Participation of the conjugated diene part for potent cytotoxicity of callystatin A, a spongean polyketide

Article abstract of DOI:10.1016/S0968-0896(00)00199-1

5-epi, 10-epi, 8-Deethyl, and 10-demethyl analogues of callystatin A, a potent cytotoxic spongean polyketide, were synthesized to elucidate structure-requirement for cytotoxic potency. Inversion of the asymmetric center at C-10 in callystatin A minimally

Full text of DOI:10.1016/S0968-0896(00)00199-1

Bioorganic & Medicinal Chemistry 8 (2000) 2651±2661
Participation of the Conjugated Diene Part for Potent
Cytotoxicity of Callystatin A, a Spongean Polyketide
Nobutoshi Murakami, Masanori Sugimoto, Tatsuo Nakajima, Motoyuki Kawanishi,
Yasuhiro Tsutsui and Motomasa Kobayashi*
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565±0871, Japan
Received 29 May 2000; accepted 18 July 2000
AbstractÐ5-epi, 10-epi, 8-Deethyl, and 10-demethyl analogues of callystatin A, a potent cytotoxic spongean polyketide, were syn-
thesized to elucidate structure-requirement for cytotoxic potency. Inversion of the asymmetric center at C-10 in callystatin A
minimally a€ected the activity, while lack of the 10-methyl group in callystatin A decreased cytotoxicity. In addition, the C-5 epi-
mer and the 8-deethyl analogue of callystatin A showed weaker cytotoxicity. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
Although some other related anti-tumor antibiotics
designated as the leptomycin family were found from
Streptomyces sp. as mentioned above, their structure±
activity relationships have not so far been investigated
because of ambiguous stereostructures. Thus, we
explored the structure±activity relationship of callystatin
A (1) in order to analyze the structure-requirement for
the potent cytotoxicity of 1 and the inhibitory activity of
NES-dependent export of nuclear protein. This paper
deals with the participation of two conjugated diene
parts from C-5 to C-15 in the cytotoxic activity of 1
(Chart 1).
During the course of our search for new biologically
active principles from marine organisms, we found an
extremely potent cytotoxic polyketide, callystatin A (1,
IC50=0.022 nM against KB cells), from the marine
sponge Callyspongia truncata.1 We determined the
absolute stereostructure of 1 by the physicochemical
properties and comparative analysis of the CD spectra
with those of the synthesized model compounds.2 In
addition, we achieved the ®rst total synthesis of 1 and
con®rmed the absolute stereostructure of 1.3 The planar
structure of callystatin A (1), having an a,b-unsaturated-
d-lactone, two conjugated dienes, and a b-hydroxy-
ketone moiety, was very alike those of several anti-tumor
antibiotics (e.g., leptomycin,4 kazusamycin,5 anguino-
mycin,6 leptofuranin7) previously isolated from Strep-
tomyces sp. As for these anti-tumor antibiotics, only
planar structures have been elucidated. Among them,
leptomycin B (2) was recently shown to inhibit nuclear
export signal (NES)-dependent transport of proteins from
the nucleus to cytoplasm through prevention of direct
binding between NES and the chromosome main-
tenance region 1 (CRM1) protein.8 Hence, we undertook
to clarify the absolute stereostructure of leptomycin B (2)
and succeeded in establishing it by the ®rst total synth-
esis.9 As a result, it was found that callystatin A (1)
possessed the same stereostructure as leptomycin B (2).
Additionally, we revealed that callystatin A (1) also
exhibited the same biological properties as 2.10
Chemistry
First of all, our interest was focused on the contribution
of the two asymmetric centers (C-5 and C-10) to the
potent cytotoxicity of 1, since three conjugated chro-
mophores (an a,b-unsaturated-d-lactone and two con-
jugated dienes) of 1 were shown to be characteristically
oriented to indicate an intense CD spectrum.2 Thus, 5-
epi- (20) and 10-epi-callystatin A (12) were synthesized
and evaluated for their cytotoxicity. According to the
synthetic protocol of 1, 10-epi-callystatin A (12) was
synthesized from the condensation of segment C1±C6
(3a), segment C7±C12 (4b), and segment C13±C22 (10) as
shown in Scheme 1. The segment C7±C12 (4b) was pre-
pared as shown in Scheme 2. Namely, the known alco-
hol 13b was converted by p-methoxybenzyl (PMB) ether
protection and subsequent n-Bu4NF treatment to a pri-
mary alcohol 14b. Swern oxidation11 of 14b followed by
Still±Wittig reaction12 gave a conjugated Z-ester 15b
*Corresponding author. Tel.: +81-6-6879-8215; fax: +81-6-6879-8129;
e-mail: kobayasi@phs.osaka-u.ac.jp
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00199-1

2652
N. Murakami et al. / Bioorg. Med. Chem. 8 (2000) 2651±2661
Chart 1.
predominantly (Z:E=16:1). The ole®nic geometry was
established by the NOE enhancements observed between
H-9 and H-23. The ester 15b was successively submitted
to diisobutylaluminum hydride (DIBAL-H) reduction
and bromination using CBr4 and PPh3 in the presence
of 2,6-lutidine13 to provide an allylic bromide. Finally,
treatment of this bromide with Bu3P furnished the
desired tributylphosphorus ylide 4b.
isopropyl acetal moiety gave a keto-lactol, which was
further oxidized by Ag2CO3±Celite17 to construct an
a,b-unsaturated d-lactone moiety. Finally, removal of
the t-butyldimethylsilyl (TBS) group by HF:pyridine
(5:1) treatment18 furnished 10-epi-callystatin A (12).
In the course of synthesis of 12, the removal of the PMB
group of 5b partly involved hydrolysis of the acetal
portion to a€ord a diol (7) together with 6b. In addition,
the dimeric macrocyclic acetal (9) was also formed dur-
ing SiO2 column separation of 6b, secondarily. Both 7
and 9 were, therefore, converted to the acetal 6b by
treatment with pyridinium p-toluenesulfonate (PPTS) in
isopropanol. However, the reaction on a large scale
reduced the yield of the desired alcohol 6b in spite of the
subsequent acidic conversion.
The two segments (3a and 4b) were coupled in the pre-
sence of LiCH2S(O)CH3 to a€ord a 6-E conjugated diene
(5b) with complete stereoselectivity.14 After deprotec-
tion of the PMB group in 5b with 2,3-dichloro-5,6-
dicyano-1,4-benzoquinone (DDQ),15 the resulting alco-
hol 6b was subjected to Swern oxidation to give a seg-
ment C1±C12 (8b). A second Wittig reaction using allylic
tributylphosphorus ylide (10) also constructed a 12,14-
conjugated diene portion selectively to give 11 in moder-
ate yield. Tetrapropylammonium perruthenate (TPAP)
oxidation16 of 11 followed by hydrolytic cleavage of the
This ®nding led us to modify a synthetic route of 5-epi-
callystatin A (20), in which a lactone moiety was con-
structed before the condensation with segment C13±C22
Scheme 1. Reagents and conditions: (a) LiCH2S(O)CH3, toluene, 78 ꢀC, rt, 5b: 72% from 4b, 11: 71% from 6b; (b) DDQ, CH2Cl2: 0.5% aq
NaHCO3 (9:1), 82%; (c) SiO2 columm chromatography; (d) PPTS, PrOH, 72%; (e) (COCl)2, DMSO, Et3N, CH2Cl2, 78 ꢀC; (f) TPAP, NMO,
i
CH2Cl2; (g) Dowex HCR-W2, acetone:H2O (5:1), 40 ꢀC; (h) Ag2CO3±Celite, PhH, 50 ꢀC, three steps 65%; (i) HF:Py (5:1), THF, 90%.

N. Murakami et al. / Bioorg. Med. Chem. 8 (2000) 2651±2661
2653
Scheme 2. Reagents and conditions: (a) PMBBr, NaH, THF, 96%; (b) nBu4NF, THF, 97%; (c) (COCl)2, DMSO, Et3N, CH2Cl2;
(d) R3OCOCH(R1)PO(OCH2CF3)2, KN(SiMe3)2, 18-crown-6, THF, 78±0 ꢀC, 15a: two steps 92% (Z:E=12:1), 15c: two steps quant. (Z:E=7.7:1),
15d: two steps 95% (Z:E=5:1); (e) DIBAL-H, CH2Cl2, 78 ꢀC; (f) CBr4, PPh3, 2,6-lutidine, CH3CN; (g) Bu3P, CH3CN, three steps 4a: 92%,
4c: 85%, 4d: 98%; (h) KH, PMBCl, THF, 77%.
(10) as depicted in Scheme 3. Deprotection of the iso-
propyl acetal group in 5a with acidic ion-exchange resin
(Dowex HCR-W2, H+ form) and subsequent oxidation
using Ag2CO3±Celite gave a lactone, whose PMB group
was readily cleaved by treatment with DDQ to furnish
the desired alcohol 17a in 82%, three steps. Then, 17a
was converted to segment C1±C12 (18a) by Dess±Martin
oxidation.19 The lactone aldehyde (18a) was coupled
with segment C13±C22 (10) under the reaction conditions
established by us3 to give a lactone-alcohol (19a) having
the same carbon framework as callystatin A (1). Oxida-
tion of 19a using Dess±Martin periodinane provided a
ketone without concomitant isomerization on the asym-
metric centers. Finally, the TBS group was removed by
use of HF:pyridine (5:1) to give 5-epi-callystatin A (20).
In the ®nal deprotection of the TBS group giving cal-
lystatin A (1) and/or the two analogues (12, 20), a minor
stereoisomer was produced in any case. However, the
chemical structure of the stereoisomer was not estab-
lished due to its almost superimposable NMR spectra.
Fortunately, the stereoisomer obtained in the deprotec-
tion of 19-O-TBS-callystatin A was found to be identical
with 5-epi-callystatin A (20) by CD and HPLC analyses.
Since the 10-epi-analogue (12) was not detected in this
reaction, the isomerization would mainly proceed via a
C-5 carbocation intermediate formed by cleavage of the
lactone ring. Taking into account an exploration of the
structure±activity relationship around the b-hydroxy-
ketone moiety in 1 by synthetic means, it should be
noted that the overall yield from 5a to 20 was improved
from 34 to 46%.
Next, we synthesized 8-deethyl (21) and 10-demethyl
analogues (22) and assessed their cytotoxicity as the ®rst
step toward a search for more simpli®ed lead com-
pounds. The same protocol utilizing the corresponding
segment C7±C12 (4c and 4d) as in the synthesis of 5-epi-
callystatin A (20), as illustrated in Scheme 4, furnished
8-deethyl- (21) and 10-demethylcallystatin A (22). The
two segments 4c and 4d were prepared from 13a and
1,4-butanediol (16), respectively, as shown in Scheme 2.
Biological properties and discussion
The cytotoxic activity against KB cells of 5-epi-cal-
lystatin A (20) was signi®cantly reduced to 7.6 nM, while
10-epi-callystatin A (12) showed almost the same activity
(IC50=0.076 nM) as that of callystatin A (1). Further-
more, 8-deethyl (21: IC50=7.9 nM) and 10-demethyl ana-
logues (22: IC50=1.4 nM) approximately exhibited 360-
Scheme 3. Reagents and conditions: (a) Dowex HCR-W2, acetone:H2O (5:1), 40 ꢀC; (b) Ag2CO3±Celite, PhH, 50 ꢀC, two steps 91%; (c) DDQ,
CH2Cl2:tBuOH:pH 6.9 phosphate bu€er (90:1:9), 91%; (d) Dess±Martin periodinane, CH2Cl2; (e) 10, LiCH2S(O)CH3, toluene, 20 ꢀC, two steps
62%; (f) Dess±Martin periodinane, CH2Cl2; (g) HF:Py (5:1), THF, two steps 90%.

2654
N. Murakami et al. / Bioorg. Med. Chem. 8 (2000) 2651±2661
Scheme 4. Reagents and conditions: (a) 3a, LiCH2S(O)CH3, toluene, 20 ꢀC; 5c: 75%; 5d: 81%; (b) Dowex; HCR-W2, acetone:H2O (5:1), 40 ꢀC;
(c) Ag2CO3±Celite, PhH, 50 ꢀC; (d) DDQ, CH2Cl2:tBuOH:pH 6.9 phosphate bu€er (90:1:9); 17c: three steps 81%, 17d: three steps 76%; (e) Dess±
Martin periodinane, CH2Cl2; (f) 10, LiCH2S(O)CH3, toluene, 20 ꢀC, 19c: two steps 66%, 19d: two steps 65%; (g) Dess±Martin periodinane,
CH2Cl2; (h) HF:Py (5:1), THF; 21: two steps 85%, 22: two steps 80%.
and 60-fold weaker cytotoxicity as compared with that
of callystatin A (1), respectively.
On the other hand, the CD spectra of callystatin A (1)
and the three analogues (12, 20 and 22) are depicted in
Figure 1. As for 1 and the two epimers (12, 20), split
Cotton e€ects at 230 and 250 nm due to the interaction
between p±p* transition of the two conjugated diene
chromophores were distinctly observed and their sign
re¯ected the con®guration at C-10 as reported pre-
viously.2,20 It is, therefore, presumed that these three
compounds have predominant conformations, in which
two conjugated diene moieties divided by C-10 are
optimally arranged. In contrast, the CD pro®le of 10-
demethyl analogue (22) suggests that the absence of a
10-methyl group permits the single bond between C-10
and C-11 to rotate ¯exibly.
Figure 1. CD spectra of callystatin A (1) and 10-epi (12), 5-epi (20),
and 10-demethyl (22) analogues in MeOH.
Based on these ®ndings, the following structure±activity
relationship of 1 is assumed: (1) 5R-con®guration and an
8-alkyl functional group are important structural factors
for the potent cytotoxicity of 1; (2) the cytotoxicity of 1 is
minimally a€ected by the con®guration at C-10; (3) lack
of a 10-methyl group reduced the cytotoxic activity of 1;
(4) the potent cytotoxicity of 1 is not a€ected by the
con®guration of the asymmetric C-10 carbon but by the
spatial arrangement between the a,b-unsaturated-d-lac-
tone and 6,8-conjugated diene moieties.
NES sequence and are transported in cytoplasm by CRM1
protein thus ful®ll their inherent function.21,22 Taking the
above-described ®ndings into consideration comprehen-
sively, callystatin A (1) is presumed to prevent MAPKKs
from binding toward CRM1 to restrict their location in
the nucleus, which may relate to the remarkably potent
cytotoxicity of 1. In fact, the inhibitors of MAPKKs
suppressed the growth of several human epithelial
tumor lines both in vitro and in vivo.23,24 Therefore,
preventing the function of this kinase would be con-
sidered to lead to potential anti-cancer chemotherapy.
Mitogen-activated protein kinase kinase (MAPKK) is a
threonine/tyrosine speci®c kinase belonging to the MAP
kinase cascade that begins with the stimulation of the
proto-oncoprotein Ras in response to a wide variety of
extracellular stimuli such as expression of some onco-
genes. It was found that MAPKKs which contain an
In summary, we have analyzed the participation of the
two asymmetric centers at C-5 and C-10 in callystatin A
(1), and the two alkyl residues at C-8 and C-10, in the
ecacy of potent cytotoxicity using synthetic analogues,

N. Murakami et al. / Bioorg. Med. Chem. 8 (2000) 2651±2661
2655
indicating that 5-R con®guration and 8-ethyl residue con-
tribute in particular. Investigation of the participation of
the b-hydroxyketone part on structure requirement for the
potent cytotoxicity of callystatin A (1) aiming at a search
for promising leads is currently in progress.
Removal of p-methoxybenzyl group in 5b. A solution of
5b (9.9 mg, 0.024 mmol) in CH2Cl2:0.5% aqueous
NaHCO3 (9:1, 0.8 mL) was treated with 2,3-dichloro-
5,6-dicyano-1,4-benzoquinone (DDQ) (8.2 mg, 0.036
mmol) at rt for 40 min. The reaction mixture was
poured into saturated aqueous NaHCO3, then the
whole was extracted with Et2O. The Et2O extract was
washed with saturated aqueous NaHCO3, then dried
over MgSO4. Removal of solvent from the Et2O extract
under reduced pressure gave a product, which was pur-
i®ed by column chromatography (SiO2 3 g, n-hex-
ane:EtOAc:Et2NH=7:1:0.08) to furnish a mixture of 6b
(5.8 mg, 82%), 7 (0.2 mg, 4%), and 9 (0.3 mg, 6%).
Because of lability of the diol 7, acidic transformation
to 6b as described below was carried out without char-
acterization. The same conversion on a large scale (5b,
416 mg, 1.00 mmol) was carried out to give 6b (71.0 mg,
24%), 7 (20.1 mg, 8%), and 9 (49.1 mg, 21%). Com-
pound 6b: colorless oil; IR nmax (KBr) cm 1: 3621, 2969,
Experimental
The following instruments were used to obtain physical
data: a JASCO DIP-370 digital polarimeter for speci®c
rotations; a Hitachi 330 spectrophotometer for UV
spectra; a JASCO FT/IR-5300 infrared spectrometer for
IR spectra; a JEOL JMS SX-102 mass spectrometer for
FAB±MS and EI±MS; a JASCO J-720W circular dichro-
ism spectrometer for CD spectra; a JEOL JNM LA-500
(500 MHz), a JEOL JNM-AL300, and JNM-EX270
NMR spectrometers for 1H NMR25 and 13C NMR spec-
tra (1H NMR: CDCl3 solution with tetramethylsilane
(TMS) as an internal standard unless otherwise speci®ed.
13C NMR: CDCl3 solution with CHCl3 (dc 77.0) as an
internal standard unless otherwise speci®ed). HPLC was
performed using a Hitachi L-6000 pump equipped with
Hitachi L-4000H UV detector. Silica gel (Merck 60±230
mesh) and pre-coated thin layer chromatography (TLC)
plates (Merck, 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-anis-
aldehyde 25 mL, c-H2SO4 25 mL, AcOH 5 mL, EtOH
425 mL) with subsequent heating.
1
1653, 1458, 1181, 1100, 1032, 999. H NMR (500 MHz,
CDCl3) d: 0.99 (3H, d, J=6.6 Hz, 10-CH3), 1.05 (3H, t,
J=7.4 Hz, 8-CH2CH3), 1.17, 1.24 (both 3H, d, J=
6.2 Hz, 1-OCH(CH3)2), 1.45, 1.66 (both 1H, m, H-11),
2.07 (2H, m, H-4), 2.20 (2H, q, J=7.4 Hz, 8-CH2CH3),
2.79 (1H, m, H-10), 3.58 (2H, t, J=6.4 Hz, H-12), 4.00
(1H, m, 1-OCH(CH3)2), 4.50 (1H, m, H-5), 5.15 (1H, br
s, H-1), 5.74 (2H, m, H-2, H-6), 6.00 (1H, dd, J=9.7,
5.2 Hz, H-3), 6.62 (1H, d, J=15.9 Hz, H-7). FAB±MS
m/z: 317 (M+Na)+. FAB±HRMS m/z: calcd for
C18H30O3Na: 317.2092; found: 317.2104. Compound 9:
1
colorless oil; IR nmax (KBr) cm : 2969, 1663, 1458,
1
1181, 1110, 1042. H NMR (500 MHz, CDCl3) d: 1.00
(6H, d, J=6.8 Hz, 10-CH3), 1.06 (6H, t, J=7.4 Hz, 8-
CH2CH3), 1.45, 1.65 (both 2H, m, H-11), 2.05 (4H, m,
H-4), 2.20 (4H, q, J=7.4 Hz, 8-CH2CH3), 2.76 (2H, m,
H-10), 3.56 (4H, t, J=6.6 Hz, H-12), 4.60 (2H, m, H-5),
5.05 (2H, br s, H-1), 5.74 (4H, m, H-2, H-6), 6.04 (2H,
dd, J=9.7, 5.2 Hz, H-3), 6.68 (2H, d, J=15.9 Hz, H-7).
FAB±MS m/z: 475 (M+Li)+. FAB±HRMS m/z: calcd
for C30H44LiO4: 475.3400; found: 475.2671.
Condensation of 3a and 4b giving 5b. A solution of n-
BuLi (1.54 M in n-hexane, 0.79 mL, 1.22 mmol) was
added to a solution of DMSO (0.35 mL) in dry toluene
(6.1 mL) at rt, then the whole was stirred for 45 min. A
solution of 4b (264 mg, 0.49 mmol) and 3a (207 mg,
1.22 mmol) in dry toluene (5.0 mL) was added to the
solution of dimesylcarbanion at 78 ꢀC, then the whole
was stirred warming from 60 ꢀC to 0 ꢀC overnight. The
reaction mixture was poured into H2O, then the whole
was extracted with Et2O. The Et2O extract was washed
with saturated aqueous NaCl, then dried over MgSO4.
Removal of solvent from the Et2O extract under
reduced pressure gave a product, which was puri®ed
by column chromatography (SiO2 7 g, n-hexane:
Et2O=15:1) to furnish 5b (144 mg, 72%). Compound
5b: colorless oil; IR nmax (KBr) cm 1: 2968, 1612, 1514,
Acidic treatment of 7 or 9 giving 6b. A solution of 9
i
(7.0 mg, 0.015 mmol) in dry PrOH (4.0 mL) was treated
with pyridinium p-toluenesulfonate (PPTS) (5.0 mg,
0.020 mmol) at rt for 2 h. The reaction mixture was
poured into saturated aqueous NaHCO3, then the
whole was extracted with EtOAc. The EtOAc extract
was successively washed with saturated aqueous
NaHCO3 and saturated aqueous NaCl, then dried over
MgSO4. Removal of solvent from the EtOAc extract in
the presence of a slight amount of pyridine under
reduced pressure gave 6b (6.3 mg, 72%). Similarly,
compound 7 (4.3 mg, 0.017 mmol) was converted to 6b
(2.6 mg, 52%).
1
1367, 1248. H NMR (500 MHz, CDCl3) d: 0.97 (3H, d,
J=6.6Hz, 10-CH3), 1.05 (3H, t, J=7.4 Hz, 8-CH2CH3),
1.17, 1.24 (both 3H, d, J=6.2Hz, 1-OCH(CH3)2), 1.43,
1.70 (both 1H, m, H-11), 2.02 (2H, m, H-4), 2.19 (2H, q,
J=7.4Hz, 8-CH2CH3), 2.80 (1H, m, H-10), 3.35 (2H, m,
H-12), 3.80 (3H, s, CH2PhOCH3), 4.01 (1H, m, 1-
OCH(CH3)2), 4.36, 4.39 (both 1H, d, J=11.3 Hz, CH2
PhOCH3), 4.52 (1H, m, H-5), 5.11 (1H, br s, H-1), 5.72±
5.76 (2H, m, H-2, H-6), 5.98 (1H, m, H-3), 6.61 (1H, d,
J=15.6 Hz, H-7), 6.86, 7.23 (both 2H, d, J=8.4 Hz,
CH2PhOCH3). EI±MS m/z: 414 (M+, 6.3), 121 (100).
EI±HRMS m/z: calcd for C26H38O4: 414.2770; found:
414.2767.
Swern oxidation of 6b followed by Wittig reaction with
10 giving 11. DMSO (0.023 mL) was added to a solu-
tion of (COCl)2 (0.020 mL, 0.165 mmol) in dry CH2Cl2
(2.7 mL) at 78 ꢀC, then the whole was stirred for
20 min. After adding a solution of 6b (16.2 mg, 0.055
mmol) in dry CH2Cl2 (2.0 mL) to the reaction mixture
at 78 ꢀC, the whole was stirred for 30 min. Then, the
reaction mixture was treated with Et3N (0.061 mL,

2656
N. Murakami et al. / Bioorg. Med. Chem. 8 (2000) 2651±2661
0.44 mmol) at 78 ꢀC for 2 h. After the reaction mixture
was diluted with dry Et2O, a ®ltrate given through
Na2SO4 column was concentrated under reduced pres-
sure to a€ord an aldehyde 8b. A solution of n-BuLi
(1.54 M in n-hexane, 0.28 mL, 0.43 mmol) was added to
a solution of DMSO (0.19 mL) in dry toluene (6.4 mL)
at rt, then the whole was stirred for 60 min. A solution
of 8b and 10 (103 mg, 0.17 mmol) in dry toluene
(3.0 mL) was added to the solution of dimesylcarbanion
at 78 ꢀC, then the whole was stirred warming from
78 ꢀC to 0 ꢀC overnight. Work up in the same man-
ner as preparation for compound 5b gave a product,
which was puri®ed by column chromatography (SiO2 1
g, n-hexane:EtOAc=30:1) to furnish 11 (24.0 mg, 71%).
Compound 11: colorless oil; IR nmax (KBr) cm 1: 3534,
2934, 1462, 1379, 1260, 1098, 1018. 1H NMR (500 MHz,
CDCl3) d: 0.08 (6H, s, 19-OSi(CH3)2C(CH3), 0.80±1.06
(34H, m, 1-OCH(CH3)2, 8-CH2CH3, 10-CH3, 16-CH3,
18-CH3, 19-OSi(CH3)2C(CH3)3, 20-CH3, H-21, H-22),
1.20 (2H, m, H-20, 21), 1.70 (3H, s, 14-CH3), 1.72 (1H,
m, H-18), 2.04±2.21 (6H, m, H-4, 8-CH2CH3, H-11),
2.57 (1H, m, H-10), 2.67 (1H, m, H-16), 3.35 (1H, d,
J=9.2 Hz, H-19), 3.63 (1H, t, J=3.7 Hz, H-17), 4.02
(1H, m, 1-OCH(CH3)2), 4.49 (1H, m, H-5), 5.05 (1H, d,
J=9.8 Hz, H-15), 5.12 (1H, br s, H-1), 5.16 (1H, d,
J=10.1 Hz, H-9), 5.47 (1H, dt, J=15.3, 7.9 Hz, H-12),
5.75 (2H, m, H-2, H-6), 5.98 (2H, m, H-3, H-13), 6.56
(1H, d, J=15.9 Hz, H-7). FAB±MS m/z: 639
(M+Na)+. FAB±HRMS m/z: calcd for C38H68O4SiNa:
639.4784; found: 639.4751.
pressure gave a product, which was puri®ed by HPLC
(column; COSMOSIL 5SL (10 mm i.d.Â250 mm);
mobile phase; n-hexane:EtOAc=2:1; detection; UV
(l=250 nm); ¯ow rate; 3.0 mL/min) to furnish 10-epi-
callystatin A (12, 1.3 mg, 90%).26 10-epi-Callystatin A
(12): colorless oil; [a]D 288.9ꢀ (c=0.05, MeOH, 26 ꢀC).
UV lmax (MeOH) nm (e) : 242 (30900), 300 (1300). CD
(MeOH) nm (Áe) : 334 ( 0.3), 300 ( 22.0), 268 ( 4.8),
254 ( 13.6), 242 (0), 229 (+25.0), 210 (+13.1). IR nmax
(KBr) cm 1: 3563, 2965, 2928, 1730, 1713, 1456, 1377,
1
1262, 1084, 1051, 1020. H NMR (500 MHz, CDCl3) d:
0.84 (3H, t, J=7.4 Hz, H-22), 0.89 (3H, d, J=6.6 Hz,
20-CH3), 0.97 (3H, d, J=6.4 Hz, 10-CH3), 1.04 (3H, t,
J=7.4 Hz, 8-CH2CH3), 1.08 (1H, m, H-21), 1.11 (3H, d,
J=7.1 Hz, 18-CH3), 1.14 (3H, d, J=6.7 Hz, 16-CH3),
1.32 (1H, m, H-21), 1.40 (1H, m, H-20), 1.80 (3H, s, 14-
CH3), 2.08 (2H, dd, J=7.4, 6.7 Hz, H-11), 2.18 (2H, q,
J=7.4 Hz, 8-CH2CH3), 2.46 (2H, m, H-4), 2.67 (1H, m,
H-10), 2.84 (1H, dq, J=4.3, 7.1 Hz, H-18), 3.56 (1H, m,
H-19), 3.64 (1H, dq, J=10.0, 6.7 Hz, H-16), 5.00 (1H,
ddd, J=7.6, 7.6, 6.4 Hz, H-5), 5.13 (1H, d, J=10.0 Hz,
H-15), 5.25 (1H, d, J=9.7 Hz, H-9), 5.58 (1H, dt,
J=15.8, 7.4 Hz, H-12), 5.76 (1H, dd, J=6.4, 16.0 Hz,
H-6), 6.01 (1H, d, J=15.8 Hz, H-13), 6.07 (1H, d,
J=10.2 Hz, H-2), 6.64 (1H, d, J=16.0 Hz, H-7), 6.90
(1H, dt, J=10.2, 4.8 Hz, H-3). 13C NMR (125 MHz,
CDCl3) dC: 10.9 (q, C-22), 11.0 (q, 18-CH3), 13.1 (q,
14-CH3), 13.4 (q, 8-CH2CH3), 14.3 (q, 20-CH3), 16.3 (q,
16-CH3), 20.9 (q, 10-CH3), 25.7 (t, C-21), 26.4 (t, 8-
CH2CH3), 30.0 (t, C-4), 32.2 (d, C-10), 36.7 (d, C-20),
40.8 (t, C-11), 45.7 (d, C-16, C-18), 74.5 (d, C-19), 78.6
(d, C-5), 121.7 (d, C-2), 124.8 (d, C-6), 127.9 (d, C-12),
128.3 (d, C-15), 129.8 (d, C-7), 135.2 (d, C-13), 135.3 (s,
C-8), 136.2 (s, C-14), 137.1 (d, C-9), 144.6 (d, C-3),
164.2 (s, C-1), 216.4 (s, C-17). FAB±MS m/z: 457
(M+H)+. FAB±HRMS m/z: calcd for C29H45O4:
457.3318; found: 457.3332.
Conversion from 11 to 10-epi-callystatin A (12). N-Methyl
morpholine N-oxide (NMO) (13.1 mg, 0.112 mmol) was
added to a solution of 11 (17.2 mg, 0.028 mmol) in
CH2Cl2 (2.8 mL), then the whole was stirred at rt for
10 min. The reaction mixture was treated with tetra-
propylammonium perruthenate (TPAP) (11.8 mg,
0.034 mmol) at rt for 36 h. After dilution with Et2O, a
®ltrate given through SiO2 column was concentrated
under reduced pressure to give a product, which was
puri®ed by column chromatography (SiO2 1 g, n-hexane:
EtOAc=10:1) to furnish a ketone (17.1 mg). A solution
of the ketone (4.2 mg, 0.0068 mmol) in acetone:H2O
(5:1) (1.2 mL) was treated with Dowex HCR-W2 (H+
form) (30 mg) at 40 ꢀC for 5 h. After removing the resi-
due by ®ltration, the ®ltrate was poured into saturated
aqueous NaHCO3 and the whole was extracted with
Et2O. The Et2O extract was washed with saturated
aqueous NaHCO3, then dried over MgSO4. Removal of
solvent from the Et2O extract under reduced pressure
gave a lactol. A solution of the lactol in dry benzene
(1.7 mL) was treated with Ag2CO3 (94 mg, 0.34 mmol)
and Celite (47 mg) at 45 ꢀC for 6 h in the dark. After
removing the residue by ®ltration with Na2SO4, the ®l-
trate was concentrated under reduced pressure to give a
product, which was puri®ed by column chromatography
(SiO2 1 g, n-hexane:EtOAc=5:1) to furnish a lactone
(2.5 mg, 65%). To a solution of the lactone (1.9 mg,
0.0033 mmol) in dry THF (2.0 mL) was added HF:pyr-
idine (5:1) (0.7 mL) at 0 ꢀC, then the whole was stirred at
rt for 80 h. The reaction mixture was neutralized with
NaHCO3 at 0 ꢀC, then the residue was removed by ®ltra-
tion. Removal of solvent from the ®ltrate under reduced
Conversion from 13b to 14b. To a solution of 13b
(21.0 g, 61.4 mmol) in dry THF (150 mL) was added
NaH (3.0 g, 123 mmol) at 0 ꢀC, then the whole mixture
was stirred at rt for 15 min. The reaction mixture was
treated with p-methoxybenzyl bromide (PMBBr)
(15.0 mL, 74.6 mmol) at rt in the dark for 24 h. The
reaction mixture was poured into saturated aqueous
NaCl, then 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
(SiO2 750 g, n-hexane:EtOAc=30:1) to a€ord a PMB
ether (27.3 g, 96%). Tetrabutylammonium ¯uoride
(1.0 M in THF, 118 mL, 118 mmol) was added to a
solution of PMB ether (27.2 g, 58.9 mmol) in dry THF
(240 mL), then the whole mixture was stirred at rt for
17 h. The reaction mixture was worked up in the same
manner as preparation for PMB ether of 13b to give a
product, which was puri®ed by column chromatography
(SiO2 350 g, n-hexane:EtOAc=5:2) to furnish 14b (12.8 g,
97%). Compound 14b: colorless oil; [a]D
7.9ꢀ
(c=2.34, CHCl3, 25 ꢀC). IR nmax (KBr) cm 1: 3487,
1
2928, 1514, 1248. H NMR (270 MHz, CDCl3) d: 0.91
(3H, d, J=6.9 Hz, 10-CH3), 1.65 (2H, m, H-11), 1.78
(1H, m, H-10), 3.37±3.61 (4H, m, H-9, H-12), 3.80 (3H,

N. Murakami et al. / Bioorg. Med. Chem. 8 (2000) 2651±2661
2657
s, OCH3), 4.45 (2H, s, CH2PhOCH3), 6.88, 7.25 (both
2H, d, J=8.6 Hz, CH2PhOCH3). FAB±MS m/z: 225
(M+H)+. FAB±HRMS m/z: calcd for C13H21O3:
225.1491; found: 225.1538.
poured into saturated aqueous NaCl, then extracted
with n-hexane:Et2O (1:1). The organic layer was washed
with saturated aqueous NaCl, then dried over Na2SO4.
Removal of solvent from this extract under reduced
pressure gave a product, which was puri®ed by column
chromatography (SiO2 10 g, n-hexane:EtOAc=10:1) to
furnish a bromide. A solution of the bromide in dry
CH3CN (4.0 mL) was treated with tri-n-butylphosphine
(0.71 mL, 2.85 mmol) at 0 ꢀC for 2 h. The reaction mix-
ture was evaporated under reduced pressure to a€ord a
product, which was puri®ed by column chromatography
(SiO2 10 g, CHCl3:MeOH=50:1) to furnish 4b (515 mg,
quant.). Because of lability of this tributylphosphonium
bromide, next transformation was carried out without
further puri®cation and characterization.
Conversion from 14b to 15b. DMSO (0.89 mL) was
added to a solution of (COCl)2 (0.55mL, 6.29mmol) in
dry CH2Cl2 (24 mL) at 78ꢀC, then the whole was stirred
for 20 min. After adding a solution of 14b (470 mg,
2.10 mmol) in dry CH2Cl2 (2.0 mL) to the reaction mix-
ture at 78 ꢀC, the whole was stirred for 30 min. Then,
the reaction mixture was treated with Et3N (2.3 mL,
16.8 mmol) at 78 ꢀC for 1 h. The reaction mixture was
poured into H2O, then the whole was extracted with
Et2O. The Et2O extract was washed with saturated
aqueous NaCl, then dried over MgSO4. Removal of
solvent from the Et2O extract under reduced pressure
gave an aldehyde. After 18-Crown-6 ether/CH3CN
complex (5.55 g, 21.0 mmol) was added to a solution of
EtOCOCHEtPO(OCH2CF3)2 (1.89 g, 5.25 mmol) in dry
THF (71 mL) at rt, the reaction mixture was treated
with potassium bis(trimethylsilyl)amide (0.5 M in tolu-
ene, 8.40 mL, 4.20 mmol) at 78 ꢀC for 5 min. Then, a
solution of aldehyde in dry THF (23 mL) was added and
the whole was stirred warming from 78 ꢀC to 0 ꢀC
overnight. The reaction mixture was poured into satu-
rated aqueous NH4Cl, then the whole was extracted
with Et2O. The Et2O extract was washed with saturated
aqueous NaCl, then dried over Na2SO4. Removal of
solvent from the Et2O extract under reduced pressure
gave a product, which was puri®ed by column chromato-
graphy (SiO2 12g, n-hexane:Et2O=10:1) to furnish 15b
Preparation of 15c. After 18-Crown-6 ether/CH3CN
complex (15.0 g, 56.7 mmol) was added to a solution of
MeOCOCH2PO(OCH2CF3)2 (3.43 mL, 16.2 mmol) in
dry THF (35 mL) at rt, the reaction mixture was treated
with potassium bis(trimethylsilyl)amide (0.5 M in tolu-
ene, 30.8 mL, 16.0 mmol) at 78 ꢀC for 10 min. Then, a
solution of the aldehyde (1.8 g, 8.1 mmol), which was
prepared from 14a for synthesis of 15a, in dry THF
(35 mL) was added and the whole was stirred warming
from 78 ꢀC to 0 ꢀC overnight. Work up in the same
manner as preparation for 15a gave a product, which
was puri®ed by column chromatography (SiO2 90 g, n-
hexane:EtOAc=3:1) to furnish 15c (2.25 g, quant.,
Z:E=7.7:1). Compound 15c: colorless oil; [a]D +12.6ꢀ
(c=3.83, CHCl3, 25 ꢀC). IR nmax (KBr) cm : 2953,
1
1
2861, 1723, 1514, 1248. H NMR (500 MHz, CDCl3) d:
(620mg, 92%, E:Z=12:1). Compound 15b: colorless oil,
1
1.03 (3H, d, J=6.8 Hz, 10-CH3), 1.66 (2H, m, H-11),
3.43 (2H, m, H-12), 3.61 (1H, m, H-10), 3.70 (3H, s, 7-
OCH3), 3.80 (3H, s, CH2PhOCH3), 4.39 (2H, s,
CH2PhOCH3), 5.71 (1H, dd, J=11.4, 0.8 Hz, H-8), 6.00
(1H, dd, J=11.4, 10.3Hz, H-9), 6.86, 7.24 (both 2H, d,
J=8.6Hz, CH2PhOCH3). FAB±MS m/z: 301 (M+Na)+.
FAB±HRMS m/z: calcd for C16H22O4Na: 301.1416;
found: 301.1409.
[a]D +2.0 ꢀ (c=0.75, CHCl3, 25ꢀC). IR nmax (KBr) cm
:
1
2967, 1713, 1512, 1248. H NMR (270 MHz, CDCl3) d:
0.99 (3H, d, J=6.3Hz, 10-CH3), 1.00 (3H, t, J=7.5 Hz, 8-
CH2CH3), 1.28 (3H, t, J= 7.3 Hz, OCH2CH3), 1.61 (2H,
m, H-11), 2.25 (2H, q, J=7.3 Hz, 8-CH2CH3), 3.10 (1H,
m, H-10), 3.41 (2H, t, J=7.1 Hz, H-12), 3.80 (3H, s,
OCH3), 4.18 (2H, q, J=7.1 Hz, OCH2CH3), 4.39 (2H, s,
CH2PhOCH3), 5.55 (1H, d, J=10.2Hz, H-9), 6.86, 7.24
(both 2H, d, J=8.6Hz, CH2PhOCH3). FAB±MS m/z: 321
(M+H)+. FAB±HRMS m/z: calcd for C19H29O4:
321.2066; found: 321.1996.
Conversion from 15c to 4c. The same procedure as pre-
paration for 4a was conducted using 15c (1530 mg,
5.50 mmol) giving a product, which was puri®ed by
column chromatography (SiO2 20 g, n-hexane:EtOAc=
2:1) to furnish an allyl alcohol (233 mg, 85%). The allyl
alcohol (150 mg, 0.60 mmol) was treated in the same
manner as preparation for 15a to give a product, which
was puri®ed by column chromatography (SiO2 6 g,
benzene:Et2O=100:1) to furnish a bromide. The same
treatment of the bromide as preparation for 4a a€orded
a product, which was puri®ed by washing with n-hexane
to furnish 4c (309 mg, quant.). Because of lability of this
tributylphosphonium bromide, next transformation was
carried out without further puri®cation and character-
ization.
Conversion from 15b to 4b. A solution of 15b (590 mg,
1.85 mmol) in dry CH2Cl2 (9 mL) was treated with di-
isobutylaluminum hydride (DIBAL-H) (1.5 M in toluene,
2.7 mL, 4.06 mmol) at 78 ꢀC for 15 min. After the
reaction mixture was diluted with Et2O, saturated aque-
ous NaCl and 4.0 N aqueous NaOH were added. The
reaction mixture was stirred vigorously until stop of
precipitate-formation, then the residue was removed by
®ltration. After drying the ®ltrate over Na2SO4, removal
of solvent from the ®ltrate under reduced pressure gave
a product, which was puri®ed by column chromato-
graphy (SiO2 10g, n-hexane:EtOAc=5:1) to furnish an
allyl alcohol (472mg, 92%). Triphenylphosphine (622mg,
2.37mmol) was added to a solution of the allyl alcohol
(264mg, 0.95mmol) in dry CH3CN (14 mL) at rt, then
2,6-lutidine (0.03 mL, 0.28 mmol) and CBr4 (962 mg,
2.85 mmol) were added at 0 ꢀC. The whole was further
stirred at rt for 15 min. The reaction mixture was
Conversion from 1,4-butanediol (16) to 15d. A solution
of 1,4-butanediol (16) (3.0 g, 33.3 mmol) in dry THF
(300 mL) was treated with KH (1.47 g, 36.7 mmol) at
0 ꢀC for 10 min. To the reaction mixture was added p-
methoxybenzyl chrolide (PMBCl) (4.54 mL, 33.3 mmol),
then the whole was stirred at rt for 1 h. Work up in the

2658
N. Murakami et al. / Bioorg. Med. Chem. 8 (2000) 2651±2661
1
same manner as preparation for 14a except for using
EtOAc in extraction gave a product, which was puri®ed
by column chromatography (SiO2 100 g, n-hexane:
EtOAc=1:1) to furnish mono-PMB ether (5.42 g, 77%).
After the same treatment for oxidation of mono-PMB
ether (932 mg, 4.44 mmol) as preparation for 15a, the
reaction mixture was extracted with CH2Cl2. The
CH2Cl2 extract was washed with saturated aqueous
NaCl:5.0% aqueous HCl (1:1), then dried over MgSO4.
Removal of solvent from the CH2Cl2 extract under
reduced pressure gave an aldehyde. The same procedure
as preparation for 15a was conducted using EtO-
COCHEtPO(OCH2CF3)2 (3.43 g, 9.5 mmol) to give a
product, which was puri®ed by column chromatography
(SiO2 25 g, n-hexane:EtOAc=5:1) to furnish 15d (1.29 g,
95%, Z:E=5:1). Compound 15d: colorless oil; IR nmax
(KBr) cm 1: 2969, 2849, 1709, 1613, 1462, 1375, 1300,
1051, 961. H NMR (500 MHz, CDCl3) d: 1.00 (3H, d,
J=6.7 Hz, 10-CH3), 1.05 (3H, t, J=7.3 Hz, 8-CH2CH3),
1.48 (1H, m, H-11), 1.63 (1H, m, H-11), 2.19 (2H, q,
J=7.3 Hz, 8-CH2CH3), 2.50 (2H, m, H-4), 2.80 (1H, m,
H-10), 3.58 (2H, m, H-12), 5.01 (1H, ddd, J=7.3, 7.3,
6.7 Hz, H-5), 5.23 (1H, d, J=9.8 Hz, H-9), 5.78 (1H, dd,
J=6.7, 15.9 Hz, H-6), 6.06 (1H, d, J=9.8 Hz, H-2), 6.70
(1H, d, J=15.9 Hz, H-7), 6.92 (1H, dd, J=9.8, 4.3 Hz,
H-3). FAB±MS m/z: 251 (M+H)+. FAB±HRMS m/z:
calcd for C15H23O3: 251.1648; found: 251.1651.
Dess±Martin oxidation of 17a followed by Wittig reac-
tion with 10 giving 19a. A solution of 17a (10 mg,
0.04 mmol) in dry CH2Cl2 (2.2 mL) was treated with
Dess±Martin periodinane (54 mg, 0.12 mmol) at rt for
30 min. The reaction mixture was poured into saturated
aqueous NaHCO3:saturated aqueous Na2S2O3 (1:1),
then the whole was extracted with Et2O. The Et2O
extract was washed with saturated aqueous NaCl:satu-
rated aqueous Na2S2O3 (1:1), then dried over MgSO4.
Removal of solvent from the Et2O extract under
reduced pressure gave 18a. A solution of n-BuLi
(1.53 M in n-hexane, 0.06 mL, 0.097 mmol) was added to
a solution of DMSO (0.07 mL) in dry toluene (1.3 mL)
at rt, then the whole was stirred for 45 min. A solution
of 18a and 10 (60.8 mg, 0.098 mmol) in dry toluene
(3.0 mL) was added to the solution of dimesylcarbanion
at 78 ꢀC, then the whole was stirred warming from
60 ꢀC to 0 ꢀC overnight. Work up in the same manner
as preparation for 5b gave a product, which was puri®ed
by column chromatography (SiO2 1 g, n-hexane:
EtOAc=3:1) to furnish 19a (14.2 mg, 62%). Compound
1
1248, 1094. H NMR (500 MHz, CDCl3) d: 1.00 (3H, t,
J=7.6 Hz, 8-CH2CH3), 1.29 (3H, t, J=7.0 Hz, OCH2
CH3), 1.73 (2H, m, H-11), 2.30 (2H, m, H-10), 2.48 (2H,
q, J=7.6 Hz, 8-CH2CH3), 3.46 (2H, dd, J= 6.7, 6.4 Hz,
H-12), 3.80 (3H, s, CH2PhOCH3), 4.19 (2H, q, J=
7.0 Hz, OCH2 CH3), 4.43 (2H, s, CH2PhOCH3), 5.83
(1H, t, J=7.6 Hz, H-9), 6.87, 7.25 (both 2H, d, J=8.5Hz,
CH2PhOCH3). FAB±MS m/z: 329 (M+Na)+. FAB±
HRMS m/z: calcd for C18H26O4Na: 329.1728; found:
329.1723.
Conversion from 15d to 4d. The same procedure as
preparation for 4a was conducted using 15d (375mg,
1.23mmol) giving a product, which was puri®ed by
column chromatography (SiO2 7 g, n-hexane:EtOAc=5:2)
to furnish an allyl alcohol (318 mg, 98%). The allyl alco-
hol (243 mg, 0.92 mmol) was treated in the same manner
as preparation for 4a to give a product, which was puri-
®ed by column chromatography (SiO2 13g, benzene:
Et2O=100:1) to furnish a bromide. The same treatment
of the bromide as preparation for 4a a€orded a product,
which was puri®ed by washing with n-hexane to furnish
4d (300 mg, quant.). Because of lability of this tributyl-
phosphonium bromide, next transformation was carried
out without further puri®cation and characterization.
1
19a: colorless oil; H NMR (500 MHz, CDCl3) d: 0.07,
0.08 (both 3H, s, 19-OSi(CH3)2 C(CH3)3), 0.83 (3H, d,
J=7.4 Hz, 20-CH3), 0.84 (3H, d, J=7.3 Hz, 18-CH3),
0.87 (3H, t, J=7.3 Hz, H-22), 0.91 (9H, s, 19-
OSi(CH3)2C(CH3)3), 0.98 (3H, d, J=7.3 Hz, 16-CH3),
1.08 (1H, m, H-21), 1.03 (3H, d, J=6.7 Hz, 10-CH3),
1.05 (3H, t, J=7.4 Hz, 8-CH2CH3), 1.58 (3H, m, H-20,
21), 1.72 (3H, s, 14-CH3), 1.76 (1H, m, H-18), 2.07 (2H,
m, H-11), 2.18 (2H, q, J=7.3 Hz, 8-CH2CH3), 2.48 (2H,
m, H-4), 2.58 (1H, m, H-16), 2.67 (1H, m, H-10), 3.36
(1H, d, J=9.2 Hz, H-19), 3.64 (1H, dd, J=7.9, 3.7 Hz,
H-17), 5.00 (1H, ddd, J=7.4, 7.4, 6.7 Hz, H-5), 5.07
(1H, d, J=9.8 Hz, H-15), 5.27 (1H, d, J=9.8 Hz, H-9),
5.49 (1H, dt, J=15.3, 7.3 Hz, H-12), 5.76 (1H, dd,
J=6.6, 15.9 Hz, H-6), 5.99 (1H, d, J=15.3 Hz, H-13),
6.08 (1H, d, J=9.8 Hz, H-2), 6.65 (1H, d, J=15.9 Hz,
H-7), 6.90 (1H, dt, J=9.8, 4.9 Hz, H-3). FAB±MS m/z:
573 (M+H)+. FAB±HRMS m/z: calcd for C35H61O4Si:
573.4340; found: 573.4337.
Conversion from 5a to 17a. A solution of 5a (153 mg,
0.37 mmol) in acetone:H2O (5:1) (45 mL) was treated
with Dowex HCR-W2 (H+ form) (1.97 g) at 40 ꢀC for
5 h. Work up in the same manner as preparation for 12
gave a lactol. A solution of the lactol in dry benzene
(60 mL) was treated with Ag2CO3 (5.08 g, 18.4 mmol)
and Celite (2.53 g) at 45 ꢀC for 6 h in the dark. Work up
in the same manner as preparation for 12 gave a pro-
duct, which was puri®ed by column chromatography
(SiO2 9 g, n-hexane:EtOAc=3:1) to furnish a lactone
(124 mg, 91%). A solution of the lactone (104 mg,
0.28 mmol) in CH2Cl2:t-BuOH:pH 6.9 phospate bu€er
(90:1:9) (13.5 mL) was treated with DDQ (127 mg,
0.56 mmol) at rt for 30 min. Work up in the same manner
as preparation for 6b except for using EtOAc in extrac-
tion gave a product, which was puri®ed by column
chromatography (SiO2 10 g, n-hexane:EtOAc=1:1) to
furnish 17a (64 mg, 91%). Compound 17a: colorless oil;
[a]D 55.3 ꢀ (c=1.94, CHCl3, 20 ꢀC). IR nmax (KBr)
cm 1: 3414, 2963, 2928, 2874, 1725, 1456, 1381, 1246,
Conversion from 19a to 5-epi-callystatin A (20). A solu-
tion of 19a (9.2 mg, 0.016 mmol) in dry CH2Cl2 (1.8 mL)
was treated with Dess±Martin periodinane (24 mg, 0.57
mmol) at rt for 1.5 h. The reaction mixture was worked
up in the same manner as preparation for 18a to give a
ketone. To a solution of the ketone in dry THF (5.0 mL)
was added HF:pyridine (5:1) (1.67 mL) at 0 ꢀC, then the
whole was stirred at rt for 80 h. Work up in the same
manner as preparation for 12 gave a product, which was
puri®ed by HPLC (column: COSMOSIL 5SL (10 mm
i.d.Â250 mm), mobile phase: n-hexane:EtOAc=2:1,

N. Murakami et al. / Bioorg. Med. Chem. 8 (2000) 2651±2661
2659
detection: UV (l=250 nm), ¯ow rate: 3.0 mL/min) to
furnish 5-epi-callystatin A (20, 6.1 mg, 90%).26 5-epi-
Callystatin A (20): colorless oil; [a]D 71.4ꢀ (c=0.27,
MeOH, 25 ꢀC). UV lmax (MeOH) nm (e): 244 (22500),
296 (2200). CD (MeOH) nm (Áe): 330 (0), 300 ( 24.6),
270 (0), 252 (+57.5), 233 (0), 222 ( 17.4), 210 ( 13.1).
IR nmax (KBr) cm 1: 3475, 2925, 2857, 1725, 1459, 1381,
found: 409.2303. Compound 5d: colorless oil; IR nmax
(KBr) cm 1: 2969, 2930, 2878, 1400, 1248, 1100, 1032, 1001.
1H NMR (500 MHz, CDCl3) d: 1.05 (3H, t, J=7.3 Hz,
8-CH2CH3), 1.17, 1.24 (both 3H, d, J=6.1 Hz, 1-
OCH(CH3)2), 1.63 (2H, m, H-11), 2.02±2.32 (6H, m, H-
4, H-10, 8-CH2CH3), 3.44 (2H, t, J=6.1 Hz, H-12), 3.80
(3H, s, CH2PhOCH3), 4.01 (1H, m, 1-OCH(CH3)2),
4.42 (2H, s, CH2PhOCH3), 4.51 (1H, m, H-5), 5.12 (1H,
s, H-1), 5.36 (1H, t, J=7.3 Hz, H-9), 5.75 (2H, m, H-2,
6), 6.00 (1H, m, H-3), 6.60 (1H, d, J=15.9 Hz, H-7),
6.87, 7.25 (both 2H, d, J=8.5 Hz, CH2PhOCH3). FAB±
MS m/z: 423 (M+Na)+. FAB±HRMS m/z: calcd for
C25H36O4Na: 423.2512; found: 423.2523.
1
1247, 1018, 965. H NMR (500 MHz, CDCl3) d: 0.85
(3H, t, J=7.4 Hz, H-22), 0.89 (3H, d, J=6.6 Hz, 20-
CH3), 0.96 (3H, d, J=6.6 Hz, 10-CH3), 1.04 (3H, t,
J=7.4 Hz, 8-CH2CH3), 1.07 (1H, m, H-21), 1.11 (3H, d,
J=7.1 Hz, 18-CH3), 1.14 (3H, d, J=6.6 Hz, 16-CH3),
1.36 (1H, m, H-21), 1.40 (1H, m, H-20), 1.81 (3H, s, 14-
CH3), 2.08 (2H, t, J=7.9 Hz, H-11), 2.18 (2H, q, J=
7.4 Hz, 8-CH2CH3), 2.47 (2H, m, H-4), 2.67 (1H, m, H-
10), 2.86 (1H, dq, J=4.2, 7.1 Hz, H-18), 3.57 (1H, dd,
J=6.9, 4.2 Hz, H-19), 3.65 (1H, dq, J=10.0, 6.6 Hz, H-
16), 5.00 (1H, ddd, J=7.7, 7.4, 6.6 Hz, H-5), 5.13 (1H,
d, J=10.0 Hz, H-15), 5.25 (1H, d, J=9.8 Hz, H-9), 5.59
(1H, dt, J=15.9, 7.9 Hz, H-12), 5.76 (1H, dd, J=6.6,
15.9 Hz, H-6), 6.01 (1H, d, J=15.9 Hz, H-13), 6.07 (1H,
dd, J=9.8, 1.9 Hz, H-2), 6.65 (1H, d, J=15.9 Hz, H-7),
6.90 (1H, dt, J=9.8, 4.2 Hz, H-3). 13C NMR (125 MHz,
CDCl3) dC: 10.9 (q, C-22), 11.1 (q, 18-CH3), 13.1 (q, 14-
CH3), 13.4 (q, 8-CH2CH3), 14.2 (q, 20-CH3), 16.2 (q,
16-CH3), 20.7 (q, 10-CH3), 25.8 (t, C-21), 26.4 (t, 8-
CH2CH3), 30.1 (t, C-4), 32.1 (d, C-10), 36.7 (d, C-20),
40.7 (t, C-11), 45.7 (d, C-16, C-18), 74.5 (d, C-19), 78.6
(d, C-5), 121.7 (d, C-2), 124.8 (d, C-6), 127.7 (d, C-12),
128.3 (d, C-15), 129.7 (d, C-7), 135.3 (d, C-13), 135.4 (s,
C-8), 136.2 (s, C-14), 137.2 (d, C-9), 144.6 (d, C-3),
164.0 (s, C-1), 216.4 (s, C-17). FAB±MS m/z: 457
(M+H)+. FAB±HRMS m/z: calcd for C29H45O4:
457.3318; found: 457.3330.
Preparation of 17c and 17d. A solution of 5c (154 mg,
0.40 mmol) in acetone:H2O (5:1) (47 mL) was treated
with Dowex HCR-W2 (H+ form) (2.0 g) at 40 ꢀC for
5 h. Work up in the same manner as preparation for 12
gave a lactol. A solution of the lactol in dry benzene
(61 mL) was treated with Ag2CO3 (2.2 g, 8.0 mmol) and
Celite (1.1 g) at 45 ꢀC for 6 h in the dark. Work up in the
same manner as preparation for 12 gave a product,
which was puri®ed by column chromatography (SiO2
5 g, n-hexane:EtOAc=2:1) to furnish
a
lactone
(130 mg). A solution of the lactone in CH2Cl2:t-BuOH:
pH 6.9 phospate bu€er (90:1:9) (17.5 mL) was treated
with DDQ (173 mg, 0.76 mmol) at rt for 30 min. Work
up in the same manner as preparation for 17a gave a
product, which was puri®ed by column chromatography
(SiO2 4 g, n-hexane:EtOAc=1:1) to furnish 17c (68.4
mg, 81%). The same procedure as preparation for 17c
was conducted using 5d (100 mg, 0.25 mmol) giving a
product, which was puri®ed by column chromatography
(SiO2 5 g, n-hexane:EtOAc=3:2) to furnish 17d (44.8
mg, 76%). Compound 17c: colorless oil; [a]D +77.5 ꢀ
(c=0.70, CHCl3, 25 ꢀC). IR nmax (KBr) cm 1: 3402,
2961, 2930, 1715, 1383, 1250, 1046. 1H NMR (500 MHz,
CDCl3) d: 1.01 (3H, d, J=6.6 Hz, 10-CH3), 1.47 (1H, m,
H-11), 1.65 (1H, m, H-11), 2.09 (1H, s, 12-OH), 2.47
(2H, m, H-4), 2.84 (1H, m, H-10), 3.57 (1H, m, H-12),
3.61 (1H, m, H-12), 4.99 (1H, ddd, J=10.7, 6.6, 5.1 Hz, H-
5), 5.33 (1H, t, J=10.4 Hz, H-9), 5.73 (1H, dd, J=6.6,
15.2Hz, H-6), 5.97 (1H, dd, J=11.2, 10.4 Hz, H-8), 6.04
(1H, d, J=9.9, Hz, H-2), 6.67 (1H, dd, J=15.2, 11.2Hz,
H-7), 6.91 (1H, ddd, J=9.9, 5.1, 3.0 Hz, H-3). FAB±MS
m/z: 223 (M+H)+. FAB-HRMS m/z: calcd for
C13H19O3: 223.1334; found: 223.1320. Compound 17d:
colorless oil; [a]D +27.0 ꢀ (c=1.74, CHCl3, 20 ꢀC). IR
nmax (KBr) cm 1: 3437, 2967, 2934, 2874, 1719, 1383,
Preparation of 5c and 5d. A solution of n-BuLi (1.54 M
in n-hexane, 7.7 mL, 12.0 mmol) was added to a solution
of DMSO (0.42 mL) in dry toluene (7.1 mL) at rt, then
the whole was stirred for 45 min. A solution of 4c
(309 mg, 0.60 mmol) and 3a (254 mg, 1.49 mmol) in dry
toluene (6.9 mL) was added to the solution of dimesyl-
carbanion at 78 ꢀC, then the whole was stirred warm-
ing from 60 ꢀC to 0 ꢀC overnight. Work up in the same
manner as preparation for 5b gave a product, which was
puri®ed by column chromatography (SiO2 50 g, n-hex-
ane:EtOAc=8:1) to furnish 5c (172 mg, 75%). The same
procedure as preparation for 5c was conducted using 4d
(213 mg, 0.40 mmol) and 3a (171 mg, 1.00 mmol) giving a
product, which was puri®ed by column chromatography
1
(SiO2 7 g, n-hexane:EtOAc=10:1) to furnish 5d (130 mg,
1
1248, 1055, 1020, 966. H NMR (500 MHz, CDCl3) d:
81%). Compound 5c: colorless oil; IR nmax (KBr) cm
:
1.06 (3H, t, J= 7.3 Hz, 8-CH2CH3), 1.66 (2H, m, H-11),
2.20 (2H, q, J=7.3 Hz, 8-CH2CH3), 2.27 (2H, m, H-10),
2.49 (2H, m, H-4), 3.65 (2H, t, J=6.7 Hz, H-12), 5.02
(1H, ddd, J=7.3, 7.3, 7.3 Hz, H-5), 5.47 (1H, t,
J=7.3 Hz, H-9), 5.78 (1H, dd, J=7.3, 15.9 Hz, H-6),
6.07 (1H, d, J=9.8, Hz, H-2), 6.70 (1H, d, J=15.9 Hz,
H-7), 6.91 (1H, dt, J=9.8, 4.3 Hz, H-3). FAB±MS m/z:
259 (M+Na)+. FAB±HRMS m/z: calcd for
C14H20O3Na: 259.1310; found: 259.1329.
2926, 2899, 2864, 1512, 1248, 1101, 1032, 993. 1H NMR
(300 MHz, CDCl3) d: 0.92 (3H, d, J=6.7 Hz, 10-CH3),
1.09, 1.14 (both 3H, d, J=6.1 Hz, 1-OCH(CH3)2), 1.40
(1H, m, H-11), 1.60 (1H, m, H-11), 1.96 (2H, m, H-4),
2.77 (1H, m, H-10), 3.30 (2H, m, H-12), 3.73 (3H, s,
CH2PhOCH3), 3.89±3.97 (1H, m, 1-OCH(CH3)2), 4.28,
4.34 (both 1H, d, J=11.6 Hz, CH2PhOCH3), 4.41 (1H,
m, H-5), 5.04 (1H, s, H-1), 5.13 (1H, t, J=10.4 Hz, H-9),
5.66 (2H, m, H-2, 6), 5.90 (2H, m, H-3, 8), 6.50 (1H, dd,
J=15.3, 10.7 Hz, H-7), 6.79, 7.17 (both 2H, d, J=8.5
Hz, CH2PhOCH3). FAB±MS m/z: 409 (M+Na)+.
FAB±HRMS m/z: calcd for C24H34O4Na: 409.2555;
Preparation of 19c and 19d. A solution of 17c (3.6 mg,
0.016 mmol) in dry CH2Cl2 (0.43 mL) was treated with
Dess±Martin periodinane (20.2 mg, 0.048 mmol) at rt

2660
N. Murakami et al. / Bioorg. Med. Chem. 8 (2000) 2651±2661
for 30 min. Work up in the same manner as preparation
for 18a gave 18c. A solution of n-BuLi (1.54 M in n-
hexane, 0.065 mL, 0.043 mmol) was added to a solution
of DMSO (0.18 mL) in dry toluene (0.5 mL) at rt, then
the whole was stirred for 45 min. A solution of 18c
(3.6 mg, 0.016 mmol) and 10 (16 mg, 0.026 mmol) in dry
toluene (0.5 mL) was added to the solution of dimesyl-
carbanion at 78 ꢀC, then the whole was stirred warm-
ing from 60 ꢀC to 0 ꢀC overnight. Work up in the same
manner as preparation for 19a gave a product, which
was puri®ed by column chromatography (SiO2 2 g, n-
hexane:EtOAc=4:1) to furnish 19c (5.9 mg, 66%). The
same procedure as preparation for 19c was conducted
using 17d (4.0 mg, 0.017 mmol) giving a product, which
was puri®ed by column chromatography (SiO2 4 g, n-
hexane:EtOAc=3:1) to furnish 19d (6.2 mg, 65%).
Compound 19c: colorless oil; [a]D +59.9 ꢀ (c=0.25,
CHCl3, 20ꢀC). IR nmax (KBr) cm 1: 3501, 2963, 2928,
solution of the ketone in dry THF (0.78 mL) was added
HF:pyridine (5:1) (0.49 mL) at 0 ꢀC, then the whole was
stirred at rt for 80 h. Work up in the same manner as
preparation for 12 gave a product, which was puri®ed
by HPLC (column: COSMOSIL 5SL (10 mm i.d.
Â250 mm), mobile phase: n-hexane:EtOAc=2:1, detec-
tion: UV (l=250 nm), ¯ow rate: 3.0 mL/min) to furnish
8-deethylcallystatin A (21, 2.2 mg, 85%).26 The same
procedure as preparation for 21 was conducted using
19d (6.0 mg, 0.011 mmol) giving a product, which was
puri®ed by HPLC (n-hexane:EtOAc=2:1) to furnish 10-
demethylcallystatin A (22, 3.2 mg, 80%).26 8-Deethyl-
callystatin A (21): colorless oil; [a]D 85.7 ꢀ (c=0.22,
MeOH, 27 ꢀC). UV lmax (MeOH) nm (e): 231 (26700),
289 (3070). CD (MeOH) nm (Áe): 331 (0), 300 ( 16.1),
268 (0), 247 (+31.7), 228 (0), 220 ( 7.1), 210 ( 3.7). IR
nmax (KBr) cm 1: 3503, 2957, 2928, 2855, 1732, 1715,
1
1458, 1381, 1258, 1032. H NMR (500 MHz, CDCl3) d:
1
1725, 1460, 1383, 1256, 1053, 837. H NMR (500MHz,
0.85 (3H, t, J=6.3 Hz, H-22), 0.88 (3H, d, J=6.7 Hz,
20-CH3), 0.99 (3H, d, J=6.7 Hz, 10-CH3), 1.10 (1H, m,
H-21), 1.11 (3H, d, J=7.3 Hz, 18-CH3), 1.14 (3H, d,
J=6.7 Hz, 16-CH3), 1.16±1.46 (2H, m, H-20, 21), 1.83
(3H, s, 14-CH3), 2.10 (2H, t, J=6.7 Hz, H-11), 2.47 (2H,
m, H-4), 2.70 (1H, m, H-10), 2.85 (1H, dq, J=7.3,
4.5 Hz, H-18), 3.57 (1H, dd, J=6.7, 4.5 Hz, H-19), 3.65
(1H, dq, J=10.2, 6.7 Hz, H-16), 4.97 (1H, ddd, J=10.5,
6.4, 4.4 Hz, H-5), 5.13 (1H, d, J=10.2 Hz, H-15), 5.35
(1H, dd, J=11.1, 10.2 Hz, H-9), 5.59 (1H, dt, J=15.2,
6.7 Hz, H-12), 5.72 (1H, dd, J=15.2, 6.4 Hz, H-6), 5.95
(1H, t, J=11.1 Hz, H-8), 6.03 (1H, d, J=15.2 Hz, H-
13), 6.06 (1H, dt, J=9.6, 1.7 Hz, H-2), 6.62 (1H, dd,
J=15.2, 11.1 Hz, H-7), 6.89 (1H, ddd, J=9.6, 4.4,
3.7 Hz, H-3). 13C NMR (125 MHz, CDCl3) dC: 10.9 (q,
C-22), 11.2 (q, 18-CH3), 13.1 (q, 14-CH3), 14.2 (q, 20-
CH3), 16.1 (q, 16-CH3), 20.6 (q, 10-CH3), 25.8 (t, C-21),
29.9 (t, C-4), 32.8 (d, C-10), 36.7 (d, C-20), 40.5 (t, C-
11), 45.7 (d, C-16), 45.8 (d, C-18), 74.5 (d, C-19), 77.8
(d, C-5), 121.7 (d, C-2), 125.9 (d, C-6), 127.5 (d, C-12),
128.5 (d, C-15), 128.7 (d, C-9), 128.9 (d, C-7), 135.5 (d,
C-13), 136.2 (s, C-14), 140.3 (d, C-8), 144.6 (d, C-3),
163.9 (s, C-1), 216.4 (s, C-17). FAB±MS m/z: 429
(M+H)+. FAB±HRMS m/z: calcd for C27H41O4:
429.3005; found: 429.2991. 10-Demethylcallystatin A
(22): colorless oil; [a]D 186.2ꢀ (c=0.37, MeOH, 21 ꢀC).
UV lmax (MeOH) nm (e): 243 (36900), 291 (3600). CD
CDCl3) d: 0.07, 0.08 (both 3H, s, 19-OSi(CH3)2C(CH3)3),
0.83 (3H, t, J=7.3 Hz, H-22), 0.85 (3H, d, J=7.3 Hz,
20-CH3), 0.87 (3H, d, J=7.3 Hz, 18-CH3), 0.97 (9H, s,
19-OSi(CH3)2C(CH3)3), 0.99 (3H, d, J=6.7 Hz, 16-
CH3), 1.03 (3H, d, J=6.1 Hz, 10-CH3), 0.95±1.15 (3H,
m, H-20, H-21), 1.54 (1H, m, H-18), 1.72 (3H, s, 14-
CH3), 2.09 (2H, m, H-11), 2.45 (2H, m, H-4), 2.58 (1H,
m, H-16), 2.69 (1H, m, H-10), 3.36 (1H, br d, J=9.2 Hz,
H-19), 3.63 (1H, m, H-17), 4.97 (1H, ddd, J=9.1, 6.7,
6.4 Hz, H-5), 5.07 (1H, d, J=10.4 Hz, H-15), 5.37 (1H,
dd, J=11.0, 10.4 Hz, H-9), 5.49 (1H, dt, J=15.9,
7.3 Hz, H-12), 5.72 (1H, dd, J=15.3, 6.7 Hz, H-6), 5.94
(1H, t, J=11.0 Hz, H-8), 5.99 (1H, d, J=15.9 Hz, H-
13), 6.01 (1H, d, J=9.8 Hz, H-2), 6.63 (1H, dd, J=15.3,
11.0 Hz, H-7), 6.89 (1H, ddd, J=9.8, 4.9, 3.7 Hz, H-3).
FAB±MS m/z: 567 (M+Na)+. FAB±HRMS m/z: calcd
for C33H56O4SiNa: 567.3846; found: 567.3836. Com-
pound 19d: colorless oil; [a]D +14.9 ꢀ (c=0.84, CHCl3,
21 ꢀC). IR nmax (KBr) cm 1: 3511, 2959, 2930, 2851,
1726, 1462, 1383, 1252, 1055, 1020, 964. 1H NMR
(500 MHz, CDCl3) d: 0.07, 0.08 (both 3H, s, 19-
OSi(CH3)2C(CH3)3), 0.82 (3H, d, J=6.7 Hz, 20-CH3),
0.83 (3H, d, J=6.7 Hz, 18-CH3), 0.87 (3H, t, J=6.7 Hz,
H-22), 0.91 (9H, s, 19-OSi (CH3)2C(CH3)3), 1.03 (3H, d,
J=6.7 Hz, 16-CH3), 1.05 (3H, t, J=7.3 Hz, 8-CH2CH3),
1.09 (1H, m, H-21), 1.54 (2H, m, H-20, H-21), 1.73 (3H,
s, 14-CH3), 1.75 (1H, m, H-18), 2.15±2.31 (6H, m, 8-
CH2CH3, H-10, H-11), 2.47 (2H, m, H-4), 2.57 (1H, m,
H-16), 3.37 (1H, br d, J=9.2 Hz, H-19), 3.64 (1H, dd,
J=3.7, 4.3 Hz, H-17), 5.00 (1H, ddd, J=7.3, 7.3, 6.7 Hz,
H-5), 5.09 (1H, d, J=9.8 Hz, H-15), 5.47 (1H, t,
J=7.3 Hz, H-9), 5.55 (1H, dt, J=15.9, 6.7 Hz, H-12),
5.77 (1H, dd, J=15.9, 6.7 Hz, H-6), 6.04 (1H, d,
J=15.9 Hz, H-13), 6.06 (1H, d, J=9.8 Hz, H-2), 6.66
(1H, d, J=15.9 Hz, H-7), 6.89 (1H, dt, J=9.8, 4.9 Hz,
H-3). FAB±MS m/z: 559 (M+H)+. FAB±HRMS m/z:
calcd for C34H59O4Si: 559.4183; found: 559.4185.
(MeOH) nm (Áe): 335 ( 0.2), 300 ( 23.2), 266 (0), 246
1
(+19.2), 220 (+2.8), 210 (+6.1). IR nmax (KBr) cm
3497, 2965, 2932, 2876, 1707, 1456, 1383, 1244, 1055,
:
1
1022, 965. H NMR (500 MHz, CDCl3) d: 0.85 (3H, t,
J=7.4 Hz, H-22), 0.90 (3H, d, J=6.7 Hz, 20-CH3), 1.05
(3H, t, J=7.4 Hz, 8-CH2CH3), 1.07 (1H, m, H-21), 1.12
(3H, d, J=7.1 Hz, 18-CH3), 1.15 (3H, d, J=6.7 Hz, 16-
CH3), 1.31±1.44 (2H, m, H-20, H-21), 1.83 (3H, s, 14-
CH3), 2.20 (2H, t, J=6.7 Hz, H-11), 2.26 (4H, m, 8-
CH2CH3, H-10), 2.47 (2H, m, H-4), 2.86 (1H, dq,
J=4.3, 7.1 Hz, H-18), 3.57 (1H, m, H-19), 3.65 (1H, dq,
J=10.2, 6.7 Hz, H-16), 5.00 (1H, ddd, J=7.4, 7.4,
6.7 Hz, H-5), 5.16 (1H, d, J=10.2 Hz, H-15), 5.45 (1H,
t, J=7.6 Hz, H-9), 5.66 (1H, dt, J=15.7, 6.7 Hz, H-12),
5.77 (1H, dd, J=6.7, 16.0 Hz, H-6), 6.05 (1H, d,
J=15.7 Hz, H-13), 6.07 (1H, d, J=9.8 Hz, H-2), 6.65
(1H, d, J=16.0 Hz, H-7), 6.90 (1H, dt, J=9.8, 4.3 Hz,
H-3). 13C NMR (125 MHz, CDCl3) dC: 10.9 (q, C-22),
Preparation of 8-deethylcallystatin A (21) and 10-deme-
thylcallystatin A (22). A solution of 19c (3.1 mg,
0.006 mmol) in dry CH2Cl2 (1.0 mL) was treated with
Dess±Martin periodinane (7.3 mg, 0.17 mmol) at rt for
1.5 h. The reaction mixture was worked up in the same
manner as preparation for 20 to give a ketone. To a

N. Murakami et al. / Bioorg. Med. Chem. 8 (2000) 2651±2661
2661
11.1 (q, 18-CH3), 13.0 (q, 14-CH3), 13.3 (q, 8-CH2CH3),
14.3 (q, 20-CH3), 16.3 (q, 16-CH3), 25.8 (t, C-21), 26.4
(t, 8-CH2CH3), 27.5 (t, C-11), 30.1 (t, C-4), 32.9 (t, C-
10), 36.7 (d, C-20), 45.7 (d, C-18), 45.8 (d, C-16), 74.5
(d, C-19), 78.7 (d, C-5), 121.8 (d, C-2), 125.0 (d, C-6),
128.4 (d, C-15), 128.9 (d, C-12), 129.5 (d, C-7), 130.4 (s,
C-8), 134.5 (d, C-13), 136.1 (s, C-14), 136.9 (d, C-9),
144.5 (d, C-3), 164.0 (s, C-1), 216.4 (s, C-17). FAB±MS
m/z: 443 (M+H)+. FAB±HRMS m/z: calcd for
C28H43O4: 443.3161; found: 443.3154.
5. Komiyama, K.; Okada, K.; Oka, H.; Tomisaka, S.;
Miyano, T.; Funayama, S.; Umezawa, I. J. Antibiot. 1985, 38,
220.
6. Hayakawa, Y.; Adachi, K.; Komeshima, N. J. Antibiot.
1987, 40, 1349.
7. Hayakawa, Y.; Sohda, K.; Seto, H. J. Antibiot. 1996, 49,
980.
8. Kudo, N.; Wol€, B.; Sekimoto, T.; Schreiner, E. P.;
Yoneda, Y.; Yanagida, M.; Horinouchi, S.; Yoshida, M. Exp.
Cell Res. 1998, 242, 540.
9. Kobayashi, M.; Wang, W.; Tsutsui, Y.; Sugimoto, M.;
Murakami, N. Tetrahedron Lett. 1998, 39, 8291.
10. Murakami, N.; Sugimoto, M.; Nakajima, T.; Higuchi, K.;
Aoki, S.; Yoshida, M.; Kudo, N.; Kobayashi, M. Abstracts of
Papers, 41st Symposium on the Chemistry of Natural Products;
p. 229, Nagoya, October 1999; Chem. Abstr. 1999, 776311.
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Bioassay. Human epidermoid carcinoma KB cells were
cultured in RPMI 1640 medium with 0.58 mg/mL of
glutamine, 50 mg/mL of kanamycin sulfate, supple-
mented with 10% fetal bovine serum. Cytotoxic activity
was measured by means of MTT colorimetric assay
performed in 96-well plates. Equal numbers of cells
(2Â104) were inoculated into each well with 100 mL of
the culture medium, then a 100 mL solution of each tes-
ted compound was added to each well. After 72 h incu-
bation under a 5% CO2 atmosphere at 37 ꢀC, 25 mL of
MTT solution (2 mg/mL in PBS) was added to each well
and incubated for a further 3 h. The medium was
removed by aspiration, then the resulting formazan was
dissolved with 200 mL of DMSO. The percentage of cell
growth inhibition was calculated from the absorbance
at 540 nm and IC50 value was determined by linear
interpolation from the inhibition curve.
Acknowledgements
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1901.
23. Zhao, A.; Lee, S. H.; Mojena, M.; Jenkins, R. G.; Patrick,
D. R.; Huber, H. E.; Goetz, M. A.; Hensens, O. D.; Zink, D.
L.; Vilella, D.; Dombrowski, A. W.; Lingham, R. B.; Huang,
L. J. Antibiot. 1999, 52, 1086.
The authors are grateful to the Naito Foundation, the
Houansha Foundation, and the Ministry of Education,
Science, Sports, and Culture of Japan for ®nancial sup-
port.
24. Dudley, D. T.; Pang, L.; Decker, S. J.; Bridges, A. J.;
Saltiel, A. R. Proc. Natl. Acad. Sci. USA 1995, 92, 7686.
25. Compound 3a consists of two diastereomers on C-1 in a
ratio of 8:1. Since all compounds with 1-acetal and/or 8-ene
functions except for the four analogues (12, 20, 21 and 22)
contained stereoisomers, the NMR data are assigned with
respect to each major isomer in signi®cant proportion. Num-
bering used for assignment of NMR data is in accordance with
that of callystatin A (1).
References and Notes
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K.; Aoki, S.; Kobayashi, M. Tetrahedron Lett. 1997, 38, 5533.
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26. The yields were determined by taking into account that
minor 8-E isomers given by Still±Wittig condensation were
removed with ®nal HPLC separation.