Synthesis and biological activities of the tris-oxazole macrolactone analogs of mycalolides

Article abstract of DOI:10.1016/j.tet.2012.08.012

Mycalolides are tris-oxazole macrolides isolated from the marine sponge Mycale sp., which shows cytotoxic, antifungal, and actin-depolymerizing activities. To develop an efficient synthetic route of mycalolides and to evaluate its functional mechanism of

Full text of DOI:10.1016/j.tet.2012.08.012

Tetrahedron 68 (2012) 8753e8760
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Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis and biological activities of the tris-oxazole macrolactone analogs
of mycalolides
*
Masaki Kita , Hirotaka Oka, Akihiro Usui, Tomoya Ishitsuka, Yuzo Mogi, Hidekazu Watanabe,
*
Hideo Kigoshi
Department of Chemistry, Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8571, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 July 2012
Received in revised form 4 August 2012
Accepted 5 August 2012
Available online 15 August 2012
Mycalolides are tris-oxazole macrolides isolated from the marine sponge Mycale sp., which shows cy-
totoxic, antifungal, and actin-depolymerizing activities. To develop an efficient synthetic route of my-
calolides and to evaluate its functional mechanism of biological activities, tris-oxazole macrolactone
analogs of mycalolides were synthesized through the use of ring-closing metathesis (RCM). The pres-
ence/absence of protecting groups at C3, solvent polarity, and reaction temperature significantly affected
the stereoselectivity of RCM (E/Z¼2.5/1.0e1.0/2.5). The 19E- and 19Z-stereoisomers both exhibited
moderate cytotoxicity against tumor cells, but neither showed significant actin-depolymerizing prop-
erties or antimycotic activity against pathogenic fungi. Thus, both the side-chain (actin-binding) moiety
and the macrolactone moiety were suggested to be essential for the potent biological activities of the
parent molecules.
Keywords:
Natural products
Structureeactivity relationships
Ring-closing metathesis
Actin depolymerization
Antimycotic activity
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
intercalate into the hydrophobic cleft between subdomains 1 and 3
of actin. Furthermore, aplyronine A (4), which shows potent anti-
Various actin-depolymerizing agents have been found in marine
invertebrates, and some show extremely potent cytotoxicity. They
are considered to be useful for the development of novel pharma-
cological tools for analyzing actin-mediated cell functions, such as
muscle contraction, cell motility, and cytokinesis. Among them,
mycalolide B (1) is a unique tris-oxazole macrolide that was iso-
lated from the marine sponge Mycale sp. (Fig. 1),1 which exhibits
various biological activities, such as cytotoxicity against B16 mel-
anoma cells, inhibition of actomyosin Mg2þ-ATPase,2 and anti-
mycotic activity against several pathogenic fungi.3 It has been
suggested that several antifungal compounds might serve as
tumor activities against several cancers, also possesses an actin-
binding side-chain part similar to that of mycalolides.11e13 Photo-
affinity labeling experiments14 and X-ray crystallographic analy-
sis15 have also established that the interactions of
actineaplyronines are closely related to those of mycalolides.
Meanwhile, the macrocyclic functional groups of aplyronines, i.e.,
the C7 trimethylserine and C9 hydroxyl groups, have been shown to
be essential for its potent cytotoxicity.16 Thus, we expected that the
structurally unique macrolactone moieties in mycalolides and re-
lated tris-oxazole macrolides could serve as a pharmacophore that
enable them to specifically interact with biomacromolecules to
exhibit various biological activities.
a
chemical defense against microbial attack for marine in-
vertebrates that lack cellular immune systems.4
Due to their complexity and unique biological activities, there
have been several synthetic studies on tris-oxazole macrolides.17
Recently, total syntheses of mycalolide A18 and ulapualide A19
have been described. We previously synthesized the C22eC35
Many tris-oxazole macrolides that are structurally related to
mycalolides have been isolated, such as ulapualides,5 halichon-
dramides,6 jaspisamides,7 and kabiramides;8 and all of these ex-
hibit potent actin-depolymerizing properties. X-ray analyses of the
actinekabiramide C,9 actinejaspisamide A,9 and actineulapualide
A complexes10 revealed that their side-chain parts (C25eC35)
part
2 of mycalolide B, which showed significant actin-
depolymerizing activity corresponding to that of 1, but no cyto-
toxicity.20 However, mycalolide derivatives that consist of the tris-
oxazole macrolactone part alone have not yet been synthesized and
characterized. It was expected that ulapualides and halichon-
dramide could incorporate several metal ions (i.e., Fe, Co, Cu) to
form coordination complexes, in which the oxazole N atoms and
the O atoms of C3 and C7 oxygenated functional groups may
* Corresponding authors. Tel.: þ81 29 853 4526; fax: þ81 29 853 4313(M.K.); tel./
fax: þ81 29 853 4313(H.K.); e-mail addresses: mkita@chem.tsukuba.ac.jp (M. Kita),
kigoshi@chem.tsukuba.ac.jp (H. Kigoshi).
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2012.08.012

8754
M. Kita et al. / Tetrahedron 68 (2012) 8753e8760
Fig. 1. Structures of mycalolide B and its derivatives.
coordinate to metals.21 Thus, it is possible that the lactone moiety of
mycalolides and related tris-oxazole macrolides might serve as an
ionophore, which could contribute to their unique bioactivities.
Here we describe the synthesis of tris-oxazole macrolactone ana-
logs 3 through the use of ring-closing metathesis (RCM) along with
their biological activities.
2.2. Synthesis of the tris-oxazole macrolactone analogs of
mycalolides
Based on the finding that olefin metathesis is a useful method
for connecting the C19eC20 double bonds in mycalolide analogs,23
we considered that the assembly of two fragments via Ni/Cr-
mediated NozakieHiyamaeKishi coupling24 at C6eC7 and RCM
at C19 could efficiently afford 3. The synthesis started with syn-
homoallylic alcohol 6, which was prepared from methyl (S)-(þ)-3-
hydroxy-2-methylpropionate (5)25 (Scheme 1). Methylation of
compound 6 by methyl trifluoromethanesulfonate (MeOTf) (90%)
and removal of the tert-butyldiphenylsilyl (TBDPS) group by tetra-
n-butylammonium fluoride (TBAF) gave primary alcohol 8 (96%).
Hydrolysis of methyl ester in 923b with LiOH afforded carboxylic
acid 10 (80%), which was condensed with 8 by the Yamaguchi
procedure to afford ester 11 in 92% yield. Fragment coupling be-
tween 11 and aldehyde 1223b by a Ni/Cr-mediated reaction (73%)
was followed by oxidation of the C7 allylic alcohol with
DesseMartin periodinane (DMP) to afford the RCM precursor 14
(94%). Alternatively, removal of the TBDPS group of 14 by TBAF
with acetic acid (AcOH) gave another RCM precursor 15 in 72%
yield.
2. Results and discussion
2.1. Molecular modeling studies
First, we estimated the three-dimensional structure of the
macrolactone part of 1 by using a molecular modeling method. A
conformational analysis and density functional theory (DFT) cal-
culation of tris-oxazole macrolactone analogs 3 revealed that the
19E isomer is lower in energy than the 19Z isomer by 5.4 kJ/mol at
the B3LYP/6-31G(d) level of theory. (Fig. 2)22 This difference was
almost identical to that for (E)- and (Z)-2-(1-butenyl)oxazoles
(6.2 kJ/mol). Conjugated tris-oxazoles, the C7 conjugated ketones,
and the C19 olefin moieties in both (E)- and (Z)-3 have an almost
planar orientation, which suggested that their conformations are
considerably fixed. The distances between the C3 hydroxyl H atom
With the key intermediates 14 and 15 in hand, RCM reactions
were examined (Table 1). Despite the calculation results that 19E-
macrolactone 3 was lower in energy than its 19Z-isomer, treat-
ment of 14 with 30 mol % of second generation HoveydaeGrubbs
catalyst26 in refluxing toluene (1 mM) gave tris-oxazole lactone 16
as a separable 1:1.9 mixture of E- and Z-isomers in 61% yield
(entry 1). Similarly, the reaction at 40 ꢀC in hexane preferred the Z-
ꢀ
and two oxazole N atoms of (E)-3 were less than 3.1 A, which
strongly indicated the presence of intramolecular hydrogen bond-
ing. In contrast, the C3 hydroxyl group in (Z)-3 did not interact with
the tris-oxazole moiety. These significant differences in the ster-
eostructures of E- and Z-isomers led us to synthesize both isomers
and compare their biological activities.

M. Kita et al. / Tetrahedron 68 (2012) 8753e8760
8755
expected that the RCM precursor 15 would have a specific confor-
mation to prefer the formation of the E-isomer, due to intra-
molecular hydrogen bonding between the hydroxyl H atom and
oxazole N atom(s), which might enable terminal olefins to move
toward each other.
2.3. Biological activities
While the E- and Z-stereoisomers of tris-oxazole macrolactone
analogs 3 both exhibited moderate cytotoxicity against human
epithelial carcinoma HeLa S3 cells, they were approximately 100
times less cytotoxic than mycalolide B (1) (Table 2). Furthermore,
they exhibited no significant actin-depolymerizing effects. In ad-
dition, neither (E)- nor (Z)-3 exhibited in vitro antimycotic activity
against several pathogenic fungi (Aspergillus fumigatus, Candida
albicans, and Trichophyton mentagrophytes) or normal fungi (Mucor
hiemalis and Rhizopus nigricans) at 30 mg/mL. Thus, both the side-
chain and the macrolactone moieties were suggested to be essen-
tial for the potent cytotoxicity or antimycotic activity of the parent
molecules.30
3. Conclusions
In conclusion, we concisely synthesized tris-oxazole macro-
lactone analogs of mycalolides by using NozakieHiyamaeKishi
coupling at C6eC7, esterification, and RCM at C19 as key steps. We
established that both the side-chain (actin-binding) moiety and the
macrolactone moiety were essential for the potent biological ac-
tivities of the parent molecules. Further studies on the structur-
eeactivity relationships for mycalolides and related actin-targeting
natural products as well as their mechanisms of action are currently
underway.
4. Experimental section
4.1. General
All chemicals were obtained commercially unless otherwise
noted. Organic solvents and reagents for moisture-sensitive re-
actions were distilled by the standard procedure. Anhydrous
CH2Cl2, tetrahydrofuran (THF), and N,N-dimethylformamide (DMF)
were obtained commercially. Column chromatography was per-
Fig. 2. Optimized structures and relative energies of 3 at the B3LYP/6-31G(d) level of
theory. Values in (E)-3 represent the calculated distances between the C3 hydroxyl H
atom and the three oxazole N atoms (A).
ꢀ
formed using silica gel BW-820MH or FL60D (75e200 or 45e75
Fuji Silysia Co., Aichi, Japan) or a Yamazen preparative silica gel
(40 m). All moisture-sensitive reactions were performed under an
mm,
m
atmosphere of argon or nitrogen, and the starting materials were
azeotropically dried with benzene before use. Merck precoated
silica gel 60 F254 plates were used for thin layer chromatography
(TLC).
isomer (E/Z¼1.0/2.5) (entry 2).27 With the use of benzotrifluoride
as a solvent,28 the RCM reaction was completed in 1 h, and the
yield of the E-isomer was improved (entry 3). Interestingly, the E/Z
product ratio was reversed at lower temperature in the same
solvent (entry 4) or in ethyl acetate (entry 5). Furthermore,
refluxing conditions in dichloromethane gave the best results
(82%, E/Z¼1.8/1.0) (entry 6).29 These results suggested that both
the solvent polarity and reaction temperature significantly af-
fected the stereoselectivity of the RCM reaction of 14, and that the
formation of C]C bonds in 16 would take place under kinetic
control.23b
Finally, removal of the TBDPS groups of the stereoisomers (E)-
and (Z)-16 by TBAF with AcOH afforded tris-oxazole macrolactones
(E)- and (Z)-3 in respective yields of 87% and 91% (Scheme 2).
For comparison, we also examined the RCM reaction of C3 hy-
droxyl analog 15 (Scheme 3). While the reaction was slower than
that of silyl ether 14 (see Table 1, entry 6), refluxing conditions in
dichloromethane (1 mM) gave macrolactone 3 in 72% yield, and the
E-selectivity was slightly improved (E/Z¼2.5/1.0). Thus, it is
4.2. Spectroscopic analysis
1H and 13C nuclear magnetic resonance (NMR) spectra were
recorded on a Bruker Biospin AVANCE 600 spectrometer (600 MHz
for 1H and 150 MHz for 13C), or a JEOL EX-270 spectrometer
(270 MHz for 1H). Chemical shifts were reported in parts per mil-
lion (ppm) with coupling constants (J) in hertz relative to the sol-
vent peaks, dH 7.26 (residual CHCl3) and dC 77.0 for CDCl3,
respectively. Optical rotations were measured with a JASCO DIP-
1000 polarimeter using the sodium D line. Infrared (IR) spectra
were recorded on a JASCO FT/IR-230 spectrometer. High-resolution
electrospray ionization mass spectra (HR-ESIMS) were measured
on a QSTAR Pulsar i spectrometer (Applied Biosystems) or an
AccuTOF CS spectrometer (JEOL).

8756
M. Kita et al. / Tetrahedron 68 (2012) 8753e8760
Scheme 1. Synthesis of the RCM precursors 14 and 15. Reagents and conditions: (a) MeOTf, 2,6-di-tert-butylpyridine, CH2Cl2, rt, 90%; (b) TBAF, THF, rt, 96%; (c) LiOH, THF/H2O, 40 ꢀC,
80%; (d) 2,4,6-Cl3C6H2COCl, Et3N, THF, rt, then DMAP, benzene, rt, 92%; (e) CrCl2, NiCl2, THF/DMF (3:1), rt, 73%; (f) DesseMartin periodinane, pyridine, CH2Cl2, 0 ꢀC, 94%; (g) TBAF,
AcOH, THF, rt, 72%.
4.3. Synthesis and spectroscopic data of mycalolide
derivatives
Table 1
Ring-closing metathesis of 14
4.3.1. Methyl ether 7. To a stirred solution of (ꢁ)-secondary alcohol
6 (787 mg, 2.14 mmol, >99%ee, syn/anti¼93/7) in dry CH2Cl2
(4.8 mL) were added methyl trifluoromethanesulfonate (0.72 mL,
6.4 mmol) and 2,6-di-tert-butylpyridine (1.5 mL, 6.4 mmol) at 0 ꢀC
under a nitrogen atmosphere. After being stirred for 20 h at room
temperature, the reaction mixture was quenched with satd sodium
bicarbonate aq (15 mL) at 0 ꢀC, stirred for 45 min at room tem-
perature, and extracted with CH2Cl2 (5 mLꢂ4). The combined ex-
tracts were washed with water and brine, dried with Na2SO4, and
concentrated. The crude material was purified with a Yamazen
preparative SiO2 column (30 g, hexane/EtOAc¼100/0 to 95/5) to
give methyl ether 7 (737 mg, 90%, syn/anti¼93/7) as a colorless oil.
Compound 7: Rf 0.41 (hexane/EtOAc¼9:1); ½a D25
þ9.0 (c 1.09,
ꢃ
Entry Reaction conditionsa
Yield of 16 (%) E/Z ratio Yield of dimer (%)
CHCl3); 1H NMR (600 MHz, CDCl3)
d 7.72e7.65 (m, 4H), 7.46e7.36
1
2
3
4
5
6
Toluene, reflux, 1.5 h 61
1.0/1.9
1.0/2.5
1.0/1.3
1.3/1.0
1.2/1.0
1.8/1.0
5
9
7
6
9
5
(m, 6H), 5.81 (ddt, J¼17.2, 10.2, 7.0 Hz, 1H), 5.08 (d, J¼17.2 Hz, 1H),
5.05 (d, J¼10.2 Hz, 1H), 3.69 (dd, J¼10.0, 7.2 Hz, 1H), 3.54 (dd,
J¼10.0, 6.1 Hz, 1H), 3.45 (dt, J¼3.9, 6.8 Hz, 1H), 3.36 (s, 3H), 2.36
(dddt, J¼13.9, 7.0, 6.8, 1.2 Hz, 1H), 2.23 (dddt, J¼13.9, 7.0, 6.8, 1.0 Hz,
1H), 1.87 (dddq, J¼7.8, 6.1, 3.9, 7.0 Hz, 1H), 1.08 (s, 9H), 0.89 (d,
Hexane, 40 ꢀC, 24 h
C6H5CF3, reflux, 1 h
C6H5CF3, 40 ꢀC, 24 h
EtOAc, 40 ꢀC, 9 h
73
69
77
78
82
CH2Cl2, reflux, 9 h
a
Concentration¼1 mM. Second HoveydaeGrubbs catalyst (30 mol %) was used.
J¼7.0 Hz, 3H); 13C NMR (150 MHz, CDCl3)
d 135.6 (d, 4C), 135.5 (d),

M. Kita et al. / Tetrahedron 68 (2012) 8753e8760
8757
Scheme 2. Synthesis of tris-oxazole macrolactones 3. Reagents and conditions: (a) TBAF, AcOH, THF, rt, 87% for (E)-3 and 91% for (Z)-3.
added 1 M lithium hydroxide aq (3.6 mL, 3.6 mmol) at 0 ꢀC. After
being stirred for 23 h at 40 ꢀC, the reaction mixture was acidi-
fied with 1 M HCl aq (5 mL) and extracted with Et2O (5 mLꢂ4).
The combined extracts were washed with brine, dried with
Na2SO4, and concentrated. The crude material was purified with
a SiO2 column (10 g, hexane/EtOAc¼9/1 to 5/1) to give carboxylic
acid 10 (234 mg, 80%, E/Z¼6.7/1) as a colorless oil. 10: Rf 0.42
Scheme 3. Ring-closing metathesis of 15.
(hexane/EtOAc¼2:1);
(270 MHz, CDCl3)
½ ꢃ
a 2D5
þ33.7 (c 1.22, CHCl3); 1H NMR
Table 2
Biological activities of mycalolide analogs
d
7.75e7.64 (m, 4H), 7.50e7.35 (m, 6H), 6.38
(dt, J¼14.4, 7.6 Hz, 1H), 5.98 (d, J¼14.4 Hz, 1H), 4.18 (tt, J¼5.9,
5.9 Hz, 1H), 2.49 (dd, J¼6.7, 6.5 Hz, 2H), 2.30e2.16 (m, 2H), 1.06
(s, 9H). COOH signal was not observed; 13C NMR (150 MHz,
Compound
Cytotoxicity (HeLa S3)
IC50 g/mL)
Actin-depolymerizing
(
m
activity EC50 (m
M)a
Mycalolide B (1)
2
(E)-3
(Z)-3
0.020
>10b
2.4
1.4b
2.7b
>30
>30
CDCl3)
d 177.1 (s), 141.5 (d), 135.9 (d, 4C), 133.4 (s, 2C), 129.9 (d),
129.8 (d), 127.7 (d, 4C), 78.2 (d), 68.8 (d), 43.1 (t), 41.2 (t), 26.9
(q, 3C), 19.3 (s); IR (CHCl3) 3073, 3054, 3010, 2961, 2933, 2897,
2860, 1712, 1606, 1589, 1472, 1428, 1363, 1221, 1111, 951, 822,
1.9
a
Activity was monitored by measuring the fluorescence intensity of pyrenyl ac-
tin. Values indicate the concentrations required to depolymerize F-actin (3 mM) to
786, 704 cmꢁ1
;
HRMS (ESI) m/z 517.0701 (calcd for
50% of its control amplitude.
C22H27INaO3Si [MþNa]þ,
D
þ2.9 mmu).
b
See Ref. 20.
4.3.4. Ester 11. To a stirred solution of carboxylic acid 10 (234 mg,
134.0 (s), 133.9 (s), 129.5 (d, 2C), 127.6 (d, 4C), 116.5 (t), 80.6 (d), 65.9
(t), 57.9 (q), 38.7 (d), 35.6 (t), 26.9 (q, 3C),19.3 (s),10.9 (q); IR (CHCl3)
3073, 3009, 2962, 2932, 2859, 1640, 1589, 1472, 1428, 1389, 1362,
1217, 1112, 1088, 998, 918, 824, 705 cmꢁ1; HRMS (ESI) m/z 405.2207
0.473 mmol) in dry THF (8.6 mL) under a nitrogen atmosphere were
added triethylamine (72
mL, 0.52 mmol) and 2,4,6-trichlorobenzoyl
chloride (81 L, 0.52 mmol). After the mixture was stirred for 5 h at
m
room temperature, the precipitate was removed by centrifugation.
After the precipitate was washed with dry THF (4 mL), the com-
bined filtrates and washings were concentrated in vacuo and dis-
solved in dry benzene (11.8 mL). To the activated ester solution was
added a solution of primary alcohol 8 (75.3 mg, 0.522 mmol, syn/
(calcd for C24H34NaO2Si [MþNa]þ,
D
ꢁ1.9 mmu).
4.3.2. Primary alcohol 8. To
a
stirred solution of 7 (1.01 g,
2.64 mmol, syn/anti¼93/7) in dry THF (18 mL) was added a 1 M
solution of tetra-n-butylammonium fluoride in THF (3.2 mL,
3.2 mmol) at 0 ꢀC under a nitrogen atmosphere. After being stirred
for 4 h at room temperature, the reaction mixture was quenched
with satd NH4Cl aq (10 mL), and extracted with Et2O (10 mLꢂ3).
The combined extracts were washed with brine, dried with Na2SO4,
and concentrated. The crude material was purified with a SiO2
column (40 g, hexane/Et2O¼9/1 to 1/1) to give primary alcohol 8
(366 mg, 96%, syn/anti¼93/7) as a colorless oil. Compound 8: Rf 0.15
anti¼93/7)
and
N,N-dimethyl-4-aminopyridine
(124 mg,
1.02 mmol) in dry benzene (4 mL) under a nitrogen atmosphere.
After being stirred for 2 h at room temperature, the reaction mix-
ture was quenched with satd sodium bicarbonate aq (10 mL), and
extracted with EtOAc (3 mLꢂ5). The combined extracts were
washed with brine, dried with Na2SO4, and concentrated. The crude
oil was purified with a SiO2 column (FL60D, 9 g, hexane/EtOAc¼49/
1 to 4/1) to give ester 11 (269 mg, 92%, syn/anti¼99/1, E/Z¼6.1/1) as
(hexane/Et2O¼7:3); ½a D25
ꢃ
þ17.2 (c 1.31, CHCl3); 1H NMR (600 MHz,
a colorless oil. Compound 11: Rf 0.60 (hexane/EtOAc¼5:1); ½a D25
ꢃ
CDCl3)
d
5.81 (ddt, J¼17.0, 10.7, 7.2 Hz, 1H), 5.10 (d, J¼17.0 Hz, 1H),
þ26.8 (c 1.31, CHCl3); 1H NMR (600 MHz, CDCl3)
d 7.68e7.64 (m,
5.04 (d, J¼10.7 Hz,1H), 3.65 (dd, J¼10.7, 7.1 Hz,1H), 3.58 (dd, J¼10.7,
4.6 Hz, 1H), 3.38 (s, 3H), 3.36 (dt, J¼3.4, 6.6 Hz, 1H), 2.56 (br s, 1H),
2.38 (dddt, J¼14.0, 7.2, 6.6, 1.3 Hz, 1H), 2.20 (dddt, J¼14.0, 7.2, 6.6,
1.1 Hz, 1H), 1.95 (m, 1H), 0.89 (d, J¼7.1 Hz, 3H); 13C NMR (150 MHz,
4H), 7.46e7.37 (m, 6H), 6.38 (dt, J¼14.5, 7.4 Hz, 1H), 5.95 (d,
J¼14.5 Hz, 1H), 5.76 (ddt, J¼17.2, 10.1, 7.1 Hz, 1H), 5.10 (d, J¼17.2 Hz,
1H), 5.06 (d, J¼10.1 Hz, 1H), 4.20 (tt, J¼5.9, 5.9 Hz, 1H), 3.99 (dd,
J¼10.7, 6.8 Hz, 1H), 3.87 (dd, J¼10.7, 6.8 Hz, 1H), 3.31 (s, 3H), 3.16
(dt, J¼3.7, 6.5 Hz, 1H), 2.45 (dd, J¼6.5, 5.9 Hz, 2H), 2.34 (m, 1H),
2.27e2.15 (m, 3H), 1.90 (m, 1H), 1.04 (s, 9H), 0.87 (d, J¼6.9 Hz, 3H);
CDCl3)
d 135.2 (d), 116.8 (t), 83.8 (d), 66.3 (t), 57.6 (q), 37.0 (d), 34.6
(t), 11.1 (q); IR (CHCl3) 3632, 3484, 3081, 3009, 2979, 2935, 2832,
1641, 1460, 1428, 1380, 1361, 1236, 1192, 1083, 1032, 918, 780, 773,
726 cmꢁ1; HRMS (ESI) m/z 167.1050 (calcd for C8H16NaO2 [MþNa]þ,
13C NMR (150 MHz, CDCl3)
d 170.9 (s), 141.8 (d), 135.9 (d, 2C), 135.8
(d, 2C), 134.9 (d), 133.6 (s), 133.5 (s), 129.8 (d, 2C), 127.7 (d, 2C),127.6
(d, 2C), 117.1 (t), 80.9 (d), 77.8 (d), 69.0 (d), 66.8 (t), 58.0 (q), 43.1 (t),
41.5 (t), 35.4 (d), 35.3 (t), 26.9 (q, 3C), 19.2 (s), 11.0 (q); IR (CHCl3)
3074, 3054, 3010, 2962, 2933, 2897, 2860, 2829, 1729, 1641, 1472,
D
þ0.2 mmu).
4.3.3. Carboxylic acid 10. To a stirred solution of (þ)-methyl ester
9 (302 mg, 0.593 mmol, 99%ee, E/Z¼6.7/1) in THF (3 mL) was
1428, 1363, 1233, 1190, 1105, 998, 952, 920, 822, 735, 704 cmꢁ1
;

8758
M. Kita et al. / Tetrahedron 68 (2012) 8753e8760
HRMS (ESI) m/z 643.1729 (calcd for C30H41INaO4Si [MþNa]þ,
þ1.3 mmu).
D
14.1 (q), 11.0 (q); IR (CHCl3) 3163, 3027, 3010, 2933, 2859, 1729,
1694, 1665, 1637, 1542, 1462, 1428, 1376, 1308, 1111, 978, 943, 919,
823, 703 cmꢁ1; HRMS (ESI) m/z 844.3618 (calcd for C46H55N3NaO9Si
4.3.5. Allylic alcohol 13. To a stirred solution of (ꢁ)-aldehyde 12
(23.3 mg, 70.8 mol) and ester 11 (65.9 mg, 106
[MþNa]þ,
D
þ1.3 mmu).
m
m
mol, E/Z¼6.1/1) in
degassed dry THF/DMF (3:1, 1.3 mL) under an argon atmosphere
was added a 99:1 (w/w) mixture of chromium chloride (II)/nickel
4.3.7. Secondary alcohol 15. To a stirred solution of ketone 14
(22.7 mg, 27.6 mol) in THF (10.2 mL) under a nitrogen atmosphere
at 0 ꢀC were added a 1 M solution of tetra-n-butylammonium
fluoride in THF (0.86 mL, 0.86 mmol) and acetic acid (49 L). After
m
chloride (II) (56.5 mg, CrCl2 455 mmol and NiCl2 4.36 mmol). After
being stirred for 14 h, the reaction mixture was diluted with EtOAc
(4 mL), satd ammonium chloride aq (4 mL), and water (1 mL), and
extracted with EtOAc (4 mLꢂ3). The combined extracts were
washed with brine, dried with Na2SO4, and concentrated. The crude
oil was purified with a SiO2 column (3.1 g, hexane/CHCl3/EtOAc¼1/
1/0, 3/7/0, 0/1/0, 0/4/1 and 0/3/1) to give allylic alcohol 13 (42.7 mg,
73%, 5E-isomer only, ca. 3:2 diastereomer mixture at C7) as a col-
m
being stirred for 18 h at room temperature, the reaction mixture
was diluted with satd sodium bicarbonate aq (10 mL) and extracted
with EtOAc (5 mLꢂ4). The combined extracts were washed with
brine, dried with Na2SO4, and concentrated. The crude oil was
purified with SiO2 columns (FL60D 1.2 g, CHCl3/EtOAc¼4/1 to 1/1;
FL60D 0.4 g, EtOAc) to give secondary alcohol 15 (11.6 mg, 72%) as
orless oil. Compound 13: Rf 0.50 (hexane/acetone¼7:3); ½a D25
ꢃ
þ5.7
a colorless oil. Compound 15: Rf 0.66 (CHCl3/MeOH¼10:1); ½a D25
ꢃ
(c 0.93, CHCl3); 1H NMR (600 MHz, CDCl3)
d
8.32 (s, 0.4H), 8.32 (s,
ꢁ39.7 (c 0.46, CHCl3); 1H NMR (600 MHz, CDCl3)
d 8.34 (s, 1H), 8.33
0.6H), 8.31 (s, 0.6H), 8.30 (s, 0.4H), 7.72e7.61 (m, 5H), 7.44e7.32 (m,
6H), 6.67 (dd, J¼17.7, 11.2 Hz, 1H), 6.34 (d, J¼17.7 Hz, 1H), 5.77 (d,
J¼11.2 Hz, 1H), 5.74 (m, 1H), 5.55 (m, 1H), 5.39 (m, 1H), 5.08 (d,
J¼17.2 Hz, 1H), 5.03 (d, J¼10.2 Hz, 1H), 4.30e4.26 (m, 0.4H), 4.26 (d,
J¼7.9 Hz, 0.6H), 4.25e4.19 (m, 0.6H), 4.17 (d, J¼7.8 Hz, 0.4H), 4.01
(m, 1H), 3.97 (m, 1H), 3.84 (m, 1H), 3.30 (s, 1.2H), 3.30 (s, 1.8H), 3.29
(s, 1.8H), 3.28 (s, 1.2H), 3.16 (m, 1H), 2.51e2.43 (m, 2H), 2.36e2.12
(m, 6H), 1.89 (m, 1H), 1.02 (s, 9H), 0.87 (d, J¼6.8 Hz, 1.2H), 0.86 (d,
J¼6.8 Hz, 1.8H), 0.75 (d, J¼7.1 Hz, 1.2H), 0.65 (d, J¼6.9 Hz, 1.8H); 13C
(s, 1H), 7.71 (s, 1H), 6.97 (dt, J¼15.8, 7.3 Hz, 1H), 6.67 (dd, J¼17.7,
11.5 Hz, 1H), 6.34 (d, J¼17.7 Hz, 1H), 6.29 (d, J¼15.8 Hz, 1H), 5.78 (d,
J¼11.5 Hz, 1H), 5.77 (ddt, J¼17.8, 10.0, 7.3 Hz, 1H), 5.11 (d, J¼17.8 Hz,
1H), 5.06 (d, J¼10.0 Hz, 1H), 4.42 (d, J¼9.6 Hz, 1H), 4.21 (m, 1H), 4.12
(dd, J¼10.9, 7.0 Hz,1H), 4.05 (dd, J¼10.9, 6.5 Hz, 1H), 3.56 (dq, J¼9.6,
7.1 Hz, 1H), 3.35 (s, 3H), 3.34 (m, 1H), 3.23 (dt, J¼3.5, 6.5 Hz, 1H),
3.19 (s, 3H), 2.55 (dd, J¼16.6, 3.1 Hz, 1H), 2.48 (dd, J¼16.6, 9.2 Hz,
1H), 2.52e2.40 (m, 2H), 2.36 (m, 1H), 2.21 (m, 1H), 2.04 (m, 1H),
0.93 (d, J¼7.0 Hz, 3H), 0.90 (d, J¼7.1 Hz, 3H); 13C NMR (150 MHz,
NMR (150 MHz, CDCl3)
d
171.34 [171.27], 161.90 [161.89], 156.0,
CDCl3) d 202.0 (s), 172.4 (s), 161.9 (s), 156.0 (s), 155.6 (s), 142.8 (d),
155.3 [155.0], 141.0 [140.6], 138.96 [138.95], 138.43 [138.39], 136.7
[136.5], 135.8 (4C), 134.9 [134.8], 134.3, 134.0 [133.9], 133.7 (2C),
131.54 [131.50], 130.7, 129.7 [129.6], 127.8, 127.5 (4C), 126.4 [124.2],
122.5, 117.0, 80.9, 80.1 [79.9], 75.7 [73.0], 70.2 [70.1], 66.6, 58.0
[57.5], 57.2 [56.9], 42.5, 42.1, 41.5 [41.4], 40.0, 39.8, 35.3, 26.9, 19.2,
12.0, 11.4, 11.02 [11.01] (sprit signals derived from the C7 di-
astereomer were shown in parenthesis); IR (CHCl3) 3673, 3462,
3167, 3072, 3029, 3009, 2966, 2933, 2899, 2859, 2828, 1729, 1651,
1542, 1462, 1428, 1380, 1308, 1238, 1218, 1191, 1111, 980, 942, 918,
822 cmꢁ1; HRMS (ESI) m/z 846.3741 (calcd for C46H57N3NaO9Si
139.8 (s), 139.0 (d), 138.6 (d), 137.2 (d), 134.8 (d), 132.9 (d), 131.6 (s),
130.7 (s), 124.2 (t), 122.6 (d), 117.6 (t), 81.3 (d), 77.7 (d), 67.1 (d), 66.8
(t), 58.0 (q), 57.0 (q), 46.8 (d), 41.0 (t), 39.4 (t), 35.2 (d), 35.1 (t), 14.2
(q), 11.1 (q); IR (CHCl3) 3674, 3481, 3168, 3079, 3010, 2934, 2826,
1720, 1665, 1628, 1542, 1459, 1377, 1233, 1222, 1210, 1187, 1092, 980,
943, 919, 787, 771, 756, 741, 731 cmꢁ1; HRMS (ESI) m/z 606.2411
(calcd for C30H37N3NaO9 [MþNa]þ,
D
ꢁ1.6 mmu).
4.3.8. Macrolactone 16. To a stirred solution of ketone 14 (7.8 mg,
9.5 mol) in dry CH2Cl2 (8.8 mL) under a nitrogen atmosphere was
added a 2 mM solution of second generation HoveydaeGrubbs
catalyst in dry CH2Cl2 (1.4 mL, 2.8 mol). After being stirred for 9 h
m
[MþNa]þ,
D
ꢁ2.1 mmu).
m
4.3.6. RCM precursor 14. To a stirred solution of allylic alcohol 13
(26.1 mg, 28.3 mol) in dry CH2Cl2 (0.63 mL) under a nitrogen at-
mosphere were added pyridine (26 L) and DesseMartin period-
inane (20.2 mg, 47.6
mol). After being stirred for 2.5 h at 0 ꢀC, the
at reflux, the reaction mixture was concentrated. The crude mate-
rial was purified with a SiO2 column (FL60D 0.5 g, hexane/
EtOAc¼2/1 to 1/1) to give macrolactone (E)-16 (4.0 mg, 53%), its
stereoisomer (Z)-16 (2.2 mg, 29%), and its dimer (0.8 mg, 5%) as
m
m
m
reaction mixture was quenched with a 1:1:1 mixture of satd so-
dium thiosulfate aq/satd sodium bicarbonate aq/water (5 mL), and
extracted with CH2Cl2 (2 mLꢂ4). The combined extracts were
washed with brine, dried with Na2SO4, and concentrated. The crude
oil was purified with a SiO2 column (FL60D 2 g, hexane/CHCl3¼1/1
to 0/1) to give ketone 14 (24.6 mg, 94%) as a colorless oil. Com-
colorless oil. (E)-16: Rf 0.16 (CHCl3/EtOAc¼4:1); ½a D25
ꢁ38.1 (c 0.63,
ꢃ
CHCl3); 1H NMR (600 MHz, CDCl3)
d 8.13 (s, 1H), 8.08 (s, 1H),
7.71e7.66 (m, 4H), 7.66 (s, 1H), 7.44e7.35 (m, 6H), 7.07 (td, J¼7.6,
15.8 Hz, 1H), 6.94 (td, J¼7.2, 16.2 Hz, 1H), 6.35 (d, J¼15.8 Hz, 1H),
5.97 (d, J¼16.2 Hz, 1H), 4.45 (m, 1H), 4.36 (d, J¼9.6 Hz, 1H), 4.13 (qd,
J¼7.0, 9.6 Hz, 1H), 4.00 (dd, J¼6.8, 10.4 Hz, 1H), 3.92 (dd, J¼6.8,
10.4 Hz, 1H), 3.39 (m, 1H), 3.35 (s, 3H), 3.09 (s, 3H), 2.77 (m, 1H),
2.72 (dd, J¼5.6, 15.6 Hz, 1H), 2.66 (dd, J¼6.8, 15.6 Hz, 1H), 2.45 (m,
2H), 2.42 (m, 1H), 2.06 (ddtq, J¼2.0, 6.8, 6.8, 6.8 Hz, 1H), 1.02 (s, 9H),
0.88 (d, J¼6.8 Hz, 3H), 0.81 (d, J¼7.0 Hz, 3H); 13C NMR (150 MHz,
pound 14: Rf 0.42 (CHCl3/EtOAc¼4:1); ½a D25
ꢃ
ꢁ5.5 (c 0.54, CHCl3); 1H
NMR (600 MHz, CDCl3)
d 8.332 (s, 1H), 8.327 (s, 1H), 7.70 (s, 1H),
7.70e7.65 (m, 4H), 7.44e7.35 (m, 6H), 6.83 (dt, J¼15.8, 7.6 Hz, 1H),
6.67 (dd, J¼17.7, 11.3 Hz, 1H), 6.34 (d, J¼17.7 Hz, 1H), 6.12 (d,
J¼15.8 Hz, 1H), 5.78 (d, J¼11.3 Hz, 1H), 5.75 (ddt, J¼17.2, 10.2, 7.1 Hz,
1H), 5.07 (d, J¼17.2 Hz, 1H), 5.04 (d, J¼10.2 Hz, 1H), 4.40 (d,
J¼9.7 Hz, 1H), 4.32 (tt, J¼6.1, 5.9 Hz, 1H), 4.01 (dd, J¼10.8, 6.8 Hz,
1H), 3.86 (dd, J¼10.8, 6.9 Hz, 1H), 3.44 (dq, J¼9.7, 7.1 Hz, 1H), 3.31 (s,
3H), 3.21e3.13 (m,1H), 3.17 (s, 3H), 2.51 (dd, J¼15.1, 6.1 Hz,1H), 2.46
(dd, J¼15.1, 6.1 Hz, 1H), 2.47e2.35 (m, 2H), 2.32 (m, 1H), 2.19 (m,
1H), 1.90 (m, 1H), 1.04 (s, 9H), 0.87 (d, J¼6.9 Hz, 3H), 0.84 (d,
CDCl3)
d 203.2 (s), 171.1 (s), 162.7 (s), 156.5 (s), 155.6 (s), 143.3 (d),
139.6 (d), 139.0 (s), 137.4 (d), 137.3 (d), 137.2 (d), 135.9 (d, 2C), 135.83
(d, 2C), 135.0 (d), 133.7 (s), 133.6 (s), 131.5 (s), 130.3 (s), 129.9 (d),
129.7 (d), 127.7 (d, 2C), 127.6 (d, 2C), 116.3 (d), 79.7 (d), 76.7 (d), 68.7
(d), 67.5 (t), 57.7 (q), 56.2 (q), 42.6 (d), 41.7 (t), 40.3 (t), 35.0 (d), 32.4
(t), 26.9 (t, 3C),19.2 (s), 14.9 (q), 9.3 (q); IR (CHCl3) 3160, 3005, 2932,
2856, 1731, 1662, 1560, 1485, 1424, 1220, 1209, 1181, 1104, 997, 979,
909, 821, 790, 721, 703 cmꢁ1; HRMS (ESI) m/z 816.3320 (calcd for
J¼7.1 Hz, 3H); 13C NMR (150 MHz, CDCl3)
d 201.7 (s), 170.9 (s), 161.9
(s), 156.0 (s), 155.5 (s), 142.8 (d), 140.0 (s), 139.0 (d), 138.5 (d), 137.2
(d), 135.9 (d, 2C), 135.8 (d, 2C), 134.8 (d), 133.5 (s), 133.4 (s), 132.9
(d), 131.6 (s), 130.7 (s), 129.9 (d), 129.8 (d), 127.7 (d, 4C), 124.2 (t),
122.6 (d), 117.1 (t), 80.9 (d), 77.6 (d), 69.2 (d), 66.8 (t), 58.0 (q), 57.0
(q), 47.0 (d), 41.6 (t), 40.0 (t), 35.3 (d), 35.3 (t), 26.9 (q, 3C), 19.2 (s),
C44H51N3O9SiNa [MþNa]þ,
D
þ2.8 mmu). (Z)-16: Rf 0.21 (CHCl3/
EtOAc¼4:1); ½a 2D5
ꢃ
ꢁ67.8 (c 0.57, CHCl3); 1H NMR (600 MHz, CDCl3)
d
8.13 (s, 1H), 8.10 (s, 1H), 7.69e7.62 (m, 4H), 7.61 (s, 1H), 7.44e7.35
(m, 6H), 6.84 (td, J¼6.8, 16.0 Hz, 1H), 6.43 (td, J¼7.6, 11.8 Hz, 1H),
6.33 (d, J¼11.8 Hz, 1H), 6.01 (d, J¼16.0 Hz, 1H), 4.38e4.29 (m, 1H),

M. Kita et al. / Tetrahedron 68 (2012) 8753e8760
8759
4.35 (d, J¼8.4 Hz, 1H), 4.16 (dd, J¼5.2, 10.8 Hz, 1H), 3.95 (m, 1H),
3.94 (dd, J¼7.6, 10.8 Hz, 1H), 3.50e3.41 (m, 2H), 3.37 (s, 3H), 3.16 (s,
3H), 2.88 (m, 1H), 2.60 (dd, J¼6.4, 15.8 Hz, 1H), 2.53 (dd, J¼6.4,
15.8 Hz, 1H), 2.40 (m, 2H), 2.19 (m, 1H), 1.07 (d, J¼6.8 Hz, 3H), 1.02
3.08(dddd, J¼16.2, 7.5, 6.6,1.4 Hz,1H, H-21a),2.60(dd,J¼16.0, 3.2 Hz,
1H, H-2b), 2.51 (dd, J¼16.0, 9.5 Hz,1H, H-2a), 2.46e2.41 (m, 2H, H-4),
2.15 (m, 1H, H-23), 1.08 (d, J¼7.0 Hz, 3H, C23-Me), 1.01 (d, J¼7.1 Hz,
3H, C8-Me); 13C NMR (150 MHz, CDCl3)
d 202.2 (s, C-7),172.2 (s, C-1),
(s, 9H), 0.93 (d, J¼6.8 Hz, 3H); 13C NMR (150 MHz, CDCl3)
d
202.5 (s),
161.8 (s, C-18), 156.6 (s, C-15), 155.6 (s, C-12), 143.4 (d, C-5), 140.4 (d,
C-20), 139.7 (s, C-10), 137.9 (d, C-14), 137.7 (d, C-17), 136.8 (d, C-11),
133.9 (d, C-6),131.4 (s, C-13),130.6 (s, C-16),114.6 (d, C-19), 81.0 (d, C-
22), 78.2 (d, C-9), 67.0 (d, C-3), 66.8 (t, C-24), 58.1 (q, C22-OMe), 57.3
(q, C9-OMe), 44.3 (d, C-8), 41.2 (t, C-2), 39.6 (t, C-4), 37.0 (d, C-23), 31.4
(t, C-21), 13.5 (q, C8-Me), 12.7 (q, C23-Me); IR (CHCl3) 3675, 3167,
3026, 3007, 2931, 2846,1724,1663,1557,1459,1377,1279,1261,1220,
1182, 1100, 1013, 975, 918 cmꢁ1; HRMS (ESI) m/z 578.2091 (calcd for
170.9 (s), 161.7 (s), 156.6 (s), 155.7 (s), 142.7 (d), 141.1 (d), 139.3 (s),
137.6 (d), 137.4 (d), 137.0 (d), 135.80 (d, 2C), 135.79 (d, 2C), 134.7 (d),
133.7 (s), 133.5 (s), 131.6 (s), 130.7 (s), 129.83 (d), 129.78 (d), 127.71
(d, 2C), 127.67 (d, 2C), 114.3 (d), 81.7 (d), 77.6 (d), 69.0 (d), 66.9 (t),
58.3 (q), 56.9 (q), 44.0 (d), 41.0 (t), 39.4 (t), 36.8 (d), 31.9 (t), 26.9 (q,
3C), 19.2 (s), 14.0 (q), 12.6 (q); IR (CHCl3) 3162, 3005, 2932, 2856,
1730, 1663, 1558, 1458, 1424, 1179, 1103, 980, 917, 820, 790, 758,
739, 702 cmꢁ1; HRMS (ESI) m/z 816.3318 (calcd for C44H51N3O9SiNa
C28H33N3NaO9 [MþNa]þ,
D
ꢁ2.3 mmu).
[MþNa]þ,
D
þ2.4 mmu).
4.3.11. Ring-closing metathesis of 15. To a stirred solution of sec-
ondary alcohol 15 (5.8 mg, 9.9 mol) in dry CH2Cl2 (9.2 mL) under
4.3.9. Mycalolide macrolactone analog (E)-3. To a stirred solution of
m
silyl ether (E)-16 (7.7 mg, 9.7
m
mol) in THF (3.7 mL) under a nitro-
a nitrogen atmosphere was added a 2 mM solution of second
generation HoveydaeGrubbs catalyst in dry CH2Cl2 (1.5 mL,
3.0 mmol). After being stirred for 37 h at reflux, the reaction mixture
gen atmosphere at 0 ꢀC were added a 1 M solution of tetra-n-
butylammonium fluoride in THF (0.29 mL, 0.29 mmol) and acetic
acid (16.5
m
L, 0.29 mmol). After being stirred for 60 h at room
was concentrated. The crude material was purified with SiO2 col-
umns (FL60D 0.5 g, CHCl3/MeOH¼100/1 to 10/1; FL60D 0.5 g,
hexane/EtOAc¼3/7 to 1/9) to give macrolactone (E)-3 (2.9 mg, 52%),
its stereoisomer (Z)-3 (1.1 mg, 20%), and its dimer (0.8 mg, 7%) as
colorless oils.
temperature, the reaction mixture was diluted with satd sodium
bicarbonate aq (5 mL), and extracted with EtOAc (5 mLꢂ3). The
combined extracts were washed with brine, dried with Na2SO4, and
concentrated. The crude oil was purified with a SiO2 column (FL60D
0.5 g, hexane/EtOAc¼3/7 to 1/9) to give (E)-lactone 3 (4.7 mg, 87%)
as a colorless oil. (E)-3: Rf 0.30 (hexane/EtOAc¼1/9); ½a D25
ꢁ54 (c
ꢃ
4.4. Bioassay of mycalolide B and its synthetic analogs
0.38, CHCl3); 1H NMR (600 MHz, CDCl3)
d 8.12 (s, 1H, H-14), 8.08 (s,
The cytotoxicities of the probes and model compounds
were measured by the MTT (3-[4,5-dimethylthiazol-2-
yl]-2,5-diphenyltetrazolium bromide) method.19a The actin-
depolymerizing activities of the compounds were measured
based on their ability to attenuate the fluorescence of pyrene-
conjugated (pyrenyl) actin, as previously described.12a Anti-
mycotic assays against several fungi were conducted at Ricerca
Biosciences Inc. (Taipei, Taiwan).
1H, H-17), 7.66 (s, 1H, H-11), 7.05 (dt, J¼15.8, 7.7 Hz, 1H, H-20), 7.04
(dt, J¼15.9, 7.5 Hz, 1H, H-5), 6.35 (dt, J¼15.8, 1.6 Hz, 1H, H-19), 6.23
(dt, J¼15.9, 1.3 Hz, 1H, H-6), 4.40 (d, J¼8.9 Hz, 1H, H-9), 4.34 (m, 1H,
H-3), 4.16 (dd, J¼10.7, 8.2 Hz, 1H, H-24b), 4.10 (m, 1H, eOH), 4.04
(dd, J¼10.7, 5.9 Hz, 1H, H-24a), 4.00 (dt, J¼8.9, 7.0 Hz, 1H, H-8), 3.53
(ddd, J¼10.2, 4.7, 2.6 Hz, 1H, H-22), 3.39 (s, 3H, C22-OMe), 3.15 (s,
3H, C9-OMe), 2.78 (dddd, J¼14.8, 7.7, 4.7, 1.6 Hz, 1H, H-21b), 2.71
(dd, J¼15.8, 2.9 Hz, 1H, H-2b), 2.61 (dd, J¼15.8, 10.0 Hz, 1H, H-2a),
2.57e2.54 (m, 2H, H-4), 2.41 (dddd, J¼14.8, 10.2, 7.7, 1.6 Hz, 1H, H-
21a), 2.05 (m, 1H, H-23), 0.93 (d, J¼6.9 Hz, 3H, C23-Me), 0.88 (d,
Acknowledgements
J¼7.0 Hz, 3H, C8-Me); 13C NMR (150 MHz, CDCl3)
d 202.6 (s, C-7),
172.3 (s, C-1), 162.7 (s, C-18), 156.5 (s, C-15), 155.5 (s, C-12), 143.6 (d,
C-5), 139.8 (d, C-20), 139.2 (s, C-10), 137.4 (d, C-11), 137.3 (d, C-14),
137.2 (d, C-17), 134.2 (d, C-6), 131.3 (s, C-13), 130.1 (s, C-16), 116.3 (d,
C-19), 79.2 (d, C-22), 77.1 (d, C-9), 67.2 (t, C-24), 66.9 (d, C-3), 57.9
(q, C9-OMe), 56.6 (q, C22-OMe), 43.6 (d, C-8), 41.5 (t, C-2), 40.4 (t, C-
4), 35.7 (d, C-23), 33.2 (t, C-21), 13.8 (q, C8-Me), 9.5 (q, C23-Me); IR
(CHCl3) 3690, 3167, 3026, 3003, 2929, 2846, 1719, 1661, 1609, 1563,
1458, 1386, 1261, 1215, 1091, 1100, 1023, 977, 918 cmꢁ1; HRMS (ESI)
This work was supported by Grants-in-Aid for Scientific Re-
search from JSPS (21681028 and 21651091 for M.K.; 23102014 and
23310148 for H.K.). Support was also provided by the Naito Foun-
dation, the Uehara Memorial Foundation, and the Takeda Science
Foundation. We thank the Kaneka Corporation for their gift of
methyl (S)-(þ)-3-hydroxy-2-methylpropionate.
Supplementary data
m/z 578.2087 (calcd for C28H33N3NaO9 [MþNa]þ,
D
ꢁ2.7 mmu).
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.tet.2012.08.012.
4.3.10. Mycalolide macrolactone analog (Z)-3. Toastirredsolutionof
silyl ether (Z)-16 (3.3 mg, 4.2 mol) in THF (1.6 mL) under a nitrogen
atmosphere at 0 ꢀC were added a 1 M solution of tetra-n-buty-
lammonium fluoride in THF (0.125 mL, 125 mol) and acetic acid
(7.1 L, 125 mol). After being stirred for 48 h at room temperature,
m
References and notes
m
m
m
1. (a) Fusetani, N.; Yasumuro, K.; Matsunaga, S.; Hashimoto, K. Tetrahedron Lett.
1989, 30, 2809e2812; (b) Wada, S.; Matsunaga, S.; Saito, S.; Fusetani, N.;
Watabe, S. J. Biochem. 1998, 123, 946e952; (c) Matsunaga, S.; Liu, P.; Celatka,
C. A.; Panek, J. S.; Fusetani, N. J. Am. Chem. Soc. 1999, 121, 5605e5606; (d)
Phuwapraisirisan, P.; Matsunaga, S.; van Soest, R. W. M.; Fusetani, N. J. Nat.
Prod. 2002, 65, 942e943; (e) Tsukamoto, S.; Koimaru, K.; Ohta, T. Mar. Drugs
2005, 3, 29e35.
2. (a) Hori, M.; Saito, S.; Shin, Y.; Ozaki, H.; Fusetani, N.; Karaki, H. FEBS Lett. 1993,
322, 151e154; (b) Saito, S.; Watabe, S.; Ozaki, H.; Fusetani, N.; Karaki, H. J. Biol.
Chem. 1994, 269, 29710e29714.
3. (a) Matsunaga, S.; Sugawara, T.; Fusetani, N. J. Nat. Prod. 1998, 61, 1164e1167; (b)
Rashid, M. A.; Gustafson, K. R.; Cardeilina, J. H., II; Boyd, M. R. J. Nat. Prod. 1995,
58, 1120e1125.
4. Kubanek, J.; Jensen, P. R.; Keifer, P. A.; Sullards, M. C.; Collins, D. O.; Fenical, W.
Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 6916e6921.
the reaction mixture was diluted with satd sodium bicarbonate aq
(5 mL), and extracted with EtOAc (5 mLꢂ4). The combined extracts
were washed with brine, dried with Na2SO4, and concentrated. The
crude oil was purified with a SiO2 column (FL60D 0.5 g, hexane/
EtOAc¼1/9) to give (Z)-lactone 3 (2.1 mg, 91%) as a colorless oil. (Z)-3:
Rf 0.39 (hexane/EtOAc¼1/9); ½a D25
ꢃ
ꢁ71 (c 0.36, CHCl3); 1H NMR
(600 MHz, CDCl3) d 8.17 (s,1H, H-14), 8.14 (s,1H, H-17), 7.63 (s,1H, H-
11), 6.91(dt, J¼16.1, 6.7 Hz,1H,H-5), 6.42(dt, J¼11.7, 7.5 Hz,1H, H-20),
6.35 (d, J¼11.7 Hz, 1H, H-19), 6.18 (d, J¼16.1 Hz, 1H, H-6), 4.37 (d,
J¼7.5 Hz, 1H, H-9), 4.18 (m, 1H, H-3), 4.16 (dd, J¼10.9, 5.3 Hz, 1H, H-
24b), 4.09 (dd, J¼10.9, 5.6 Hz, 1H, H-24a), 3.89 (qd, J¼7.1, 7.5 Hz, 1H,
H-8), 3.75 (br s,1H, H-22), 3.51 (br s,1H, eOH), 3.40 (s, 3H, C22-OMe),
3.36 (dddd, J¼16.2, 7.5, 4.7, 1.4 Hz, 1H, H-21b), 3.24 (s, 3H, C9-OMe),
5. Roesener, J. A.; Scheuer, P. J. J. Am. Chem. Soc. 1986, 108, 846e847.
6. (a) Matsunaga, S.; Fusetani, N.; Hashimoto, K.; Koseki, K.; Noma, M.; Noguchi,
H.; Sankawa, U. J. Org. Chem. 1989, 54, 1360e1363; (b) Kernan, M. R.; Molinski,

8760
M. Kita et al. / Tetrahedron 68 (2012) 8753e8760
T. F.; Faulkner, D. J. J. Org. Chem. 1988, 53, 5014e5020; (c) Chung, S.-C.;
Lee, S.-H.; Jang, K. H.; Park, W.; Jeon, J.-E.; Oh, H.; Shin, J.; Oh, K.-B. Bioorg. Med.
Chem. Lett. 2011, 21, 3198e3201.
18. (a) Liu, P.; Panek, J. S. J. Am. Chem. Soc. 2000, 122, 1235e1236; (b) Panek, J. S.;
Liu, P. J. Am. Chem. Soc. 2000, 122, 11090e11097.
19. (a) Pattenden, G.; Ashweek, N. J.; Baker-Glenn, C. A. G.; Walker, G. M.; Yee, J. G.
K. Angew. Chem., Int. Ed. 2007, 46, 4359e4363; (b) Chattopadhyay, S. K.;
Pattenden, G. Tetrahedron Lett. 1998, 39, 6095e6098.
20. (a) Suenaga, K.; Kimura, T.; Kuroda, T.; Matsui, K.; Miya, S.; Kuribayashi, S.;
Sakakura, A.; Kigoshi, H. Tetrahedron 2006, 62, 8278e8290; (b) Kuroda, T. Ph.D.
thesis, University of Tsukuba, 2008.
21. Maddock, J.; Pattenden, G.; Wight, P. G. J. Comput. Aided Mol. Des. 1993, 7,
573e586.
22. Molecular modeling studies and DFT calculations were conducted using Spar-
tan’08 for Windows. Energetically stable conformers were obtained by using
the Monte Carlo method via the iterative application of MMFFs (Merck Mo-
lecular Force Fields). Ten of the most-minimized conformations for each ste-
reoisomer were refined by geometry optimization. Their minimum energies
were confirmed by frequency calculations.
23. (a) Kimura, T.; Kuribayashi, S.; Sengoku, T.; Matsui, K.; Ueda, S.; Hayakawa, I.;
Suenaga, K.; Kigoshi, H. Chem. Lett. 2007, 36, 1490e1491; (b) Kita, M.; Watanabe,
H.; Ishitsuka, T.; Mogi, Y.; Kigoshi, H. Tetrahedron Lett. 2010, 51, 4882e4885.
24. (a) Jin, H.; Uenishi, J.; Christ, W. J.; Kishi, Y. J. Am. Chem. Soc. 1986, 108,
5644e5646; (b) Takai, K.; Tagashira, M.; Kuroda, T.; Oshima, K.; Utimoto, K.;
Nozaki, H. J. Am. Chem. Soc. 1986, 108, 6048e6050; (c) Okude, Y.; Hirano, S.;
Hiyama, T.; Nozaki, H. J. Am. Chem. Soc. 1977, 99, 3179e3181.
25. (a) BouzBouz, S.; Cossy, J. Org. Lett. 2004, 6, 3469e3472; (b) Hafner, A.;
Duthaler, R. O.; Marti, R.; Rihs, J.; Rothe-Streit, P.; Schwarzenbach, F. J. Am.
Chem. Soc. 1992, 114, 2321e2336.
26. (a) Kingsbury, J. S.; Harrity, J. P. A.; Bonitatebus, P. J.; Hoveyda, A. H. J. Am. Chem.
Soc. 1999, 121, 791e799; (b) Garber, S. B.; Kingsbury, J. S.; Gray, B. L.; Hoveyda,
A. H. J. Am. Chem. Soc. 2000, 122, 8168e8179.
7. Kobayashi, J.; Murata, O.; Shigemori, H. J. Nat. Prod. 1993, 56, 787e791.
8. (a) Matsunaga, S.; Fusetani, N.; Hashimoto, K. J. Am. Chem. Soc. 1986, 108,
847e849; (b) Tanaka, J.; Yan, Y.; Choi, J.; Bai, J.; Klenchin, V. A.; Rayment, I.;
Marriott, G. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 13851e13856.
9. Klenchin, V. A.; Allingham, J. S.; King, R.; Tanaka, J.; Marriott, G.; Rayment, I. Nat.
Struct. Biol. 2003, 10, 1058e1063.
10. Allingham, J. S.; Tanaka, J.; Marriott, G.; Rayment, I. Org. Lett. 2004, 6, 597e599.
11. (a) Yamada, K.; Ojika, M.; Ishigaki, T.; Yoshida, Y.; Ekimoto, H.; Arakawa, M. J.
Am. Chem. Soc. 1993, 115, 11020e11021; (b) Ojika, M.; Kigoshi, H.; Ishigaki, T.;
Tsukada, I.; Tsuboi, T.; Ogawa, T.; Yamada, K. J. Am. Chem. Soc. 1994, 116,
7441e7442; (c) Kigoshi, H.; Suenaga, K.; Mutou, T.; Ishigaki, T.; Atsumi, T.;
Ishiwata, H.; Sakakura, A.; Ogawa, T.; Ojika, M.; Yamada, K. J. Org. Chem. 1996, 61,
5326e5351; (d) Kitamura, K.; Teruya, T.; Kuroda, T.; Kigoshi, H.; Suenaga, K.
Bioorg. Med. Chem. Lett. 2009, 19, 1896e1898; (e) Ohno, O.; Morita, M.;
Kitamura, K.; Teruya, T.; Kita, M.; Kigoshi, H.; Suenaga, K. submitted for
publication.
12. Reviews: (a) Yamada, K.; Ojika, M.; Kigoshi, H.; Suenaga, K. Nat. Prod. Rep. 2009,
26, 27e43; (b) Yamada, K.; Ojika, M.; Kigoshi, H.; Suenaga, K. Proc. Jpn. Acad. Ser.
B 2010, 86, 176e189.
13. Preparation of biotin and fluorescent derivatives of aplyronines: (a) Kita, M.;
Hirayama, Y.; Sugiyama, M.; Kigoshi, H. Angew. Chem., Int. Ed. 2011, 50,
9871e9874; (b) Kita, M.; Yoneda, K.; Hirayama, Y.; Yamagishi, K.; Saito, Y.;
Sugiyama, Y.; Miwa, Y.; Ohno, O.; Morita, M.; Suenaga, K.; Kigoshi, H. Chem-
BioChem 2012, 13, 1754e1758.
14. Kuroda, T.; Suenaga, K.; Sakakura, A.; Handa, T.; Okamoto, K.; Kigoshi, H. Bio-
conjugate Chem. 2006, 17, 524e529.
15. Hirata, K.; Muraoka, S.; Suenaga, K.; Kuroda, K.; Kato, K.; Tanaka, H.; Yamamoto,
M.; Takata, M.; Yamada, K.; Kigoshi, H. J. Mol. Biol. 2006, 356, 945e954.
16. (a) Suenaga, K.; Kamei, N.; Okugawa, Y.; Takagi, M.; Akao, A.; Kigoshi, H.;
Yamada, K. Bioorg. Med. Chem. Lett. 1997, 7, 269e274; (b) Kigoshi, H.; Suenaga,
K.; Takagi, M.; Akao, A.; Kanematsu, K.; Kamei, N.; Okugawa, Y.; Yamada, K.
Tetrahedron 2002, 58, 1075e1102.
27. An example of Z-preferential RCM reaction in hexane: Wang, B.; Forsyth, C. J.
Org. Lett. 2006, 8, 5223e5226.
28. Ogawa, A.; Curran, D. P. J. Org. Chem. 1997, 62, 450e451.
29. Under the same conditions, the RCM reaction of the whole part (C1eC35) of the
mycalolide B synthetic intermediate was not completed (E/Z¼2:1, w30% yield).
See Ref. 23b.
17. Reviews: (a) Yeung, K.-S.; Paterson, I. Angew. Chem., Int. Ed. 2002, 41,
4632e4653; (b) Chattopadhyay, S. K.; Pattenden, G. J. Chem. Soc., Perkin Trans. 1
2000, 2429e2454 and references are therein.
30. Recently, (19Z)-halichondramide was isolated from the marine sponge Chon-
drosia sp., which exhibited both actin-depolymerizing and antimycotic activi-
ties comparable to those of halichondramide (19E isomer). See Ref. 6c.