Synthesis of 15,20-triamide analogue with polar substituent on the phenyl ring of arenastatin A, an extremely potent cytotoxic spongean depsipeptide

Article abstract of DOI:10.1016/j.bmc.2006.07.019

In order to increase metabolic stability and water solubility of arenastatin A, an extremely potent cytotoxic depsipeptide from the Okinawan marine sponge of Dysidea arenaria, several 15,20-triamide analogues with a polar substituent on the phenyl ring were synthesized. The 15,20-triamide analogues with a polar substituent (24, 30, and 31) showed increased solubility to MeOH and stronger cytotoxicity against KB cells in comparison with the parental 15,20-triamide analogue (2). Furthermore, the diethylamine analogue (30) exhibited in vivo anti-tumor activity against subcutaneously implanted murine sarcoma.

Full text of DOI:10.1016/j.bmc.2006.07.019

Bioorganic & Medicinal Chemistry 14 (2006) 7446–7457
Synthesis of 15,20-triamide analogue with polar substituent on the
phenyl ring of arenastatin A, an extremely potent cytotoxic
spongean depsipeptide
Naoyuki Kotoku,a Tomoya Kato,a Fuminori Narumi,a Emiko Ohtani,a Sayo Kamada,a
Shunji Aoki,a Naoki Okada,b Shinsaku Nakagawab and Motomasa Kobayashia,*
aDepartment of Natural Product Chemistry, Graduate School of Pharmaceutical Sciences, Osaka University, Japan
bDepartment of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Osaka University,
1-6 Yamada-oka, Suita, Osaka 565-0871, Japan
Received 12 June 2006; revised 3 July 2006; accepted 7 July 2006
Available online 31 July 2006
Abstract—In order to increase metabolic stability and water solubility of arenastatin A, an extremely potent cytotoxic depsipeptide
from the Okinawan marine sponge of Dysidea arenaria, several 15,20-triamide analogues with a polar substituent on the phenyl ring
were synthesized. The 15,20-triamide analogues with a polar substituent (24, 30, and 31) showed increased solubility to MeOH and
stronger cytotoxicity against KB cells in comparison with the parental 15,20-triamide analogue (2). Furthermore, the diethylamine
analogue (30) exhibited in vivo anti-tumor activity against subcutaneously implanted murine sarcoma.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
insoluble in polar solvents such as MeOH, DMSO,
and water, so it could not be applied to in vivo biolog-
ical evaluation (see Fig. 1).4
In the course of our search for bioactive substances from
marine organisms, we have isolated and characterized
arenastatin A (1), a cyclic depsipeptide having extremely
potent cytotoxic activity (IC50 = 5 pg/mL) against KB
cells, from the Okinawan marine sponge of Dysidea
arenaria.1 Thereafter, we achieved the total synthesis
of 12 and elucidated the activity of 1 to be ascribable
to the inhibition of microtubule assembly by binding
to the rhizoxin/maytansine site on tubulin.3 On the other
hand, 1 was found to exhibit only marginal in vivo anti-
tumor activity in the case of intravenous administration,
because of a rapid metabolism of the 15,20-ester linkage
in 1 in mouse serum. In order to overcome this biologi-
cal lability of 1, we have synthesized 15,20-triamide ana-
logue (2), in which the labile ester function of 1 was
replaced by an amide moiety, and found that analogue
2 showed sufficient stability in serum and moderate
cytotoxicity (IC50 = 6 ng/mL). However, 2 was almost
O
O
O
O
X
HN
HN
15
O
OMe
20
O
arenastatin A (1): X = O
15,20-triamide analogue (2): X = NH
O
O
O
O
O
HN
HN
Cl
O
OMe
21
O
R'
R
Keywords: Arenastatin A; Depsipeptide; Marine sponge; Structure–
activity relationship.
cryptophycin 1 (3): R = Me, R' = H
cryptophycin 52 (4): R = R' = Me
*
Corresponding author. Tel.: +81 6 6879 8215; fax: +81 6 6879
8219; e-mail: kobayasi@phs.osaka-u.ac.jp
Figure 1.
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.07.019

N. Kotoku et al. / Bioorg. Med. Chem. 14 (2006) 7446–7457
7447
Cryptophycins, a family of closely related depsipeptides,
have been found from terrestrial cyanobacterium of
Nostoc sp.5 Some of them, such as cryptophycin 1 (3)
having a C21-methyl group which prevents hydrolysis
of the 15,20-ester function, exhibited not only potent
cytotoxicity but also excellent in vivo anti-tumor activity.
Due to their potent bioactivity and synthetically attrac-
tive structure, many synthetic studies have been
reported, including the total synthesis of these natural
products and structure–activity relationship (SAR) stud-
ies through the synthesis of several analogues.6 Among
them, cryptophycin 52 (4), a synthetic analogue with a
gem-dimethyl group on the C-21 position, has been
selected for clinical development.7 Recently, the SAR
study of cryptophycin 52 (4) by Al-awar and co-workers
revealed that substitution on the phenyl ring of the left
side of the molecule was well tolerated and some ana-
logues with improved aqueous solubility showed potent
anti-tumor activity.8 This information led us to synthe-
size the 15,20-triamide analogues with a polar substitu-
ent on the phenyl ring, in order to improve their
solubility and to evaluate their biological activity. Here
we report the full details of our synthetic study and
biological evaluation.
(DMAP) to give an ester 17. After deprotection of the
TBS group of 17 by tetra-n-butylammonium fluoride
(TBAF) and following Dess–Martin oxidation of the
alcohol 18, the resulting aldehyde 6 was subjected to
Horner–Emmons reaction with segment BD (7)4 to give
an a,b-unsaturated amide (19). After deprotection of the
N-Boc group and O-(2-trimethylsilyl)ethyl group of 19
by trifluoroacetic acid (TFA), HCl/Et2O treatment of
the resulting amine 20 and intramolecular macrocycliza-
tion by using diphenylphosphoryl azide (DPPA) affor-
ded the objective cyclic depsipeptide 5 in good yield
(Scheme 2).
2.4. Heck reaction—preliminary study
With the required cyclic peptide 5 in hand, the introduc-
tion of an aryl moiety with a polar substituent was
examined. As a similar synthetic strategy using the Heck
reaction has been reported by Georg’s group,10 we first
tried a coupling reaction between aryl iodide 21 and the
cyclic peptide 5 according to their method. Thus, 5 and
21 were treated with Pd(OAc)2 and Et3N in hot CH3CN,
but the reaction hardly proceeded and resulted in recov-
ery of the starting material. In order to find better reac-
tion conditions, we examined the model reaction using
compound 17, as shown in Scheme 3 and Table 1. It
was revealed that the condition using an inorganic base
and a phosphine ligand was effective for this reaction
(entry 2), and the reaction was further activated by the
addition of tetra-n-butylammonium chloride to give de-
sired coupling product 22 in good yield (entry 3).11
Either DMF and CH3CN can be used for the reaction
solvent (entry 4).
2. Chemistry
2.1. Synthetic strategy
Figure 2 shows our strategy toward synthesis of 15,20-
triamide analogues with a polar substituent. Relatively
unstable b-7,8-epoxide should be introduced in the final
step by the oxidation of olefin. In order to prepare var-
ious analogues with a polar substituent on the phenyl
ring efficiently, the aryl moiety was introduced through
Heck reaction between aryl halide and a cyclic depsipep-
tide 5 having a terminal olefin. This approach made the
synthesis flexible for incorporation of various aryl sub-
stituents. The cyclic peptide 5 would be synthesized in
a similar manner with our synthesis of 15,20-triamide
analogue (2),4 namely, sequential condensation of four
segments A–D (8–11) and macrocyclization at the
15,20-amide bond.
2.5. Synthesis of ether and ester analogues
With the use of the conditions developed above, Heck
coupling between cyclic peptide 5 and aryl iodide 21
proceeded to give 23, but the yield of the coupling prod-
uct 23 was not satisfactory. After further investigation,
we found that the addition of water accelerated the reac-
tion and gave the coupling product 23 in improved yield
(54%). Finally, treatment of 23 with dimethyldioxirane
at ꢀ20 ꢁC gave the desired 15,20-triamide analogue 24
having b-epoxide, after separation of the a/b mixture
(a/b = 1:2.6) by reversed-phase HPLC.12 Analogue 27
having an ester moiety was synthesized from 23 in three
steps, for example, deprotection of the MOM group of
23, condensation of the resulting alcohol 25 with N-
Boc-glycine, and epoxidation of 26 by dimethyldioxira-
ne (Scheme 4).
2.2. Preparation of segment A
For the stereoselective synthesis of segment A, we
decided to use the same strategy with our total synthe-
sis of arenastatin A (1) as shown in Scheme 1.2b Evans’
syn-selective asymmetric aldol reaction of chiral N-
crotonyl oxazolidinone (12)9 and an aldehyde 13 pro-
ceeded stereoselectively to afford the desired (2R,3S)-
adduct 14 in good yield. Removal of the chiral auxilia-
ry with LiBH4 and reductive deoxygenation of the
resulting primary hydroxyl group of 15 gave segment
A (8).
2.6. Synthesis of amine and ammonium analogues
Next, we examined synthesis of the analogues having
an amine moiety on the phenyl ring. We planned that
such analogues would be efficiently prepared from
benzyl chloride 28, by treatment with the correspond-
ing amines. First, we executed a direct approach to
synthesize the key compound 28 through Heck reac-
tion between cyclic peptide 5 and 4-iodobenzyl chlo-
ride. However, the objective product could not be
obtained. So, the analogues having amine moieties
2.3. Synthesis of cyclic depsipeptide
Segment A (8) and commercially available N-Boc-L-leu-
cine (9) were coupled by 1,3-dicyclohexylcarbodiimide
(DCC) in the presence of 4-dimethylaminopyridine

7448
N. Kotoku et al. / Bioorg. Med. Chem. 14 (2006) 7446–7457
X
8
5
O
O
5
6
R
7
O
R
O
O
HN
O
O
HN
20
NH HN
O
OMe
NH HN
O
OMe
O
O
15
15,20-triamide analogue with polar substituent
(R: water-soluble substituent)
O
P
MeO
MeO
O
OTBS
O
HN
OH
O
O
segment A (8)
TMS
O
HN
O
7
OMe
NHBoc
HO
O
O
6
NHBoc
segment C (9)
TMS
CbzHN
HO
O
NH2
O
O
OMe
segment B (11)
segment D (10)
Figure 2. Retrosynthetic analysis.
Ph
Me
O
Me
O
O
N
a
O
OTBS
N
+
Ph
OHC
O
3
OTBS
O
13
2
12
OH
14
OR
OH
b, c
d
OTBS
OTBS
OH
15 : R = H
16 : R = Ts
8
Scheme 1. Reagents and conditions: (a) Bu2BOTf, Et3N, CH2Cl2, ꢀ78 ꢁC, 91%; (b) Bu3B, AcOH, LiBH4, THF, 88%; (c) TsCl, pyridine, 90%;
(d) NaBH4, DMSO, 73%.
O
OR
O
O
c-e
a, b
HN
OTBS
O
O
NHR
HN
OH
8
OMe
O
O
NHBoc
R'O
17: R = TBS
18: R = H
19: R = Boc, R' = 2-trimethylsilylethyl
20: R = R' = H
O
f
O
O
HN
NH HN
O
OMe
O
5
Scheme 2. Reagents and conditions: (a) 9, DCC, DMAP, CH2Cl2, 100%; (b) TBAF, AcOH, THF, 100%; (c) Dess–Martin periodinane, CH2Cl2;
(d) 7, NaH, THF, 75% (two steps); (e) TFA, CH2Cl2; HCl/Et2O; (f) DPPA, NaHCO3, DMF, 89% (two steps).

N. Kotoku et al. / Bioorg. Med. Chem. 14 (2006) 7446–7457
7449
I
MOMO
OTBS
OTBS
21
MOMO
O
O
O
O
Pd(OAc)2 (5 mol%)
Phosphine, additive
NaHCO3, 80 ˚C
NHBoc
NHBoc
22
17
Scheme 3.
Table 1. Heck reaction between 17 and 21a
3. Biological evaluation
3.1. In vitro cell growth inhibition
Entry
PPh3
(10 mol%)
n-Bu4NCl
(100 mol%)
Solvent
Yield
(%)
1
2
3
4
ꢀ
+
+
+
ꢀ
ꢀ
+
+
DMF
DMF
DMF
CH3CN
n.d.
45
The growth inhibitory effect of the synthesized 15-tria-
mide analogues with a polar substituent on the phenyl
ring against KB 3-1 cells was evaluated as summarized
in Table 2, together with their solubility in MeOH. All
analogues showed better solubility than the parental
15,20-triamide analogue (2). In the case of 31, having
a piperazine moiety at the phenyl ring, 85-fold solubility
was observed.
77
79
a All reaction was performed in the presence of Pd(OAc)2 (5 mol%) and
NaHCO3 (250 mol%) at 80 ꢁC.
were synthesized from benzyl alcohol 25, as shown in
Scheme 5. Benzyl alcohol 25 was converted to the cor-
responding chloride 28 in good yield, by treatment
with MsCl and LiCl.13 Oxidation of 28 with dim-
ethyldioxirane and the following HPLC separation
gave b-epoxide 29,12 and subsequent treatment with
diethylamine or piperazine afforded the corresponding
tertiary amine analogues (30, 31) without affecting
the labile epoxide moiety. Quaternary ammonium
analogues (32 and 33), aiming at improved water sol-
ubility by their constantly charged structure, were
also prepared by the treatment of 29 with triethyl-
amine and 1,4-diazabicyclo[2.2.2]octane (DABCO),
respectively.
Among them, diethylamine analogue (30) exhibited the
most potent cytotoxic activity (IC50 = 0.18 ng/mL),
which was 30-fold greater than that of 2. MOM-ether
derivative (24) also showed increasing activity, 10 times
as potent as 2. On the other hand, quaternary ammo-
nium analogues (32 or 33) showed disappointing
results in terms of both cytotoxicity and solubility in
MeOH.
3.2. In vivo test for anti-tumor activity
The in vivo anti-tumor testing of diethylamine analogue
(30), possessing both potent cytotoxicity and good solu-
O
O
RO
O
O
HN
O
O
HN
a
NH HN
O
OMe
NH HN
5
O
OMe
O
O
23: R = MOM
c
25: R = H
O
d
26: R =
NHBoc
O
O
b
RO
O
O
HN
NH HN
O
OMe
O
O
NHBoc
27: R =
24: R = MOM
Scheme 4. Reagents and conditions: (a) 21, Pd(OAc)2, Ph3P, NaHCO3, Bu4NCl, DMF/H2O, 80 ꢁC, 54%; (b) dimethyldioxirane, CH2Cl2/MeOH,
53% for 24; 48% for 27; (c) 6 M HCl, MeOH, 72%; (d) N-Boc-Gly, DCC, DMAP, CH2Cl2, 100%.

7450
N. Kotoku et al. / Bioorg. Med. Chem. 14 (2006) 7446–7457
O
O
O
R
O
O
HN
Cl
O
O
HN
b
NH HN
O
OMe
NH HN
29
O
OMe
O
O
25: R = OH
a
28: R = Cl
O
+
30: R = NEt2
O
O
32: R = NEt3
c
R
O
HN
NH
N
31: R =
33: R =
NH HN
O
OMe
N
N
O
Scheme 5. Reagents and conditions: (a) MsCl, LiCl, c-collidine, DMF, 93%; (b) dimethyldioxirane, CH2Cl2/MeOH, 60%; (c) amine, DMF, 85% for
30; 80% for 31; 86% for 32; 85% for 33.
Table 2. In vitro growth inhibitory activity of arenastatin A analogues
and their solubility in MeOH
4. Summary
From the SAR study of 15,20-triamide analogues with a
polar substituent on the phenyl ring, the analogues with
diethylamine or MOM-ether substituent (30 or 24,
respectively) were found to exhibit more than 10-fold
potent cytotoxic activity against KB cells in comparison
with that of the parental 15,20-triamide analogue (2).
Furthermore, diethylamine analogue (30) showed to
exhibit in vivo anti-tumor activity against subcutaneous-
ly implanted murine sarcoma.
Compound
IC50 (ng/mL)
Solubility (mg/mL)
2
24
27
30
31
32
33
6
0.2
2
0.61
2.16
0.18
1.5
19
2.6
13
17
7.2
4.9
44
5. Experimental
5.1. General
bility in MeOH, was carried out. Murine sarcoma S180
cells were implanted subcutaneously, and the testing
compound was administered intraperitoneally every
other day for 2 weeks. The effectiveness of the testing
compound was determined by measuring the median tu-
mor diameter. It was found that diethylamine analogue
(30) inhibited the growth of tumor at the dose of 1 mg/
kg, exhibiting comparable efficacy as that of doxorubi-
cin (positive control) (Table 3). Even though diethyl-
amine analogue (30) showed toxicity at the 5 mg/kg
dose, 1 mg/kg administration of 30 showed no signifi-
cant acute toxicity such as body weight loss. As expect-
ed, arenastatin A analogue with improved stability and
water solubility could be a promising candidate for
anti-tumor agent.
All the reaction solvents were distilled prior to use. The
following instruments were used to obtain physical data:
a JASCO DIP-370 digital polarimeter for specific rota-
tions; a JASCO FT/IR-5300 infrared spectrometer for
IR spectra; a Waters Q-Tof Ultima API mass spectrom-
eter for ESI-TOF MS; a JEOL JNM AL-500 NMR
1
spectrometer for H and 13C NMR using tetramethylsi-
lane as an internal standard. HPLC was performed
using a Hitachi L-6000 pump equipped with Hitachi
L-4000H UV detector. Silica gel (Fuji Silysia BW-200
or Chromatorexꢂ-NH2) and pre-coated thin-layer chro-
matography (TLC) plates (Merck, 60F254) were used for
column chromatography and TLC. Spots on TLC plates
were detected by spraying acidic p-anisaldehyde solution
(p-anisaldehyde: 25 mL, c-H2SO4: 25 mL, AcOH: 5 mL,
and EtOH: 425 mL) with subsequent heating.
Table 3. In vivo anti-tumor effect of analogue 30
Compound
Dose (mg/kg)a
Tumor size (mm)b
30
1
0.5
11.7 1.7
12.7 2.3
5.1.1. (4R,5S)-3-{(2R)-2-[(1S)-3-(tert-Butyldimethylsila-
nyloxy)-1-hydroxypropyl]-but-3-enoyl}-4-methyl-5-phenyl-
oxazolidin-2-one (14). Under argon atmosphere, a THF
solution of Bu2BOTf (1.0 M, 7.3 mL, 7.3 mmol) was
added dropwise to a solution of 12 (1.5 g, 6.1 mmol) in
CH2Cl2 (23 mL) at ꢀ78 ꢁC. After being stirred for
10 min, Et3N (1.3 mL, 9.2 mmol) was added to the reac-
Doxorubicin
Control
5
3
11.4 2.4
11.5 1.6
13.1 2.0
a Ip administration (days 3, 5, 7, 9, 11, and 13).
b Mean SD (n = 8).

N. Kotoku et al. / Bioorg. Med. Chem. 14 (2006) 7446–7457
7451
tion mixture, and the whole mixture was stirred at this
temperature for 20 min, then warmed to 0 ꢁC and stirred
for 30 min. After the reaction mixture was cooled again
to ꢀ78 ꢁC, 13 (1.4 g, 7.3 mmol) was added dropwise to
the reaction mixture, and the whole mixture was stirred
at this temperature for 2 h, then warmed to 0 ꢁC and
stirred for 1 h. The reaction was quenched with water,
and the whole mixture was extracted with EtOAc. The
EtOAc extract was washed with saturated aqueous
NaCl, then dried over Na2SO4. Removal of the solvent
from the EtOAc extract under reduced pressure gave a
crude product, which was purified by column chroma-
tography (SiO2; CHCl3) to give 14 (2.41 g, 91%) as col-
orless oil.
18.1, ꢀ5.5 (2C). ESI-MS: m/z 283 (M + Na)+. HR-
ESI-MS: m/z 283.1705, calcd for C13H28O3SiNa. Found:
283.1709. Anal. Calcd for C13H28O3Si: C, 59.95; H,
10.84. Found: C, 59.76; H, 10.52.
5.1.3.
(2R)-2-[(1S)-3-(tert-Butyldimethylsilanyloxy)-1-
hydroxypropyl]-but-3-enyl toluene-4-sulfonate (16). p-
TsCl (1.37 g, 7.2 mmol) was added to a solution of 15
(1.80 g, 6.9 mmol) in pyridine (10 mL) at 0 ꢁC. The
whole mixture was stirred at this temperature for
30 min, then warmed to rt and stirred for 5 h. The reac-
tion was quenched with saturated aqueous NaHCO3,
and the whole mixture was extracted with EtOAc. The
EtOAc extract was washed with saturated aqueous
NaCl, then dried over Na2SO4. Removal of the solvent
from the EtOAc extract under reduced pressure gave a
crude product, which was purified by column chroma-
tography (SiO2; n-hexane/EtOAc 10:1) to give 16
(2.57 g, 90%) as colorless oil.
20
Compound 14: ½aꢁ +45 (c 1.46, CHCl3). IR (KBr):
D
3491, 2930, 2858, 1782, 1699 cmꢀ1
.
1H NMR
(500 MHz, CDCl3) d: 7.37–7.44 (m, 3H), 7.30 (d,
J = 6.7 Hz, 2H), 6.06 (ddd, J = 8.5, 10.4, 17.1 Hz, 1H),
5.68 (d, J = 7.3 Hz, 1H), 5.33 (d, J = 10.4 Hz, 1H),
5.32 (d, J = 17.1 Hz, 1H), 4.81 (dq, J = 6.7, 7.3 Hz,
1H), 4.54 (dd, J = 4.3, 8.5 Hz, 1H), 4.24–4.27 (m, 1H),
3.80–3.90 (m, 2H), 3.67 (d, J = 1.8 Hz, 1H), 1.75–1.82
(m, 1H), 1.65–1.70 (m, 1H), 0.89 (d, J = 6.7 Hz, 3H),
0.90 (s, 9H), 0.08 (s, 6H). 13C NMR (125 MHz, CDCl3)
d: 173.2, 152.6, 133.3, 131.9, 128.9 (2C), 128.8 (2C),
125.7, 120.4, 78.9, 71.3, 61.7, 55.0, 53.0, 36.3, 25.9
(3C), 18.2, 14.3, ꢀ5.5 (2C). ESI-MS: m/z 456
(M + Na)+. HR-ESI-MS: m/z 456.2182, calcd for
C23H35NO5SiNa. Found: 456.2180. Anal. Calcd for
C23H35NO5Si: C, 63.71; H, 8.14; N, 3.23. Found: C,
63.62; H, 8.11; N, 3.21.
20
Compound 16: ½aꢁ +20 (c 1.70, CHCl3). IR (KBr):
D
3495, 2955, 2858, 1599, 1469 cmꢀ1
.
1H NMR
(500 MHz, CDCl3) d: 7.79 (d, J = 8.5 Hz, 2H), 7.33 (d,
J = 8.5 Hz, 2H), 5.70 (ddd, J = 8.5, 10.4, 17.7 Hz, 1H),
5.19 (dd, J = 1.2, 10.4 Hz, 1H), 5.12 (dd, J = 1.2,
17.7 Hz, 1H), 4.30 (dd, J = 7.3, 9.8 Hz, 1H), 4.02 (dd,
J = 6.7, 9.8 Hz, 1H), 3.99–4.01 (m, 1H), 3.86 (dt,
J = 4.3, 9.8 Hz, 1H), 3.78 (dd, J = 3.1, 9.8 Hz, 1H),
3.33 (br s, 1H), 2.44 (s, 3H), 2.40–2.45 (m, 1H), 1.69
(ddt, J = 4.3, 9.8, 14.0 Hz, 1H), 1.43–1.48 (m, 1H),
0.88 (s, 9H), 0.06 (s, 6H). 13C NMR (125 MHz, CDCl3)
d: 144.7, 133.1, 133.0, 129.8 (2C), 128.0 (2C), 119.7, 70.4,
70.4, 62.8, 49.3, 36.1, 25.9 (3C), 21.6, 18.1, ꢀ5.6 (2C).
ESI-MS: m/z 437 (M + Na)+. HR-ESI-MS: m/z
437.1794, calcd for C20H34O5SiSNa. Found: 437.1810.
Anal. Calcd for C20H34O5SiS: C, 57.93; H, 8.27; S,
7.73. Found: C, 57.68; H, 8.10; S, 7.74.
5.1.2. (2R,3S)-5-(tert-Butyldimethylsilanyloxy)-2-vinyl-
pentane-1,3-diol (15). Bu3B solution (1.0 M in THF,
13.8 mL, 13.8 mmol) was added to a solution of 14
(5.0 g, 11.5 mmol) and AcOH (2.0 mL, 34.5 mmol) in
THF (30 mL) at 0 ꢁC under argon atmosphere, and
the whole mixture was stirred for 30 min, then cooled
to ꢀ78 ꢁC. LiBH4 (2.0 M in THF, 11.5 mL, 23.0 mmol)
was added dropwise to the reaction mixture and stirred
at this temperature for 4 h, then warmed to 0 ꢁC and
stirred for 30 min. The reaction mixture was successively
treated with H2O (10 mL), 3N NaOH (10 mL), and 30%
H2O2 (10 mL) at 0 ꢁC. After being stirred for 15 min, the
whole mixture was extracted with EtOAc. The EtOAc
extract was washed with saturated aqueous NaCl, then
dried over Na2SO4. Removal of the solvent from the
EtOAc extract under reduced pressure gave a crude
product, which was purified by column chromatography
(SiO2; n-hexane/EtOAc 3:1) to give 15 (2.55 g, 88%) as
colorless oil.
5.1.4. (3S,4R)-1-(tert-Butyldimethylsilanyloxy)-4-methyl-
hex-5-en-3-ol (8). A solution of 16 (346 mg, 0.83 mmol)
in DMSO (1.6 mL) was treated with NaBH4 (63 mg,
1.7 mmol) at rt for 2 h. The reaction was quenched
with saturated aqueous NaHCO3 and the whole mix-
ture was extracted with EtOAc. The EtOAc extract
was washed with saturated aqueous NaCl, then dried
over Na2SO4. Removal of the solvent from the EtOAc
extract under reduced pressure gave a crude product,
which was purified by column chromatography (SiO2;
n-hexane/EtOAc 10:1) to give 8 (148 mg, 73%) as color-
less oil.
20
Compound 8: ½aꢁ +9 (c 0.67, CHCl3). IR (KBr): 3506,
D
2957, 2858, 1464, 1255 cmꢀ1
.
1H NMR (500 MHz,
20
Compound 15: ½aꢁ +1.3 (c 2.30, CHCl3). IR (KBr):
CDCl3) d: 5.80–5.87 (m, 1H), 5.05–5.08 (m, 2H), 3.89
(dt, J = 4.9, 10.4 Hz, 1H), 3.79–3.85 (m, 1H), 3.66–3.71
(m, 1H), 3.22 (d, J = 1.8 Hz, 1H), 2.21–2.28 (m, 1H),
1.62–1.66 (m, 2H), 1.04 (d, J = 6.7 Hz, 3H), 0.88 (s,
9H), 0.08 (s, 6H). 13C NMR (125 MHz, CDCl3) d:
140.8, 115.1, 75.0, 62.8, 44.0, 35.6, 25.9 (3C), 18.2,
15.8, ꢀ5.5 (2C). ESI-MS: m/z 267 (M + Na)+. HR-
ESI-MS: m/z 267.1756, calcd for C13H28O2SiNa. Found:
267.1758. Anal. Calcd for C13H28O2Si: C, 63.87; H,
11.55. Found: C, 63.65; H, 11.29.
D
3391, 2930, 2858, 1639, 1471 cmꢀ1
.
1H NMR
(500 MHz, CDCl3) d: 5.87 (ddd, J = 8.5, 10.4, 17.1 Hz,
1H), 5.22 (dd, J = 1.2, 10.4 Hz, 1H), 5.15 (dd, J = 1.2,
17.1 Hz, 1H), 4.10 (dt, J = 2.4, 9.8 Hz, 1H) 3.92 (dt,
J = 4.3, 14.0 Hz, 1H), 3.83 (dt, J = 3.7, 10.4 Hz, 1H),
3.73–3.79 (m, 3H), 2.65 (br s, 1H), 2.34 (m, 1H), 1.80
(ddt, J = 4.3, 9.8, 14.6 Hz, 1H), 1.52–1.57 (m, 1H),
0.90 (s, 9H), 0.08 (s, 6H). 13C NMR (125 MHz, CDCl3)
d: 135.3, 118.3, 73.8, 64.5, 63.1, 51.2, 35.7, 25.9 (3C),

7452
N. Kotoku et al. / Bioorg. Med. Chem. 14 (2006) 7446–7457
5.1.5. (1S,2R)-1-[2-(tert-Butyldimethylsilanyloxy)ethyl]-
2-methylbut-3-enyl (2S)-2-tert-butoxycarbonylamino-4-
methylpentanoate (17). N-Boc-L-leucine (9, 1.87 g,
7.5 mmol), DMAP (910 mg, 7.5 mmol), and DCC
(2.58 g, 12.5 mmol) were successively added to a solu-
tion of 8 (630 mg, 2.5 mmol) in CH2Cl2 (10 mL), then
the whole mixture was stirred at rt for 2 h. 5% HCl
was added to the reaction mixture, then diluted with
Et2O. The whole mixture was filtered to remove the pre-
cipitate, then the filtrate was extracted with Et2O. The
Et2O extract was washed with saturated aqueous NaCl,
then dried over Na2SO4. Removal of the solvent from
the Et2O extract under reduced pressure gave a crude
product, which was purified by column chromatography
(SiO2; n-hexane/EtOAc 10:1) to give 17 (1.15 g, 100%) as
colorless oil.
Dess–Martin periodinane (14 mg, 0.042 mmol) at rt for
2 h. Then saturated aqueous NaHCO3 and saturated
aqueous Na2S2O3 were added to the reaction mixture
and stirred for 30 min. The whole mixture was extracted
with EtOAc. The EtOAc extract was washed with satu-
rated aqueous NaCl, then dried over MgSO4. Removal
of the solvent from the EtOAc extract under reduced
pressure gave a crude aldehyde 6.
NaH (2.5 mg, 0.105 mmol) was added to a solution of
segment BD (7) (22 mg, 0.042 mmol) in THF (1 mL)
at ꢀ10 ꢁC, then the whole mixture was stirred for
30 min. A solution of the above aldehyde 6 in THF
(0.3 mL) was added dropwise to the reaction mixture,
then the whole mixture was stirred at 0 ꢁC for 1 h. The
reaction was quenched with saturated aqueous NH4Cl,
then the whole mixture was extracted with EtOAc.
The EtOAc extract was washed with saturated aqueous
NaCl, then dried over Na2SO4. Removal of the solvent
from the EtOAc extract under reduced pressure gave a
crude product, which was purified by column chroma-
tography (SiO2; n-hexane/EtOAc 2:1) to give 19
(12 mg, 75% in two steps) as yellow oil.
20
Compound 17: ½aꢁ ꢀ26 (c 1.49, CHCl3). IR (KBr):
D
3373, 2959, 2860, 1716, 1501 cmꢀ1
.
1H NMR
(500 MHz, CDCl3) d: 5.69–5.76 (m, 1H), 5.00–5.07 (m,
3H), 4.86 (d, J = 8.5 Hz, 1H), 4.25–4.35 (m, 1H),
3.54–3.65 (m, 2H), 2.42–2.49 (m, 1H), 1.69–1.79 (m,
3H), 1.56–1.62 (m, 1H), 1.42–1.48 (m, 1H), 1.44 (s,
9H), 1.01 (d, J = 7.3 Hz, 3H), 0.95 (d, J = 6.1 Hz, 3H),
0.94 (d, J = 6.7 Hz, 3H), 0.88 (s, 9H), 0.03 (s, 6H). 13C
NMR (125 MHz, CDCl3) d: 173.0, 155.6, 139.2, 115.8,
79.6, 75.0, 59.5, 52.4, 42.1, 41.5, 34.4, 28.4 (3C), 25.9
(3C), 24.9, 23.0, 21.9, 18.3, 15.8, ꢀ5.4 (2C). ESI-MS:
m/z 480 (M + Na)+. HR-ESI-MS: m/z 480.3121, calcd
for C24H47NO5SiNa. Found: 480.3113. Anal. Calcd
for C24H47NO5Si: C, 62.98; H, 10.35; N, 3.06. Found:
C, 62.93; H, 10.24; N, 3.09.
20
Compound 19: ½aꢁ +3.3 (c 0.79, CHCl3). IR (KBr):
D
3279, 3076, 2957, 1716, 1512 cmꢀ1
.
1H NMR
(500 MHz, CDCl3) d: 7.11 (d, J = 8.5 Hz, 2H), 6.80 (d,
J = 8.5 Hz, 2H), 6.72 (dt, J = 6.7, 15.8 Hz, 1H), 6.55
(d, J = 7.3 Hz, 1H), 6.25 (brs, 1H), 5.84 (d,
J = 15.8 Hz, 1H), 5.70 (dt, J = 9.2, 17.7 Hz, 1H), 5.04–
5.08 (m, 2H), 4.88–4.93 (m, 2H), 4.54–4.59 (m, 1H),
4.19–4.24 (m, 1H), 4.08–4.16 (m, 2H), 3.77 (s, 3H),
3.44–3.49 (m, 1H), 3.32–3.36 (m, 1H), 2.92–3.08 (m,
2H), 2.29–2.46 (m, 5H), 1.69–1.75 (m, 2H), 1.55–1.61
(m, 1H), 1.45 (s, 9H), 1.02 (d, J = 6.7 Hz, 3H), 0.96 (t,
J = 7.9 Hz, 2H), 0.93 (m, 6H), 0.02 (s, 9H). 13C NMR
(125 MHz, CDCl3) d: 172.3, 172.1, 170.8, 165.4, 158.6,
155.6, 139.3, 138.7, 130.3 (2C), 128.8, 125.9, 116.4,
114.1 (2C), 80.0, 76.1, 63.0, 55.2, 55.0, 54.8, 41.4, 41.0,
37.6, 34.8, 34.0, 33.5, 28.4 (3C), 24.9, 23.0, 21.8, 17.3,
16.3, ꢀ1.5 (3C). ESI-MS: m/z 754 (M + Na)+. HR-
ESI-MS: m/z 754.4075, calcd for C38H61N3O9SiNa.
Found: 754.4075. Anal. Calcd for C38H61N3O9Si: C,
62.35; H, 8.40; N, 5.74. Found: C, 62.06; H, 8.35; N,
5.68.
5.1.6.
(1S,2R)-1-(2-Hydroxyethyl)-2-methylbut-3-enyl
(2S)-2-tert-butoxycarbonylamino-4-methylpentanoate (18).
A solution of 17 (500 mg, 1.1 mmol) and AcOH
(0.37 mL, 6.6 mmol) in THF (10 mL) was treated with
TBAF (1.0 M in THF, 3.3 mL, 3.3 mmol) at 0 ꢁC for
4 h. Removal of the solvent from the reaction mixture
under reduced pressure gave a crude product, which
was purified by column chromatography (SiO2; n-hex-
ane/EtOAc 3:1) to give 18 (374 mg, 100%) as colorless
oil.
20
Compound 18: ½aꢁ ꢀ47 (c 1.01, CHCl3). IR (KBr):
D
3368, 2962, 2874, 1711, 1518 cmꢀ1
.
1H NMR
(500 MHz, CDCl3) d: 5.69–5.77 (m, 1H), 5.04–5.09 (m,
3H), 4.86 (d, J = 7.9 Hz, 1H), 4.21–4.26 (m, 1H), 3.60–
3.64 (m, 2H), 2.58 (br s, 1H), 2.38-2.45 (m, 1H), 1.80–
1.86 (m, 1H), 1.65–1.79 (m, 2H), 1.57–1.62 (m, 1H),
1.47–1.49 (m, 1H), 1.44 (s, 9H), 1.04 (d, J = 6.7 Hz,
3H), 0.96 (d, J = 6.7 Hz, 3H), 0.95 (d, J = 6.7 Hz, 3H).
13C NMR (125 MHz, CDCl3) d: 174.6, 155.7, 139.1,
116.0, 80.1, 75.0, 58.5, 52.3, 42.1, 41.4, 34.6, 28.3 (3C),
24.9, 22.9, 21.8, 16.3. ESI-MS: m/z 366 (M + Na)+.
HR-ESI-MS: m/z 366.2256, calcd for C18H33NO5SiNa.
Found: 366.2255.
5.1.8. (3S,10R,16S)-3-Isobutyl-10-(4-methoxybenzyl)-16-
[(1R)-1-methylallyl]-1-oxa-4,8,11-triazacyclohexadec-13-
ene-2,5,9,12-tetraone (5). A solution of 19 (16 mg,
0.022 mmol) in CH2Cl2 (0.5 mL) was treated with
TFA (1 mL) at rt for 3 h. Removal of the solvent from
the reaction mixture under reduced pressure gave a
TFA salt of 20. Then the TFA salt was treated with
HCl in Et2O (1 mL, 4·) to furnish a HCl salt. DPPA
(7 lL, 0.033 mmol) and NaHCO3 (9.2 mg, 0.11 mmol)
were added to a solution of the HCl salt in DMF
(0.5 mL) at 0 ꢁC, and the whole mixture was stirred at
this temperature for 10 h. The reaction mixture was
poured into water, and the whole mixture was extracted
with EtOAc. The EtOAc extract was washed with satu-
rated aqueous NaCl, then dried over Na2SO4. Removal
of the solvent from the EtOAc extract under reduced
pressure gave a crude product, which was purified by
5.1.7. (1S,2R)-1-(3-{(1R)-2-(4-Methoxyphenyl)-1-[2-(2-
trimethylsilanylethoxycarbonyl)ethylcarbamoyl]ethylcar-
bamoyl}allyl)-2-methylbut-3-enyl (2S)-2-tert-butoxycar-
bonylamino-4-methylpentanoate (19). A solution of 18
(7.3 mg, 0.021 mmol) in CH2Cl2 (1 mL) was treated with

N. Kotoku et al. / Bioorg. Med. Chem. 14 (2006) 7446–7457
7453
column chromatography (SiO2; CHCl3/MeOH 30:1) to
give 5 (10 mg, 89%) as a white powder.
5.1.10. (3S,10R,16S)-3-Isobutyl-10-(4-methoxybenzyl)-16-
(1S)-1-[(2R,3R)-{3-[4-(methoxymethoxymethyl)phenyl]
oxiranyl}ethyl]-1-oxa-4,8,11-triazacyclohexadec-13-ene-2,
5,9,12-tetraone (24). A solution of 23 (5.0 mg, 7.5 lmol)
in CH2Cl2–MeOH (3:1, 0.3 mL) was treated with dim-
ethyldioxirane (0.074 M in acetone, 0.3 mL) at ꢀ20 ꢁC
for 2 h. Removal of the solvent from the reaction mix-
ture under reduced pressure gave a crude product, which
was purified by HPLC (COSMOSIL 5C18-AR-II,
MeOH/H2O 65:35) to give 24 (2.7 mg, 53%) as a white
powder.
20
Compound 5: ½aꢁ +33 (c 0.81, CHCl3). IR (KBr): 3294,
D
3074, 2957, 1736, 1658 cmꢀ1
.
1H NMR (500 MHz,
CDCl3) d: 7.08 (d, J = 8.5 Hz, 2H), 6.98 (d, J = 6.7 Hz,
1H), 6.82 (d, J = 8.5 Hz, 2H), 6.72 (ddd, J = 3.7, 11.0,
14.6 Hz, 1H), 6.15 (d, J = 8.5 Hz, 1H), 5.64–5.72 (m,
2H), 5.71 (d, J = 14.6 Hz, 1H), 5.05–5.23 (m, 3H),
4.59–4.63 (m, 1H), 4.54 (dd, J = 6.7, 8.5 Hz, 1H),
3.80–3.82 (m, 1H), 3.78 (s, 3H), 3.25–3.30 (m, 1H),
3.12 (dd, J = 4.9, 14.6 Hz, 1H), 2.97 (dd, J = 7.9,
14.6 Hz, 1H), 2.28–2.42 (m, 5H), 1.56–1.65 (m, 1H),
1.47 (t, J = 6.7 Hz, 2H), 1.04 (d, J = 6.7 Hz, 3H), 0.92
(d, J = 6.7 Hz, 3H), 0.90 (d, J = 6.1 Hz, 3H). 13C
NMR (125 MHz, CDCl3) d: 173.1, 171.6, 170.1, 165.0,
158.8, 142.1, 138.7, 130.1 (2C), 128.2, 124.9, 116.5,
114.3 (2C), 75.9, 55.3, 55.0, 50.6, 42.3, 41.8, 36.0, 35.8,
34.5, 34.2, 24.8, 22.6, 22.0, 16.5. ESI-MS: m/z 536
(M + Na)+. HR-ESI-MS: m/z 536.2737, calcd for
C28H39N3O6Na. Found: 536.2741.
20
Compound 24: ½aꢁ +47 (c 0.23, CHCl3). IR (KBr):
D
3288, 2928, 1738, 1660, 1543 cmꢀ1
.
1H NMR
(500 MHz, CDCl3) d: 7.35 (d, J = 7.9 Hz, 2H), 7.22 (d,
J = 7.9 Hz, 2H), 7.08 (d, J = 8.5 Hz, 2H), 6.98 (d,
J = 7.9 Hz, 1H), 6.83 (d, J = 8.5 Hz, 2H), 6.71 (ddd,
J = 4.0, 11.0, 14.6 Hz, 1H), 5.91 (d, J = 9.2 Hz, 1H),
5.67 (d, J = 14.6 Hz, 1H), 5.52 (d, J = 6.1 Hz, 1H),
5.26–5.29 (m, 1H), 4.71 (s, 2H), 4.60 (s, 2H), 4.57–4.59
(m, 1H), 4.50 (dd, J = 5.5, 9.8 Hz, 1H), 3.79 (s, 3H),
3.75–3.82 (m, 1H), 3.69 (d, J = 1.2 Hz, 1H), 3.42 (s,
3H), 3.21–3.26 (m, 1H), 3.13 (dd, J = 4.9, 14.6 Hz,
1H), 2.95 (dd, J = 7.9, 14.6 Hz, 1H), 2.91 (dd, J = 1.2,
7.3 Hz, 1H), 2.52–2.55 (m, 1H), 2.30–2.43 (m, 3H),
1.77–1.83 (m, 1H), 1.53–1.61 (m, 1H), 1.37–1.42 (m,
1H), 1.28–1.32 (m, 1H), 1.14 (d, J = 6.7 Hz, 3H), 0.86
(d, J = 6.1 Hz, 3H), 0.84 (d, J = 6.7 Hz, 3H). 13C
NMR (125 MHz, CDCl3) d: 173.1, 171.7, 170.6, 164.8,
158.8, 141.4, 138.4, 136.7, 130.4 (2C), 128.2 (2C),
128.0, 125.8 (2C), 125.1, 114.4 (2C), 95.7, 75.0, 68.7,
63.1, 58.8, 55.4, 55.3, 55.1, 50.5, 41.4, 40.5, 36.6, 35.8,
34.4, 34.0, 24.7, 22.6, 21.6, 13.3. ESI-MS: m/z 702
(M + Na)+. HR-ESI-MS: m/z 702.3367, calcd for
C37H49N3O9Na. Found: 702.3342.
5.1.9. (3S,10R,16S)-3-Isobutyl-10-(4-methoxybenzyl)-16-
{(1R)-3-[4-(methoxymethoxymethyl)phenyl]-1-methylal-
lyl}-1-oxa-4,8,11-triazacyclohexadec-13-ene-2,5,9,12-tet-
raone (23). Compound 21 (140 mg, 0.50 mmol),
Pd(OAc)2 (4.5 mg, 0.02 mmol), PPh3 (5.2 mg,
0.02 mmol), n-Bu4NCl (83 mg, 0.3 mmol), and NaHCO3
(42 mg, 0.50 mmol) were successively added to a solu-
tion of 5 (100 mg, 0.20 mmol) in DMF-H2O (9:1,
4 mL), then the whole mixture was stirred at 80 ꢁC for
40 h. Water was added to the reaction mixture, then
the whole mixture was extracted with EtOAc. The
EtOAc extract was washed with saturated aqueous
NaCl, then dried over Na2SO4. Removal of the solvent
from the EtOAc extract under reduced pressure gave a
crude product, which was purified by column chroma-
tography (SiO2; CHCl3/MeOH 30:1) and further puri-
fied by HPLC (COSMOSIL 5C18-AR-II, MeOH/H2O
4:1) to give 23 (71 mg, 54%) as a white powder.
5.1.11.
(3S,10R,16S)-16-{(1R)-3-[4-(Hydroxymethyl)-
phenyl]-1-methylallyl}-3-isobutyl-10-(4-methoxybenzyl)-
1-oxa-4,8,11-triazacyclohexadec-13-ene-2,5,9,12-tetraone
(25). A solution of 23 (10 mg, 15 lmol) in MeOH
(0.5 mL) was treated with 6 N HCl (0.3 mL) at 0 ꢁC
for 3 h. Saturated aqueous NaHCO3 was added to the
reaction mixture, then the whole mixture was extracted
with EtOAc. The EtOAc extract was washed with satu-
rated aqueous NaCl, then dried over Na2SO4. Removal
of the solvent from the EtOAc extract under reduced
pressure gave a crude product, which was purified by
column chromatography (SiO2; CHCl3/MeOH 20:1) to
give 25 (6.6 mg, 72%) as a white powder.
20
Compound 23: ½aꢁ +62 (c 0.20, CHCl3). IR (KBr):
D
3288, 2951, 1736, 1658, 1541 cmꢀ1
.
1H NMR
(500 MHz, CDCl3) d: 7.27–7.32 (m, 4H), 7.08 (d,
J = 8.5 Hz, 2H), 6.98 (d, J = 7.9 Hz, 1H), 6.82 (d,
J = 8.5 Hz, 2H), 6.73 (ddd, J = 3.7, 11.0, 14.6 Hz,
1H), 6.39 (d, J = 15.9 Hz, 1H), 6.02 (dd, J = 7.3,
15.9 Hz, 1H), 5.99–6.02 (m, 1H), 5.70 (d, J = 14.6 Hz,
1H), 5.59 (d, J = 4.9 Hz, 1H), 5.11–5.14 (m, 1H), 4.68
(s, 2H), 4.59–4.62 (m, 1H), 4.57 (s, 2H), 4.52 (dd,
J = 7.3, 8.5 Hz, 1H), 3.78 (s, 3H), 3.75–3.82 (m, 1H),
3.40 (s, 3H), 3.23–3.28 (m, 1H), 3.12 (dd, J = 4.9,
14.0 Hz, 1H), 2.97 (dd, J = 7.9, 14.0 Hz, 1H), 2.49–
2.56 (m, 2H), 2.30–2.41 (m, 3H), 1.47–1.55 (m, 1H),
1.36 (t, J = 7.3 Hz, 2H), 1.12 (d, J = 7.3 Hz, 3H), 0.75
(d, J = 6.1 Hz, 6H). 13C NMR (125 MHz, CDCl3) d:
173.2, 171.6, 170.6, 165.0, 158.8, 141.9, 137.3, 136.3,
131.4, 130.3, 130.1 (2C), 128.2 (3C), 126.2 (2C),
125.0, 114.3 (2C), 95.6, 76.1, 68.8, 55.4, 55.3, 55.0,
50.7, 42.2, 41.7, 36.4, 35.8, 34.5, 34.1, 24.7, 22.3,
21.8, 17.1. ESI-MS: m/z 686 (M + Na)+. HR-ESI-MS:
m/z 686.3417, calcd for C37H49N3O8Na. Found:
686.3424.
20
Compound 25: ½aꢁ +87 (c 0.20, CHCl3/MeOH = 10:1).
D
IR (KBr): 3452, 3285, 2959, 1732, 1660, 1628, 1539,
1
1512 cmꢀ1. H NMR (500 MHz, CDCl3) d: 7.29–7.34
(m, 4H), 7.08 (d, J = 8.5 Hz, 2H), 6.95 (d, J = 6.7 Hz,
1H), 6.82 (d, J = 8.5 Hz, 2H), 6.72 (ddd, J = 3.7, 11.0,
14.6 Hz, 1H), 6.40 (d, J = 15.9 Hz, 1H), 6.01 (dd,
J = 8.5, 15.9 Hz, 1H), 5.83 (d, J = 8.5 Hz, 1H), 5.70 (d,
J = 14.6 Hz, 1H), 5.49 (d, J = 6.7 Hz, 1H), 5.13–5.16
(m, 1H), 4.68 (s, 2H), 4.59–4.62 (m, 1H), 4.52 (dd,
J = 7.3, 8.5 Hz, 1H), 3.78 (s, 3H), 3.79–3.83 (m, 1H),
3.49 (s, 1H), 3.22–3.27 (m, 1H), 3.12 (dd, J = 4.9,
14.6 Hz, 1H), 2.97 (dd, J = 7.9, 14.6 Hz, 1H), 2.50–
2.56 (m, 2H), 2.31–2.38 (m, 3H), 1.48–1.55 (m, 1H),

7454
N. Kotoku et al. / Bioorg. Med. Chem. 14 (2006) 7446–7457
1.36 (t, J = 7.3 Hz, 2H), 1.13 (d, J = 7.3 Hz, 3H), 0.76 (d,
J = 6.1 Hz, 6H). 13C NMR (125 MHz, CDCl3) d: 173.1,
171.6, 170.6, 164.9, 158.8, 141.9, 140.2, 136.3, 131.4,
130.3, 130.1 (2C), 128.1, 127.3 (2C), 126.4 (2C), 125.0,
114.3 (2C), 76.1, 65.1, 55.3, 55.0, 50.6, 42.2, 41.7, 36.4,
35.8, 34.5, 34.1, 24.7, 22.3, 21.8, 17.2. ESI-MS: m/z
642 (M + Na)+. HR-ESI-MS: m/z 642.3155, calcd for
C35H45N3O7Na. Found: 642.3146.
3.96 (d, J = 5.5 Hz, 2H), 3.79 (s, 3H), 3.75–3.83 (m,
1H), 3.66 (d, J = 1.8 Hz, 1H), 3.21–3.26 (m, 1H), 3.13
(dd, J = 4.9, 14.6 Hz, 1H), 2.95 (dd, J = 7.9, 14.6 Hz,
1H), 2.94 (dd, J = 1.8, 7.3 Hz, 1H), 2.52–2.55 (m, 1H),
2.31–2.43 (m, 3H), 1.77–1.81 (m, 1H), 1.48–1.53 (m,
1H), 1.43 (s, 9H), 1.36–1.39 (m, 1H), 1.28–1.31 (m,
1H), 1.14 (d, J = 7.3 Hz, 3H), 0.85 (d, J = 6.7 Hz, 3H),
0.83 (d, J = 6.7 Hz, 3H). 13C NMR (125 MHz, CDCl3)
d: 173.1, 171.7, 170.6, 170.2, 164.9, 158.8, 156.1, 141.3,
137.2, 135.7, 130.1 (2C), 128.8 (2C), 128.1, 126.0 (2C),
125.2, 114.4 (2C), 81.0, 75.0, 66.6, 63.2, 58.7, 55.3,
55.1, 50.5, 42.5, 41.4, 40.5, 36.6, 35.8, 34.4, 34.1, 28.3
(3C), 24.7, 22.6, 21.6, 13.3. ESI-MS: m/z 815
(M + Na)+. HR-ESI MS: m/z 815.3843, calcd for
C42H56N4O11Na. Found: 815.3859.
5.1.12. 4-((2R,3R)-3-{(1S)-1-[(3S,10R,16S)-3-Isobutyl-
10-(4-methoxybenzyl)-2,5,9,12-tetraoxo-1-oxa-4,8,11-tri-
azacyclohexadec-13-en-16-yl]ethyl}oxiranyl)benzyl tert-
butoxycarbonylaminoacetate
(27).
N-Boc-glycine
(6.0 mg, 0.03 mmol), DMAP (0.9 mg, 8 lmol), and
DCC (6.6 mg, 0.03 mmol) were successively added to a
solution of 25 (9.0 mg, 14 lmol) in CH2Cl2 (0.14 mL),
and the whole mixture was stirred at rt for 3 h. After fil-
tration through a Celite pad with Et2O as eluent, remov-
al of the solvent from the filtrate under reduced pressure
gave a crude product, which was purified by column
chromatography (SiO2; CHCl3/MeOH 20:1) to give gly-
cine ester 26 (11 mg, 100%) as a white powder.
5.1.13. (3S,10R,16S)-16-{(1R)-3-[4-(Chloromethyl)phen-
yl]-1-methylallyl}-3-isobutyl-10-(4-methoxybenzyl)-1-oxa-
4,8,11-triazacyclohexadec-13-ene-2,5,9,12-tetraone (28).
2,4,6-Collidine (3 lL, 0.024 mmol) and MsCl (1 lL,
0.015 mmol) were added to a solution of 25 (6.0 mg,
9.6 lmol) in DMF (0.5 mL), then the whole mixture
was stirred for 15 min under argon atmosphere. LiCl
(4.9 mg, 0.115 mmol) was added to the reaction mixture,
and the whole mixture was further stirred for 6 h. The
reaction was quenched with 5% HCl, and the whole
mixture was extracted with EtOAc. The EtOAc extract
was washed with saturated aqueous NaCl, then dried
over Na2SO4. Removal of the solvent from the EtOAc
extract under reduced pressure gave a crude product,
which was purified by column chromatography (SiO2;
CHCl3/MeOH 20:1) to give 28 (5.7 mg, 93%) as a white
powder.
20
Compound 26: ½aꢁ +42 (c 0.64, CHCl3). IR (KBr):
D
3290, 2957, 1736, 1660, 1628, 1541, 1514 cmꢀ1
.
1H
NMR (500 MHz, CDCl3) d: 7.28–7.31 (m, 5H), 7.07
(d, J = 8.5 Hz, 2H), 7.05–7.07 (m, 1H), 6.81 (d,
J = 8.5 Hz, 2H), 6.73 (ddd, J = 3.7, 11.6, 14.6 Hz, 1H),
6.38 (d, J = 15.9 Hz, 1H), 6.34–6.37 (m, 1H), 6.03 (dd,
J = 8.5, 15.9 Hz, 1H), 5.80 (br s, 1H), 5.72 (d,
J = 14.6 Hz, 1H), 5.13 (s, 2H), 5.11–5.29 (m, 1H),
5.00–5.05 (m, 1H), 4.58–4.62 (m, 1H), 4.48–4.53 (m,
1H), 3.93 (d, J = 4.9 Hz, 2H), 3.78 (s, 3H), 3.75–3.83
(m, 1H), 3.22–3.27 (m, 1H), 3.11 (dd, J = 4.9, 14.6 Hz,
1H), 2.97 (dd, J = 7.9, 14.6 Hz, 1H), 2.49–2.56 (m,
2H), 2.29–2.41 (m, 3H), 1.48–1.53 (m, 1H), 1.43 (s,
9H), 1.29–1.39 (m, 1H), 1.12 (d, J = 6.7 Hz, 3H), 0.73
(d, J = 5.5 Hz, 3H), 0.72 (d, J = 4.9 Hz, 3H). 13C
NMR (125 MHz, CDCl3) d: 173.2, 171.8, 170.8, 170.3,
165.1, 158.7, 155.7, 142.0, 137.1, 134.4, 131.1, 131.0,
130.1 (2C), 128.8 (2C), 128.1, 126.4 (2C), 124.9, 114.2
(2C), 80.1, 76.0, 66.8, 55.2, 55.1, 50.6, 42.5, 42.2, 41.4,
36.3, 35.8, 34.3, 34.2, 28.3 (3C), 24.6, 22.3, 21.7, 17.1.
ESI-MS: m/z 799 (M + Na)+. HR-ESI-MS: m/z
799.3894, calcd for C42H56N4O10Na. Found: 799.3909.
20
Compound 28: ½aꢁ +62 (c 0.22, CHCl3/MeOH 1:1). IR
D
(KBr): 3290, 2961, 2928, 1736, 1658, 1637, 1545,
1
1514 cmꢀ1. H NMR (500 MHz, CDCl3) d: 7.29–7.35
(m, 4H), 7.08 (d, J = 8.5 Hz, 2H), 6.97 (d, J = 6.7 Hz,
1H), 6.82 (d, J = 8.5 Hz, 2H), 6.72 (ddd, J = 3.7, 11.0,
14.6 Hz, 1H), 6.39 (d, J = 15.9 Hz, 1H), 6.03 (dd,
J = 8.5, 15.9 Hz, 1H), 5.90 (m, 1H), 5.70 (d,
J = 14.6 Hz, 1H), 5.54 (m, 1H), 5.11–5.16 (m, 1H),
4.59–4.61 (m, 1H), 4.56 (s, 2H), 4.50–4.53 (m, 1H),
3.78 (s, 3H), 3.77–3.83 (m, 1H), 3.22–3.27 (m, 1H),
3.13 (dd, J = 4.9, 14.6 Hz, 1H), 2.98 (dd, J = 7.9,
14.6 Hz, 1H), 2.50–2.57 (m, 2H), 2.33–2.41 (m, 3H),
1.47–1.52 (m, 1H), 1.32–1.36 (m, 2H), 1.13 (d,
J = 6.7 Hz, 3H), 0.75 (d, J = 6.1 Hz, 6H). 13C NMR
(125 MHz, CDCl3) d: 173.2, 171.6, 170.7, 165.0, 158.8,
141.9, 137.0, 136.7, 131.1, 131.1, 130.1 (2C), 128.8
(2C), 128.0, 126.5 (2C), 125.0, 114.4 (2C), 76.1, 55.3,
55.0, 50.7, 46.0, 42.2, 41.6, 36.4, 35.8, 34.5, 34.2, 24.7,
22.3, 21.8, 17.2. ESI-MS: m/z 660 (M + Na)+. HR-
ESI-MS: m/z 660.2816, calcd for C35H44ClN3O6Na.
Found: 660.2813.
A solution of the compound 26 (11 mg, 16 lmol) in
CH2Cl2 (0.5 mL) was treated with dimethyldioxirane
(0.074 M in acetone, 0.5 mL) at 0 ꢁC for 3 h. Removal
of the solvent from the reaction mixture gave a crude
product, which was purified by HPLC (COSMOSIL
5C18-MS-II, CH3CN/H2O 3:2) to give 27 (6.1 mg,
48%) as a white powder.
20
Compound 27: ½aꢁ +54 (c 0.51, CHCl3). IR (KBr):
D
3294, 2961, 2932, 1738, 1664, 1514 cmꢀ1
.
1H NMR
(500 MHz, CDCl3) d: 7.35 (d, J = 7.9 Hz, 2H), 7.23 (d,
J = 7.9 Hz, 2H), 7.08 (d, J = 8.5 Hz, 2H), 6.97 (d,
J = 7.9 Hz, 1H), 6.83 (d, J = 8.5 Hz, 2H), 6.70 (ddd,
J = 4.0, 11.6, 14.6 Hz, 1H), 5.88 (d, J = 9.2 Hz, 1H),
5.67 (d, J = 14.6 Hz, 1H), 5.55 (d, J = 6.1 Hz, 1H),
5.26–5.29 (m, 1H), 5.18 (s, 2H), 5.02 (br s, 1H),
4.57–4.61 (m, 1H), 4.47–4.51 (dt, J = 5.5, 9.2 Hz, 1H),
5.1.14. (3S,10R,16S)-16-((1S)-1-{(2R,3R)-3-[4-(Chloro-
methyl)phenyl]oxiranyl}ethyl)-3-isobutyl-10-(4-methoxyb-
enzyl)-1-oxa-4,8,11-triazacyclohexadec-13-ene-2,5,9,12- tet-
raone (29). A solution of 28 (80 mg, 0.12 mmol) in
CH2Cl2–MeOH (1:1, 2 mL) was treated with dim-
ethyldioxirane (0.074 M in acetone, 1 mL) at ꢀ30 ꢁC
for 5 h. Removal of the solvent from the reaction mix-

N. Kotoku et al. / Bioorg. Med. Chem. 14 (2006) 7446–7457
7455
ture under reduced pressure gave a crude product, which
was purified by HPLC (COSMOSIL 5C18-MS-II,
MeOH/H2O 7:3) to give 29 (41 mg, 50%) as a white
powder.
5.1.16. (3S,10R,16S)-3-Isobutyl-10-(4-methoxybenzyl)-16-
((1S)-1-{(2R,3R)-3-[4-(piperazin-1-ylmethyl)phenyl]oxira-
nyl}ethyl)-1-oxa-4,8,11-triazacyclohexadec-13-ene-2,5,9,
12-tetraone (31). A solution of 29 (4.3 mg, 7.9 lmol) in
DMF (0.5 mL) was treated with piperadine (50 lL, ex-
cess) at rt for 12 h. Removal of the solvent from the
reaction mixture under reduced pressure gave a crude
product, which was purified by column chromatography
(Chromatorexꢂ-NH2; CH3CN ! MeOH) to give 31
(3.7 mg, 80%) as a white powder.
20
Compound 29: ½aꢁ +51 (c 0.17, CHCl3/MeOH 1:1). IR
D
(KBr): 3290, 2955, 1738, 1660, 1637, 1541, 1514 cmꢀ1
.
1H NMR (500 MHz, CDCl3) d: 7.39 (d, J = 7.9 Hz,
2H), 7.23 (d, J = 7.9 Hz, 2H), 7.08 (d, J = 8.5 Hz,
2H), 6.97 (d, J = 6.7 Hz, 1H), 6.83 (d, J = 8.5 Hz,
2H), 6.70 (ddd, J = 3.7, 11.0, 14.6 Hz, 1H), 5.80 (d,
J = 9.2 Hz, 1H), 5.67 (d, J = 14.6 Hz, 1H), 5.49 (d,
J = 6.7 Hz, 1H), 5.26–5.29 (m, 1H), 4.59 (s, 2H),
4.57–4.61 (m, 1H), 4.50 (dt, J = 5.4, 9.2 Hz, 1H), 3.79
(s, 3H), 3.77–3.83 (m, 1H), 3.68 (d, J = 1.8 Hz, 1H),
3.21–3.26 (m, 1H), 3.13 (dd, J = 4.9, 14.6 Hz, 1H),
2.95 (dd, J = 7.9, 14.6 Hz, 1H), 2.90 (dd, J = 1.8,
7.3 Hz, 1H), 2.53–2.55 (m, 1H), 2.31–2.43 (m, 3H),
1.77–1.81 (m, 1H), 1.47–1.52 (m, 1H), 1.36–1.42 (m,
1H), 1.27–1.32 (m, 1H), 1.14 (d, J = 7.3 Hz, 3H), 0.86
(d, J = 6.7 Hz, 3H), 0.84 (d, J = 6.7 Hz, 3H). 13C
NMR (125 MHz, CDCl3) d: 173.1, 171.7, 170.5,
164.8, 157.3, 141.3, 137.9, 137.1, 130.0 (2C), 129.0
(2C), 128.0, 126.1 (2C), 125.2, 114.4 (2C), 74.9, 63.2,
58.6, 55.3, 55.0, 50.4, 45.8, 41.4, 40.4, 36.6, 35.8, 34.4,
34.0, 24.7, 22.6, 21.6, 13.3. ESI-MS: m/z 676
(M + Na)+. HR-ESI-MS: m/z 676.2765, calcd for
C35H44ClN3O7Na. Found: 676.2766.
20
Compound 31: ½aꢁ +43 (c 0.22, CHCl3). IR (KBr):
D
3287, 2949, 1738, 1660, 1537, 1514 cmꢀ1
.
1H NMR
(500 MHz, CDCl3) d: 7.32 (d, J = 7.9 Hz, 2H), 7.17 (d,
J = 7.9 Hz, 2H), 7.07 (d, J = 8.5 Hz, 2H), 6.99 (d,
J = 6.7 Hz, 1H), 6.82 (d, J = 8.5 Hz, 2H), 6.71 (ddd,
J = 3.7, 11.0, 14.6 Hz, 1H), 5.88 (d, J = 9.2 Hz, 1H),
5.67 (d, J = 14.6 Hz, 1H), 5.49 (d, J = 6.7 Hz, 1H),
5.26–5.30 (m, 1H), 4.57–4.61 (m, 1H), 4.50 (dt, J = 5.4,
9.2 Hz, 1H), 3.79 (s, 3H), 3.77–3.83 (m, 1H), 3.66 (d,
J = 1.8 Hz, 1H), 3.49 (s, 2H), 3.20–3.25 (m, 1H), 3.12
(dd, J = 4.9, 14.6 Hz, 1H), 2.96 (dd, J = 8.5, 14.6 Hz,
1H), 2.92 (dd, J = 1.8, 6.7 Hz, 1H), 2.90 (t, J = 4.9 Hz,
1H), 2.52–2.55 (m, 1H), 2.30–2.45 (m, 7H), 1.75–1.79
(m, 1H), 1.53–1.61 (m, 1H), 1.29–1.43 (m, 2H), 1.14
(d, J = 6.7 Hz, 3H), 0.86 (d, J = 6.7 Hz, 3H), 0.84 (d,
J = 6.7 Hz, 3H). 13C NMR (125 MHz, CDCl3) d:
173.1, 171.8, 170.5, 164.8, 158.8, 141.5, 138.8, 135.4,
130.1 (2C), 129.5 (2C), 128.0, 125.6 (2C), 125.1, 114.4
(2C), 75.0, 63.4, 63.1, 59.0, 55.3, 55.0, 54.6 (2C), 50.5,
46.2 (2C), 41.4, 40.6, 36.6, 35.8, 34.4, 34.0, 24.7, 22.6,
21.6, 13.4. ESI-MS: m/z 704 (M)+. HR-ESI-MS: m/z
704.4023, calcd for C39H54N5O7. Found: 704.4015.
5.1.15. (3S,10R,16S)-16-((1S)-1-{(2R,3R)-3-[4-(Diethyl-
aminomethyl)phenyl]oxiranyl}ethyl)-3-isobutyl-10-(4-meth-
oxybenzyl)-1-oxa-4,8,11-triazacyclohexadec-13-ene-2,5,
9,12-tetraone (30). A solution of 29 (3.2 mg, 4.8 lmol) in
DMF (1 mL) was treated with diethylamine (25 lL, ex-
cess) at rt for 3 h. Removal of the solvent from the reac-
tion mixture gave a crude product, which was purified
by column chromatography (SiO2; CHCl3/MeOH 10:1)
to give 30 (2.8 mg, 85%) as a white powder.
5.1.17. Triethyl-[4-((2R,3R)-3-{(1S)-1-[(3S,10R,16S)-3-
isobutyl-10-(4-methoxybenzyl)-2,5,9,12-tetraoxo-1-oxa-4,
8,11-triazacyclohexadec-13-en-16-yl]ethyl}oxiranyl) ben-
zyl]ammonium chloride (32). A solution of 29 (5.2 mg,
7.9 lmol) in DMF (1 mL) was treated with triethyl-
amine (50 lL, excess) at rt for 24 h. Removal of the
solvent from the reaction mixture under reduced pres-
sure gave a crude product, which was purified by
20
Compound 30: ½aꢁ +33 (c 0.24, CHCl3). IR (KBr):
D
3292, 2966, 2928, 1738, 1660, 1631, 1539 cmꢀ1
.
1H
NMR (500 MHz, CDCl3) d: 7.35 (d, J = 7.9 Hz, 2H),
7.18 (d, J = 7.9 Hz, 2H), 7.07 (d, J = 8.5 Hz, 2H), 6.99
(d, J = 7.9 Hz, 1H), 6.82 (d, J = 8.5 Hz, 2H), 6.71
(ddd, J = 3.6, 11.0, 14.6 Hz, 1H), 5.94 (d, J = 9.2 Hz,
1H), 5.68 (d, J = 14.6 Hz, 1H), 5.56 (d, J = 6.7 Hz,
1H), 5.26–5.30 (m, 1H), 4.59 (m, 1H), 4.49 (dd,
J = 5.5, 9.2 Hz, 1H), 3.78 (s, 3H), 3.75–3.83 (m, 1H),
3.66 (d, J = 1.8 Hz, 1H), 3.60 (s, 3H), 3.19-3.25 (m,
1H), 3.13 (dd, J = 4.9, 14.6 Hz, 1H), 2.96 (dd, J = 7.9,
14.6 Hz, 1H), 2.92 (dd, J = 1.8, 6.7 Hz, 1H), 2.52–2.57
(m, 5H), 2.31–2.45 (m, 3H), 1.74–1.81 (m, 1H), 1.52–
1.61 (m, 1H), 1.37–1.43 (m, 1H), 1.29–1.34 (m, 1H),
1.13 (d, J = 6.7 Hz, 3H), 1.08 (t, J = 6.7 Hz, 6H), 0.86
(d, J = 6.7 Hz, 3H), 0.84 (d, J = 6.7 Hz, 3H). 13C
NMR (125 MHz, CDCl3) d: 173.1, 172.0, 171.0,
165.1, 158.6, 141.0, 139.0, 131.6 (2C), 130.1 (2C),
128.8 (2C), 128.4, 128.2, 126.5 (2C), 125.3, 114.2 (2C),
77.2 (2C), 74.9, 63.5, 58.3, 55.5, 55.3, 55.2, 50.6, 45.9
(2C), 41.3, 40.3, 36.6, 35.7, 34.3, 34.1, 24.8, 22.7, 21.8,
13.5, 8.6 (2C). ESI-MS: m/z 691 (M + H)+. HR-ESI-
MS: m/z 691.4071, calcd for C39H55N4O7. Found:
691.4099.
column
chromatography
(Chromatorexꢂ-NH2;
CH3CN ! MeOH) to give 32 (4.8 mg, 86%) as a white
powder.
20
Compound 32: ½aꢁ +32 (c 0.34, CHCl3). IR (KBr):
D
3391, 3172, 2957, 2916, 1738, 1657, 1514, 1467 cmꢀ1
.
1H NMR (500 MHz, CDCl3) d: 7.61 (d, J = 7.9 Hz,
2H), 7.36 (d, J = 7.9 Hz, 2H), 7.21 (br s, 1H), 7.14 (d,
J = 8.5 Hz, 2H), 7.03–7.12 (m, 1H), 6.78 (d,
J = 8.5 Hz, 2H), 6.63 (ddd, J = 3.7, 10.4, 14.6 Hz, 1H),
6.55–6.66 (m, 1H), 5.85 (d, J = 14.6 Hz, 1H), 5.26 (br
d, J = 11.0 Hz, 1H), 4.78 (ABq, J = 14.0 Hz, 2H), 4.56
(m, 1H), 4.46 (m, 1H), 3.79 (s, 3H), 3.75–3.82 (m, 1H),
3.73 (d, J = 1.8 Hz, 1H), 3.42 (d-like, J = 6.7 Hz, 6H),
3.18–3.26 (m, 1H), 3.11 (dd, J = 4.9, 14.6 Hz, 1H),
2.97 (dd, J = 8.5, 14.6 Hz, 1H), 2.87 (dd, J = 1.8,
7.9 Hz, 1H), 2.29–2.52 (m, 4H), 1.74–1.78 (m, 1H),
1.55–1.63 (m, 1H), 1.50 (t, J = 6.7 Hz, 9H), 1.40–1.45
(m, 1H), 1.28–1.33 (m, 1H), 1.15 (d, J = 7.3 Hz, 3H),
0.88 (d, J = 6.7 Hz, 3H), 0.87 (d, J = 6.7 Hz, 3H). 13C
NMR (125 MHz, CDCl3) d: 173.3, 172.0, 170.7, 165.6,

7456
N. Kotoku et al. / Bioorg. Med. Chem. 14 (2006) 7446–7457
158.7, 140.3, 140.2, 133.3 (2C), 131.0, 130.4 (2C), 127.4,
126.8 (2C), 126.3, 114.3 (2C), 75.1, 64.0, 61.2, 58.6, 56.0,
55.5, 53.3 (3C), 51.1, 41.8, 40.6, 37.0, 35.9, 35.0, 34.7,
25.1, 22.9, 22.3, 14.0, 8.6 (3C). ESI-MS: m/z 719 (M)+.
HR-ESI-MS: m/z 719.4384, calcd for C41H59N4O7.
Found: 719.4404.
proliferation was determined by MTT colorimetric
assay.
5.4. In vivo anti-tumor experiment
Murine sarcoma cells, S180 (2 · 105 cells/body), were
implanted subcutaneously into the right ventral flank
of ddY mice (5 weeks old). Three days after implanta-
tion, analogue 30 or doxorubicin was administered at
various doses on every other day (total 6 times), by ip
injection as a suspension in 1% CMC. Tumor diameter
was measured with calipers. Tumor size was evaluated
by the larger diameter.
5.1.18. 1-[4-(2R,3R)-3-{(1S)-1-[(3S,10R,16S)-3-Isobutyl-
10-(4-methoxybenzyl)-2,5,9,12-tetraoxo-1-oxa-4,8,11-tri-
azacyclohexadec-13-en-16-yl]ethyl}oxiranyl]benzyl]-4-aza-
1-azoniabicyclo[2.2.2]octane (33). A solution of 29
(5.0 mg, 7.9 lmol) in DMF (0.5 mL) was treated with
DABCO (50 lL, excess) at rt for 24 h. Removal of the
solvent from the reaction mixture under reduced
pressure gave a crude product, which was purified
by column chromatography (Chromatorexꢂ-NH2;
CH3CN ! MeOH) to give 33 (4.9 mg, 85%) as a white
powder.
Acknowledgments
This study was financially supported by Grant-in-Aid
for Scientific research from the Ministry of Education,
Science, Sport and Culture of Japan. The authors are
also grateful to the Takeda Science Foundation and
the Tokyo Biochemical Research Foundation for finan-
cial support.
20
Compound 33: ½aꢁ +26 (c 0.35, CHCl3). IR (KBr):
D
3402, 3285, 3252, 2959, 2928, 1738, 1668, 1537,
1514 cmꢀ1 1H NMR (500 MHz, CDCl3) d: 7.62 (d,
.
J = 7.9 Hz, 2H), 7.29 (d, J = 7.9 Hz, 2H), 7.11 (d,
J = 8.5 Hz, 2H), 7.17 (br s, 1H), 6.79 (d, J = 8.5 Hz,
2H), 6.67 (ddd, J = 3.7, 11.0, 14.6 Hz, 1H), 6.62–6.79
(m, 1H), 6.56 (m, 1H), 5.80 (d, J = 14.6 Hz, 1H), 5.27
(br d, J = 11.0 Hz, 1H), 4.99 (ABq, J = 12.8 Hz, 2H),
4.56–4.60 (m, 1H), 4.45 (dt, J = 5.4, 9.2 Hz, 1H), 3.76
(s, 3H), 3.73–3.78 (m, 1H), 3.70 (s, 1H), 3.18-3.25 (m,
7H), 3.10 (dd, J = 4.9, 14.6 Hz, 1H), 3.01 (dd, J = 8.5,
14.6 Hz, 1H), 2.86 (d, J = 6.7 Hz, 1H), 2.50–2.55 (m,
1H), 2.33–2.46 (m, 9H), 1.75–1.81 (m, 1H), 1.58–1.62
(m, 1H), 1.49–1.55 (m, 1H), 1.38–1.44 (m, 1H), 1.14
(d, J = 6.7 Hz, 3H), 0.88 (d, J = 6.7 Hz, 3H), 0.86 (d,
J = 6.7 Hz, 3H). 13C NMR (125 MHz, CDCl3) d:
173.1, 171.9, 171.0, 165.2, 158.6, 140.9, 139.9, 133.9
(2C), 130.2 (2C), 128.0, 126.6, 126.4 (2C), 125.4,
114.2 (2C), 74.9, 67.2, 63.6, 58.1, 55.5, 55.3, 52.2
(3C), 50.7, 45.5 (3C), 41.4, 40.3, 36.7, 35.7, 34.5, 34.2,
24.8, 22.7, 21.9, 13.5. ESI-MS: m/z 730 (M)+. HR-
ESI-MS: m/z 730.4180, calcd for C41H56N5O7. Found:
730.4206.
References and notes
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183–191; (b) Morita, K.; Koiso, Y.; Hashimoto, Y.;
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5.2. Solubility test in MeOH
A saturated solution of the test compounds in MeOH
was sonicated at 25 ꢁC for 10 min, then filtered through
the filter (pore size = 0.45 lm, Millex-FH, Millipore).
The solubility was determined through analyzing UV
(230 nm) absorption of the filtrate, by calculating from
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of arenastatin A (1) at 230 nm.
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5.3. In vitro biological evaluation
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KB 3-1 cells were cultured in RPMI-1640 medium sup-
plemented with 10% heat-inactivated fetal bovine serum,
kanamycin (50 lg/mL), and L-glutamine (4 lM). Cells
were plated into 96-well microplates at 5 · 103 cells/
100 lL assay medium/well, and various concentration
of test compounds were added to each well as 1 lL
DMSO solution. The plates were incubated at 37 ꢁC in
a humidified atmosphere of 5% CO2 for 72 h, and cell

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12. The stereochemistry of the epoxy moieties of 24, 27, and
29 was established by comparison of the proton chemical
shifts due to the 8-H and 6-Me groups from the same
behavior observed between arenastatin A (1, 8-H: d 3.68,
6-Me: d 1.14) and the a-epoxy isomer of 1 (d 3.59, 1.05).
Namely, an obvious difference in chemical shifts of the 8-
H and 6-Me groups was observed; the signals of 24, 27,
and 29 appeared in lower field (24: d 3.69, 1.14; 27: d 3.66,
1.14; 29: d 3.68, 1.14) than those of the a-isomers (240: d
3.59, 1.04; 270: d 3.59, 1.04; 290: d 3.59, 1.04).
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