Total synthesis and biological evaluation of (+)-neopeltolide and its analogues

Article abstract of DOI:10.1002/chem.200901675

The stereocontrolled total synthesis of the originally proposed (1) and correct (2) structures of (+)-neopeltolide, a novel marine macrolide natural product with highly potent antiproliferative activity against several cancer cell lines as well as potent antifungal activity, has been achieved by exploiting a newly developed SuzukiMiyaura coupling/ring-closing metathesis strategy. Alkylborate 44, which was generated in situ from iodide 34, was coupled with enol phosphate 8 by a Suzuki-Miyaura coupling. Ring-closing metathesis of the derived diene 45 followed by stereoselective hydrogenation afforded tetrahydropyran 47 as a single stereoisomer in high overall yield from 34. Our convergent strategy enabled us to construct the 14-membered macrolactone core structure of 2 in a rapid and efficient manner. Total synthesis and biological evaluation of synthetic intermediates and designed synthetic analogues, performed to establish the structure-activity relationships of 2, led to the discovery of a structurally simple yet potent cytotoxic analogue, 9-demethylneopeltolide (54).

Full text of DOI:10.1002/chem.200901675

FULL PAPER
DOI: 10.1002/chem.200901675
Total Synthesis and Biological Evaluation of (+)-Neopeltolide and Its
Analogues
Haruhiko Fuwa,*[a] Asami Saito,[a] Shinya Naito,[a] Keiichi Konoki,[b]
Mari Yotsu-Yamashita,[b] and Makoto Sasaki*[a]
Abstract: The stereocontrolled total
synthesis of the originally proposed (1)
and correct (2) structures of (+)-neo-
peltolide, a novel marine macrolide
natural product with highly potent anti-
proliferative activity against several
cancer cell lines as well as potent anti-
fungal activity, has been achieved by
exploiting a newly developed Suzuki–
Miyaura coupling/ring-closing metathe-
sis strategy. Alkylborate 44, which was
generated in situ from iodide 34, was
coupled with enol phosphate 8 by a
Suzuki–Miyaura coupling. Ring-closing
metathesis of the derived diene 45 fol-
lowed by stereoselective hydrogenation
afforded tetrahydropyran 47 as a single
stereoisomer in high overall yield from
34. Our convergent strategy enabled us
to construct the 14-membered macro-
lactone core structure of 2 in a rapid
and efficient manner. Total synthesis
and biological evaluation of synthetic
intermediates and designed synthetic
analogues, performed to establish the
structure–activity relationships of 2, led
to the discovery of a structurally simple
yet potent cytotoxic analogue, 9-deme-
thylneopeltolide (54).
Keywords: macrocycles
·
natural
products · ring-closing metathesis ·
Suzuki–Miyaura coupling
synthesis
· total
Introduction
0.62 mgmLÀ1). The gross structure including the relative ste-
reochemistry of neopeltolide was initially proposed as 1
based on extensive 2D NMR spectroscopic analysis
(Figure 1).[2] However, the stereochemical assignment of the
Secondary metabolites from marine organisms continue to
be a rich source of novel, biologically active, structurally in-
teresting natural products.[1] In 2007, Wright et al. reported
the isolation of a novel marine macrolide natural product
designated as neopeltolide from a deep-water sponge of the
family Neopeltidae, collected off the north-west coast of Ja-
maica.[2] Neopeltolide displays significant in vitro antiproli-
ferative activity against several cancer-cell lines including
the A549 human lung adenocarcinoma, the NCI/ADR-RES
ovarian carcinoma, and the P388 murine leukemia cells with
low nanomolar IC50 values (IC50 =half maximal inhibitory
concentration). Furthermore, this natural product shows
potent antifungal activity against pathogenic yeast Candida
albicans (minimum inhibitory concentration (MIC)
[a] Prof. Dr. H. Fuwa, A. Saito, S. Naito, Prof. Dr. M. Sasaki
Graduate School of Life Sciences, Tohoku University
1–1 Tsutsumidori-amamiya, Aoba-ku, Sendai 981-8555 (Japan)
Fax : (+81)22-717-8896
Figure 1. Proposed and correct structures of (+)-neopeltolide.
C11 and C13 stereogenic centers was later revised independ-
ently by Panek and co-workers[3] and Scheidt and co-work-
ers[4] through total synthesis. At the same time, the absolute
configuration of neopeltolide was unambiguously deter-
mined to be the structure represented by 2. The structural
characteristics of 2 are 1) the 2,4,6-trisubstituted tetrahydro-
E-mail: hfuwa@bios.tohoku.ac.jp
[b] Prof. Dr. K. Konoki, Prof. Dr. M. Yotsu-Yamashita
Graduate School of Agricultural Science, Tohoku University
1–1 Tsutsumidori-amamiya, Aoba-ku, Sendai 981-8555 (Japan)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200901675.
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pyran subunit embedded in the 14-membered macrolactone
core and 2) the highly unsaturated oxazole-containing side
chain. These biological and structural aspects of neopelto-
lide spurred the interest of synthetic organic chemists. As a
consequence, seven total syntheses, including ours, and two
formal syntheses have been recorded to date.[5–12] In addi-
tion, Kozmin and co-workers have recently reported that
synthetic neopeltolide targets cytochrome bc1 complex and
may inhibit mitochondrial Adenosine triphosphate synthe-
sis.[8]
Herein, we describe in detail the total synthesis of the
originally proposed (1) and correct (2) structures of neopel-
tolide by exploiting our newly developed Suzuki–Miyaura
coupling/ring-closing metathesis strategy for the synthesis of
multi-substituted tetrahydropyrans.[7,13] Our efficient and
convergent strategy was amenable for rapid assembly of the
14-membered macrolactone core of 1 and 2 from readily
available acyclic precursors. Moreover, we successfully ela-
borated the designed synthetic analogues by virtue of the
convergence of our strategy and evaluated their biological
activity to establish the structure–activity relationships of
this biologically intriguing natural substance.
Results and Discussion
Synthesis plan: We initially targeted the proposed structure
1 of neopeltolide because the correct structure 2 had not yet
been elucidated at the outset of our work. According to the
literature precedents on the synthesis of the structurally re-
lated marine macrolide leucascandrolide A,[14,15] we planned
to incorporate the oxazole-containing side chain at the final
step of the total synthesis through Mitsunobu esterifica-
tion[16] of alcohol 3 and the known acid 4[15] (Scheme 1). We
envisaged the 14-membered macrolactone core structure of
3 to be derived from tetrahydropyran 5 via Yamaguchi lac-
tonization.[17] The 2,4,6-trisubstituted tetrahydropyran subu-
nit within 5 could be derived from endocyclic enol ether 6
by stereoselective hydrogenation. We envisioned that assem-
bly of acyclic enol phosphate 8 and alkylborate 9 (generated
in situ from iodide 10) via a Suzuki–Miyaura coupling/ring-
closing metathesis sequence[18–22] would lead to endocyclic
enol ether 6 via diene 7.
Scheme 1. Our synthetic strategy for the proposed structure 1 of neopel-
tolide.
1
600 MHz H NMR analysis). Reduction of 14 with diisobu-
tylaluminum hydride (DIBALH) afforded allylic alcohol 15
in 100% yield. It was also possible to synthesize 15 directly
from 13 through cross-metathesis with cis-2-butene-1,4-diol
by using G-II in 75% yield (E:Z=ca. 7:1, by 600 MHz
1H NMR analysis).[27] Sharpless asymmetric epoxidation of
15 gave hydroxy epoxide 16 as a single diastereomer in 95%
yield. Iodination of 16 followed by zinc reduction of the de-
rived iodo epoxide led to allylic alcohol 17 in 87% yield for
the two steps. Protection of the hydroxy group of 17 as its
benzyloxymethyl (BOM) ether, cleavage of the MPM ether,
and subsequent acylation afforded acetate 18 in 98% yield
for the three steps. Finally, enolization of 18 with KHMDS
(HMDS=hexamethyldisilazane) in the presence of diphe-
Synthesis of acyclic enol phosphate 8: The synthesis of acy-
clic enol phosphate 8 commenced with Keck asymmetric al-
lylation[23] of aldehyde 11,[15p] giving homoallylic alcohol 12
in 93% yield with greater than 95% ee (Scheme 2). Protec-
tion of 12 as its p-methoxyphenylmethyl (MPM) ether was
best accomplished by using MPMOC
presence of 10 mol% of Sc
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
flate),[24] leading to the MPM ether 13 in 77% yield. This
was followed by cross-metathesis[25] with methyl acrylate
(5 mol% Grubbsꢁ second-generation catalyst (benzylidene-
nylphosphoryl
chloride
(Hexamethylphosphoramide
(HMPA), THF, À788C) furnished enol phosphate 8.
A
Synthesis of iodide 10: Our synthesis of iodide 10 started
with asymmetric allylation of the known aldehyde 19[28] by
using (À)-Ipc2B
(allyl)[29] (Ipc=isopinocampheyl) in diethyl
ACHTUNGTRENNUNG
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Evaluation of (+)-Neopeltolide and Its Analogues
FULL PAPER
ether at À788C, giving homoallylic alcohol 20 in 94% yield
(Scheme 3). Methylation of 20 (MeOTf, 2,6-di-tert-butylpyri-
dine, 90%) was followed by ozonolysis of the double bond
(O3; then Ph3P) to deliver aldehyde 22 in 100% yield.
Asymmetric allylation of 22 with (+)-Ipc2BACHTNUTRGNEUNG(allyl) (diethyl
ether, À788C) led to the homoallylic alcohol 23 in 90%
yield. Hydrogenation of 23 followed by protection of the
C13 hydroxy group as its MPM ether (MPMOC
ACHTUNGTRNEN(UNG =NH)CCl3,
La(OTf)3, toluene), and subsequent desilylation with tetra-
AHCTUNGTRENNUNG
butylammonium fluoride (TBAF) gave alcohol 24 in 66%
yield for the three steps. Iodination of 24 under standard
conditions afforded iodide 10 in 100% yield.
Construction of the 2,4,6-trisubstituted tetrahydropyran sub-
unit: After synthesizing the requisite fragments, we next in-
vestigated their assembly and construction of the 2,4,6-tri-
substituted tetrahydropyran subunit by the Suzuki–Miyaura
coupling/ring-closing metathesis sequence (Scheme 4 and
Table 1). Alkylborate 9 was prepared in situ from iodide 10
according to the Marshall protocol[30] with a slight modifica-
tion. Thus, treatment of 10 with tBuLi in the presence of B-
MeO-9-BBN (9-BBN=9-borabicycloACTHNUTRGNEUNG[3,3,l] nonane) in di-
ethyl ether at À788C followed by the addition of THF and
warming of the reaction mixture to room temperature clean-
ly generated alkylborate 9. Without isolation, 9 was coupled
Scheme 2. Synthesis of acyclic enol phosphate 8. BINOL=2,2’-dihy-
droxy-1,1’-binaphthyl, DDQ=2,3-dichloro-5,6-dicyano-1,4-benzoquinone,
DET=diethyl tartrate, MS=molecular sieves,
Scheme 3. Synthesis of iodide 10.
Scheme 4. Synthesis of the 2,4,6-trisubstituted tetrahydropyran subunit.
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H. Fuwa, M. Sasaki et al.
Table 1. Optimization of Suzuki–Miyaura Coupling of 8 and 9.[a]
went ring-closing metathesis in the presence of 10 mol% of
G-II in toluene (20 mm) at 708C, giving rise to endocyclic
enol ether 6 in 67% overall yield from iodide 10. Stereose-
lective hydrogenation of 6 (H2 (0.8 MPa), 10% Pd/C, 1:1
EtOAc/MeOH) proceeded cleanly to afford tetrahydropyr-
an 5 in 81% yield as a single stereoisomer. The newly gener-
ated C7 stereogenic center was unambiguously confirmed by
NOE experiments as shown. Thus, the 2,4,6-trisubstituted
tetrahydropyran subunit of 1 was successfully constructed
from readily available acyclic fragments 8 and 10 in a con-
vergent and efficient manner.[31]
Entry 8 [equiv] Pd catalyst
Base
Temp Yield of 6
[8C]
[%]
1
2
3
4
1.8
1.4
1.2
1.4
[Pd
A
N
aq Cs2CO3
(dppf)·CH2Cl2] aq Cs2CO3
50
50
38
34
Decomp.
67
N
[Pd
[Pd
aq NaHCO3 50
aq Cs2CO3 RT
[a] All reactions were carried out by using 10 mol% of Pd catalyst and
3 equivalents of base in DMF at indicated temperature. Yields are based
on iodide 10 and were determined after conversion to 6.
with enol phosphate 8 under the influence of a palladium
catalyst and a base. We initially examined the Suzuki–
Miyaura coupling of 9 with 8 (prepared from 1.8 equivalents
of acetate 18) by using aqueous Cs2CO3 and 10 mol% of
Completion of the total synthesis of the proposed structure
1 of (+)-neopeltolide: With the key tetrahydropyran 5 in
hand, we proceeded to complete the total synthesis of 1
(Scheme 5). Deprotection of the triisopropylsilyl (TIPS)
group of 5 gave the alcohol 25 (TBAF, THF, 97%), which
was reacted further to form methyl ester 26 in 91% overall
yield by a two-stage oxidation (SO3·pyridine [O]; then
NaClO2) and subsequent esterification with TMSCHN2
(TMS=trimethylsilyl). Removal of the MPM group fol-
lowed by saponification with TMSOK[32] gave hydroxy acid
27 in 85% yield for the two steps. Macrolactonization of 27
under Yamaguchi conditions (2,4,6-Cl3C6H2COCl, Et3N,
[PdACHTUNGTRENNUNG(PPh3)4] in N,N-dimethylformamide (DMF) at 508C.
Subsequent ring-closing metathesis of the derived enol ether
7 was achieved with 10 mol% of G-II in toluene (20 mm) at
708C. The desired endocyclic enol ether 6 was isolated in
38% yield from iodide 10 (Table 1, entry 1). The intermolec-
ular Suzuki–Miyaura coupling of 8 and 9 predominated over
the possible intramolecular Heck reaction of 8 as we could
not identify any products derived from the latter pathway.
We noticed that a small amount (ca. 20%) of acetate 18 was
recovered after the Suzuki–
Miyaura coupling, implying that
the degradation of enol phos-
phate 8 to acetate 18 occurred
under the basic reaction condi-
tions. Therefore, we set out to
investigate the optimal condi-
tions for the Suzuki–Miyaura
coupling of enol phosphate 8
and alkylborate 9, and the re-
sults are summarized in Table 1.
The use of 10 mol% of [PdCl2-
ACHTUNGTRENNUNG
6
(Table 1, entry 2). Changing the
base to aqueous NaHCO3 did
not afford the intermediate
diene 7 at all, and only degra-
dation of
8
was observed
(Table 1, entry 3). We finally
found that the yield of endocy-
clic enol ether 6 could be dra-
matically improved simply by
running the reaction at room
temperature (Table 1, entry 4).
Thus, coupling of alkylborate 9
with enol phosphate 8 (pre-
pared from 1.4 equivalents of
acetate 18) under the influence
of 10 mol% [PdACHTUNGTRENNUNG(PPh3)4] and
aqueous Cs2CO3 in DMF at
room temperature gave diene 7.
The formed diene 7 then under- Scheme 5. Completion of the total synthesis of the proposed structure 1 of neopeltolide.
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Evaluation of (+)-Neopeltolide and Its Analogues
FULL PAPER
THF; then 4-dimethylaminopyridine (DMAP), toluene,
808C) afforded the 14-membered lactone 28 in 97% yield.
Hydrogenolysis of the benzyloxymethyl (BOM) ether of 28
delivered alcohol 3 in 100% yield. Finally, Mitsunobu
esterification of 3 with the known acid 4[15] under standard
conditions (diisopropyl azodicarboxylate (DIAD), Ph3P,
benzene, room temperature) furnished the proposed struc-
ture 1 of neopeltolide in 86% yield. The spectroscopic prop-
erties of 1 did not match those of the natural product (that
is, the correct structure 2) but were in close agreement with
the data for 1 reported by Panek and co-workers[3] and
Scheidt and co-workers.[4]
Total synthesis of the correct structure 2 of (+)-neopelto-
lide: After recognizing the correct structure of (+)-neopel-
tolide, which was independently reported by Panek and co-
workers[3] and Scheidt and co-workers,[4] we focused our at-
tention on the synthesis of the correct structure 2. To this
end, we synthesized iodide 34 from aldehyde 19 (Scheme 6).
Scheme 7. Improved synthesis of iodide 40.
reduction (98% overall yield). Acetalization of 35 with p-
methoxybenzaldehyde dimethylacetal/camphorsulfonic acid
(CSA) followed by DIBALH reduction gave alcohol 36 in
100% yield for the two steps. Oxidation of 36 with Dess–
Martin periodinane delivered aldehyde 37 in 97% yield. For
installation of the C11 stereogenic center, chelation-con-
trolled allylation of 37 was employed. Thus, exposure of 37
to allyltrimethylsilane in the presence of MgBr2·OEt2 in
CH2Cl2 at 08C led to the desired homoallylic alcohol 38 in
99% yield with an excellent diastereoselectivity (d.r.=ca.
14:1). Methylation of homoallylic alcohol 38 gave methyl
ether 39 in 100% yield. Hydroboration of 39 with 9-BBN-H
followed by oxidative workup delivered alcohol 40 in 98%
yield. A two-stage oxidation of 40 (Dess–Martin periodi-
nane; then NaClO2) followed by condensation of the de-
rived acid with (S)-4-benzyl-2-oxazolidinone (41)[34] afforded
imide 42 in 85% yield for the three steps. Diastereoselective
alkylation of 42 (NaHMDS, MeI, THF, À788C)[35] led to me-
thylated product 43 in 91% yield as a single stereoisomer
Scheme 6. Synthesis of iodide 34.
Asymmetric allylation of 19 with (+)-Ipc2BACHTNUTRGNEUNG(allyl) (diethyl
ether, À788C) gave homoallylic alcohol 29, which was con-
verted to aldehyde 31 by methylation and ozonolysis. A
second asymmetric allylation by using (À)-Ipc2B
ACHTUNGTRENUN(NG allyl) (di-
ethyl ether, À788C) delivered homoallylic alcohol 32, which
was further reacted to form iodide 34 as described for the
synthesis of 10.
Meanwhile, we have also developed an improved synthe-
sis of iodide 34, which allowed us to prepare multigram
quantities of 34 (Scheme 7). The synthesis started with the
known diol 35,[33] which was readily available from ethyl 3-
oxohexanoate by asymmetric hydrogenation and subsequent
1
(judged by 600 MHz H NMR) after flash column chroma-
tography on silica gel. Removal of the chiral auxiliary with
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H. Fuwa, M. Sasaki et al.
NaBH4 in aqueous THF[36] gave alcohol 33 in 100% yield,
which was iodinated to furnish iodide 34 in 97% yield.
Completion of the synthesis of
2 is illustrated in
Scheme 8. Lithiation of iodide 34 with tBuLi in the presence
of B-MeO-9-BBN generated alkylborate 44, which was cou-
pled with enol phosphate 8 in the presence of aqueous
Cs2CO3 and 10 mol% of [PdACHTNURTGNEUNG(PPh3)4] in DMF at room tem-
perature. Ring-closing metathesis of the resultant diene 45
by using 10 mol% of G-II in toluene (5 mm)[37] at 708C fur-
nished endocyclic enol ether 46 in 78% yield from 34. Ste-
reoselective hydrogenation of 46 (H2 (0.8 a), 10% Pd/C, 2:1
EtOAc/MeOH) afforded tetrahydropyran 47 as a single ste-
reoisomer in 81% yield. Desilylation of 47 gave alcohol 48
(97%), which was elaborated to methyl ester 49 by a two-
stage oxidation and esterification (89%, three steps). De-
protection of the MPM group and subsequent saponification
delivered hydroxy acid 50 in 92% yield for the two steps.
Macrolactonization of 50 under Yamaguchi conditions fur-
nished 14-membered lactone 51 in 100% yield. Hydrogeno-
lysis of the BOM group of 51 led to alcohol 52 in 100%
yield. Finally, Mitsunobu esterification of 52 with acid 4
(DIAD, Ph3P, benzene) furnished synthetic (+)-neopeltolide
(2) in 61% yield. The spectroscopic properties (1H NMR,
13C NMR, HRMS, [a]D) of synthetic 2 were in full accord-
ance with those of the natural product.[2]
Synthesis of structural analogues of 2 and their biological
evaluation: Intrigued by the fascinating biological activities
of neopeltolide, we next turned our attention to the elucida-
tion of structure–activity relationships of this natural sub-
stance. To probe the influence of the peripheral substituents
of the 14-membered macrolactone on biological activity, we
designed and synthesized 11-demethoxyneopeltolide (53)
and 9-demethylneopeltolide (54) (Figure 2).
Figure 2. Synthetic analogues for investigation on the structure-activity
relationships.
The synthesis of 11-demethoxyneopeltolide (53) started
with tosylation of the known alcohol 55[28] followed by alky-
lation with allylmagnesium bromide in the presence of CuBr
to give olefin 56 (Scheme 9). Hydroboration of 56 with
Cy2BH delivered alcohol 57, which was oxidized to give al-
dehyde 58. Asymmetric allylation of 58 by using (À)-Ipc2B-
(allyl) (diethyl ether, À788C) provided homoallylic alcohol
Scheme 8. Completion of the total synthesis of the correct structure 2 of
neopeltolide.
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Evaluation of (+)-Neopeltolide and Its Analogues
FULL PAPER
Scheme 9. Total synthesis of 11-demethoxyneopeltolide (53). Cy2BH=dicyclohexylborane.
59. Conversion of 59 to iodide 61 and subsequent elabora-
tion to 11-demethoxyneopeltolide (53) were performed as
described for the synthesis of 2.
Cs2CO3, [PdAHCTUNGTRENNNUG
The synthesis of 9-demethylneopeltolide (54) was easily
achieved by a standard B-alkyl Suzuki–Miyaura coupling
(Scheme 10). Thus, hydroboration of olefin 39 with 9-BBN-
H generated alkylborane 71, which was coupled with enol
We next evaluated the cytotoxic property of the correct
structure (2) of neopeltolide, the originally proposed struc-
phosphate
8 under the optimized conditions (aqueous
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H. Fuwa, M. Sasaki et al.
Scheme 10. Total synthesis of 9-demethylneopeltolide (54).
ture (1) of neopeltolide (that is, 11,13-bis-epi-neopeltolide),
11-demethoxyneopeltolide (53), 9-demethylneopeltolide
(54), and synthetic intermediates 3, 4, 28, 51, and 52 against
P388 murine leukemia cells, as summarized in Table 2. As
expected, synthetic 2 exhibited potent cytotoxicity with an
IC50 value of 0.899 nm. In contrast, synthetic 1, which has
the opposite configurations at the C11 and C13 stereogenic
centers, showed a tenfold lower activity than 2 (IC50
10.1 nm). The cytotoxicity of 11-demethoxy analogue 53 was
only fourfold lower than that of 2 (IC50 3.70 nm). These data
suggested that the change of the stereochemistry at the C13
stereogenic center and deletion of the C11 methoxy sub-
stituent are tolerated without significantly affecting the cyto-
toxicity. On the other hand, 9-demethyl analogue 54 exhibit-
ed cytotoxicity that is equipotent to or slightly more potent
than that of 2 (IC50 0.813 nm). Owing to its simplified struc-
ture and synthetic accessibility, 54 is an attractive starting
point for detailed investigations on the structure–activity re-
lationships and biological activities of 2. The synthetic inter-
mediates lacking the oxazole-containing side chain (3, 28,
51, and 52) were completely inactive at a concentration of
100 nm. These results clearly indicated that the highly unsa-
turated oxazole side chain is indispensable for the potent cy-
totoxicity of 2, which is in accordance with the very recent
report from Maier and co-workers.[5b] The oxazole-contain-
ing side chain 4 itself was also inactive at 100 nm,[38] suggest-
ing that the 14-membered macrolactone moiety is also im-
portant for the potent cytotoxicity.
Table 2. Cytotoxicity of synthetic 1, 2 and analogues against P388 murine
leukemia cells.
Compound
IC50 [nM]
Relative potency
1
2
3
4
28
51
52
53
54
10.1
0.0890
1
—
—
—
—
—
0.243
1.11
0.899
>100
>100
>100
>100
>100
3.70
0.813
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Evaluation of (+)-Neopeltolide and Its Analogues
FULL PAPER
corded on a JEOL JMS-700 spectrometer, and ESI-TOF mass spectra
were measured on a Bruker microTOFfocus spectrometer.
Conclusions
Enol phosphate 8: To a solution of acetate 18 (39.3 mg, 0.0846 mmol) in
THF (1 mL) were added HMPA (0.044 mL, 0.25 mmol) and diphenyl-
phosphoryl chloride (0.021 mL, 0.10 mmol). The resultant mixture was
cooled to À788C and treated with KHMDS (0.5m solution in toluene,
0.22 mL, 0.11 mmol). After being stirred at À788C for 25 min, the reac-
tion was quenched with 3% NH4OH and diluted with diethyl ether. The
resultant mixture was allowed to warm to room temperature over 20 min
and extracted with EtOAc. The organic layer was washed with brine,
dried (Na2SO4), filtered, and concentrated under reduced pressure. The
crude enol phosphate 8 was used immediately in the next reaction with-
out further purification.
In the present study, total synthesis of the originally pro-
posed (1) and correct (2) structures of (+)-neopeltolide has
been accomplished. Our newly developed Suzuki–Miyaura
coupling/ring-closing metathesis strategy has proven to be
highly efficient for the synthesis of the 2,4,6-trisubstituted
tetrahydropyran subunit of neopeltolide from readily avail-
able acyclic fragments. The convergent nature of our syn-
thetic strategy allowed us to elaborate two synthetic ana-
logues, 11-demethoxyneopeltolide (53) and 9-demethylneo-
peltolide (54), in a rapid and efficient manner. Biological
evaluation of synthetic 1, 2, 53, and 54 as well as several syn-
thetic intermediates elucidated the structure–activity rela-
tionships of the peripheral substituents of the 14-membered
macrolactone of neopeltolide. Our designed 9-demethylneo-
peltolide (54) was equipotent to or slightly more active than
the parent 2. In terms of synthetic efficiency, the simplified
analogue 54 represents a good starting point for further in-
vestigations on the structure–activity relationships. Thus, the
chemistry described herein lays the foundation for the
design and synthesis of even-more-potent structural ana-
logues and molecular probes for the investigation of the bio-
logical mode of action of neopeltolide. In addition, our
Suzuki–Miyaura coupling/ring-closing metathesis strategy
will find its use in the synthesis of structurally related
marine macrolide natural products. Further studies along
these lines are currently underway in our laboratory and
will be reported shortly.
Endocyclic enol ether 46: tBuLi (1.58m solution in pentane, 0.450 mL,
0.711 mmol) was added to a solution of iodide 34 (102.5 mg, 0.236 mmol)
and B-MeO-9-BBN (1.0m solution in hexane, 0.61 mL, 0.61 mmol) in di-
ethyl ether (2.3 mL) at À788C. The resultant mixture was stirred at
À788C for 5 min before it was diluted with THF (2.3 mL) and allowed to
warm to room temperature over 1 h. To this mixture was added 3m aque-
ous Cs2CO3 (0.236 mL, 0.708 mmol), and the resultant mixture was
stirred at room temperature for 15 min. To this mixture were added a so-
lution of enol phosphate 8 (prepared from 142 mg (0.306 mmol) of ace-
tate 18) in DMF (1 mL
+ 1.3 mL rinse) and [PdCAHTUNTGNRUEGN(PPh3)4] (27.3 mg,
0.0236 mmol). The resultant mixture was stirred at room temperature for
17 h. The mixture was diluted with H2O and extracted with EtOAc. The
organic layer was washed with brine, dried (Na2SO4), and concentrated
under reduced pressure. Purification of the residue by flash chromatogra-
phy on silica gel (2.5 to 5% EtOAc/hexane) gave enol ether 45 (206 mg).
This material was unstable so that it was immediately used in the next re-
action without further purification.
A solution of Grubbsꢁ 2nd-generation catalyst (20 mg, 0.024 mmol) in tol-
uene (7 mL) was added to a solution of the above material 45 (206 mg)
in toluene (40 mL). The resultant solution was stirred at 708C for 1 h.
The mixture was allowed to cool to room temperature under air for 1 h
and then concentrated under reduced pressure. Purification of the resi-
due by flash chromatography on silica gel (3 to 10% EtOAc/hexanes)
gave endocyclic enol ether 46 (133.3 mg, 78% from 34) as a colorless oil:
[a]2D0 =+16.7 (c=1.00 in benzene); 1H NMR (500 MHz, C6D6): d=7.38–
7.29 (m, 3H), 7.19 (d, J=7.5 Hz, 2H), 7.13–7.07 (m, 1H), 6.85 (d, J=
7.5 Hz, 2H), 4.85 (brs, 1H), 4.74 (ddd, J=7.0, 7.0, 7.0 Hz, 2H), 4.59 (s,
2H), 4.52 (d, J=6.0 Hz, 1H), 4.42 (d, J=6.0 Hz, 1H), 4.39 (m, 1H), 4.18
(m, 1H), 3.89 (m, 1H), 3.76 (m, 1H), 3.70 (m, 1H), 3.64 (m, 1H), 3.32 (s,
3H), 3.25 (s, 3H), 2.22–2.12 (m, 2H), 2.02 (dd, J=6.5, 17.5 Hz, 1H),
1.97–1.85 (m, 2H), 1.84–1.68 (m, 5H), 1.60 (m, 1H), 1.55–1.38 (m, 3H),
1.22 (ddd, J=5.5, 8.0, 13.5 Hz, 1H), 1.17–1.05 (m, 22H), 1.03 (d, J=
6.5 Hz, 3H), 0.93 ppm (t, J=7.0 Hz, 3H); 13C NMR (125 MHz, C6D6):
d=159.6, 155.2, 138.8, 132.0, 129.4 (2C), 128.5, 128.5, 128.3, 127.9, 127.6,
114.0 (2C), 100.2, 93.3, 76.1, 75.8, 71.6, 70.7, 69.7, 69.3, 59.8, 56.0, 54.7,
42.6, 41.8, 40.6, 38.7, 37.0, 35.4, 27.7, 20.3, 18.7, 18.3 (6C), 14.6, 12.3 ppm
Experimental Section
General methods: All reactions that were sensitive to moisture and/or air
were carried out under an atmosphere of argon in dry, freshly distilled
solvents under anhydrous conditions by using oven-dried glassware
unless otherwise noted. Anhydrous dichloromethane (CH2Cl2), dimethyl-
sulfoxide (DMSO), and N,N-dimethylformamide (DMF) were purchased
from Kanto Chemical Co. Inc. and used directly without further drying.
Anhydrous tetrahydrofuran, diethyl ether, and toluene were purchased
from Wako Pure Chemical Industries, Ltd. and further purified by a
Glass Contour solvent purification system under an atmosphere of argon
immediately prior to use. Hexamethylphosphoramide (HMPA) was dis-
tilled from calcium hydride under reduced pressure. Triethylamine, 2,6-
lutidine, and methanol were distilled from calcium hydride under an at-
mosphere of argon. Tetrakis(triphenylphosphine)palladium(0) was pre-
pared according to the literature procedure (D. R. Coulson, Inorg. Synth.
1972, 13, 121). All other chemicals were purchased at highest commercial
grade and used directly. Analytical thin-layer chromatography was per-
formed by using E. Merck silica gel 60 F254 plates (0.25 mm thickness).
Flash column chromatography was carried out by using Kanto Chemical
silica gel 60N (40–100 mesh, spherical, neutral) or Fuji Silysia silica gel
BW-300 (200–400 mesh). Optical rotations were recorded on a JASCO P-
1020 digital polarimeter. IR spectra were recorded on a JASCO FT/IR-
4100 spectrometer. 1H and 13C NMR spectra were recorded on a Varian
Unity INOVA-500 or INOVA-600 spectrometer, and chemical shift
values are reported in ppm (d) downfield from tetramethylsilane with
reference to the internal residual solvent [1H NMR, CHCl3 (7.24), C6HD5
(7.15), CHD2OD (3.31); 13C NMR, CDCl3 (77.0), C6D6 (128.0), CD3OD
(49.8)]. Coupling constants (J) are reported in Hertz (Hz). The following
abbreviations were used to designate the multiplicities: s=singlet; d=
doublet; m=multiplet; br=broad. EI and FAB mass spectra were re-
(3C); IR (film): n˜ =2940, 2866, 1670, 1613, 1513, 1462, 1094, 820 cmÀ1
;
HRMS (ESI): m/z: calcd for C43H70O7SiNa: 749.4783 [M+Na]+; found:
749.4759.
Tetrahydropyran 47: 10% Pd/C (6 mg) was added to a solution of endo-
cyclic enol ether 46 (29.9 mg, 0.0411 mmol) in EtOAc/MeOH (2:1, v/v,
1 mL). The resultant heterogeneous mixture was stirred vigorously at
room temperature under an atmosphere of hydrogen (0.8 MPa). After
5.5 h, the reaction mixture was filtered through Celite and the filtrate
was concentrated under reduced pressure. Purification of the residue by
flash chromatography on silica gel (5 to 10% EtOAc/hexanes) gave tetra-
hydropyran 47 (24.4 mg, 81%) as a colorless oil: [a]1D7 =+3.1 (c=2.00 in
CHCl3); 1H NMR (500 MHz, CDCl3): d=7.35–7.20 (m, 7H), 6.84 (d, J=
8.5 Hz, 2H), 4.80 (s, 2H), 4.59 (s, 2H), 4.46 (d, J=11.0 Hz, 1H), 4.36 (d,
J=11.0 Hz, 1H), 3.83–3.68 (m, 6H), 3.54 (m, 1H), 3.48–3.38 (m, 2H),
3.36–3.28 (m, 1H), 3.23 (s, 3H), 1.97–1.80 (m, 3H), 1.74–1.28 (m, 10H),
1.26–0.94 (m, 26H), 0.90 (d, J=7.5 Hz, 3H), 0.88 ppm (t, J=6.5 Hz, 3H);
13C NMR (125 MHz, CDCl3): d=159.0, 137.9, 131.2, 129.3 (2C), 128.4
(2C), 127.9 (2C), 127.7, 113.7 (2C), 92.3, 75.8, 75.5, 73.2, 72.9, 72.0, 70.4,
69.4, 59.6, 56.3, 55.2, 44.0, 42.6, 40.2, 39.6, 39.2, 38.8, 36.6, 25.8, 19.7, 18.3,
Chem. Eur. J. 2009, 15, 12807 – 12818
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www.chemeurj.org
12815

H. Fuwa, M. Sasaki et al.
18.0 (6C), 14.3, 11.9 ppm (3C); IR (film): n˜ =2941, 2866, 1613, 1514,
1H), 2.40 (dd, J=5.0, 15.0 Hz, 1H), 2.00 (m, 1H), 1.90 (m, 1H), 1.80–
1.50 (m, 4H), 1.50–1.40 (m, 3H), 1.40–1.26 (m, 2H), 1.26–1.13 (m, 3H),
1.09 (m, 1H), 0.92 (t, J=7.0 Hz, 3H), 0.87 ppm (d, J=6.5 Hz, 3H);
13C NMR (125 MHz, CDCl3): d=171.5, 137.8, 128.4 (2C), 127.8 (2C),
127.7, 92.5, 77.6, 73.2, 72.8, 72.2, 69.5, 68.5, 56.6, 51.6, 43.2, 41.12, 41.10,
40.0, 39.3, 38.8, 38.0, 25.9, 19.7, 18.8, 14.1 ppm; IR (film): n˜ =3489, 2932,
1740, 1376, 1149, 1091, 1040, 736, 698 cmÀ1; HRMS (ESI): m/z: calcd for
C27H44O7SiNa: 503.2979 [M+Na]+; found: 503.2965.
1463, 1247, 1093, 1041, 820, 736 cmÀ1
; HRMS (ESI): calcd for
C43H72O7SiNa: 751.4940 [M+Na]+; found: 751.4986.
Alcohol 48: To a solution of tetrahydropyran 47 (97.5 mg, 0.134 mmol) in
THF (1.5 mL) was added TBAF (1.0m solution in THF, 0.40 mL,
0.40 mmol). The resultant solution was stirred at room temperature for
80 min before it was concentrated under reduced pressure. Purification of
the residue by flash chromatography on silica gel (15 to 60% EtOAc/hex-
anes) gave alcohol 48 (74.3 mg, 97%) as a colorless oil: [a]1D7 =+14.8 (c=
1.00 in CHCl3); 1H NMR (500 MHz, CDCl3): d=7.35—7.20 (m, 7H),
6.85 (d, J=8.0 Hz, 2H), 4.80 (s, 2H), 4.59 (s, 2H), 4.47 (d, J=11.0 Hz,
1H), 4.36 (d, J=11.0 Hz, 1H), 3.81–3.68 (m, 6H), 3.56–3.44 (m, 2H),
3.44–3.35 (m, 2H), 3.23 (s, 3H), 2.68 (brs, 1H), 1.97–1.86 (m, 2H), 1.80–
1.62 (m, 3H), 1.62–1.10 (m, 12H), 0.90 (d, J=5.5 Hz, 3H), 0.88 ppm (t,
J=7.5 Hz, 3H); 13C NMR (125 MHz, CDCl3): d=159.0, 137.8, 131.1,
129.4 (2C), 128.4 (2C), 127.8 (2C), 127.7, 113.7 (2C), 92.4, 75.8, 75.7, 75.6,
73.8, 72.8, 70.5, 69.5, 61.1, 56.5, 55.2, 43.7, 42.6, 40.2, 38.8, 38.5, 37.9, 36.5,
26.2, 19.8, 18.3, 14.3 ppm; IR (film): n˜ =3457, 2932, 1514, 1248, 1087,
1039, 821, 698 cmÀ1; HRMS (ESI): m/z: calcd for C34H52O7SiNa: 595.3605
[M+Na]+; found: 595.3650.
TMSOK (31.8 mg, 0.246 mmol) was added to a solution of the above al-
cohol (39.4 mg, 0.0820 mmol) in diethyl ether (1 mL) at 08C. The resul-
tant solution was stirred at room temperature for 2 h 45 min. After cool-
ing to 08C, the mixture was acidified with 1m aqueous HCl (pH value of
approximately 4). The mixture was extracted with CHCl3, and the com-
bined organic layers were dried (Na2SO4) and concentrated under re-
duced pressure. Purification of the residue by flash chromatography on
silica gel (30 to 60% EtOAc/hexanes) gave the hydroxy acid 50 (38.2 mg,
100%) as a colorless oil: [a]1D7 =+11.9 (c=1.00 in CHCl3); 1H NMR
(500 MHz, CDCl3): d=7.36–7.25 (m, 5H), 4.80 (s, 2H), 4.59 (s, 2H), 3.98
(m, 1H), 3.84–3.72 (m, 2H), 3.51 (m, 1H), 3.39 (m, 1H), 3.33 (s, 3H),
2.50–2.40 (m, 2H), 2.02–1.85 (m, 3H), 1.81–1.69 (m, 2H), 1.56–1.17 (m,
10H), 1.08 (ddd, J=4.0, 8.5, 13.5 Hz, 1H), 0.90 (t, J=7.0 Hz, 3H),
0.88 ppm (d, J=6.5 Hz, 3H), (one proton missing due to the H/D ex-
change of the carboxylic acid proton); 13C NMR (125 MHz, CDCl3): d=
173.5, 137.7, 128.4 (2C), 127.9 (2C), 127.8, 92.5, 78.2, 75.6, 72.7, 72.5, 69.5,
68.8, 56.9, 43.7, 41.2, 40.6, 40.0, 39.0, 38.5, 37.9, 28.0, 20.7, 18.6, 14.1 ppm;
IR (film): n˜ =3460, 2932, 1728, 1455, 1378, 1149, 1090, 1040, 740,
Methyl ester 49: Et3N (0.086 mL, 0.62 mmol) and SO3·pyridine (79.6 mg,
0.500 mmol) were added to
a solution of alcohol 48 (71.3 mg,
0.124 mmol) in 1:1 CH2Cl2/DMSO (v/v, 2 mL) at 08C. The resultant solu-
tion was stirred at 08C for 0.5 h before it was diluted with diethyl ether.
The mixture was washed successively with 1m aqueous HCl, saturated
aqueous NaHCO3, and brine, dried (Na2SO4) and then concentrated
under reduced pressure. The residual aldehyde (77.2 mg) was used in the
next reaction without further purification.
699 cmÀ1
; HRMS (ESI): m/z: calcd for C26H42O7SiNa: 489.2828
[M+Na]À; found: 489.2807.
Macrolactone 51: Et3N (0.068 mL, 0.49 mmol) and 2,4,6-Cl3C6H2COCl
(0.051 mL, 0.33 mmol) were added to a solution of hydroxy acid 50
(38.2 mg, 0.0819 mmol) in THF (3 mL) at 08C. The resultant mixture was
stirred at room temperature for 1.5 h before it was diluted with toluene
(30 mL). This mixture was added dropwise to a solution of DMAP
(200 mg, 1.64 mmol) in toluene (100 mL) at 808C over a period of 7 h.
The resultant mixture was further stirred at 808C for 1 h. After cooling
to room temperature, the reaction mixture was washed successively with
1m aqueous HCl, saturated aqueous NaHCO3, and brine, dried (Na2SO4),
and concentrated under reduced pressure. Purification of the residue by
flash chromatography on silica gel (5 to 20% EtOAc/hexanes) gave mac-
rolactone 51 (36.8 mg, 100%) as a colorless oil: [a]1D8 =+16.7 (c=0.50 in
CHCl3); 1H NMR (500 MHz, CDCl3): d=7.36–7.26 (m, 5H), 5.17–5.09
(m, 1H), 4.80 (s, 2H), 4.59 (s, 2H), 3.81–3.67 (m, 2H), 3.55 (m, 1H), 3.29
(s, 3H), 2.58 (dd, J=4.5, 14.0 Hz, 1H), 2.41 (dd, J=10.0, 14.0 Hz, 1H),
1.96 (m, 1H), 1.89–1.78 (m, 2H), 1.68 (m, 1H), 1.60–1.52 (m, 2H), 1.52–
1.43 (m, 2H), 1.43–1.17 (m, 7H), 1.11 (m, 1H), 0.95 (d, J=6.5 Hz, 3H),
0.89 ppm (t, J=7.0 Hz, 3H); 13C NMR (125 MHz, CDCl3): d=170.8,
137.7, 128.5 (2C), 127.9 (2C), 127.8, 92.5, 78.7, 75.5, 73.2, 72.9, 72.4, 69.5,
56.3, 44.1, 42.3, 42.2, 40.0, 39.1, 38.0, 36.9, 31.2, 25.5, 19.0, 13.9 ppm; IR
(film): n˜ =2923, 1731, 1373, 1263, 1091, 1039, 737, 698 cmÀ1; HRMS
(ESI): m/z: calcd for C26H40O6Na: 471.2717 [M+Na]+; found: 471.2705.
2-Methyl-2-butene
(0.263 mL,
2.48 mmol),
NaH2PO4
(16.4 mg,
0.136 mmol), and NaClO2 (39.3 mg, 0.434 mmol) were added to a solu-
tion of the above material (77.2 mg) in tBuOH/H2O (5:1, v/v, 1.8 mL) at
08C. The resultant mixture was stirred at room temperature for 75 min.
The mixture was cooled to 08C and acidified with 1m aqueous HCl (pH
value of approximately 4). The resultant mixture was extracted with
CHCl3, and the combined organic layers were washed with brine, dried
(Na2SO4), and then concentrated under reduced pressure. The residual
acid (80.0 mg) was used in the next reaction without further purification.
TMSCHN2 (2.0m solution in hexane, 0.186 mL, 0.372 mmol) was added
to a solution of the above material (80.0 mg) in MeOH/benzene (1:1, v/v,
2 mL) at 08C. The resultant mixture was stirred at room temperature for
20 min before it was concentrated under reduced pressure. Purification of
the residue by flash chromatography on silica gel (10 to 30% EtOAc/hex-
anes) gave methyl ester 49 (66.1 mg, 89% for the three steps) as a color-
less oil: [a]1D7 =+16.4 (c=1.00 in CHCl3); 1H NMR (500 MHz, CDCl3):
d=7.35–7.24 (m, 7H), 6.85 (d, J=8.0 Hz, 2H), 4.79 (s, 2H), 4.59 (s, 2H),
4.47 (d, J=11.0 Hz, 1H), 4.36 (d, J=11.0 Hz, 1H), 3.84–3.74 (m, 4H),
3.70 (m, 1H), 3.63 (s, 3H) 3.54 (m, 1H), 3.44–3.32 (m, 2H), 3.22 (s, 3H),
2.54 (dd, J=7.5, 14.5 Hz, 1H), 2.38 (dd, J=5.0, 14.5 Hz, 1H), 2.00 (m,
1H), 1.90 (m, 1H), 1.80 (m, 1H), 1.60–1.30 (m, 8H), 1.25–1.05 (m, 4H),
0.89 (t, J=7.0 Hz, 3H), 0.86 ppm (d, J=6.5 Hz, 3H); 13C NMR
(125 MHz, CDCl3): d=171.5, 159.0, 137.8, 131.2, 129.3 (2C), 128.4 (2C),
127.8 (2C), 127.7, 113.7 (2C), 92.4, 75.7, 75.5, 73.3, 72.9, 72.2, 70.4, 69.5,
56.2, 55.2, 51.5, 43.6, 42.4, 41.1, 40.1, 38.8, 38.0, 36.6, 25.8, 19.4, 18.3,
14.3 ppm; IR (film): n˜ =2952, 1740, 1514, 1249, 1091, 1039, 821, 739,
699 cmÀ1; HRMS (ESI): m/z: calcd for C35H52O8SiNa: 623.3554 [M+Na]+
; found: 623.3517.
Alcohol 52: 20% Pd(OH)2/C (4.3 mg) was added to a solution of macro-
lactone 51 (8.6 mg, 0.019 mmol) in THF/MeOH (1:1, v/v, 2.0 mL). The re-
sultant homogeneous mixture was stirred vigorously at room temperature
under an atmosphere of hydrogen. After 40 min, the reaction mixture
was filtered through Celite, and the filtrate was concentrated under re-
duced pressure to give alcohol 52 (6.4 mg, 100%) as a colorless oil:
[a]1D8 =+18.3 (c=0.14 in CHCl3); 1H NMR (500 MHz, CDCl3): d=5.13
(m, 1H), 3.84–3.67 (m, 2H), 3.55 (m, 1H), 3.29 (s, 3H), 3.16 (m, 1H),
2.60 (dd, J=4.0, 14.5 Hz, 1H), 2.41 (dd, J=11.0, 14.5 Hz, 1H), 1.99–1.91
(m, 1H), 1.89–1.79 (m, 2H), 1.74–1.63 (m, 1H), 1.63–1.25 (m, 9H), 1.25–
1.07 (m, 3H), 0.97 (d, J=6.5 Hz, 3H), 0.89 ppm (t, J=7.5 Hz, 3H);
13C NMR (125 MHz, CDCl3): d=170.8, 78.6, 75.5, 73.2, 72.3, 68.1, 56.3,
44.0, 42.3, 42.2, 41.9, 40.7, 40.0, 36.9, 31.2, 25.6, 19.0, 13.9 ppm; IR (film):
n˜ =3423, 2923, 2870, 1731, 1457, 1247, 1089, 794 cmÀ1; HRMS (ESI): m/z:
calcd for C18H32O5Na: 351.2142 [M+Na]+; found: 351.2151.
Hydroxy acid 50: 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ;
8.5 mg, 0.038 mmol) was added to a solution of methyl ester 49 (20.5 mg,
0.0341 mmol) in CH2Cl2/pH 7 buffer (10:1, v/v, 1.1 mL) at 08C. The resul-
tant mixture was stirred at room temperature for 75 min before it was
quenched with saturated aqueous NaHCO3 at 08C. The mixture was ex-
tracted with EtOAc, and the organic layer was washed with brine, dried
(Na2SO4), and concentrated under reduced pressure. Purification of the
residue by flash chromatography on silica gel (10 to 50% EtOAc/hex-
anes) gave an alcohol (15.1 mg, 92%) as a colorless oil: [a]1D7 =+15.5 (c=
1.00 in CHCl3); 1H NMR (500 MHz, CDCl3): d=7.35–7.25 (m, 5H), 4.80
(s, 2H), 4.59 (s, 2H), 3.90–3.75 (m, 2H), 3.74–3.66 (m, 1H), 3.64 (s, 3H),
3.53 (m, 1H), 3.40–3.28 (m, 4H), 3.00 (brs, 1H), 2.54 (dd, J=8.5, 15.0 Hz,
(+)-Neopeltolide (2): DIAD (1.9m solution in toluene, 0.031 mL,
0.059 mmol) was added to a solution of alcohol 52 (6.4 mg, 0.020 mmol),
acid 4 (10.6 mg, 0.038 mmol), and Ph3P (15.3 mg, 0.0585 mmol) in ben-
12816
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ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 12807 – 12818

Evaluation of (+)-Neopeltolide and Its Analogues
FULL PAPER
zene (2.3 mL). The resultant mixture was stirred at room temperature for
1 h before it was concentrated under reduced pressure. Purification of
the residue by flash chromatography on silica gel (10 to 40% EtOAc/hex-
[6] S. K. Woo, M. S. Kwon, E. Lee, Angew. Chem. 2008, 120, 3286–
3288; Angew. Chem. Int. Ed. 2008, 47, 3242–3244.
[7] Part of this work has been communicated: H. Fuwa, S. Naito, T.
Goto, M. Sasaki, Angew. Chem. 2008, 120, 4815–4817; Angew.
Chem. Int. Ed. 2008, 47, 4737–4739.
[8] O. A. Ulanovskaya, J. Janjic, M. Suzuki, S. S. Sabharwal, P. T. Schu-
macker, S. J. Kron, S. A. Kozmin, Nat. Chem. Biol. 2008, 4, 418–424.
[9] I. Paterson, N. A. Miller, Chem. Commun. 2008, 4708–4710.
[10] R. Kartika, T. R. Gruffi, R. E. Taylor, Org. Lett. 2008, 10, 5047–
5050.
[11] T. Wangyang, P. E. Floreancig, Angew. Chem. 2009, 121, 4637–4641;
Angew. Chem. Int. Ed. 2009, 48, 4567–4571.
[12] For a review: J. Gallon, S. Reymond, J. Cossy, C. R. Chim. 2008, 11,
1463–1476.
[13] Application of the Suzuki–Miyaura coupling/ring-closing metathesis
strategy to the synthesis of spiroacetals has also been described by
us: H. Fuwa, M. Sasaki, Org. Lett. 2008, 10, 2549–2552.
[14] Isolation and structure elucidation: M. D’Ambrosio, A. Guerriero,
C. Debitus, F. Pietra, Helv. Chim. Acta 1996, 79, 51–60.
anes) gave (+)-neopeltolide 2 (7.0 mg, 61%) as a colorless oil: [a]D18
=
+26.8 (c=0.24 in CH3OH); 1H NMR (500 MHz, CD3OD): d=7.67 (s,
1H), 6.37 (ddd, J=8.0, 8.0, 12.0 Hz, 1H), 6.28 (ddd, J=2.5, 2.5, 12.0 Hz,
1H), 6.04 (ddd, J=6.0, 6.0, 11.5 Hz, 1H), 5.89 (app. d, J=12.0 Hz, 1H),
5.22–5.13 (m, 2H), 4.30 (d, J=4.5 Hz, 2H), 4.08 (m, 1H), 3.67 (brt, J=
9.0 Hz, 1H), 3.65 (s, 3H), 3.56 (brt, J=9.5 Hz, 1H), 3.28 (s, 3H), 3.03
(ddd, J=6.5, 8.0, 8.0 Hz, 2H), 2.72 (d, J=7.5 Hz, 2H), 2.69 (dd, J=4.0,
10.0 Hz, 1H), 2.29 (dd, J=10.0, 15.0 Hz, 1H), 1.86 (dd, J=11.5, 15.0 Hz,
1H), 1.82 (brd, J=11.5 Hz, 1H), 1.74–1.64 (m, 2H), 1.62–1.47 (m, 4H),
1.47–1.22 (m, 6H), 1.12 (ddd, J=1.5, 11.0, 12.5 Hz, 1H), 0.97 (d, J=
6.5 Hz, 3H), 0.94 ppm (t, J=7.0 Hz, 3H), (one proton missing due to the
H/D exchange); 13C NMR (125 MHz, CD3OD): d=173.1, 166.8, 161.9,
159.6, 150.0, 142.3, 139.2, 135.9, 129.3, 121.7, 115.9, 77.1, 77.0, 73.9, 71.3,
69.2, 56.4, 52.6, 45.2, 43.5, 43.2, 41.0, 37.9, 37.4, 36.2, 32.6, 29.0, 26.4, 26.0,
20.0, 14.1 ppm; IR (film): n˜ =3275, 2915, 2372, 1716, 1636, 1419, 1249,
1179, 1083 cmÀ1; HRMS (ESI): m/z: calcd for C31H46N2O9Na: 613.3096
[M+Na]+; found: 613.3111.
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Cytotoxicity assays: Cytotoxicity of each compound against P388 murine
leukemia cells was evaluated as the IC50 value by using the WST-8 (2-(2-
methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetra-
zolium monosodium salt) assay. P388 cells were cultured for 2–3 days in
10% FBS/RPMI1640 medium supplemented with 100 unitsmLÀ1 of peni-
cillin and 100 mgmLÀ1 of streptomycin (Pen Strep, Invitrogen, Carlsbad,
California), and maintained at 5% CO2/Air atmosphere in a CO2 incuba-
tor. The cells were harvested and the concentration was adjusted to be
50000 cellsmLÀ1. Cell suspension (198 mL) was distributed to wells of a
96-well microplate and 2 mL of a sample solution dissolved in 10% aque-
ous DMSO was added to each well. The mixture was cultured for 24–
48 h and then treated for 3 h with 5 mL of WST-8 (Cell Counting Kit-8,
Dojindo, Kumamoto, Japan). The UV absorbance at 405 nm and 650 nm
were measured by using a microplate reader (Microplate Reader Model
680, Biorad, Hercules, California). The relative absorbance was calculat-
ed by subtracting UV absorbance at 650 nm from that at 405 nm. The rel-
ative absorbance for 0% and 100% cell viability were obtained with
100 nm okadaic acid and 10% aqueous DMSO, respectively.
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Received: June 18, 2009
Published online: October 30, 2009
12818
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