Total synthesis and structure-activity investigation of the marine natural product neopeltolide

Article abstract of DOI:10.1021/ja904604x

The total synthesis and biological evaluation of neopeltolide and analogs are reported. The key bond-forming step utilizes a Lewis acid-catalyzed intramolecular macrocyclization that installs the tetrahydropyran ring and macrocycle simultaneously. Independent of each other, neither the macrolide nor the oxazole side chain substituents of neopeltolide can inhibit the growth of cancer cell lines. The biological data of the analogs indicate that alterations to either the ester side chain or the stereochemistry of the macrolide result in a loss of biological activity.

Full text of DOI:10.1021/ja904604x

Published on Web 08/06/2009
Total Synthesis and Structure-Activity Investigation of the
Marine Natural Product Neopeltolide
Daniel W. Custar,† Thomas P. Zabawa,† John Hines,‡ Craig M. Crews,‡ and
Karl A. Scheidt*,†
Department of Chemistry, Center for Molecular InnoVation and Drug DiscoVery, Northwestern
UniVersity, EVanston, Illinois, 60208, and Department of Molecular, Cellular, and
DeVelopmental Biology, Department of Chemistry, and Department of Pharmacology, Yale
UniVersity, New HaVen, Connecticut 06520
Received June 11, 2009; E-mail: scheidt@northwestern.edu
Abstract: The total synthesis and biological evaluation of neopeltolide and analogs are reported. The key
bond-forming step utilizes a Lewis acid-catalyzed intramolecular macrocyclization that installs the
tetrahydropyran ring and macrocycle simultaneously. Independent of each other, neither the macrolide
nor the oxazole side chain substituents of neopeltolide can inhibit the growth of cancer cell lines. The
biological data of the analogs indicate that alterations to either the ester side chain or the stereochemistry
of the macrolide result in a loss of biological activity.
Introduction
Background. Natural products continue to be excellent
sources for new drug candidates, especially in the area of
anticancer therapeutics.1-3 In many instances, the limited
amount of natural material that is typically isolated severely
limits the ability of the cancer research community to investigate
important lead compounds. Organic synthesis can facilitate the
preparation of sufficient quantities of target compounds when
natural sources are scarce or no longer available. Additionally,
synthetic routes to molecules can be manipulated to create
simplified analogs of the target compound that may have the
same or enhanced biological activity. Total synthesis can also
confirm the structural assignment and absolute stereochemistry
of a natural product, which was required for neopeltolide 1.4
In 2007, Wright and co-workers reported the structure of a
new, highly cytotoxic, natural product neopeltolide 2, which
was isolated from sponges most closely related to the genus
Daedalopelta Sollas.5 The key structural features of neopeltolide
include a trisubstituted 2,6-cis-tetrahydropyran moiety, which
is embedded in a 14-membered macrolactone containing six
stereogenic centers. The pyran subunit possesses a highly
functionalized oxazole substituent that is identical to the side
chain of leucascandrolide A. The molecular formula and tricyclic
nature of the molecule were determined by high resolution mass
spectrometry (HRMS) and 13C NMR spectroscopy. The planar
Figure 1. Neopeltolide structures.
structure was elucidated by the analysis of one (1D)- and two-
1
dimensional (2D) H NMR spectroscopy, including advanced
TOCSY and COSY experimentation. The relative stereochem-
istry of neopeltolide was originally proposed based on 1D and
2D NOESY spectra, which ultimately lead to the incorrect
structural assignment. The key protons on the macrolide (H-3,
H-7, H-9, H-11, and H-13) were assigned to all be syn in
relationship with each other, all adopting a pseudoaxial orienta-
tion (Figure 1). The absolute stereochemistry of the molecule
could not be initially established due to the lack of available
material.
† Northwestern University.
‡ Yale University.
(1) Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2004, 67, 1216–1238.
(2) Newman, D. J.; Cragg, G. M. Curr. Med. Chem. 2004, 11, 1693–
1713.
(3) Newman, D. J.; Cragg, G. M.; Snader, K. M. J. Nat. Prod. 2003, 66,
1022–1037.
(4) Custar, D. W.; Zabawa, T. P.; Scheidt, K. A. J. Am. Chem. Soc. 2008,
130, 804–805.
Neopeltolide potently inhibits proliferation of a number of
cancer cell lines. Although it exhibits IC50 values in the low
nanomolar to subnanomolar range, the efficacy with which
(5) Wright, A. E.; Botelho, J. C.; Guzman, E.; Harmody, D.; Linley, P.;
McCarthy, P. J.; Pitts, T. P.; Pomponi, S. A.; Reed, J. K. J. Nat. Prod.
2007, 70, 412–416.
9
12406 J. AM. CHEM. SOC. 2009, 131, 12406–12414
10.1021/ja904604x CCC: $40.75  2009 American Chemical Society

Marine Natural Product Neopeltolide
A R T I C L E S
Scheme 1. Synthetic Plan
Scheme 2. Scandium(III) Triflate Methodology
neopeltolide inhibits cancer cell growth varies, with some cell
lines completely inhibited and others only partially inhibited.5,6
Based on that limited efficacy and data showing an accumulation
of cells in the G1 phase, Wright hypothesized that neopeltolide
may be at least partially cytostatic to these cell lines rather than
simply cytotoxic. Additionally, neopeltolide is also a potent
inhibitor of the fungal pathogen Candida albicans (MIC value
of 0.625 µg/mL), which severely and adversely affects the health
of AIDS patients, potentially resulting in death. Preliminary
investigations into the mechanism of action suggest that it does
not interact with either tubulin or actin. Recently, Kozmin, Kron
and co-workers have advanced that the cellular target of
neopeltolide is the cytochrome bc1 complex and that the
molecule inhibits mitochondrial ATP synthesis. However, their
investigations seemingly employed racemic material and it is
unclear if and how this aspect impacts their biological studies.6
The macrolide ring of neopeltolide has similarities to the
macrocycles found in the callipeltosides,7 lyngbyaloside,8,9
lyngbouilloside,10 and the aurisides.11 The key distinction
between these natural products is the C-7 hemiketal functionality
as opposed to the reduced ether of the tetrahydropyran ring
system present in neopeltolide. Additionally, several of these
molecules exhibit modest to slight cytotoxicity as opposed to
the high biological activity of neopeltolide. Leucascandrolide
A is also structurally similar and has comparable activity to
neopeltolide.12 Preliminary biological studies of leucascandrolide
A have shown that: (i) the macrolide core is essential for tumor
cytotoxicity, and (ii) the oxazole side chain is important for
fungistatic activity. Due to the interesting structural aspects, as
well as the high biological activity, neopeltolide has garnered
the attention of the synthetic community and has resulted in
several other total syntheses.6,13-19
Synthesis Plan. An important goal of our synthesis was to
develop a highly convergent and flexible synthetic route with
the ability to easily prepare analogs. Excision of the oxazole
side chain affords macrocyclic core 3 (Scheme 1), which
contains an embedded tetrahydropyran that we envisioned
forming through our previously developed Lewis acid-catalyzed
cyclization.20 This methodology allows for a highly diastereo-
selective (>20:1 dr) cyclization of ꢀ-hydroxy dioxinone 9 with
aldehyde 10 in the presence of a catalytic amount of Sc(OTf)3
to furnish bicyclic heterocycle 11 (Scheme 2). Heating 11 in
the presence of H2O resulted in the formation of a 2,6-cis-
tetrahydropyranone. Notably, our studies have indicated that the
use of optically enriched ꢀ-hydroxy dioxinone 9 in the cycliza-
tion results in complete retention of optical purity. Utilizing this
methodology, the tetrahydropyran core could be constructed as
a single diastereomer in two different manners: (1) through an
(6) Ulanovskaya, O. A.; Janjic, J.; Suzuki, M.; Sabharwal, S. S.;
Schumacker, P. T.; Kron, S. J.; Kozmin, S. A. Nat. Chem. Biol. 2008,
4, 418–424.
(13) Youngsaye, W.; Lowe, J. T.; Pohlki, F.; Ralifo, P.; Panek, J. S. Angew.
Chem., Int. Ed. 2007, 46, 9211–9214.
(7) Zampella, A.; DAuria, M. V.; Minale, L.; Debitus, C.; Roussakis, C.
J. Am. Chem. Soc. 1996, 118, 11085–11088.
(14) Fuwa, H.; Naito, S.; Goto, T.; Sasaki, M. Angew. Chem., Int. Ed. 2008,
47, 4737–4739.
(8) Luesch, H.; Yoshida, W. Y.; Harrigan, G. G.; Doom, J. P.; Moore,
R. E.; Paul, V. J. J. Nat. Prod. 2002, 65, 1945–1948.
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J. Nat. Prod. 1997, 60, 1057–1059.
(15) Paterson, I.; Miller, N. A. Chem. Commun. 2008, 4708–4710.
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(17) Woo, S. K.; Kwon, M. S.; Lee, E. Angew. Chem., Int. Ed. 2008, 47,
3242–3244.
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925–928.
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(19) Tu, W.; Floreancig, P. E. Angew. Chem., Int. Ed. 2009, 48, 4567–
4571.
(11) Sone, H.; Kigoshi, H.; Yamada, K. J. Org. Chem. 1996, 61, 8956–
8960.
(12) DAmbrosio, M.; Guerriero, A.; Debitus, C.; Pietra, F. HelV. Chim.
Acta 1996, 79, 51–60.
(20) Morris, W. J.; Custar, D. W.; Scheidt, K. A. Org. Lett. 2005, 7, 1113–
1116.
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A R T I C L E S
Custar et al.
Table 1. Optimization of Aldol Reactiona
Scheme 3. Dioxinone Fragment Synthesisa
entry
solvent
R-BINOL (mol %)
additive
yield (%)b
ee (%)c
1
2
3
4
5
6
7
8
9
THF
THF
THF
toluene
CH2Cl2
THF
THF
THF
THF
THF
THF
THF
8
8
4 Å MS
4 Å MS
4 Å MS
4 Å MS
4 Å MS
3 Å MSf
3 Å MS
3 Å MSf,g
4 Å MSg
4 Å MSg
4 Å MS
4 Å MS
50
53
60
30
-
65
66
65
57
66
63
64
73d,e
71d
78d,e
71
-
68
63
81
74
80
10
10
10
10
10
10
10
15
10
15
a Conditions: (a) Ti(i-OPr)4, (R)-BINOL, 4 Å sieves, THF, 63%, 88%
ee. (b) TBSOTf, 2,6-lutidine, CH2Cl2, 91%. (c) pPTs, MeOH, 83%. (d)
PDC, DMF, 97%.
10
11
12
88
60
little to improve the yield or enantioselectivity. In the end we
observed that 10 mol % Ti(i-OPr)4, 10 mol % (R)-BINOL,
flame-dried powdered 4 Å molecular sieves and a higher
precomplexation concentration (0.5 M) resulted in our best
results (Table 1, entry 11). The resultant ꢀ-hydroxy dioxinone
was converted to a bis-silyl ether and a selective deprotection
of the primary TBS-ether with pPTs was accomplished to furnish
primary alcohol 16 (Scheme 3). Oxidation to the carboxylic acid
was achieved with PDC in a 97% yield.
Alcohol Fragment. We next turned our attention toward the
synthesis of the alcohol fragment, which began with a Noyori
reduction of ethyl 3-oxohexanoate 17 (Scheme 4>).32,33 Using
(R)-tol-BINAP as the chiral ligand, the C-13 stereocenter was
set, affording the ꢀ-hydroxyester in 94% yield and 97% ee. The
ester was then converted to the Weinreb amide followed by
PMB protection of the secondary alcohol to give rise to amide
18. Next, iodide 19, which was furnished by using Myers’
pseudoephedrine controlled alkylation methodology,34 under-
went lithium-halogen exchange and addition to Weinreb amide
18.35 The yield of this reaction was moderate as a result of a
competing elimination of the PMB-alcohol to produce the R,ꢀ-
unsaturated ketone. Various conditions were attempted to render
the reaction less basic (i.e., CeCl3),36 unfortunately the yield
remained moderate. The PMB protecting group was removed
using DDQ and the resulting ꢀ-hydroxy ketone underwent an
Evans-Tishchenko anti-reduction with SmI2 and benzaldehyde.37
The anti-relationship was confirmed by Rychnovsky’s 13C NMR
spectroscopic analysis.38,39 Methylation of the secondary alcohol
and subsequent hydrolysis of the benzyl ester yielded alcohol
8 to complete the second main fragment.
a Reactions performed under N2 at 0.17
M (precomplexation
concentration 0.53 M) with Ti(i-OPr)4 and ligand after 60 min, reaction
cooled to -78 °C followed by addition of 13 and 14. b Isolated yields
after column chromatography. c Determined by chiral HPLC. d Precom-
plexation concentration 0.21 M. e Slowly warmed to 23 °C. f MS pellets
(3 Å). g Dried in a reduced pressure oven at 145 °C for 2 days.
intermolecular cyclization followed by a Yamaguchi macrolac-
tonization to create the macrocycle,21 or (2) through a new
intramolecular cyclization that would simultaneously form the
macrocycle. At the time of our initial publication, we were aware
of only a single report in the literature in which a Prins
cyclization was employed to provide a pyran-containing mac-
rocycle.22 Since we reported the synthesis of neopeltolide several
other research groups have successfully utilized the Prins
cyclization strategy as a key step in their syntheses.17,23-26 The
precursors to both cyclization pathways are carboxylic acid 7
and alcohol 8.
Results and Discussion
Carboxylic Acid Fragment. The synthesis began with mono-
protection of 1,3-propanediol as a silyl ether followed by
subsequent oxidation with TPAP27 to afford aldehyde 13, the
precursor to the vinylogous aldol reaction. Using the procedure
as reported by Scettri and co-workers,28,29 the aldol reaction
with aldehyde 13 and dienoxy silane 1430,31 resulted in moderate
yields and enantioselectivities (Table 1, entries 1 and 2). Varying
the solvent from THF and trying alternative drying agents did
(21) Inanaga, J.; Hirata, K.; Saeki, H.; Katsuki, T.; Yamaguchi, M. Bull.
Chem. Soc. Jpn. 1979, 52, 1989–1993.
Oxazole Fragment. The synthesis of oxazole 4 was based
upon the routes independently reported by Leighton40 and
Kozmin,41 with minor modifications. Carbamate 23 was pre-
pared from the addition of propargylamine 22 to methyl
chloroformate, followed by a carboxylation and subsequent
(22) Schulteelte, K. H.; Hauser, A.; Ohloff, G. HelV. Chim. Acta 1979, 62,
2673–2680.
(23) Bahnck, K. B.; Rychnovsky, S. D. J. Am. Chem. Soc. 2008, 130,
13177–13181.
(24) Boeckman Jr, R. K.; Potenza, J. C.; Enholm, E. J. J. Org. Chem. 1987,
52, 469–472.
(25) Hoye, T. R.; Danielson, M. E.; May, A. E.; Zhao, H. Angew. Chem.,
Int. Ed. 2008, 47, 9743–9746.
(32) Noyori, R.; Ohta, M.; Hsiao, Y.; Kitamura, M.; Ohta, T.; Takaya, H.
J. Am. Chem. Soc. 1986, 108, 7117–7119.
(26) Wender, P. A.; DeChristopher, B. A.; Schrier, A. J. J. Am. Chem.
Soc. 2008, 130, 6658–6659.
(33) Deng, L. S.; Huang, X. P.; Zhao, G. J. Org. Chem. 2006, 71, 4625–
4635.
(27) Ley, S. V.; Norman, J.; Griffith, W. P.; Marsden, S. P. Synthesis 1994,
639–666.
(34) Myers, A. G.; Yang, B. H.; Chen, H.; McKinstry, L.; Kopecky, D. J.;
Gleason, J. L. J. Am. Chem. Soc. 1997, 119, 6496–6511.
(35) Nahm, S.; Weinreb, S. M. Tetrahedron Lett. 1981, 22, 3815–3818.
(36) Kurosu, M.; Kishi, Y. Tetrahedron Lett. 1998, 39, 4793–4796.
(37) Evans, D. A.; Hoveyda, A. H. J. Am. Chem. Soc. 1990, 112, 6447–
6449.
(28) De Rosa, M.; Acocelli, M. R.; Villano, R.; Soriente, A.; Scettri, A.
Tetrahedron Lett. 2003, 44, 6087–6090.
(29) De Rosa, M.; Acocelli, M. R.; Villano, R.; Soriente, A.; Scettri, A.
Tetrahedron: Asymmetry 2003, 14, 2499–2502.
(30) Singer, R. A.; Carreira, E. M. J. Am. Chem. Soc. 1995, 117, 12360–
12361.
(38) Rychnovsky, S. D.; Rogers, B.; Yang, G. J. Org. Chem. 1993, 58,
3511–3515.
(31) Casiraghi, G.; Zanardi, F.; Appendino, G.; Rassu, G. Chem. ReV. 2000,
100, 1929–1972.
(39) Rychnovsky, S. D.; Skalitzky, D. J. Tetrahedron Lett. 1990, 31, 945–
948.
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Marine Natural Product Neopeltolide
A R T I C L E S
Scheme 6. Intermolecular Scandium(III) Triflate Methodologya
Scheme 4. Alcohol Fragment Synthesisa
a Conditions: (a) RuCl2(PhH)2, (R)-tol-BINAP, H2, EtOH, 94%, 97% ee.
(b) MeN(H)OMe ·HCl, i-PrMgCl, THF, -20 °C, 85%. (c) PMB-imidate,
pPTs, cyclohexane, CH2Cl2, 0 °C, 80%. (d) t-BuLi, pentane/Et2O, -78 °C,
50%. (e) DDQ, pH 7 buffer, CH2Cl2, 84%. (f) SmI2, PhCHO, THF, 91%.
(g) MeOTf, DTBMP, CH2Cl2, 88%. (h) K2CO3, MeOH, 86%.
Scheme 5. Oxazole Fragment Synthesisa
a Conditions: (a) TMSCH2N2, CH2Cl2, MeOH, 90%. (b) HF ·pyridine,
THF, 97%. (c) HF ·pyridine, THF, 99%. (d) TEMPO, H5C6I(OAc)2, CH2Cl2,
99%. (e) Sc(OTf)3, CaSO4, CH2Cl2, 55%. (f) DMSO, H2O, 87%.
was utilized to deprotect 28 which was subsequently oxidized
to aldehyde 29 with TEMPO.46 Next, subjecting both dioxinone
27 and aldehyde 29 to our cyclization conditions afforded the
bicyclic dioxinone as a single diastereomer in a 55% yield,
which upon heating in wet DMSO formed pyranone 6. While
convinced that this route would lead us to the completion of
the proposed structure of neopeltolide, we were also interested
in examining the scope of our scandium(III) triflate-catalyzed
cyclization for the more elegant and demanding intramolecular
macrocyclization route.
a Conditions: (a) ClCO2Me, dioxane, satd NaHCO3, 89%. (b) n-BuLi,
CO2, THF, -78 °C. (c) Lindlar’s catalyst, quinoline, atm H2, EtOAc, 70%
over two steps (d) i-BuOCOCl, N-Me-Morpholine, Ser-OMe ·HCl, 71%.
(e) DAST, CH2Cl2, -20 °C; BrCCl3, DBU, 0 °C, 62%. (f) DIBAL, THF,
0
°C, 85%. (g) CBr4, PPh3, 2,6-lutidine, CH3CN, 80%. (h)
The two main fragments, 7 and 8, were coupled using
Yamaguchi’s esterification protocol (Scheme 7).21 Global
deprotection of the silyl ethers proceeded smoothly with the
use of HF ·pyridine and selective oxidation of the primary
alcohol was accomplished with TEMPO to generate acyclic
aldehyde 5. In the key step, 10 mol % of scandium(III) triflate
promoted the macrocyclization of 5 to furnish the fully
elaborated 14-membered tricyclic ring 30 in 25% yield (unop-
timized) and greater than 20:1 dr. Formation of the tetrahydro-
pyranone was accomplished by heating 30 in wet DMSO and
the pyranone was selectively reduced with NaBH4 to place the
requisite alcohol in the equatorial position. Much to our surprise,
a Mitsunobu reaction47 of the macrocyclic pyran with oxazole
4 produced a compound (2) whose spectra were similar, but
not identical, to the natural product. Throughout the course of
the synthesis, extensive NOE experiments on advanced inter-
mediates and the final molecule all indicated that the key protons
on the macrolide where syn to each other, as was reported by
Wright and co-workers (Figure 2).5
n-Bu3SnCHdCH2, Pd2dba3, tri(2-furyl)phosphine, THF, reflux, 83%. (i)
9-BBN, THF, H2O2, 68%. (j) Dess-Martin, CH2Cl2, 98%. (k)
(F3CCH2O)2P(O)CH2CO2Me, 18-c-6, KHMDS, THF, 72%. (l) 1.0 M LiOH,
THF, 80%.
reduction with Lindlar’s catalyst (Scheme 5). Carbamate 23 was
then coupled with L-serine methyl ester hydrochloride and
converted to the oxazole.42 The resultant ester was reduced with
DIBAL to afford alcohol 24, which underwent subsequent
bromination to generate a primary bromide. A Stille coupling
was employed to couple vinyltributyltin to the primary bro-
mide.43 Subsequent hydroboration of the terminal olefin fol-
lowed by a Dess-Martin oxidation afforded aldehyde 26.44 The
cis-alkene was then obtained by a Still-Gennari olefination45
and consequent hydrolysis of the methyl ester delivered the
oxazole side chain.
Coupling the Fragments. We first attempted to utilize our
scandium(III) triflate-promoted cyclization methodology in an
intermolecular fashion followed by closure of the macrocycle
using a Yamaguchi macrolactonization. Therefore, carboxylic
acid 7 was converted into the methyl ester by addition of
(trimethylsilyl)diazomethane (Scheme 6). Meanwhile, HF ·pyridine
At this point, we hypothesized that two potential scenarios
could account for the discrepancies evident in the comparison
of our synthetic material and the isolated natural product. First,
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A R T I C L E S
Custar et al.
Scheme 7. Fragment Coupling and Initial Synthesisa
a Conditions: (a) 8, 2,4,6-trichlorobenzoyl chloride, Et3N, DMAP, THF, 80%. (b) HF·pyridine, THF, 93%. (c) TEMPO, H6C5I(OAc)2, CH2Cl2, 92%. (d)
Sc(OTf)3, CaSO4, MeCN, 25%. (e) DMSO, H2O, 130 °C, 99%. (f) NaBH4, MeOH, 0 °C, 71%. (g) DIAD, Ph3P, 4, benzene, 80%.
Scheme 8. Oxonia-Cope Pathway
Figure 2. Observed NOEs for synthetic compounds.
our macrocyclization, which forms the tricyclic macrocycle,
could have potentially proceeded through an oxonia-Cope
mechanism48-50 to give the inverted stereochemistry at the C-3
and C-7 positions (Scheme 8). This possibility could be the
function of ring strain in the macrocycle that would result in
the undesired configuration being the more thermodynamically
stable product. Our other hypothesis was that we had success-
fully synthesized the proposed structure and that this structure
differed in some way from the actual configuration of
neopeltolide.
selective oxidation of the primary alcohol furnished aldehyde
33 (Scheme 9). The resultant bicyclic dioxinone was then
generated upon subjection of 33 with our scandium(III) triflate-
catalyzed cyclization conditions and did not provide matching
NMR spectra to that of 30. The material was decarboxylated
and extensive NOE experimentation was done on the resulting
pyranone indicating that the C-3 and C-7 positions had been
inverted (Figure 3). This data indicated that our scandium(III)
triflate cyclization reaction did not epimerize the C-3 position
and, therefore the stereochemistry about the embedded tetrahy-
dropyran ring was not inverted via the oxonia-Cope pathway
in the initial synthesis. Nevertheless, we decided to finish the
synthetic route with the inverted pyranone due to the ease of
the last two steps. The reduction of pyranone with NaBH4
followed by the Mitsunobu addition of oxazole 4, again did
not produce neopeltolide. Additional NOE experimentation
performed on 34 confirmed the relative stereochemistry of the
protons on the macrolide (Figure 3). We could now exclude
the hypothesis that an oxonia-Cope process affected our
Synthesis of a Second Diastereomer (34). We wanted to
examine the viability of the oxonia-Cope pathway by synthesiz-
ing the enantiomer of carboxylic acid ent-7, couple this to
alcohol 8 and determine if the same tricyclic macrocycle is
formed after our cyclization reaction. Toward this end, we
synthesized the enantiomer of carboxylic acid fragment ent-7
(Scheme 9). Therefore, utilizing (S)-BINOL for the vinylogous
aldol reaction we installed the ꢀ-hydroxy substituent with the
opposite configuration. With access to both enantiomers, 15 and
ent-15, a Mosher ester analysis confirmed that we had correctly
assigned the stereochemistry at the hydroxyl position.51 The
dioxinone afforded the carboxylic acid ent-7 through the same
protection, deprotection and oxidation sequence as described
above.
The coupling of fragments ent-7 and 8, following Yamagu-
chi’s procedure, removal of the silyl protecting groups, and
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Marine Natural Product Neopeltolide
A R T I C L E S
Scheme 9. Synthesis of Neopeltolide Diastereomer 34a
a Conditions: (a) Ti(i-OPr)4, (S)-BINOL, 4 Å sieves, THF, 63%, 88% ee. (b) TBSOTf, 2,6-lutidine, CH2Cl2, 91%. (c) pPTs, MeOH, 83%. (d) PDC, DMF,
97%. (e) 2,4,6-trichlorobenzoyl chloride, Et3N, DMAP, THF, 76%. (f) HF ·pyridine, THF, 80%. (g) TEMPO, H6C5I(OAc)2, CH2Cl2, 98%. (h) Sc(OTf)3,
CaSO4, MeCN, 20%. (i) DMSO, H2O, 130 °C, 98%. (j) NaBH4, MeOH, 0 °C, 70%. (k) DIAD, Ph3P, 4, benzene, 82%.
Scheme 10. Synthesis of New Alcohol Fragmenta
a Conditions: (a) RuCl2(PhH)2, (S)-tol-BINAP, H2, EtOH, 94%, 96% ee.
(b) MeN(H)OMe·HCl, i-PrMgCl, THF, s20 °C, 69%. (c) PMB-imidate,
pPTs, cyclohexane, CH2Cl2, 0 °C, 97%. (d) 19, t-BuLi, pentane/Et2O, s78
°C, 28%. (e) DDQ, pH7 buffer, CH2Cl2, 92%. (f) SmI2, PhCHO, THF,
80%. (g) MeOTf, DTBMP, CH2Cl2. (h) K2CO3, MeOH, 48% over two-
steps.
Figure 3. Observed NOEs for synthetic compounds.
fewer steps to obtain 36 (Scheme 11). The reduction of 18 and
consequent addition of iodide 19 to the resulting aldehyde
produced a 1:1 mixture of inseparable diastereomers. Fortu-
nately, upon methylation of the secondary alcohol, the diaster-
eomers were separated and PMB ether 39 was isolated in 34%
yield over the two steps as a single diastereomer. Hydrolysis
of the benzyl ester followed by inversion of the resultant alcohol
via Mitsunobu reaction generated alcohol 36.
Acyclic aldehyde 40 can be accessed by coupling newly
synthesized alcohol 36 and carboxylic acid 7 followed by
deprotection and a selective oxidation. Our scandium(III) triflate-
Figure 4. Our structural reassignment of neopeltolide 1.
catalyzed macrocyclization produced the tricyclic macrocycle
synthetic route, validating that our initial synthesis afforded the
reported structure of neopeltolide.
in 40% yield. Despite the modest yield, this reaction is
remarkable due to the high degree of complexity generated in
this single step. Decarboxylation of 41 followed by a subsequent
reduction and Mitsunobu reaction with 4, gratifyingly, furnished
Completion of Neopeltolide. After careful consideration of
the original isolation data in conjunction with our synthetic
efforts, we postulated that the correct structure for neopeltolide
would be derived from alcohol diastereomer 36 where the C11
and C13 substituents are inverted (Figure 4). Accessing 36 was
accomplished by using (S)-tol-BINAP for the Noyori reduction
of ethyl 3-oxahexanoate (Scheme 10). The same reaction
sequence was employed on this enantiomer to provide alcohol
36. Additionally, the undesired enantiomer of Weinreb amide
18, from the original synthesis (Scheme 4), could be carried
forward due to the availability of the material and it required
1
neopeltolide (1). Our H and 13C NMR spectra match those
reported in the literature for the isolated natural product.
Additionally, the 2D NOESY, HRMS and optical rotation data
confirmed a successful synthesis of the natural (+)-enantiomer
of neopeltolide (1).
Biological Evaluation. With a highly convergent and flexible
route to access the natural product and related compounds, we
sought to explore the biological activity and to determine the
structure-activity relationships of neopeltolide (1), the two
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A R T I C L E S
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Scheme 11. Completion of the Synthesisa
a Conditions: (a) DIBAL, CH2Cl2, -78 °C, 67%. (b) 19, t-BuLi, pentane/Et2O, -78 °C. (c) MeOTf, DTBMP, CH2Cl2, 34% over two-steps, single
diastereomer. (d) DDQ, pH7 buffer, CH2Cl2, 83%. (e) 4-NO2-C6H5, DIAD, PPh3, benzene, 73%. (f) K2CO3, MeOH, 71%. (g) 7, 2,4,6-trichlorobenzoyl
chloride, Et3N, DMAP, THF, 82%. (h) HF ·pyridine, THF, 93%. (i) TEMPO, H6C5I(OAc)2, CH2Cl2, 99%. (j) Sc(OTf)3, CaSO4, MeCN, 40%. (k) DMSO,
H2O, 130 °C, 82%. (l) NaBH4, MeOH, 0 °C, 96%. (m) DIAD, Ph3P, 4, benzene, 76%.
Scheme 12. Synthesis of Neopeltolide Analogs
diastereomers (2 and 34), three advanced intermediates (4, 42
and 43), and finally, two analogs of neopeltolide (44 and 45).
The two analogs were readily prepared from a Mitsunobu
reaction of pyran 43 with the corresponding acid (Scheme 12).
First, to confirm that synthetic neopeltolide 1 we had created
was identical to the natural product itself, we evaluated its
biological activity against several tumor cell lines (Figure 5a).
Against the human breast adenocarcinoma line MCF-7, our
structurally reassigned neopeltolide potently inhibited cell
proliferation (as assayed by incorporation of [3H]-thymidine)
with an IC50 value of 2.2 ( 0.1 nM (Figure 5a).52 Furthermore,
the synthetic neopeltolide inhibited proliferation of the murine
leukemia line P388 even more potently, with an IC50 of 0.6 (
0.2 nM. These IC50 values are nearly identical to those reported
by Wright and co-workers in their report on the isolation of
neopeltolide from Daedalopelta sp., validating our revised
structure as the correct one;5 conversely, diastereomer 2, which
corresponded to the originally proposed structure, was ap-
proximately 100-fold less active in both cell lines (Table 2).
While neopeltolide completely inhibited the proliferation of
Figure 5. (a) Activity of synthetic neopeltolide 1 against cancer cell lines. (b) Activity against cultured primary human cells.
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Marine Natural Product Neopeltolide
A R T I C L E S
Table 2. Activity of Neopeltolide (1) and Structural Analogs To Inhibit Proliferation of Human Cancer Cellsa
MCF-7 cells
P-388 cells
analog
efficacy (at 10 mM)
potency (IC50)
efficacy (at 10 mM)
potency (IC50)
neopeltolide (1)
84% inhibition
86% inhibition
88% inhibition
78% inhibition
33% inhibition
nominal inhibition
nominal inhibition
nominal inhibition
2.2 ( 0.1 nM
219 ( 59.1 nM
328 ( 28.4 nM
5.8 ( 4.7 µM
>10 µM
100% inhibition
100% inhibition
100% inhibition
75% inhibition
25% inhibition
10% inhibition
nominal inhibition
nominal inhibition
0.6 ( 0.2 nM
42.3 ( 14.4 nM
216 ( 62.3 nM
3.3 ( 2.0 nM
>10 µM
diastereomer 1 (2)
diastereomer 2 (34)
benzoyl ester (44)
octanoyl ester (45)
oxazole side chain (4)
core pyran-alcohol (43)
core pyranone (42)
>10 µM
>10 µM
>10 µM
>10 µM
>10 µM
>10 µM
a IC50 values represent the mean ( SEM of three experiments.
P388 cells, there remained a small level of growth (10-12%)
of the MCF-7 cells that was refractory to neopeltolide. More
surprisingly, however, was our finding that despite its potent
effect on P388 and MCF-7 cells, neopeltolide was minimally
actiVe or inactiVe against the four other cell lines that we
tested (Figure 5a): the human cervical carcinoma line HeLa,
the rat adrenal tumor line PC12, the human epidermal
carcinoma line KB and the human lung carcinoma line A549.
We detected no dose-dependent growth inhibition in A549
or HeLa cells, and minimal inhibition of PC12 and KB cells
was observed only at 10 µM neopeltolide. Limited effective-
ness of neopeltolide at blocking growth of different cell lines,
including A549 cells, has been recently reported5,6 but in
these cases there was no rightward shift in IC50 value. Our
discoVery of entirely resistant cell lines was therefore
surprising. Neopeltolide appears to be selectiVe with respect
to which cancer cells it inhibits.
We have initiated structure-activity studies in order to begin
identification of the biologically important structural attributes
of neopeltolide. The molecule can be divided structurally into
two main components: the core macrolide 43 and the oxazole-
containing side chain 4. Our synthetic scheme provided us with
variants of both of these components as well as a pair of
stereoisomers (2, 34) of the full molecule. In addition, we
synthesized a pair of ester variants of the side chain by replacing
the oxazole-containing side chain with either a benzoyl (44) or
octanoyl group (45). In total, the set of analogs enabled us to
evaluate the importance of both the side chain as well as the
core macrolide by testing the activity of the analogs in the
MCF-7 and P-388 cells (Table 2). The results were similar in
both of the cell lines. By themselves, both the oxazole-containing
side chain (4) and the core macrolide (with either the pyran 43
or pyranone 42 functionality) are completely without activity
and do not inhibit the proliferation of either cell line even at
maximum concentrations. As with elimination of the oxazole-
containing side chain, substitution of this native portion with
the octanoyl (45) or benzoyl (44) groups also abolished the
vast majority of activity, with growth inhibition observed at
only the maximum concentration: 10 µM of the octanoyl ester
inhibits 25% of P-388 cell growth and 33% of MCF-7 cell
growth, while the benzoyl ester is somewhat more effective
at the same concentration and inhibits 75% and 78%,
respectively. It has been shown that the olefin in the side
chain, which is approximated in the benzoyl ester (44) but
not the octanoyl ester (45), plays a significant role in
inhibiting proliferation.53 Both the octanoyl and benzoyl side
chains are also significantly shorter in length than the natural
side chain. This tends to support the recent finding that overall
side chain length has importance;53 alternatively, the oxazole
ring itself may be paramount, although that possibility
remains to be firmly established experimentally. Thus, while
other pharmacophores may reside apart from the olefin, the
importance of the side chain for full activity is clearly
demonstrated by the data presented herein and in agreement
with the results of others.12,53
These data also reinforce that the core macrolide is necessary
for full growth inhibition. As with the side chain, elimination
of the macrolide from the molecule also eliminates activity while
smaller scale alterations of its structure also markedly reduce
potency. Diastereomer 1 (2), in which the stereochemistry of
C13 and C11 were inverted, is 84-fold and 100-fold less potent
than neopeltolide in P-388 and MCF-7 cells, respectively. The
effects of stereocenter inversion appear to be cumulative, as
inversion of C11 alone is reported to result in a 10-fold reduction
of activity.53 Diastereomer 2 (34), which in addition to the
inversions at C13 and C11 also contained inversions at C3, C5
and C7, is an additional 5-fold less potent than diastereomer 1
(2) in P-388 cells but only 50% less potent in MCF-7 cells.
The reason why the additional inversions of C3, C5 and C7
have a greater impact on activity in P-388 cells is not known;
however, in either cell line the additional reduction in potency
is noteworthy. Taken together, the results show that the activity
of the molecule is not easily ascribed to any single pharma-
cophore; rather, full activity is reliant on the concerted contribu-
tions of many structural featuressboth in the core macrolide
(40) Hornberger, K. R.; Hamblett, C. L.; Leighton, J. L. J. Am. Chem.
Soc. 2000, 122, 12894–12895.
(41) Wang, Y.; Janjic, J.; Kozmin, S. A. J. Am. Chem. Soc. 2002, 124,
13670–13671.
(42) Phillips, A. J.; Uto, Y.; Wipf, P.; Reno, M. J.; Williams, D. R. Org.
Lett. 2000, 2, 1165–1168.
(43) Stille, J. K. Angew. Chem., Int. Ed. 1986, 25, 508–523.
(44) Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113, 7277–7287.
(45) Still, W. C.; Gennari, C. Tetrahedron Lett. 1983, 24, 4405–4408.
(46) DeMico, A.; Margarita, R.; Parlanti, L.; Vescovi, A.; Piancatelli, G.
J. Org. Chem. 1997, 62, 6974–6977.
(47) Mitsunobu, O.; Yamada, M. Bull. Chem. Soc. Jpn. 1967, 40, 2380–
&.
(48) Rychnovsky, S. D.; Marumoto, S.; Jaber, J. J. Org. Lett. 2001, 3, 3815–
3818.
(49) Roush, W. R.; Dilley, G. J. Synlett 2001, 955–959.
(50) Jasti, R.; Rychnovsky, S. D. J. Am. Chem. Soc. 2006, 128, 13640–
13648.
(51) Dale, J. A.; Mosher, H. S. J. Am. Chem. Soc. 1968, 90, 3732–3740.
(52) For all details and procedures for biological evaluations of neopeltolide
and analogs, see Supporting Information.
(53) Vintonyak, V. V.; Kunze, B.; Sasse, F.; Maier, M. E. Chem.sEur. J.
2008, 14, 11132–11140.
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and in the side chainsof which the removal of any one is
detrimental to activity to some degree.
be important for full biological activity. Further investigations
of neopeltolide and related analogs to enhance their properties
as potential chemotherapeutic agents are ongoing. These
studies reinforce the vital role that total synthesis continues
to play in determining the actual structures of natural products
and the viability of these molecules to be used as lead
compounds in a pharmacological setting.
Conclusion
We have completed the total synthesis of neopeltolide and
related compounds by employing an aggressive intramolecular
Sc(III)-catalyzed cyclization between a dioxinone and an in situ
generated oxocarbenium ion to furnish the THP and the
macrocycle in a single step. The flexible and convergent
synthetic route allowed for facile construction of two diaster-
eomers that ultimately lead to the structural revision and absolute
stereochemical determination of neopeltolide. The biological
evaluation of synthetic neopeltolide also confirmed that we
had correctly reassigned the structure. Neopeltolide has a
variable degree of impact on the growth of different cancer
cells that may not be useful against all forms of cancer. The
basis for this variation inhibition is not clear, but indicates
that neopeltolide may interact selectively with specific
(cancer) cells and not adversely affect normal cells in the
body, which is suggested by its lack of effect on cultured
primary cells. Both the core macrolide and the oxazole side
chain bound together play a role in mediating the effect of
neopeltolide: neither component on its own is active. The
substitution of the side chain and the stereochemistry of the
macrolide ring carbons, especially C11 and C13, appear to
Acknowledgment. This work has been supported by the Sloan
Foundation, American Cancer Society (Research Scholar Award
to K.A.S.), AstraZeneca, GlaxoSmithKline, Boehringer-Ingelheim,
and the H-Foundation. C.M.C. gratefully acknowledges the support
of the NIH (GM62120). Funding for the NU Integrated Molecular
Structure Education and Research Center (IMSERC) has been
furnished in part by the NSF (CHE-9871268). We thank FM-
CLithium, Sigma-Aldrich, Wacker Chemical Company, and Pres-
sure Chemical Company for generous reagent support, Dr. Thad
Franczyk for assistance with the details of catalytic hydrogenation,
and Professor Regan Thomson for helpful discussions.
Supporting Information Available: Experimental procedures
and spectral data for new compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
JA904604X
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