtered off. The
mother liquor was extracted with AcOEt, and the organic layer was
washed with water and brine, dried with Na2SO4, and evaporated. The
residue was combined with the solid already obtained, and washed
with hexane to give 26a (595 mg) as a colorless solid.
To a suspension of 26a (550 mg) was added N,O-bis-
(trimethylsilyl)acetamide (1.05 mL, 4.28 mmol) under N2 atmos-
phere, and the mixture was stirred for 30 min at room temperature to
give a clear solution. Then N,N-dimethylaminopyridine (34.8 mg,
0.285 mmol) and di-tert-butyl carbonate (0.497 mL, 2.14 mmol) were
added, and the solution was stirred another 1 h at room temperature.
The solvent was evaporated, and the residue was purified by silica gel
chromatography (CHCl3/MeOH = 20:1) to obtain 27a (578 mg, 1.19
mmol, 78%) as a pale brown foam. [α]D22 +120.2° (c = 0.50, CHCl3);
1H NMR (CDCl3, 600 MHz) δ 1.41 (9H, s), 1.53 (9H, s), 1.72−1.75
(1H, m), 2.21−2.24 (1H, m), 2.62−2.66 (1H, m), 3.42 (3H, s), 6.46
(1H, s), 7.28−7.36 (3H, m), 7.45−7.48 (2H, m); 13C NMR (CDCl3,
125 MHz) δ 19.36, 27.78, 27.85, 30.19, 33.79, 38.80, 84.95, 85.15,
111.58, 127.85, 128.46, 130.00, 138.94, 148.35, 148.56, 148.56, 161.07,
162.90, 173.19; HRMS (ESI) calcd for C25H32N3O7: 486.2235 [(M +
H)+], found 486.2237.
(1R,2S)-2-(2-(Bis(tert-butoxycarbonyl)amino)-1-methyl-6-oxo-
1 , 6 - d i h y d r o p y r i m i d i n - 4 - y l ) - 1 - ( 3 - b r o m o p h e n y l ) -
cyclopropanecarboxylic Acid (27b). 26b (2.88 g) was obtained as a
colorless solid from 25b (3.85 g, 6.66 mmol) by the same procedure
used to prepare 26a. Then 27b (2.99 g, 5.30 mmol, 80% from 25b)
was obtained as an orange brown foam from 26b (2.85 g) by the same
procedure used to prepare 27a. [α]D22 +107.3° (c = 0.30, CHCl3); 1H
NMR (CDCl3, 600 MHz) δ 1.42 (9H, s), 1.48 (9H, s), 1.71−1.75
(1H, m), 2.22−2.25 (1H, m), 2.60−2.64 (1H, m), 3.42 (3H, s), 6.47
(1H, s), 7.21 (1H, t, J = 7.8 Hz), 7.40 (1H, d, J = 7.8 Hz), 7.43 (1H, d,
J = 7.8 Hz), 7.60 (1H, s); 13C NMR (CDCl3, 125 MHz) δ 19.40,
27.79, 27.86, 30.21, 33.47, 38.46, 85.20, 85.34, 111.62, 122.21, 128.75,
129.96, 130.89, 133.00, 141.38, 148.53, 148.58, 148.68, 161.06, 162.94,
172.80; HRMS (ESI) calcd for C25H31BrN3O7: 564.1340 [(M + H)+],
found 564.1345.
2-Bis(tert-butoxycarbonyl)amino-3-methyl-6-[(1R,2S)-2-(3-
bromophenyl)cyclopropyl]pyrimidin-4(3H)-one (ent-28b). ent-28b
(122 mg, 0.235 mmol, 52%) was obtained as a pale gray solid from
ent-27b (253 mg, 0.447 mmol) by the same procedure used to prepare
22
28a. [α]D −8.8° (c = 0.50, CHCl3); HRMS (ESI) calcd for
C24H31BrN3O5: 520.1442 [(M + H)+], found 520.1442.
2-Bis(tert-butoxycarbonyl)amino-3-methyl-6-[(1S,2R)-2-(4-
bromophenyl)cyclopropyl]pyrimidin-4(3H)-one (28c). 28c (355 mg,
0.681 mmol, 38%) was obtained as a colorless solid from 27c (1.02 g,
22
1.80 mmol) by the same procedure used to prepare 28a. [α]D
1
−17.8° (c = 0.50, CHCl3); H NMR (CDCl3, 600 MHz) δ 1.37 (9H,
m), 1.40 (9H, m), 1.44−1.49 (1H, m), 1.61−1.65 (1H, m), 2.33−2.38
(1H, m), 2.56−2.61 (1H, m), 3.31 (3H, s), 6.09 (1H, s), 7.01 (2H, d, J
= 8.4 Hz), 7.28 (2H, d, J = 8.4 Hz); 13C NMR (CDCl3, 125 MHz) δ
10.80, 25.26, 26.18, 27.75, 27.79 29.97, 84.61, 84.88, 110.81, 120.32,
130.95, 131.07, 135.54, 147.93, 148.17, 148.31, 162.46, 162.59; HRMS
(ESI) calcd for C24H31BrN3O5: 520.1442 [(M + H)+], found
520.1450.
(1S,2R)-2-(2-(Bis(tert-butoxycarbonyl)amino)-1-methyl-6-oxo-
1 , 6 - d i h y d r o p y r i m i d i n - 4 - y l ) - 1 - ( 3 - b r o m o p h e n y l ) -
cyclopropanecarboxylic Acid (ent-27b). ent-26b (897 mg) was
obtained as a colorless solid from ent-25b (1.27 g, 2.20 mmol) by the
same procedure used to prepare 26a. Then ent-27b (790 mg, 1.40
mmol, 78% from ent-25b) was obtained as a yellow brown foam from
2-Bis(tert-butoxycarbonyl)amino-6-((1S,2R)-2-(3-bromophenyl)-
2-(pyridin-2-ylthio)cyclopropyl)-3-methylpyrimidin-4(3H)-one (29).
[α]D +22.0° (c = 0.30, CHCl3); H NMR (CDCl3, 600 MHz) δ
1.39 (9H, s), 1.45 (9H, s), 1.89−1.93 (1H, m), 2.34−2.37 (1H, m),
2.88−2.91 (1H, m), 3.43 (3H, s), 6.24 (1H, s), 6.97 (1H, t, J = 7.8
22
1
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Hz), 7.02 (1H, dd, J = 7.6, 4.8 Hz), 7.18 (1H, d, J = 7.8 Hz), 7.18 (1H,
d, J = 7.8 Hz), 7.26 (1H, d, J = 7.6 Hz), 7.48 (1H, dt, J = 7.6, 0.9 Hz),
7.59 (1H, s), 8.50 (1H, dd, J = 4.8, 0.9 Hz); 13C NMR (CDCl3, 125
MHz) δ 21.23, 27.81, 27.90, 30.11, 33.37, 36.43, 84.71, 84.85, 111.98,
120.46, 121.80, 122.60, 128.74, 129.38, 130.34, 133.21, 136.29, 141.04,
148.24, 148.35, 148.52, 149.79, 158.42, 160.96, 162.47; HRMS (ESI)
calcd for C29H33BrN4O5SNa: 651.1247 [(M + Na)+], found 651.1260.
2-Amino-3-methyl-6-((1S,2R)-2-phenylcyclopropyl)pyrimidin-
4(3H)-one (6) from 28a. To a solution of 28a (31.2 mg, 0.0707
mmol) in CH2Cl2 (0.3 mL) was added trifluoroacetic acid (0.3 mL),
and the mixture was stirred for 30 min at room temperature. The
solvent was evaporated, and the residue was partitioned between
AcOEt and 5% K2CO3 solution. The organic layer was washed with
water and brine, dried with Na2SO4, and evaporated. The residue was
purified by silica gel chromatography (AcOEt/MeOH = 20:1) to
2-Amino-6-((1R,2S)-2-(biphenyl-3-yl)cyclopropyl)-3-methylpyri-
midin-4(3H)-one Hydrochloride (ent-32). ent-32 (33.6 mg, 0.950
mmol, 76%) was obtained as a colorless solid from ent-28b (65.3 mg,
0.125 mmol) and phenylboronic acid (45.9 mg, 0.376 mmol) by the
22
same procedure used to prepare 32 (method A). [α]D +67.0° (c =
0.30, MeOH); HRMS (ESI) calcd for C20H20N3O: 318.1601 [(M +
H)+], found 318.1601.
2-Amino-6-((1S,2R)-2-(3′-methoxybiphenyl-3-yl)cyclopropyl)-3-
methylpyrimidin-4(3H)-one Hydrochloride (33). 33 (35.3 mg, 0.920
mmol, 72%) was obtained as a colorless solid from 28b (66.5 mg,
0.128 mmol) and 3-methoxyphenylboronic acid (29.1 mg, 0.192
mmol) by the same procedure used to prepare 32 (method A). mp
186−189 °C; [α]D22 −56.0° (c = 0.30, MeOH); 1H NMR (DMSO-d6,
500 MHz) δ 1.44−1.51 (1H, m), 1.97−2.03 (1H, m), 2.32−2.38 (1H,
m), 2.78−2.85 (1H, m), 3.13 (3H, s), 3.83 (3H, s), 5.71 (1H, s), 6.93
(1H, d, J = 8.0 Hz), 7.15 (1H, s), 7.19 (1H, d, J = 8.0 Hz), 7.21 (1H, d,
J = 8.0 Hz), 7.31 (1H, t, J = 8.0 Hz), 7.35 (1H, t, J = 8.0 Hz), 7.46
(1H, d, J = 8.0 Hz), 7.50 (1H, s); 13C NMR (CD3OD, 125 MHz) δ
10.29, 26.62, 27.42, 28.72, 55.86, 102.30, 113.77, 113.92, 120.49,
126.91, 128.43, 129.04, 129.86, 130.94, 137.31, 142.49, 143.56, 153.26,
154.19, 160.36, 161.63; HRMS (ESI) calcd for C21H22N3O2: 348.1707
1
obtain 6 (12.8 mg, 0.0530 mmol, 75%) as a colorless solid. H NMR
and LCMS data were identical to those obtained by the previous
procedure. Optical purity: 94.8% ee (column: Daicel CHIRALPAK
AY-H 4.6 mm × 250 mm; eluent: MeCN (0.1% diethylamine), 1.0
mL/min, 40 °C, 259 nm; retention time: 4.2 min).
2-Amino-6-((1S,2R)-2-(3-bromophenyl)cyclopropyl)-3-methylpyr-
imidin-4(3H)-one (31). To a solution of 28b (31.2 mg, 0.0707 mmol)
in CH2Cl2 (0.3 mL) was added trifluoroacetic acid (0.3 mL), and the
mixture was stirred for 30 min at room temperature. The solvent was
evaporated, and the residue was partitioned between AcOEt and 5%
K2CO3 solution. The organic layer was washed with water and brine,
dried with Na2SO4, and evaporated. The residue was purified by silica
gel chromatography (AcOEt/MeOH = 20:1) to obtain a free form of
31 (60.6 mg). To a solution of the free form of 31 (55.5 mg) in
dioxane (1.1 mL) was added 4 M hydrogen chloride in dioxane (0.130
mL), and the resulting suspension was stirred for 1 h at room
temperature. The precipitate was filtered off and washed with AcOEt
to give 31 (51.4 mg, 0.144 mmol, 81%) as a colorless solid. mp 218−
224 °C (dec); [α]D20 −41.3° (c = 0.30, MeOH); 1H NMR (DMSO-d6,
500 MHz) δ 1.41−1.48 (1H, m), 1.87−1.93 (1H, m), 2.28−2.34 (1H,
m), 2.73−2.78 (1H, m), 3.15 (3H, s), 5.64 (1H, s), 7.15−7.23 (2H,
m), 7.36 (1H, d, J = 8.4 Hz), 7.44 (1H, s), 8.47 (1H, br s); 13C NMR
(CD3OD, 125 MHz) δ 10.28, 22.73, 26.76, 28.76, 102.27, 123.32,
128.64, 131.12, 131.24, 133.06, 139.57, 152.99, 154.28, 160.36; HRMS
(ESI) calcd for C14H15BrN3O: 320.0393 [(M + H)+], found 320.0388.
2-Amino-6-((1S,2R)-2-(3-bromophenyl)cyclopropyl)-3-methylpyr-
imidin-4(3H)-one Hydrochloride (ent-31). ent-31 (26.5 mg, 0.0743
mmol, 80%) was obtained as a colorless solid from ent-28b (48.3 mg,
[ ( M
+
H ) + ] , f o u n d 3 1 8 . 1 7 0 5 ; A n a l . C a l c d f o r
C21H21N3O2·HCl·0.3H2O: C, 64.79; H, 5.85; N, 10.79. Found: C,
64.80; H, 5.83; N, 10.92.
2-Amino-6-((1R,2S)-2-(3′-methoxybiphenyl-3-yl)cyclopropyl)-3-
methylpyrimidin-4(3H)-one Hydrochloride (ent-33). ent-33 (19.8
mg, 0.0516 mmol, 59%) was obtained as a colorless solid from ent-28b
(45.4 mg, 0.0872 mmol) and 3-methoxyphenylboronic acid (19.9 mg,
0.131 mmol) by the same procedure used to prepare 32 (method A).
[α]D22 +52.0° (c = 0.30, MeOH); HRMS (ESI) calcd for C21H22N3O2:
348.1707 [(M + H)+], found 348.1707; Anal. Calcd for
C21H21N3O2·HCl·0.1H2O: C, 65.40; H, 5.80; N, 10.90. Found: C,
65.29; H, 5.85; N, 10.97.
2-Amino-6-((1S,2R)-2-(biphenyl-4-yl)cyclopropyl)-3-methylpyri-
midin-4(3H)-one (34). 34 (32.8 mg, 0.103 mmol, 98%) was obtained
as a light brown solid from 28c (54.8 mg, 0.105 mmol) and
phenylboronic acid (19.3 mg, 0.158 mmol) by the same procedure
used to prepare 32 (method A). mp 179−182 °C; [α]D20 −66.7° (c =
1
0.30, CHCl3); H NMR (CDCl3, 600 MHz) δ 1.38−1.43 (1H, m),
1.65−1.70 (1H, m), 2.19−2.24 (1H, m), 2.56−2.61 (1H, m), 3.26
(3H, s), 4.69 (2H, s), 5.72 (1H, s), 7.22 (1H, d, J = 7.8 Hz), 7.29 (1H,
t, J = 7.8 Hz), 7.39 (1H, d, J = 7.8 Hz), 7.41 (1H, s), 7.42 (2H, d, J =
7.5 Hz), 7.55 (2H, d, J = 7.5 Hz); 13C NMR (CDCl3, 125 MHz) δ
10.82, 25.42, 25.56, 27.47, 101.76, 126.32, 126.82, 127.08, 128.73,
129.58, 136.58, 138.66, 140.77, 153.89, 162.36, 164.22; HRMS (ESI)
calcd for C20H19N3ONa: 345.1420 [(M + Na)+], found 345.1420.
2-Amino-3-methyl-6-((1S,2R)-2-(2′-methylbiphenyl-4-yl)-
cyclopropyl)pyrimidin-4(3H)-one Hydrochloride (35). 35 (20.2 mg,
0.0549 mmol, 50%) was obtained as a colorless solid from 28c (57.3
mg, 0.110 mmol) and 2-methylphenylboronic acid (22.5 mg, 0.165
mmol) by the same procedure used to prepare 36 (method B). mp
20
0.0928 mmol) by the same procedure used to prepare 31. [α]D
+46.0° (c = 0.30, MeOH); HRMS (ESI) calcd for C14H15BrN3O:
320.0393 [(M + H)+], found 320.0389.
Suzuki−Miyaura Coupling: Typical Procedures. Method A
(Conventional Heating): 2-Amino-6-[(1S,2R)-2-(biphenyl-3-yl)-
cyclopropyl]-3-methylpyrimidin-4(3H)-one Hydrochloride (32). To
a solution of 28b (100 mg, 0.193 mmol) in dioxane (1.1 mL) and
water (0.5 mL) was added phenylboronic acid (35.2 mg, 0.289 mmol),
potassium carbonate (80 mg, 0.578 mmol), and dichlorobis-
(triphenylphosphine)palladium (6.8 mg, 0.00964 mmol), and the
mixture was heated at 100 °C for 4.5 h. The reaction mixture was
partitioned between AcOEt and water. The organic layer was washed
with water and brine, dried with Na2SO4, and evaporated. The residue
was purified by silica gel chromatography (CHCl3/MeOH = 50:1) to
obtain the free form of 32. This was dissolved in dioxane (0.7 mL),
and 4 M hydrogen chloride in dioxane (0.089 mL, 0.356 mmol) was
added. The precipitate was filtered and washed with AcOEt to give 32
(25.7 mg, 0.0726 mmol, 61%) as a colorless solid. mp 205−208 °C;
20
1
165−168 °C; [α]D −50.7° (c = 0.30, MeOH); H NMR (CD3OD,
600 MHz) δ 1.60−1.65 (1H, m), 1.78−1.82 (1H, m), 2.13 (3H, s),
2.32−2.37 (1H, m), 2.88−2.93 (1H, m), 3.27 (3H, s), 5.58 (1H, s),
7.09−7.24 (6H, m), 7.27 (2H, d, J = 8.4 Hz); 13C NMR (CD3OD, 125
MHz) δ 10.56, 20.53, 22.47, 27.15, 28.70, 102.08, 126.87, 128.43,
129.84, 130.14, 130.47, 131.34, 135.40, 136.25, 142.16, 142.74, 153.81,
154.24, 160.43; HRMS (ESI) calcd for C21H22N3O: 332.1757 [(M +
H)+], found 332.1758; Anal. Calcd for C21H21N3O·HCl·0.3H2O: C,
67.57; H, 6.10; N, 11.26; Cl, 9.50. Found: C, 67.43; H, 6.07; N, 11.41;
Cl, 9.13.
[α]D22 −64.0° (c = 0.30, MeOH); H NMR (DMSO-d6, 500 MHz) δ
1
Method B (Microwave Heating): 2-Amino-3-methyl-6-((1S,2R)-2-
(3′-methylbiphenyl-4-yl)cyclopropyl)pyrimidin-4(3H)-one Hydro-
chloride (36). To a solution of 28c (55.5 mg, 0.107 mmol) in
dioxane (1.1 mL) and water (0.5 mL) was added 3-methylphenylbor-
onic acid (21.8 mg, 0.160 mmol), potassium carbonate (44.2 mg,
0.320 mmol), and dichlorobis(triphenylphosphine)palladium (3.7 mg,
0.0053 mmol), and the mixture was heated at 150 °C under
microwave irradiation for 30 min. The reaction mixture was
partitioned between AcOEt and water. The organic layer was washed
1.45−1.50 (1H, m), 1.94−2.02 (1H, m), 2.32−2.37 (1H, m), 2.77−
2.85 (1H, m), 3.13 (3H, s), 5.69 (1H, s), 7.21 (1H, d, J = 7.5 Hz), 7.32
(1H, t, J = 7.5 Hz), 7.36 (1H, t, J = 7.5 Hz), 7.45 (3H, t, J = 7.3 Hz),
7.50 (1H, s), 7.63 (2H, d, J = 7.3 Hz), 8.46 (1H, br s); 13C NMR
(CD3OD, 125 MHz) δ 10.31, 22.89, 27.41, 28.69, 102.15, 126.75,
128.06, 128.47, 128.54, 128.92, 129.85, 129.92, 137.46, 142.10, 142.54,
154.24, 154.37, 160.72; HRMS (ESI) calcd for C20H20N3O: 318.1601
[(M + H)+], found 318.1602.
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with water and brine, dried with Na2SO4, and evaporated. The residue
was purified by silica gel chromatography (AcOEt/MeOH = 10:1) to
obtain the free form of 36. This was dissolved in dioxane (0.7 mL),
and 4 M hydrogen chloride in dioxane (0.080 mL, 0.322 mmol) was
added. After the evaporation of the solvent, the residue was solidified
with AcOEt. The solid was filtered and washed with AcOEt to give 36
(29.2 mg, 0.0794 mmol, 74%) as a colorless solid. mp 173−178 °C;
[α]D −68.0° (c = 0.30, MeOH); H NMR (CD3OD, 600 MHz) δ
1.58−1.63 (1H, m), 1.77−1.81 (1H, m), 2.32−2.37 (1H, m), 2.37
(3H, s), 2.84−2.89 (1H, m), 3.26 (3H, s), 5.64 (1H, s), 7.13 (1H, d, J
= 7.8 Hz), 7.27 (1H, t, J = 7.8 Hz), 7.28 (2H, d, J = 7.8 Hz), 7.33 (1H,
d, J = 7.8 Hz), 7.37 (1H, s), 7.50 (2H, d, J = 7.8 Hz); 13C NMR
(CD3OD, 125 MHz) δ 10.42, 21.58, 22.76, 27.14, 28.72, 102.12,
124.92, 127.82, 128.45, 129.11, 129,80, 130.42, 135.76, 139.61, 141.23,
141.70, 153.71, 154.28, 160.55; HRMS (ESI) calcd for C21H22N3O:
332.1757 [(M + H)+], found 332.1759; Anal. Calcd for
C21H21N3O·HCl·0.1H2O: C, 68.23; H, 6.05; N, 11.37; Cl, 9.59.
Found: C, 68.07; H, 6.00; N, 11.56; Cl, 9.35.
154.03, 162.25, 164.01; HRMS (ESI) calcd for C18H17N3O2Na:
330.1213 [(M + Na)+], found 330.1215.
2-Amino-3-methyl-6-((1S,2R)-2-(4-(5-methylthiophen-2-yl)-
phenyl)cyclopropyl)pyrimidin-4(3H)-one (42). 42 (24.8 mg, 0.0735
mmol, 82%) was obtained as a colorless solid from 28c (46.9 mg,
0.0901 mmol) and 5-methylthiophene-2-ylboronic acid (19.2 mg,
0.135 mmol) by the same procedure used to prepare 36 (method B).
mp 207−210 °C; [α]D20 −72.3° (c = 0.30, CHCl3); 1H NMR (CDCl3,
500 MHz) δ 1.35−1.40 (1H, m), 1.62−1.67 (1H, m), 2.16−2.22 (1H,
m), 2.48 (3H, s), 2.51−2.57 (1H, m), 3.25 (3H, s), 4.68 (2H, br s),
5.73 (1H, s), 6.68 (1H, dd, J = 3.5, 1.0 Hz), 7.03 (1H, d, J = 3.5 Hz),
7.12 (2H, d, J = 8.2 Hz), 7.35 (2H, d, J = 8.2 Hz); 13C NMR (CDCl3,
125 MHz) δ 10.70, 15.44, 25.44, 25.53, 27.45, 101.93, 122.43, 124.68,
126.13, 129.54, 132.36, 136.36, 139.10, 141.91, 153.82, 162.28, 164.08;
HRMS (ESI) calcd for C19H19N3OSNa: 360.1141 [(M + Na)+], found
360.1141.
20
1
2-Amino-3-methyl-6-((1S,2R)-2-(4-(4-methylthiophen-2-yl)-
phenyl)cyclopropyl)pyrimidin-4(3H)-one Hydrochloride (43). 43
(22.3 mg, 0.0596 mmol, 66%) was obtained as a colorless solid
from 28c (46.9 mg, 0.0901 mmol) and 4,4,5,5-tetramethyl-2-(4-
methylthiophen-2-yl)-1,3,2-dioxaborolane (30.3 mg, 0.135 mmol) by
the same procedure used to prepare 36 (method B). mp 193−196 °C;
2-Amino-3-methyl-6-((1S,2R)-2-(4′-methylbiphenyl-4-yl)-
cyclopropyl)pyrimidin-4(3H)-one Hydrochloride (37). 37 (26.7 mg,
0.0726 mmol, 67%) was obtained as a colorless solid from 28c (56.5
mg, 0.109 mmol) and 4-methylphenylboronic acid (22.1 mg, 0.163
mmol) by the same procedure used to prepare 36 (method B). mp
20
1
[α]D −68.7° (c = 0.30, MeOH); H NMR (CD3OD, 500 MHz) δ
1.57−1.62 (1H, m), 1.75−1.80 (1H, m), 2.24 (3H, s), 2.30−2.36 (1H,
m), 2.81−2.86 (1H, m), 3.26 (3H, s), 5.64 (1H, s), 6.91 (1H, s), 7.16
(1H, s), 7.21 (2H, d, J = 8.3 Hz), 7.49 (2H, d, J = 8.3 Hz); 13C NMR
(CD3OD, 125 MHz) δ 10.37, 15.80, 22.81, 27.11, 28.75, 102.21,
121.27, 126.33, 126.51, 130.48, 134.79, 135.91, 139.99, 144.59, 153.54,
154.30, 160.54; HRMS (ESI) calcd for C18H18N3OS: 338.1322 [(M +
H)+], found 338.1324.
20
223−228 °C (dec); [α]D −78.0° (c = 0.30, MeOH); 1H NMR
(CD3OD, 600 MHz) δ 1.58−1.63 (1H, m), 1.77−1.81 (1H, m), 2.31−
2.35 (1H, m), 2.35 (3H, s), 2.84−2.89 (1H, m), 3.26 (3H, s), 5.64
(1H, s), 7.21 (2H, d, J = 8.4 Hz), 7.27 (2H, d, J = 8.4 Hz), 7.45 (2H, d,
J = 8.4 Hz), 7.50 (2H, d, J = 8.4 Hz); HRMS (ESI) calcd for
C21H22N3O: 332.1757 [(M + H)+], found 332.1759; Anal. Calcd for
C21H21N3O·HCl·0.2H2O: C, 67.90; H, 6.08; N, 11.31; Cl, 9.54.
Found: C, 67.80; H, 6.06; N, 11.41; Cl, 9.38.
2-Amino-6-((1S,2R)-2-(4-(6-methoxypyridin-2-yl)phenyl)-
cyclopropyl)-3-methylpyrimidin-4(3H)-one (44). 44 (9.2 mg, 0.026
mmol, 30%) was obtained as a colorless solid from 28c (46.3 mg,
0.0890 mmol) and 2-methoxy-6-(4,4,5,5-tetramethyl-1,3,2-dioxabor-
olan-2-yl)pyridine (62.8 mg, 0.267 mmol) by the same procedure used
to prepare 36 (method B). mp 188−190 °C; [α]D20 −61.0° (c = 0.10,
2-Amino-3-methyl-6-((1S,2R)-2-(4-(thiophen-2-yl)phenyl)-
cyclopropyl)pyrimidin-4(3H)-one Hydrochloride (39). 39 (23.2 mg,
0.0645 mmol, 65%) was obtained as a colorless solid from 28c (51.6
mg, 0.0992 mmol) and thiophen-2-ylboronic acid (19.0 mg, 0.149
mmol) by the same procedure used to prepare 36 (method B). mp
1
CHCl3); H NMR (CDCl3, 500 MHz) δ 1.38−1.44 (1H, m), 1.67−
20
1
203−208 °C; [α]D −74.3° (c = 0.30, MeOH); H NMR (CD3OD,
500 MHz) δ 1.58−1.63 (1H, m), 1.76−1.81 (1H, m), 2.31−2.36 (1H,
m), 2.82−2.88 (1H, m), 3.26 (3H, s), 5.65 (1H, s), 7.06 (1H, dd, J =
5.1, 3.8 Hz), 7.23 (2H, d, J = 8.3 Hz), 7.33−7.35 (2H, m), 7.53 (2H, d,
J = 8.3 Hz); 13C NMR (CDCl3, 125 MHz) δ 10.39, 22.85, 27.07,
28.75, 102.24, 124.26, 125.91, 126.57, 129.19, 130.51, 134.61, 136.10,
144.90, 153.79, 154.33, 160.60; HRMS (ESI) calcd for C18H18N3OS:
324.1165 [(M + H)+], found 324.1167; Anal. Calcd for
C18H17N3OS·HCl·0.3H2O: C, 59.19; H, 5.13; N, 11.50. Found: C,
59.23; H, 5.17; N, 11.75.
1.71 (1H, m), 2.20−2.26 (1H, m), 2.56−2.62 (1H, m), 3.25 (3H, s),
4.01 (3H, s), 4.65 (2H, br s), 5.75 (1H, s), 6.64 (1H, dd, J = 8.2, 0.6
Hz), 7.25 (2H, d, J = 8.3 Hz), 7.29 (1H, dd, J = 7.4, 0.6 Hz), 7.59 (1H,
dd, J = 8.3, 7.4 Hz), 7.88 (2H, d, J = 8.3 Hz); 13C NMR (CDCl3, 125
MHz) δ 10.72, 25.63, 25.70, 27.46, 53.20, 102.04, 108.89, 112.47,
125.95, 129.34, 136.65, 138.39, 139.14, 153.79, 154.42, 162.28, 163.69,
163.96; HRMS (ESI) calcd for C20H20N4O2Na: 371.1479 [(M +
Na)+], found 371.1482.
2-Amino-6-((1S,2R)-2-(4-(2-methoxypyridin-4-yl)phenyl)-
cyclopropyl)-3-methylpyrimidin-4(3H)-one (45). 45 (23.5 mg, 0.0675
mmol, 78%) was obtained as a light brown solid from 28c (45.1 mg,
0.0867 mmol) and 2-methoxypyridin-4-ylboronic acid (19.9 mg, 0.130
mmol) by the same procedure used to prepare 36 (method B). mp
224−226 °C; [α]D20 −57.3° (c = 0.30, CHCl3); 1H NMR (CDCl3, 500
MHz) δ 1.39−1.44 (1H, m), 1.67−1.71 (1H, m), 2.20−2.26 (1H, m),
2.56−2.62 (1H, m), 3.27 (3H, s), 3.97 (3H, s), 4.64 (2H, br s), 5.72
(1H, s), 6.91 (1H, dd, J = 1.7, 0.8 Hz), 7.07 (1H, dd, J = 5.4, 1.7 Hz),
7.25 (2H, d, J = 8.2 Hz), 7.44 (2H, d, J = 8.2 Hz), 8.17 (1H, dd, J =
5.4, 0.8 Hz); 13C NMR (CDCl3, 125 MHz) δ 10.74, 25.44, 25.64,
27.48, 53.51, 102.02, 108.01, 115.10, 126.17, 129.90, 135.65, 138.65,
147.16, 150.87, 153.73, 162.24, 163.99, 164.92; HRMS (ESI) calcd for
C20H21N4O2: 349.1659 [(M + H)+], found 349.1662.
2-Amino-3-methyl-6-((1S,2R)-2-(4-(thiophen-3-yl)phenyl)-
cyclopropyl)pyrimidin-4(3H)-one Hydrochloride (40). 40 (29.6 mg,
0.0823 mmol, 89%) was obtained as a colorless solid from 28c (48.1
mg, 0.0924 mmol) and thiophen-3-ylboronic acid (17.7 mg, 0.139
mmol) by the same procedure used to prepare 36 (method B). mp
20
214−220 °C (dec); [α]D −71.7° (c = 0.30, MeOH); 1H NMR
(CD3OD, 500 MHz) δ 1.57−1.63 (1H, m), 1.76−1.81 (1H, m), 2.30−
2.36 (1H, m), 2.82−2.88 (1H, m), 3.26 (3H, s), 5.64 (1H, s), 7.24
(2H, d, J = 8.4 Hz), 7.40 (1H, dd, J = 5.1, 1.3 Hz), 7.45 (1H, dd, J =
5.1, 2.9 Hz), 7.56 (2H, d, J = 8.4 Hz), 7.57 (1H, dd, J = 2.9, 1.3 Hz);
HRMS (ESI) calcd for C18H18N3OS: 324.1165 [(M + H)+], found
324.1166.
2-Amino-6-((1S,2R)-2-(4-(furan-2-yl)phenyl)cyclopropyl)-3-meth-
ylpyrimidin-4(3H)-one (41). 41 (26.0 mg, 0.0846 mmol, 90%) was
obtained as a colorless solid from 28c (48.7 mg, 0.0936 mmol) and
furan-2-ylboronic acid (15.7 mg, 0.140 mmol) by the same procedure
used to prepare 36 (method B). mp 160−162 °C; [α]D20 −74.0° (c =
2-Amino-6-((1S,2R)-2-(4-(6-methoxypyrazin-2-yl)phenyl)-
cyclopropyl)-3-methylpyrimidin-4(3H)-one (46). 46 (20.3 mg, 0.0581
mmol, 67%) was obtained as a light yellow powder from 28c (45.1 mg,
0.0867 mmol) and 2-methoxy-6-(4,4,5,5-tetramethyl-1,3,2-dioxabor-
olan-2-yl)pyrazine (30.7 mg, 0.130 mmol) by the same procedure used
to prepare 36 (method B). mp 192−195 °C; [α]D20 −64.0° (c = 0.10,
1
0.30, CHCl3); H NMR (CDCl3, 500 MHz) δ 1.35−1.41 (1H, m),
1
1.64−1.69 (1H, m), 2.17−2.23 (1H, m), 2.53−2.59 (1H, m), 3.25
(3H, s), 4.67 (2H, br s), 5.72 (1H, s), 6.42−6.45 (1H, m), 6.56 (1H, d,
J = 3.2 Hz), 7.16 (2H, d, J = 8.1 Hz), 7.42 (1H, s), 7.48 (2H, d, J = 8.1
Hz); 13C NMR (CDCl3, 125 MHz) δ 10.65, 25.40, 25.63, 27.45,
101.87, 104.50, 111.62, 123.15, 128.67, 129.46, 136.67, 141.76, 153.82,
CHCl3); H NMR (CDCl3, 500 MHz) δ 1.40−1.46 (1H, m), 1.67−
1.73 (1H, m), 2.22−2.28 (1H, m), 2.57−2.62 (1H, m), 3.26 (3H, s),
4.10 (3H, s), 4.73 (2H, br s), 5.72 (1H, s), 7.27 (2H, d, J = 8.1 Hz),
7.87 (2H, d, J = 8.1 Hz), 8.11 (1H, s), 8.54 (1H, s); 13C NMR
(CDCl3, 125 MHz) δ 10.74, 25.65, 25.72, 27.49, 53.38, 101.96, 126.07,
8852
dx.doi.org/10.1021/jm3011405 | J. Med. Chem. 2012, 55, 8838−8858

Journal of Medicinal Chemistry
Article
127.70, 132.80, 133.23, 133.84, 139.42, 148.72, 153.80, 159.77, 162.26,
163.89; HRMS (ESI) calcd for C19H19N5O2Na: 372.1431 [(M +
Na)+], found 372.1430.
2-Amino-6-((1S,2R)-2-(3′-(cyclopropylmethoxy)biphenyl-4-yl)-
cyclopropyl)-3-methylpyrimidin-4(3H)-one Hydrochloride (51). 51
(16.8 mg, 0.0396 mmol, 46%) was obtained as a colorless solid from
28c (44.6 mg, 0.0857 mmol) and 3-cyclopropylmethoxyphenylboronic
acid (24.7 mg, 0.129 mmol) by the same procedure used to prepare 36
(method B). mp 152−155 °C; [α]D −55.3° (c = 0.30, MeOH); H
NMR (CD3OD, 500 MHz) δ 0.33−0.37 (2H, m), 0.59−0.64 (2H, m),
1.21−1.30 (1H, m), 1.60−1.64 (1H, m), 1.77−1.82 (1H, m), 2.31−
2.37 (1H, m), 2.84−2.90 (1H, m), 3.26 (3H, s), 5.65 (1H, s), 6.86
(1H, ddd, J = 8.0, 2.4, 1.0 Hz), 7.07 (1H, dd, J = 2.4, 1.7 Hz), 7.12
(1H, ddd, J = 8.0, 1.7, 1.0 Hz), 7.28 (2H, d, J = 8.8 Hz), 7.29 (1H, t, J
= 8.0 Hz), 7.51 (2H, d, J = 8.8 Hz); 13C NMR (CD3OD, 125 MHz) δ
3.56, 10.43, 11.28, 22.77, 27.12, 28.73, 73.84, 102.19, 114.26, 114.50,
120.20, 127.87, 130.41, 130.91, 136.00, 141.00, 143.16, 153.60, 154.27,
160.51, 161.00; HRMS (ESI) calcd for C24H26N3O2: 388.2020 [(M +
H)+], found 388.2021.
2-Amino-6-((1S,2R)-2-(3′-ethylbiphenyl-4-yl)cyclopropyl)-3-meth-
ylpyrimidin-4(3H)-one Hydrochloride (52). 52 (24.2 mg, 0.0634
mmol, 73%) was obtained as a colorless solid from 28c (45.0 mg,
0.0865 mmol) and 3-ethylphenylboronic acid (19.5 mg, 0.130 mmol)
by the same procedure used to prepare 36 (method B). mp 175−178
°C; [α]D20 −62.7° (c = 0.30, MeOH); 1H NMR (CD3OD, 500 MHz)
δ 1.25 (3H, t, J = 7.6 Hz), 1.61−1.66 (1H, m), 1.77−1.82 (1H, m),
2.32−2.37 (1H, m), 2.68 (2H, q, J = 7.6 Hz), 2.85−2.91 (1H, m), 3.26
(3H, s), 5.65 (1H, s), 7.16 (1H, d, J = 7.6 Hz), 7.28 (2H, d, J = 8.2
Hz), 7.30 (1H, t, J = 7.6 Hz), 7.36 (1H, d, J = 7.6 Hz), 7.38 (1H, s),
7.51 (2H, d, J = 8.2 Hz); 13C NMR (CD3OD, 125 MHz) δ 10.42,
16.32, 22.70, 27.15, 28.73, 29.97, 102.18, 125.21, 127.35, 127.86,
127.98, 129.91, 130.41, 135.71, 141.35, 141.77, 146.15, 153.46, 154.25,
160.47; HRMS (ESI) calcd for C22H24N3O: 346.1914 [(M + H)+],
found 346.1914.
2-Amino-6-((1S,2R)-2-(3′-methoxybiphenyl-4-yl)cyclopropyl)-3-
methylpyrimidin-4(3H)-one Hydrochloride (47). 47 (21.0 mg, 0.0547
mmol, 51%) was obtained as a colorless solid from 28c (55.4 mg,
0.106 mmol) and 3-methoxyphenylboronic acid (24.3 mg, 0.160
mmol) by the same procedure used to prepare 36 (method B). mp
20
1
20
1
151−154 °C; [α]D −67.7° (c = 0.30, MeOH); H NMR (CD3OD,
500 MHz) δ 1.58−1.64 (1H, m), 1.77−1.82 (1H, m), 2.31−2.37 (1H,
m), 2.37 (3H, s), 2.84−2.90 (1H, m), 3.26 (3H, s), 3.82 (3H, s), 5.65
(1H, s), 6.88 (1H, dd, J = 7.8, 2.5 Hz), 7.08 (1H, dd, J = 2.5, 1.7 Hz),
7.08 (1H, dd, J = 7.8, 1.7 Hz), 7.28 (2H, d, J = 8.2 Hz), 7.31 (1H, t, J =
7.8 Hz), 7.51 (2H, d, J = 8.2 Hz); 13C NMR (CD3OD, 125 MHz) δ
10.42, 22.75, 27.13, 28.74, 55.76, 102.17, 113.54, 113.76, 120.23,
127.88, 130.43, 130.93, 136.03, 140.96, 143.18, 153.53, 154.25, 160.50,
161.62; HRMS (ESI) calcd for C21H22N3O2: 348.1707 [(M + H)+],
found 348.1709; Anal. Calcd for C21H21N3O2·HCl·0.3H2O: C, 64.79;
H, 5.85; N, 10.79; Cl, 9.11. Found: C, 64.70; H, 5.87; N, 10.81; Cl,
9.10.
2-Amino-6-((1S,2R)-2-(3′-ethoxybiphenyl-4-yl)cyclopropyl)-3-
methylpyrimidin-4(3H)-one Hydrochloride (48). 48 (22.5 mg, 0.0565
mmol, 63%) was obtained as a colorless solid from 28c (46.6 mg,
0.0895 mmol) and 3-ethoxyphenylboronic acid (22.3 mg, 0.134
mmol) by the same procedure used to prepare 36 (method B). mp
20
1
153−155 °C; [α]D −63.7° (c = 0.30, MeOH); H NMR (CD3OD,
500 MHz) δ 1.39 (3H, t, J = 7.1 Hz), 1.58−1.64 (1H, m), 1.77−1.82
(1H, m), 2.31−2.37 (1H, m), 2.84−2.90 (1H, m), 3.26 (3H, s), 4.07
(2H, q, J = 7.1 Hz), 5.65 (1H, s), 6.87 (1H, ddd, J = 8.1, 2.0, 0.8 Hz),
7.07 (1H, dd, J = 2.0, 1.4 Hz), 7.11 (1H, ddd, J = 8.1, 1.4, 0.8 Hz), 7.28
(2H, d, J = 8.4 Hz), 7.30 (1H, t, J = 8.1 Hz), 7.51 (2H, d, J = 8.4 Hz);
13C NMR (CD3OD, 125 MHz) δ 10.42, 15.23, 22.74, 27.12, 28.74,
2-Amino-3-methyl-6-((1S,2R)-2-(3′-propylbiphenyl-4-yl)-
cyclopropyl)pyrimidin-4(3H)-one Hydrochloride (53). 53 (26.5 mg,
0.0669 mmol, 76%) was obtained as a colorless solid from 28c (45.7
mg, 0.0878 mmol) and 4,4,5,5-tetramethyl-2-(3-propylphenyl)-1,3,2-
dioxaborolane (32.4 mg, 0.132 mmol) by the same procedure used to
64.55, 102.19, 114.13, 114.36, 120.15, 127.87, 130.41, 130.91, 135.99,
141.01, 143.15, 153.48, 154.25, 160.48, 160.90; HRMS (ESI) calcd for
C22H24N3O2: 362.1863 [(M + H)+], found 364.1863. Anal. Calcd for
C22H23N3O2·HCl·0.4H2O: C, 65.23; H, 6.17; N, 10.37; Cl, 8.75.
Found: C, 65.31; H, 6.22; N, 10.60; Cl, 8.50.
20
prepare 36 (method B). mp 177−182 °C; [α]D −66.3° (c = 0.30,
1
MeOH); H NMR (CD3OD, 500 MHz) δ 0.95 (3H, t, J = 7.6 Hz),
2-Amino-3-methyl-6-((1S,2R)-2-(3′-propoxybiphenyl-4-yl)-
cyclopropyl)pyrimidin-4(3H)-one Hydrochloride (49). 49 (15.7 mg,
0.0381 mmol, 38%) was obtained as a colorless solid from 28c (51.7
mg, 0.0993 mmol) and 3-propoxyphenylboronic acid (26.8 mg, 0.149
mmol) by the same procedure used to prepare 36 (method B). mp
1.59−1.64 (1H, m), 1.67 (3H, sextet, J = 7.6 Hz), 1.77−1.82 (1H, m),
2.32−2.37 (1H, m), 2.63 (2H, t, J = 7.6 Hz), 2.85−2.91 (1H, m), 3.26
(3H, s), 5.65 (1H, s), 7.14 (1H, d, J = 7.6 Hz), 7.28 (2H, d, J = 8.2
Hz), 7.30 (1H, t, J = 7.6 Hz), 7.36 (1H, d, J = 7.6 Hz), 7.37 (1H, s),
7.51 (2H, d, J = 8.2 Hz); 13C NMR (CD3OD, 125 MHz) δ 10.43,
14.13, 22.70, 25.86, 27.14, 28.73, 39.14, 102.20, 125.25, 127.86,
127.97, 128.63, 129.81, 130.41, 141.34, 141.68, 144.46, 153.42, 154.25,
160.46; HRMS (ESI) calcd for C23H26N3O: 360.2070 [(M + H)+],
found 360.2073; Anal. Calcd for C23H25N3O·HCl·0.3H2O: C, 68.83;
H, 6.68; N, 10.47; Cl 8.83. Found: C, 68.91; H, 6.58; N, 10.76; Cl
8.62.
20
1
161−164 °C; [α]D −74.0° (c = 0.10, MeOH); H NMR (CD3OD,
500 MHz) δ 1.05 (3H, t, J = 7.4 Hz), 1.60−1.64 (1H, m), 1.76−1.84
(3H, m), 2.31−2.37 (1H, m), 2.84−2.90 (1H, m), 3.26 (3H, s), 3.95
(2H, t, J = 6.4 Hz), 5.65 (1H, s), 6.87 (1H, ddd, J = 8.0, 2.6, 1.0 Hz),
7.07 (1H, dd, J = 2.6, 1.6 Hz), 7.11 (1H, ddd, J = 8.0, 1.6, 1.0 Hz), 7.28
(2H, d, J = 8.2 Hz), 7.30 (1H, t, J = 8.0 Hz), 7.51 (2H, d, J = 8.2 Hz);
13C NMR (CD3OD, 125 MHz) δ 10.42, 10.91, 22.72, 23.75, 27.12,
2-Amino-3-methyl-6-((1S,2R)-2-(3′-(methylthio)biphenyl-4-yl)-
cyclopropyl)pyrimidin-4(3H)-one Hydrochloride (54). 54 (20.5 mg,
0.0513 mmol, 57%) was obtained as a colorless solid from 28c (46.6
mg, 0.0895 mmol) and 3-(methylthio)phenylboronic acid (22.6 mg,
0.134 mmol) by the same procedure used to prepare 36 (method B).
28.75, 70.63, 102.19, 114.13, 114.38, 120.13, 127.87, 130.42, 130.91,
135.98, 141.03, 143.15, 153.46, 154.23, 160.49, 161.09; HRMS (ESI)
calcd for C23H26N3O2: 376.2020 [(M + H)+], found 376.2020.
2-Amino-6-((1S,2R)-2-(3′-isopropoxybiphenyl-4-yl)cyclopropyl)-
3-methylpyrimidin-4(3H)-one Hydrochloride (50). 50 (26.4 mg,
0.0641 mmol, 75%) was obtained as a colorless solid from 28c (44.6
mg, 0.0857 mmol) and 3-isopropoxyphenylboronic acid (23.1 mg,
0.129 mmol) by the same procedure used to prepare 36 (method B).
20
mp 154−159 °C; [α]D −67.7° (c = 0.30, MeOH); 1H NMR
(CD3OD, 500 MHz) δ 1.59−1.64 (1H, m), 1.77−1.82 (1H, m), 2.32−
2.38 (1H, m), 2.50 (3H, s), 2.84−2.90 (1H, m), 3.26 (3H, s), 5.65
(1H, s), 7.22 (1H, ddd, J = 6.0, 2.6, 2.0 Hz), 7.29 (2H, d, J = 8.1 Hz),
7.32−7.34 (2H, m), 7.42 (1H, dd, J = 2.6, 1.3 Hz), 7.51 (2H, d, J = 8.1
Hz); 13C NMR (CD3OD, 125 MHz) δ 10.43, 15.74, 22.80, 27.12,
28.75, 102.13, 124.60, 125.78, 126.41, 127.87, 130.40, 130.52, 136.23,
140.58, 140.80, 142.41, 153.66, 154.27, 160.56; HRMS (ESI) calcd for
C21H22N3OS: 364.1478 [(M + H)+], found 364.1480.
20
mp 168−171 °C; [α]D −60.0° (c = 0.30, MeOH); 1H NMR
(CD3OD, 500 MHz) δ 1.32 (6H, d, J = 6.1 Hz), 1.58−1.64 (1H, m),
1.77−1.82 (1H, m), 2.31−2.37 (1H, m), 2.84−2.90 (1H, m), 3.26
(3H, s), 4.64 (1H, septet, J = 6.1 Hz), 5.65 (1H, s), 6.86 (1H, ddd, J =
7.9, 2.5, 0.8 Hz), 7.05 (1H, dd, J = 2.5, 1.7 Hz), 7.10 (1H, ddd, J = 7.9,
1.7, 0.8 Hz), 7.28 (2H, d, J = 8.0 Hz), 7.29 (1H, t, J = 7.9 Hz), 7.50
(2H, d, J = 8.0 Hz); 13C NMR (CD3OD, 125 MHz) δ 10.42, 22.41,
22.73, 27.12, 28.74, 71.05, 102.19, 115.67, 115.77, 120.18, 127.85,
130.42, 130.94, 135.97, 141.01, 143.22, 153.45, 154.25, 159.75, 160.48;
HRMS (ESI) calcd for C23H26N3O2: 376.2020 [(M + H)+], found
376.2023; Anal. Calcd for C23H25N3O2·HCl·0.4H2O: C, 65.91; H,
6.44; N, 10.03; Cl 8.46. Found: C, 66.02; H, 6.39; N, 10.19; Cl 8.42.
2-Amino-6-((1S,2R)-2-(3′-(ethylthio)biphenyl-4-yl)cyclopropyl)-3-
methylpyrimidin-4(3H)-one Hydrochloride (55). 55 (28.3 mg, 0.0684
mmol, 76%) was obtained as a colorless solid from 28c (47.0 mg,
0.0903 mmol) and 3-(ethylthio)phenylboronic acid (24.7 mg, 0.135
mmol) by the same procedure used to prepare 36 (method B). mp
20
1
141−144 °C; [α]D −62.7° (c = 0.30, MeOH); H NMR (CD3OD,
500 MHz) δ 1.30 (3H, t, J = 7.3 Hz), 1.59−1.65 (1H, m), 1.77−1.82
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a
Scheme 5. Synthesis of Reference Compounds with Ethylene Linker (4, 5, 38, and 61)
a
(a) 2.5 equiv of Boc2O, 0.3 equiv of DMAP, CH2Cl2, rt, 72% (63), 92% (64); (b) R-B(OH)2, K2CO3, PdCl2(PPh3)2, dioxane, H2O, 100 °C
(Method A), 150 °C under microwave, 30 min (Method B).
(1H, m), 2.32−2.38 (1H, m), 2.85−2.91 (1H, m), 2.98 (2H, q, J = 7.3
Hz), 3.26 (3H, s), 5.65 (1H, s), 7.28 (1H, dt, J = 7.6, 1.7 Hz), 7.30
(2H, d, J = 8.1 Hz), 7.34 (1H, t, J = 7.6 Hz), 7.37 (1H, dt, J = 7.6, 1.7
Hz), 7.49 (1H, t, J = 1.7 Hz), 7.51 (2H, d, J = 8.1 Hz); 13C NMR
(CD3OD, 125 MHz) δ 10.45, 14.81, 22.76, 27.11, 28.27, 28.75, 102.20,
125.38, 127.88, 128.21, 128.81, 130.44, 130.52, 136.24, 138.85, 140.53,
142.48, 153.43, 154.26, 160.48; HRMS (ESI) calcd for C22H24N3OS:
378.1635 [(M + H)+], found 378.1638; Anal. Calcd for
C22H23N3OS·HCl·0.2H2O: C, 63.28; H, 5.89; N, 10.06; S, 7.68; Cl,
8.49. Found: C, 63.49; H, 5.89; N, 10.12; S, 7.39; Cl, 8.37.
(33.1 mg, 0.0806 mmol, 76%) was obtained as a light brown solid
from 28c (55.2 mg, 0.106 mmol) and 3-acetoamidophenylboronic acid
(28.5 mg, 0.159 mmol) by the same procedure used to prepare 36
20
1
(method B). mp 163−166 °C; [α]D −58.0° (c = 0.30, MeOH); H
NMR (CD3OD, 500 MHz) δ 1.59−1.65 (1H, m), 1.78−1.83 (1H, m),
2.14 (3H, s), 2.33−2.38 (1H, m), 2.85−2.91 (1H, m), 3.26 (3H, s),
5.66 (1H, s), 7.30 (2H, d, J = 8.1 Hz), 7.30 (1H, d, J = 7.9 Hz), 7.35
(1H, t, J = 7.9 Hz), 7.41 (1H, d, J = 7.9 Hz), 7.52 (2H, d, J = 8.1 Hz),
7.87 (1H, s); 13C NMR (CD3OD, 125 MHz) δ 10.38, 22.64, 23.93,
27.11, 28.78, 102.27, 119.55, 120.10, 123.56, 130.34, 130.46, 136.07,
140.45, 140.80, 142.38, 153.02, 154.16, 160.32, 171.86; HRMS (ESI)
calcd for C22H23N4O2: 375.1816 [(M + H)+], found 375.1816.
4′-((1R,2S)-2-(2-Amino-1-methyl-6-oxo-1,6-dihydropyrimidin-4-
yl)cyclopropyl)biphenyl-3-carboxamide Hydrochloride (60). 60
(29.0 mg, 0.0731 mmol, 68%) was obtained as a colorless solid
from 28c (55.8 mg, 0.107 mmol) and 3-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)benzamide (39.7 mg, 0.161 mmol) by the same
2-Amino-6-((1S,2R)-2-(3′-(methoxymethyl)biphenyl-4-yl)-
cyclopropyl)-3-methylpyrimidin-4(3H)-one Hydrochloride (56). 56
(27.1 mg, 0.0681 mmol, 77%) was obtained as a colorless solid from
28c (46.2 mg, 0.0888 mmol) and 3-(methoxymethyl)phenylboronic
acid (22.1 mg, 0.133 mmol) by the same procedure used to prepare 36
20
1
(method B). mp 183−185 °C; [α]D −65.0° (c = 0.30, MeOH); H
NMR (CD3OD, 500 MHz) δ 1.59−1.65 (1H, m), 1.78−1.83 (1H, m),
2.32−2.38 (1H, m), 2.85−2.91 (1H, m), 3.26 (3H, s), 3.40 (3H, s),
4.50 (2H, s), 5.65 (1H, s), 7.28−7.31 (3H, m), 7.39 (1H, t, J = 7.8
Hz), 7.50 (1H, ddd, J = 7.8, 1.7, 1.3 Hz), 7.52−7.55 (3H, m); 13C
NMR (CD3OD, 125 MHz) δ 10.42, 22.71, 27.13, 28.73, 58.43, 75.62,
102.19, 127.19, 127.25, 127.87, 127.94, 129.99, 130.49, 135.99, 140.17,
140.90, 141.88, 153.40, 154.24, 160.44; HRMS (ESI) calcd for
C22H24N3O2: 362.1863 [(M + H)+], found 362.1866.
20
procedure used to prepare 36 (method B). mp 165−169 °C; [α]D
1
−61.7° (c = 0.30, MeOH); H NMR (CD3OD, 500 MHz) δ 1.60−
1.66 (1H, m), 1.79−1.84 (1H, m), 2.34−2.40 (1H, m), 2.87−2.93
(1H, m), 3.26 (3H, s), 5.66 (1H, s), 7.33 (2H, d, J = 8.2 Hz), 7.51
(1H, t, J = 7.8 Hz), 7.60 (2H, d, J = 8.2 Hz), 7.77 (1H, ddd, J = 7.8,
1.7, 1.1 Hz), 7.82 (1H, ddd, J = 7.8, 1.7, 1.1 Hz), 8.09 (1H, t, J = 1.7
Hz); 13C NMR (CD3OD, 125 MHz) δ 10.42, 22.65, 27.11, 28.77,
102.25, 127.13, 127.54, 127.96, 130.20, 130.61, 131.21, 135.55, 136.44,
140.12, 142.10, 153.01, 154.17, 160.32, 172.31; HRMS (ESI) calcd for
C21H20N4O2Na: 383.1479 [(M + Na)+], found 383.1481.
2-(4′-((1R,2S)-2-(2-Amino-1-methyl-6-oxo-1,6-dihydropyrimidin-
4-yl)cyclopropyl)biphenyl-3-yl)acetonitrile Hydrochloride (57). 57
(25.7 mg, 0.0654 mmol, 73%) was obtained as a colorless solid from
28c (46.7 mg, 0.0897 mmol) and 2-(3-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)phenyl)acetonitrile (32.7 mg, 0.135 mmol) by the
same procedure used to prepare 36 (method B). mp 184−186 °C;
Synthesis of Reference Compounds (Scheme 5). Starting
materials 2, 30, and 62 were prepared according to the literature or
patent.6b,c
20
1
[α]D −59.7° (c = 0.30, MeOH); H NMR (CD3OD, 500 MHz) δ
1.59−1.65 (1H, m), 1.78−1.83 (1H, m), 2.33−2.39 (1H, m), 2.85−
2.91 (1H, m), 3.26 (3H, s), 3.95 (2H, s), 5.65 (1H, s), 7.30−7.35 (3H,
m), 7.44 (1H, t, J = 7.7 Hz), 7.53−7.58 (4H, m); 13C NMR (CD3OD,
125 MHz) δ 10.44, 22.74, 23.56, 27.10, 28.74, 102.21, 127.33, 127.51,
127.91, 128.07, 130.57, 130.69, 133.08, 136.38, 140.34, 142.66, 153.42,
154.27, 160.47; HRMS (ESI) calcd for C22H21N4O: 357.1710 [(M +
H)+], found 357.1713.
2-Amino-6-(2-(biphenyl-3-yl)ethyl)-3-methylpyrimidin-4(3H)-one
(4).6b,c 4 (33.9 mg, 0.111 mmol, quant) was obtained as a colorless
solid from 63 (53.6 mg, 0.105 mmol) and phenylboronic acid (19.3
mg, 0.158 mmol) by the same procedure used to prepare 32 (method
A). Spectrum data were found to be identical to those in the literature.
2-Amino-6-(2-(3′-methoxybiphenyl-3-yl)ethyl)-3-methylpyrimi-
din-4(3H)-one (5).6b,c 5 (36.9 mg, 0.110 mmol, 100%) was obtained as
a colorless solid from 63 (56.0 mg, 0.110 mmol) and 3-
methoxyphenylboronic acid (25.1 mg, 0.165 mmol) by the same
procedure used to prepare 32 (method A). Spectrum data were found
to be identical to those in the literature.
2-Amino-6-(2-(biphenyl-4-yl)ethyl)-3-methylpyrimidin-4(3H)-one
(38).6c 38 (19.2 mg, 0.0629 mmol, 56%) was obtained as colorless
solid from 64 (57.0 mg, 0.112 mmol) and phenylboronic acid (20.5
mg, 0.168 mmol) by the same procedure used to prepare 36 (method
B). mp 213−216 °C; 1H NMR (CDCl3, 500 MHz) δ 2.70 (2H, dd, J =
9.2, 7.0 Hz), 2.97 (2H, dd, J = 9.2, 7.0 Hz), 3.41 (3H, s), 5.05 (2H, br
s), 5.84 (1H, s), 7.28 (2H, d, J = 8.2 Hz), 7.30−7.34 (1H, m), 7.40−
7.44 (2H, m), 7.51 (2H, d, J = 8.2 Hz), 7.55−7.59 (2H, m); 13C NMR
(CDCl3, 125 MHz) δ 27.59, 33.72, 38.93, 101.87, 126.99, 127.10,
127.17, 128.35, 128.74, 128.78, 139.97, 140.22, 140.95, 154.81, 162.68,
166.55; HRMS (ESI) calcd for C19H20N3O: 306.1601 [(M + H)+],
found 306.1606.
2-Amino-6-((1S,2R)-2-(3′-(2-hydroxyethyl)biphenyl-4-yl)-
cyclopropyl)-3-methylpyrimidin-4(3H)-one Hydrochloride (58). 58
(30.7 mg, 0.0771 mmol, 77%) was obtained as a colorless solid from
28c (52.4 mg, 0.101 mmol) and 3-(2-hydroxyethyl)phenylboronic acid
(25.1 mg, 0.151 mmol) by the same procedure used to prepare 36
20
1
(method B). mp 166−168 °C; [α]D −65.7° (c = 0.30, MeOH); H
NMR (CD3OD, 500 MHz) δ 1.59−1.64 (1H, m), 1.77−1.82 (1H, m),
2.32−2.37 (1H, m), 2.84−2.91 (1H, m), 2.86 (2H, t, J = 7.0 Hz), 3.26
(3H, s), 3.78 (2H, t, J = 7.0 Hz), 5.65 (1H, s), 7.19 (1H, d, J = 7.6
Hz), 7.29 (2H, d, J = 8.2 Hz), 7.33 (1H, t, J = 7.6 Hz), 7.40 (1H, d, J =
7.6 Hz), 7.43 (1H, s), 7.52 (2H, d, J = 8.2 Hz); 13C NMR (CD3OD,
125 MHz) δ 10.42, 22.74, 27.14, 28.72, 40.29, 64.21, 102.17, 125.71,
127.87, 128.57, 129.13, 129.92, 130.43, 135.80, 141.11, 141.20, 141.79,
153.53, 154.26, 160.48; HRMS (ESI) calcd for C22H23N3O2Na:
384.1681 [(M + Na)+], found 384.1683; Anal. Calcd for
C22H23N3O2·HCl·0.5H2O: C, 64.94; H, 6.19; N, 10.33; Cl 8.71.
Found: C, 64.98; H, 6.09; N, 10.43; Cl 8.48.
2-Amino-6-(2-(3′-(ethylthio)biphenyl-4-yl)ethyl)-3-methylpyrimi-
din-4(3H)-one (61). 61 (32.4 mg, 0.0886 mmol, 95%) was obtained as
colorless solid from 64 (47.7 mg, 0.0938 mmol) and 3-(methylthio)-
phenylboronic acid (25.6 mg, 0.141 mmol) by the same procedure
N-(4′-((1R,2S)-2-(2-Amino-1-methyl-6-oxo-1,6-dihydropyrimidin-
4-yl)cyclopropyl)biphenyl-3-yl)acetamide Hydrochloride (59). 59
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1
used to prepare 36 (method B). mp 130−132 °C; H NMR (CDCl3,
500 MHz) δ 1.34 (3H, t, J = 7.3 Hz), 2.68−2.72 (2H, m), 2.95−3.00
(2H, m), 2.99 (2H, d, J = 7.3 Hz), 3.41 (3H, s), 5.11 (2H, br s), 5.83
(1H, s), 7.27 (2H, d, J = 8.3 Hz), 7.28 (1H, dt, J = 7.5, 1.7 Hz), 7.34
(1H, t, J = 7.5 Hz), 7.37 (1H, dt, J = 7.5, 1.7 Hz), 7.49 (2H, d, J = 8.3
Hz), 7.50 (1H, t, J = 1.7 Hz); 13C NMR (CDCl3, 125 MHz) δ 14.42,
27.61, 27.69, 33.68, 38.80, 101.76, 124.57, 127.17, 127.53, 127.58,
128.81, 129.17, 137.14, 138.55, 140.47, 141.63, 154.88, 162.68, 166.37;
HRMS (ESI) calcd for C21H24N3OS: 366.1635 [(M + H)+], found
366.1640; Anal. Calcd for C21H23N3OS·0.2H2O: C, 68.34; H, 6.39; N,
11.38; S, 8.69. Found: C, 68.59; H, 6.21; N, 11.61; S, 8.31.
amino-n-capronic acid, Eu = europium cryptate) and 1 μL of
recombinant human BACE1 protein (R & D systems) for 3 h at 30
°C in the 96 well half area plate (black color plate, Costar). The
substrate peptide was synthesized by reacting with Biotin-XSEVNL-
DAEFREDSGC (Peptide Institute) and cryptate TBPCOOH mono
SMP (CIS bio international). The final concentrations of the substrate
peptide and recombinant human BACE1 protein were 18 nM and 7.4
nM, respectively. The enzymatic reaction was performed in sodium
acetate buffer (50 mM sodium acetate (pH 5.0), 0.008% Triton X-
100). After the reaction, a 50 μL of 8.0 μg/mL Streptavidin-XL665
(CIS bio international) dissolved in phosphate buffer (160 mM
K2HPO4−H2PO4 (pH 7.0), 0.008% TritonX-100, 0.8 M KF) was
added to each well and incubated for l h at 30 °C. Then, the
fluorescence intensity (excitation wavelength 320 nm, emission
wavelength 620 and 665 nm) in each well was measured using a
Wallac 1420 multilabel counter (Perkin-Elmer life sciences). The
enzymatic activity was calculated by each fluorescence intensity ratio
([ratio of fluorescence at 665 nm to that at 620 nm] × 10,000).
Crystallography. Crystallization of apo human BACE1 was
performed by the sitting-drop vapor diffusion method at 20 °C. ∼3
mg/mL of human BACE1 prepared as above was mixed with an equal
volume of mother liquor containing 120 mM sodium citrate pH 6.5,
200 mM ammonium iodide, and 28−30% w/v polyethylene glycol
5000 monomethyl ether (PEG 5000 MME). In order to prepare 6-
bound crystals, apo crystals were soaked in a 50 mM solution of 6,
10% (v/v) dimethyl sulfoxide (DMSO), 90 mM sodium citrate pH
6.6, 180 mM ammonium iodide, and 25.2% (w/v) PEG 5000 MME
for 20 min. For 33, apo crystals were soaked in 10 mM 33, 10% (v/v)
DMSO, 90 mM sodium citrate pH 6.6, 180 mM ammonium iodide,
and 25.2% (w/v) PEG 5000 MME for 20 min. For 36, apo crystals
were soaked in 10 mM 36, 10% (v/v) DMSO, 90 mM sodium citrate
pH 5.0, and 25.2% (w/v) PEG 5000 MME for 15 min. Inhibitor
soaked crystals were transferred into cryoprotectant (0.1 M sodium
citrate pH 6.6, 0.2 M ammonium iodide, 28% w/v PEG 5000 MME,
20% v/v glycerol or 0.1 M sodium citrate pH 5.0, 28% w/v PEG5000
MME, 20% v/v glycerol) for a few seconds and were flash-frozen prior
to data collection.
Diffraction data were collected at 100 K using a MicroMax-007 HF
X-ray generator (Rigaku). Data reduction was performed with
HKL2000,28 iMosflm,29 and SCALA.30 Structures were solved by
molecular replacement using apo BACE1 structure (PDB ID: 1W50)
as a search model. Model rebuilding and structure refinement were
performed with COOT31 and Refmac5,30 respectively. The structure
factors and coordinates of 6, 33, and 36 are available in accession
numbers 3VV6, 3VV7, and 3VV8, respectively.
Molecular Modeling. Figure 1: Conformations of compounds 6,
ent-6, 7, and ent-7 were energy-minimized on Molecular Operating
Environment (MOE), 2011.10,15 with default settings using the
MMFF94x force field16 and three minimization algorithms (steepest
descent method, conjugate gradient method, truncated Newton
method) until the root-mean-square gradient falls less than 0.05.
The resulting structures were superimposed with that of 3 in PDB
code 2VA5 on an aminopyrimidone ring.
2-Bis(tert-butoxycarbonyl)amino-6-(3-bromophenethyl)-3-meth-
ylpyrimidin-4(3H)-one (63). 63 (275 mg, 0.541 mmol, 72%) was
obtained as a colorless solid from 30 (233 mg, 0.756 mmol) by the
1
same procedure used to prepare 25a. H NMR (CDCl3, 600 MHz) δ
1.46 (9H, s), 2.80 (2H, dd, J = 9.0, 6.6 Hz), 2.93 (2H, dd, J = 9.0, 6.6
Hz), 3.42 (3H, s), 6.21 (1H, s), 7.10 (1H, d, J = 7.6 Hz), 7.14 (1H, t, J
= 7.6 Hz), 7.33 (1H, d, J = 7.6 Hz), 7.35 (1H, s); 13C NMR (CDCl3,
125 MHz) δ 27.90, 30.08, 33.48, 38.17, 84.96, 111.40, 122.57, 127.00,
129.44, 130.05, 131.42, 142.80, 148.46, 149.02, 162.85, 164.58; HRMS
(ESI) calcd for C23H31BrN3O5: 508.1442 [(M + H)+], found
508.1443.
2-Bis(tert-butoxycarbonyl)amino-6-(4-bromophenethyl)-3-meth-
ylpyrimidin-4(3H)-one (64). 64 (261 mg, 0.514 mmol, 92%) was
obtained as a colorless solid from 62 (172 mg, 0.558 mmol) by the
1
same procedure used to prepare 25a. H NMR (CDCl3, 500 MHz) δ
1.47 (9H, s), 2.79 (2H, dd, J = 8.6, 7.4 Hz), 2.92 (2H, dd, J = 8.6, 7.4
Hz), 3.42 (3H, s), 6.20 (1H, s), 7.05 (2H, d, J = 8.4 Hz), 7.38 (2H, d, J
= 8.4 Hz); 13C NMR (CDCl3, 125 MHz) δ 27.89, 30.77, 33.26, 38.26,
84.95, 111.46, 120.06, 130.14, 131.56, 139.40, 148.48, 148.98, 162.83,
164.63; HRMS (ESI) calcd for C23H31BrN3O5: 508.1442 [(M + H)+],
found 508.1453.
Protein Expression and Purification. A cDNA fragment
encoding human BACE1 (hBACE1) residues 43−454 was cloned
into pET28a and expressed in E. coli BL21(DE3) cells. The hBACE1
protein was expressed as insoluble inclusion bodies and was refolded
by a modified method using iFOLD protein Refolding System 3
(Novagen). The refolded protein was applied on a Q-Sepharose FF
column. The fractions with BACE catalytic activity were collected and
applied to a His Trap HP column followed by a HiLoad Q Sepharose
HP column and Superdex 200 column for further purification. After
thrombin digestion, a HisTrap Benzamine FF column was used for
removal of thrombin. The flow through fraction was loaded onto a His
trap column, and the purified hBACE1 protein was >98% purity on
SDS-PAGE. The buffer was exchanged into 20 mM Tris-HCl (pH
8.0), 150 mM NaCl, 2 mM DTT.
ELISA (High-Sensitive Enzyme Assay). In vitro high-sensitive
BACE1 activity assays were performed using substrate peptides from
the American Peptide Company, Inc. (Sunnyvale, CA) and human
BACE1 prepared as above. The substrate peptide sequence was
SEVNLDAEFRHDSGYEK-biotin. Peptides and inhibitors were
dissolved in dimethyl sulfoxide (DMSO), and dissolved peptides
were stored at −20 °C. The substrate peptide was captured on a
streptavidin 96-well-format plate (Nunc) at 25 nM (100 μL scale
reaction). The reaction buffer was 50 mM sodium acetate, pH 4.5,
containing 0.25 mg/mL bovine serum albumin (BSA). The reactions
were started by adding 70 μL of reaction buffer, 10 μL of inhibitor
solution or DMSO, and 20 μL of BACE1 (final 0.45 nM) in each well.
After 3 h of incubation at 25 °C, reaction mixtures except cleaved and
uncleaved substrates were washed out with TBST (Tris-buffered saline
containing 0.1% of Tween-20). The cleaved product on plates was
detected with horseradish peroxidase (HRP)-conjugated 82E1, which
is a monoclonal antibody specific for the N-terminal end generated by
BACE1 cleavage (82E1; IBL Co., Ltd., Gunma, Japan), like the general
ELISA assay. The quantifications of HRP activity were carried out by a
colorimetric method using TMB substrate (Thermo Scientific, Inc.).
Homogeneous Time-Resolved Fluorescence (HTRF) Assay.
0.5 μL of the test compounds (dissolved in dimethyl sulfoxide) was
incubated with 48.5 μL of the fluorescence-quenching peptide
substrate solution (Biotin-XSEVNLDAEFRHDSGC-Eu: X = ε-
Figure 5: The crystal structure of a complex between BACE1 and 6
(PDB code: 3VV6) was used for a molecular modeling with MOE. A
molecular structure of compound 32 was constructed by editing the
molecule of 6 extracted from the complex. Energy-minimization of 32
was performed under the constraint of fixing the positions of atoms in
aminopyrimidone, the cyclopropane ring, and the middle benzene ring
with default settings as was done in Figure 1. Then, the ligand 6
complexed with the BACE1 protein was replaced with the energy-
minimized structure of 32.
All calculations were performed on Panasonic CF-V8.
ASSOCIATED CONTENT
■
S
* Supporting Information
LCMS (ESI-LRMS) data and plausible reaction mechanism for
the production of ent-28 and 29. This material is available free
of charge via the Internet at http://pubs.acs.org.
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protease memapsin 2 cleaves the β-secretase site of β-amyloid
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(3) (a) Luo, Y.; Bolon, B.; Kahn, S.; Bennett, B. D.; Babu-Khan, S.;
Denis, P.; Fan, W.; Kha, H.; Zhang, J.; Gong, Y.; Martin, L.; Louis, J.-
C.; Yan, Q.; Richards, W. G.; Citron, M.; Vassar, R. Mice deficient in
BACE1, the Alzheimer’s β-secretase, have normal phenotype and
abolished β-amyloid generation. Nature Neurosci. 2001, 4, 231−232.
(b) Cai, H.; Wang, Y.; McCarthy, D.; Wen, H.; Borchelt, D. R.; Price,
D. L.; Wong, P. C. BACE1 is the major β-secretase for generation of
Aβ peptides by neurons. Nature Neurosci. 2001, 4, 233−234.
Accession Codes
3VV6, 3VV7, 3VV8.
AUTHOR INFORMATION
Corresponding Author
■
*S.Y.: E-mail address: shuji.yonezawa@shionogi.co.jp. Tele-
phone number: +81-11-700-4702. Fax: +81-11-700-4714. S.S.:
E-mail address: shu@pharm.hokudai.ac.jp. Telephone & fax
number: +81-11-706-3769.
Notes
(4) For recent reviews, see: (a) Albert, J. S. Progress in the
development of β-secretase Inhibitors for Alzheimer’s disease. Prog.
Med. Chem. 2009, 48, 133−161. (b) Hamada, Y.; Kiso, Y. Recent
progress in the drug discovery of non-peptidic BACE1 inhibitors.
Expert Opin. Drug. Discovery 2009, 4, 391−416. (c) Iserloh, U.;
Cumming, J. N. Peptidomimetic BACE1 inhibitors for treatment of
Alzheimer’s disease: design and evolution. Methods Princ. Med. Chem.
2010, 45 (AsparticAcid Proteases as Therapeutic Targets), 441−479.
(d) Cole, D. C.; Bursavich, M. G. Nonpeptide BACE1 inhibitors:
design and synthesis. Methods Princ. Med. Chem. 2010, 45 (Aspartic
Acid Proteases as Therapeutic Targets), 481−509. (e) Ghosh, A. K.;
Brindisi, M.; Tang, J. Developing β-secretase inhibitors for treatment
of Alzheimer’s disease. J. Neurochem. 2012, 120 (Suppl. 1), 71−83.
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B. M.; Stout, S. L.; Timm, D. E.; LaBell, E. S.; Gonzales, C. R.;
Nakano, M.; Jhee, S. S.; Yen, M.; Ereshefsky, L.; Lindstrom, T. D.;
Calligaro, D. O.; Cocke, P. J.; Hall, D. G.; Friedrich, S.; Citron, M.;
Audia, J. E. Robust central reduction of amyloid-β in humans with an
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Mr. Rikio Ikenishi (Shionogi Techno Advance
Research Co., Ltd.) for elemental analysis, Ms. Izumi Fujisaka
(Shionogi Techno Advance Research Co., Ltd.) for measuring
the optical rotation, Ms. Naomi Umesako (Shionogi & Co.,
Ltd.), and the members of the Instrumental Analysis Division,
Equipment Management Center, Creative Research Institution,
Hokkaido University for the high-resolution mass spectroscopy
analysis. This investigation was supported by a Grant-in-Aid for
Scientific Research (21390028) from the Japan Society for the
Promotion of Science.
ABBREVIATIONS USED
■
AVN, 2,2′-azobisvaleronitrile; BSA, N,O-bistrimethylsilylaceta-
mide; Bpin, 4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl; CDI,
carbonyl diimidazole; HRP, horseradish peroxidase; HTRF,
homogeneous time-resolved fluorescence; LRMS, low-resolu-
tion mass spectrometry; MMFF94x, Merck molecular force
field 94x; MME, monomethyl ether; MOE, Molecular
Operating Environment; MS4A, molecular sieves 4A; TBDPS,
tert-butyldiphenylsilyl; TBST, tris-buffered saline containing
0.1% of Tween-20; TPAP, tetrapropylammonium perruthenate;
sAPPβ, soluble amyloid precursor protein β
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Edwards, P. D.; de Beer, T.; Folmer, R. H. A. Discovery of a novel
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Med. Chem. 2007, 50, 5903−5911. (b) Edwards, P. D.; Albert, J. S.;
Sylvester, M.; Aharony, D.; Andisik, D.; Callaghan, O.; Campbell, J. B.;
Carr, R. A.; Chessari, G.; Congreve, M.; Frederickson, M.; Folmer, R.
H. A.; Geschwindner, S.; Koether, G.; Kolmodin, K.; Krumrine, J.;
Mauger, R. C.; Murray, C. W.; Olsson, L.-L.; Patel, S.; Spear, N.; Tian,
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Edwards, P.; Empfield, J. R.; Frederickson, M.; Koether, G. M.;
Krumrine, J.; Mauger, R.; Murray, C. W.; Patel, S.; Sylvester, M.;
Throner, S. Preparation of substituted 2-aminopyrimidin-4-ones for
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