Synthesis of stable analogs in blood and conformational analysis of arenastatin A, a potent cytotoxic spongean depsipeptide

Article abstract of DOI:10.1016/S0040-4020(01)00339-8

In order to produce stable analogs in blood of arenastatin A, a potent cytotoxic depsipeptide from the marine sponge Dysidea arenaria, we synthesized four analogs in which the 15-20 ester linkage was modified. Among them, the carba analog and 20-deoxo analog showed stability in serum. The conformation of arenastatin A and its three analogs were analyzed by distance-restrained molecular dynamic calculation to elucidate a three-dimensional stereostructure contributing to the extremely potent cytotoxicity of arenastatin A.

Full text of DOI:10.1016/S0040-4020(01)00339-8

TETRAHEDRON
Pergamon
Tetrahedron 57 32001) 4323±4336
Synthesis of stable analogs in blood and conformational analysis of
arenastatin A, a potent cytotoxic spongean depsipeptide
Nobutoshi Murakami, Satoru Tamura, Weiqi Wang, Tatsuya Takagi and Motomasa Kobayaship
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka, 565-0871, Japan
Received 28 February 2001; accepted 23 March 2001
AbstractÐIn order to produce stable analogs in blood of arenastatin A, a potent cytotoxic depsipeptide from the marine sponge Dysidea
arenaria, we synthesized four analogs in which the 15-20 ester linkage was modi®ed. Among them, the carba analog and 20-deoxo analog
showed stability in serum. The conformation of arenastatin A and its three analogs were analyzed by distance-restrained molecular dynamic
calculation to elucidate a three-dimensional stereostructure contributing to the extremely potent cytotoxicity of arenastatin A. q 2001
Elsevier Science Ltd. All rights reserved.
Bioassay-guided separation disclosed a cyclic depsipeptide,
arenastatin A 31), with extremely potent cytotoxicity against
tumor cell lines 3IC50: 5 pg/ml against KB cells) as a minute
amount of principle from the Okinawan marine sponge,
Dysidea arenaria.1,2 Thereafter, we achieved the total
synthesis3,4 of 1 by using four segments and found that
arenastatin A 31) displayed cytotoxicity by inhibiting
microtubule assembly through binding to the rhizoxin/
maytansine site on tubulin.5,6 Although 1 was found to
exhibit in vivo anti-tumor activity by ip±ip administration
371% ILS at 5 mg/kg dose against P388 mice), ip±iv
administration reduced its potency signi®cantly because of
instability in mouse serum. Under the assumption that
this regrettable biological behavior is ascribable to
metabolic degradation of the ester linkages in 1, we
synthesized three amide analogs, in which one or two
ester moieties were replaced by an amide group.
Assessment of their stability elucidated that the 15,20-
ester linkage in arenastatin A 31) must be subject to
degradation in blood.7 On the other hand, despite the nearly
complete stability and potent cytotoxicity of triamide analog
32), poor solubility in polar solvents 3such as MeOH,
DMSO, and water) made 2 inapplicable to in vivo anti-
tumor test.7
Chart 1.
Keywords: arenastatin A; depsipeptide; marine metabolite; restrained molecular dynamics.
Corresponding author. Tel. 181-6-6879-8215; fax: 181-6-6879-8219; e-mail: kobayasi@phs.osaka-u.ac.jp
p
0040±4020/01/$ - see front matter q 2001Elsevier Science Ltd. All rights reserved.
PII: S0040-4020301)00339-8

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N. Murakami et al. / Tetrahedron 57+2001) 4323±4336
In this context, we synthesized four analogs [carba analog
33), 20-deoxo analog 34), thio analog 35), and 15-dimethyl
analog 36)], which were expected to exhibit good stability in
serum, and evaluated their biological property 3Chart 1). In
addition, conformational analyses of 1±4 were performed
by molecular dynamic calculation with the distance
restraints obtained from their NOESY spectra in order to
search for a contributive three-dimensional stereostructure
to the potent cytotoxic activity of 1. This paper deals with
the synthesis and biological property of the four analogs
33±6) of arenastatin A 31)8 and the conformational
comparison of 1±4.
position provided the desired cyclic depsipeptide i. The
modi®ed part of the synthesis of arenastatin A 31) is as
follows.
After protecting the hydroxyl group in segment A 37) as a
methoxymethyl 3MOM) ether, removal of the t-butyl-
diphenylsilyl 3TBDPS) group with tetra-n-butylammonium
¯uoride 3TBAF) afforded a primary alcohol 8 in 92% yield
for two steps. Dess±Martin oxidation of 8 followed by
Horner±Emmons reaction with segment B in the presence
of NaH provided an a,b-unsaturated amide, which was
treated with pyridinium p-toluenesulfonate 3PPTS) in
t-BuOH to give segment A-B 39) in 82% yield for three
steps. On the other hand, condensation of segments C 310)
and
D 311) with 3-33-dimethylaminopropyl)-1-ethyl-
1. Synthesis and biological property of carba ꢀ3)and 20-
deoxo analogs ꢀ4)8
carbodiimide hydrochloride 3EDCI´HCl) in the presence
of 4-dimethylaminopyridine 3DMAP) followed by
deprotection of the benzyl ester using Pd-C under hydrogen
atmosphere gave segment C-D 312). Subsequently,
segments A-B 39) and C-D 312) were connected with
EDCI´HCl and DMAP to afford 13. We executed the
macrolactamization of 13 followed by epoxidation as
reported before.4 Namely, simultaneous cleavage of the
two protecting groups in 13 followed by HCl treatment
afforded a seco amino acid as a hydrochloride salt,
which was further subjected to intramolecular macro-
lactamization by use of diphenylphosphorus azide 3DPPA)
and NaHCO3 to give a cyclic depsipeptide 14 in 92%
yield from 13. Finally, epoxidation of 14 was successfully
treated with dimethyldioxirane to furnish the desired
arenastatin A 31) predominantly in a ratio of ca. 2:1
3Scheme 1).
In comparison with arenastatin A 31), the triamide analog
32) exhibited about 1000-fold weaker cytotoxicity against
KB cells. Taking into consideration the contribution of the
respective 20-carbonyl residue and 15,20-ether oxygen, we
undertook the synthesis of carba 33) and 20-deoxo analogs
34). After reporting the total synthesis of 1, we further
modi®ed the synthetic protocol to establish a more facile
route. Chart 2 illustrates our present outline for the synthesis
of arenastatin A 31) and its analogs. Namely, introduction of
the 7,8-epoxy portion was carried out in the ®nal stage
and the precursory cyclic depsipeptide i was disconnected
into four segments A±D. After construction of segments
A-B and C-D, both segments were connected through
5,14-ester linkage. Removal of the protecting group of
ii followed by macrolactamization at the C-22,23
Chart 2.

N. Murakami et al. / Tetrahedron 57+2001) 4323±4336
4325
Scheme 1. Synthesis of arenastatin A 31). Reagents and conditions: 3a) MOMCl, iPr2NEt, CH2Cl2; 3b) TBAF, THF, 92% 2 steps; 3c) Dess±Martin periodinane,
CH2Cl2, 08C; 3d) segment B, NaH, THF, 08C; 3e) PPTS, t-BuOH, re¯ux, 82% 3 steps; 3f) EDCI´HCl, DMAP, CH2Cl2, 97%; 3g) H2, Pd-C, MeOH, 89%;
3h) EDCI´HCl, DMAP, CH2Cl2, quant.; 3i) TFA, CH2Cl2; 3j) HCl±Et2O; 3k) DPPA, NaHCO3, DMF, 08C, 3 steps, 92%; and 3l) dimethyldioxirane, CH2Cl2, 08C,
61%.
Carba analog 33), in which a methylene group is substituted
for the oxygen atom linked to C-15 of 1, was synthesized as
illustrated in Scheme 2. The key segment C-D 320) was
prepared by Sn2 nucleophilic substitution of the carbanion
of segment D 318) for segment C 316) and the following
decarboxylation reaction. Thus, treatment of 3R)-leucinic
acid 315) with trimethylsilyldiazomethane followed by
tri¯uoromethanesulfonylation using Tf2O and 2,6-lutidine
gave segment C 316). Carbonyldiimidazole induced the
coupling of N-Boc-b-alanine 311) and lithium enolate
Scheme 2. Synthesis of carba analog 33). Reagents and conditions: 3a) CHN2TMS, Et2O±MeOH 35:1); 3b) Tf2O, 2,6-lutidine, 2208C, 2 steps 95%; 3c) LDA,
THF, 2788C; 3d) 11, carbonyldiimidazole, THF, 2 steps 83%; 3e) NaH, THF, 2788C, 71%; 3f) TFA; 3g) 3Boc)2O, Et3N, CH2Cl2; 3h) benzene, re¯ux; 3i) LiOH,
THF±H2O 33:1), 4 steps 84%; 3j) 2,4,6-trichlorbenzoyl chloride, Et3N, THF; 3k) 9, Et3N DMAP toluene, 2 steps 97%; 3l) TFA, CH2Cl2; 3m) HCl±Et2O;
3n) DPPA, NaHCO3, DMF, 08C, 3 steps 66%; and 3o) dimethyldioxirane, CH2Cl2, 2108C, 66%.

4326
N. Murakami et al. / Tetrahedron 57+2001) 4323±4336
Scheme 3. Synthesis of 20-deoxo analog 34). Reagents and conditions: 3a) 9, EDCI´HCl, DMAP, CH2Cl2, quant.; 3b) TFA, CH2Cl2; 3c) HCl±Et2O; 3d) DPPA,
NaHCO3, DMF, 08C, 3 steps 74%; and 3e) dimethyldioxirane, CH2Cl2, 65%.
generated from t-butyl acetate 317) to provide segment D
318) in 83% yield. Condensation of 16 and 18 in the
presence of NaH in THF afforded a diastereomeric mixture
19 in 71% yield in a ratio of ca. 2:1. Removal of the t-butyl
group in 19 and subsequent re-introduction of a Boc group
gave a half-ester, which was subjected to decarboxylation
under re¯ux in benzene to yield a ketoester. Saponi®cation
of the resulting ketoester by LiOH furnished the desired
segment C-D 320) with high enantioselectivity in a ratio
of 30:1in 84% overall yield through four steps. The
C-15R con®guration in segment C-D 320) was con®rmed
by Kusumi's method,9 for determining the con®guration at
a chiral center adjacent to a carboxyl group. Thus, 20 was
converted to 3S)- and 3R)-phenylglycine methyl ester
acid 23 was prepared from d-leucine according to
TenBrink's procedure.13 The resulting carboxylic acid 23
was coupled with 9 using EDCI´HCl in the presence of
DMAP in CH2Cl2 to give a conjugated product 24 quantita-
tively. After removal of the two protecting groups followed
by conversion to a hydrochloride salt, the resulting seco acid
was also cyclized intramolecularly to give a cyclic
depsipeptide 25 in moderate overall yield 374%). Treatment
of 25 with dimethyldioxirane furnished 20-deoxo analog
34) as
a
major oxygenated product 3a-epoxide:
b-epoxideˆ1:2).
The synthesized two analogs were evaluated for stability in
mouse serum, and the results are shown in Fig. 1. Carba 33)
and 20-deoxo analog 34) displayed nearly complete stability
in mouse serum as well as moderate cytotoxic ef®cacy
against KB cells 3IC50, 3: 70 ng/ml, 4: 40 ng/ml),
respectively. It should be noted that the two analogs had
higher solubility applicable to in vivo anti-tumor test than
that of the triamide analog 32).8
1
3PGME) amides 320a, 20b) and the respective H NMR
data were compared.8
Condensation of segment A-B 39) and segment C-D 320)
with EDCI´HCl, which afforded the coupled product in
good yield in the syntheses of arenastatin A 31) and the
other analogs 34±6), proceeded in poor yield in the synthesis
of 3. Activation of the carboxyl group using isopropenyl
chloroformate 3IPCF), diphenylphosphoryl azide 3DPPA),
and PyBOP10 also gave unsatisfactory results. Examination
of various activating reagents for the condensation of 9 and
20 led to the ®nding that usage of 2,4,6-trichlorobenzoyl
chloride11 and Et3N furnished the desired diester 21 in
97% yield. Conversion from 21 to a hydrochloride salt
followed by macrolactamization afforded a cyclic depsi-
peptide 22 in 66% overall yield through three steps along
with a stereo isomer 333%). The chemical structure of this
isomer was deduced to be the epimer at C-24 by
comparison of the proton signals due to 2-H, 3-H, 25-
H2 3d: 5.84, 6.51, 3.20, 2.97) from those 3d: 5.72, 6.44,
3.24, 2.91) of the congener obtained during the synthe-
sis of 24-epi-arenastatin A. Although the epimerization
might have occurred in the stage of condensation of 9
and 20, the presence of C-24 epimer could not be detected
from the NMR spectrum of 21. Epoxidation of 22 with
dimethyldioxirane afforded the desired carba analog 33) as
a major product together with an a-epoxide in a ratio of
ca. 2:1.12
Figure 1. Stability of arenastatin A 31) and its analogs 32±4) in mouse
serum. Each sample 310 ml of 0.1mg/ml solution) was treated with fresh
mouse serum 3100 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 remaining
amounts of 1, 2, 3, and 4.
Scheme 3 shows the synthesis of 20-deoxo analog 34)
lacking the 20-carbonyl group of 1. An alkoxy carboxylic

N. Murakami et al. / Tetrahedron 57+2001) 4323±4336
4327
Scheme 4. Synthesis of thio analog 35). Reagents and conditions: 3a) Piv±Cl, Et3N, THF, 08C; 3b) NaSH, 15-crown-5, 08C; 3c) 26, 3 steps 51%; 3d) 9,
EDCI´HCl, DMAP, CH2Cl2, 82%; 3e) TFA, CH2Cl2; 3f) HCl±Et2O; 3g) DPPA, NaHCO3, DMF, 08C, 3 steps 58%; and 3h) dimethyldioxirane, CH2Cl2, 2208C,
31%.
2. Synthesis and biological property of thio ꢀ5)and 15-
dimethyl analogs ꢀ6)
Thus, treatment of 11 with pivaloyl chloride and Et3N
afforded a mixed anhydride, which was subjected to substi-
tution reaction with NaSH in the presence of 15-crown-5 to
generate a sodium salt of thiocarboxylic acid. Without
puri®cation, the sodium salt was directly coupled with 26
to give segment C-D 327) in 51% yield from 11. The con-
densation between segments A-B 39) and C-D 327) afforded
28 in 82% yield. Subsequent removal of the protecting
groups and macrolactamization under the same conditions
in the synthesis of 1 provided 29, which was submitted
to epoxidation using dimethyldioxirane to furnish thio
analog 35).
Judging from the reduction of cytotoxicity observed in 3 and
4 relative to arenastatin A 31), it was presumed that the
15,20-ester function must be required to exert potent cyto-
toxic activity. On the other hand, cryptophycin 1, the
congener of 1, bearing the methyl residue on C-21was
reported by Moore's group14 to display excellent in vivo
anti-tumor effect. We assumed that the different biological
behavior between arenastatin A 31) and cryptophycin 1was
derived from steric hindrance around the 15,20-ester
linkage. Therefore, we further designed thio analog 35)
and 15-dimethyl analog 36), assuming that replacement of
the ester function by a thioester group or steric hindrance
due to two methyl residues on C-15 may prevent metabolic
degradation in blood in mice.
The synthesis of 15-dimethyl analog 36) was performed
using a benzyl ester of 2-hydroxyisobutyric acid 331) in
the same manner as that of arenastatin A 31) as shown in
Scheme 5.
The analog 35) showed potent cytotoxicity against KB cells
3IC50:0.9 ng/ml); nevertheless, the degradation rate in
mouse serum was higher than that of 1. On the contrary,
15-dimethyl analog 36) was somewhat more stable than 1 in
mouse serum; however, the biological potency of 6 was
reduced 3IC50: 200 ng/ml against KB cells) 3Fig. 2).
The synthetic protocol of thio analog 35), in which the C-15
ester oxygen was replaced by a sulfur atom, is summarized
in Scheme 4. The key intermediate, segment C-D 327), was
synthesized by Sn2 nucleophilic substitution of thio-
carboxylic acid generated in situ from N-Boc-b-alanine
311) and 3R)-2-bromo-4-methylpentanoic acid 326).15
Scheme 5. Synthesis of 15-dimethyl analog 36). Reagents and conditions: 3a) BnBr, NaHCO3, TBAI, CH2Cl2, 92%; 3b) 11, EDCI´HCl, DMAP, 3CH2Cl)2, 95%;
3c) H2, Pd-C, MeOH, 88%; 3d) 9, EDCI´HCl, DMAP, CH2Cl2, 96%; 3e) TFA, CH2Cl2; 3f) HCl±Et2O; 3g) DPPA, NaHCO3, DMF, 08C, 3 steps 90%; and 3h)
dimethyldioxirane, CH2Cl2, 08C, 60%.

4328
N. Murakami et al. / Tetrahedron 57+2001) 4323±4336
In the ®rst instance, the NOESY spectra of 1±4 in d6-DMSO
were measured to obtain distance restraints.16 The observed
NOE cross peaks were classi®ed into three classes
depending on their intensities as strong, medium, or weak
and then translated into distance restraints 3upper bound of
Ê
distance: 2.8, 3.5, and 5.0 A), respectively. Based on the
NOE data, an initial structure was built with a complete
random array of atoms and re®ned by energy-minimization
using the BFGS method.17 Then, the re®ned structure was
subjected to 50 cycles of molecular dynamic calculation.
The 50 structures obtained were re®ned by energy-
minimization once again. Among the resulting conformers,
acceptable conformers were selected on the basis of the
criterion that torsion angles of the amide linkages and
enone moieties were within ^158.
The average value of pairwise root-mean-square distance
deviation 3RMSD) for the backbone 316-membered ring)
heavy atoms and the restraint violations of arenastatin A
31) and the three analogs 32±4) are summarized in Table
1. Each compound exhibited not only good convergence on
the 16-membered ring portions, but also good compatibility
with NOE information. Since the two ester linkages in
arenastatin A 31) were clari®ed to play an important role
for potent cytotoxicity from our structure-activity relation-
ship study, we compared the 16-membered ring structure of
each compound focusing on the two torsion angles
[C4±C5±O±C14 and C14±C15±O±C20].
Figure 2. Stability of arenastatin A 31), thio analog 35), and 15-dimethyl
analog 36) in mouse serum.
3. Conformational analysis for arenastatin A ꢀ1)and
three analogs ꢀ2±4)
The foregoing large difference in cytotoxic outcome among
arenastatin A 31) and the analogs of 1 led us to presume that
a conclusive conformation of the 16-membered ring portion
in 1 is contributive to biological potency. We, therefore,
undertook analysis of the conformational features with
respect to arenastatin A 31) and the three analogs 32±4) by
molecular dynamic calculation with distance restraints
using SYBYL 6.5 3Tripos Ass., St Louis, MO).
In each compound, a characteristic cluster consisting of
predominant conformers appeared as depicted in Fig. 3.
Furthermore, the % abundance of conformers belonging to
Table 1. Structural statistics and distance restraint violations
Arenastatin A 31)
Triamide 32)
Carba 33)
20-Deoxo 34)
Number of accepted conformers
Ê
43
0.62^0.22
0.18^0.27
3150
0.42^0.17
0.56^0.45
47
0.44^0.210.62
1.20^1.94
a
RMSD 3A) for backbone heavy atom
Restraint violation 3kcal/mol)a,b
^0.22
0.67^0.41
a
The given numbers are the mean^standard deviation. Backbone means 16-membered ring.
When the calculated distance is greater than the distance restraint, the energy incurred is 2003dmeasure2drestrain)2.
b
Figure 3. Distribution of conformers obtained by molecular dynamic calculation.

N. Murakami et al. / Tetrahedron 57+2001) 4323±4336
4329
Figure 4. The lowest energy structures of arenastatin A 31) and the three analogs 32±4).
each cluster was estimated by Boltzmann distribution. Apart
from the three analogs 32±4), arenastatin A 31) adopted the
torsion-1angle in the vicinity of 1608, leading to a speci®c
conformation.
otherwise speci®ed]. Silica gel 3Merck 60±230 mesh) and
pre-coated thin layer chromatography 3TLC) plates
3Merck, Kiesel gel, 60F254) were used for column chromato-
graphy and TLC. Spots on TLC plates were detected by
spraying vanillin/H2SO4 3vanillin 5 g, H2SO4 95 ml) or
acidic p-anisaldehyde solution 3p-anisaldehyde 25 ml,
H2SO4 25 ml, AcOH 5 ml, EtOH 425 ml) with subsequent
heating.
Fig. 4 illustrates the lowest energy structure of each
compound, which was contained in the respective predomi-
nant cluster. With respect to arenastatin A 31), the carbonyl
residue at C-20 was pointed toward the outside region of the
16-membered ring. In contrast, the directions of the C-20
carbonyl groups of 2 and 3 were deviated from that of 1.
Namely, the C-20 carbonyl residue of triamide analog 32)
was oriented to the downward region of the macrocyclic
ring. In the case of carba analog 33), the C-20 carbonyl
function was turned toward the inside of the 16-membered
ring moiety. As for the C-14 carbonyl groups, that of 20-
deoxo analog 34) pointed toward a different spatial region.
4.1. Conversion from 7 to 8
A solution of segment A 37, 250 mg, 0.56 mmol) in dry
CH2Cl2 310 ml) was treated with methoxymethylchloride
3130 ml) in the presence of iPr2NEt 3400 ml) for 10 h. The
reaction mixture was poured into aqueous saturated NaCl,
and then the whole was extracted with EtOAc. The EtOAc
extract was washed with 5% HCl, aqueous saturated
NaHCO3, and aqueous saturated NaCl, then dried over
MgSO4. Removal of solvent from the EtOAc extract
under reduced pressure gave a MOM ether. A solution of
the MOM ether in dry THF 35.6 ml) was treated with tetra-
n-butylammonium ¯uoride 3TBAF, 1.0 M in THF, 0.8 ml)
for 3 h. The reaction mixture was poured into water, then the
whole was extracted with EtOAc. The EtOAc extract was
washed with water, then dried over MgSO4. Removal of
solvent from the EtOAc extract under reduced pressure
gave a product, which was puri®ed by column chromato-
graphy 3SiO2: 8 g, hexane:EtOAcˆ2:1) to furnish 8
3130 mg, 92% 2 steps).
The present conformational analysis led us to presume
that the characteristic spatial orientation of the C-20
carbonyl residue and the 16-membered ring structure
in 1 would be associated with rigid binding to the
active site in tubulin to exert the extremely potent cyto-
toxicity of 1. A program to develop new analogs utilizing
this template structure of arenastatin A 31), obtained by the
present molecular dynamic calculation, is proceeding in our
laboratory.
4. Experimental
8: Colorless oil, [a]23D232.78 3cˆ0.40 in CHCl3). IR 3KBr):
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 3500 MHz) NMR
spectrometer for H NMR spectra [CDCl3 solution with
tetramethylsilane 3TMS) as an internal standard unless
1
3422, 2953, 2885, 1599 cm21. H NMR d: 7.40±7.17 35H,
m, 10, 11, 12-H), 6.40 31H, d, Jˆ15.8 Hz, 8-H), 6.15 31H,
dd, Jˆ7.6, 15.8 Hz, 7-H), 4.70 32H, m, CH2OCH3), 3.83±
3.70 33H, m, 3, 5-H), 3.42 33H, s, CH2OCH3), 2.60 31H, m,
6-H), 2.40 31H, m, OH), 1.75 32H, m, 4-H), 1.13 33H, d,
1

4330
N. Murakami et al. / Tetrahedron 57+2001) 4323±4336
Jˆ6.9 Hz, 13-H). FABMS m/z: 273 [M1Na]1. FABHRMS
m/z: Calcd for C15H22O31Na: 273.1467. Found: 273.1442.
atmosphere at room temperature for 2 h. The reaction
mixture was ®ltered through Celite, then removal of solvent
from the ®ltrate gave a product, which was puri®ed by
column chromatography 3SiO2: 8 g, CHCl3:MeOHˆ7:1)
to furnish segment C-D 312, 260 mg, 89%).
4.2. Conversion from 8 to 9
A solution of 8 375 mg, 0.30 mmol) in dry CH2Cl2 35 ml)
was treated with Dess±Martin periodinane 3500 mg) for 1h
at 08C. After adding aqueous saturated NaHCO3 34 ml) and
Na2S2O3 34 ml), the reaction mixture was further stirred for
1h at room temperature. The whole was extracted with
EtOAc, and then the EtOAc extract was dried over
MgSO4. Removal of solvent from the EtOAc extract
under reduced pressure gave an aldehyde. A solution of
segment B 3260 mg, 0.56 mmol) in dry THF 35.5 ml) was
treated with NaH 367 mg, ca. 60% in paraf®n liquid) for 1h
at 08C. To this reaction mixture was added a solution of the
aldehyde in dry THF 31.5 ml), then the whole was stirred for
20 min at 08C. The reaction mixture was quenched with
aqueous saturated NH4Cl, and then the whole was extracted
with EtOAc. The EtOAc extract was dried over MgSO4.
Removal of solvent from the EtOAc extract under reduced
pressure gave a crude product, which was puri®ed by
column chromatography 3SiO2: 8 g, hexane:EtOAcˆ5:1)
to furnish an amide 3136 mg). A solution of the amide in
dry t-BuOH 35 ml) was treated with PPTS 3600 mg) for 6 h
under re¯ux. The whole was extracted with EtOAc, and then
the EtOAc extract was washed with aqueous saturated NaCl
and dried over MgSO4. Removal of solvent from the EtOAc
extract under reduced pressure gave a product, which was
puri®ed by column chromatography 3SiO2: 8 g, hexane:
EtOAcˆ3:1) to furnish segment A-B 39, 120 mg, 82% 3
step).
12: A white powder, [a]22D224.08 3cˆ1.1 in CHCl3). IR
3KBr): 3371, 2959, 1734, 1523 cm21. 1H NMR d: 4.98 31H,
brs, 22-NH), 4.16 31H, t, Jˆ7.3 Hz, 15-H), 3.43 32H, m,
22-H), 2.53 32H, t, Jˆ6.3 Hz, 21-H), 1.80±1.60 33H, m,
16, 17-H), 1.41 39H, s, tBu), 0.98, 0.96 3both 3H, d,
Jˆ6.7 Hz, 18, 19-H), FABMS m/z: 326 [M1Na]1.
FABHRMS m/z: Calcd for C14H25NO61Na: 326.1580.
Found: 326.1583.
4.4. Condensation of segment A-B ꢀ9)and C-D ꢀ12)
giving 13
A solution of 9 3140 mg, 0.26 mmol) and 12 3160 mg,
0.54 mmol) in dry CH2Cl2 34 ml) was treated with
EDCI´HCl 3320 mg) in the presence of DMAP 334 mg) at
room temperature for 1h. The reaction mixture was poured
into aqueous saturated NH4Cl, and then the whole was
extracted with EtOAc. The EtOAc extract was washed
with 5% HCl, aqueous saturated NaHCO3, and aqueous
saturated NaCl, and then dried over MgSO4. Removal of
solvent from the EtOAc extract under reduced pressure
gave a product, which was puri®ed by column chromato-
graphy 3SiO2: 7 g, hexane:EtOAcˆ3:1) to furnish 13
3190 mg, quant.).
13: Colorless oil, [a]22D23.38 3cˆ1.2 in CHCl3). IR 3KBr):
2969, 1711, 1670, 1610, 1512 cm21. 1H NMR d: 7.38±7.30
35H, m, 10, 11, 12-H), 7.02 32H, d, Jˆ8.5 Hz, 27-H), 6.79
32H, d, Jˆ8.5 Hz, 28-H), 6.76 31H, m, 3-H), 6.40 31H, d,
Jˆ15.8 Hz, 8-H), 6.01 31H, dd, Jˆ8.5, 15.8 Hz, 7-H), 5.88
31H, d, Jˆ15.9 Hz, 2-H), 5.02 31H, q-like, Jˆca. 6 Hz, 5-H),
4.92 31H, dd, Jˆ3.7, 10.4 Hz, 15-H), 4.87 31H, dt, Jˆ7.5,
6.1Hz, 24-H), 4.18 32H, m, C H2CH2TMS), 3.76 33H, s, 29-
OMe), 3.35 32H, brd, Jˆ6.1Hz, 22-H), 3.08 32H, m, 25-H),
2.56 35H, m, 4, 6, 21-H), 1.64 33H, m, 16, 17-H), 1.42 39H,
s, tBu), 1.10 33H, d, Jˆ7.4 Hz, 13-H), 0.96 32H, m,
CH2CH2TMS), 0.85 33H, d, Jˆ6.1Hz), 0.81 33H, d,
Jˆ6.1 Hz, 18, 19-H), 0.03 39H, s, TMS). FABMS m/z:
9: Colorless oil, [a]20D221.28 3cˆ0.33 in CHCl3). IR 3KBr):
1
3366, 2955, 1738, 1668, 1631, 1514 cm21. H NMR d:
7.39±7.20 35H, m, 10, 11, 12-H), 7.01 32H, d, Jˆ8.6 Hz,
27-H), 6.9131H, m, 3-H), 6.80 32H, d, Jˆ8.6 Hz, 28-H),
6.46 31H, d, Jˆ15.8 Hz, 8-H), 6.12 31H, dd, Jˆ8.6, 15.8 Hz,
7-H), 5.913H1, d,
Jˆ7.6 Hz, 24-NH), 5.88 31H, d,
Jˆ15.5 Hz, 2-H), 4.86 31H, ddd, Jˆ5.3, 5.3, 7.6 Hz, 24-
H), 4.22 32H, m, CH2CH2TMS), 3.77 33H, s, 29-OMe),
3.65 31H, m, 5-H), 3.09 32H, d-like, Jˆ5.3 Hz, 25-H),
2.45±2.33 33H, m, 4, 6-H), 1.14 33H, d, Jˆ6.8 Hz, 13-H),
0.94 32H, t, Jˆ8.6 Hz, CH2CH2TMS), 0.04 39H, s, TMS).
FABMS m/z: 524 [M1H]1. FABHRMS m/z: Calcd for
C30H41NO2Si1H: 524.2832: Found: 524.2857.
809
[M1H]1.
FABHRMS
m/z:
Calcd
for
C44H64N2O10Si1H: 809.4409. Found: 809.4429.
4.3. Preparation for segment C-D ꢀ12)
4.5. Deprotection of 13 followed by macrolactamization
giving 14
A solution of segment C 310, 220 mg, 1.0 mmol) and
segment D 311, 490 mg, 2.5 mmol) in dry CH2Cl2 310 ml)
were treated with EDCI´HCl 31.2 g) in the presence of
DMAP 3320 mg) at room temperature for 3 h. The reaction
mixture was poured into aqueous saturated NH4Cl, and then
the whole was extracted with EtOAc. The EtOAc extract
was washed with 5% HCl, aqueous saturated NaHCO3, and
aqueous saturated NaCl, then dried over MgSO4. Removal
of solvent from the EtOAc extract under reduced pressure
gave a product, which was puri®ed by column chromato-
graphy 3SiO2: 25 g, hexane:EtOAcˆ12:1) to furnish a
diester 3380 mg, 97%). To a solution of this diester
3380 mg) in MeOH 320 ml) was added 10% Pd-C
3100 mg), then the reaction mixture was stirred under H2
A solution of 13 388 mg, 0.11 mmol) in dry CH2Cl2 32 ml)
was treated with TFA 32 ml) at room temperature for 5 h to
give a seco amino acid as a TFA salt, which was treated with
saturated HCl gas±Et2O three times to furnish an HCl salt.
A solution of HCl salt in dry DMF 320 ml) was treated with
DPPA 345 ml) and NaHCO3 345 mg) at 08C for 48 h. The
reaction mixture was poured into aqueous saturated NH4Cl,
and 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 3SiO2: 7 g,
hexane:EtOAcˆ1:3) to furnish 14 359 mg, 92%).

N. Murakami et al. / Tetrahedron 57+2001) 4323±4336
4331
14: A white powder, [a]25D117.58 3cˆ0.14 in CHCl3). IR
3KBr): 2920, 1741, 1728, 1651, 1602, 1516 cm21. 1H NMR
d: 7.34±7.20 35H, m, 10, 11, 12-H), 7.11 32H, d, Jˆ8.5 Hz,
27-H), 6.8132H, d, Jˆ8.5 Hz, 28-H), 6.7131H, ddd, Jˆ4.5,
10.1, 15.1 Hz, 3-H), 6.40 31H, d, Jˆ16.0 Hz, 8-H), 6.01
31H, dd, Jˆ9.3, 16.0 Hz, 7-H), 5.73 31H, d, Jˆ15.1 Hz,
2-H), 5.57 31H, d, Jˆ7.9 Hz, 24-NH), 5.05 31H, m, 5-H),
4.90 31H, dd, Jˆ3.5, 9.6 Hz, 15-H), 4.63 31H, ddd, Jˆ5.3,
7.2, 7.9 Hz, 24-H), 3.78 33H, s, 29-OMe), 3.53 31H, m,
22-Ha), 3.44 31H, m, 22-Hb), 3.16 31H, dd, Jˆ5.3,
14.3 Hz, 25-Ha), 3.05 31H, dd, Jˆ7.2, 14.3 Hz, 25-Hb),
2.54, 2.37 3total 5H, m, 4, 6, 21-H), 1.63, 1.33 3total 3H,
m, 16, 17-H), 1.13 33H, d, Jˆ6.9 Hz, 13-H), 0.75 33H, d,
Jˆ6.6 Hz), 0.72 33H, d, Jˆ6.4 Hz, 18, 19-H). FABMS m/z:
591[M 1H]1. FABHRMS m/z: Calcd for C34H42N2O71H:
591.3070. Found: 591.3072.
treated with carbonyldiimidazole 32.4 g) for 30 min at 08C,
and then the reaction mixture was stirred for 3 h at room
temperature. The reaction mixture was added to a solution
of lithium enolate of 17, which was prepared by treatment of
t-butyl acetate 35.4 ml) with a solution of LDA 32.0 M,
20 ml) for 1h at 2788C, and then the whole was stirred
for 30 min. The reaction mixture was poured into aqueous
saturated NH4Cl, and then the whole was extracted with
EtOAc. The EtOAc extract was washed with aqueous
saturated NH4Cl, then dried over MgSO4. Removal of
solvent from the EtOAc extract under reduced pressure
gave a product, which was puri®ed by column chromato-
graphy 3SiO2: 150 g, hexane:EtOAcˆ5:1) to furnish 18
32.9 g, 83%).
18: Colorless oil, IR 3KBr): 2920, 1728, 1708, 1518 cm21
.
1H NMR d: 4.95 31H, brs, 22-NH), 3.38 32H, q-like, Jˆca.
5 Hz, 22-H), 3.36 32H, s, 150-H), 2.76 32H, t, Jˆ5.5 Hz,
21-H), 1.49, 1.42 3both 9H, s, tBu£2). FABMS m/z: 288
[M1H]1. FABHRMS m/z: Calcd for C14H25NO51H:
288.1811. Found: 288.1814.
4.6. Epoxidation of 14 giving 1
A solution of 14 327 mg, 0.046 mmol) in CH2Cl2 320 ml)
was treated with dimethyldioxirane 30.074 M in acetone,
10 ml) for 3 h at 08C. Removal of solvent from the reaction
mixture under reduced pressure gave a product, which was
puri®ed by HPLC [column: YMC-Pack ODS 320 mm
4.8. Preparation for diester 19
i.d.£250 mm), MeOH:H2Oˆ75:25,
mobile
phase;
A solution of 3R)-leucinic acid 315, 1.3 g, 9.7 mmol) in Et2O
35 ml)-MeOH 31ml) was treated with CHN 2TMS 32.0 M in
hexane, 5 ml) for 1h. Removal of solvent from the reaction
mixture under reduced pressure gave a methyl ester. A
solution of 2,6-lutidine 31.0 ml) in dry CH2Cl2 310 ml)
was treated with tri¯uoromethanesulfonic anhydirde
31.4 ml) for 10 min at 2208C. A solution of the methyl
ester in CH2Cl2 33 ml) was added to the reaction mixture,
and then the whole was stirred at 2208C for 15 min and at
room temperature for 1h. The reaction mixture was ®ltered,
and then the ®ltrate was dried over MgSO4. Removal of
solvent from the ®ltrate under reduced pressure gave a
crude tri¯ate 16, which was not further puri®ed because of
its instability. A solution of 18 31.1 g, 3.8 mmol) in dry THF
320 ml) was treated with NaH 3130 mg) for 40 min at 08C.
After the reaction mixture was cooled to 2788C, a solution
of 16 in THF 33 ml) was added to the reaction mixture and
the whole was stirred for 48 h at 2788C. The reaction
mixture was poured into aqueous saturated NH4Cl, and
then the whole was extracted with EtOAc and dried
over MgSO4. Removal of solvent from the EtOAc extract
under reduced pressure gave a product, which was puri®ed
by column chromatography 3SiO2:40 g, hexane:
EtOAcˆ10:1) to furnish 19 31.1 g, 71%) as a mixture of
two diastereomers.
detection] to furnish 1 317 mg, 61%).
1: A white powder, [a]18D135.58 3cˆ0.21in CHCl ). IR
3
3KBr): 2928, 1742, 1680, 1660, 1614, 1535, 1514 cm21. 1H
NMR d: 7.42±7.23 35H, m, 10, 11, 12-H), 7.10 32H, d,
Jˆ8.5 Hz, 27-H), 6.80 32H, d, Jˆ8.5 Hz, 28-H), 6.70 31H,
ddd, Jˆ4.9, 10.4, 15.3 Hz, 3-H), 5.70 31H, d, Jˆ15.3 Hz,
2-H), 6.62 31H, brd, Jˆ7.3 Hz, 24-NH), 5.19 31H, ddd,
Jˆ3.6, 5.5, 9.7 Hz, 5-H), 4.89 31H, dd, Jˆ3.7, 10.4 Hz,
15-H), 4.73 31H, q-like, Jˆca. 7 Hz, 24-H), 3.77 33H, s,
29-OMe), 3.68 31H, d, Jˆ1.8 Hz, 8-H), 3.48 31H, m,
22-Ha), 3.44 31H, m, 22-Hb), 3.13 31H, dd, Jˆ5.9,
14.0 Hz, 25-Ha), 3.02 31H, dd, Jˆ7.3, 14.0 Hz, 25-Hb),
2.92 31H, dd, Jˆ1.8, 7.9 Hz, 7-H), 2.53 3total 4H, m,
4-H, 21-H), 1.80 31H, m, 6-H), 1.67, 1.29 3total 3H, m,
16-H, 17-H), 1.29 31H, m, 16-Hb), 1.14 33H, d, Jˆ7.3 Hz,
1
13-H), 0.84, 0.82 3both 3H, d, Jˆ6.1 Hz, 18, 19-H). H
NMR 3d6-DMSO) d: 8.20 31H, d, Jˆ8.5 Hz, 24-NH),
7.38±7.30 35H, m, 10, 11, 12-H), 7.21 31H, m, 22-NH),
7.12 32H, d, Jˆ8.5 Hz, 27-H), 6.82 32H, d, Jˆ8.5 Hz,
28-H), 6.42 31H, ddd, Jˆ3.5, 10.2, 15.1 Hz, 3-H), 5.77
31H, d, Jˆ15.1 Hz, 2-H), 5.12 31H, ddd, Jˆ3.6, 5.2,
10.6 Hz, 5-H), 4.97 31H, dd, Jˆ3.9, 9.4 Hz, 15-H), 4.27
31H, m, 24-H), 3.88 31H, d, Jˆ1.8 Hz, 8-H), 3.70 33H, s,
29-OMe), 3.37 31H, m, 22-Ha), 3.24 31H, m, 22-Hb), 2.99
31H, dd, Jˆ1.8, 7.4 Hz, 7-H), 2.97 31H, dd, Jˆ4.5, 12.3 Hz,
25-Ha), 2.68 31H, m, 4-Ha), 2.65 31H, m, 25-Hb), 2.61 31H,
m, 21-Ha), 2.27 32H, m, 21-Hb, 4-Hb), 1.83 31H, m, 6-H),
1.56 31H, m, 17-H), 1.50 31H, m, 16-Ha), 1.24 31H, m, 16-
Hb), 1.05 33H, d, Jˆ7.3 Hz, 13-H), 0.78 33H, d, Jˆ6.8 Hz),
0.76 33H, d, Jˆ6.4 Hz, 18, 19-H). FABMS m/z: 607
[M1H]1. FABHRMS m/z: Calcd for C34H42N2O81H:
607.3019. Found: 607.3015.
19: Colorless oil, IR 3KBr): 2925, 1728, 1712, 1698,
1
1510 cm21. H NMR d 3major diastereomer): 4.95 31H,
brs, 22-NH), 3.72 31H, d, Jˆ4.5 Hz, 150-H), 3.67 33H, s,
COOMe), 3.36 32H, m, 22-H), 3.19 31H, dt, Jˆ4.5, 10.0 Hz,
15-H), 2.71 32H, m, 21-H), 1.60±1.51 33H, m, 16, 17-H),
1.46, 1.42 3both 9H, s, tBu£2), 0.94, 0.88 3both 3H, d,
1
Jˆ6.5 Hz, 18, 19-H). H NMR d 3minor diastereomer):
4.95 31H, brs, 22-NH), 3.74 31H, d, Jˆ3.8 Hz, 150-H),
3.69 33H, s, COOMe), 3.36 32H, m, 22-H), 2.80±2.75
33H, m, 15, 21-H), 1.60±1.51 33H, m, 16, 17-H), 1.48,
1.43 3both 9H, s, tBu£2), 0.93, 0.86 3both 3H, d,
6
[1MH]1.
4.7. Coupling between 11 and t-butyl acetate ꢀ17)giving
segment D ꢀ18)
Jˆ6.5 Hz, 18, 19-H). FABMS m/z: 41
A solution of 11 32.5 g, 13 mmol) in dry THF 315 ml) was

4332
N. Murakami et al. / Tetrahedron 57+2001) 4323±4336
FABHRMS m/z: Calcd for C21H37NO71H: 416.2726.
Found: 416.2632.
m, 16-Hb), 0.95 33H, d, Jˆ6.7 Hz), 0.89 33H, d,
Jˆ6.1 Hz, 18, 19-H). FABMS m/z: 449 [M1H]1.
FABHRMS m/z: Calcd for C24H37N2O61H: 449.2651.
Found: 449.2672.
4.9. Preparation for segment C-D ꢀ20)
A solution of 19 3390 mg, 0.94 mmol) in TFA 34 ml) was
stirred for 2 h at room temperature. Removal of TFA from
the reaction mixture gave an aminocarboxylic acid. A
solution of the aminocarboxylic acid in dry CH2Cl2 35 ml)
was treated with Et3N 3530 ml) and 3Boc)2O 3330 mg) for
3 h at room temperature. The reaction mixture was poured
into aqueous NaH2PO4, then the whole was extracted with
EtOAc. The EtOAc extract was washed with aqueous
saturated NaCl, then dried over MgSO4. Removal of solvent
from the EtOAc extract under reduced pressure gave a half-
ester. A solution of the half-ester in benzene 310 ml) was
heated under re¯ux for 5 h. Removal of solvent from the
reaction mixture under reduced pressure gave a product,
which was puri®ed by column chromatography 3SiO2:
20 g, hexane:EtOAcˆ5:1) to furnish a ketoester 3250 mg,
84%). A solution of the ketoester in THF±H2O 33:1, 9 ml)
was treated with 1N LiOH 32.4 ml) for 1 h at room tempera-
ture. The reaction mixture was acidi®ed with 5% HCl to pH
3, and then the whole was extracted with EtOAc. The
EtOAc extract was dried over MgSO4. Removal of solvent
from the EtOAc extract under reduced pressure gave a
product, which was puri®ed by column chromatography
3SiO2: 10 g, benzene: acetoneˆ5:1) to furnish 20 3240 mg,
quant.).
4.11. Preparation for ꢀR)-PGME amide of 20
Segment C-D 320, 6.5 mg, 0.021mmol) was converted to
20b 36.5 mg, 66%) in the same manner as preparation for
20a.
20b: Colorless oil, [a]22D115.08 3cˆ0.58 in CHCl3). IR
3KBr): 2953, 2851, 1740, 1642, 1561, 1515 cm21 1H
.
NMR d: 7.36±7.20 35H, m, Ph), 6.75 31H, d, Jˆ6.7 Hz,
amide-NH), 5.45 31H, d, Jˆ6.7 Hz, PGME-a-H), 5.16
31H, brs, 22-NH), 3.71 33H, s, COOMe), 3.40 32H, brd,
Jˆ5.5 Hz, 22-H), 2.88 31H, m, 150-Ha), 2.87 31H, m,
15-H), 2.65 32H, m, 21-H), 2.43 31H, m, 150-Hb),1.60
31H, m, 16-Ha), 1.50 31H, m, 17-H), 1.42 39H, s, tBu),
1.12 31H, m, 16-Hb), 0.87 33H, d, Jˆ6.7 Hz), 0.83 33H, d,
Jˆ6.1 Hz, 18, 19-H). FABMS m/z: 449 [M1H]1.
FABHRMS m/z: Calcd for C24H37N2O61H: 449.2651.
Found: 449.2686.
4.12. Condensation of segment A-B ꢀ9)with segment C-D
ꢀ20)
A solution of 20 375 mg, 0.25 mmol) and Et3N 370 ml) in dry
THF 32.5 ml) was treated with 2,4,6-trichlorobenzoyl-
chloride 344 ml) for 3 h at room temperature. The solvent
of the reaction mixture was removed, then to the residue was
added the solution of 9 347 mg, 0.090 mmol) in dry toluene
35 ml). After addition of Et3N 370 ml) and DMAP 32.0 mg),
the reaction mixture was stirred for 2 h at room temperature.
Work-up in the same manner as preparation for 13 gave a
product, which was puri®ed by column chromatography
3SiO2: 4 g, benzene: acetoneˆ50:1) to furnish 21 370 mg,
97%).
20: Colorless oil, [a]22D13.88 3cˆ2.5 in CHCl3). IR 3KBr):
3362, 2959, 1714, 1683, 1521 cm21. 1H NMR d: 4.95 31H,
brs, 22-NH), 3.28 32H, m, 22-H), 2.87 31H, m, 15-H), 2.74
31H, dd, Jˆ9.9, 18.0 Hz, 150-Ha), 2.59 32H, m, 21-H), 2.43
31H, brd, Jˆ18.0 Hz, 150-Hb), 1.59±1.49 33H, m, 16, 17-H),
1.40 39H, s, tBu), 0.87, 0.83 3both 3H, d, Jˆ6.5 Hz, 18, 19-
H). FABMS m/z: 324 [M1Na]1. FABHRMS m/z: Calcd for
C15H27NO51Na: 324.1787. Found: 324.1798.
21: Colorless oil, [a]22D20.838 3cˆ5.2 in CHCl3). IR 3KBr):
4.10. Preparation for ꢀS)-PGME amide of 20
1
3360, 2959, 1731, 1716, 1672, 1608, 1512 cm21. H NMR
d: 7.38±7.30 35H, m, 10, 11, 12-H), 7.20 31H, m, 22-NH),
7.02 32H, d, Jˆ8.6 Hz, 27-H), 6.80 31H, m, 3-H), 6.79 32H,
d, Jˆ8.6 Hz, 28-H), 6.39 31H, d, Jˆ16.0 Hz, 8-H), 6.05
31H, dd, Jˆ8.6, 16.0 Hz, 7-H), 5.92 31H, d, Jˆ15.0 Hz,
2-H), 4.98 31H, m, 5-H), 4.88 31H, m, 24-H), 4.20 32H, m,
CH2CH2TMS), 3.76 33H, s, 29-OMe), 3.25 32H, m, 22-H),
3.08 32H, m, 25-H), 2.88, 2.59, 2.50 3total 6H, m, 4, 6, 15,
21-H), 2.77 31H, dd, Jˆ10.4, 17.7 Hz, 150-Ha), 2.42 31H,
dd, Jˆ4.2, 17.7 Hz, 150-Hb), 1.77±1.47 33H, m, 16, 17-H),
1.41 39H, s, tBu), 1.09 33H, d, Jˆ6.7 Hz, 13-H), 0.96 32H,
m, CH2CH2TMS), 0.82 33H, d, Jˆ6.7 Hz), 0.79 33H,
d, Jˆ6.1 Hz, 18 or 19-H), 0.03 39H, s, TMS). FABMS
m/z: 807 [M1H]1. FABHRMS m/z: Calcd for
C45H66N2O9Si1H: 807.4616. Found: 807.4630.
To a solution of segment C-D 320, 5.5 mg, 0.018 mmol)and
3S)-PGME 34 mg, 0.023 mmol) in dry DMF 30.2 ml) was
added Py-BOP 311 mg), 1-hydroxybenzotriazole 33 mg)
and N-methylmorpholine 36 ml) at 08C, and then the
whole was stirred for 2 h at room temperature. The reaction
mixture was diluted with benzene 31ml) and EtOAc 31ml),
and then the whole was washed with 5% HCl, aqueous
saturated NaHCO3, and aqueous saturated NaCl, then
dried over Na2SO4. Removal of solvent from the organic
layer gave a product, which was puri®ed by column chroma-
tography 3SiO2: 600 mg, hexane:EtOAcˆ2:1) to furnish
20a 36.0 mg, 79%).
20a: Colorless oil, [a]22D23.98 3cˆ0.55 in CHCl3). IR
3KBr): 2948, 2851, 1740, 1639, 1560, 1515 cm21 1H
.
NMR d: 7.36±7.20 35H, m, Ph), 6.67 31H, d, Jˆ6.7 Hz,
amide-NH), 5.50 31H, d, Jˆ6.7 Hz, PGME-a-H), 4.88
31H, brs, 22-NH), 3.73 33H, s, COOMe), 3.25 32H, m,
22-H), 2.82 31H, m, 15-H), 2.80 31H, m, 150-Ha), 2.56
31H, dt, Jˆ17.5, 5.5 Hz, 21-Ha), 2.46 31H, dt, Jˆ17.5,
5.5 Hz, 21-Hb), 2.39 31H, dd, Jˆ8.0, 14.0 Hz, 150-Hb),
1.61 32H, m, 16-Ha, 17-H), 1.41 39H, s, tBu), 1.20 31H,
4.13. Deprotection of 21 followed by macrolactamization
giving 22
A solution of 21 340 mg, 0.050 mmol) in dry CH2Cl2
31.5 ml) was treated with TFA 33 ml) at room temperature
for 8 h to give a seco amino acid as a TFA salt, which was
treated with saturated HCl gas±Et2O three times to furnish

N. Murakami et al. / Tetrahedron 57+2001) 4323±4336
4333
an HCl salt. A solution of the HCl salt in dry DMF 310 ml)
was treated with DPPA 314 ml) and NaHCO3 313 mg) at 08C
for 48 h. Work-up in the same manner as preparation for 14
gave a product, which was puri®ed by column chromato-
graphy 3SiO2: 4 g, benzene: acetoneˆ6:1) to furnish 22
320 mg, 66%).
d, Jˆ6.7 Hz), 0.70 33H, d, Jˆ6.4 Hz, 18, 19-H). FABMS
m/z: 605 [M1H]1. FABHRMS m/z: Calcd for
C35H44N2O71H: 605.3227. Found: 605. 3224.
4.15. Condensation of segment A-B ꢀ9)with segment C-D
ꢀ23)
22: A white powder, [a]22D128.08 3cˆ0.20 in CHCl3). IR
3KBr): 2920, 1735, 1714, 1660, 1602, 1530 cm21. 1H NMR
d: 7.34±7.20 35H, m, 10, 11, 12-H), 7.10 32H, d, Jˆ8.6 Hz,
27-H), 6.82 32H, d, Jˆ8.6 Hz, 28-H), 6.7131H, ddd, Jˆ4.8,
10.4, 15.5 Hz, 3-H), 6.39 31H, d, Jˆ16.0 Hz, 8-H), 6.06
31H, dd, Jˆ8.6, 16.0 Hz, 7-H), 5.75 31H, d, Jˆ15.5 Hz,
2-H), 5.52 31H, d, Jˆ7.4 Hz, 24-NH), 5.11 31H, m, 5-H),
4.63 31H, ddd, Jˆ5.5, 7.3, 7.4 Hz, 24-H), 3.79 33H, s,
29-OMe), 3.67 31H, m, 22-Ha), 3.20 31H, m, 22-Hb), 3.11
31H, dd, Jˆ5.5, 14.7 Hz, 25-Ha), 3.05 31H, dd, Jˆ7.3,
14.7 Hz, 25-Hb), 2.88 32H, m, 15-H, 150-Ha), 2.59 31H,
m, 21-Ha), 2.54 31H, m, 21-Hb), 2.50±2.30 33H, m, 4,
6-H), 2.35 31H, m, 150-Hb), 1.65±1.40 33H, m, 16, 17-H),
1.13 33H, d, Jˆ7.3 Hz, 13-H), 0.73 36H, d, Jˆ6.7 Hz, 18,
19-H). FABMS m/z: 589 [M1H]1. FABHRMS m/z: Calcd
for C35H44N2O61H: 589.3277. Found: 589.3285.
A solution of 9 371mg, 0.13 mmol) and 23 379 mg,
0.27 mmol) in dry CH2Cl2 32 ml) was treated with
EDCI´HCl 3160 mg) in the presence of DMAP 317 mg) at
room temperature for 1h. Work-up in the same manner as
preparation for 13 gave a product, which was puri®ed by
column chromatography 3SiO2: 5 g, hexane:EtOAcˆ3:1) to
furnish 24 3110 mg, quant.).
24: Colorless oil, [a]21D15.58 3cˆ0.99 in CHCl3). IR 3KBr):
2935, 1734, 1637, 1560, 1514 cm21. 1H NMR d: 7.32±7.27
35H, m, 10, 11, 12-H), 7.21 31H, brt, Jˆca. 6 Hz, 22-NH),
7.0132H, d, Jˆ8.5 Hz, 27-H), 6.8132H, d, Jˆ8.5 Hz,
28-H), 6.78 31H, m, 3-H), 6.40 31H, d, Jˆ15.8 Hz, 8-H),
6.04 31H, dd, Jˆ8.5, 15.8 Hz, 7-H), 5.85 31H, d, Jˆ15.3 Hz,
2-H), 5.13 31H, brs, 24-NH), 5.05 31H, m, 5-H), 4.84 31H,
q-like, Jˆca. 6 Hz, 24-H), 4.17 32H, m, CH2CH2TMS), 3.80
31H, dd, Jˆ3.7, 9.8 Hz, 15-H), 3.77 33H, s, 29-OMe), 3.54
31H, m, 22-Ha), 3.30 31H, m, 22-Hb), 3.18 32H, m, 20-H),
3.08 32H, m, 25-H), 2.60 31H, m, 6-H), 2.50 32H, m, 4-H),
1.71 33H, m, 17, 21-H), 1.56 31H, m, 16-Ha), 1.43 39H, s,
tBu), 1.38 31H, m, 16-Hb), 1.11 33H, d, Jˆ6.7 Hz, 13-H),
0.97 32H, m, CH2CH2TMS), 0.84, 0.79 3both 3H, d,
Jˆ6.7 Hz, 18, 19-H), 0.04 39H, s, TMS). FABMS m/z:
4.14. Epoxidation of 22 giving 3
A solution of 22 35.5 mg, 0.0090 mmol) in CH2Cl2 33 ml)
was treated with dimethyldioxirane 30.074 M in acetone,
3.0 ml) for 3 h at 2108C. Removal of solvent from the
reaction mixture under reduced pressure gave a product,
which was puri®ed by HPLC [column; COSMOSIL
796
[M1H]1.
FABHRMS
m/z:
Calcd
for
5C18AR
310 mm
i.d.£250 mm),
mobile
phase;
C44H66N2O9Si1H: 796.1118. Found: 796.1129.
CH3CN:H2O:CH2Cl2ˆ60:55:0.1] to furnish 3 33.6 mg,
66%).
3: A white powder, [a]22D159.08 3cˆ0.15 in CHCl3). IR
3KBr): 2930, 2860, 1728, 1720, 1696, 1668, 1514 cm21. 1H
NMR d: 7.40±7.25 35H, m, 10, 11, 12-H), 7.18 31H, m,
22-NH), 7.09 32H, d, Jˆ8.5 Hz, 27-H), 6.82 32H, d,
4.16. Deprotection of 24 followed by macrolactamization
giving 25
Deprotection of 24 3110 mg, 0.13 mmol) and subsequent
conversion to an HCl salt and macrolactamization was
carried out in the same manner as preparation for 14 to
give a product, which was puri®ed by column chromato-
graphy 3SiO2: 7 g, benzene:acetoneˆ6:1) to furnish 25
356 mg, 74%).
Jˆ8.5 Hz, 28-H), 6.713H1 , ddd,
Jˆ8.0, 14.7, 15.3 Hz,
3-H), 5.73 31H, d, Jˆ15.3 Hz, 2-H), 5.50 31H, d,
Jˆ7.3 Hz, 24-NH), 5.25 31H, m, 5-H), 4.65 31H, ddd,
Jˆ5.5, 7.3, 7.4 Hz, 24-H), 3.79 33H, s, 29-OMe), 3.69
31H, d, Jˆ1.9 Hz, 8-H), 3.63 31H, m, 22-Ha), 3.14 31H,
m, 22-Hb), 3.10 31H, dd, Jˆ5.5, 14.0 Hz, 25-Ha), 3.05
31H, dd, Jˆ7.4, 14.0 Hz, 25-Hb), 2.94 31H, dd, Jˆ1.9,
7.3 Hz, 7-H), 2.90 31H, dd, Jˆ8.5, 16.2 Hz, 150-Ha), 2.87
31H, m, 15-H), 2.61 31H, m, 21-Ha), 2.54±2.47 33H, m, 4-H,
21-Hb), 2.33 31H, brd, Jˆ16.2 Hz, 150-Hb), 1.65±1.40 33H,
m, 16, 17-H), 1.40 31H, m, 6-H), 1.13 33H, d, Jˆ7.4 Hz,
13-H), 0.86, 0.83 3both 3H, d, Jˆ6.8 Hz, 18, 19-H). 1H NMR
3d6-DMSO) d: 8.10 31H, d, Jˆ8.0 Hz, 24-NH), 7.34±7.23
35H, m, 10, 11, 12-H), 7.33 31H, m, 22-NH), 7.05 32H, d,
Jˆ8.5 Hz, 27-H), 6.76 32H, d, Jˆ8.5 Hz, 28-H), 6.30 31H,
ddd, Jˆ3.2, 11.0, 15.2 Hz, 3-H), 5.73 31H, d, Jˆ15.2 Hz,
2-H), 5.05 31H, ddd, Jˆ3.8, 5.5, 10.6 Hz, 5-H), 4.15 31H, m,
24-H), 3.84 31H, d, Jˆ1.8 Hz, 8-H), 3.64 33H, s, 29-OMe),
3.21 31H, m, 22-Ha), 3.09 31H, m, 22-Hb), 2.86 32H, m,
7-H, 25-Ha), 2.70 31H, m, 150-Ha), 2.57 32H, m, 4-Ha,
25-Hb), 2.47 31H, m, 150-Hb), 2.42 31H, m, 21-Ha), 2.28
32H, m, 4-Hb, 21-Hb), 1.75 31H, m, 6-H), 1.37 31H, m,
17-H), 1.21 31H, m, 16-Ha), 1.05 31H, m, 15-H), 0.95
33H, d, Jˆ7.3 Hz, 13-H), 0.78 31H, m, 16-Hb), 0.74 33H,
25: A white powder, [a]23D177.28 3cˆ0.27 in CHCl3). IR
3KBr): 2934, 1743, 1657, 1626, 1538, 1514 cm21. 1H NMR
d: 7.32±7.19 35H, m, 10, 11, 12-H), 7.13 32H, d, Jˆ8.5 Hz,
27-H), 6.85 31H, ddd, Jˆ4.3, 11.6, 15.0 Hz, 3-H), 6.80 32H,
d, Jˆ8.5 Hz, 28-H), 6.56 31H, brs, 22-NH), 6.39 31H, d,
Jˆ15.9 Hz, 8-H), 6.00 31H, dd, Jˆ9.2, 15.9 Hz, 7-H),
5.74 31H, d, Jˆ8.0 Hz, 24-NH), 5.70 31H, d, Jˆ15.0 Hz,
2-H), 5.18 31H, ddd, Jˆ2.4, 6.1, 11.6 Hz, 5-H), 4.66 31H,
q-like, Jˆca. 8Hz, 24-H), 3.77 33H, s, 29-OMe), 3.76 31H,
m, 22-Ha), 3.63 31H, dd, Jˆ3.4, 10.1 Hz, 15-H), 3.25 31H,
m, 22-Hb), 3.19 31H, dd, Jˆ7.0, 14.6 Hz, 25-Ha), 3.10 31H,
m, 20-Ha), 2.90 31H, dd, Jˆ7.9, 14.6 Hz, 25-Hb), 2.86 31H,
m, 20-Hb), 2.55 32H, m, 4-Ha, 6-H), 2.29 31H, m, 4-Hb),
1.81 32H, m, 21-H), 1.73, 1.46, 1.23 3total 3H, m, 16, 17-H),
1.13 33H, d, Jˆ6.7 Hz, 13-H), 0.79, 0.70 3both 3H, d,
Jˆ6.7 Hz, 18, 19-H). FABMS m/z: 577 [M1H]1.
FABHRMS m/z: Calcd for C34H44N2O61H: 577.3278.
found: 577.3264.

4334
N. Murakami et al. / Tetrahedron 57+2001) 4323±4336
4.17. Epoxidation of 25 giving 4
15-H), 3.42 32H, m, 22-H), 2.88 32H, t, Jˆ6.1Hz, 21-H),
1.86 31H, ddd, Jˆ7.3, 7.3, 14.0 Hz, 16-Ha), 1.71 31H, m,
17-H), 1.59 31H, m, 16-Hb), 1.44 39H, s, tBu), 0.96, 0.92
3both 3H, d, Jˆ6.4 Hz, 18, 19-H). FABMS m/z: 342
[M1Na]1. FABHRMS m/z: Calcd for C14H25NO5S1Na:
342.1351. Found: 342.1361.
A solution of 25 312 mg, 0.020 mmol) in CH2Cl2 37 ml) was
treated with dimethyldioxirane 30.074 M in acetone, 4.0 ml)
for 4 h at room temperature. Removal of solvent from the
reaction mixture under reduced pressure gave a product,
which was puri®ed by HPLC [column; YMC-Pack ODS
320 mm i.d.£250 mm), mobile phase; MeOH:H2Oˆ75:25]
to furnish 4 37.6 mg, 65%).
4.19. Condensation of segment A-B ꢀ9)with segment C-D
ꢀ27)
4: A white powder, [a]18D144.18 3cˆ0.11 in CHCl3). IR
3KBr): 2928, 1745, 1660, 1614, 1535, 1514 cm21. 1H NMR
d: 7.36±7.23 35H, m, 10, 11, 12-H), 7.12 32H, d, Jˆ8.5 Hz,
27-H), 6.84 31H, ddd, Jˆ3.7, 11.0, 14.6 Hz, 3-H), 6.81 32H,
d, Jˆ8.5 Hz, 28-H), 6.52 31H, brd, Jˆ6.7 Hz, 22-NH), 5.67
31H, d, Jˆ14.6 Hz, 2-H), 5.66 31H, d, Jˆ8.5 Hz, 24-NH),
5.33 31H, ddd, Jˆ2.4, 5.5, 9.2 Hz, 5-H), 4.66 31H, q-like,
Jˆca. 8 Hz, 24-H), 3.77 33H, s, 29-OMe), 3.76 31H, m,
22-Ha), 3.67 31H, d, Jˆ1.8 Hz, 8-H), 3.60 31H, dd, Jˆ3.1,
10.4 Hz, 15-H), 3.26 31H, m, 22-Hb), 3.18 31H, dd, Jˆ7.3,
14.6 Hz, 25-Ha), 3.08 31H, m, 20-Ha), 2.92 31H, dd, Jˆ1.8,
7.9 Hz, 7-H), 2.89 31H, m, 25-Hb), 2.84 31H, m, 20-Hb),
2.59 31H, m, 4-Ha), 2.37 31H, m, 4-Hb), 1.78 33H, m, 6,
21-H), 1.64, 1.49, 1.23 3total 3H, m, 16, 17-H), 1.14 33H, d,
Jˆ73 Hz, 13-H), 0.84, 0.83 3both 3H, d, Jˆ6.7 Hz, 18,
19-H). 1H NMR 3d6-DMSO) d: 8.47 31H, m, 24-NH),
7.40±7.25 35H, m, 10, 11, 12-H), 7.38 31H, m, 22-NH),
7.10 32H, d, Jˆ8.5 Hz, 27-H), 6.80 32H, d, Jˆ8.5 Hz,
28-H), 6.48 31H, ddd, Jˆ3.8, 9.5, 14.8 Hz, 3-H), 5.84
31H, d, Jˆ14.8 Hz, 2-H), 5.14 31H, m, 5-H), 4.38 31H,
q-like, Jˆca. 7 Hz, 24-H), 3.86 31H, d, Jˆ1.8 Hz, 8-H),
3.70 33H, s, 29-OMe), 3.58 31H, m, 15-H), 3.38 31H, m,
22-Ha), 3.24 31H, m, 20-Ha), 2.91 33H, m, 7-H, 20-Hb,
25-Ha), 2.77 31H, m, 22-Hb), 2.67 32H, m, 4-Ha, 25-Hb),
2.23 31H, m, 4-Hb), 1.78 31H, m, 6-H), 1.67 31H, m, 17-H),
1.58 32H, m, 21-H), 1.25 32H, m, 16-H), 1.03 33H, d,
Jˆ6.7 Hz, 13-H), 0.82 33H, d, Jˆ6.8 Hz), 0.78 33H, d,
Jˆ6.3 Hz, 18, 19-H). FABMS m/z: 593 [M1H]1.
FABHRMS m/z: Calcd for C34H44N2O71H: 593.3277.
Found: 593.3217.
Condensation of 9 310 mg, 0.019 mmol) and 27 312 mg,
0.038 mmol) was carried out in the same manner as prepara-
tion for 13 to give a product, which was puri®ed by column
chromatography 3SiO2: 1.5 g, hexane:EtOAcˆ3:1) to
furnish 28 313 mg, 82%).
28: Colorless oil, [a]30D22.88 3cˆ0.85 in CHCl3). IR 3KBr):
1
2965, 2928, 1714, 1695, 1534, 1516 cm21. H NMR d:
7.34±7.20 35H, m, 10, 11, 12-H), 7.01 32H, d, Jˆ8.7 Hz,
27-H), 6.80 32H, d. Jˆ8.7 Hz, 28-H), 6.75 31H, m, 3-H),
6.40 31H, d, Jˆ15.7 Hz, 8-H), 6.13 31H, d, Jˆ7.4 Hz,
24-NH), 6.04 31H, dd, Jˆ8.4, 15.7 Hz, 7-H), 5.87 31H, d,
Jˆ15.4 Hz, 2-H), 4.98 31H, m, 5-H), 4.86 31H, m, 24-H),
4.18 33H, m, 15-H, CH2CH2TMS), 3.77 33H, s, 29-OMe),
3.32 32H, m, 22-H), 3.09 32H, m, 25-H), 2.71, 2.61 3total
3H, m, 4, 6-H), 2.49 32H, m, 21-H), 1.77±1.47 33H, m, 16,
17-H), 1.41 39H, s, tBu), 1.10 33H, d, Jˆ7.0 Hz, 13-H), 0.97
32H, m, CH2CH2TMS), 0.81, 0.78 3both 3H, d, Jˆ6.4 Hz,
18, 19-H), 0.03 39H, s, TMS). FABMS m/z: 825 [M1H]1.
FABHRMS m/z: Calcd for C44H64N2O9SSi1H: 825.4180.
Found: 825.4176.
4.20. Deprotection of 28 followed by macrolactamization
giving 29
Deprotection of 28 321mg, 0.026 mmol) and subsequent
conversion to an HCl salt and macrolactamization was
carried out in the same manner as preparation for 14 to
give a product, which was puri®ed by column chromato-
graphy 3SiO2: 800 mg, hexane:EtOAcˆ1:1) to furnish 29
39.5 mg, 58%).
4.18. Preparation for segment C-D ꢀ27)
29: A white powder, [a]30D132.08 3cˆ0.30 in CHCl3). IR
3KBr): 2925, 1730, 1718, 1685, 1533, 1516 cm21. 1H NMR
d: 7.34±7.20 35H, m, 10, 11, 12-H), 7.09 32H, d, Jˆ8.5 Hz,
27-H), 6.82 32H, d, Jˆ8.5 Hz, 28-H), 6.69 31H, ddd, Jˆ3.8,
10.6, 14.7 Hz, 3-H), 6.38 31H, d, Jˆ15.8 Hz, 8-H), 6.02
31H, dd, Jˆ8.5, 15.8 Hz, 7-H), 5.78 31H, dd, Jˆ1.2,
14.7 Hz, 2-H), 5.54 31H, d, Jˆ7.2 Hz, 24-NH), 5.14 31H,
ddd, Jˆ3.0, 6.5, 10.9 Hz, 5-H), 4.66 31H, ddd, Jˆ5.3, 7.2,
7.4 Hz, 24-H), 4.09 31H, dd, Jˆ6.9, 8.9 Hz, 15-H), 3.79
33H, s, 29-OMe), 3.65 31H, ddd, Jˆ4.6, 8.4, 17.4 Hz,
22-Ha), 3.34 31H, ddd, Jˆ3.6, 7.9, 17.4 Hz, 22-Hb), 3.11
31H, dd, Jˆ5.3, 14.3 Hz, 25-Ha), 3.02 31H, dd, Jˆ7.4,
14.3 Hz, 25-Hb), 2.74, 2.56, 2.45 3total 5H, m, 4, 6,
21-H), 1.65±1.40 33H, m, 16, 17-H), 1.13 33H, d,
Jˆ6.8 Hz, 13-H), 0.71, 0.70 3both 3H, d, Jˆ6.1Hz, 18,
19-H). FABMS m/z: 607 [M1H]1. FABHRMS m/z:
Calcd for C34H42N2O6S1H: 607.2842. Found: 607.2845.
A solution of 11 350 mg, 0.27 mmol) in dry THF 32 ml) was
treated with Et3N 338 ml) and pivaloyl chloride 334 ml) at
08C for 1h, and then to the reaction mixture was added
NaSH 328 mg) and 15-crown-5 368 ml) at 08C. The whole
mixture was stirred for 1h, and then a solution of 26 380 mg,
0.41mmol) in dry THF 30.5 ml) was added to the reaction
mixture. After stirring at room temperature overnight, the
reaction mixture was quenched with aqueous saturated
NaHCO3, and then the whole was washed with Et2O. The
water phase was acidi®ed with 1N NaHSO4, and 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 HPLC [column; YMC-Pack ODS-A 310 mm
i.d.£250 mm), mobile phase; MeOH:H2Oˆ75:25] to
furnish 27 340 mg, 51%).
27: A pale yellow powder, [a]30D251.08 3cˆ3.5 in CHCl3).
4.21. Epoxidation of 29 giving 5
1
IR 3KBr): 3377, 2959, 2928, 1695, 1530, 1510 cm21. H
NMR d: 4.95 31H, brs, 22-NH), 4.22 31H, t, Jˆ7.3 Hz,
A solution of 29 33.5 mg, 0.0058 mmol) in CH2Cl2 31ml)

N. Murakami et al. / Tetrahedron 57+2001) 4323±4336
4335
was treated with dimethyldioxirane 30.074 M in acetone,
2.0 ml) for 3 h at 2208C. Removal of solvent from
the reaction mixture under reduced pressure gave a product,
which was puri®ed by HPLC [column; YMC-Pack ODS-A
310 mm i.d.£250 mm), mobile phase; MeOH:H2Oˆ75:25]
to furnish 5 31.2 mg, 31%).
0.4 mmol) was carried out in the same manner as prepara-
tion for 13 to give a product, which was puri®ed by column
chromatography 3SiO2: 7 g, hexane:EtOAcˆ2:1) to furnish
33 3120 mg, 96%).
33: Colorless oil, [a]21D26.18 3cˆ0.30 in CHCl3). IR 3KBr):
1
2972, 1740, 1669, 1639, 1530, 1512 cm21. H NMR d:
5: A white powder, [a]30D148.08 3cˆ0.10 in CHCl3). IR
3KBr): 2925, 1732, 1716, 1685, 1535, 1516 cm21. 1H NMR
d: 7.34±7.20 35H, m, 10, 11, 12-H), 7.00 32H, d, Jˆ8.0 Hz,
27-H), 6.83 32H, d, Jˆ8.0 Hz, 28-H), 6.67 31H, ddd, Jˆ6.0,
8.0, 14.9 Hz, 3-H), 5.76 31H, d, Jˆ14.9 Hz, 2-H), 5.52 31H,
d, Jˆ7.4 Hz, 24-NH), 5.11 31H, m, 5-H), 4.65 31H, q-like,
Jˆca. 8 Hz, 24-H), 4.2131H, dd, Jˆ6.2, 8.5 Hz, 15-H), 3.79
33H, s, 29-OMe), 3.77 31H, d, Jˆ2.4 Hz, 8-H), 3.65 31H, m,
22-Ha), 3.33 31H, m, 22-Hb), 3.09 31H, dd, Jˆ5.5, 14.5 Hz,
25-Ha), 3.0131H, dd, Jˆ8.6, 14.5 Hz, 25-Hb), 2.93 31H, dd,
Jˆ2.4, 6.9 Hz, 7-H), 2.75 31H, m, 21-Ha), 2.53 31H, m,
21-Hb), 2.30 32H, m, 4-H), 1.65±1.40 33H, m, 16, 17-H),
1.40 31H, m, 6-H), 1.11 33H, d, Jˆ7.3 Hz, 13-H), 0.88, 0.86
3both 3H, d, Jˆ7.6 Hz, 18, 19-H). FABMS m/z: 623
[M1H]1. FABHRMS m/z: Calcd for C34H42N2O7S1H:
623.2791. Found: 623.2790.
7.38±7.30 35H, m, 10, 11, 12-H), 7.01 32H, d, Jˆ8.3 Hz,
27-H), 6.79 32H, d, Jˆ8.3 Hz, 28-H), 6.75 31H, m, 3-H),
6.4131H, d, Jˆ15.8 Hz, 8-H), 6.17 31H, brs, 24-NH), 6.04
31H, dd, Jˆ8.5, 15.8 Hz, 7-H), 5.86 31H, d, Jˆ15.6 Hz,
2-H), 5.20 31H, brs, 22-NH), 5.04 31H, m, 5-H), 4.86 31H,
m, 24-H), 4.23±4.15 32H, m, CH2CH2TMS), 3.77 33H, s,
29-OMe), 3.30 32H, m, 22-H), 3.09 32H, m, 25-H), 2.64
31H, m, 6-H), 2.49 34H, m, 4, 21-H), 1.50 36H, s, 16,
17-H), 1.41 39H, s, tBu), 1.11 33H, d, Jˆ6.7 Hz, 13-H),
0.98 32H, m, CH2CH2TMS), 0.04 39H, s, TMS). FABMS
m/z: 781[M 1H]1. FABHRMS m/z: Calcd for
C42H60N2O10Si1H: 781.4096. Found: 781.4040.
4.24. Deprotection of 33 followed by macro-
lactamization giving 34
Deprotection of 33 370 mg, 0.09 mmol) and subsequent
conversion to an HCl salt and macrolactamization was
carried out in the same manner as preparation for 14 to
give a product, which was puri®ed by column chromato-
graphy 3SiO2: 6 g, hexane:EtOAcˆ1:3) to furnish 34
346 mg, 90%).
4.22. Preparation for segment C-D ꢀ32)
A solution of 2-hydroxyisobutyric acid 330, 280 mg,
2.7 mmol) and NaHCO3 3230 mg) in CH2Cl2 35 ml) and
water 35 ml) was treated with benzyl bromide 3BnBr,
0.35 ml) and tetra-n-butylammonium iodide 3TBAI,
980 mg) at room temperature for 2 days. After removal of
about half volume of solvent from reaction mixture, the
whole was extracted with Et2O. The Et2O extract was
dried over MgSO4. Removal of solvent from the Et2O
extract under reduced pressure gave a product, which was
puri®ed by column chromatography 3SiO2: 3 g, hexane:
EtOAcˆ4:1) to furnish a benzyl ester 331, 480 mg, 92%).
A solution of 31 3200 mg, 1.0 mmol) and 11 3490 mg,
2.5 mmol) in dry 3CH2Cl)2 310 ml) was treated with
EDCI´HCl 31.2 g) in the presence of DMAP 3320 mg) at
room temperature for 6 h. Work-up in the same manner as
preparation for 13 gave a product, which was puri®ed by
column chromatography 3SiO2: 25 g, hexane:EtOAcˆ12:1)
to furnish a diester 3360 mg, 95%). To a solution of the
diester 3350 mg, 0.92 mmol) in MeOH 320 ml) was added
10% Pd-C 3100 mg), and then the reaction mixture was
stirred under H2 atmosphere at room temperature for 2 h.
The reaction mixture was ®ltered through Celite, and then
removal of solvent from the ®ltrate gave a product, which
was puri®ed by column chromatography 3SiO2: 1 g,
CHCl3:MeOHˆ7:1) to furnish 32 3230 mg, 88%).
34: A white powder, [a]21D139.08 3cˆ0.75 in CHCl3). IR
3KBr): 2930, 1738, 1676, 1640, 1528, 1514 cm21. 1H NMR
d: 7.34±7.20 35H, m, 10, 11, 12-H), 7.12 32H, d, Jˆ8.5 Hz,
27-H), 6.80 32H, d, Jˆ8.5 Hz, 28-H), 6.69 31H, ddd, Jˆ6.1,
8.5, 15.3 Hz, 3-H), 6.41 31H, d, Jˆ15.9 Hz, 8-H), 6.03 31H,
dd, Jˆ8.6, 15.9 Hz, 7-H), 5.79 31H, d, Jˆ15.3 Hz, 2-H),
5.75 31H, d, Jˆ9.2 Hz, 24-NH), 4.98 31H, ddd, Jˆ2.4,
5.5, 11.0 Hz, 5-H), 4.82 31H, dt, Jˆ9.2, 6.7 Hz, 24-H),
3.76 33H, s, 29-OMe), 3.5131H, ddd, Jˆ5.5, 6.1, 14.1 Hz,
22-Ha), 3.36 31H, dt, Jˆ14.1, 5.5 Hz, 22-Hb), 3.11 32H, m,
25-H), 2.56 31H, m, 6-H), 2.50±2.38 34H, m, 4, 21-H), 1.56,
1.36 3both 3H, s, 16, 17-H), 1.12 33H, d, Jˆ6.7 Hz, 13-H).
FABMS m/z: 563 [M1H]1. FABHRMS m/z: Calcd for
C32H38N2O71H: 563.2757. Found: 563.2778.
4.25. Epoxidation of 34 giving 6
A solution of 34 315 mg, 0.027 mmol) in CH2Cl2 315 ml)
was treated with dimethyldioxirane 30.074 M in acetone,
7 ml) for 8 h. Removal of solvent from the reaction mixture
under reduced pressure gave a product, which was puri®ed
by HPLC [column; COSMOSIL 5C18AR 310 mm
i.d.£250 mm), mobile phase; MeOH:H2Oˆ65:35] to
furnish 6 39.3 mg, 60%).
32: A white powder, IR 3KBr): 3362, 2980, 1720,
1
1520 cm21. H NMR d: 5.07 31H, brs, 22-NH), 3.40 32H,
m, 22-H), 2.54 32H, t, Jˆ6.0 Hz, 21-H), 1.60 36H, s, 16,
17-H), 1.44 39H, s, tBu). FABMS m/z: 298[M1Na]1.
FABHRMS m/z: Calcd for C12H21NO61Na: 298.1267.
Found: 298.1281.
6: A white powder, [a]21D140.08 3cˆ0.80 in CHCl3). IR
3KBr): 2932, 1739, 1676, 1646, 1533, 1514 cm21. 1H NMR
d: 7.34±7.20 35H, m, 10, 11, 12-H), 7.11 32H, d, Jˆ8.6 Hz,
27-H), 6.80 32H, d, Jˆ8.6 Hz, 28-H), 6.68 31H, ddd, Jˆ6.1,
8.5, 15.3 Hz, 3-H), 5.77 31H, d, Jˆ15.3 Hz, 2-H), 5.62 31H,
d, Jˆ8.8 Hz, 24-NH), 5.08 31H, ddd, Jˆ3.0, 5.6, 11.3 Hz,
5-H), 4.83 31H, dt, Jˆ8.8, 6.8 Hz, 24-H), 3.77 33H, s, 29-
OMe), 3.68 32H, d, Jˆ1.9 Hz, 8-H), 3.46 31H, m, 22-Ha),
4.23. Condensation of segment A-B ꢀ9)with segment C-D
ꢀ32)
Condensation of 9 385 mg, 0.16 mmol) and 32 3110 mg,

4336
N. Murakami et al. / Tetrahedron 57+2001) 4323±4336
7. Murakami, N.; Wang, W.; Ohyabu, N.; Ito, T.; Tamura, S.;
Aoki, S.; Kobayashi, M.; Kitagawa, I. Tetrahedron 2000, 56,
9121±9128.
3.41 31H, m, 22-Hb), 3.11 32H, d, Jˆ6.8 Hz, 25-H), 2.88
31H, dd, Jˆ1.9, 7.6 Hz, 7-H), 2.75 31H, m, 4-Ha), 2.43 33H,
m, 4-Hb, 21-H), 1.80 31H, sextet, Jˆca. 6.5 Hz, 6-H), 1.50,
1.24 3both 3H, s, 16, 17-H), 1.13 33H, d, Jˆ6.9 Hz, 13-H).
FABMS m/z: 579 [M1H]1. FABHRMS m/z: Calcd for
C32H38N2O81H: 579.2706. Found: 579.2722.
8. Part of this paper was reported as
a preliminary
communication; Murakami, N.; Wang, W.; Tamura, S.;
Kobayashi, M. Bioorg. Med. Chem. Lett. 2000, 10, 1823±
1826.
9. Nagai, Y.; Kusumi, T. Tetrahedron Lett. 1995, 36, 1853±
1856.
4.26. Conformational analysis
10. Coste, J.; Le-Nguyen, D.; Castro, B. Tetrahedron Lett. 1990,
31, 205±208.
The NOESY spectra were measured by Varian Inova 600
3600 MHz for 1H) at 248C in d6-DMSO, and a total of 48 3for
1), 47 3for 2), 33 3for 3), and 43 3for 4) NOEs were used as
distance restraints for molecular dynamic calculations,
respectively. The pro-chiral methylene proton and methyl
proton were treated as pseudo-atoms18 and additional
distance terms were imposed to the upper bound of distance
in the following manner. As the NOEs observed between the
protons less than ®ve covalent bonds, the methyl and
11. Inanaga, J.; Hirata, K.; Saeki, H.; Katsuki, T.; Yamaguchi, M.
Bull. Chem. Soc. Jpn. 1979, 52, 1989±1993.
12. The stereochemistry of the epoxy moiety of 3 was established
by comparison of the proton chemical shifts due to the 8-H and
6-Me group from the same behavior observed between arena-
statin A 31, 8-H: d 3.68, 6-Me: d 1.14) and the a-epoxy isomer
of 1 3d 3.59, 1.05). Namely, an obvious difference in chemical
shifts of the 8-H and 6-Me groups was observed; the signals of
3 appeared in lower ®eld 3d 3.69, 1.13) than those 3d 3.59,
1.04) of the a-isomer. This physicochemical property enabled
us to establish stereochemistry on the 7, 8-epoxy moieties of
the other analogs 34±6); 4: d 3.67, 1.14, the a-epoxy isomer of
4: d 3.59, 1.03, 5: d 3.77, 1.11, the a-epoxy isomer of 5: d
3.57, 1.05, 6: d 3.68, 1.13, the a-epoxy isomer of 6: d 3.58,
1.05.
Ê
methylene groups were allowed to have 0.9 and 0.6 A of
distance terms, respectively. In the case of NOE via
Ê
more than four covalent bonds, 1.3 and 1.0 A of distance
terms were added to the methyl and methylene groups,
respectively. The calculation was performed using
SYBYL 6.5 3Tripos) on SGI R10000-O2 3Silicon Graphics)
with Tripos force ®eld.19 The initial structure built up with
reference to the NOE data was energy-minimized without
restraints using the BFGS method until the derivative
13. TenBrink, R. E. J. Org. Chem. 1987, 52, 418±422.
14. Golakoti, T.; Ogino, J.; Heltzel, C. E.; Husebo, T. L.; Jenson,
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.
Ê
became less than 0.005 kcal/mol A or the iteration steps
reached 20,000. This re®ned structure was subjected to 50
cycles of simulated annealing in vacuo with distance
restraints. Simulated annealing was executed for 500 fs at
700 K at an initial equilibrium step, and then the tempera-
ture was exponentially cooled down to 100 K over 1500 fs.
The resulting 50 structures, which were extracted at each
cycle end, were subjected to 20,000 steps of BFGS energy
minimization with the restraints until the derivative became
15. Chang, S.-C.; Gil-Av, E.; Charles, R.; J. Chromatogr, . 289.
1984, 53±63.
16. The NMR data of triamide analog 32) taken in d6-DMSO are
listed. d: 8.27 31H, d, Jˆ8.6 Hz, 15-NH), 8.21 31H, d, Jˆ7.9
Hz, 24-NH), 7.38±7.26 35H, m, 10, 11, 12-H), 7.28 31H, m,
22-NH), 7.10 32H, d, Jˆ8.6 Hz, 27-H), 6.80 32H, d, Jˆ8.6 Hz,
28-H), 6.36 31H, ddd, Jˆ4.3, 11.6, 15.3 Hz, 3-H), 5.75 31H, d,
Jˆ15.3 Hz, 2-H), 5.10 31H, brdd, Jˆ5.5, 11.6 Hz, 5-H), 4.35
31H, ddd, Jˆ5.5, 8.6, 9.7 Hz, 15-H), 4.18 31H, ddd, Jˆ3.7, 7.9,
11.6 Hz, 24-H), 3.87 31H, d, Jˆ1.9 Hz, 8-H), 3.69 33H, s,
29-OMe), 3.50 31H, m, 22-Ha), 2.96 33H, m, 7-H, 22-Hb,
25-Ha), 2.62 32H, m, 4-Ha, 25-Hb), 2.24 32H, m, 4-Hb,
21-Ha), 2.13 31H, m, 21-Hb), 1.79 31H, q-like, Jˆca. 7 Hz,
6-H), 1.47 31H, m, 17-H), 1.28 31H, m, 16-Ha), 1.11 31H, m,
16-Hb), 1.03 33H, d, Jˆ6.8 Hz, 13-H), 0.75, 0.74 3both 3H, d,
Jˆ6.7 Hz, 18, 19-H).
Ê
less than 0.005 kcal/mol A. During both the simulated
annealing and the secondary energy minimization,
Ê 2
200 kcal/A of force constant was employed as the violation
of the restraint.
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