Synthesis and biological evaluation of a focused library of beauveriolides

Article abstract of DOI:10.1016/j.bmcl.2008.06.054

Fungal beauveriolide III (1b), discovered as an inhibitor of lipid droplet accumulation in mouse macrophages and showing antiatherogenic activity in mouse model, consists of l-Phe, l-Ala, d-allo-Ile, and (3S, 4S)-3-hydroxy-4-methyloctanoic acid moieties. A combinatorial library of beauveriolide analogues focusing on l-Ala and d-allo-Ile of 1b was synthesized by combinatorial synthesis. Among them, d-Ala analogues consisting of A{2} improved their solubility, while those with 7{1, 3, 2},7{2, 3, 1}, and 7{2, 3, 2} were 20 times more potent than 1b.

Full text of DOI:10.1016/j.bmcl.2008.06.054

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4397–4400
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of a focused library of beauveriolides
Kenichiro Nagai a, Takayuki Doi b, Taichi Ohshiro c, Toshiaki Sunazuka a, Hiroshi Tomoda c,
b
a,
*
¯
Takashi Takahashi , Satoshi Omura
a Kitasato Institute for Life Sciences and Graduate School of Infection Control Sciences, Kitasato University and The Kitasato Institute, 5-9-1 Shirokane, Minato-ku,
Tokyo 108-8641, Japan
b Department of Applied Chemistry, Tokyo Institute of Technology, 2-12-1 Ookayama, Meguro, Tokyo 152-8552, Japan
c School of Pharmacy, 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:
Fungal beauveriolide III (1b), discovered as an inhibitor of lipid droplet accumulation in mouse macro-
Received 15 May 2008
Revised 16 June 2008
Accepted 17 June 2008
Available online 20 June 2008
phages and showing antiatherogenic activity in mouse model, consists of
(3S, 4S)-3-hydroxy-4-methyloctanoic acid moieties. A combinatorial library of beauveriolide analogues
focusing on -Ala and -allo-Ile of 1b was synthesized by combinatorial synthesis. Among them, -Ala
analogues consisting of A{2} improved their solubility, while those with 7{1,3,2},7{2,3,1}, and 7{2,3,2}
were 20 times more potent than 1b.
L-Phe, L-Ala, D-allo-Ile, and
L
D
D
Keywords:
Ó 2008 Elsevier Ltd. All rights reserved.
Cyclic depsipeptide
Focused library
Antiatherogenic activity
ACAT
Lipid droplet accumulation in macrophages is a critical stage for
plaque formation, which limits blood flow and rupture of blood
vessels, ultimately leading to the development of atherosclerosis
in the arterial wall. Thus, inhibitors for lipid droplet accumulation
in macrophages would be useful for the treatment of atherosclero-
sis.1 Beauveriolides are a family of cyclic depsipeptides isolated
from a culture broth of Beauveria sp. FO-6979 during the course
of our screening program for inhibitors of lipid droplet accumula-
tion in mouse macrophages. In particular, beauveriolides I (1a) and
III (1b) were found to dose-dependently reduce the number and
size of lipid droplets in mouse macrophages without cytotoxicity,
and to inhibit cholesteryl ester (CE) synthesis with IC50 values of
4
3
R
O
O
O
Beauveriolide I (1a)
Beauveriolide III (1b)
R
NH
HN
O
N
O
H
Figure 1. Structures of beauveriolides I and III.
tionships.5a In the 3-hydroxy-4-methyloctanoic acid moiety, the
importance of stereochemistry of the 3-hydroxyl and 4-methyl
groups has been investigated. The configuration of the 3S hydroxyl
group is essential for the activity because isomers with an inverted
hydroxyl group at C-3 were inactive, while the stereochemistry of
the methyl group at C-4 did not affect the inhibition of CE synthe-
sis.5b Naturally occurring beauveriolides IV, V, and VI in which
0.78 and 0.41 l
M, respectively (Fig. 1).2a,b,3 In addition, beauverio-
lides decreased atherogenic lesions in the aorta and heart when
administrated orally to apoE and LDL receptor knockout mice. This
mode of action has been examined indicating that 1a and 1b inhi-
bit CE synthesis by blocking acyl-CoA:cholesterol acyltransferase
(ACAT) activity to suppress foam cell formation.2c
Synthetic ACAT inhibitors including amides, ureas, and imida-
zoles do not have structural similarity to beauveriolides, which
are expected to be promising lead compounds for the treatment
of atherosclerosis.4 We established a method for combinatorial
synthesis of beauveriolide analogues using a 2-chlorotrityl chloride
linker and evaluated their inhibitory activity against CE synthesis
in mouse macrophages to elucidate partial structure–activity rela-
L
-Phe is replaced with
L
-Val show remarkably decreased activity.2d
With regard to the -Phe moiety, p- and m-chloro substituents on
L
the phenyl ring enhanced the activity. Furthermore, the diphenyl
alanine analogue was 10-fold more potent than 1b.5 These results
indicate that the phenyl group can be replaced by aromatic rings to
increase the activity. To date, the effects of substituents on
and -Leu parts in 1a (or -allo-Ile in 1b) are unclear. Herein, we
report on the synthesis of beauveriolide analogues focusing on
the -Ala and -Leu (or -allo-Ile) parts and evaluation of their
ACAT inhibitory activity in cell- and enzyme-based assays.
L-Ala
D
D
L
D
D
* Corresponding author. Fax: +81 3 3444 8360.
¯
E-mail address: omura-s@kitasato.or.jp (S. Omura).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.054

4398
K. Nagai et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4397–4400
Building block A{1-4
}
Building block B{1-3
}
Building block C{1-4}
FmocNH
CO2H
FmocNH
CO2H
O
O
O
1
FmocNH
CO2H
2
CO2H
CO2H
O
O
1
1
2
O
H2N
BocHN
BocHN
BocHN
BocHN
2
HO
FmocNH
CO2H
FmocNH
CO2H
FmocNH
FmocNH
CO2H
CO2H
3
4
O
CO2H
CO2H
O
4
O
3
3
Figure 2. Building blocks A, B, and C for a combinatorial library of beauveriolides.
To examine the effect of the
L
-Ala and
D
-Leu parts, we designed
was loaded onto 2-chlorotrityl chloride resin. Next, deprotection
of the Fmoc group in 3, followed by coupling with building block
B afforded dipeptide 4. After subsequent deprotection, depsipep-
tide 5 was obtained by coupling of the resulting amine and build-
ing block C using PyBrop. Treatment of 5 with 4 M HCl in 1,4-
dioxane cleaved the C–O bond between the carboxylic acid and lin-
ker concomitantly with the removal of the Boc group to release
depsipeptide 6, which was subjected to cyclization with EDCI
and i-Pr2NEt under high dilution conditions to provide cyclic dep-
sipeptide 7. Each compound was identified by LCMS analysis and
purified by preparative reversed-phase HPLC.
a 48-member library of beauveriolides consisting of building
blocks A{1–4}, B{1–3}, and C{1–4} as shown in Figure 2. The library
includes 1a and 1b as standards. Building block C{1–4} was pre-
pared in good yield by condensation of a known benzyl ester 2
and Boc-D-amino acids under DCC-DMAP conditions followed by
the removal of the benzyl group (Scheme 1).6
Cyclic products 7 were prepared in the same way as the first
generation beauveriolide library (Scheme 2). Building block A
We tested the effect of beauveriolide analogue 7 on ACAT activ-
ity by measuring the CE synthesis of lipid droplets in a cell-based
assay using mouse macrophages and in an enzyme assay using
mouse liver microsomes.2 These results are summarized in Table
1. Inhibition of ACAT activity in the macrophage assay was in good
agreement with that in the enzyme assay. In the building block A,
O
O
OH
O
a,b
R
O
OH
OBn
BocHN
D-Ala analogues consisting of A{2} showed a similar activity com-
2
pared to -Ala analogues consisting of A{1} and improved their sol-
L
C1 (R = (1S)-1-methylpropyl, 85%)
C2 (R = (1R)-1-methylpropyl, 90%)
C3 (R = 2-methylpropyl, 89%)
C4 (R = benzyl, 92%)
ubility in assay media. Cyclic compounds from A{3} and A{4} tend
to decrease the inhibitory activity. However, 7{3,2,4}, 7{3,3,1},
7{3,3,2}, and 7{3,3,4} inhibited CE synthesis in macrophage at
IC50 values of 0.11–0.16 lM. These data indicate that fluorescent
or biotin labeled groups could be introduced via the hydroxyl
Scheme 1. Synthesis of building block C. Reagents and conditions: (a) Boc-D-amino
acid-OH, DCC, DMAP, CH2Cl2, 0 °C to rt, 22 h; (b) 10% Pd/C, H2, EtOH, rt, 12 h.
FmocHN
R2
Cl
Cl
=
Cl
b,c
b,d
a
NHFmoc
NH
O
O
O
R1
R1
O
O
3
4
2-Chlorotrityl Cl resin
O
O
O
O
O
O
O
O
O
R3
R3
f
R3
HCl·H2N
HO2C
e
NH
R2
NH
R2
NH
HN
R2
BocHN
N
N
NH
O
O
O
O
O
O
H
H
R1
R1
R1
5
6
7
Scheme 2. Synthesis of beauveriolide analogues. Reagents and conditions: (a) Block A{1–4}, i-Pr2NEt, CH2Cl2, rt, 2 h; (b) 20% piperidine/DMF, rt, 1 h; (c) Block B{1–3}, DIPCI,
HOBt, CH2Cl2/DMF (4:1), rt, 2 h; (d) Block C{1–4}, PyBrop, i-Pr2NEt, CH2Cl2/DMF (4:1), rt, 1.5 h; (e) 4 M HCl in 1,4-dioxane, rt, 2 h; (f) EDCIÁHCl, i-Pr2NEt, CH2Cl2, rt, 2 h.

K. Nagai et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4397–4400
4399
Table 1
A combinatorial library of beauveriolide analogues 7
7{A,B,C}
Yield
(%)a
Enzyme assayb % of
Cell-based assayc Rt
IC50 M)
(min)d (m/z)e
MS
7{A,B,C} Yield
Enzyme assayb % of
Cell-based assayc Rt
IC50 M)
(min)d (m/z)e
MS
inhibition at 20
l
M
(l
(%)a
inhibition at 20
l
M
(l
7{1,1,1}f 13
7{1,1,2}
7{1,1,3}g 24
91
80
95
76
20
70
87
47
95
88
83
71
94
96
98
94
99
91
96
60
91
97
93
75
0.4
1.7
0.6
7.5
8.19
8.22
8.29
8.21
8.40
8.42
8.47
8.37
8.78
8.80
8.85
8.75
8.13
8.15
8.22
8.22
7.59
7.61
7.69
7.63
8.74
8.77
8.83
8.74
488
488
488
522
488
488
488
522
564
564
564
598
488
488
488
522
488
488
488
522
564
564
564
598
7{3,1,1} 10
7{3,1,2} 10
78
99
93
79
12
64
40
95
98
99
70
96
64
52
43
11
10
27
4
12.0
7.6
12.3
9.3
>20
>20
>20
0.11
0.11
0.11
>20
0.16
>20
>20
>20
>20
>20
>20
>20
>20
7.67
7.69
7.75
7.71
7.79
7.80
7.83
7.83
8.28
8.35
8.40
8.34
7.28
7.32
7.39
7.39
7.43
7.44
7.50
7.51
7.86
7.89
7.95
7.93
504
504
504
538
504
504
504
538
580
580
580
614
531
531
531
565
531
531
531
565
607
607
607
641
8
7{3,1,3}
8
7{1,1,4}
7{1,2,1}
7{1,2,2}
7{1,2,3}
7{1,2,4}
7{1,3,1}
7{1,3,2}
7{1,3,3}
7{1,3,4}
7{2,1,1}
7{2,1,2}
7{2,1,3}
7{2,1,4}
7{2,2,1}
7{2,2,2}
7{2,2,3}
7{2,2,4}
7{2,3,1}
7{2,3,2}
7{2,3,3}
7{2,3,4}
18
16
20
15
13
12
23
16
12
12
11
12
18
11
9
7{3,1,4} 10
7{3,2,1} 14
>20
>20
>20
>20
7{3,2,2}
7{3,2,3} 12
7{3,2,4}
6
9
0.04
7{3,3,1} 19
7{3,3,2} 20
7{3,3,3} 17
7{3,3,4} 19
0.02
0.08
0.08
0.8
0.65
0.48
10.0
0.9
2.5
1.0
7{4,1,1}
7{4,1,2}
9
8
7{4,1,3} 11
7{4,1,4}
7{4,2,1} 10
8
7{4,2,2}
7{4,2,3}
7{4,2,4}
8
8
6
7
16
16
21
21
14
0.3
8
0.02
0.02
0.07
0.24
7{4,3,1} 17
7{4,3,2} 17
7{4,3,3} 17
7{4,3,4} 16
94
97
98
97
2.3
2.0
2.0
0.9
a
Isolated yield by automated preparative HPLC (UV at 215 nm, Waters Symmetry C18 5
lm, 19 Â 50 mm with a linear gradient of 10% acetonitrile containing 0.1% formic
acid/aqueous 0.1% formic acid to 100% acetonitrile containing 0.1% formic acid for over 20 min at a flow rate of 10 mL/min).
b
Assay for ACAT activity in microsomes prepared from mouse liver microsomes.
Assay for CE synthesis of lipid droplet accumulation in macrophages.
c
d
Retention time (UV at 215 nm, Waters Symmetry C18 5
lm, 4.6 Â 50 mm with a linear gradient of 10% acetonitrile containing 0.1% formic acid/aqueous 0.1% formic acid
to 100% acetonitrile containing 0.1% formic acid for over 12 min at a flow rate of 1.0 mL/min).
e
Positive ion electrospray MS data, [M+H]+ were recorded.
Beauveriolide III (1b).
Beauveriolide I (1a).
f
g
O
O
O
O
O
O
O
O
O
NH
NH
NH
HN
O
HN
O
HN
O
N
N
N
O
O
O
H
H
H
7{1, 3, 2
}
7{2, 3, 1
}
7{2, 3, 2
}
Figure 3. Structures of beauveriolide analogues 7{1,3,2}, 7{2,3,1}, and 7{2,3,2}.
group of the serine without losing potency. Interestingly, D-Phe
Acknowledgments
analogues derived from B{2} were inactive except for 7{2,2,1},
7{2,2,2}, 7{2,2,3}, 7{2,2,4}, and 7{3,2,4} in the cell-based assay.
These results suggest that displacement of L-Phe with D-Phe might
cause a different conformation, thus decreasing the affinity for
ACAT. Diphenyl alanine analogues from B{3} enhanced the activity
as previously reported.5a It is considered that the diphenyl group
plays a critical role in inhibition of CE synthesis in macrophages.
Building block C had little impact on the activity. Among them,
7{1,3,2}, 7{2,3,1}, and 7{2,3,2} were the most potent inhibitors
This work was supported by the Program for Promotion of Fun-
damental Studies in Health Sciences of the National Institute of
Biomedical Innovation (NIBIO), by grant-in-aid for Scientific Re-
search (B) 18390008 (H.T.) and Scientific Research on Priority
Areas 18032028 (T.D.) from the Ministry of Education, Culture,
Sports, Science and Technology, Japan, and by the Hoh-ansya Foun-
dation, Japan.
with the same IC50 value of 0.020
In conclusion, we have synthesized a second beauveriolide li-
brary focusing on the -Ala and -allo-Ile moieties using our
combinatorial strategy. Inhibitory activity of the combinatorial li-
brary was tested to elucidate structure–activity relationships.
Based on these results, further new analogues have been inves-
tigated. The in vivo biological activity will be reported
elsewhere.
lM (Fig. 3).
References and notes
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1979, 76, 333; (b) Brown, M. S.; Goldstein, J. L.; Krieger, M.; Ho, Y. K.; Anderson,
R. G. J. Cell Biol. 1979, 82, 597.
2. (a) Namatame, I.; Tomoda, H.; Si, S.; Yamaguchi, Y.; Masuma, R.; Omura, S. J.
Antibiot. 1999, 52, 1; (b) Namatame, I.; Tomoda, H.; Tabata, N.; Si, S.; Omura, S. J.
Antibiot. 1999, 52, 7; (c) Namatame, I.; Tomoda, H.; Ishibashi, S.; Omura, S. Proc.
Natl. Acad. Sci. U.S.A. 2004, 101, 737; (d) Matsuda, D.; Namatame, I.; Tomoda, H.;
Kobayashi, S.; Zocher, R.; Kleinkauf, H.; Omura, S. J. Antibiot. 2004, 57, 1.
L
D

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K. Nagai et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4397–4400
3. Mochizuki, K.; Ohmori, K.; Tamura, H.; Shizuri, Y.; Nishiyama, S.; Miyoshi, E.;
Yamamura, S. Bull. Chem. Soc. Jpn. 1993, 66, 3041.
CD3OD = 4:1) d (ppm): 7.25–7.12 (m, 5H), 4.93 (dt, J = 8.5, 4.5 Hz, 1H),
4.58 (t, J = 8.0 Hz, 1H), 4.29 (t, J = 8.0 Hz, 1H), 3.82 (q, J = 7.0 Hz, 1H), 3.05
(dd, J = 13.5, 8.0 Hz, 1H), 2.94 (dd, J = 13.5, 8.0 Hz, 1H), 2.46 (dd, J = 14.5,
5.5 Hz, 1H), 2.40 (dd, J = 14.5, 8.5 Hz, 1H), 2.06 (m, 1H), 1.55–1.43 (m, 3H),
1.36–1.25 (m, 3H), 1.23 (d, J = 7.0 Hz, 3H), 1.22–1.09 (m, 2H), 1.00 (m,
1H), 0.90 (d, J = 7.0 Hz, 3H), 0.88 (d, J = 7.0 Hz, 3H), 0.84 (t, J = 7.0 Hz, 3H),
4. Sliskovic, D. R.; Picard, J. A.; Krause, B. R. Prog. Med. Chem. 2002, 39, 121.
5. (a) Nagai, K.; Doi, T.; Sekiguchi, T.; Namatame, I.; Sunazuka, T.; Tomoda, H.;
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I.; Nagai, K.; Sekiguchi, T.; Doi, T.; Takahashi, T.; Akasaka, K.; Rudel, L. L.;
Tomoda, H.; Omura, S. J. Org. Chem. 2006, 71, 7643; (c) Tomoda, H.; Doi, T. Acc.
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0.83 (d, J = 7.0 Hz, 3H). 13C NMR (75.4 MHz, CDCl3/CD3OD = 4:1)
d (ppm):
172.2, 171.8, 171.5, 170.1, 136.6, 129.2 (2Â), 128.7 (2Â), 127.1, 76.4, 57.0,
52.7, 49.0, 36.1, 35.8, 35.7, 30.9, 29.5, 25.0, 23.0, 22.34, 22.26, 15.6, 15.0,
14.0.
6. Selected spectral data. 7{1,1,1} (Beauveriolide I) HRFABMS: calcd for
C
27H42N3O5 [M+H]+ 488.3124, found 488.3125. 1H NMR (300 MHz, CDCl3/