Conformationally restricted analog and biotin-labeled probe based on beauveriolide III

Article abstract of DOI:10.1016/j.bmcl.2011.10.045

A conformationally restricted oxazoline analog 7 was designed on the basis of a SAR study of beauveriolide III (2) and its analogs reported previously. Conformational analysis by molecular mechanics calculation suggested that the three side chains of 7 mo

Full text of DOI:10.1016/j.bmcl.2011.10.045

Bioorganic & Medicinal Chemistry Letters 22 (2012) 696–699
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Conformationally restricted analog and biotin-labeled probe based
on beauveriolide III
Takayuki Doi a, , Terushige Muraoka b, Taichi Ohshiro c, Daisuke Matsuda d, Masahito Yoshida a,
⇑
b
e
d,
⇑
¯
Takashi Takahashi , Satoshi Omura , Hiroshi Tomoda
a Tohoku University, Graduate School of Pharmaceutical Sciences, 6-3 Aza-aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan
b Tokyo Institute of Technology, Department of Applied Chemistry, 2-12-1 Ookayama, Meguro, Tokyo 152-8552, Japan
c Jichi Medical University, Shimotsuke, Tochigi, Japan
d Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
e Kitasato Institute for Life Sciences and Graduate School of Infection Control Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A conformationally restricted oxazoline analog 7 was designed on the basis of a SAR study of beauverio-
lide III (2) and its analogs reported previously. Conformational analysis by molecular mechanics calcula-
tion suggested that the three side chains of 7 mostly occupy the same spaces as those of 2. The analog 7
Received 20 September 2011
Revised 12 October 2011
Accepted 13 October 2011
Available online 21 October 2011
was synthesized by peptide coupling of the D-cyclohexylglycine-containing ester 11 and D-Ser-containing
dipeptide 12, macrolactamization, and cyclodehydration of 6 for the construction of an oxazoline ring.
The bicyclic 7 exhibited potential inhibitory activity for cholesteryl ester synthesis similar to that by 2.
These results revealed biologically important 3D spaces of the three side chains in inhibitory activity
for cholesteryl ester synthesis. In addition, we accomplished the synthesis of a biotin-labeled probe 8
by copper-catalyzed (3+2) cycloaddition of a biotin-containing alkyne 16 and azido-containing beauve-
riolide analog 15 prepared from 6.
Keywords:
Beauveriolide
ACAT inhibitor
Cyclic depsipeptide
Oxazoline
Ó 2011 Elsevier Ltd. All rights reserved.
Molecular probe
Beauveriolides I (1) and III (2), isolated from Beauveria sp.
FO-6979, are selective inhibitors against acyl–CoA: cholesterol acyl-
transferase (ACAT), which catalyzes cholesteryl ester (CE) synthesis.
These beauveriolides inhibit CE synthesis of lipid-droplet accumula-
analogs of beauveriolides is highly attractive in drug discovery be-
cause beauveriolides I and III have proven orally active in athero-
sclerogenic mouse models, reducing atherogenic lesions of the
artery and heart in apolipoprotein E knockout mice and low-density
tion in macrophages at half maximal inhibitory concentration (IC50
)
of 0.78 and 0.41 l
M, respectively (Fig. 1).1,2 Exploration of potent
O
O
O
O
O
O
NH
4
NH
HN
O
HN
O
N
O
O
O
O
O
O
3
N
H
O
O
R
H
NH
NH
OH
HN
O
HN
O
7
6
N
N
O
O
H
H
R
beauveriolide I (1)
IC50, 0.78 μM
R =
R =
3 (R = β-Me)
4 (R = α-Me)
5 (R = β-CH2OH) IC50, 0.11 μM
IC50, 0.040 μM
IC50, 0.020 μM
O
O
O
NH
beauveriolide III (2)
IC50, 0.41 μM
HN
N
O
N
O
H
Figure 1. Structures and CE synthesis inhibitory activity of beauveriolides I and III
as well as their analogs.
O
N
N
O
O
NH
H
S
HN
N
N
H
H
8
H
⇑
Corresponding authors. Fax: +81 22 795 6864.
E-mail addresses: doi_taka@mail.pharm.tohoku.ac.jp (T. Doi), tomodah@pharm.
kitasato-u.ac.jp (H. Tomoda).
Figure 2. Synthetic key intermediate 6, conformationally restricted oxazoline
analog 7, and biotin-labeled probe 8.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.10.045

T. Doi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 696–699
697
Figure 3. Conformational analysis of 2 and 7. (a) Superimposed 100 conformers from the global minimum of 2 in water. (b) Superimposed 100 conformers from the global
minimum of 7 in water. (c) Global minimum of 2 in water. (d) Global minimum of 7 in water. (e) Superimposed global minima of 2 (orange) and 7 (cyan).
lipoprotein receptor knockout mice.1c We have already reported the
combinatorial synthesis of various analogs of beauveriolides to elu-
cidate structure–activity relationships and to discover potent ana-
logs such as 3, which possesses a diphenylalanine instead of a
phenylalanine moiety.3 This compound exhibits a CE synthesis
inhibitory activity 10-fold greater than that exhibited by the natural
products.3a It was also found that the 1-methylpentyl group and
sec-butyl group are important for biological activity, whereas mod-
ification of the methyl group in the Ala has a minor influence on the
labeling group can be incorporated via the hydroxy group of the
Ser moiety without losing potency. We report here the synthesis
of a new template compound 6, a conformationally restricted
oxazoline analog 7, and a biotin-labeled probe 8, prepared by
copper(I)-catalyzed (3+2) cycloaddition of an azido analog with an
alkyne-containing biotin tag (Fig. 2).
We initially performed conformational searches of 2 and the
oxazoline analog 7 using MacroModel 9.7 with 10,000-step mixed
torsional/low-mode sampling with an OPLS2005 force field and a
generalized Born/solvent-accessible surface area (GB/SA) solvent
model.4,5 The calculations were conducted both in vacuo and in
water. In both cases, similar conformations were obtained. Several
ring conformations were observed in 2 (Fig. 3a), whereas two dom-
inant ring conformations were observed in 7 (Fig. 3b). Note that a
activity.3d For example,
solubility in assay media and an activity similar to that by
logs. An -Ser analog 5 inhibited CE synthesis in macrophages at an
IC50 value of 0.11 M although its activity decreased slightly com-
pared with 3. Thus, we expected that a fluorescence- or biotin-
D-Ala analogs such as 4 exhibited improved
L-Ala ana-
L
l

698
T. Doi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 696–699
0.31
l
M) similar to that by 5 (0.11
l
M), possessing
D-allo-IIe and L-
1) DIC, cat. DMAP
CH2Cl2, 0 ºC (91%)
OH
O
Ser.9 Therefore, the sec-butyl group in
D
-allo-IIe was replaced by a
O
O
HO
+
CO2H
CO2Bn 2) H2, Pd/C,
EtOH (98%)
cyclohexyl group without loss of activity. More interestingly, the
oxazoline analog 7 retained a high level of inhibitory activity, with
an IC50 value of 0.28
NHBoc
NHBoc
9
10
lM compared with that of the parent natural
11
product 2 (0.41 M). On the basis of the conformational analysis
l
described above, it is concluded that 7 must have a 3D structure
similar to that of 2 to be active for inhibition of CE synthesis.
Next, we converted 6 to the azido-containing analog 15. Tosyla-
tion of 6 with TsCl and Et3N in the presence of Me3NÁHCl10 pro-
vided the tosylate 14 in 69% yield (Scheme 2). Nucleophilic
substitution of the tosyl group with NaN3 afforded azide 15 in
90% yield. In general, elimination of the leaving group is often ob-
served in Ser derivatives.11 The rigidity of the macrocyclic ring of
14 probably prevented the elimination of the tosyl group. As a re-
sult, azido displacement occurred in high yield. Finally, copper(I)-
catalyzed (3+2) cycloaddition of azide 15 and biotin-containing
terminal alkyne 16 was achieved using CuSO4 and sodium ascor-
bate in t-BuOH–H2O (1:1) at 50 °C to furnish the biotin-labeled
probe 8 in 78% yield.12,13 The compound 8 exhibited inhibitory
activity against acyl–CoA: cholesterol acyltransferase1 (ACAT1)
O
O
O
NHBoc
Ph
1) 4 M HCl, dioxane
NH
BnO2C
2) 11, EDCI, HOBt, DIEA,
CH2Cl2–DMF
N
H
Ph
Ph
Ph
NHBoc
BnO2C
O
N
H
OH
O
90% yield from 11
12
OH
13
1) H2, Pd/C, EtOH (94%)
2) 4 M HCl, dioxane
DAST, CH2Cl2,
–78 ºC
7
6
3) HATU, HOAt, DIEA
CH2Cl2
(62% in 2 steps)
89%
Scheme 1. Synthesis of 6 and bicyclo oxazoline analog 7.
with an IC50 value of 0.8
not inhibit CE synthesis in the cell-based assay below a concentra-
tion of 10 M because of low cell membrane permeability.
lM in the enzyme assay, although it did
1) TsCl, Et3N,
Me3N•HCl,
CH2Cl2 (69%)
O
O
O
l
NH
In summary, we have demonstrated the design, synthesis, and
biological investigation of a conformationally restricted oxazoline
analog 7, which was found to be potentially active for inhibiting
CE synthesis. A conformational analysis suggested that 7 has a
3D structure similar to that of beauveriolide III (2). These results
indicate that information about the biologically important 3D
spaces of the three side chains would be useful in the design of
non-peptidic mimetics for drug discovery. We have also accom-
plished the synthesis of a biotin-labeled beauveriolide derivative.
Further studies using the biotin-labeled molecular probe are in
progress in our laboratories.
6
2) NaN3, DMF
(90%)
HN
O
N
O
H
X
14 (X = Ts)
15 (X = N3)
O
O
O
NH
H
S
HN
N
H
N
H
H
16
8
CuSO4, Na ascorbate,
t-BuOH–H2O (1:1)
(78%)
Acknowledgements
50 ºC
Scheme 2. Synthesis of a biotin-labeled probe 8.
This study was supported by a Grant-in-Aid (No. 23310145)
from MEXT and The Novartis Foundation (Japan) for the Promotion
of Science.
bicyclic system including the oxazoline ring restricted the confor-
mations of the macrocyclic ring. Interestingly, the three side chains
of 7 (cyclohexyl, 1-methylpentyl, and diphenylmethyl groups)
mostly occupied the same spaces as those of 2 (sec-butyl, 1-meth-
ylpentyl, and phenylmethyl groups; Fig. 3a and b). Therefore, the
synthesis and biological investigation of the oxazoline analog 7
can give considerable insight about structure–activity relation-
ships in the design of inhibitors of CE synthesis.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.10.045.
The synthesis of the bicyclic oxazoline analog 7 is illustrated in
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