Synthesis of amide analogs of arenastatin A

Article abstract of DOI:10.1016/S0040-4020(00)00766-3

In order to probe the metabolism of arenastatin A, a potent cytotoxic depsipeptide from the marine sponge Dysidea arenaria, we synthesized three analogs in which the ester linkages were replaced by amide bonds. Triamide analog-II and tetraamide analog, both of which contained a 15,20-amide linkage, showed stability in serum. However, arenastatin A and triamide analog-I, which both contained a 15,20-ester moiety, were readily metabolized in serum. Among the three amide analogs, only triamide analog-II exhibited potent cytotoxic activity against KB cells. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00766-3

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 9121±9128
Synthesis of Amide Analogs of Arenastatin A
Nobutoshi Murakami, Weiqi Wang, Naoki Ohyabu, Takeshi Ito, Satoru Tamura, Shunji Aoki,
*
Motomasa Kobayashi and Isao Kitagawa
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan
This paper is dedicated to Professor P. J. Scheuer, Hawaii University, on the occasion of his 85th birthday
Received 20 April 2000; accepted 27 July 2000
AbstractÐIn order to probe the metabolism of arenastatin A, a potent cytotoxic depsipeptide from the marine sponge Dysidea arenaria, we
synthesized three analogs in which the ester linkages were replaced by amide bonds. Triamide analog-II and tetraamide analog, both of which
contained a 15,20-amide linkage, showed stability in serum. However, arenastatin A and triamide analog-I, which both contained a 15,20-
ester moiety, were readily metabolized in serum. Among the three amide analogs, only triamide analog-II exhibited potent cytotoxic activity
against KB cells. q 2000 Elsevier Science Ltd. All rights reserved.
Introduction
metabolic breakdown, we synthesized three amide analogs
(2, 4, 5) in which one or two ester moieties were replaced by
an amide group. This paper describes the synthesis and
biological activities of these amide analogs (2, 4, 5)
(Chart 1).
During the course of our investigation in a search for new
biologically active substances from marine organisms, we
isolated and characterized an extremely potent cytotoxic
(IC50 5 pg/ml against KB cells) depsipeptide named arena-
statin A (1) from the Okinawan marine sponge Dysidea
arenaria.1,2 In addition, we established the total synthesis
of 1 and elucidated its structure-activity relationship among
several synthesized stereoisomers.3,4 Arenastatin A (1) was
also shown to inhibit microtubule assembly and bind to the
rhizoxin/maytansine site on tubulin speci®cally.5,6
Syntheses of amide analogs
According to our synthetic protocol of arenastatin A (1),4 we
synthesized three amide analogs (2, 4, 5). The strategic
disconnections of the three analogs (2, 4, 5) are summarized
as depicted in Chart 2. Since arenastatin A (1) having an
epoxy moiety adjacent to a phenyl group as well as a cyclic
diester structure is fairly unstable under both acidic and
alkaline conditions,4 the epoxy function was introduced at
the ®nal stage of the synthesis.
Arenastatin A (1) bears a close structural similarity to the
potent anti-tumor depsipeptide cryptophycin 1 (3), which
was isolated from a cyanobacterium of Nostoc sp.7 The
ring portions and overall frameworks of the two compounds
are identical, except that arenastatin A (1) lacks an aryl
chlorine and a methyl on C-21 which are found in crypto-
phycin 1 (3). Cryptophycin 1 (3) exhibits potent in vivo anti-
tumor activity.8 Arenastatin A (1) was found to exhibit in
vivo anti-tumor activity (71% ILS at 5 mg/kg dose against
P388) by ip±ip administration, but little activity by ip±iv
administration. In addition, when placed in fresh mouse
serum, arenastatin A (1) was found to be readily metabo-
lized. These observations led us to presume that one of the
ester linkages in 1 is hydrolyzed (presumably enzymati-
cally) in the case of intravenous administration, leading to
loss of anti-tumor activity. In order to elucidate the speci®c
portions of arenastatin A (1) which were susceptible to
Scheme 1 illustrates the synthetic route of triamide analog-I
(2) having a 5,14-amide function in place of the ester link-
age in 1. Introduction of an amino group at C-5 in segment
A (6)4 with S-con®guration was carried out by Mitsunobu
reaction,9 followed by SN2-type nucleophilic substitution
reaction of an azide group. The treatment of 6 with benzoic
acid in the presence of tributylphosphine and diethyl-
azodicarboxylate (DEAD) afforded a 5R-benzoate, which
was then saponi®ed by NaOMe in MeOH to give an alcohol
13 in 68% yield. After mesylation of 13, the resulting mesyl-
ate was treated with NaN3 in DMF to provide a 5S-azide,
which was then converted to the desired segment A0 (7) by
LiAlH4 reduction in 87% yield from 13. Segments A0 (7)
and C±D (14)10 were conjugated by 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide hydrochloride (EDCI´HCl)
and 4-dimethylaminopyridine (DMAP) to give an amide,
the methoxymethyl (MOM) group of which was removed
by Me2BBr11 treatment to provide 15 in 72% yield. The
Keywords: depsipeptide; cytotoxin; marine metabolite; marine sponge.
* Corresponding author. Tel.: 181-6-6879-8215; fax: 181-6-6879-8219;
e-mail: kobayasi@phs.osaka-u.ac.jp
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00766-3

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N. Murakami et al. / Tetrahedron 56 (2000) 9121±9128
Chart 1.
primary alcohol in 15 was converted by Dess±Martin
oxidation12 to the corresponding aldehyde, which was
further subjected to Horner±Emmons reaction with segment
B (8)4 to afford the diamide 16 in 96% yield. Removal of
both protecting groups in 16 by tri¯uoroacetic acid (TFA)
and subsequent HCl treatment gave a seco aminocarboxylic
acid as an HCl salt. The seco aminocarboxylic acid was
subjected to intramolecular macrolactamization by use of
diphenylphosphorus azide (DPPA)13,14 in the presence of
NaHCO3 in DMF to afford a cyclic depsipeptide 17 in
93% yield from 16. Epoxidation of 17 using dimethyl-
dioxirane proceeded stereoselectively to give triamide
analog-I (2). The stereochemistry of the 7R,8R-epoxy func-
tion in 2 was con®rmed by comparison of the chemical
shifts of the C6-methyl proton (d 1.06) and C8-proton (d
3.62) of 2 in CDCl3±CD3OD (5:1) with those of 1 and its
epoxy isomer. Thus, the C6-methyl proton and C8-proton in
1 were observed at d 1.07 and d 3.65 in CDCl3±CD3OD
(5:1), while those of the epoxy isomer of 1 were observed at
d 1.00 and d 3.56.
Chart 2.

N. Murakami et al. / Tetrahedron 56 (2000) 9121±9128
9123
Scheme 1. Synthesis of triamide analog-I (2). Reagents and conditions: (a) PBu3, DEAD, benzoic acid, THF; (b) NaOMe±MeOH, 2 steps 68%; (c) MsCl,
Et3N, CH2Cl2; (d) NaN3, DMF; (e) LiAlH4, Et2O, 08C, 3 steps 91%; (f) 14, EDCI´HCl, DMAP, CH2Cl2; (g) Me2BBr, CH2Cl2, 2788C, 2 steps 72%; (h) Dess±
Martin periodinane, CH2Cl2; (i) 8, DBU, LiCl, CH3CN, 2 steps 96%; (j) TFA, CH2Cl2; (k) HCl±ether; (l) DPPA, NaHCO3, DMF, 08C, 3 steps 93%; (m)
dimethyldioxirane, CH2Cl2±MeOH (3:1), quant.
Triamide analog-II (4), in which an amide function was
substituted for the 15,20-ester linkage in 1, was synthesized
as shown in Scheme 2. Condensation of segment A (6) and
segment C0 (10) in the presence of dicyclohexylcarbodi-
imide (DCC) and DMAP afforded an ester, the MOM
group of which was removed by Me2BBr treatment to
give 18. Dess±Martin oxidation of the primary alcohol in
18 provided an aldehyde, which was subjected to Horner±
Emmons reaction with the phosphonate 193 to give an
unsaturated amide 20. Simultaneous deprotection of the
t-butoxycarbonyl and the 2-trimethylsilylethyl groups
using TFA and subsequent HCl treatment gave an HCl
salt of a seco aminocarboxylic acid, which was then
cyclized intramolecularly by use of DPPA and NaHCO3 to
give a cyclic depsipeptide 21. Finally, dimethyldioxirane
oxidation of 21 furnished the desired triamide analog-II
(4)6,15 and its epoxy isomer in a ratio of 2:1. The stereo-
chemistry of the 7,8-epoxy function in 4 and the epoxy
isomer of 4 were also con®rmed by comparison of the
chemical shifts of the C6-methyl proton and C8-proton in
CDCl3. Thus, the C6-methyl proton and C8-proton of 4 and
its epoxy isomer were observed at d 1.14, 1.04 and d 3.67,
3.59 in CDCl3, respectively. On the other hand, the C6-
methyl proton and C8-proton of 1 and its epoxy isomer
were observed at d 1.14, 1.05 and d 3.68, 3.59 in CDCl3,
respectively.
Additionally, tetraamide analog (5), in which both ester
linkages in 1 were replaced by amide bonds, was prepared
in a similar manner as for the synthesis of 2 (Scheme 3). The
Scheme 2. Synthesis of triamide analog-II (4). Reagents and conditions: (a) 10, DCC, DMAP, CH2Cl2; (b) Me2BBr, CH2Cl2, 2788C, 2 steps 92%; (c) Dess±
Martin periodinane, CH2Cl2; (d) NaH, THF, 2 steps 76%; (e) TFA, CH2Cl2; (f) HCl±ether; (g) DPPA, NaHCO3, DMF, 08C, 3 steps 68%; (h) dimethyl-
dioxirane, CH2Cl2±MeOH (2:1), 62%.

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N. Murakami et al. / Tetrahedron 56 (2000) 9121±9128
Scheme 3. Synthesis of tetraamide analog (5). Reagents and conditions: (a) 10, DCC, DMAP, CH2Cl2, 82%; (b) 10% HCl±MeOH; (c) 12, EDCI´HCl, DMAP,
CH2Cl2, 2 steps 77%; (d) Dess±Martin periodinane, CH2Cl2; (e) 8, DBU, LiCl, CH3CN, 2 steps 93%; (f) TFA, CH2Cl2; (g) HCl±ether; (h) DPPA, NaHCO3,
DMF, 08C, 3 steps 87%; (i) dimethyldioxirane, CHCl3±MeOH (3:1), 2308C, 61%.
stereochemistry of the 7R,8R-epoxy function in 5 was also
con®rmed by comparison of the chemical shifts of the C6-
methyl proton (d 1.01) and C8-proton (d 3.82). Thus, the
C6-methyl proton and C8-proton in 1 were observed at d
1.05 and d 3.82 in d6-DMSO, respectively.
one of the ester linkages contributed to the loss of activity
during intravenous administration. In order to test this
presumption, the stability of arenastatin A (1) was compared
with each of the three analogs in mouse serum. The
compounds were allowed to incubate in mouse serum,
then extracted and the remaining compound was evaluated
by HPLC. Arenastatin A (1) and triamide analog-I (2), both
of which contain the 15,20-ester linkage, were rapidly
metabolized in mouse serum. In contrast, triamide analog-
II (4) and tetraamide analog (5), in which the 15,20-ester is
replaced by an amide functional group, were recovered
unchanged from the mouse serum (Fig. 1).
Discussion
When tested for cytotoxicity against KB cells, triamide
analog-II (4) exhibited potent cytotoxicity (IC50 6 ng/ml),
although this was 103 weaker than the activity of 1.
Triamide analog-I (2) and tetraamide analog (5) showed
only weak cytotoxic activity of 4 and 6 mg/ml, respectively.
As mentioned earlier, it was suspected that the hydrolysis of
The results found with arenastatin A (1) and these three
analogs suggest the following observations regarding cyto-
toxicity: (1) substitution of the 5,14-ester linkage by an
amide results in a loss of cytotoxicity, (2) the 15,20-ester
linkage is readily metabolized in serum, and (3) replacement
of the 15,20-ester by an amide function results in reduced
metabolism of the compound. Recently, the Lilly research
group16 synthesized a 15,20-amide analog of cryptophycin 1
(3) and reported that this analog exhibited a similar cyto-
toxicity as that of 3.
In spite of exhibiting potent cytotoxicity, triamide analog-II
(4) was poorly soluble in polar solvents such as H2O, MeOH
and DMSO to be applied to in vivo anti-tumor tests.17
A
program aiming at producing analogs of arenastatin A (1)
for further anti-tumor and cytotoxic evaluation, and which
will overcome these solubility limitations, is currently in
progress in our laboratory.
Experimental
The following instruments were used to obtain physical
data: a Jasco DIP-181 digital polarimeter for speci®c
rotations; a Hitachi 260-30 infrared spectrometer for IR
spectra; a JEOL JNM Lambda-500 (500 MHz) NMR
Figure 1. Stability of arenastatin A (1) and amide analogs (2, 4, 5) in mouse
serum. Each sample (10 ml of 0.1 mg/ml solution in DMSO) was treated
with fresh mouse serum (100 ml) and incubated at 378C for 0, 10, 30, 60,
300 min, respectively. After extraction of the reaction mixture with EtOAc,
each extract was analyzed by reversed phase HPLC to determine the remain
of 1, 2, 4, and 5.
1
spectrometer for H NMR spectra [CDCl3 solution with
tetramethylsilane (TMS) as an internal standard unless
otherwise speci®ed]. Silica gel (Merck 60±230 mesh) and
pre-coated thin layer chromatography (TLC) plates (Merck,

N. Murakami et al. / Tetrahedron 56 (2000) 9121±9128
9125
Kiesel gel, 60F254) were used for column chromatography
and TLC. Spots on TLC plates were detected by spraying
vanillin/H2SO4 (vanillin 5 g, c-H2SO4 95 ml) or acidic
p-anisaldehyde solution (p-anisaldehyde 25 ml, c-H2SO4
25 ml, AcOH 5 ml, EtOH 425 ml) with subsequent heating.
Amidation of segment C±D (14) with segment A0 (7). A
solution of segment A0 (7, 71 mg, 0.30 mmol) and segment
C±D (14, 130 mg, 0.43 mmol) in dry CH2Cl2 (2.0 ml) was
treated with EDCI´HCl (170 mg, 0.89 mmol) in the
presence of DMAP (27 mg, 0.22 mmol) at room tempera-
ture for 1 h. The reaction mixture was poured into water,
then the whole was extracted with EtOAc. The EtOAc
extract was washed with 5% HCl, saturated aqueous
NaHCO3, and saturated aqueous NaCl, then dried over
Na2SO4. Removal of solvent from the EtOAc extract
under reduced pressure gave a product, which was puri®ed
by column chromatography (SiO2 8 g, n-hexane±
EtOAcˆ1:1) to furnish an amide. (123 mg, 78%). A solu-
tion of the amide (40 mg, 0.075 mmol) in dry CH2Cl2
(0.75 ml) was treated with Me2BBr (1.67 M in CH2Cl2,
130 ml) at 2788C for 1 h. After a mixture of THF and
aqueous saturated NaHCO3 (2:1) was dropped into the
reaction mixture, the whole was extracted with EtOAc.
The EtOAc extract was dried over Na2SO4. Removal of
solvent from the EtOAc extract under reduced pressure
gave a product, which was puri®ed by column chromato-
graphy (SiO2 2 g, n-hexane±EtOAcˆ1:1) to furnish 15
(33.6 mg, 92%). 15: Colorless oil, [a]2D3ˆ244.98 (cˆ0.51
in CHCl3). IR (KBr): 3306, 2962, 1736, 1718, 1695, 1664,
1523 cm21. 1H NMR d: 7.42±7.22 (5H, m, Ph), 6.41 (1H, d,
Jˆ15.9 Hz, 8-H), 6.14 (1H, dd, Jˆ7.9, 15.9 Hz, 7-H), 6.02
(1H, brd, Jˆ6.1 Hz, 5-NH), 5.08 (1H, dd, Jˆ3.1, 12.2 Hz,
15-H), 4.93 (1H, brs, 22-NH), 4.13 (1H, m, 5-H), 3.67 (1H,
dt, Jˆ12.2, 4.3 Hz, 3-Ha), 3.53 (1H, dt, Jˆ12.2, 3.1 Hz,
3-Hb), 3.22 (2H, m, 22-H), 2.54 (1H, m, 6-H), 2.44 (1H,
m, 21-Ha), 2.34 (1H, m, 21-Hb), 2.01 (1H, m, 4-Ha), 1.69±
1.48 (4H, m, 4-Hb, 16, 17-H), 1.43 (9H, s, tBu), 1.15 (3H, d,
Jˆ6.7 Hz, 13-H), 0.85, 0.84 (both 3H, d-like, Jˆca. 6.0 Hz,
18, 19-H). FAB-MS m/z: Calcd for C27H43N2O16 ˆ491.6495.
Found: 491.6493 (M1H)1.
Inversion of hydroxy group in 6. DEAD (480 ml,
3.0 mmol) was added to a THF solution (2.0 ml) of 6
(250 mg, 1.0 mmol), PBu3 (760 ml, 3.0 mmol) and benzoic
acid (360 mg, 3.0 mmol) at 08C, then the whole was stirred
at 408C for 8 h. The reaction mixture was evaporated in
vacuo to afford a product, which was puri®ed by column
chromatography (SiO2 50 g, n-hexane±Et2Oˆ10:1) to give
a benzoate (320 mg). A solution of the benzoate in MeOH
(5 ml) was treated with 28% NaOMe±MeOH (2.5 ml) at
room temperature for 2 h. The reaction mixture was poured
into saturated aqueous NH4Cl, then the whole was extracted
with EtOAc. The EtOAc extract was dried over Na2SO4.
Removal of solvent from the EtOAc extract under reduced
pressure gave a crude product, which was puri®ed by
column chromatography (SiO2 10 g, n-hexane±
EtOAcˆ3:1) to furnish 13 (170 mg, 68%). 13: Colorless
oil, [a]2D6ˆ139.98 (cˆ2.0 in CHCl3). IR (KBr): 3441,
1
2928, 1450, 1151, 1107 cm21. H NMR d: 7.33±7.16 (5H,
m, Ph), 6.36 (1H, d, Jˆ15.8 Hz, 8-H), 6.11 (1H, dd, Jˆ7.9,
15.8 Hz, 7-H), 4.56 (2H, s, OCH2OCH3), 3.74±3.56 (3H, m,
3, 5-H), 3.30 (3H, s, OCH2OCH3), 2.38 (1H, m, 6-H), 1.76
(1H, m, 4-Ha), 1.67 (1H, m, 4-Hb), 1.09 (3H, d, Jˆ6.6 Hz,
13-H). FAB-MS m/z: Calcd for C15H22LiO13 ˆ257.1729.
Found: 257.1754 (M1Li)1.
Preparation for segment A0 (7). A solution of 13 (145 mg,
0.58 mmol) in dry CH2Cl2 (5.8 ml) was treated with Et3N
(160 ml, 1.2 mmol) and methansulfonyl chloride (54 ml,
0.7 mmol) for 10 min under cooling in an ice-water bath.
The reaction mixture was poured into saturated aqueous
NH4Cl, then the whole was extracted with EtOAc. The
EtOAc extract was dried over Na2SO4. Removal of solvent
from the EtOAc extract under reduced pressure gave a crude
mesylate. A solution of this mesylate in dry DMF (5.8 ml)
was treated with NaN3 (110 mg, 1.7 mmol) for 3 h at 908C.
After removing the residue by ®ltration, the ®ltrate was
poured into the water, then the whole was extracted with
EtOAc. The EtOAc extract was washed with aqueous satu-
rated NaCl and dried over Na2SO4. Removal of solvent from
the EtOAc extract under reduced pressure gave a product,
which was puri®ed by column chromatography (SiO2 10 g,
n-hexane±Et2Oˆ15:1) to furnish an azide (145 mg, 91%).
A solution of azide compound (82 mg, 0.30 mmol) in dry
Et2O (2.9 ml) was stirred in the presence of LiAlH4 (34 mg,
0.90 mmol) at 08C for 1 h. The reaction mixture was succes-
sively treated with water saturated Et2O, 3 drops of 1N
NaOH, and then the whole was ®ltered. Removal of solvent
from the ®ltrate under reduced pressure gave segment A0 (7,
71 mg, quant.). 7: Colorless oil, [a]2D3ˆ117.78 (cˆ2.39 in
CHCl3). IR (KBr): 3306, 1736, 1718, 1523 cm21. 1H NMR
d:7.37±7.18 (5H, m, Ph), 6.44 (1H, d, Jˆ15.9 Hz, 8-H),
6.14 (1H, dd, Jˆ8.4, 15.9 Hz, 7-H), 4.63 (2H, s,
OCH2OCH3), 3.69 (2H, m, 3-H), 3.37 (3H, s, OCH2OCH3),
2.88 (1H, ddd, Jˆ3.7, 9.0, 12.6 Hz, 5-H), 2.33 (1H, m, 6-H),
1.89 (1H, m, 4-Ha), 1.54 (1H, ddt, Jˆ9.0, 14.1, 5.9 Hz,
4-Hb), 1.14 (3H, d, Jˆ6.6 Hz, 13-H). FAB-MS m/z: Calcd
for C15H24NO12 ˆ250.1807. Found: 250.1742 (M1H)1.
Conversion from 15 to 16. A solution of 15 (10.5 mg,
0.021 mmol) in dry CH2Cl2 (0.4 ml) was treated with
Dess±Martin periodinane (45.6 mg, 0.11 mmol) for
30 min at room temperature. Saturated aqueous NaHCO3
and saturated aqueous Na2S2O3 were successively dropped
into the reaction mixture, then the whole was stirred for
30 min. After extracting with EtOAc, the EtOAc extract
was dried over Na2SO4. Removal of solvent from the
EtOAc extract under reduced pressure gave a crude alde-
hyde. A solution of segment B (8, 19 mg, 0.04 mmol) in dry
CH3CN (1.9 ml) was treated with LiCl (8.8 mg, 0.2 mmol)
and DBU (6 ml, 0.04 mmol) at room temperature for
10 min, the crude aldehyde was added to the solution with
CH2Cl2 (0.5 ml). After further 10 min stirring, the reaction
mixture was poured into saturated aqueous NaCl, then the
whole was extracted with EtOAc. The EtOAc extract was
dried over Na2SO4. Removal of solvent from the EtOAc
extract under reduced pressure gave a product, which was
puri®ed by column chromatography (SiO2 2 g, n-hexane±
EtOAcˆ1:1) to furnish 16 (16.6 mg, 96%). 16: Colorless
oil, [a]2D3ˆ110.68 (cˆ0.04 in CHCl3). IR (KBr): 2959,
1
1739, 1718, 1655, 1637 cm21. H NMR d: 7.38±7.22 (5H,
m, Ph), 7.03 (2H, d, Jˆ8.6 Hz, 27-H), 6.81 (2H, d,
Jˆ8.6 Hz, 28-H), 6.76 (1H, m, 3-H), 6.39 (1H, d,
Jˆ15.8 Hz, 8-H), 6.15 (1H, brs, 5-NH), 6.06 (1H, dd,
Jˆ8.3, 15.8 Hz, 7-H), 5.90 (1H, d, Jˆ8.3 Hz, 24-NH),
5.89 (1H, d, Jˆ15.5 Hz, 2-H), 5.04 (1H, m, 22-NH), 5.04

9126
N. Murakami et al. / Tetrahedron 56 (2000) 9121±9128
(1H, dd, Jˆ4.6, 12.9 Hz, 15-H), 4.85 (1H, q-like, Jˆca.
6.0 Hz, 24-H), 4.19 (2H, m, OCH2CH2TMS), 3.99 (1H, m,
5-H), 3.77 (3H, s, 30-H), 3.19 (2H, m, 22-H), 3.08 (2H, d,
Jˆ5.9 Hz, 25-H), 2.56 (1H, m, 6-H), 2.54±2.23 (4H, m, 4,
21-H), 1.68±1.40 (3H, m, 16, 17-H), 1.42 (9H, s, tBu), 1.12
(3H, d, Jˆ6.6 Hz, 13-H), 0.97 (2H, t-like, Jˆca. 8.5 Hz,
OCH2CH2TMS), 0.82 (6H, d, Jˆ5.3 Hz, 18, 19-H),
0.04 (9H, s, TMS). FAB-MS m/z: Calcd for
C44H66N3O9Si1ˆ808.4568. Found: 808.4576 (M1H)1.
50 mg, 0.2 mmol) and segment C0 (10, 92 mg, 0.4 mmol)
in CH2Cl2 (4 ml) was treated with DCC (206 mg, 1.0 mmol)
and DMAP (73 mg, 0.6 mmol) at room temperature for 2 h.
To the reaction mixture 10% HCl was added until the whole
was neutralized. Then, the whole was extracted with Et2O.
The Et2O extract was dried over Na2SO4. Removal of
solvent from the Et2O extract under reduced pressure gave
a crude product, which was puri®ed by column chromato-
graphy (SiO2 15 g, n-hexane±EtOAcˆ6:1) to furnish an
ester (89 mg, 96%). A solution of the ester (46 mg,
0.099 mmol) in dry CH2Cl2 (1.0 ml) was treated with
Me2BBr (1.67 M in CH2Cl2, 190 ml) at 2788C for 20 min.
The mixture of THF and aqueous saturated NaHCO3 (2:1)
was dropped into the reaction mixture. The whole was
extracted with EtOAc. The EtOAc extract was dried over
Na2SO4. Removal of solvent from the EtOAc extract
under reduced pressure gave a crude product, which was
puri®ed by column chromatography (SiO2 4 g, n-hexane±
EtOAcˆ3:1) to furnish 18 (40 mg, 96%). 18: Colorless
oil, [a]2D5ˆ218.58 (cˆ1.1 in CHCl3). IR (KBr): 2962,
Deprotection of 16 followed by macrolactamization
giving 17. A solution of 16 (16.6 mg, 0.02 mmol) in dry
CH2Cl2 (0.4 ml) was treated with TFA (0.8 ml) under cool-
ing in an ice-water bath for 4 h to give a seco amino acid as
TFA salt. A solution of the TFA salt was treated with satu-
rated HCl gas±Et2O three times to furnish an HCl salt. A
solution of the HCl salt in dry DMF (2.6 ml) was treated
with DPPA (5 ml, 0.024 mmol) and NaHCO3 (9 mg,
0.1 mmol) at 08C for 44 h. The reaction mixture was poured
into aqueous saturated NH4Cl, then the whole was extracted
with EtOAc, and the EtOAc extract was dried over Na2SO4.
Removal of solvent from the EtOAc extract under reduced
pressure gave a product, which was puri®ed by column
chromatography (SiO2 1 g, CHCl3±MeOHˆ30:1) to furnish
17 (11.3 mg, 93%). 17: A white powder, [a]1D9ˆ139.78
(cˆ0.07 in CHCl3±MeOHˆ5:1). IR (KBr): 2922, 1797,
1
1712, 1367, 1168 cm21. H NMR d: 7.31±7.07 (5H, m,
Ph), 6.31 (1H, d, Jˆ15.8 Hz, 8-H), 5.99 (1H, dd, Jˆ8.6,
15.8 Hz, 7-H), 5.00 (1H, ddd, Jˆ3.0, 5.9, 10.5 Hz, 5-H),
4.73 (1H, d, Jˆ7.6 Hz, 15-NH), 4.11 (1H, m, 15-H),
3.55 (2H, m, 3-H), 2.49 (1H, m, 6-H), 1.83 (1H, m,
4-Ha), 1.68-1.23 (4H, m, 4-Hb, 16, 17-H), 1.33 (9H,
s, tBu), 1.03 (3H, d, Jˆ6.9 Hz, 13-H), 0.76, 0.70
(both 3H, d, Jˆ6.6 Hz, 18, 19-H). FAB-MS m/z:
Calcd for C24H37NNaO15 ˆ442.2569. Found: 442.2546
(M1Na)1.
1
1743, 1651, 1514 cm21. H NMR (CDCl3±CD3ODˆ5:1)
d: 7.31±7.04 (5H, m, Ph), 7.00 (2H, d, Jˆ8.5 Hz, 27-H),
6.67 (2H, d, Jˆ8.5 Hz, 28-H), 6.47 (1H, ddd, Jˆ4.9, 10.4,
15.3 Hz, 3-H), 6.23 (1H, d, Jˆ15.9 Hz, 8-H), 5.90 (1H, dd,
Jˆ9.2, 15.9 Hz, 7-H), 5.65 (1H, d, Jˆ15.3 Hz, 2-H), 4.73
(1H, dd, Jˆ5.5, 9.8 Hz, 15-H), 4.39 (1H, dd, Jˆ5.5, 9.2 Hz,
24-H), 3.93 (1H, m, 5-H), 3.63 (3H, s, 30-H), 3.31 (1H, m,
22-Ha), 3.21 (1H, m, 22-Hb), 3.03 (1H, dd, Jˆ5.5, 14.0 Hz,
25-Ha), 2.73 (1H, dd, Jˆ9.2, 14.0 Hz, 25-Hb), 2.37 (4H, m,
4-Ha, 6, 21-H), 2.04 (1H, m, 4-Hb), 1.41±1.11 (3H, m,
16,17-H), 0.99 (3H, d, Jˆ6.7 Hz, 13-H), 0.54, 0.53 (both
3H, d, Jˆ6.7 Hz, 18, 19-H). FAB-MS m/z: Calcd for
C34H44N3O16 ˆ590.3228. Found: 590.3229 (M1H)1.
Dess±Martin oxidation of 18 followed by Horner±
Emmons reaction giving 20. A solution of 18 (36 mg,
0.086 mmol) in dry CH2Cl2 (1.7 ml) was treated with
Dess±Martin periodinane (110 mg, 0.26 mmol) at room
temperature for 1.5 h. The reaction mixture was worked
up in the same manner as preparation for 16 to give a
crude aldehyde. After a solution of segment B±D (19,
70 mg, 0.13 mmol) in dry THF (1.3 ml) was treated with
NaH (20 mg, 60% in oil, 0.5 mmol) at 2108C for 1 h, the
crude aldehyde was added to the solution with CH2Cl2
(0.4 ml). And the whole was stirred for 30 min. The reaction
mixture was poured into saturated aqueous NH4Cl, then the
whole was extracted with EtOAc. The EtOAc extract was
dried over Na2SO4. Removal of solvent from the EtOAc
extract under reduced pressure gave a product, which was
puri®ed by column chromatography (SiO2 5 g, n-hexane±
EtOAcˆ1:1) to furnish 20 (52.5 mg, 76%). 20: Colorless
oil, [a]2D4ˆ131.98 (cˆ0.79 in CHCl3). IR (KBr): 3285,
Epoxidation of 17 giving 2. A solution of 17 (2.5 mg,
0.004 mmol) in CH2Cl2±MeOH (3:1, 0.1 ml) was treated
with dimethyldioxirane (0.074 M in acetone, 0.1 ml) for
6 h. Removal of solvent of the reaction mixture under
reduced pressure gave 2 (2.6 mg, quant.). 2: A white
powder, [a]2D0ˆ113.58 (cˆ0.06 in CHCl3±MeOHˆ5:1).
1
IR (KBr): 2932, 1747, 1657, 1635, 1537 cm21. H NMR
(CDCl3±CD3ODˆ5:1) d: 7.34±7.08 (5H, m, Ph), 7.07
(2H, d, Jˆ8.5 Hz, 27-H), 6.73 (2H, d, Jˆ8.5 Hz, 28-H),
6.54 (1H, ddd, Jˆ4.9, 11.6, 15.9 Hz, 3-H), 5.76 (1H, d,
Jˆ15.9 Hz, 2-H), 4.83 (1H, dd, Jˆ7.3, 10.4 Hz, 15-H),
4.42 (1H, dd, Jˆ4.9, 9.8 Hz, 24-H), 4.03 (1H, m, 5-H),
3.66 (3H, s, 30-H), 3.62 (1H, d, Jˆ1.8 Hz, 8-H), 3.53 (1H,
m, 22-Ha), 3.22 (1H, m, 22-Hb), 3.03 (1H, dd, Jˆ4.9,
14.6 Hz, 25-Ha), 2.89 (1H, dd, Jˆ1.8, 7.9 Hz, 7-H), 2.70
(1H, dd, Jˆ9.8, 14.6 Hz, 25-Hb), 2.52 (1H, m, 4-Ha), 2.43
(2H, m, H-21), 2.14 (1H, m, 4-Hb), 1.58 (1H, m, 6-H), 1.47
(2H, m, 16-Ha, 17-H), 1.27 (1H, m, 16-Hb), 1.06 (3H, d,
Jˆ6.7 Hz, 13-H), 0.68, 0.64 (both 3H, d, Jˆ6.1 Hz, 18,
19-H). FAB-MS m/z: Calcd for C34H44N3O17 ˆ606.7410.
Found: 606.7431 (M1H)1.
1
2957, 1730, 1658, 1631, 1512 cm21. H NMR d: 7.33±
7.19 (5H, m, Ph), 7.12 (2H, d, Jˆ8.6 Hz, 27-H), 6.80 (2H,
d, Jˆ8.6 Hz, 28-H), 6.77 (1H, m, 3-H), 6.56 (1H, d,
Jˆ7.7 Hz, 24-NH), 6.40 (1H, d, Jˆ15.8 Hz, 8-H), 6.26
(1H, brs, 22-NH), 6.05 (1H, dd, Jˆ8.6, 15.8 Hz, 7-H),
5.86 (1H, d, Jˆ15.8 Hz, 2-H), 4.98 (1H, m, 5-H), 4.87
(1H, d, Jˆ8.6 Hz, 15-NH), 4.57 (1H, q-like, Jˆca. 7 Hz,
24-H), 4.21 (1H, m, 15-H), 4.13 (2H, m, OCH2CH2TMS),
3.77 (3H, s, 30-H), 3.47 (1H, m, 22-Ha), 3.34 (1H, m,
22-Hb), 3.01 (2H, d, Jˆ6.0 Hz, 25-H), 2.61 (1H, m, 6-H),
2.50 (2H, m, 21-H), 2.38 (2H, m, 4-H), 1.52-1.22 (3H, m,
16, 17-H), 1.44 (9H, s, tBu), 1.11 (3H, d, Jˆ6.8 Hz, 13-H),
0.96 (2H, t, Jˆ8.6 Hz, OCH2CH2TMS), 0.87, 0.81 (both
3H, d, Jˆ6.4 Hz, 18, 19-H), 0.04 (9H, s, TMS). FAB-MS
Conversion from 6 to 18. A solution of segment A (6,

N. Murakami et al. / Tetrahedron 56 (2000) 9121±9128
9127
m/z: Calcd for C44H66N3O9Si1ˆ808.4568. Found: 808.4592
(M1H)1.
(1H, d, Jˆ1.7 Hz, 8-H), 3.23 (1H, m, 22-Hb), 3.14 (1H, dd,
Jˆ5.1, 14.5 Hz, 25-Ha), 2.98 (1H, dd, Jˆ7.9, 14.5 Hz, 25-
Hb), 2.90 (1H, dd, Jˆ1.7, 7.7 Hz, 7-H), 2.54 (1H, m, 4-Ha),
2.43 (1H, m, 4-Hb), 2.35 (2H, m, 21-H), 1.81 (1H, m, 6-H),
1.53 (1H, m, 17-H), 1.35 (1H, m, 16-Ha), 1.30 (1H, m,
16-Hb), 1.04 (3H, d, Jˆ6.8 Hz, 13-H), 0.90, 0.87 (both
3H, d, Jˆ6.6 Hz, 18,19-H). FAB-MS m/z: Calcd for
C34H43LiN3O17 ˆ612.3261. Found: 612.3255 (M1Li)1.
Deprotection of 20 followed by macrolactamization
giving 21. Deprotection of 20 (50 mg, 0.062 mmol) and
subsequent conversion to HCl salt and macrolactamization
was carried out in the same manner as preparation for 17 to
give a crude product, which was puri®ed by HPLC [column;
COSMOSIL 5C18 AR (10 mm i.d.£250 mm), mobile phase;
CH3CN±H2O±CH2Cl2ˆ60:40:1, detection; UV (lˆ
220 nm), ¯ow rate; 2.0 ml/min] to furnish 21 (25 mg,
68%). 21: A white powder, [a]2D4ˆ140.88 (cˆ0.35 in
CHCl3). IR (KBr): 3294, 2957, 1736, 1662, 1541,
Conversion from 7 to 22. A solution of segment A0 (7,
60 mg, 0.24 mmol) and segment C0 (10, 86 mg,
0.36 mmol) in dry CH2Cl2 (2.0 ml) was treated with DCC
(190 mg, 0.96 mmol) in the presence of DMAP (28 mg,
0.24 mmol) at 08C for 2 h. The reaction mixture was worked
up in the same manner as preparation for 18 to give a crude
product, which was puri®ed by column chromatography
(SiO2 7 g, n-hexane±EtOAcˆ5:1) to furnish an amide
(92 mg, 82%). A solution of the amide (77 mg, 0.18
mmol) was treated with 10% dry HCl±MeOH for 2 h.
Removal of solvent from the reaction mixture under reduced
pressure gave a crude amide. A solution of the crude amide
and segment D0 (12, 34 mg, 0.18 mmol) in dry CH2Cl2
(5.0 ml) was treated with EDCI´HCl (51.4 mg, 0.24 mmol)
and DMAP (26.4 mg, 0.22 mmol) at 08C for 2 h. Usual
work-up was carried out in the same manner as preparation
for 15. Removal of solvent from the EtOAc extract under
reduced pressure gave a product, which was puri®ed by
column chromatography (SiO2 3 g, CHCl3±MeOHˆ50:1)
to furnish 22 (63 mg, 77%). 22: Colorless oil, [a]2D4ˆ
232.68 (cˆ0.33 in CHCl3). IR (KBr): 2955, 1691, 1637,
1
1514 cm21. H NMR d: 7.36±7.19 (5H, m, Ph), 7.07 (2H,
d, Jˆ8.6 Hz, 27-H), 6.96 (1H, m, 22-NH), 6.80 (2H, d,
Jˆ8.6 Hz, 28-H), 6.71 (1H, ddd, Jˆ3.6, 10.5, 14.8 Hz,
3-H), 6.39 (1H, d, Jˆ15.8 Hz, 8-H), 6.01 (1H, dd, Jˆ8.5,
15.8 Hz, 7-H), 5.90 (1H, d, Jˆ8.5 Hz, 15-NH), 5.70 (1H, d,
Jˆ14.8 Hz, 2-H), 5.64 (1H, d, Jˆ6.9 Hz, 24-NH), 5.12 (1H,
ddd, Jˆ2.6, 6.6, 11.2 Hz, 5-H), 4.61 (1H, m, 15-H), 4.51
(1H, m, 24-H), 3.79 (1H, m, 22-Ha), 3.78 (3H, s, 30-H), 3.30
(1H, m, 22-Hb), 3.12 (1H, dd, Jˆ4.9, 14.2 Hz, 25-Ha), 2.96
(1H, dd, Jˆ8.3, 14.2 Hz, 25-Hb), 2.51 (1H, m, 4-Ha), 2.37
(4H, m, 4-Hb, 6, 21-H), 1.75±1.22 (3H, m, 16, 17-H), 1.13
(3H, d, Jˆ6.9 Hz, 13-H), 0.74 (6H, d, Jˆ6.6 Hz, 18, 19-H).
FAB-MS m/z: Calcd for C34H44N3O16 ˆ590.3228. Found:
590.3229 (M1H)1.
Epoxidation of 21 giving 4. A solution of 21 (4.7 mg,
0.008 mmol) in CH2Cl2±MeOH (2:1, 0.3 ml) was treated
with dimethyldioxirane (0.074 M in acetone, 0.8 ml) for
1 h. Removal of solvent of the reaction mixture under
reduced pressure gave a product, which was puri®ed by
HPLC [column; COSMOSIL 5C18 AR (20 mm
i.d.£250 mm), mobile phase; CH3CN±H2O±CH2Cl2ˆ
50:50:1, detection; UV (lˆ240 nm), ¯ow rate; 3.0 ml/
min] to furnish 4 (3.0 mg, 62%) and its epoxy isomer in
2:1 ratio. 4: A white powder, [a]1D7ˆ118.68 (cˆ0.14 in
CHCl3±MeOHˆ1:1). IR (KBr): 3287, 2928, 1660, 1635,
1
1545 cm21. H NMR d: 7.33±7.19 (5H, m, Ph), 6.42 (1H,
d, Jˆ15.8 Hz, 8-H), 6.12 (1H, dd, Jˆ7.9, 15.8 Hz, 7-H),
6.09 (2H, m, 5, 15-NH), 5.04 (1H, m, 22-NH), 4.26 (1H,
q-like, Jˆca. 7 Hz, 15-H), 4.04 (1H, m, 5-H), 3.49 (1H,
m, 3-Ha), 3.38 (1H, m, 3-Hb), 3.35 (2H, m, 22-H), 2.50
(1H, m, 6-H), 2.37 (2H, t-like, Jˆca. 6 Hz, 21-H), 1.98
(1H, m, 4-Ha), 1.63-1.36 (4H, m, 4-Hb, 16, 17-H), 1.43
t
(9H, s, Bu), 1.16 (3H, d, Jˆ6.6 Hz, 13-H), 0.79, 0.77
(both 3H, d, Jˆ6.3 Hz, 18, 19-H). FAB-MS m/z: Calcd for
C27H44N3O15 ˆ490.3262. Found: 490.3260. (M1H)1.
1
1539, 1514 cm21. H NMR d:7.29±7.15 (5H, m, Ph), 7.07
(2H, d, Jˆ8.6 Hz, 27-H), 6.94 (1H, brd, Jˆ6.5 Hz, 22-NH),
6.82 (2H, d, Jˆ8.6 Hz, 28-H), 6.70 (1H, ddd, Jˆ3.9, 7.3,
15.0 Hz, 3-H), 5.82 (1H, d, Jˆ5.0 Hz, 15-NH), 5.68 (1H, d,
Jˆ15.0 Hz, 2-H), 5.54 (1H, d, Jˆ6.4 Hz, 24-NH), 5.28 (1H,
m, 5-H), 4.60 (1H, q-like, Jˆca. 7 Hz, 24-H), 4.50 (1H, m,
15-H), 3.78 (3H, s, 30-H), 3.77 (1H, m, 22-Ha), 3.67 (1H, d,
Jˆ1.7 Hz, 8-H), 3.23 (1H, m, 22-Hb), 3.12 (1H, dd, Jˆ5.1,
14.5 Hz, 25-Ha), 2.96 (1H, dd, Jˆ8.0, 14.5 Hz, 25-Hb), 2.92
(1H, dd, Jˆ1.7, 7.7 Hz, 7-H), 2.55 (1H, m, 4-Ha), 2.42 (1H,
m, 4-Hb), 2.35 (2H, m, 21-H), 1.80 (1H, m, 6-H), 1.52 (1H,
m, 17-H), 1.37 (1H, m, 16-Ha), 1.29 (1H, m, 16-Hb), 1.14
(3H, d, Jˆ6.8 Hz, 13-H), 0.85, 0.83 (both 3H, d, Jˆ6.6 Hz,
18, 19-H). FAB-MS m/z: Calcd for C34H43LiN3O17 ˆ
612.3261. Found: 612.3250 (M1Li)1. Epoxy isomer of 4:
A white powder, [a]D17ˆ119.58 (cˆ0.11 in CHCl3±
MeOHˆ1:1). IR (KBr): 3279, 2910, 1667, 1633, 1534,
Dess±Martin oxidation of 22 followed by Horner±
Emmons reaction. A solution of 22 (59 mg, 0.12 mmol)
in dry CH2Cl2 (2 ml) was treated with Dess±Martin periodi-
nane (102 mg, 0.24 mmol) for 30 min at room temperature.
The reaction mixture was worked up in the same manner as
preparation for 16 to give a crude aldehyde. After a solution
of segment B (8, 91 mg, 0.19 mmol) in dry CH3CN (3 ml)
was treated with LiCl (16.3 mg, 0.38 mmol) and DBU
(32 ml, 0.21 mmol) for 10 min at room temperature, the
crude aldehyde was added to the solution with CH2Cl2
(0.5 ml). Then the reaction mixture was stirred for 10 min.
The reaction mixture was worked up in the same manner as
preparation for 16. Removal of solvent from the EtOAc
extract under reduced pressure gave a product, which was
puri®ed by column chromatography (SiO2 7 g, n-hexane±
EtOAcˆ6:1) to furnish 23 (89 mg, 92%). 23: Colorless oil,
[a]2D4ˆ221.98 (cˆ2.9 in CHCl3). IR (KBr): 3312, 2957,
1
1514 cm21. H NMR d: 7.29±7.15 (5H, m, Ph), 7.07 (2H,
d, Jˆ8.6 Hz, 27-H), 6.96 (1H, brd, Jˆ6.5 Hz, 22-NH), 6.83
(2H, d, Jˆ8.6 Hz, 28-H), 6.71 (1H, ddd, Jˆ3.9, 7.3,
15.0 Hz, 3-H), 5.84 (1H, d, Jˆ5.0 Hz, 15-NH), 5.68 (1H,
d, Jˆ15.0 Hz, 2-H), 5.54 (1H, d, Jˆ6.4 Hz, 24-NH), 5.27
(1H, m, 5-H), 4.60 (1H, q-like, Jˆca. 7.0 Hz, 24-H), 4.50
(1H, m, 15-H), 3.78 (3H, s, 30-H), 3.77 (1H, m, 22-Ha), 3.59
1
1734, 1691, 1643 cm21. H NMR d: 7.37±7.18 (5H, m,
Ph), 7.08 (2H, d, Jˆ8.6 Hz, 27-H), 6.81 (2H, d, Jˆ8.6 Hz,
28-H), 6.69 (2H, m, 3-H,15-NH), 6.41 (1H, d, Jˆ15.8 Hz,
8-H), 6.40 (1H, m, 5-NH), 6.23 (1H, d, Jˆ7.7 Hz, 24-NH),
6.08 (1H, dd, Jˆ8.6, 15.8 Hz, 7-H), 5.78 (1H, d, Jˆ15.2 Hz,

9128
N. Murakami et al. / Tetrahedron 56 (2000) 9121±9128
2-H), 5.16 (1H, brs, 22-NH), 4.79 (1H, q-like, Jˆca. 7 Hz,
24-H), 4.31 (1H, m, 15-H), 4.20 (2H, m, OCH2CH2TMS),
4.05 (1H, m, 5-H), 3.77 (3H, s, 30-H), 3.30 (2H, m, 22-H),
3.11 (1H, dd, Jˆ5.9, 14.1 Hz, 25-Ha), 3.02 (1H, dd, Jˆ6.1,
14.1 Hz, 25-Hb), 2.45 (1H, m, 6-H), 2.37±2.24 (4H, m, 4,
21-H), 1.68±1.40 (3H, m, 16, 17-H), 1.39 (9H, s, tBu), 1.14
(3H, d, Jˆ6.6 Hz, 13-H), 0.88 (2H, m, OCH2CH2TMS),
0.79, 0.78 (both 3H, d, Jˆ6.3 Hz, 18, 19-H), 0.04 (9H,
s, TMS). FAB-MS m/z: Calcd for C44H66LiN4O8Si1ˆ
813.4810. Found: 813.4811 (M1Li)1.
CH3CN±H2O (1:1, 100 ml), then an aliquot (45 ml) was
analyzed by reversed phase HPLC [column; mightysil
RP-18 GP (4.6 mm i.d.£150 mm), mobile phase; CH3CN±
H2Oˆ50:50, detection; UV (lˆ220 nm), ¯ow rate; 1.0 ml/
min] to determine the remain of 1, 2, 4, and 5.
Acknowledgements
The authors are grateful to Dr K. Sugita, the Research
Laboratory of Shionogi Pharmaceutical Co., Ltd., for in
vivo anti-tumor test. The authors are also grateful to the
Naito Foundation, the Houansha Foundation, and the
Ministry of Education, Sciences, Sports, and Culture of
Japan for ®nancial support.
Deprotection of 23 followed by macrolactamization.
Compound 23 (26 mg, 0.033 mmol) was converted to a
macrolactam 24 in the same manner as preparation for 21.
The crude product was puri®ed by HPLC [column;
COSMOSIL 5C18 AR (10 mm i.d.£250 mm), mobile
phase; CH3CN±EtOH±H2O±CH2Cl2ˆ40:10:50:0.5, detec-
tion; UV (lˆ220 nm), ¯ow rate; 3.0 ml/min] to furnish 24
(16.5 mg, 87%). 24: A white powder, [a]1D8ˆ128.98
(cˆ0.09 in CHCl3±MeOHˆ5:1). IR (KBr): 2962, 1651,
References
1639, 1633, 1556 cm21
.
FAB-MS m/z: Calcd for
1. Kobayashi, M.; Aoki, S.; Ohyabu, N.; Kurosu, M.; Wang, W.;
Kitagawa, I. Tetrahedron Lett. 1994, 35, 7969±7972.
2. Kobayashi, M.; Kurosu, M.; Ohyabu, N.; Wang, W.; Fujii, S.;
Kitagawa, I. Chem. Pharm. Bull. 1994, 42, 2196±2198.
3. Kobayashi, M.; Kurosu, M.; Wang, W.; Kitagawa, I. Chem.
Pharm. Bull. 1994, 42, 2394±2396.
C34H45N4O15 ˆ589.3390. Found: 589.3361 (M1H)1. This
compound was characterized by the data of IR and
FAB-MS spectrum because of its extremely low solubility
in various solvent for NMR measurement.
Epoxidation of 24 giving 5. A solution of 24 (4.3 mg
0.007 mmol) in CHCl3±MeOH (3:1, 1.0 ml) was treated
with dimethyldioxirane (0.074 M in acetone, 0.2 ml) for
1 h at 2308C. Removal of solvent from the reaction mixture
under reduced pressure gave a product, which was puri®ed
by HPLC [column; COSMOSIL 5C18 AR (10 mm
i.d.£250 mm), mobile phase; CH3CN±H2Oˆ50:50, detec-
tion; UV (lˆ220 nm), ¯ow rate; 2.5 ml/min] to furnish 5
(2.7 mg, 61%). 5: A white powder, [a]1D9ˆ119.68 (cˆ0.06
in CHCl3±MeOHˆ5:1). IR (KBr): 1637, 1554, 1516,
4. Kobayashi, M.; Wang, W.; Ohyabu, N.; Kurosu, M.; Kitagawa,
I. Chem. Pharm. Bull. 1995, 43, 1598±1600.
5. Koiso, Y.; Morita, K.; Kobayashi, M.; Wang, W.; Ohyabu, N.;
Iwasaki, S. Chem. Biol. Interact. 1996, 102, 183±191.
6. Morita, K.; Koiso, Y.; Hashimoto, Y.; Kobayashi, M.; Wang,
W.; Ohyabu, N.; Iwasaki, S. Biol. Pharm. Bull. 1997, 20, 171±174.
7. Trimurtulu, G.; Ohtani, I.; Patterson, G. M. L.; Moore, R. E.;
Corbett, T. H.; Valeriote, F. A.; Demchik, L. J. Am. Chem. Soc.
1994, 116, 4729±4737.
8. Golakoti, T.; Ogino, J.; Heltzel, C. E.; Husebo, T. L.; Jensen,
C. M.; Larsen, L. K.; Patterson, G. M. L.; Moore, R. E.; Mooberry,
S. L.; Corbett, T. H.; Valeriote, F. A. J. Am. Chem. Soc. 1995, 117,
12030±12049.
1381 cm21
.
1H NMR (d6-DMSO) d: 8.27 (1H, d,
Jˆ7.9 Hz, 24-NH), 7.97 (1H, d, Jˆ9.8 Hz, 5-NH), 7.90
(1H, d, Jˆ7.9 Hz, 15-NH), 7.37±7.16 (5H, m, Ph), 7.13
(2H, d, Jˆ8.5 Hz, 27-H), 7.07 (1H, brd, Jˆ8.5 Hz, 22-
NH), 6.82 (2H, d, Jˆ8.5 Hz, 28-H), 6.39 (1H, ddd, Jˆ3.1,
11.0, 14.6 Hz, 3-H), 5.73 (1H, d, Jˆ14.6 Hz, 2-H), 4.28
(1H, m, 15-H), 4.22 (1H, m, 24-H), 4.03 (1H, m, 5-H),
3.82 (1H, d, Jˆ2.0 Hz, 8-H), 3.70 (3H, s, 30-H), 3.66 (1H,
m, 22-Ha), 3.47 (1H, m, 22-Hb), 2.99 (1H, dd, Jˆ2.0,
7.0 Hz, 7-H), 2.96 (1H, dd, Jˆ4.0, 12.0 Hz, 25-Ha), 2.69
(1H, m, 25-Hb), 2.52 (2H, m, 4-Ha, 21-Ha), 2.16 (1H, m,
21-Hb), 2.09 (1H, m, 4-Hb), 1.50 (2H, m, 6-H, 16-Ha), 1.38
(1H, m, 17-H), 1.17 (1H, m, 16-Hb), 1.01 (3H, d, Jˆ6.8 Hz,
13-H), 0.74, 0.69 (both 3H, d, Jˆ6.7 Hz, 18, 19-H). FAB-
MS m/z: Calcd for C34H44N4NaO16 ˆ627.3158. Found:
627.3151 (M1Na)1.
9. Mitsunobu, O.; Yamada, M. Bull. Chem. Soc. Jpn 1967, 40,
2380±2382.
10. Segment C±D (14) was prepared by condensation of 9 and 12
using EDCI´HCl and DMAP followed by debenzylation with 10%
Pd±C under hydrogen atmosphere.
11. Guindon, Y.; Yoakim, C.; Morton, H. E. Tetrahedron Lett.
1983, 24, 2969±2972.
12. Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48, 4155±4156.
13. Shioiri, T.; Ninomiya, K.; Yamada, S. J. Am. Chem. Soc. 1972,
94, 6203±6205.
14. Brady, S. F.; Freidinger, R. M.; Paleveda, W. J.; Colton, C. D.;
Homnick, C. F.; Whitter, W. L.; Curley, P.; Nutt, R. F.; Veber, D. F.
J. Org. Chem. 1987, 52, 764±769.
15. Kobayashi, M.; Ohyabu, N.; Wang, W.; Kitagawa, I. Proceed-
ings of 116th Annual Meeting of the Japanese Society of
Pharmaceutical Sciences, 1996; p.198.
Evaluation for stability of 1, 2, 4, and 5 in mouse serum.
Each sample (10 ml of 0.1 mg/ml solution in DMSO) was
treated with fresh mouse serum (100 ml) and incubated at
378C for 0, 10, 30, 60, 300 min, respectively. After extrac-
tion of the reaction mixture with EtOAc (500 ml), the
solvent from each EtOAc (400 ml) extract was removed
by nitrogen stream. Each residue was dissolved with
16. Norman, B. H.; Hemscheidt, T.; Schultz, R. M.; Andis, S. L.
J. Org. Chem. 1998, 63, 5288±5294.
17. For example, the solubility of arenastatin A (1) and its amide
analogs (2, 4, 5) in MeOH were as follows; 1: 2.8 mg/ml, 2:
1.1 mg/ml, 4: 0.20 mg/ml, 5: 0.028 mg/ml.