Concise synthesis and biological assessment of (+)-neopeltolide and a 16-member stereoisomer library of 8,9-dehydroneopeltolide: Identification of pharmacophoric elements

Article abstract of DOI:10.1002/chem.201300664

We describe herein a concise synthesis of (+)-neopeltolide, a marine macrolide natural product that elicits a highly potent antiproliferative activity against several human cancer cell lines. Our synthesis exploited the powerful bond-forming ability and high functional group compatibility of olefin metathesis and esterification reactions to minimize manipulations of oxygen functionalities and to maximize synthetic convergency. Our findings include a chemoselective olefin cross-metathesis reaction directed by H-bonding, and a ring-closing metathesis conducted under non-high dilution conditions. Moreover, we developed a 16-member stereoisomer library of 8,9-dehydroneopeltolide to systematically explore the stereostructure-activity relationships. Assessment of the antiproliferative activity of the stereoisomers against A549 human lung adenocarcinoma, MCF-7 human breast adenocarcinoma, HT-1080 human fibrosarcoma, and P388 murine leukemia cell lines has revealed marked differences in potency between the stereoisomers. This study provides comprehensive insights into the structure-activity relationship of this important antiproliferative agent, leading to the identification of the pharmacophoric structural elements and the development of truncated analogues with nanomolar potency. SAR of (+)-neopeltolide: A modular synthetic route to (+)-neopeltolide, a potent antiproliferative marine macrolide, was established by exploiting the esterification/olefin metathesis strategy, and a 16-member stereoisomer library of 8,9-dehydeoneopeltolide was developed to elucidate the stereostructure- activity relationships (see figure). Copyright

Full text of DOI:10.1002/chem.201300664

DOI: 10.1002/chem.201300664
Concise Synthesis and Biological Assessment of (+)-Neopeltolide and a 16-
Member Stereoisomer Library of 8,9-Dehydroneopeltolide: Identification of
Pharmacophoric Elements
Haruhiko Fuwa,*[a] Masato Kawakami,[a] Kenkichi Noto,[a] Takashi Muto,[a] Yuto Suga,[a]
Keiichi Konoki,[b] Mari Yotsu-Yamashita,[b] and Makoto Sasaki[a]
Abstract: We describe herein a concise
synthesis of (+)-neopeltolide, a marine
macrolide natural product that elicits a
highly potent antiproliferative activity
against several human cancer cell lines.
Our synthesis exploited the powerful
bond-forming ability and high function-
al group compatibility of olefin meta-
thesis and esterification reactions to
minimize manipulations of oxygen
functionalities and to maximize syn-
thetic convergency. Our findings in-
clude a chemoselective olefin cross-
metathesis reaction directed by H-
bonding, and a ring-closing metathesis
conducted under non-high dilution con-
ditions. Moreover, we developed a 16-
member stereoisomer library of 8,9-de-
hydroneopeltolide to systematically ex-
plore the stereostructure–activity rela-
tionships. Assessment of the antiproli-
ferative activity of the stereoisomers
against A549 human lung adenocarci-
noma, MCF-7 human breast adenocar-
cinoma, HT-1080 human fibrosarcoma,
and P388 murine leukemia cell lines
has revealed marked differences in po-
tency between the stereoisomers. This
study provides comprehensive insights
into the structure–activity relationship
of this important antiproliferative
agent, leading to the identification of
the pharmacophoric structural ele-
ments and the development of truncat-
ed analogues with nanomolar potency.
Keywords: antiproliferative activi-
ty · macrocycles · stereochemistry ·
structure–activity relationships · to-
tal synthesis
Introduction
because of their complex structure, which contains multiple
stereogenic centers.
Naturally occurring macrocycles such as cyclic peptides and
polyketide macrolides represent a unique class of biological-
ly-relevant small molecules. They display multiple functional
groups with certain degrees of conformational flexibility
along their macrocyclic backbone. Hence, they have a more
accessible surface area compared with ordinary small organ-
ic molecules.[1] These characteristic features of macrocycles
allow them to effectively interact with the extended shallow
surface of target proteins and also make them potentially
useful compounds for modulating protein–protein interac-
tions.[1,2] Many naturally occurring or natural-product-de-
rived macrocycles are known to have drug-like physico-
chemical and pharmacokinetic properties, even though they
violate the Lipinski Rule of Five.[3] Nonetheless, macrocycles
have been less exploited as scaffolds in medicinal chemistry
The significance of the relationship between chirality and
biological activity of natural products and pharmaceuticals
has long been recognized.[4,5] Moreover, it has recently been
argued that the molecular complexity arising from the pres-
ence of sp3 hybridized carbon atoms and stereogenic centers
(i.e., shape diversity[6]) may be important for the success of
molecular probe development and drug discovery endeav-
ors.[7,8] It is known that the stereoisomerism of cyclic pepti-
des significantly affects their molecular topology, physico-
chemical property, and biological activity.[9] By analogy to
cyclic peptides, it is conceivable that the investigation of the
stereostructure–activity relationship (SSAR) of polyketide
macrolides may provide comprehensive information about
their structure–activity relationship (SAR) and may provide
opportunities for discovering new biologically active small
molecules. However, SSAR studies of natural polyketide
macrolides have rarely been reported, possibly because of
synthetic intractability.[10,11]
[a] Prof. Dr. H. Fuwa, M. Kawakami,+ K. Noto,+ T. Muto, Y. Suga,
Prof. Dr. M. Sasaki
Graduate School of Life Sciences, Tohoku University
2-1-1 Katahira, Aoba-ku, Sendai 980-8577 (Japan)
E-mail: hfuwa@m.tohoku.ac.jp
(+)-Neopeltolide (1, Figure 1) is a 14-membered marine
macrolide, isolated from a deep-water lithistid sponge that
belongs to the family Neopeltidae, by Wright and co-work-
ers.[12] The gross structure and relative stereochemistry were
assigned on the basis of extensive 2D NMR analyses. Later,
Panek[13] and Scheidt[14] independently revised the stereo-
chemical assignments of the C11 and C13 stereogenic cen-
ters and established the absolute configuration of 1 by
[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)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201300664.
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FULL PAPER
hydroneopeltolide
Figure 1).[18]
Our
(3a,
synthetic
avenue to 1 exploited olefin
metathesis[19] and esterification
reactions as the versatile and
robust key bond-forming proc-
esses to maximize synthetic
convergency; this also allowed
us to access various synthetic
analogues of 1 in a concise
manner. The preparation and
coupling
of
stereoisomeric
building blocks followed by the
construction of the macrocyclic
framework generated 16 stereo-
isomers of 8,9-dehydroneopel-
Figure 1. Structures of (+)-neopeltolide (1), (+)-leucascandrolide A (2), and (À)-8,9-dehydroneopeltolide
(3a).
chemical synthesis. A structurally related marine macrolide,
(+)-leucascandrolide A (2, Figure 1), has also been report-
ed.[15] (+)-Neopeltolide inhibits the proliferation of several
cancer cell lines, including P388 murine leukemia cells,
A549 human lung adenocarcinoma cells, and NCI-ADR-
RES human ovarian sarcoma cells, at nanomolar concentra-
tions and elicits antifungal activity against pathogenic yeast
Candida albicans with a minimum inhibitory concentration
value of 0.62 mgmLÀ1. Kozmin and co-workers showed that
mitochondrial complex III is a molecular target of 1.[16] The
characteristic structure and intriguing biological aspects of 1
attracted the interest of the synthetic community, and more
than ten total and formal syntheses of this natural product
have been reported to date.[17] Furthermore, the SAR of 1
has been investigated by the evaluation of synthetic ana-
logues. Previous studies proposed that the macrolactone and
oxazole-containing side chain are required for inhibiting the
proliferation of cancer cells.[14b,17f,o] Maier et al. provided a
detailed SAR analysis of the oxazole-containing side chain,
illustrating the importance of the distance between the mac-
rolactone and the oxazole ring.[17f] Recently, the Floreancig/
Day group described that the oxazole and methyl carbamate
present in the side chain are essential for maintaining potent
antiproliferative activity.[17o] These reports suggest that it
would be quite difficult to bring about structural alterations
of the oxazole-containing side chain without affecting the bi-
ological activity. By contrast, it has been shown that the
macrolactone tolerates structural changes to some extent.
As independently described by Maier,[17f] Scheidt,[14b] and
our group,[17j] some diastereomers of 1 (e.g., 5-epi-neopelto-
lide, 11-epi-neopeltolide, and 11,13-di-epi-neopeltolide)
show approximately 10- to 100-fold diminished activity com-
pared with 1. We have also shown that the C9 methyl group
can be omitted with minimal loss of activity.[17j] The Florean-
cig/Day group reported that the C8–C9 region tolerates
moderate structural modifications.[17o] However, the pharma-
cophoric elements of 1 still remain elusive, because previous
studies only evaluated a limited set of specific synthetic ana-
logues employing different cancer cell lines.[14b,16,17f,j,o]
tolide (3a). Assessment of the antiproliferative activity of
the stereoisomer library provided a systematic insight into
the SSAR of this intriguing natural product for the first
time and ultimately led to the identification of the pharma-
cophoric structural elements of 1 and the development of
truncated analogues with nanomolar activity.
Results and Discussion
Synthetic plan: A prerequisite for the efficient synthesis of
complex polyketide natural products is a high degree of ster-
eocontrol to establish asymmetric carbon centers and to
minimize oxygen functional group manipulations. With this
in mind, we planned our synthetic avenue toward 1 as sum-
marized in Scheme 1. As described before,[17c,j] we envi-
Herein, we describe the concise synthesis of 1 and the
construction of a 16-member stereoisomer library of 8,9-de-
Scheme 1. Proposed retrosynthesis of 1. BOM=benzyloxymethyl.
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H. Fuwa et al.
sioned the attachment of the oxazole-containing side chain
4[20] to the macrolactone domain of 1 at the final stage of
the total synthesis by Mitsunobu esterification.[21] The mac-
rolactone domain of 1 can in turn be obtained from diene
6a by a ring-closing olefin metathesis (RCM) reaction[19]
and stereoselective hydrogenation of the resultant macrocy-
cle 5a. However, RCM of 6a may not be straightforward
because several literature precedents describe the difficulties
associated with the formation of macrocyclic trisubstituted
olefins by RCM.[22] In addition, it was unclear at the outset
of this study whether the sterically encumbered styryl group
within 6a was sufficiently reactive to participate in RCM.
Nevertheless, we planned to accept the challenging nature
of RCM because the styryl group was considered indispen-
sable for the efficient synthesis of the tetrahydropyran frag-
ment (see below). On the other hand, we envisaged that the
stereoselective hydrogenation of the trisubstituted olefin
within 5a would provide 1 with the correct configuration at
the C9 stereogenic center because molecular modeling sug-
gested that the Re face of the trisubstituted olefin would be
sterically more hindered than the Si face. Diene 6a would
be derived from carboxylic acid 7 and alcohol 8 by esterifi-
cation. To minimize manipulations of oxygen functionalities,
such as oxidation/reduction and protection/deprotection se-
quences, we envisioned the synthesis of 7 from a,b-unsatu-
rated ester 9 by intramolecular oxa-conjugate cyclization
under thermodynamic control.[23] Ester 9 would be available
by olefin cross-metathesis[24] of homoallylic alcohol 10 with
methyl acrylate. Finally, 10 can be synthesized from trans-
cinnamaldehyde by asymmetric acetate aldol reaction.
Scheme 2. Synthesis of carboxylic acid 7. d.r. = diastereomer ratio, RT =
room temperature, DBU=1,8-diazabicycloACHTNUTGRNEUNG[5.4.0]undec-7-ene, TMS=tri-
methylsilyl, Mes=2,4,6-trimethylphenyl, Cy=cyclohexyl.
Synthesis of carboxylic acid 7: We devised a practical syn-
thesis of carboxylic acid 7 that hinged on the asymmetric
acetate aldol reaction and diastereoselective 1,3-anti reduc-
tion, as summarized in Scheme 2. The synthesis commenced
with the condensation of trans-cinnamaldehyde with a titani-
um enolate derived from chiral thiazolidinethione 11 under
Nagao conditions[25] to deliver alcohol 12 as an 11:1 mixture
(two steps). The methanolysis of the propionyl group using
K2CO3 in methanol at room temperature and concomitant
intramolecular oxa-conjugate cyclization provided tetrahy-
dropyran 17, albeit with low diastereoselectivity (cis/trans
1
1
of diastereomers (600 MHz H NMR analysis), from which
ꢀ2:1 by 600 MHz H NMR analysis). However, the expo-
the desired product was readily separated by flash column
chromatography using silica gel. Direct amidation of 12 with
MeONHMe·HCl and imidazole gave Weinreb amide 13.
The addition of allylmagnesium chloride to 13 provided b,g-
unsaturated ketone 14, which was subjected to Evans–Tish-
chenko 1,3-anti reduction (SmI2, EtCHO, THF, À108C)[26] to
yield alcohol 10 with greater than 20:1 diastereoselectivity
sure of this material to excess DBU in toluene at 1008C en-
riched the thermodynamically more stable 2,6-cis isomer
and allowed the isolation of stereochemically pure 17 in
53% overall yield from 16. Finally, the hydrolysis of the
methyl ester[29] afforded carboxylic acid 7 in quantitative
yield.
1
(600 MHz H NMR analysis). The olefin cross-metathesis of
Synthesis of alcohol 8: Alcohol 8 could be conveniently pre-
pared in a straightforward manner from commercially avail-
able (R)-epichlorohydrin (Scheme 3). The addition of 2-
lithio-1,3-dithiane gave epoxide 18,[30] which was treated
with EtMgBr in the presence of CuI to provide alcohol 19.
The subsequent protection of the hydroxy group as its MPM
ether 20 followed by removal of the dithiane furnished alde-
hyde 21, which was methallylated with methallyltrimethylsi-
lane and MgBr2·OEt2 under chelate-control[31] to afford ho-
moallylic alcohol 22 with approximately 15:1 diastereoselec-
10 with methyl acrylate under the influence of the Grubbs
second-generation catalyst (G-II)[27] proceeded without inci-
dent to preferentially afford a,b-unsaturated ester 15 over
the corresponding RCM product (not shown). The olefin
cross-metathesis would presumably be facilitated by an in-
tramolecular H-bonding between the C5 hydroxy group and
one of the two chlorine atoms of an intermediary ruthenium
alkylidene complex.[28] The protection of the C5 hydroxy
group (BOMCl, iPr2NEt, nBu4NI, (CH2Cl)2, 1208C under
microwave irradiation) gave BOM ether 16 in 68% yield
1
tivity, as estimated by 600 MHz H NMR. The methylation
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Synthesis and Biological Assessment of (+)-Neopeltolide
FULL PAPER
Scheme 3. Synthesis of alcohol 8. MPM=para-methoxyphenylmethyl,
DDQ=2,3-dichloro-5,6-dicyanobenzoquinone.
of the resultant hydroxy group and subsequent deprotection
of the MPM group by using DDQ yielded alcohol 8.
Completion of the total synthesis: With the requisite frag-
ments available, we focused our attention on their coupling
and the formation of the 14-membered macrocycle
(Scheme 4). The esterification of carboxylic acid 7 with alco-
hol 8 under Yamaguchi conditions[32] proceeded without in-
cident to give diene 6a.
We investigated the challenging RCM of diene 6a under
various conditions (Table 1). Preliminary experiments had
shown that the addition of 1,4-benzoquinone[33] was critical
for suppressing undesired olefin isomerization during RCM.
The treatment of 6a with G-II (10 mol%) in the presence of
Scheme 4. Completion of the total synthesis of 1. DMAP=4-dimethyla-
minopyridine, DIAD=diisopropyl azodicarboxylate.
1,4-benzoquinone in CH2Cl2 (1 mm) at 408C gave only a
trace amount of macrolactone 5a, and the starting material
6a was recovered in 86% yield (Table 1, entry 1). Thus, we
attempted to run the RCM under rigorous conditions. Al-
though we could isolate 5a in 42% yield (along with recov-
ered 6a in 41% yield), when RCM was performed in
(CH2Cl)2 (1 mm) at 808C, a portionwise addition of G-II
(15 mol%ꢁ4) was necessary because of the instability of
the catalytic species under these high temperature condi-
tions (Table 1, entry 2). Changing the solvent to toluene
(1 mm) and running the reaction at 1008C gave 5a in 50%
yield, but 6a was still recovered in 29% yield (Table 1,
entry 3). We found that the conversion of the RCM reaction
could be significantly improved by adding a toluene solution
of G-II (30 mol%) through a syringe pump over 6 h
(Table 1, entry 4). We were able to isolate 5a in 85% yield
by increasing the substrate concentration to 3 mm (Table 1,
entry 5). More importantly, RCM of 6a could be achieved
even under non-high dilution conditions (e.g., 10–40 mm
substrate concentrations; Table 1, entries 6 and 7). In con-
trast to the above result, the use of the second-generation
Hoveyda–Grubbs catalyst (HG-II)[34] was completely inef-
fective for the present RCM, presumably because of the
bulkiness of HG-II (Table 1, entry 8). After RCM, the
Table 1. Screening of reaction conditions.[a]
Entry
Catalyst
[mol%]
Conditions
Yield
[%]
RSM[b]
[%]
ACHTUNGTRENNUNG
1
2
3
4
5
6
7
8
G-II (10)
CH2Cl2 (1 mm), 408C
ACHTUGNTRENUN(NG CH2Cl)2 (1 mm), 808C
Trace
42
86
41
29
G-II (60)[c]
G-II (60)[c]
G-II (30)[d]
G-II (30)[d]
G-II (30)[e]
G-II (30)[d]
HG-II (10)
toluene (1 mm), 1008C
toluene (1 mm), 1008C
toluene (3 mm), 1008C
toluene (10 mm), 1008C
toluene (40 mm), 1008C
toluene (1 mm), 1008C
50
75
85
82
75
0
5
trace
trace
trace
88
[a] All reactions were performed in the presence of 1,4-benzoquinone
(0.2–0.6 equiv). [b] RSM=recovered starting material. [c] G-II was
added in four portions. [d] G-II was added as a toluene solution through
a syringe pump over 6 h. [e] G-II was added as a toluene solution
through a syringe pump over 12 h.
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H. Fuwa et al.
served significant amounts of dimer 25 and olefin cross-
metathesis product 26 as side products of the treatment of
23 with G-II, even under high dilution conditions (CH2Cl2
(3 mm), 408C). Despite the high catalyst loading required,
our finding provides the basis for practical RCM for the
construction of the neopeltolide macrolactone.
As shown in Scheme 4, the hydrogenation of macrolac-
tone 5a with concomitant hydrogenolysis of the BOM group
provided alcohol 27 in 93% yield as a single stereoisomer
1
(d.r.>20:1 determined by 600 MHz H NMR). As anticipat-
ed by preliminary molecular modeling, the hydrogenation of
the trisubstituted olefin occurred from the less hindered Si
face of the molecule. Finally, the Mitsunobu esterification
with the known acid 4[20] furnished 1 in 85% yield. The spec-
troscopic data and specific rotation value of synthetic 1 were
in full accordance with those of the natural product.[12]
Development of a 16-member stereoisomer library: Having
established the concise synthetic access to 1, our interest
turned to the SSAR of 1. However, it was not practical to
synthesize all 26 =64 possible stereoisomers of 1. We thought
it prudent to reduce the number of library members to a
manageable level with solution-phase parallel synthesis.
We focused our attention on stereoisomers of 8,9-dehy-
droneopeltolide (3a). Our previous study had shown that
deletion of the C9 methyl group did not affect the antiproli-
ferative activity of 1 against P388 murine leukemia cells, im-
plying that the C9 position would be structurally modifia-
ble.[17j] Furthermore, we planned to fix the relative stereo-
chemistry of the C3 and C7 substituents of the tetrahydro-
pyran in a cis formation, because it is known that 14-mem-
bered macrolide natural products usually contain 2,6-cis-
substituted tetrahydropyran.[35] Consequently, 16 stereoisom-
ers of 8,9-dehydroneopeltolide (i.e., 3a–p) were selected as
the candidates for our stereoisomer library (Table 2).
Scheme 5. Plausible explanation for the RCM of diene 6a.
minor C11 diastereomer that originated from methallylation
of 21 (Scheme 3) could be removed by flash column chro-
matography using silica gel.
Our rationale for the remarkable propensity of 6a to un-
dergo RCM is summarized in Scheme 5. Because the phenyl
group blocks the formation of ruthenium alkylidene species
such as A, RCM starts with metathesis between the 1,1’-dis-
ubstituted olefin and G-II to generate ruthenium alkylidene
B. It is conceivable that B undergoes neither dimerization
nor CM with styrene because the formation of tri- and tetra-
substituted olefins by olefin cross-metathesis using G-II is
known to be particularly difficult.[27] Accordingly, B reacts
intramolecularly to furnish the macrocycle 5a with the con-
comitant release of an equimolar amount of styrene. The
importance of the substitution pattern of the olefins was
demonstrated by the RCM of diene 23 (Scheme 6). We ob-
Table 2. Stereochemistry of (À)-8,9-dehydroneopeltolide (3a) and ster-
eoisomers 3b–p.
Compound
Stereochemistry
Compound
Stereochemistry
3a
3b
3c
3d
3e
3 f
3g
3h
3R,5S,7S,11S,13S
3R,5S,7S,11R,13S
3R,5S,7S,11S,13R
3R,5S,7S,11R,13R
3S,5R,7R,11R,13R
3S,5R,7R,11S,13R
3S,5R,7R,11R,13S
3S,5R,7R,11S,13S
3i
3j
3R,5R,7S,11S,13S
3R,5R,7S,11R,13S
3R,5R,7S,11S,13R
3R,5R,7S,11R,13R
3S,5S,7R,11R,13R
3S,5S,7R,11S,13R
3S,5S,7R,11R,13S
3S,5S,7R,11S,13S
3k
3l
3m
3n
3o
3p
Scheme 6. RCM of diene 23.
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Synthesis and Biological Assessment of (+)-Neopeltolide
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Scheme 7. Synthesis of 16 stereoisomers of 8,9-dehydroneopeltolide 3a–p. DMP=Dess–Martin periodinane, l-Selectride=lithium tri-sec-butyl-
(hydrido)borate.
ACHTUNGTRENNUNG
The synthesis of 8,9-dehydroneopeltolide stereoisomers
lectivity was uniformly moderate, and in some cases, axial
alcohols were obtained as the minor diastereomer. We could
not account for the observed diastereoselectivity; however,
Paterson et al. have reported a similar result in their total
synthesis of 2.[39] Finally, alcohols 29a–p were esterified with
4 under Mitsunobu conditions[21] to furnish 16 stereoisomers
of 8,9-dehydroneopeltolide (i.e., 3a–p). The stereochemistry
of 3a–p is summarized in Table 2. Note that 3e–h and 3m–p
are the enantiomers of 3a–d and 3i–l, respectively. Each
pair of enantiomers showed exactly the same spectroscopic
properties and a good match of specific rotation value
except for sign of rotation.[40] Each of the eight diastereom-
was carried out on the basis of the total synthesis of 1, as
summarized in Scheme 7. The readily available building
blocks, carboxylic acids 7/ent-7 and alcohols 8/ent-8 and 28/
ent-28, were sufficient for the synthesis of eight stereoisom-
ers of 8,9-dehydroneopeltolide (3a). The syntheses of ent-7
and ent-8 were achieved in much the same way as described
for 7 and 8, respectively.[36] Alcohols 8/ent-8 were trans-
formed into alcohols 28/ent-28 by the inversion of the C13
stereogenic center through the Mitsunobu reaction[21] [ i) p-
NO2C6H4CO2H, diethyl azodicarboxylate, Ph3P, THF, RT;
ii) aqueous NaOH, MeOH/THF, RT]. The esterfication of
all possible combinations of carboxylic acids 7/ent-7 with al-
cohols 8/ent-8 or 28/ent-28 produced dienes 6a–h in excel-
lent yields. The subsequent RCM under the influence of G-
II (30–60 mol%) in toluene at 1008C delivered macrolac-
tones 5a–h in good yields. Here we observed that the reac-
tivity of dienes 6a–h depends on their stereochemistry, and
some require a higher catalyst loading to complete RCM.
The removal of the BOM group of 5a–h under mild
acidic conditions[37] gave alcohols 29a–h. To prepare the ad-
ditional eight stereoisomers at the C5 stereogenic center,[38]
alcohols 29a–h were oxidized to provide the respective ke-
tones. We anticipated that the reduction of the ketones with
l-Selectride (THF, À908C) would exclusively yield the cor-
responding axial alcohols 29i–p. However, the diastereose-
1
ers 3a–d and 3i–l show H and 13C NMR spectra that are
easily distinguishable from those of other diastereomers.[41]
Antiproliferative activity and SSAR. Previous reports de-
scribe some inconsistent results for the antiproliferative ac-
tivity of 1. For example, Wright, Maier, and Kozmin have in-
dependently reported that 1 elicits potent antiproliferative
activity against A549 cells,[12,16,17f] whereas Scheidt has claim-
ed that 1 is inactive against the same cell line.[14b] According-
ly, we first examined the effect of 1 on A549 cells and found
that cell viability was not only dose-dependent but also
strongly time-dependent, as shown in Figure 2A. At 48 and
72 h, cell viability did not fall below 75 and 60%, respective-
ly, even at higher concentrations. The inhibition curve gave
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H. Fuwa et al.
Table 3. Antiproliferative activity (IC50 [mm]) of 3a–p against A549,
MCF-7, HT-1080, and P388 cells.[a]
Compound
A549
MCF-7
HT-1080
0.00051
P388
3a
3b
3c
3d
3e
3 f
3g
3h
3i
0.00050
0.0055
0.0097
0.22
0.18
0.22
0.059
0.22
2.8
0.033
0.00072
0.0022
0.0077
0.076
0.054
0.069
0.016
0.036
9.9
0.019
0.055
0.68
0.55
0.49
0.15
0.46
0.031
0.044
0.37
0.22
0.43
0.098
0.25
5.1
>10
3j
3k
3l
3m
3n
3o
3p
0.60
6.3
5.8
7.5
1.9
0.71
7.0
8.4
0.67
2.6
0.40
1.8
1.1
0.93
0.66
0.55
>10
4.1
>10
>10
2.5
>10
>10
>10
1.4
6.8
1.5
1.6
[a] Antiproliferative activity was evaluated by the WST-8 colorimetric
assay. Assays were performed in triplicate, at least three times. For de-
tails of assay procedure, see the Supporting Information.
3.55, 3.36, and 1.14 mm, respectively. Stereoisomers 3a–p
showed differential growth inhibitory activity against each
of the four cell lines. It appeared that A549, MCF-7, and
HT-1080 cells were more sensitive than P388 cells toward
stereochemical alterations of the macrolactone domain. The
IC50 values for MCF-7 cells were generally higher than those
of A549 and P388 cells.
It is clear that the stereoisomers with an axially oriented
C5 oxazole-containing side chain on the tetrahydropyran
ring (i.e., 3a–h) were considerably more potent than the cor-
responding C5 epimers (i.e., 3i–p), regardless of the stereo-
chemistry of the macrolactone domain. Our observation is
in line with that of Maier et al.,[17f] who reported that 5-epi-
neopeltolide is approximately 30-fold less active than the
parent natural product against A549 cells, although in our
case, 3i was 5600 times less active than 3a against the same
cell line. Because the tetrahydropyran and oxazole are
linked with a flexible hinge, we were intrigued by the find-
ing that the “5-epi” series of stereoisomers showed only mi-
cromolar activity.
Another general observation is that stereoisomers con-
taining a (3R,5S,7S)-configured tetrahydropyran (i.e., 3a–c)
are more active than the respective enantiomers (i.e., 3e–g).
A remarkable difference in potency between 3a and its
enantiomer 3e was observed (approximately 1000-fold for
A549 and HT-1080, 17-fold for MCF-7, and 70-fold for P388
cells). This result was unexpected because both enantiomers
of 2 have been reported to elicit comparable growth inhibi-
tory activity against A549 cells.[16] In contrast, 3d and its
enantiomer 3h showed almost equal activity.
Figure 2. Growth inhibition curves of 1. Incubation time: red, 48 h; blue,
72 h; green, 96 h; purple, 120 h. (A) A549 cells. (B) MCF-7 cells.
(C) P388 cells.
a typical sigmoidal pattern upon extending the incubation
time to 96 or 120 h, although 1 still did not completely elimi-
nate the cell viability. The same trend was observed for
MCF-7 human breast adenocarcinoma cell line (Figure 2B).
Myxothiazol, a mitochondrial complex I inhibitor, has been
recently reported to show similar time- and dose-dependent
antiproliferative activity against mouse P815 mastocytoma
cells.[42] By contrast, a sigmoidal inhibition curve was ob-
tained upon incubation of P388 cells with varying concentra-
tions of 1 for 48 h (Figure 2C). From these experiments, we
established suitable culture conditions for each cell line, and
the IC50 values of 1 against A549, MCF-7, and P388 cells
were determined to be 1.6, 19, and 0.50 nm, respectively.
The antiproliferative activity of 16 stereoisomers of 8,9-
dehydroneopeltolide (3a–p) was next evaluated; the results
are summarized in Table 3. The average IC50 values of 3a–p
against A549, MCF-7, HT-1080, and P388 cells were 1.81,
Among stereoisomers 3a–d, the most potent was 3a. Al-
though 3b and 3c still retained appreciable potency, 3d
showed a significantly reduced activity. These results indi-
cate that the C11 and C13 stereogenic centers have a syner-
gistic rather than additive effect on the activity of 3a–d. On
the other hand, stereoisomers 3e–h inhibited the prolifera-
8106
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Chem. Eur. J. 2013, 19, 8100 – 8110

Synthesis and Biological Assessment of (+)-Neopeltolide
FULL PAPER
tion of the cancer cell lines with comparable potency regard-
less of the C11 and C13 configurations, and their activities
were also similar to that of 3d. Because the overall shapes
of the macrolactone domains of 3d–h must be different
from each other, these biological results point to the possi-
bility that the macrocyclic structure is not a prerequisite for
activity. Meanwhile, in previous studies,[14b,17j] the oxazole-
containing side chain alone did not exhibit antiproliferative
activity against P388 cells. We postulate that the tetrahydro-
pyran ring and oxazole-containing side chain comprise the
minimal structure responsible for the nanomolar activity.
Next, we designed and evaluated a series of structurally
simplified analogues to further our understanding of the
SAR of 1 (Figure 3).[43] In our previous study, the antiproli-
Although recognizing the importance of the C13 n-propyl
group, we were encouraged by the fact that 31 still showed
submicromolar activity and designed a further series of trun-
cated analogues 32–36. Gratifyingly, 32 inhibited the growth
of A549 cells at a single-digit micromolar concentration,
showing that our postulate drawn from the SSAR analysis
that the macrocyclic backbone is not essential for the activi-
ty of 1 is in fact accurate. Moreover, increasing the length of
the ester group of 32 was beneficial for potentiating activity.
In particular, n-hexyl ester 34 elicited growth inhibitory ac-
tivity against A549 cells at a nanomolar concentration,
being even more potent than most of the stereoisomers of
8,9-dehydroneopeltolide (see Table 3). It appears that the n-
hexyl group of 34 mimics the C13 n-propyl side chain of 1.
Although further biological investigations are required to
address whether 1 and 34 actually demonstrate the same
mechanism of action, these results are in accordance with
our postulate that the C13 n-propyl group is important for
reinforcing the antiproliferative activity of 1.
This study identified the pharmacophoric elements of 1,
as summarized in Figure 4. The C5 oxazole-containing side
chain must be axially disposed on the tetrahydropyran ring
to exhibit nanomolar activity. The absolute configuration of
Figure 3. Structures and antiproliferative activity (IC50 values for the
A549 cell line) of structurally simplified analogues.
Figure 4. Summary of SAR.
ferative activity of 9-demethylneopeltolide and 11-deme-
thoxyneopeltolide against P388 cells was found to be virtual-
ly comparable with that of 1.[17j] On the basis of our previous
results and the SSAR analysis discussed above, we designed
compounds 30 and 31 to determine whether the peripheral
C9, C11, and C13 substituents can be removed from the
macrocyclic backbone. Significantly, the growth inhibitory
activity of 30 against A549 cells was comparable to that of 1
and 3a–c and approximately 40 times more potent than 3d.
Thus, it is most likely that the C9 and C11 substituents are
not specifically involved in the target binding, but the con-
figuration of the C11 and C13 stereogenic centers is impor-
tant for defining the overall shape of the macrocyclic back-
bone. Meanwhile, 31 showed an approximately 40-fold re-
duced activity compared with 30. This and the above results
indicate that the C13 n-propyl group substantially potenti-
ates the activity of 1 and that the macrocyclic backbone ap-
pears to control the orientation of the C13 n-propyl group,
thus allowing it to acquire low nanomolar potency.
the tetrahydropyran domain is crucial for inhibiting cancer
cell growth at low nanomolar concentrations. The macrocy-
clic backbone controls the overall conformation and orienta-
tion of the C13 n-propyl group, making it suitable for ac-
quiring low nanomolar potency. On the other hand and in
line with previous reports,[17j,o] the C9–C11 domain tolerates
structural modifications and is possibly not involved in
target binding. This observation appears reasonable when
one considers the structural difference between 1 and 2.[44]
Most importantly, and in opposition to previous studies, the
minimal structure required for activity was identified to be
the tetrahydropyran ring appended with the oxazole-con-
taining side chain.
Chem. Eur. J. 2013, 19, 8100 – 8110
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
8107

H. Fuwa et al.
(Nos. 24102507 and 23102016) from the Ministry of Education, Culture,
Sports, Science and Technology (MEXT), Japan, and by the Novartis
Foundation (Japan) for the Promotion of Science.
Conclusion
We have achieved a concise synthesis of a naturally occur-
ring antiproliferative agent, (+)-neopeltolide, in which
olefin metathesis and esterification reactions were exploited
as the key bond-forming processes. We took advantage of
the high chemoselectivity and wide functional group toler-
ance of these transformations to minimize manipulations of
oxygen functionalities.
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preparation of stereoisomeric building blocks, carboxylic
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We thank Ms. Asami Saito (Tohoku University) for her contribution to
the initial stage of this work. This work was supported in part by a
Grant-in-Aid for Young Scientists (A) (No. 23681045) and a Grant-in-
Aid for Scientific Research (A) (No. 21241050) from the Japan Society
for the Promotion of Science (JSPS), by Grants-in-Aid for Scientific Re-
search on Innovative Areas “Chemical Biology of Natural Products”
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FULL PAPER
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Received: February 20, 2013
Published online: April 19, 2013
8110
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Chem. Eur. J. 2013, 19, 8100 – 8110