The total synthesis and structure-activity relationships of a highly cytotoxic depsipeptide kulokekahilide-2 and its analogs

Article abstract of DOI:10.1016/j.tet.2011.10.094

We successfully completed the total synthesis of kulokekahilide-2, a cytotoxic depsipeptide from the Cephalaspiedean mollusk Phillinopsis speciosa. We have revised the absolute stereochemistry of kulokekahilide-2 to 21-l-Ala, 24-d-MePhe, 37-l-Ile, 43-d-Ala, 15-d-Hica, and 5S,6S,7S-Dtda. We also investigated the cause of the mis-assignment of the configuration in the originally proposed kulokekahilide-2 and concluded that methanolysis using MeONa caused partial racemization, which led to the mis-assignment. The structure-activity relationships of kulokekahilide-2 and its analogs indicate the importance of an l-amino acid at position 21.

Full text of DOI:10.1016/j.tet.2011.10.094

Tetrahedron 68 (2012) 659e669
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The total synthesis and structureeactivity relationships of a highly cytotoxic
depsipeptide kulokekahilide-2 and its analogs
,
Yuuki Takada a, Masahiro Umehara a, Ryosuke Katsumata b, Yoichi Nakao b, Junji Kimura a
*
a Department of Chemistry and Biological Science, College of Science and Engineering, Aoyama Gakuin University, 5-10-1 Fuchinobe, Chuo-ku, Sagamihara,
Kanagawa 252-5258, Japan
b Department of Chemistry and Biochemistry, School of Advanced Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 October 2011
Received in revised form 26 October 2011
Accepted 27 October 2011
Available online 3 November 2011
We successfully completed the total synthesis of kulokekahilide-2, a cytotoxic depsipeptide from the
Cephalaspiedean mollusk Phillinopsis speciosa. We have revised the absolute stereochemistry of
kulokekahilide-2 to 21-L-Ala, 24-D-MePhe, 37-L-Ile, 43-D-Ala, 15-D-Hica, and 5S,6S,7S-Dtda. We also in-
vestigated the cause of the mis-assignment of the configuration in the originally proposed
kulokekahilide-2 and concluded that methanolysis using MeONa caused partial racemization, which led
to the mis-assignment. The structureeactivity relationships of kulokekahilide-2 and its analogs indicate
Keywords:
the importance of an L-amino acid at position 21.
Kulokekahilide-2
Cyclic depsipeptide
Cytotoxicity
Ó 2011 Elsevier Ltd. All rights reserved.
Total synthesis
Structureeactivity relationships
1. Introduction
we successfully completed the total synthesis of an array of analogs
with plausible stereochemistries (1c: 21- -Ala, 24- -MePhe, and
-MePhe, and 43- -Ala, 1e: 21- -Ala,
-Ala). By comparison of the spectral data
L
D
Marine organisms are well known as a rich source of bioactive
compounds that can be potential drug leads.1 Among their various
bioactivities, cytotoxicity is a desirable characteristic, and numer-
ous marine cytotoxic compounds have been reported.2 Cytotoxic
cyclic depsipeptides, as exemplified by aurilide isolated from the
Japanese sea hare Dolabella auricularia3 and palau’amide from the
marine cyanobacterium Lyngbya majuscula,4 are potential anti-
43-
L
-Ala, and 1d: 21-
L
-Ala, 24-
D
D
D
L
24-
L
-MePhe, and 43-
with those of the natural compound, we conclude that the absolute
stereostructure of kulokekahilide-2 is 1d (Fig. 1).5c
In this paper, we describe the details of the total synthesis of
kulokekahilide-2 and its analogs, which allowed us to revise the
configuration of the proposed structure from 1b to 1d. The cause of
the mis-assignment of the initial stereochemistry was also in-
vestigated. Finally, structureeactivity relationships (SARs) are
discussed using the kulokekahilide-2 analogs prepared in this
study.
cancer leads.
A related 26-membered cyclic depsipeptide,
kulokekahilide-2 (1), which was isolated from a Cephalaspidean
mollusk Philinopsis speciosa, also showed potent cytotoxicity
against several cell lines (IC50 values ranging from 4.2 to 59.1 nM
against P388, SK-OV-3, MDA-MB-435, and A-10 cells).5a The
structure of kulokekahilide-2 was originally proposed as 1b com-
posed of five amino acids (
-isoleucine (37- -Ile), N-methylglycine (MeGly), and
phenylalanine (24- -MePhe)) as well as two hydroxy acids (
2-hydroxyisocaproic acid (15-
L
- and
D
-alanine (43-
L
-Ala, 21-
D-Ala),
2. Result and discussion
L
L
L
-N-methyl-
L
D-
2.1. Total synthesis of kulokekahilide-2
D
-Hica) and (5S,6S,7S,2E,8E)-
2,6,8-trimethyl-5,7-dihydroxy-2,8-decadienoic acid (Dtda)).5a In-
vestigation into its stereochemistry using synthetic 1b revealed
that the proposed stereochemistry (1b) was incorrect.5b Therefore,
2.1.1. Synthesis by macrolactonization. Our first synthetic strategy
followed the suggested biosynthetic route, including macro-
lactonization at the final step.6 We attempted to prepare
kulokekahilide-2 (1b) by macrolactonization of the corresponding
seco acid (2) as the key reaction (Scheme 1, route A). Compound 2
was prepared by the coupling of hexapeptide (3) and dihydroxy
acid (4). Hexapeptide 3 was synthesized from five protected amino
* Corresponding author. Tel.: þ81 42 759 6229; fax: þ81 42 759 6493; e-mail
address: kimura@chem.aoyama.ac.jp (J. Kimura).
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.10.094

660
Y. Takada et al. / Tetrahedron 68 (2012) 659e669
37-L-Ile
21-Ala
R1 R2
24-MePhe
43-Ala
R5 R6
Compounds
R3
R4
24-MePhe
R3 R4
MeGly
O
H
H
Me
Me
H
Bn
Bn
H
H
H
H
Me
Me
H
Me
H
1a
1b
1c
37
N
O
24
33
N
N
H
R2
R1
HN
R5
R6
O
HN
43-Ala
21-Ala
O
O
43
21
Me
Me
Me
H
Bn
Bn
H
H
O
1
OH
O
7
O
kulokekahilide-2 (1d)
14
H
H
Me
Me
Me
O
1e
1f
H
Bn
H
H
15-D-Hica
11
Me
Bn
H
5S,6S,7S-Dtda
Fig. 1. Structures of revised kulokekahilide-2 (1d) and its analogs.
acids and
D
-Hica. Dihydroxy acid (4) was prepared by enantiose-
in 63% yield. Yamaguchi lactonization10 produced 12a with a max-
imum of only 48% yield. Deprotection of the methylthiomethyl
(MTM) group in 12a by AgNO3 afforded 1a (75%).11
lective aldol condensation as described previously.5a,b
Hexapeptide 3 was synthesized via the usual condensation steps
of peptide synthesis using 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide hydrochloride salt (EDCI-HCl)7 or benzotriazol-1-yl-
oxy-trispyrrolydino phosphonium hexafluorophosphate (PyBOP)8
in the presence of 1-hydroxy benzotriazole (HOBt) or N,N-dime-
thylaminopyridine (DMAP) (Scheme 2).
Coupling of suitably protected hexapeptide 3 and dihydroxy acid
4 in the presence of EDCI-HCl gave the corresponding depsipeptide
11 in 54% yield. When this coupling reaction was carried out using
Shiina’s reagent,9 2-methyl-6-nitrobenzoic anhydride (MNBA), and
DMAP, the yield of 11 was increased up to 67%. Thus, the combi-
nation of MNBA and DMAP was more effective in this coupling than
that of EDCI-HCl and DMAP. Treatment with HF-Py and sub-
sequently with zinc powder provided seco acid 2.
The macrolactonization of 2 was first attempted utilizing EDCI-
HCl and DMAP, but resulted in quantitative recovery of the starting
material. The coupling reagent showed only low reactivity in the
macrolactonization reaction, as was the case for the synthesis of
aurilide. When macrolactonization was implemented with a low
concentration of dichloromethane (2.0 mM) by Shiina’s reagent,
the reaction proceeded smoothly to give the cyclized product (12a)
To our great disappointment, the 1H NMR spectrum of 1a was
not identical to that of natural kulokekahilide-2 (Fig. 2). Thus, the
absolute stereochemistry of each amino acid in 1a was re-assessed
by Marfey’s analysis,12 which led to more confusion due to the re-
sults that the component amino acids from 1a were
L
-Ile,
L
-MePhe,
-Ala had
and -Ala. The loss of the expected -Ala indicated that 43-
D
L
L
completely inverted its stereochemistry; therefore, 1a was con-
cluded to be the 43-epimer of the originally proposed 1b.5b
Racemization occasionally occurs at the optically active C-ter-
minal amino acids when DMAP is used for condensation.13 The
complete inversion of the stereochemistry at C-terminal 43-Ala
from
macrolactonization step, which did not proceed with C-terminal
43- -Ala because of the steric hindrance but proceeded for the 43-
epi seco acid derived from the epimerization equilibrium between
-Ala and -Ala of 2.
L- to D-form was presumed to be caused during the final
L
L
D
2.1.2. Synthesis by macrolactamization. To confirm the structure of
1a with 43- -Ala, we synthesized 1a through the independent
D
route B by macrolactamization of the linear precursor 5 (see
O
O
N
OTce
N
N
H
N
N
N
H
COOH
H2N
O
O
H
N
O
O
O
O
O
Fmoc
HN
HN
6
O
MTMO
O
O
HO
Ala1
O
OH
O
MTMO
OTBS
O
route B
HO
5
O
4
Macrolactamization
N
O
N
N
H
O
O
HN
O
O
O
HN
O
OH
Macrolactonization
O
H
N
N
O
O
N
N
H
N
H
N
O
O
N
N
H
O
O
OTce
route A
O
O
O
1
HN
3
O
O
O
O
OH
HN
MTMO
OH
OH
MTMO
OTBS
O
O
HO
2
4
Scheme 1. Retrosynthetic analysis of kulokekahilide-2.

Y. Takada et al. / Tetrahedron 68 (2012) 659e669
661
O
a
b, a
b, c
b, c
b, d
H
N
OTce
N
7
8
9
10
HCl H2N
N
N
H
O
O
OH
OH
O
O
O
O
O
O
OTce
Boc
OH
N
N
L-Ala-OTce HCl
Boc
OH
Boc
OH
Boc
OH
H
HN
N
H
N
O
O
O
3
(76 %)
(90 %)
(73 %)
(69 %)
(67 %)
OH
O
O
H
N
N
N
O
N
N
H
N
N
H
e
h
O
O
O
O
O
HN
O
OR1
O
O
HN
O
HN
O
OR2
O
OR
O
MTMO
O
O
R1 = Tce, R2 = TBS
R1 = H , R2 = H
11
2
12a R = MTM
1a R = H
f, g
i
Scheme 2. Synthesis of 1a via macrolactonization (route A). Reagents and conditions: (a) EDCI-HCl, HOBt, Et3N, CH2Cl2/DMF (1:1), rt; (b) 4 M HCl/dioxane; (c) PyBOP, i-Pr2NEt,
CH2Cl2/DMF (1:1), rt; (d) EDCI-HCl, HOBt, DMF, rt; (e) 4, EDCI-HCl, DMAP, CH2Cl2, rt, (54%), or 4, MNBA, DMAP, CH2Cl2, rt, (67%); (f) HF-pyridine/prydine/THF (1:1:4), rt, (97%); (g) Zn
powder, 1 M AcONH4, rt, (97%); (h) MNBA, DMAP, CH2Cl2, rt, (63%) or 2,4,6-trichlorobenzoyl chloride, i-Pr2NEt, benzene, rt then DMAP, benzene, rt, (48%); (i) AgNO3, 2,6-lutidine,
THF/H2O (4:1), 65 ꢀC, (75%).
Scheme 1, route B and Scheme 3).3b,c Synthesis of 1a by lactam-
ization was performed as below.
Coupling of pentapeptide 6a with protected dihydroxy acid 4 was
effected by EDCI-HCl and DMAP to furnish ester 16a (83%), which
was deprotected to yield alcohol 17a (87%). Esterification of 17a with
Pentapeptide 6a was prepared from L-Ile-OTce-HCl as the start-
ing material following standard peptide synthetic procedures.14
N-Fmoc-D-Ala instead of N-Fmoc-L-Ala gave the protected 18a (88%)
natural 1
1a
1b
1c
1d
1e
1f
Fig. 2. 1H NMR spectra of kulokekahilide-2 (1) and its analogs (1aef) in CD2Cl2.

662
Y. Takada et al. / Tetrahedron 68 (2012) 659e669
O
a
b, d
b, c
b, e
O
N
OTce
13
14a
15a
N
N
H
OTce
HCl H2N
O
O
OH
OH
O
O
O
O
Boc
OH
N
H
N
HN
Boc
OH
Boc
OH
L-Ile-OTce HCl
N
O
O
6a
OH
(88 %)
(91 %)
(80 %)
(76 %)
O
O
N
COOR1
N
OTce
N
N
H
N
N
H
H
f
h
k
l
O
O
N
O
O
O
O
O
R2
12a
1a
O
O
H
N
HN
HN
O
O
MTMO
Fmoc
OR
MTMO
HO
O
O
O
(88 %)
16a : R = TBS
17a : R = H
18a : R1 = Tce, R2 = Fmoc
5a : R1 = H, R2 = H
g
i, j
Scheme 3. Synthesis of 1a via macrolactamization (route B). Reagents and conditions: (a) EDCI-HCl, HOBt, Et3N, CH2Cl2/DMF (1:1), rt; (b) 4 M HCl/dioxane; (c) TBTU, i-Pr2NEt,
CH2Cl2/DMF (1:1), rt; (d) HBTU, i-Pr2NEt, CH2Cl2/DMF (1:1), rt; (e) EDCI-HCl, HOBt, Et3N, DMF, rt; (f) 4, EDCI-HCl, DMAP, CH2Cl2, rt, (83%); (g) HF-pyridine/prydine/THF (1:1:4), rt,
(87%); (h) EDCI-HCl, DMAP, CH2Cl2, rt, (88%); (i) Zn powder, 1 M AcONH4, rt, (75%); (j) Et2NH, MeCN, rt, (84%); (k) EDCI-HCl, HOAt, CH2Cl2/DMF (10:1), rt, (57%); (l) AgNO3, 2,6-
lutidine, THF/H2O (4:1), 65 ꢀC, (75%).
without racemization. Deprotection of 18a generated 5a and mac-
Therefore, we synthesized an array of analogs to evaluate different
rolactamization between 37-
L
-Ile and 43-
D
-Ala was carried out using
combinations of the configurations: 21-
24- . Namely, 21- -Ala, 24- -MePhe, and 43-
: 1c); 21- -Ala, 24- -MePhe, and 43- -Ala (
24- -MePhe, and 43- -Ala ( : 1e); and 21-
and 43- -Ala ( : 1f) were prepared through the same macro-
L
, 24-
D
; 21-
-Ala (abbreviated as
: 1d); 21-
-Ala, 24- -MePhe,
L
, 24-
L
; and 21-
D
,
,
EDCI-HCl and 1-hydroxy-7-azabenzotriazole (HOAt)15 to yield
cyclized 12a (57%). Deprotection of 12a at the 5-hydroxy group
afforded 1a. The NMR spectrum of the obtained 1a was identical to
D
L
D
L
L
D,
L
L
D
D
L
,
D
D
,
D
L-Ala,
L
D
L,
L,
D
D
that of the previously prepared 1a. Epimerization of 37-
L-Ile did not
D
D, D, D
occur, and the corresponding -Val in aurilide was inverted to
L
D
-Val
lactamization reaction applied for 1a and 1b (Scheme 5).
under the similar lactamization condition and yielded the byprod-
The 1H and 13C NMR data in CD2Cl2 for the major isomerof 1aef are
summarizedinTable 1. Allof the1Hand13Cspectraldatawereassigned
based on DQF-COSY, HMQC, and HMBC experiments. Comparison of
uct 6-epi-aurilide.3c
These results indicated that the macrolactamization using 37-L-
Ile as the C-terminus in 5a was more practical without racemization
than the macrolactonization using 43-Ala as the C-terminus.
the 1HNMRspectraof1aefindicatedthat1d (
L
,
D
,
D
) corresponds tothe
natural 1 (Fig. 2). From these results, we concluded that the absolute
stereostructure of 1 is 21- -Ala, 24- -MePhe, and 43- -Ala.
Therefore, we attempted to synthesize the proposed 1b (21-
24- -MePhe, and 43- -Ala, abbreviated as ) by macro-
lactamization of 18b in which 43- -Ala was moved to the N-ter-
D
-Ala,
L
D
D
L
L
D,
L,
L
Comparison of spectral data for these analogs revealed that
1aef adopt two conformers derived from the cisetrans amide
isomerism between MeGly and MePhe in CD2Cl2. The ROESY anal-
L
minus of the seco-peptide derived from 17a (Scheme 4).
O
O
O
N
OTce
N
O
N
COOTce
N
N
H
N
N
H
N
N
H
a
b-e
H
O
O
O
O
O
HN
O
O
O
N
O
O
O
O
O
O
Fmoc
H
N
HN
Fmoc
HO
HN
HN
OH
MTMO
OH
O
O
O
MTMO
O
O
O
O
17a
18b
1b
Scheme 4. Synthesis of 1b. Reagents and conditions: (a) EDCI-HCl, DMAP, CH2Cl2, rt, (95%); (b) Zn powder, 1 M AcONH4, rt; (c) Et2NH, MeCN, rt; (d) EDCI-HCl, HOAt, CH2Cl2/DMF
(10:1), rt, (70% in three steps); (e) AgNO3, 2,6-lutidine, THF/H2O (4:1), 65 ꢀC, (91%).
Deprotection and macrolactamization of 18b yielded 1b. The
NMR data of the obtained product yielded further confusion. The 1H
NMR spectrum of 1b was not identical to that of natural
kulokekahilide-2 (1). All of the stereocenters, including 43-Ala in 1b,
were confirmed, as shown in Fig. 2. Therefore, we concluded that the
proposed structure 1b in our original report was incorrect.5b
ysis indicated that only the trans conformer was seen for 1d in
DMSO-d6 and that CH3-35 and C5-OH are closely located. The
5d
presence of
D-phenylalanine and an N-methylated amino acid
might stabilize the specific ring conformation16 in DMSO, poten-
tially showing a similar conformation to that of 1d under physio-
logical conditions.17
2.1.3. Determination of the true structure of kulokekahilide-2 by
synthesis of analogs. We reexamined the stereochemistry of 1 in
comparison to that of aurilide and palau’amide. The strangest dif-
ferences were reversed configurations at positions 21 and 24 in 1b
2.2. Consideration of the mis-assignment of the absolute
stereochemistry
With the correct configuration confirmed, we next in-
vestigated the reason for the mis-assignment of the absolute
(21-
D and 24-L) to that of related compounds (21-L and 24-D).

Y. Takada et al. / Tetrahedron 68 (2012) 659e669
663
O
O
N
N
N
O
N
N
N
N
H
a, c
a, d - k
a, b
O
N
OTce
O
O
O
N
N
H
HN
O
N
OTce
O
O
Boc
N
OTce
Boc
N
H
O
O
N
N
H
O
HN
HN
HN
O
O
OH
O
O
NH
13
Boc
14a
O
15e
1e
O
O
O
N
H
a, l
a, d - k
N
OTce
O
a, b
O
O
N
N
H
HN
O
O
O
Boc
N
OTce
O
O
N
N
H
O
O
OH
O
O
NH
14c
Boc
O
15f
a, c
1f
O
O
N
OTce
N
N
O
g - k
a, d, e
H
O
N
H
O
O
O
O
N
OTce
O
O
HN
O
N
N
O
O
HN
H
O
OR
MTMO
O
O
O
OH
O
O
NH
O
Boc
15c
: R = TBS
16c
f
1c
: R = H
17c
g
O
O
N
COOR1
N
O
N
N
H
N
N
H
H
j
O
O
N
O
O
HN
O
O
O
R2
O
O
HN
HN
O
O
MTMO
OR
O
O
O
18d : R1 = Tce, R2 = Fmoc
5d : R1 = H, R2 = H
12d : R = MTM
1d : R = H
h, i
k
Scheme 5. Synthesis of analogs 1cef. Reagents and conditions: (a) 4 M HCl/dioxane; (b) Boc-L- or D-NMePhe, HBTU, i-Pr2NEt, CH2Cl2/DMF (1:1), rt; (c) Boc-L-Ala, PyBOP, i-Pr2NEt
and/or Et3N, CH2Cl2/DMF (1:1), rt; (d) D-Hica, EDCI-HCl, HOBt, Et3N, DMF, rt; (e) 4, EDCI-HCl, DMAP, CH2Cl2, rt; (f) HF-pyridine/pyridine/THF (1:1:4), 40 ꢀC; (g) Fmoc-L- or D-Ala,
EDCI-HCl, DMAP, CH2Cl2, rt; (h) Zn powder, 1 M AcONH4, rt; (i) Et2NH, MeCN, rt; (j) EDCI-HCl, HOAt, CH2Cl2/DMF (10:1), rt; (k) AgNO3, 2,6-lutidine, THF/H2O (4:1), 65 ꢀC; (l) Boc-D-
Ala, HBTU, Et3N, i-Pr2NEt, CH2Cl2/DMF (1:1), rt.
stereochemistry of kulokekahilide-2. In a previous report, the
stereochemistries of all of the amino acids as well as dihydroxy
acid 4 were confirmed to not be isomerized during the processes
of reductive degradation and acid hydrolysis.5b,c Therefore, we
suspected that the alkaline methanolysis determined the amino
acid configuration in the original structure.5a Compound 1d was
subjected to alkaline methanolysis by MeONa, and the reaction
mixture was purified by HPLC (column: COSMOSIL 5C18-MS-II,
10 mm IDꢁ250 mm, UV at 220 nm; mobile phase: 0.05% TFA in
CH3CN/H2O (60/40) at a flow rate of 2.5 mL/min), yielding three
hexapeptides as the main peaks. These peptides were acid hy-
drolyzed and examined by Marfey analysis. The first peptide
contained the same configurations of amino acids as 1d (35%),
2.3. Structureeactivity relationships (SARs)
Structureeactivity relationships were investigated based on the
cytotoxicity of the synthetic analogs 1aef. The cytotoxicities of
these analogs against two cell lines (P388 and HeLa) are summa-
rized in Table 2.
As expected, 1d with the natural conformation L, D, D exhibited
the same potent cytotoxicity (IC50 values of 16 and 3.2 ng/mL
against P388 and HeLa cells, respectively) as natural kulokekahilide-
2. Analog 1c (L, D, L) with the exact same configuration as aurilide (L,
D, L) with high cytotoxicity (IC50 values of 11 ng/mL against HeLa
cells3c) as described in Fig. 3 showed a similar cytotoxicity (IC50
values of 16 and 5.3 ng/mL, respectively).
while the second and the third contained 24-
L
-MePhe (corre-
By contrast, the cytotoxicity of 1e (
1/20the1/10th of the level (IC50 values of 400 and 39 ng/mL,
respectively) of 1d. Aurilide B ( , l, ) and C ( , l, ) also exhibited
L, l, D) was reduced to
sponding to 1e, 12%) and 43-
L
-Ala (corresponding to 1c, 20%),
respectively.
L
L
L
L
These observations suggested that the hexapeptide used for the
hydrazinolysis might have been racemized in the preceding
methanolysis; consequently, 43-Ala was misassigned as being in
a lower LC50 value of 10e130 nM against NCI-H460 and neuro-
2a mouse neuroblastoma cell lines.18 The configuration at C-
24-
D seemed critical to the cytotoxicity (Fig. 3, black line
the L-form in the subsequent Marfey analysis.
square).

Table 1
1H and 13C NMR data for the major rotamer of compound 1aef in CD2Cl2
Unit
Position
1a
1b
1c
1d
1e
1f
dH, mult., (J in Hz)a
dC
dH, mult., (J in Hz)
dC
dH, mult., (J in Hz) a
dC
dH, mult., (J in Hz) a
dC
dH, mult., (J in Hz)
dC
dH, mult., (J in Hz) a
dC
b
a
b
b
b
a
b,c
b
Dtda
1
2
3
4a
4b
5
6
7
d
167.2
126.9
141.5
31.1
d
168.2
126.2
144.6
33.0
d
168.1
126.5
142.6
31.9
d
166.9
128.2
141.9
32.5
d
d
166.5
129.4
140.3
34.0
d
d
d
d
d
d
6.98 dt 7.4, 1.3
2.30e2.34 m
2.30e2.34 m
3.75 ddd 10.6, 3.2, 3.2
2.30e2.34 m
5.05 d 11.0
d
7.17 m
2.20 m
2.43 ddd 14.9, 10.1, 7.5
4.13 m
2.19 m
5.09 d 11.0
d
6.99 bt 7.3
2.26 m
6.97 dt 8.4, 1.1
2.37 dd 14.3, 8.4
2.14 m
3.51 bdd 8.7, 5.6
2.11 m
6.91 m
140.4
7.01 bddd 8.4, 6.4, 1.3
2.25 m
2.55 ddd 14.7, 8.4, 2.1
3.59 dt 7.7, 2.1
2.16 m
1.99e2.22 m
2.37e2.51 m
3.85e3.93 m
1.99e2.22 m
4.89e4.97 m
d
70.3
40.3
83.4
132.5
126.8
13.3
12.8
11.5
11.0
170.2
73.2
41.4
71.3
40.8
82.6
132.7
126.8
13.2
12.9
11.7
10.7
170.9
73.7
41.4
3.75 dt 6.4, 4.4
2.16 m
4.98 d 11.2
d
71.3
40.6
83
72.1
41.4
83.5
133.2
125.9
13.1
12.7
11.6
11.2
170.4
72.6
40.9
74.4
40.5
85.2
133.3
125.9
13.2
13.0
13.3
11.6
170.0
73.1
41.4
5.22 d 9.8
d
5.17 d 9.3
d
8
9
132.5
127.1
13.2
12.7
11.6
12.8
169.8
73.4
41.0
5.60 m
1.62 d 6.8
1.88 s
0.86 d 6.8
1.63 s
d
5.57 m
1.62 d 6.0
1.90 s
0.80 d 6.9
1.62 s
d
5.55 qd 6.4, 1.2
1.60 bd 7.1
1.87 s
0.78 d 7.1
1.61 br s
d
5.55 qd 6.6, 1.1
1.61 dq 6.6, 0.9
1.83 bs
0.79 d 6.9
1.64 bs
d
5.45e5.50 m
1.55e1.80 m
1.52 s
0.70e0.96 m
1.88 m
d
5.57 bq 6.8
1.62 bd 6.8
1.88 bs
0.84 d 7.1
1.67 bs
d
10
11
12
13
14
15
16a
16b
17
18
19
20
21
22
NH
23
24
25a
25b
26
27
28
29
30
31
32
33
34a
34b
35
36
37
38
39a
39b
40
41
NH
42
43
44
NH
Hica
5.18 m
5.03 m
5.03 dd 9.2, 4.7
1.66 m
1.79 m
5.14 dd 10.3, 5.8
1.77 m
1.71 m
5.18e5.24 m
1.55e1.80 m
1.55e1.80 m
1.55e1.80 m
0.70e0.96 m
0.70e0.96 m
d
73.0
5.21 dd 8.2, 3.7
1.64e1.75 m
1.64e1.75 m
dc
1.60e1.73 m
1.60e1.73 m
1.60e1.73 m
0.91 d 5.2
0.91 d 5.2
d
1.60e1.72 m
1.60e1.72 m
1.60e1.72 m
0.92 d 6.0
0.92 d 6.0
d
25.0
22.0
23.3
173.1
45.0
17.8
d
25.0
21.9
23.3
172.9
45.0
17.0
d
1.66 m
25.1
21.9
23.3
173.6
45.3
17.7
d
1.69 m
24.9
21.9
23.3
173.0
45.5
17.5
d
24.9
22.1
23.3
172.7
46.0
18.1
d
0.92 d 6.4
0.93 d 6.3
d
0.91 d 6.3
0.91 d 6.3
d
0.88e0.94 m
0.88e0.94 m
d
Ala-1
4.69 m
4.65 dq 7.3, 6.8
0.81 d 6.9
6.67 d 7.3
d
4.68 dq 7.7, 7.0
0.79 d 7.0
6.86 d 7.7
d
4.71 dq 7.8, 6.7
0.87 d 6.7
7.10 d 7.8
d
4.89e4.97 m
1.24 d 6.8
7.46 d 7.2
d
4.58 dq 7.0, 6.8
1.28 d 6.8
dc
0.69 d 6.8
6.87 d 8.2
d
d
N-MePhe
170.1
53.5
35.3
170.1
53.7
35.2
171.7
54.4
36.2
172.5
56.3
35.3
d
dc
171.6
dc
5.63 m
5.52 dd 10.1, 6.0
2.97 dd 14.7, 7.8
3.06 dd 14.7, 10.1
d
5.47 dd 9.9, 6.0
3.02 dd 14.4, 6.0
3.10 dd 14.4, 9.9
d
5.62 dd 9.1, 7.0
3.25 dd 14.1, 7.0
3.07 dd 14.1, 9.1
d
5.54 dd 10.0, 6.0
3.01e3.22 m
3.01e3.22 m
d
3.02 dd 14.2, 7.7
3.15 dd 14.2, 6.7
d
3.22 m
3.22 m
d
34.9
137.0
129.9
128.6
127.5
128.6
129.9
30.4
137.3
129.7
128.5
127.2
128.5
129.7
30.4
137.0
129.7
128.6
128.8
128.6
129.7
30.6
136.9
129.9
128.6
127.2
128.6
129.9
31.2
dc
7.20e7.24 m
7.20e7.24 m
7.15e7.30 m
7.32 bd 8.0
7.25 dd 8.0, 7.1
7.21 t 7.1
7.25 dd 8.0, 7.1
7.32 bd 8.0
2.95 s
7.12e7.34 m
7.16e7.24 m
129.8
128.8
127.2
128.8
129.8
30.7
2.91 s
d
2.97 s
d
2.99 s
d
3.24 s
d
3.02 s
d
N-MeGly
170.0
52.5
168.9
52.0
168.9
52.2
d
169.7
52.9
169.7
53.8
3.52 d 17.2
4.39 d 17.2
2.86 s
3.56 d 17.4
4.24 d 17.4
2.88 s
3.54 d 17.0
3.94 d 17.0
2.96 s
4.28 bd
3.72 bd
2.71 s
d
2.98 d 16.1
4.71 d 16.1
2.71 s
3.47 d 14.1
4.29 d 14.1
2.87 s
35.1
168.3
58.0
38.3
25.1
35.1
171.7
57.9
38.6
25.1
35.6
170.5
57.6
38.1
25.2
36.3
170.8
58.8
35.8
25.1
33.5
37.2
170.7
58.2
35.5
24.8
Ile
d
d
d
d
d
4.41 dd 7.4, 7.2
1.80 m
1.12 m
4.50 dd 9.2, 6.2
1.80 m
1.48 m
4.01 dd 8.0, 8.0
1.92 m
1.29 m
4.09 dd 8.2, 7.1
1.96 m
1.48 m
4.15 t 6.8
1.55e1.80 m
0.96e1.05 m
1.21e1.33 m
0.70e0.96 m
0.70e0.96 m
6.63 bd 8.2
d
4.27 dd 9.0, 5.2
2.08 m
1.07 m
1.36e1.41 m
0.88e0.94 m
0.88e0.94 m
7.06 d 9.0
d
1.46 m
1.48 m
1.59 m
1.12 m
0.85 t 6.7
0.91 d 4.5
6.22 d 7.2
d
10.3
15.8
d
0.87 t 7.3
0.95 d 6.4
6.91 d 9.2
d
11.5
15.3
d
0.95 t 7.4
0.90 d 6.7
6.96 d 7.5
d
10.8
15.5
d
0.88 t 7.5
0.92 d 5.7
6.95 d 8.2
d
11.3
16.0
d
11.8
16.2
d
d
Ala-2
170.6
49.5
18.1
d
172.3
50.2
16.3
d
171.3
49.6
16.6
d
171.6
48.7
18.5
d
171.7
48.9
18.2
d
4.40 m
1.33 d 7.3
6.47 d 8.6
4.16 dd 7.3, 4.6
1.32 d 7.3
6.47 d 4.6
4.18 dq 7.0, 6.7
1.34 d 7.0
6.66 bd
4.44 dq 7.6, 7.1
1.35 d 7.1
6.32 d 7.6
4.44 dq 7.5, 7.4
1.32 d 7.4
7.06 bd
48.7
d
4.49 dq 8.4, 7.2
1.36 d 7.2
dc
a
Recorded at 500 MHz.
Recorded at 126 MHz.
Signals were not assigned due to overlap.
b
c

Y. Takada et al. / Tetrahedron 68 (2012) 659e669
665
Table 2
paula’amide (
L
,
D
,
L
) in nonpolar solvents. The cytotoxicity of the 24-
Cyototoxicity of kulokekahilide-2 and its analogs against two cell lines
membered isomer of 1d (IC50 values of 7.2 and 40 ng/mL against
P388 and Hela cells, respectively)5d showed equivalent activity to
that (IC50 values of 16 and 3.2 ng/mL, respectively) of 1d. Also, the
24-membered paula’amide exhibited potent cytotoxicity against
KB cells (IC50 value of 13 nM).4a On the other hand, 1b and 1c
Compound
Chirality at each residue
H-Ala-MePhe-MeGly-Ile-Ala-OH
Cell lines (IC50: ng/mL)
P388
HeLa
1a
1b
1c
1d
1e
1f
DL
DL
LD
LD
e
e
e
e
LD
LL
>10,000
10,000
16
>10,000
>10,000
5.3
containing 43-L-Ala did not undergo intramolecular ester exchange
LL
LD
LD
16
3.2
and thus did not yield any 24-membered isomers. The effect of the
ring size on the cytotoxicity and the relationship between cyto-
toxicity and intramolecular esterification will be described in more
detail in a future publication (gray line square).
LLe
400
39
DD
e
LD
4500
2000
Compound 1f (d,
P388 and HeLa cells (IC50 values of 4500 and 2000 ng/mL, re-
spectively), while 1a (d, ) and 1b (d, ) completely lost activity
D, D) showed only weak cytotoxicity against
L,
D
L, L
3. Conclusion
(IC50 value of ꢂ10,000 ng/mL against both P388 and HeLa cells).
Therefore, the configuration at C-21-L is also critical to the cyto-
We completed the total synthesis of kulokekahilide-2 (1d:
and its analogs 1aec, 1e, and 1f (1a: , 1b: , 1c: , 1e:
, and 1f: ) by macrolactamization.
We investigated the reason for the mis-assignment of the con-
L
,
D
,
D
)
toxicity (dotted line square).
D
,
L,
D
D
,
L,
L
L
,
D,
L
L
, L,
In recent years, deoxyaurilide and aurilide analogs with an
L-Lys
D
D, D, D
residue instead of 2- -MeAla, 6- and 21- -Val (corresponding to
L
L
positions 43, 37 and 21 in 1d, respectively) have been synthesized.
The cytotoxicities of these analogs were 10e100 times less
than that of aurilide. Moreover, 6-epi-aurilide with 6-D-Val instead
figuration in the original report using synthetic 1d. We concluded
that the methanolysis of 1d by MeONa caused partial racemization,
which led to the mis-assignment.
of -Val (corresponding to position 37 in 1d) showed no cytotox-
L
Evaluation of the cytotoxicities of these compounds (1aef)
showed that the 21-L and 24-D configurations of 1d are critical to
icity,3e indicating that the absolute configuration at position 37
contributes to the cytotoxicity. Therefore, the reversed stereo-
chemistry of potent cytotoxic lagnamides at the position corre-
sponding to that of C-7 in 1d is interesting.19
the cytotoxicity, while the C-43 stereochemistry has less influence.
The whole ring conformation affected by the C-21 and C-24 con-
figurations was likely more important for the cytotoxicity than the
side chain substitutions. Investigation of the target molecules for
1d and its analogs is currently in progress.
We have reported that 1d (L, D, D) undergoes intramolecular
ester exchange to yield 24-membered products, such as
35
O
O
O
O
37
N
N
N
N
O
O
O
O
N
N
H
N
N
H
N
N
H
N
N
H
24
O
O
O
O
O
O
O
O
O
HN
O
HN
HN
O
HN
O
O
O
O
O
O
O
21
43
HN
HN
HN
HN
O
O
O
O
OH
O
OH
5
OH
OH
O
7
O
O
O
O
26-memberd kulokekahilide-2 (1d)
1c
1e
1f
O
O
O
O
N
O
N
O
N
N
H
N
N
H
N
O
N
O
N
N
H
N
N
H
O
O
O
O
NH
N
O
O
O
O
O
O
O
O
O
N
O
HN
O
O
O
O
O
HN
HN
O
O
O
HN
HN
O
OH
O
OH
O
5
O
O
O
HO
HO
24-memberd kulokekahilide-2
palau'amide
aurilide B
O
1b
O
O
O
N
O
N
N
H
O
N
N
H
N
N
H
N
N
O
O
N
N
N
H
O
O
O
N
N
O
O
O
O
O
O
O
O
O
N
O
HN
O
O
O
O
O
HN
HN
O
OH
O
7
O
HN
OH
HN
O
O
OH
O
OH
R
O
O
O
O
lagunamide A, R =
lagunamide B, R =
aurilide
aurilide C
1a
potent
non-potent
Fig. 3. Structurally related cyclic depsipeptides.

666
Y. Takada et al. / Tetrahedron 68 (2012) 659e669
4. Experimental section
37.5 (d), 53.1 (t), 56.2 (d), 74.4 (t), 80.8 (s), 94.3 (s), 169.9 (s, 3C); HR-
FABMS: [MþH]þ m/z 433.1086 (m/z 433.1064 calcd for
4.1. General procedures
C16H28N2O355Cl3).
All moisture-sensitive reactions were carried out under an
argon atmosphere. The solvents used were distilled from the fol-
lowing drying reagents: tetrahydrofuran (THF: sodium benzophe-
4.2.2. Tripeptide Boc-N-Me-
13 (3.71 g, 8.57 mmol) was stirred with 4 M HCl in dioxane (20 mL) at
0 ꢀC for 1 h, and then the solvent was removed to give N-MeGly-
-Ile-
OTce HCl salt (3.10 g, 8.38 mmol, 98%). Boc-N-Me- -Phe-OH (486 mg,
1.74 mmol), i-Pr2NEt (910 L, 5.22 mmol), and HBTU (792 mg,
2.09 mmol) were added to a stirred solution of N-MeGly- -Ile-OTce
HCl salt (644 mg, 1.74 mmol) in DMF/CH2Cl2 (1:1, 6 mL), and the
mixture was stirred at room temperature for 12 h. The reaction
mixture was diluted with 1 M HCl (40 mL) and extracted with CH2Cl2
(40 mLꢁ2). The combined extracts were washed with 5% aqueous
NaHCO3 (40 mL), dried over Na2SO4, and evaporated to dryness. The
residue was subjected to flash column chromatography on silica gel
D-Phe-N-MeGly-L-Ile-OTce (14c). Dipeptide
L
ꢀ
none), dichloromethane (CH2Cl2: calcium chloride and 4-A
D
molecular sieves), dimethylformamide and dimethylsulfoxide
(DMF and DMSO: calcium hydride). The other anhydrous solvents
were purchased from Aldrich or Kanto Kagaku. N,N-Dimethylami-
nopyridine (DMAP) was recrystallized from EtOAc prior to use.
Open and flash column chromatography were performed using Fuji
Silysia silica gel BW-300 and BW-350, respectively. Analytical thin-
layer chromatography (TLC) was performed using TLC plates pre-
coated with Merck Silica gel 60 F254 (0.25 mm layer thickness). The
1H (500 MHz) and 13C (126 MHz) NMR spectra were measured on
a JEOL JNM-ECP500 spectrometer. Chemical shifts are reported in
m
L
[n-hexane/EtOAc, (8:2) to (1:1)] to give tripeptide 14c as a colorless
20
amorphous powder (882 mg, 1.48 mmol, 85%). [
a
]
þ78.9 (c 0.50,
D
d
parts per million calibrated using residual undeuterated solvent
CHCl3); IR (KBr disk) 3328, 2968, 1757, 1696, 1658, 1636, 1455, 1388,
1141, 754, 701, 575 cmꢃ1. Characteristic signals of four conformations
were observed, but some carbons could not be determined due to
as an internal reference. The following abbreviations are used to
explain the multiplicities: s¼singlet, d¼doublet, t¼triplet,
q¼quartet, qu¼quintet, m¼multiplet, br¼broadened. FABMS (Fast
Atom Bombardment Mass Spectrometry) spectra were obtained on
a JEOL MStation JMS-700A spectrometer. Nitrobenzyl alcohol (NBA)
was used as a matrix for the FABMS measurements. Polyethylene
glycol was used as a marker for HR-FABMS. Optical rotations were
measured on a PerkineElmer-340 digital spectropolarimeter. IR
spectra are reported in wavenumbers (cmꢃ1) and were measured
on a Shimadzu FTIR-8200A infrared spectrometer. HPLC was car-
ried out with a Shimadzu LC-10 liquid chromatograph equipped
with a Shimadzu SPD-10A UVevis detector and Integrator C-R6A
Chromatopac and with a JASCO 880-PV liquid chromatograph
equipped with a JASCO 875-UV UVevis detector and Sic chroma-
tocorder 12.
overlap (major rotamer). 1H NMR (500 MHz, CDCl3)
d
0.91 (t, 3H,
-H), 1.18 (m, 1H, Ile -Ha),
-Hb), 1.98 (m, 1H, Ile -H), 2.80
(s, 3H, GlyeNMe), 2.94 (dd, 1H, J¼14.0, 8.2 Hz, Phe -Ha), 3.03 (s, 3H,
PheeNMe), 3.14 (dd, 1H, J¼14.0, 7.1 Hz, Phe -Hb), 3.84 (d, 1H,
J¼14.9 Hz, Gly -Ha), 4.12 (d, 1H, J¼14.9 Hz, Gly -Hb), 4.59 (d, 1H,
J¼11.9 Hz, CH2CCl3), 4.66 (m, 1H, Ile -H), 4.88 (d, 1H, J¼11.9 Hz,
-H), 6.67 (d, 1H, J¼8.5 Hz,
J¼7.1 Hz, Ile
d
-H), 0.92 (d, 3H, J¼6.9 Hz, Ile
g
g
1.32 (s, 9H, OC(CH3)3), 1.45 (m, 1H, Ile
g
b
b
b
a
a
a
CH2CCl3), 5.35 (dd, 1H, J¼8.2, 7.1 Hz, Phe
a
IleeNH), 7.14e7.27 (m, 5H, PheAreH); 13C NMR (126 MHz, CDCl3) dC
11.5 (q),15.6 (q), 24.8 (t), 28.1 (q, 3C), 29.7 (q), 35.2 (t), 36.3 (q), 37.2 (d),
52.6 (t), 55.5 (d), 56.4 (d), 74.4 (t), 80.1 (s), 94.4 (s), 126.4 (d), 128.4 (d,
2C), 129.3 (d, 2C), 137.2 (s), 155.3 (s), 168.7 (s), 169.9 (s), 171.7 (s); HR-
FABMS: [MþNa]þ m/z 618.1675 (m/z 618.1699 calcd for
C26H38N3O365Cl237ClNa). The ratio of isomers is 10:5.7:2:1.
4.2. Synthesis of revised kulokekahilide-2 (1d)
4.2.3. Tetrapeptide
Boc-L-Ala-N-Me-D-Phe-N-MeGly-L-Ile-OTce
4.2.1. Dipeptide Boc-N-MeGly-
(1.06 mL, 11.0 mmol), DMAP (122 mg, 1.00 mmol), and EDCI-HCl
(2.30 g, 12.0 mmol) were added to a stirred solution of Boc- -Ile-
OH (2.32 mg, 10.0 mmol) in CH2Cl2 (20 mL), and the mixture was
stirred at room temperature for 1 h. The reaction mixture was
evaporated to dryness and dissolved in EtOAc (40 mL). The EtOAc
solution was washed with 1 M HCl (40 mL) and 5% aqueous NaHCO3
(40 mL), dried over MgSO4, and evaporated to dryness. The residue
was treated with 4 M HCl in dioxane (20 mL) at room temperature
L
-Ile-OTce (13). 2,2,2-Trichloroethanol
(15c). Tripeptide 14c (2.24 g, 3.76 mmol) was treated with 4 M HCl
in dioxane (8.0 mL) at 0 ꢀC for 1 h, and then the solvent was re-
L
moved to give N-Me-
3.76 mmol, 100%). Boc-
(1.31 mL, 7.52 mmol), Et3N (0.52 mL, 3.76 mmol), and HBTU
(2.10 g, 5.64 mmol) were added to a stirred solution of N-Me-
Phe-N-MeGly- -Ile-OTce HCl salt (2.02 g, 3.76 mmol) in DMF/
D
-Phe-N-MeGly-L-Ile-OTce HCl salt (2.02 g,
L-Ala-OH (1.05 g, 5.53 mmol), i-Pr2NEt
D
-
L
CH2Cl2 (1:1, 16 mL), and the mixture was stirred at room tem-
perature for 14 h. The reaction mixture was diluted with 1 M HCl
(40 mL) and extracted with CH2Cl2 (20 mLꢁ3). The combined ex-
tracts were washed with 5% aqueous NaHCO3 (20 mL), dried over
Na2SO4, and evaporated to dryness. The residue was subjected to
flash column chromatography on silica gel [n-hexane/EtOAc, (8:2)
for 2 h to yield
Boc-N-MeGly-OH (2.08 g, 11.0 mmol), Et3N (1.39
HOBt (1.49 g, 11.0 mmol), and EDCI-HCl (4.22 g, 22.0 mmol) were
added to a stirred solution of -Ile-OTce HCl salt (2.88 g, 9.64 mmol)
L-Ile-OTce HCl salt (2.88 mg, 9.64 mmol, 96%).
mL, 10.0 mmol),
L
in CH2Cl2/DMF (1:1, 20 mL), and the mixture was stirred at room
temperature for 6 h. The reaction mixture was concentrated, di-
luted with 1 M HCl (40 mL) and extracted with CH2Cl2 (40 mLꢁ2).
The combined extracts were washed with 5% aqueous NaHCO3
(40 mL), dried over MgSO4, and evaporated to dryness. The residue
was subjected to column chromatography on silica gel [n-hexane/
EtOAc, (7:3)] to give dipeptide 13 as a colorless oil (3.71 g,
to (1:1)] to give tetrapeptide 15c as a colorless powder (2.07 g,
20
3.11 mmol, 83%). [
a
]
þ48.6 (c 0.50, CHCl3); IR (KBr disk) 3320,
D
2968, 1757, 1694, 1653, 1636, 1497, 1455, 1367, 1173, 755, 719,
574 cmꢃ1; (major rotamer); 1H NMR (500 MHz, CDCl3)
0.87 (d,
3H, J¼6.9 Hz, Ala -H), 0.93 (t, 3H, J¼7.6 Hz, Ile -H), 0.96 (d, 3H,
J¼6.9 Hz, Ile -H), 1.22 (m, 1H, Ile -H), 1.39 (s, 9H, OC(CH3)3), 1.45
(m. 1H, Ile -H), 2.00 (m, 1H, Ile -H), 3.00 (s, 3H, GlyeNMe), 3.03 (s,
3H, PheeNMe), 3.05 (dd, 1H, J¼14.7, 9.2 Hz, Phe -H), 3.17 (dd, 1H,
J¼14.7, 6.9 Hz, Phe -H), 3.97 (d, 1H, J¼15.6 Hz, Gly
J¼15.6 Hz, Gly -H), 4.44 (dq, 1H, J¼8.2, 6.9 Hz, Ala
J¼11.9 Hz, CH2CCl3), 4.70 (dd, 1H, J¼8.7, 4.6 Hz, Ile
d
b
d
g
g
g
b
8.57 mmol, 86%). [
a
]
20 þ7.6 (c 1.0, CHCl3); IR (KBr disk) 3315, 2968,
b
D
1760, 1699, 1669, 1636, 1534, 1455, 1392, 1146, 767, 719, 574 cmꢃ1
;
b
a
a
a
-H), 4.06 (d, 1H,
-H), 4.62 (d, 1H,
-H), 4.90 (d, 1H,
1H NMR (500 MHz, CDCl3)
3H, J¼6.9 Hz, Ile -H), 1.20 (m, 1H, Ile
1.47 (s, 9H, OC(CH3)3), 2.03 (m, 1H, Ile
3.81 (d, 1H, J¼16.0 Hz, Gly -Ha), 3.98 (d, 1H, J¼16.0 Hz, Gly
d
0.93 (t, 3H, J¼7.3 Hz, Ile
d
-H), 0.98 (d,
a
g
g
-Ha), 1.48 (m, 1H, Ile
g
-Hb),
b
-H), 2.96 (s, 3H, GlyeNMe),
-Hb),
-H), 4.90 (d, 1H,
J¼11.9 Hz, CH2CCl3), 5.26 (d, 1H, J¼8.2 Hz, AlaeNH), 5.77 (dd, 1H,
a
a
J¼9.2, 6.9 Hz, Phe -H), 6.68 (d, 1H, J¼8.7 Hz, IleeNH), 7.14e7.27
a
4.64 (d, 1H, J¼11.9 Hz, CH2CCl3), 4.75 (m, 1H, Ile
a
(m, 5H, PheAreH); 13C NMR (126 MHz, CDCl3) dC 11.6 (q), 15.5 (q),
17.5 (q), 24.9 (t), 28.2 (q, 3C), 30.3 (q), 35.1 (t), 36.5 (q), 37.4 (d),
46.5 (d), 52.5 (t), 53.8 (d), 56.3 (d), 74.4 (t), 79.4 (s), 94.3 (s), 126.7
J¼11.9 Hz, CH2CCl3), 6.41e6.83 (br d, 1H, Ile-NH); 13C NMR
(126 MHz, CDCl3) dC 11.4 (q), 15.5 (q), 24.8 (t), 28.2 (q, 3C), 35.7 (q),

Y. Takada et al. / Tetrahedron 68 (2012) 659e669
667
(d), 128.3 (d, 2C), 129.2 (d, 2C), 136.2 (s), 155.1 (s), 168.4 (s), 170.1
(s), 171.1 (s), 173.9 (s); HR-FABMS: [MþNa]þ m/z 689.2094 (m/z
689.2071 calcd for C29H43N4O375Cl237Cl Na). The ratio of isomers is
3.1:1.
(q), 24.4 (d), 24.9 (t), 25.7 (q, 3C), 28.7 (t), 30.3 (q), 35.1 (t), 36.5 (q),
37.3 (d), 38.3 (d), 40.7 (t), 45.1 (d), 52.6 (t), 53.7 (d), 56.3 (d), 72.4
(d), 73.2 (t), 74.4 (t), 75.6 (d), 80.8 (d), 94.3 (s), 122.2 (d), 126.7 (d),
127.7 (s), 128.3 (d, 2C), 129.1 (d, 2C), 136.2 (s), 136.3 (s), 141.9 (d),
166.6 (s),168.3 (s),170.0 (s),170.1 (s),170.9 (s),172.3 (s); HR-FABMS:
35
4.2.4. Pentapeptide
(6c). Tetrapeptide 15c (2.14 g, 3.21 mmol) was treated with 4 M
D
-Hica-
L
-Ala-N-Me-
D
-Phe-N-MeGly-
L
-Ile-OTce
[MþNa]þ m/z 1101.4530 (m/z 1101.4548 calcd for C51H83N4O Cl327Cl
10
SSiNa).
HCl in dioxane (10 mL) at room temperature for 2 h, and then the
solvent was removed to give
OTce HCl salt (1.93 g, 3.20 mmol, 100%).
3.52 mmol), Et3N (446 L, 3.20 mmol), HOBt (865 mg, 6.40 mmol),
and EDCI-HCl (920 mg, 4.80 mmol) were added to a stirred solu-
tion of -Ala-N-Me- -Phe-N-Me-Gly- -Ile-OTce HCl salt (1.93 g,
3.20 mmol) in DMF (6.4 mL), and the mixture was stirred at room
temperature for 17 h. The reaction mixture was diluted with 1 M
HCl (40 mL) and extracted with EtOAc (20 mLꢁ2). The combined
extracts were washed with 5% aqueous NaHCO3 (40 mL), dried
over Na2SO4, and evaporated to dryness. The residue was sub-
jected to flash column chromatography on silica gel [n-hexane/
L
-Ala-N-Me-
D
-Phe-N-MeGly-
L
-Ile-
4.2.6. Alcohol (17c). Depsihexapeptide 16c (487 mg, 0.451 mmol)
was dissolved in HF-pyridine/pyridine/THF (1:1:4, 27 mL). The
solution was stirred at 40 ꢀC for 12 h. After cooling, the reaction
mixture was diluted with EtOAc (100 mL) and poured into satu-
rated NaHCO3 (160 mL). The mixture was separated and extracted
with EtOAc (20 mL). The combined extracts were washed with 1 M
HCl (20 mL), dried over Na2SO4, and evaporated to dryness. The
residue was subjected to flash column chromatography on silica
D
-Hica (465 mg,
m
L
D
L
gel [n-hexane/EtOAc, (7:3) to (3:7)] to give alcohol 17c as a color-
20
less powder (383 mg, 0.397 mmol, 88%). [
a
]
þ33.2 (c 0.50,
D
CHCl3); IR (KBr disk) 3321, 2961, 1696, 1642, 1523, 1457, 756,
EtOAc, (7:3) to EtOAc] to give pentapeptide 6c as a colorless
668 cmꢃ1; (major rotamer); 1H NMR (500 MHz, CDCl3)
d
0.72 (d,
20
powder (1.64 g, 2.41 mmol, 75%). [
a
]
þ37.5 (c 0.50, CHCl3); IR
3H, J¼7.0 Hz), 0.85 (d, 3H, J¼6.8 Hz), 0.90 (d, 3H, J¼5.7 Hz), 0.91 (d,
3H, J¼5.6 Hz), 0.93 (t, 3H, J¼7.4 Hz), 0.96 (d, 3H, J¼6.9 Hz), 1.20 (m,
1H), 1.46 (m, 1H), 1.61 (overlapping s and d, 6H), 1.69 (m, 2H), 1.90
(s, 3H), 2.03 (overlapping m, 3H), 2.17 (s, 3H), 2.40 (m, 1H), 2.50 (m,
1H), 3.02 (s, 3H), 3.04 (overlapping s and dd, 4H), 3.18 (dd, 1H,
J¼14.4, 6.5 Hz), 3.77 (d, 1H, J¼9.7 Hz), 3.97 (d, 1H, J¼15.1 Hz), 4.06
(d, 1H, J¼15.1 Hz), 4.15 (dt, 1H, J¼7.3, 4.6 Hz), 4.61 (d, 1H,
J¼12.0 Hz), 4.65 (s, 2H), 4.73 (m, 2H), 4.90 (d, 1H, J¼11.9 Hz), 5.23
(dd, 1H, J¼9.2, 3.8 Hz), 5.43 (q, 1H, J¼5.6 Hz), 5.80 (dd, 1H, J¼9.0,
6.5 Hz), 6.66 (d, 1H, J¼8.8 Hz), 6.77 (d, 1H, J¼7.6 Hz), 7.03 (t, 1H,
J¼7.3 Hz), 7.14e7.27 (m, 5H). A signal from one proton (OH) was
not observed. 13C NMR (126 MHz, CDCl3) dC 10.2 (q), 11.3 (q), 11.4
(q), 12.6 (q), 12.9 (q), 14.1 (q), 15.5 (q), 17.3 (q), 21.6 (q), 23.0 (q),
24.4 (d), 24.8 (t), 29.4 (t), 30.4 (q), 35.0 (t), 36.5 (q), 37.3 (d), 38.1
(d), 40.6 (t), 45.0 (d), 52.5 (t), 53.7 (d), 56.3 (d), 72.3 (d), 73.3 (t),
74.4 (t), 77.3 (d), 80.7 (d), 94.3 (s), 122.7 (d), 126.7 (d), 127.8 (s),
128.3 (d, 2C), 129.1 (d, 2C), 136.1 (s), 136.6 (s), 141.1 (d), 166.4 (s),
168.3 (s), 169.8 (s), 170.0 (s), 170.7 (s), 172.4 (s); HR-FABMS:
D
(KBr disk) 3320, 2960, 1757, 1696, 1653, 1636, 1455, 1140, 754, 720,
574 cmꢃ1; (major rotamer); 1H NMR (500 MHz, CDCl3)
d
0.93 (d,
3H, J¼6.6 Hz), 0.93 (d, 3H, J¼6.8 Hz), 0.94 (t, 3H, J¼7.2 Hz), 0.95 (d,
3H, J¼6.9 Hz), 0.96 (d, 3H, J¼6.9 Hz), 1.48 (m, 1H), 1.48 (m, 1H), 1.57
(overlapping m, 2H), 1.83 (m, 1H), 2.02 (m, 1H), 2.99 (s, 3H), 3.05
(s, 3H), 3.05 (dd, 1H, J¼13.9, 9.0 Hz), 3.18 (dd, 1H, J¼13.9, 6.9 Hz),
3.86 (d, 1H, J¼17.2 Hz), 3.99 (d, 1H, J¼17.2 Hz), 4.07 (dd, 1H, J¼10.0,
3.4 Hz), 4.62 (d, 1H, J¼11.9 Hz), 4.69 (dd, 1H, J¼8.7, 4.8 Hz), 4.78
(dd, 1H, J¼7.8, 6.8 Hz), 4.91 (d, 1H, J¼11.9 Hz), 5.73 (dd, 1H, J¼9.0,
6.9 Hz), 6.67 (d, 1H, J¼8.7 Hz), 7.13 (d, 1H, J¼7.8 Hz), 7.15e7.28 (m,
5H), A signal from one proton (OH) was not observed. 13C NMR
(126 MHz, CDCl3) dC 11.4 (q), 15.5 (q), 17.5 (q), 21.3 (q), 23.4 (q),
24.3 (d), 24.9 (t), 30.5 (q), 35.2 (t), 36.5 (q), 37.3 (d), 43.5 (t), 45.1
(d), 52.3 (t), 53.9 (d), 56.4 (d), 70.4 (d), 74.5 (t), 94.3 (s), 126.8 (d),
128.3 (d, 2C), 129.1 (d, 2C), 136.2 (s), 168.4 (s), 170.2 (s), 170.9 (s),
172.7 (s), 174.0 (s); HR-FABMS: [MþNa]þ m/z 703.2221 (m/z
703.2228 calcd for C30H45N4O375Cl237Cl Na). The ratio of isomers is
2.9:1.
[MþNa]þ
m/z
987.3654
(m/z
987.3680
calcd
for
35
C45H69N4O Cl327ClSNa).
10
4.2.5. Depsihexapeptide (16c). Dihydroxy acid
4
(167 mg,
0.400 mmol), DMAP (48.9 mg, 0.400 mmol), and EDCI-HCl (199 mg,
1.04 mmol) were added to a stirred solution of pentapeptide 6c
(407 mg, 0.598 mmol) in CH2Cl2 (1.1 mL), and the mixture was
stirred at room temperature for 22 h. The reaction mixture was
diluted with 1 M HCl (40 mL) and extracted with CH2Cl2 (20 mLꢁ2).
The combined extracts were washed with 5% aqueous NaHCO3
(40 mL), dried over Na2SO4, and evaporated to dryness. The residue
was subjected to flash column chromatography on silica gel [n-
4.2.7. Depsiheptapeptide
(18d). Fmoc-
D
-Ala-OH
(286 mg,
0.918 mmol), DMAP (56.2 mg, 0.460 mmol), and EDCI-HCl (176 mg,
0.918 mmol) were added to a stirred solution of alcohol 17c
(444 mg, 0.460 mmol) in CH2Cl2 (1.8 mL) and maintained at 0 ꢀC for
2 h and then at room temperature for 4 h. The reaction mixture was
diluted with 1 M HCl (40 mL) and extracted with EtOAc (20 mLꢁ2).
The combined extracts were washed with 5% aqueous NaHCO3
(20 mL), dried over Na2SO4, and evaporated to dryness. The residue
was subjected to flash column chromatography on silica gel [n-
hexane/EtOAc, (7:3) to (3:7)] to give depsiheptapeptide 18d as
a colorless amorphous powder (533 mg, 0.423 mmol, 92%). [a]
þ14.5 (c 0.35, CHCl3); IR (KBr disk) 3325, 2928, 1759, 1689, 1647,
1526, 1462, 1054, 836, 755, 573 cmꢃ1. Characteristic signals of two
rotamers are shown, but some protons could not be determined
due to overlap (major rotamer). 1H NMR (500 MHz, CDCl3)
hexane/EtOAc, (8:2) to (1:1)] to give depsihexapeptide 16c as
20
a colorless powder (383 mg, 0.355 mmol, 90%). [
a
]
D
þ22 (c 0.25,
20
CHCl3); IR (KBr disk) 3314, 2964, 1757, 1696, 1640, 1144, 750,
D
576 cmꢃ1; (major rotamer); 1H NMR (500 MHz, CDCl3)
d
ꢃ0.06 (s,
3H), 0.01 (s, 3H), 0.71 (d, 3H, J¼7.1 Hz), 0.82 (d, 3H, J¼6.9 Hz), 0.88
(overlapping s and m, 15H), 0.93 (t, 3H, J¼7.3 Hz), 0.96 (d, 3H,
J¼6.6 Hz), 1.20 (m, 1H), 1.33 (dd, 1H, J¼14.8, 7.0 Hz), 1.47 (m, 1H),
1.59 (br s, 3H), 1.61 (br d, 3H, J¼6.9 Hz), 1.69 (overlapping m, 2H),
1.89 (s, 3H), 2.04 (m, 2H), 2.12 (s, 3H), 2.35 (m, 2H), 3.01 (s, 3H), 3.04
(overlapping s and dd, 4H), 3.18 (dd, 1H, J¼14.5, 6.5 Hz), 3.68 (d, 1H,
J¼9.2 Hz), 3.95 (d, 1H, J¼15.1 Hz), 4.10 (overlapping d and dt, 2H),
4.54 (d, 1H, J¼11.7 Hz), 4.62 (d, 1H, J¼11.9 Hz), 4.63 (d, 1H,
J¼11.5 Hz), 4.69 (m, 2H), 4.90 (d, 1H, J¼11.9 Hz), 5.15 (m, 1H), 5.38
(q,1H, J¼6.8 Hz), 5.83 (dd,1H, J¼15.6, 6.3 Hz), 6.64 (d,1H, J¼7.6 Hz),
6.64 (d, 1H, J¼8.5 Hz), 7.00 (t, 1H, J¼6.3 Hz), 7.15e7.27 (m, 5H); 13C
NMR (126 MHz, CDCl3) dC ꢃ5.4 (q), ꢃ4.6 (q), 10.2 (q), 10.5 (q), 11.5
(q), 12.7 (q), 12.8 (q), 14.0 (q), 15.5 (q), 17.1 (s), 18.0 (q), 21.5 (q), 23.0
d
0.73e0.96 (overlapping m,18H),1.20 (m,1H),1.49 (overlapping m,
4H), 1.59 (overlapping m, 6H), 1.73 (overlapping m, 3H), 1.89 (s, 3H),
2.01 (m,1H), 2.11 (s, 3H), 2.33 (overlapping m, 3H), 3.00 (s, 3H), 3.03
(s, 3H), 3.13 (m, 1H), 3.17 (dd, 1H, J¼14.0, 6.7 Hz), 3.88 (m, 1H), 3.95
(d, 1H, J¼15.1 Hz), 4.07 (d, 1H, J¼15.1 Hz), 4.21 (dd, 1H, J¼7.1,
6.9 Hz), 4.38 (overlapping m, 3H), 4.57 (d, 1H, J¼11.8 Hz), 4.60 (d,
1H, J¼11.7 Hz), 4.64 (overlapping d and m, 2H, J¼11.7 Hz), 4.69 (dd,
1H, J¼8.7, 8.7 Hz), 4.89 (d, 1H, J¼11.9 Hz), 5.02 (d, 1H, J¼10.1 Hz),
5.16 (dd, 1H, J¼9.4, 3.7 Hz), 5.58 (br dd, 1H, J¼6.9, 6.2 Hz), 5.82 (dd,
1H, J¼8.8, 6.7 Hz), 6.63 (d, 1H, J¼8.7 Hz), 6.89 (br t, 1H, J¼7.6 Hz),

668
Y. Takada et al. / Tetrahedron 68 (2012) 659e669
7.16e7.32 (m, 7H), 7.39 (dd, 2H, J¼7.5, 7.5 Hz), 7.60 (dd, 2H, J¼6.8,
3C), 172.1 (s); HR-FABMS: [MþH]þ m/z 886.5002 (m/z 886.5000
4.2 Hz), 7.76 (d, 2H, J¼7.6 Hz); HR-FABMS: [MþNa]þ m/z 1278.4733
calcd for C46H72N5O10S).
35
(m/z 1278.4750 calcd for C63H84N5O Cl3 SNa).
13
4.2.9. Revised
kulokekahilide-2
(1d). 2,6-Lutidine
(500 mL,
4.2.8. O-MTM-cyclic depsipeptide (12d). Activated Zn powder
(1.89 g, 28.8 mmol) was added to a stirred solution of dep-
siheptapeptide 18d (519 mg, 0.414 mmol) in THF (18 mL) and 1 M
AcONH4 (3.6 mL), and the mixture was stirred at room tempera-
ture for 22.5 h. The reaction mixture was filtered through a Celite
pad, and the residue was washed with EtOAc (20 mL). The com-
bined organic layer was evaporated. The residue was dissolved in
EtOAc (20 mL), washed with 1 M HCl (20 mL), 5% NaHCO3 (20 mL),
dried over Na2SO4, and evaporated to dryness. The residue was
subjected to column chromatography on silica gel [n-hexane, n-
4.32 mmol) and AgNO3 (1.47 g, 8.68 mmol) were added to a solu-
tion of 12d (192 mg, 0.217 mmol) in THF/H2O (4:1, 6.3 mL), and the
mixture was stirred at 65 ꢀC for 1.5 h. The mixture was diluted with
EtOAc (50 mL) and filtered through a Celite pad, and the residue
was washed with EtOAc (50 mL). The filtrate and washings were
combined, then washed with 1 M HCl (30 mL), H2O (30 mL), 5%
NaHCO3 (30 mL), and brine (30 mL), dried over Na2SO4, and evap-
orated to dryness. The residual oil was subjected to flash column
chromatography on silica gel [n-hexane/EtOAc, (7:3) to (3:7), EtOAc,
CHCl3/MeOH (95:5)] to give the revised kulokekahilide-2 (1d) as
hexane/EtOAc (6:4), (3:7), EtOAc, and CHCl3/MeOH (9:1)] to give
a colorless amorphous powder (137 mg, 0.165 mmol, 76%). [
a
]
D
20 ꢃ4
20
carboxylic acid as a colorless oil (436 mg, 0.387 mmol, 94%). [
a
]
(c 0.10, MeOH); UV (MeOH) lmax 204 nm (
3
3.3ꢁ104); IR (KBr disk)
D
þ3.3 (c 0.40, CHCl3); IR (KBr disk) 3318, 2929, 1720, 1641, 1524,
3296, 2962, 1687 (sh), 1658, 1634 (sh), 1530, 1456, 1382, 1269, 1096,
752 cmꢃ1. The 1H and 13C NMR spectra were identical to those of
natural 1; HR-FABMS: [MþNa]þ m/z 848.4763 (m/z 848.4786 calcd
for C44H67N5O10Na).
1452, 1053, 755 cmꢃ1; (major rotamer); 1H NMR (500 MHz, CDCl3)
d
0.81 (d, 3H, J¼6.9 Hz),0.90 (overlapping m, 12H), 0.97 (d, 3H,
J¼6.6 Hz), 1.20 (m, 1H), 1.50 (d, 3H, J¼7.1 Hz), 1.55 (m, 1H), 1.60 (s,
3H), 1.63 (d, 3H, J¼6.6 Hz), 1.65e1.77 (overlapping m, 3H), 1.87 (s,
3H), 1.92 (m, 1H), 2.11 (s, 3H), 2.27e2.38 (overlapping m, 3H), 2.90
(s, 3H), 2.94e2.99 (m, 1H), 3.02e3.07 (m, 1H), 3.05 (s, 3H), 3.65 (d,
1H, J¼16.0 Hz), 3.90 (overlapping m, 1H), 3.95 (d, 1H, J¼16.0 Hz),
4.21 (overlapping m, 2H), 4.32e4.42 (overlapping m, 3H),
4.53e4.65 (overlapping m, 3H), 4.77 (qu, 1H, J¼6.9 Hz), 5.01 (d, 1H,
J¼10.3 Hz), 5.22 (dd, 1H, J¼9.3, 3.6 Hz), 5.58 (m, 1H), 5.72 (br dd,
1H, J¼7.6, 7.1 Hz), 6.74 (d, 1H, J¼8.0 Hz), 6.87e6.91 (m, 1H), 7.06 (d,
1H, J¼8.2 Hz), 7.15e7.25 (m, 5H), 7.30 (t, 2H, J¼7.3 Hz), 7.39 (t, 2H,
J¼7.1 Hz), 7.59 (br d, 2H, J¼7.1 Hz), 7.75 (d, 2H, J¼7.3 Hz). Diethyl-
amine (2.1 mL) was added to a stirred solution of carboxylic acid
(426 mg, 0.378 mmol) in MeCN (21 mL). The mixture was stirred at
room temperature for 4 h and evaporated to dryness. The reside
was subjected to flash column chromatography on silica gel [n-
hexane, n-hexane/EtOAc (1:1), EtOAc, and CHCl3/MeOH (95:5),
(9:1), (8:2)] to give amino acid 5d as a colorless powder (329 mg,
0.364 mmol, 96%). Next, HOAt (166 mg, 1.22 mmol) and EDCI-HCl
(234 mg, 1.22 mmol) were added to a solution of amino acid 5d
(111 mg, 0.122 mmol) in CH2Cl2/DMF (10:1, 121 mL), and the mix-
ture was stirred at room temperature for 23 h. The mixture was
then diluted with EtOAc (100 mL) and washed with 1 M HCl
(30 mLꢁ2), H2O (30 mL), 5% aqueous NaHCO3 (30 mL) and brine
(30 mL). The organic layer was dried over Na2SO4 and evaporated
to dryness. The residue was subjected to flash column chroma-
tography on silica gel [n-hexane, n-hexane/EtOAc (1:1), (3:7),
4.3. Procedures for IC50 measurements against P388 and HeLa
S3 cells
4.3.1. Leukemia cell culture. P388 cells were maintained in
RPMI1640 medium containing 10% fetal bovine serum, 100
mg/mL
kanamycine, and 10 M 2-hydroxyethyldisulfide. The cells were
m
washed and resuspended in the above-described medium to
1ꢁ104 cells/mL, and 100
m
L of this cell suspension was placed in
each well of a 96-well flat-bottom plate. Next, 100
mL of different
concentrations of each test solution (1aef in medium) were added,
and 100 mL of medium was added into control wells. The cells were
incubated for 96 h at 37 ꢀC in 5% CO2/air.
HeLa S3 cells were maintained in MEM medium (GibcoBRL)
containing 10% fetal bovine serum, 2 mg/mL gentamicine and 10 mg/
mL antibiotics adjusted to pH 7.0e7.4 by 1 M HCl. The cells were
washed and resuspended in the above medium to 1000 cells/mL,
and 200
well flat-bottom plate. The cells were incubated for 24 h at 37 ꢀC in
5% CO2/air. After incubation, 2 L of different concentrations of each
mL of this cell suspension was placed in each well of a 96-
m
test solution were added, and the cells were incubated for 96 h at
37 ꢀC in 5% CO2/air.
4.3.2. MTT assay. After terminating the cell culture, 50
mL of 3-(4,5-
EtOAc, and CHCl3/MeOH (95:5)] to give cyclic depsipeptide 12d as
dimethyl-2-tiazoyl)-2,5-diphenyl-Htetrazolium bromide (MTT) in
20
a colorless amorphous powder (83 mg, 0.0938 mmol, 77%). [
a
]
saline (1 mg/mL) was added to every well, and the plate was rein-
D
ꢃ8.9 (c 0.35, CHCl3); IR (KBr disk) 3296, 2959, 1659, 1634, 1269,
1054, 754 cmꢃ1, Characteristic signals of two rotamers are shown,
but some protons could not be determined due to overlap (major
cubated at 37 ꢀC in 5% CO2/air for an additional 3 h. The plate was
then centrifuged at 800ꢁ g for 5 min to precipitate the cells and
formazan. The supernatant was removed from every well, and
rotamer). 1H NMR (500 MHz, CDCl3)
d
0.79e0.81 (overlapping m,
100 mL of DMSO was added to dissolve the formazan crystals. The
6H), 0.87e0.97 (overlapping m, 12H), 1.22 (m, 1H), 1.46 (d, 3H,
J¼7.2 Hz), 1.61 (overlapping s, d and m, 7H), 1.68e1.79 (overlapping
m, 3H), 1.85 (s, 3H), 2.00 (m, 1H), 2.12 (s, 3H), 2.26 (m, 1H), 2.38 (m,
2H), 3.01 (overlapping s and m, 4H), 3.04 (s, 3H), 3.12 (dd, 1H,
J¼14.1, 9.0 Hz), 3.50 (d, 1H, J¼16.8 Hz), 3.82 (dt, 1H, J¼9.4, 3.0 Hz),
3.85 (d, 1H, J¼16.8 Hz), 4.25 (dd, 1H, J¼8.2, 8.2 Hz), 4.46e4.56
(overlapping m, 1H), 4.60 (s, 3H), 4.77 (dq, 1H, J¼7.1, 6.8 Hz), 5.02
(d, 1H, J¼10.4 Hz), 5.22 (dd, 1H, J¼6.7, 6.1 Hz), 5.48 (overlapping m,
2H), 6.78 (overlapping m, 2H), 6.99 (d, 1H, J¼7.0 Hz), 7.14e7.24 (m,
5H), 7.72 (d, 1H, J¼9.4 Hz); dC 9.8 (q), 10.7 (q), 11.4 (q), 12.4 (q), 12.9
(q), 13.7 (q), 15.6 (q), 16.6 (q), 17.9 (q), 21.6 (q), 23.0 (q), 24.4 (d),
24.6 (t), 28.7 (t), 30.8 (q), 34.8 (d), 35.0 (t), 35.8 (q), 36.9 (d), 39.9
(t), 44.4 (d), 48.0 (d), 51.7 (t), 54.7 (d), 57.8 (d), 72.2 (d), 73.4 (t),
74.5 (d), 81.9 (d), 126.6 (d, 2C), 128.2 (s), 128.5 (2C, d), 129.1 (2C, d),
131.6 (s), 136.1 (s), 140.4 (d), 166.6 (s), 168.1 (s), 169.5 (s), 170.6 (s,
plate was mixed on a microshaker for 10 min and then read on
a microplate reader at 510 nm. A doseeresponse curve was plotted
for each drug, and the concentration that yielded 50% inhibition of
cell growth (IC50) was calculated.
Acknowledgements
The authors thank Ms. Eriko Mori and Ms. Ei Horii at the Aoyama
Gakuin University for support in the synthesis. This work was
supported by a Grant-in-Aid for Scientific Research (KAKENHI) (No.
1931013B) and Strategic Research Foundation Grant-aided Project
for Private Universities (No. S0901031) from the Japan Society for
the Promotion of Science and the Ministry of Education, Culture,
Sports, Science and Technology (MEXT).

Y. Takada et al. / Tetrahedron 68 (2012) 659e669
669
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Supplementary data
Supplementary data related to this article can be found online at
doi:10.1016/j.tet.2011.10.094. These data include MOL files and
InChiKeys of the most important compounds described in this
article.
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