Total synthesis of potent antitumor macrolide (-)-zampanolide: An oxidative intramolecular cyclization-based strategy

Article abstract of DOI:10.1002/ejoc.201200286

A detailed account of the enantioselective total synthesis of (-)-zampanolide, a macrolide marine natural product with high anticancer activity, is described. For the synthesis of the 4-methylenetetrahydropyran unit of (-)-zampanolide, we initially relied

Full text of DOI:10.1002/ejoc.201200286

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FULL PAPER
DOI: 10.1002/ejoc.201200286
Total Synthesis of Potent Antitumor Macrolide (–)-Zampanolide: An Oxidative
Intramolecular Cyclization-Based Strategy
Arun K. Ghosh,*[a] Xu Cheng,[a] Ruoli Bai,[b] and Ernest Hamel[b]
Keywords: Natural products / Total synthesis / C–H activation / Metathesis / Stereoselective catalysis
A detailed account of the enantioselective total synthesis of
(–)-zampanolide, a macrolide marine natural product with
high anticancer activity, is described. For the synthesis of the
4-methylenetetrahydropyran unit of (–)-zampanolide, we ini-
tially relied upon an oxidative C–H activation of an alkenyl
ether and intramolecular cyclization to provide the substi-
tuted tetrahydropyran ring. However, this strategy was un-
successful. Subsequently, we found that a cinnamyl ether is
critical for the successful oxidative intramolecular cyclization
reaction. The synthesis also features a cross-metathesis reac-
tion for the construction of a trisubstituted olefin, a ring-clos-
ing metathesis to form a highly functionalized macrolactone,
and a chiral phosphoric acid promoted formation of an N-
acyl aminal to furnish (–)-zampanolide stereoselectively and
in good yield. The synthetic (–)-zampanolide had effects on
cultured cells and on tubulin assembly consistent with the
properties reported for the natural product.
Introduction
Zampanolide (1; Figure 1), an unsaturated 20-membered
macrolide with an N-acyl hemiaminal side-chain, was ini-
tially isolated by Tanaka and Higa in 1996, from the marine
sponge Fasciospongia rimosa taken from Okinawa, Japan.[1]
More recently, (–)-zampanolide has been isolated from the
Tongan marine sponge Cacospongia mycofijiensis by
Northcote and co-workers.[2] Zampanolide exhibits potent
cytotoxicity against a variety of cell lines. It has shown IC50
values of 1.1 and 2.9 nm against the SKM-1 and U937 cell
lines, respectively. Moreover, it is very active against HL-
60, 1A9, and, in particular, A2780AD cells, which show re-
sistance to paclitaxel based on overexpression of the P-gly-
coprotein.[2] Its biological mechanism of action involves the
stabilization of microtubules with enhancement of microtu-
bule assembly and blocking cell division in the G2/M phase
of the cell cycle.[2,3] Macrolide (+)-dactylolide (2; Figure 1)
has been isolated from the sponge Dactylospongia sp.[4]
Interestingly, it displays only modest cytotoxicity, however,
it contains opposite configurations of the macrolide core of
(–)-zampanolide (1). The biological relevance of the macro-
lide core of (+)-dactylolide and (–)-zampanolide is not
clear.
Figure 1. (–)-Zampanolide (1) and (+)-dactylolide (2).
Both zampanolide and dactylolide have attracted much
interest in synthesis and in the biological studies of struc-
tural variants.[3] Smith et al. first reported the total synthe-
sis of (+)-zampanolide, the unnatural antipode, and pro-
vided tentative assignment of the absolute configurations of
natural (–)-zampanolide.[5] Since then, Hoye et al. in
2003[6a] and Uenishi et al. in 2009[6b] have reported the total
synthesis of natural (–)-zampanolide. Recently, we reported
an enantioselective synthesis of (–)-zampanolide.[7] Since
the isolation of (+)-dactylolide by Riccio and co-workers in
2001,[4] a number of total syntheses and various synthetic
approaches to (+)-dactylolide have been reported.[8] The N-
acyl side-chain of (–)-zampanolide is critical to its potent
cytotoxic properties.[1,2] Previous syntheses have furnished
zampanolide by direct N-acyl aminal formation from un-
natural (–)-dactylolide, albeit in only 12% yield. Other
major products include the zampanolide epimer and bis-
acylated products.[6] This direct transformation was
achieved through a strategy promoted by an Al reagent[6a]
or catalyzed by camphorsulfonic acid (CSA),[6b] although
in both cases no stereoselectivity was observed. Herein, we
[a] Department of Chemistry and Department of Medicinal
Chemistry Purdue University,
560 Oval Drive, West Lafayette, IN 47907, USA
Fax: +1-765-496-1612
E-mail: akghosh@purdue.edu
[b] Screening Technologies Branch, Developmental Therapeutics
Program, Division of Cancer Treatment and Diagnosis,
National Cancer Institute at Frederick, National Institutes of
Health,
Frederick, MD 21702, USA
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A. K. Ghosh, X. Cheng, R. Bai, E. Hamel
FULL PAPER
report the details of our synthetic efforts that have led to a The synthesis of 5 is shown in Scheme 1. Optically active
convergent total synthesis of (–)-zampanolide. The synthe- methyl ketone 8 was prepared in five steps from l-malic
sis features a novel intramolecular oxidative cyclization re- acid, as described previously.[9] Horner–Wadsworth–
action and the organocatalyic N-acyl aminal formation of Emmons reaction of ketone 8 with NaH and tert-butyl
dactylolide, which stereoselectively furnishes (–)-zampanol- phosphorylacetate at 23 °C afforded α,β-unsaturated ester
ide and no bis-amide byproduct.
9 as a mixture of E/Z isomers (4:1). In contrast, the use
of triethyl phosphonoacetate provided 10 with lower E/Z
selectivity (2:1). The ester was reduced by exposure to ex-
cess DIBAL-H to give allyl alcohol 11. Treatment of 11
with NaH and trichloroacetonitrile in ether furnished tri-
chloroimidate 12. Initially, we attempted etherification of
Results and Discussion
Our initial retrosynthetic analysis of (–)-zampanolide (1) known alcohol 7[10] with imidate 12 in the presence of a
is outlined in Figure 2. Our synthetic strategy was to syn- number of Brønsted acids and Lewis acids such as TfOH,
thesize (–)-dactylolide (2) and carry out a stereoselective BF3·Et2O, and TMSOTf. However, these conditions re-
amidation to provide (–)-zampanolide. Strategic disconnec- sulted in decomposition of the starting materials. The use
tion of (–)-dactylolide at the C8–C9 and C1–C19 ester of TBSOTf resulted in a considerable amount of ether 13.
bonds results in the 4-methylenetetrahydropyran derivative The coupling reaction with TIPSOTf proceeded well, pro-
4 and the unsaturated carboxylic acid 3. Our plan was to viding 13 in 79% yield as a 2:1 mixture of E/Z isomers
1
form an ester with the C19 alcohol and carboxylic acid 3 (by H NMR analysis). The ester functionality of 13 was
and then carry out ring-closing metathesis to construct the converted into allylsilane derivative 5 by using the pro-
C20 macrolactone core of (–)-dactylolide. It was planned to cedure developed by Narayanan and Bunnelle.[11] We at-
functionalize 4-methylenetetrahydropyran 4 by an intramo- tempted an oxidative cyclization reaction of this allylsilane
lecular oxidative cyclization of allyl ether 5. This substrate substrate with DDQ under a variety of reaction condi-
can be synthesized from allylic alcohol 6 and optically
active β-hydroxy ester 7.
Scheme 1. Attempted synthesis of dihydropyran 4. Reagents and
conditions: a) tert-butyl dimethoxyphosphorylacetate (4.4 equiv.),
NaH (3.8 equiv.), THF, 23 °C, 17 h, 74%; b) DIBAL-H (8 equiv.),
CH2Cl2, –15 °C, 5 min, 91%; c) NaH (10 mol-%), CCl3CN
Figure 2. First-generation retrosynthetic analysis.
(1.0 equiv.), diethyl ether, 23 °C, 1 h, 98%; d) 7 (0.9 equiv.), TIP-
SOTf (0.34 equiv.), mol. sieves (4 Å), CH2Cl2, 23 °C, 12 h, 79%
(brsm); e) CeCl3 (5 equiv.), TMSCH2MgCl (5 equiv.), THF, 23 °C,
We first examined the feasibility of the oxidative cycliza-
tion of allylsilane derivative 5 to yield tetrahydropyran 4. 4 h, decomposition.
12 h, 73%; f) DDQ (2 equiv.), Lewis acid, CH2Cl2, –78 to 23 °C,
2
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Total Synthesis of Potent Antitumor Macrolide (–)-Zampanolide
tions.[12] However, we did not obtain any desired cycliza- by Tsuji–Trost reaction with
a catalytic amount of
tion. At this point, we speculated that a cinnamyl ether may [Pd(PPh3)4] and tert-butyl cinnamyl carbonate 21 to provide
be more convenient for such an oxidative cyclization reac- 22 in 71% yield.[14] The ester group in 22 was converted
tion. Therefore, we modified our synthetic strategy.
into allylsilane 17 by treatment with excess trimethyl-
Our alternative retrosynthetic analysis is illustrated in silylmethylmagnesium chloride in the presence of CeCl3, as
Figure 3. Disconnection of the N-acyl aminal side-chain described by Bunnelle and co-workers.[11] We then investi-
would provide macrolactone core 14. Further disassembly gated a variety of reaction conditions for the effective oxi-
of the macrolactone would give rise to tetrahydropyran de- dative cyclization of key substrate 17. The results are shown
rivative 15 and trisubstituted olefin 3. It was planned to in Table 1. As can be seen, our initial attempt using DDQ
synthesize
functionalized
4-methylenetetrahydro- in the presence of InCl3 as promoter resulted in the desired
pyran unit 15 from tetrahydropyran 16 by a cross-metathe- tetrahydropyran cyclization product 16 in 57% yield (en-
1
sis reaction. The tetrahydropyran 16 with a cinnamyl side- try 1) as a single diastereoisomer (by H NMR analysis).
chain would be assembled from cinnamyl ether 17 by an The cis stereochemical identity of dihydropyran 16 was es-
1
oxidative cyclization reaction. We presumed that oxidative tablished by H NMR NOESY experiments. The oxidative
activation of a cinnamyl group with DDQ would proceed cyclization presumably proceeds through the Zimmerman–
favorably compared with allylic ether 5. The oxidative cycli- Traxler transition state shown as stereochemical model 23.
zation substrate 17 can be derived from β-hydroxy ester 18. The presence of 2,6-dichloropyridine did not improve reac-
This β-hydroxy ester would be prepared in optically active tion yields (entry 2). Other Lewis acids did not offer any
form by an enantioselective hydrogenation process. The advantage (entries 3–6).[12] The cyclization reaction in the
polyene unit 3 could be obtained by Reformatsky reaction presence of CSA, however, proved to be the most effective
of bromo olefin 19 and acrolein followed by a Wittig ole- in both CH2Cl2 as well as CH3CN (entries 7 and 8). The
fination.
catalytic version of this reaction in the presence of PPTS
marginally improved the yield to 71% (entry 9). The reac-
tion was then carried out on a 4-g scale at –38 °C in
CH3CN to provide 16 in 81% yield.
Figure 3. Alternative synthetic plan for (–)-zampanolide.
In accord with our revised plan, optically active β-hy-
droxy ester 20 was synthesized based upon a Noyori hydro-
genation reaction.[13] This ester was converted into cinna-
myl ether 22, as shown in Scheme 2. Catalytic hydrogena-
tion of 20 over 10% Pd-C followed by selective protection
of the primary alcohol with TBDPSCl and imidazole in
THF at 0 °C afforded the corresponding silyl ether. The free
Scheme 2. Synthesis of tetrahydropyran 16. Reagents and condi-
tions: a) Pd-C (3 mol-%), H2, EtOH, 23 °C, 4 h; b) TBDPSCl
(1.5 equiv.), imidazole (1.5 equiv.), THF, 0 °C, 1 h, 71% in two
steps; c) 21 (2 equiv.), [Pd(PPh3)4] (5.5 mol-%), THF, reflux, 36 h,
71%; d) CeCl3 (5 equiv.), TMSCH2MgCl (5 equiv.), THF, –78 to
23 °C overnight, 81%; e) see Table 1. TBDPS = tert-butyldiphenyl-
silyl, TMS = trimethylsilyl, DDQ = 2,3-dichloro-5,6-dicyano-1,4-
secondary hydroxy was then protected as a cinnamyl ether benzoquinone, PPTS = pyridinium p-toluenesulfonate.
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FULL PAPER
Table 1. Optimization of the oxidative cyclization of 17.[a]
triphenylphosphorane to provide alkene 24 in 78% yield for
the three steps. For the planned cross-metathesis reaction
of 24, we prepared 26 by ring opening of PMB-protected
glycidyl derivative 25 with isopropenylmagnesium bromide
followed by protection of the resulting alcohol as a TES
ether. Cross-metathesis of 24 and 26 was carried out with
second-generation Grubbs catalyst[16] in CH2Cl2 at reflux
for 9 h to furnish trisubstituted olefin as an E/Z mixture of
isomers (ratio 1.7:1) in 57% combined yield. The mixture
of olefins was treated with HF·Py to remove the two silyl
groups and the resulting diols were separated by silica gel
chromatography. Olefin (Z)-27 was subjected to photo-
chemical isomerization to provide the (E)-27 isomer in 51%
yield [based on residual starting material (brsm), 68%].
The C1–C9 triene carboxylic acid 3 was synthesized by
a Reformatsky reaction as the key step. As outlined in
Scheme 4, allyl bromide 19 was prepared as an E/Z mixture
(1:1) according to the procedure of Lei and Fallis.[17] Treat-
ment of this E/Z mixture with Zn dust followed by reaction
with acrolein afforded the E-alcohol 28 (47%) and α,β-un-
saturated δ-lactone 29 (36%), which results from concomi-
tant lactonization of the Z-alcohol. These products were
separated by silica gel chromatography. Treatment of lact-
one 29 with DIBAL-H followed by reaction of the re-
sulting lactol with (ethoxycarbonylmethylene)triphenyl-
phosphorane afforded unsaturated ester 30 in 33% yield for
the two steps. The free allylic alcohol was protected as a
PMB-ether and saponification of the resulting ester fur-
nished the C1–C9 carboxylic acid fragment 3 in 62% yield
over two steps.
Entry
Acid
Solvent
Yield [%]
1
2
3
4
5
6
7
8
9
10
InCl3[b]
InCl3
AlCl3
LiClO4[b]
LiClO4
TiCl4
CSA
CSA
PPTS
PPTS
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH3CN
CH3CN
CH3CN
57
55
n.d.
41
46
n.d.
65
69
71[c]
81[d]
[a] The reaction was carried out on a 0.5 mmol scale. Reaction
conditions: acid (1.5 equiv.), mol. sieves (4 Å), at –78 °C for
CH2Cl2, at –38 °C for CH3CN, 6–8 h. [b] Performed with 2,6-
dichloropyridine (1.5 equiv.). [c] 20 mol-% DDQ and 2 equiv. CAN.
[d] Performed with 4 g of 17. CAN = ceric ammonium nitrate.
For elaboration of the C17–C20 chain of (–)-zampanol-
ide with an E-trisubstituted olefin, we initially relied upon
a cross metathesis of 16 with the corresponding olefin sub-
strate 26. However, our many attempts under a variety of
conditions and catalysts did not result in the desired cross-
metathesis product. This is possibly due to the lack of reac-
tivity of the styrene side-chain. Even our attempted cross
metathesis with ethylene resulted in no cross-metathesis
product 24. We therefore devised an alternative strategy to
install this side-chain. As shown in Scheme 3, we carried
out selective cleavage of the styrenyl double bond by dihy-
droxylation with AD-mix-α followed by diol cleavage with
NaIO4, as described previously.[15] The resulting unstable
aldehyde was subjected to Wittig reaction with methylene-
Scheme 4. Synthesis of polyene fragment 3. Reagents and condi-
tions: a) Zn (10 equiv.), acrolein (1.0 equiv.), THF, reflux, 2 h, 84%
based on effective starting material; b) DIBAL-H (1.0 equiv.),
CH2Cl2, –78 °C, 2 h; c) Ph3P=CHCOOEt (1.3 equiv.), toluene,
100 °C, 16 h, 33% two steps; d) 4-methoxybenzyl 2,2,2-trichloro-
acetimidate (1.3 equiv.), (+)-CSA (10 mol-%), mol. sieves (4 Å),
CH2Cl2, 0 to 23 °C, 24 h, 80%; e) NaOH (3.0 equiv.), EtOH/H2O,
0 to 23 °C, 12 h, 77%. DIBAL-H = diisobutylaluminium hydride,
CSA = camphorsulfonic acid.
Scheme 3. Synthesis of tetrahydropyran (E)-27. Reagents and con-
ditions: a) AD-mix-α (5 mol-% based on osmium), MeSO2NH2
(1.5 equiv.), tBuOH/H2O (1:1), 23 °C, 1 h; b) NaIO4 (1.2 equiv.),
MeOH/H2O, 23 °C, 2 h; c) Ph3P=CH2 (4.5 equiv.), THF, 40 °C,
16 h, 78% overall yield three steps; d) isopropenylMgBr, (2 equiv.),
Li2CuCl4 (5 mol-%), THF, 0 °C, 2 h, 91%; (e) TESOTf (1 equiv.),
2,6-lutidine (1.5 equiv.), CH2Cl2, –30 °C, 1 h, 94%; f) Grubbs II
catalyst (10 mol-%), 26 (10 equiv.), CH2Cl2, reflux, 9 h, 57%;
g) HF·Py, THF, 23 °C, 14 h, E/Z = 1.7:1, overall 81%; h) hv, benz-
ene, 23 °C, 4 h, 51% after two runs.
For elaboration of the macrolactone core of (–)-dactylol-
ide and (–)-zampanolide, as shown in Scheme 5, the pri-
mary alcohol in 27 was first selectively oxidized with
TEMPO and PhI(OAc)2.[18] Wittig olefination of the re-
sulting aldehyde with methylenetriphenylphosphorane af-
4
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Total Synthesis of Potent Antitumor Macrolide (–)-Zampanolide
forded olefin 31 in 60% overall yield. The secondary
alcohol in 31 was esterified under Yamaguchi conditions[19]
with carboxylic acid 3 by using 2,4,6-trichlorobenzoyl
chloride in the presence of DMAP to provide a mixture
(1:1) of diastereomeric esters 32 in excellent yield. Subjec-
tion of this mixture of diastereomers to Grubbs II catalyst
(12 mol-%) in benzene at 60 °C for 20 h furnished the corre-
sponding 20-membered macrolactone. Exposure of the
macrolactone mixture to DDQ in aqueous CH2Cl2 removed
the PMB group, providing a diastereomeric mixture (1:1 at
C7) of alcohols 14 in 65% yield over two steps. Oxidation
of 14 with the Dess–Martin reagent[20] furnished (–)-dac-
tylolide 2 in 80% yield. The spectroscopic data (1H and
13C NMR) of (–)-2 is in full agreement with natural (+)-
dactylolide except for the optical rotation of
(–)-2 {[α]2D4 = –148 (c = 0.09, MeOH)}.
Scheme 6. Model N-acyl aminal reaction. Reagents and conditions:
a) amide (10 equiv.), Cy2BCl (9 equiv.), Et3N (9 equiv.), Et2O, 0 °C,
1 h.
Scheme 5. The synthesis of (–)-dactylolide 2. Reagents and condi-
tions: a) PhI(OAc)2 (2.3 equiv.), TEMPO (0.8 equiv.), CH2Cl2,
23 °C, 8.5 h; b) PPh3CH2 (3.5 equiv.), THF, 40 °C, 12 h, 60% in
two steps; c) 2,4,6-trichlorobenzoyl chloride (1.5 equiv.), Et3N
(2.6 equiv.), DMAP (1 equiv.), toluene, 23 °C, 20 h, 91%;
d) Grubbs II catalyst (12 mol-%), benzene, 60 °C, 20 h; e) DDQ
(2.2 equiv.), CH2Cl2/water, 23 °C, 30 min, 65% in two steps;
f) Dess–Martin reagent (6.0 equiv.), NaHCO3 (13.8 equiv.),
CH2Cl2, 23 °C, 3.5 h, 80%. DMAP = 4-(dimethylamino)pyridine.
TEMPO = 2,2,6,6-tetramethylpiperidine 1-oxyl.
Previously, direct N-acyl aminal formation proceeded
only with limited success, providing (–)-zampanolide (1)
along with a mixture of C-20 diastereomers in poor yield
(ca. 12%).[6] To improve this transformation, we initially
selected cyclohexanecarbaldehyde (33) and aldehyde 34 as
model substrates. We investigated N-acyl aminal formation
by using the reaction conditions reported by Williams and
co-workers.[21] As shown in Scheme 6, our initial attempts
with both aldehyde substrates 33 and 34 and isobutyramide
35 proceeded well, providing N-acylamides 36 (90% yield)
Scheme 7. Brønsted acid catalyzed direct amidation of 2 and 38.
Reagents and conditions: a) amide 38, diphenylphosphoric acid
(10 mol-%), CH2Cl2, 23 °C, 14 h. The product distribution (1/39/40
and 37 (56% yield) as a 1:1 mixture of diastereomers. How- = 2.7:2.5:1) was measured by HPLC.
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A. K. Ghosh, X. Cheng, R. Bai, E. Hamel
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ever, our attempts to carry out this reaction with (–)-dac- sulted in (–)-1 and 39 as a 1:1 mixture of diastereomers.
tylolide (2) and (2Z,4E)-hexa-2,4-dienamide 38 were unsuc- Interestingly, the formation of the bis-amide byproduct 40
cessful.
was not observed. The spectroscopic data (1H and 13C
We subsequently planned to optimize the conditions for NMR) for synthetic (–)-zampanolide {1; [α]D = –94, (c =
direct amidation reported by Uenishi et al.[6b] Reaction of 0.08, CH2Cl2)} are in full agreement with those of the natu-
(–)-dactylolide (2) and amide 38 with pTsOH was reported ral zampanolide {ref.[1]: [α]D = –101 (c = 0.12, CH2Cl2)}.
to provide (–)-zampanolide (1; 12%), the C-20 epimeric
product (12%), and the corresponding bis-amide (23%).[6b]
In this transformation, the lack of stereoselectivity and the
formation of the bis-amide limited the yield of the desired
(–)-zampanolide. In an effort to improve the reaction yield
and minimize conversion into the bis-amide byproduct, we
Biological Activity
The biological properties of compounds 1, 39, and 40
were evaluated with purified tubulin combined with heat-
treated microtubule-associated proteins and cytotoxic ef-
investigated the reaction of (–)-2 and amide 38 in the pres-
fects were examined by using several cell lines. Paclitaxel
ence of the Brønsted acid diphenylphosphoric acid (10 mol-
was used as a control in all experiments. With tubulin, 1
%) in CH2Cl2 at 23 °C for 14 h, as depicted in Scheme 7. As
indicated by HPLC analysis, the above reaction conditions
provided (–)-zampanolide (1), its epimer 39, and bis-amide
strongly induced assembly, as has been reported pre-
viously,[2] whereas compounds 39 and 40 showed only slight
activity (data not shown). The cytotoxic activities of the
40 in a better ratio than reported for the pTsOH-catalyzed
three agents against three human cancer cell lines are sum-
reaction.[6b]
marized in Table 2. We used MCF-7 breast cancer cells and
Encouraged by this result, we investigated the effect of
two ovarian lines, OVCAR 8 and its isogenic derivative
chiral phosphoric acid TRIP 41[22] on the N-acyl aminal
NCI/ADR-RES, which, like A2780AD,[2] overexpresses P-
reaction of (–)-2 and amide 38. As shown in Scheme 8, the
reaction of (–)-2 in the presence of (S)-TRIP (41; 20 mol-
%) at 23 °C for 12 h furnished N-acyl aminal derivatives
diastereoselectively in good yields and, most significantly,
the formation of the byproduct bis-amide 40 was not ob-
served. The reaction provided (–)-zampanolide (1) in 53%
yield and epi-zampanolide 39 in 18% yield after separation
by HPLC. In contrast, the corresponding reaction with 38
glycoprotein, an important cellular drug efflux pump. Over-
expression of P-glycoprotein produces the major multidrug
resistance phenotype. P-Glycoprotein overexpressing cell
lines are of particular interest in evaluating agents with a
paclitaxel-like mechanism of action because such cell lines
are highly resistant to paclitaxel and multidrug resistance
may cause clinical loss of sensitivity to paclitaxel.
and (–)-2 in the presence of mismatched (R)-TRIP (42) re-
Table 2. Cytotoxic activity of compounds against three human can-
cer cell lines.[a]
IC50 [nm]
OVCAR 8
MCF-7
NCI/ADR-RES
1
4.0Ϯ0.5
200Ϯ0
430Ϯ200
2.0Ϯ0
20Ϯ0
25Ϯ7
39
250Ϯ70
300Ϯ0
7.5Ϯ2
750Ϯ200
750Ϯ400
Ͼ5000
40
Paclitaxel
[a] A standard deviation of 0 indicates the same value was obtained
in all experiments (usually two independent determinations).
Compound 1 shows similar cytotoxic activity to paclit-
axel in both the MCF-7 and OVCAR 8 cell lines, but it was
much more active than paclitaxel in the multidrug resistant
NCI/ADR-RES cell line. Compounds 39 and 40 were sub-
stantially less active than 1 in all the cell lines examined.
However, they were more active than paclitaxel in the NCI/
ADR-RES cells, which indicates that they, too, are not
good substrates for P-glycoprotein.
We also performed a limited study with 1, 39, and 40 in
human CA46 Burkitt lymphoma cells because this cell line
yields a high proportion of cells arrested in the G2/M phase
of the cell cycle when treated with antitubulin agents at
higher concentrations, such as 10-fold higher than the IC50.
At such high concentrations, all three compounds and pa-
clitaxel caused over 90% G2/M arrest in the Burkitt cells.
With compound 1 and paclitaxel, the agents were used at
100 nm, whereas with 39 and 40, the concentration used was
5 μm (data not presented).
Scheme 8. Synthesis of (–)-zampanolide (1) by the (S)-TRIP-cata-
lyzed amidation reaction. Reagents and conditions: a) amide 38
(3 equiv.), (S)-TRIP (20 mol-%), CH2Cl2, 23 °C, 14 h.
6
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Total Synthesis of Potent Antitumor Macrolide (–)-Zampanolide
(s, 3 H), 1.35 (s, 3 H) ppm. 13C NMR (100 MHz, CDCl3): δ =
165.6, 154.5, 119.5, 108.8, 79.6, 75.3, 69.1, 36.7, 28.1, 28.0, 26.8,
Conclusions
A detailed account of the enantioselective total synthesis
of (–)-zampanolide (1) has been described. Our initial syn-
thetic route to the zampanolide tetrahydropyran core in-
volved oxidative C–H activation of an allylic ether deriva-
tive. However, this strategy did not provide the function-
alized tetrahydropyran core for zampanolide. We sub-
sequently devised an alternative oxidative C–H activation
strategy in which a cinnamyl ether was used in place of the
allyl ether. This cyclization method provided the 4-methyl-
enetetrahydropyran derivative in a highly stereoselective
manner. The C1–C9 triene carboxylic acid unit 3 was syn-
thesized by a Reformatsky reaction as the key step. Yamag-
uchi esterification and ring-closing olefin metathesis pro-
vided the zampanolide core. Organocatalytic N-acyl aminal
formation with a chiral phosphoric acid proceeded stereose-
lectively and in good yield without the formation of the bis-
amide byproduct. The current synthesis is amenable to a
variety of structural analogues of zampanolide. The syn-
thetic zampanolide has biological properties equivalent to
those previously described for the natural product.[1,2]
26.3, 25.6 ppm. IR (thin film): ν = 2981, 1706, 1646, 1500, 1368,
˜
1254, 1221, 1072, 968, 860, 844 cm–1. MS (ESI): m/z = 279 [M +
Na]+. E isomer: [α]2D3 = –11.7 (c = 2.2, ethyl acetate). Rf = 0.28
(hexanes/ethyl acetate
=
90:10, KMnO4 stain). 1H NMR
(400 MHz, CDCl3): δ = 5.63 (d, J = 1.2 Hz, 1 H), 4.32–4.24 (m, 1
H), 4.06 (dd, J = 5.9, 8.1 Hz, 1 H), 3.57 (dd, J = 6.8, 8.1 Hz, 1 H),
2.46 (dd, J = 6.9, 13.4 Hz, 1 H), 2.27 (dd, J = 7.0, 14.1 Hz, 1 H),
2.17 (d, J = 1.3 Hz, 3 H), 1.48 (s, 9 H), 1.42 (s, 3 H), 1.36 (s, 3
H) ppm. 13C NMR (100 MHz, CDCl3): δ = 165.9, 153.4, 119.5,
109.1, 79.7, 73.8, 69.1, 44.8, 28.1, 26.9, 25.5, 18.6 ppm. IR (thin
film): ν = 2982, 1714, 1651, 1455, 1380, 1184, 1110, 1073, 862,
˜
829 cm–1. MS (ESI): m/z = 279 [M + Na]+.
Compound 11: DIBAL-H (1.18 g, 1.47 mL, 8.3 mmol) was added
to a solution of the E isomer of 9 (0.85 g, 3.3 mmol) in CH2Cl2
(20 mL) at –15 °C. The mixture was stirred at –15 °C for 5 min and
quenched with saturated NH4Cl. After stirring at room tempera-
ture for 5 min, the resulting gel was diluted with CH2Cl2 and fil-
tered through Celite. The aqueous layer was extracted with CH2Cl2
once. The combined organic layers were dried (Na2SO4) and con-
centrated. Silica gel chromatography with hexanes/diethyl ether
(2:3) as eluent yielded 11 as a colorless oil (0.56 g, 91%). [α]2D3
=
+1.5 (c = 2.6, ethyl acetate). Rf = 0.27 (hexanes/diethyl ether = 2:3,
KMnO4 stain). 1H NMR (400 MHz, CDCl3): δ = 5.63 (t, J =
6.8 Hz, 1 H), 4.27–4.18 (m, 1 H), 4.12 (d, J = 6.6 Hz, 2 H), 4.01
(dd, J = 6.0, 8.0 Hz, 1 H), 3.53 (t, J = 7.5 Hz, 1 H), 2.34 (dd, J =
7.0, 13.9 Hz, 1 H), 2.17 (dd, J = 6.2, 14.0 Hz, 1 H), 1.98 (br. s, 1
H), 1.69 (s, 3 H), 1.39 (s, 3 H), 1.32 (s, 3 H) ppm. 13C NMR
(100 MHz, CDCl3): δ = 135.0, 126.1, 108.9, 76.7, 69.1, 58.9, 43.5,
Experimental Section
General Methods: All moisture-sensitive reactions were carried out
in oven-dried flasks under argon. Anhydrous solvents were ob-
tained as follows: THF, diethyl ether, and benzene were distilled
from sodium and benzophenone, dichloromethane, pyridine, trieth-
ylamine, and diisopropylethylamine were distilled from CaH2. All
other solvents were of HPLC grade. 1H and 13C NMR spectra were
recorded with Varian INOVA300-1 and Bruker Avance ARX-400
spectrometers. NMR spectroscopic data were resolved with Mes-
trec software. IR spectra were recorded with a Perkin–Elmer spec-
trometer. Optical rotations were recorded with a Perkin–Elmer 341
polarimeter. Mass spectra were obtained at the Purdue University
Campus-wide Mass Spectrometry Center. Column chromatography
was performed with Whatman 240–400 mesh silica gel under a low
pressure of 3–5 psi. TLC was carried out with Merck silica gel 60
F254 plates. HPLC was performed with an Agilent 1100 instrument.
26.8, 25.5, 16.7 ppm. IR (thin film): ν = 3413, 2985, 2935, 1668,
˜
1455, 1371, 1216, 1157, 1062, 999, 831, 790 cm–1. MS (ESI): m/z =
209 [M + Na]+.
Compound 12: A solution of 11 (400 mg, 2.15 mmol) in diethyl
ether (1 mL) was added dropwise to a suspension of NaH (60% in
silicon oil, 10 mol-%, 8.6 mg, 0.22 mmol) in diethyl ether (1.5 mL)
at 0 °C. Then the solution was stirred at room temperature for
20 min and cooled to –40 °C. Next, CCl3CN (215 μL, 2.15 mmol)
was added. The mixture was stirred at –40 °C for 1 h and then at
room temperature for 1 h followed by the addition of MeOH
(9 μL). The solution was concentrated, suspended in hexanes, and
filtered through Celite. The filtrate was concentrated and analyzed
by 1H NMR spectroscopy. The resulting yellow oil 12 (697 mg,
98%) was sufficiently pure to be used in the next step without fur-
ther purification. [α]2D3 = –1.9 (c = 2.0, ethyl acetate). Rf = 0.28
Compound 9: A solution of tert-butyl 2-(dimethoxyphosphoryl)-
acetate (8.7 g, 38.8 mmol) in THF (10 mL) was added to a suspen-
sion of NaH (1.35 g, 60% in mineral oil, 33.7 mmol) in THF
(40 mL) at 0 °C. The mixture was stirred at 0 °C for 30 min and at
room temperature for 30 min. After cooling to 0 °C, ketone 8
(1.69 g, 10.7 mmol) in THF (10 mL) was added. After stirring at
0 °C for 10 min and at room temperature overnight, the reaction
was quenched with saturated NH4Cl. The aqueous layer was ex-
tracted with diethyl ether twice. The combined organic layers were
washed with brine, dried (Na2SO4), and concentrated. The residue
was purified by silica gel chromatography with hexanes/ethyl acet-
ate (4:1) to give a mixture of E/Z isomers, and pure ethyl acetate
was used to recover excess tert-butyl 2-(dimethoxyphosphoryl)-
acetate. The mixture was subjected to silica gel column chromatog-
raphy and eluted with hexanes/ethyl acetate (9:1) to yield the Z
(0.4 g) and E isomers (1.7 g) in an overall yield of 74%. Z isomer:
[α]2D3 = –43 (c = 2.3, ethyl acetate). Rf = 0.29 (hexanes/ethyl acetate
(hexanes/ethyl acetate
=
90:10, KMnO4 stain). 1H NMR
(400 MHz, CDCl3): δ = 8.27 (s, 1 H), 5.54 (t, J = 6.8 Hz, 1 H),
4.82 (d, J = 6.8 Hz, 2 H), 4.30–4.21 (m, 1 H), 4.02 (dd, J = 5.9,
8.1 Hz, 1 H), 3.57 (t, J = 7.5 Hz, 1 H), 2.44 (dd, J = 6.7, 13.4 Hz,
1 H), 2.25 (dd, J = 6.9, 14.0 Hz, 1 H), 1.79 (s, 3 H), 1.42 (s, 3 H),
1.36 (s, 3 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 162.6, 139.1,
120.3, 108.9, 74.2, 69.1, 65.8, 43.5, 26.9, 25.6, 17.2 ppm. IR (thin
film): ν = 3470, 3344, 2986, 1738, 1662, 1455, 1370, 1291, 1229,
˜
1157, 1068, 984, 827, 799, 650 cm–1. MS (ESI): m/z = 352 [M +
Na]+.
Compound 13: TIPSOTf (25 μL, 93 µmol) was added to a suspen-
sion of 7 (40 mg, 0.25 mmol), 12 (90 mg, 0.27 mmol), and mol. si-
eves (4 Å, 40 mg) in CH2Cl2 (1 mL) at –78 °C and the mixture was
stirred at room temperature overnight. The reddish suspension was
quenched with Et3N (0.1 mL) and concentrated. The residue was
purified by silica gel chromatography with hexanes/diethyl ether
(4:1) as eluent to give the target product 13 as a colorless oil (32 mg,
1
= 90:10, KMnO4 stain). H NMR (400 MHz, CDCl3): δ = 5.69 (s,
1 H), 4.36–4.27 (m, 1 H), 4.07 (dd, J = 6.0, 8.1 Hz, 1 H), 3.62 (dd,
J = 7.1, 8.0 Hz, 1 H), 3.07 (dd, J = 4.7, 12.7 Hz, 1 H), 2.68 (dd, J
= 7.8, 12.7 Hz, 1 H), 1.95 (d, J = 1.3 Hz, 3 H), 1.46 (s, 9 H), 1.42
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
7

Job/Unit: O20286
/KAP1
Date: 12-06-12 18:20:27
Pages: 11
A. K. Ghosh, X. Cheng, R. Bai, E. Hamel
FULL PAPER
E/Z = 2:1) containing residual 7 (20 mg). The yield was 79%
(brsm). Rf = 0.40 (hexanes/ethyl acetate = 4:1, KMnO4 stain). H
rial, but development with hexanes/ethyl acetate = 99:1 three times
1
gave a slightly higher Rf value than that of the starting material.
1
NMR (300 MHz, CDCl3): δ = 5.87–5.73 (m, 2 H), 5.57–5.35 (m, 2
H), 5.12–5.06 (m, 4 H), 4.27–3.95 (m, 12 H), 3.88–3.79 (m, 2 H),
3.57–3.43 (m, 2 H), 2.55–2.15 (m, 12 H), 1.78 (s, 1.7 H), 1.69 (s,
4.3 H), 1.41 (s, 1.7 H), 1.40 (s, 4.3 H), 1.34 (s, 6 H), 1.25 (t, J =
7.2 Hz, 6 H) ppm.
[α]2D0 = +3.5 (c = 1.2, ethyl acetate). H NMR (300 MHz, CDCl3):
δ = 7.69–7.66 (m, 4 H), 7.45–7.21 (m, 11 H), 6.59 (d, J = 16.0 Hz,
1 H), 6.24 (dd, J = 16.0, 5.7 Hz, 1 H), 4.78 (s, 2 H), 3.99–3.86 (m,
2 H), 3.81–3.74 (m, 1 H), 3.70–3.62 (m, 1 H), 2.36 (d, J = 14.8 Hz,
1 H), 2.28 (d, J = 14.4 Hz, 1 H), 1.92–1.76 (m, 2 H), 1.05 (s, 9
H) ppm. 13C NMR (75 MHz, CDCl3): δ = 144.3, 136.8, 135.5,
133.9, 133.8, 130.1, 129.5, 128.4, 127.6, 126.4, 108.9, 78.5, 75.1,
Compound 5: Finely powdered CeCl3·7H2O (0.228 g, 0.61 mmol)
was heated at 160 °C under high vacuum for 2.5 h and cooled to
room temperature under argon. The white powder was suspended
in THF (1 mL) and stirred at room temperature for 2 h. Then the
white suspension was cooled to –78 °C and to it was added
TMSCH2MgCl (0.615 mL, 1 m in diethyl ether, 0.615 mmol). The
yellow suspension was stirred at –78 °C for 1 h and 13 (40 mg,
0.12 mmol) in THF (0.4 mL) was added. Then the mixture was
stirred at –78 °C for 2 h and at room temperature overnight. The
white suspension was cooled to 0 °C, diluted with anhydrous di-
ethyl ether (5 mL) followed by the addition of HCl (1 mL, 2 m).
Then the reaction mixture was diluted with water/diethyl ether. The
aqueous layer was extracted with diethyl ether twice. The combined
organic layers were washed with brine, dried (Na2SO4), and con-
centrated. The residue was purified by silica gel chromatography
with hexanes/ethyl acetate (93:7) as eluent to give the an E/Z mix-
ture (ca. 2:1) of 5 (32 mg, 73%) as a colorless oil. Rf = 0.41 (hex-
anes/ethyl acetate = 4:1, PMA stain). MS (ESI): m/z = 389 [M +
60.1, 41.0, 40.7, 39.0, 26.8, 19.2 ppm. IR (thin film): ν = 3070,
˜
2857, 1959, 1823, 1651, 1590, 1427, 1359, 1110, 966, 892, 741, 703,
614 cm–1. MS (ESI): m/z 505 [M + Na+]. HRMS (ESI): calcd. for
C32H38O2SiNa 505.2539; found 505.2536.
Determination of the Enantiomeric Excess: TBAF (0.4 mL, 1 m in
THF, 0.4 mmol) was added to a solution of 16 (30 mg, 60.4 μmol)
in THF (1 mL). Then the solution was stirred at room temperature
for 4 h and concentrated. The residue was purified by silica gel
chromatography with hexanes/ethyl acetate (3:2, v/v) as eluent to
give the free alcohol as a colorless oil (8.0 mg, 60%) with 97% ee.
Rf = 0.35 (hexanes/ethyl acetate = 7:3, UV). [α]2D0 = +6.8 (c = 0.73,
ethyl acetate). HPLC: Daicel IC column, hexanes/IPA = 9:1,
0.5 mL/min, 254 nm, t1 = 14.1 min, t2 = 14.9 min. Peak at t2 is the
major enantiomer.1H NMR (400 MHz, CDCl3): δ = 7.39–7.23 (m,
5 H), 6.23 (dd, J = 6.1, 16.0 Hz, 1 H), 6.58 (d, J = 16.0 Hz, 1 H),
4.79 (s, 2 H), 4.03–3.99 (m, 1 H), 3.86–3.82 (m, 2 H), 3.68–3.62 (m,
1 H), 2.64 (s, 1 H), 2.36 (d, J = 13.3 Hz, 1 H), 2.23 (d, J = 13.3 Hz,
1 H), 2.20–2.08 (m, 2 H), 1.94–1.84 (m, 1 H), 1.83–1.76 (m, 1
H) ppm. 13C NMR (100 MHz, CDCl3): δ = 143.4, 130.6, 129.5,
128.4, 127.6, 126.4, 109.3, 79.0, 78.7, 61.2, 40.7, 40.4, 37.9 ppm. IR
1
Na]+. H NMR (400 MHz, CDCl3): δ = 5.90–5.80 (m, 2 H), 5.48
(t, J = 6.6 Hz, 0.66 H), 5.40 (t, J = 6.6 Hz, 1.38 H), 5.13–5.07 (m,
4 H), 5.10–5.04 (m, 4 H), 4.65 (s, 2 H), 4.59 (s, 2 H), 4.27–4.14 (m,
2 H), 4.05–3.98 (m, 6 H), 3.57–3.46 (m, 4 H), 2.46–2.37 (m, 2 H),
2.30–2.17 (m, 8 H), 2.08 (dd, J = 6.3, 14.1 Hz, 2 H), 1.78 (s, 2 H),
1.70 (s, 4 H), 1.58 (s, 1.4 H), 1.55 (s, 2.6 H), 1.42 (s, 2 H), 1.41 (4
H), 1.35 (s, 6 H), 0.02 (s, 18 H) ppm. 13C NMR (100 MHz, CDCl3):
δ = 144.5, 135.6, 135.2, 135.1, 124.8, 124.1, 116.8, 109.7, 109.6,
108.9, 108.8, 108.7, 74.6, 74.5, 69.3, 65.6, 65.4, 65.3, 43.7, 42.9,
38.5, 36.5, 27.1, 27.0, 26.9, 25.7, 25.6, 24.1, 23.2, 17.0, –1.3 ppm.
(thin film): ν = 3401, 2942, 2900, 1719, 1650, 1450, 1421, 1315,
˜
1062, 966, 746, 693 cm–1.
Catalyzed Synthesis of Compound 16 (Table 1, entry 9): A solution
of 17 (1.80 g, 3.24 mmol) in CH3CN (10 mL) was added to a sus-
pension of DDQ (0.147 g, 20 mol-%), PPTS (1.63 g, 6.48 mmol),
CAN (3.51 g, 6.48 mmol), and mol. sieves (4 Å, 3.6 g) in CH3CN
(120 mL). The suspension was stirred at –38 °C for 16 h and
quenched with Et3N (1 mL). The mixture was filtered through a
column of silica gel with hexanes/ethyl acetate (95:5, v/v) as eluent.
The concentrated product was purified by silica gel chromatog-
raphy with hexanes/ethyl acetate (95:5, v/v) as eluent to give meth-
ylenetetrahydropyran 16 as a reddish oil (1.17 g, 71%). The analyti-
cal data comply with the data reported above.
IR (thin film): ν = 3075, 2953, 1633, 1435, 1370, 1248, 1157, 1068,
˜
857, 695 cm–1.
Optimization of the Oxidative Cyclization Reaction: Solvent (4 mL),
DDQ (34 mg, 0.15 mmol), and acid (1.5 equiv.) were added to a
reaction vial charged with 17 (56 mg, 0.1 mmol) and mol. sieves
(4 Å) (0.1 g) at –38 °C under argon. Then the mixture was stirred
until full conversion, as monitored by TLC (hexanes/ethyl acetate
= 98:2, developed four times, ca. 6–8 h to reach completion). Then
the reaction was quenched with Et3N (0.2 mL) and filtered under
pressure through a pad of silica gel. The concentrated residue was
purified by silica gel chromatography with hexanes/ethyl acetate
(95:5) as eluent to give 16 as a colorless oil.
Compound 36: Et3N (56 μL, 0.4 mmol) and Cy2BCl (0.24 mL, 1 m
in hexanes, 0.24 mmol) were added in sequence to a reaction vial
charged with isobutyramide (20.9 mg, 0.24 mmol) in diethyl ether
(1.2 mL) to yield a white suspension. The mixture was stirred at
0 °C for 15 min and then cyclohexanecarbaldehyde (24 μL,
0.2 mmol) was added. After being stirred at 0 °C for 1 h, the reac-
tion mixture was loaded onto a column of silica gel with CH2Cl2
(0.5 mL) to dissolve the white precipitate. The column was eluted
with hexanes/ethyl acetate = 3:2 to give the product 36 as a white
solid (40 mg, 99%). Rf = 0.20 (hexanes/ethyl acetate = 3:2, PMA
stain). 1H NMR (400 MHz, CDCl3): δ = 6.12 (d, J = 7.1 Hz, 1 H),
5.08–5.04 (m, 1 H), 3.95 (d, J = 3.7 Hz, 1 H), 2.39–2.32 (m, 1 H),
1.78–1.65 (m, 4 H), 1.54–1.44 (m, 1 H), 1.21–0.99 (m, 12 H) ppm.
13C NMR (100 MHz, CDCl3): δ = 178.2, 77.6, 42.1, 35.6, 28.13,
Synthesis of Compound 16. Cyclization Reaction with a Stoichiomet-
ric Amount of DDQ (Table 1, Entry 10): Anhydrous acetonitrile
(100 mL) was added to a flask charged with DDQ (1.56 g,
6.87 mmol), PPTS (1.78 g, 6.87 mmol), and mol. sieves (4 Å, 9.0 g)
under argon at –38 °C. A solution of (R)-tert-butyl[3-cinnamyloxy-
5-(trimethylsilylmethyl)hex-5-enyloxy]diphenylsilane (17; 2.63 g,
4.72 mmol, 97% ee) in acetonitrile (31 mL) was added dropwise
over 5 min. The mixture was stirred at –38 °C for 3 h. The reaction
was quenched with triethylamine (3 mL) and filtered through a pad
of Celite. The filtrate was concentrated and then dissolved in ethyl
acetate, washed with brine, and dried (Na2SO4). Evaporation of the
solvent gave a residue which was purified by silica gel chromatog-
raphy with hexanes/ethyl acetate (98:2) to provide compound 16 as
a pale-yellow oil (1.81 g, 81%). Rf = 0.36 (hexanes/ethyl acetate =
95:5, UV). The Rf is almost the same as that of the starting mate-
27.9, 26.2, 25.7, 25.6, 19.5, 19.2 ppm. IR (thin film): ν = 1693,
˜
1639, 1512, 1366, 1249, 1171, 778 cm–1. MS (ESI): m/z = 222 [M
+ Na]+.
Compound 34: Li2CuCl4 (5.3 mL, 0.1 m in THF) and isoprenylmag-
nesium bromide (0.5 m in THF, 26 mL, 13 mmol) were added to a
solution of TBS glycidol (2 g, 10.6 mmol) in THF (20 mL) at
8
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Eur. J. Org. Chem. 0000, 0–0

Job/Unit: O20286
/KAP1
Date: 12-06-12 18:20:27
Pages: 11
Total Synthesis of Potent Antitumor Macrolide (–)-Zampanolide
–78 °C. After stirring at –78 °C for 15 min and at room temperature
for 2 h, crotonyl chloride (1.22 mL, 12.8 mmol) was added and the
reaction mixture was stirred at 0 °C for 5 min. Then the reaction
was quenched with water and extracted with ethyl acetate twice.
The combined organic layers were washed with sat. NaHCO3 and
brine, dried (Na2SO4), and concentrated. The residue was purified
by flash chromatography with hexanes/ethyl acetate = 97:3 as elu-
ent to give TBS ether as a colorless oil. Rf = 0.31 (hexanes/ethyl
acetate = 97:3, PMA stain). 1H NMR (400 MHz, CDCl3): δ =
6.98–6.91 (m, 1 H), 5.81 (dd, J = 1.6, 15.5 Hz, 1 H), 5.23–5.16 (m,
1 H), 4.77 (s, 1 H), 4.71 (s, 1 H), 3.66 (d, J = 5.2 Hz, 2 H), 2.36
(dd, J = 7.3, 13.9 Hz, 1 H), 2.24 (dd, J = 6.0, 13.8 Hz, 1 H), 1.86
(dd, J = 1.5, 6.9 Hz, 3 H), 1.75 (s, 3 H), 0.88 (s, 9 H), 0.02 (s, 6
H) ppm. 13C NMR (100 MHz, CDCl3): δ = 166.0, 144.4, 141.5,
122.8, 113.1, 72.2, 64.0, 38.9, 25.7, 22.5, 18.1, 17.9, –5.4 ppm. IR
three times. The combined organic layers were washed with brine,
dried (Na2SO4), and concentrated. The residue was purified by sil-
ica gel chromatography with hexanes/ethyl acetate (1:1, v/v) as elu-
ent to yield 37 as a mixture of diastereomers in the ratio of 1:1
1
(15 mg, 56%). H NMR (300 MHz, CDCl3): δ = 10.2 (s, 1 H), 9.9
(s, 1 H), 7.06–6.93 (m, 2 H), 6.70 (d, J = 7.6 Hz, 1 H), 6.60 (d, J
= 8.3 Hz, 1 H), 5.86–5.84 (m, 1 H), 5.81–5.79 (m, 1 H), 5.16–5.07
(m, 2 H), 4.82–8.81 (m, 2 H), 4.75–4.74 (m, 2 H), 2.48–2.34 (m, 4
H), 2.15–2.02 (m, 2 H), 1.90–1.87 (m, 6 H), 1.75 (s, 3 H), 1.73 (s,
3 H), 1.20–1.16 (m, 12 H) ppm. IR (thin film): ν = 3339, 1711,
˜
1659, 1531, 1444, 1293, 1183, 1103, 1045, 969, 893 cm–1. MS (ESI):
m/z = 292 [M + Na]+. Rf = 0.35 (hexanes/ethyl acetate = 1:1,
KMnO4).
Synthesis of Compounds 1, 39, and 40 by Amidation Using Diphenyl
Phosphate as Catalyst: A mixture containing dactylolide (2; 0.5 mg,
1.31 μmol), amide 38 (0.30 mg, 2.76 μmol), and diphenyl phosphate
(0.03 mg, 0.13 μmol) in CH2Cl2 (30 μL) was stirred at 23 °C for
14 h. The mixture was loaded onto a column of silica gel with hex-
anes/ethyl acetate (1:1) as eluent to give a mixture of 1, 38, 39, and
40, which was further separated by HPLC (IC, hexanes/iPrOH =
1:1, 0.5 mL/min, 254 nm). The ratio of 1/39 was close to 1:1.
(thin film): ν = 2930, 1722, 1657, 1258, 1182, 1101, 836, 777 cm–1.
˜
MS (ESI): m/z = 321 [M + Na]+.
HCl (170 μL, 2 m) and water (170 μL) were added to a solution
of TBS ether prepared as described above (50 mg, 0.168 mmol) in
MeOH/CH2Cl2 (4:1, v/v, 5 mL). Then the reaction was quenched
with sat. NaHCO3 and diluted with water. The aqueous layer was
extracted with ethyl acetate three times. The combined organic lay-
ers were washed with brine, dried (Na2SO4), and concentrated. The
residue was purified by silica gel chromatography with hexanes/
ethyl acetate (7:3) as eluent to give the alcohol as a colorless oil
(25 mg, 70%). Rf = 0.28 (hexanes/ethyl acetate = 7:3, I2 or KMnO4
Bis-amide 40: 1H NMR (500 MHz, CDCl3): δ = 7.66 (dd, J = 12.1,
14.7 Hz, 1 H), 7.48–7.41 (m, 2 H), 6.85–6.80 (m, 1 H), 6.45–6.39
(m, 3 H), 6.33 (d, J = 7.3 Hz, 1 H), 6.10 (d, J = 11.6 Hz, 1 H),
6.06–5.98 (m, 2 H), 5.93 (d, J = 14.7 Hz, 1 H), 5.92 (d, J = 16.2 Hz,
1 H), 5.68–5.61 (m, 2 H), 5.41 (d, J = 11.0 Hz, 1 H), 5.40 (d, J =
11.0 Hz, 1 H), 5.16 (d, J = 8.0 Hz, 1 H), 4.72 (s, 2 H), 4.17 (d, J =
13.6 Hz, 1 H), 3.97–3.93 (m, 1 H), 3.26 (t, J = 10.4 Hz, 1 H), 3.00
(d, J = 13.4 Hz, 1 H), 2.39–2.33 (m, 2 H), 2.24–2.19 (m, 2 H), 2.13
(d, J = 12.7 Hz, 1 H), 2.06 (d, J = 13.6 Hz, 1 H), 1.93 (d, J =
12.4 Hz, 2 H), 1.86 (d, J = 3.9 Hz, 6 H), 1.79 (s, 3 H), 1.71 (s, 3
H) ppm. MS (ESI): m/z = 611 [M + Na]+.
1
stain). H NMR (400 MHz, CDCl3): δ = 7.05–6.93 (m, 1 H), 5.85
(dd, J = 1.7, 15.5 Hz, 1 H), 5.15–5.10 (m, 1 H), 4.81 (s, 1 H), 4.76
(s, 1 H), 3.78–3.72 (m, 1 H), 3.68–3.62 (m, 1 H), 2.37 (dd, J = 7.5,
14.1 Hz, 1 H), 2.30 (dd, J = 6.0, 14.2 Hz, 1 H), 2.06 (t, J = 6.0 Hz,
1 H), 1.88 (d, J = 5.9 Hz, 3 H), 1.77 (s, 3 H) ppm. 13C NMR
(100 MHz, CDCl3): δ = 166.5, 145.3, 141.0, 122.4, 113.5, 73.1, 64.5,
39.0, 22.4, 17.9 ppm. IR (thin film): ν = 3441, 2918, 1719, 1655,
˜
1445, 1310, 1293, 1184, 1103, 1048, 969, 894, 838 cm–1. MS (ESI):
(–)-Zampanolide (1) and 39: (S)-TRIP (41; 2.7 mg, 20 mol-%) in
CH2Cl2 (0.7 mL) was added to a flask containing (–)-2 (7 mg,
18.3 μmol) and (2Z,4E)-hexa-2,4-dienamide 38 (6.1 mg, 55 μmol).
The resulting mixture was stirred at 23 °C for 12 h. After this
period the reaction was loaded on a short column of silica gel and
eluted with hexanes/ethyl acetate (3:2) to provide a crude mixture
of (S)-TRIP (41), 1, and 39. The mixture was separated by HPLC
to give (–)-zampanolide (1; 4.6 mg, 51%) and 39 (1.6 mg, 18%).
m/z = 207 [M + Na]+.
Dess–Martin reagent (0.68 mL, 0.3 m in CH2Cl2, 0.204 mmol) was
added to a suspension of the above alcohol (25 mg, 0.136 mmol)
and NaHCO3 (0.1 g, 1.2 mmol) in CH2Cl2 (5 mL). The mixture was
vigorously stirred for 2 h and quenched with a Na2S2O3/NaHCO3
solution. The aqueous layer was extracted with CH2Cl2 twice. The
combined organic layers were dried (Na2SO4) and concentrated.
The residue was filtered through a plug of silica gel with hexanes/
ethyl acetate (7:3) as eluent to give 34 as a colorless oil (21 mg,
83%). The compound was unstable and used immediately in the
next step. Rf = 0.52 (hexanes/ethyl acetate = 7:3, I2 stain or
KMnO4). 1H NMR (300 MHz, CDCl3): δ = 9.56 (s, 1 H), 7.13–
7.01 (m, 1 H), 5.92 (dd, J = 1.7, 15.6 Hz, 1 H), 5.20 (dd, J = 5.7,
9.0 Hz, 1 H), 4.85 (s, 1 H), 4.78 (s, 1 H), 2.56 (dd, J = 4.6, 14.9 Hz,
1 H), 2.45 (dd, J = 8.9, 15.8 Hz, 1 H), 1.91 (dd, J = 1.6, 6.9 Hz, 3
H), 1.76 (s, 3 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 198.3,
165.6, 146.6, 139.4, 121.4, 114.3, 76.1, 36.9, 22.3, 18.0 ppm. IR
HPLC: hexanes/iPrOH = 1:1, 0.5 mL/min, 254 nm, t(S)-TRIP
7.4 min, t38 = 12.5 min, t1 = 15.3 min, t39 = 23.3 min.
=
Zampanolide (1): Rf = 0.35 (hexanes/ethyl acetate = 1:1, UV,
PMA). [α]2D0 = –94 (c = 0.08, CH2Cl2). 1H NMR (500 MHz,
CDCl3): δ = 7.65 (dd, J = 11.6, 14.9 Hz, 1 H), 7.45–7.40 (m, 1 H),
6.85–6.79 (m, 1 H), 6.46 (t, J = 11.3 Hz, 1 H), 6.31 (d, J = 7.8 Hz,
1 H), 6.11 (d, J = 11.9 Hz, 1 H), 6.08–6.02 (m, 1 H), 5.95 (d, J =
15.0 Hz, 1 H), 5.94 (d, J = 16.4 Hz, 1 H), 5.46–5.43 (m, 2 H), 5.31–
5.27 (m, 1 H), 5.20 (d, J = 7.9 Hz, 1 H), 4.73 (s, 2 H), 4.13 (d, J =
13.0 Hz, 1 H), 3.98–3.94 (m, 1 H), 3.66 (br. s, 1 H), 3.29 (t, J =
10.5 Hz, 1 H), 3.04 (d, J = 13.6 Hz, 1 H), 2.41 (d, J = 13.6 Hz, 1
H), 2.38–2.29 (m, 1 H), 2.29–2.20 (m, 2 H), 2.14 (d, J = 13.4 Hz,
1 H), 2.09 (d, J = 13.4 Hz, 1 H), 1.97–1.91 (m, 2 H), 1.87 (d, J =
7.0 Hz, 3 H), 1.81 (s, 3 H), 1.72 (s, 3 H) ppm. 13C NMR (125 MHz,
(thin film): ν = 2943, 2851, 1722, 1656, 1444, 1377, 1294, 1263,
˜
1178, 1105, 969, 898, 837, 688 cm–1. MS (ESI): m/z = 205 [M +
Na]+.
Compound 37: Cy2BCl solution (0.20 mL, 0.6 m in hexanes,
0.12 mmol) was added to a solution of amide 35 (10.4 mg, CDCl3): δ = 197.9, 167.6, 166.8, 146.3, 143.8, 143.6, 143.5, 140.2,
0.12 mmol) and Et3N (28 μL, 0.2 mmol) in diethyl ether (0.4 mL).
The resulting white suspension was stirred at 0 °C for 15 min and
then aldehyde 34 (28.4 mg, 0.1 mmol) in diethyl ether (0.2 mL) was
added. After stirring at 0 °C for 1 h, the mixture was quenched
with THF/phosphate buffer (pH = 7.4)/30% H2O2 (1:1:1, v/v/v,
2 mL). Then the aqueous layer was extracted with ethyl acetate
140.1, 132.0, 131.4, 129.9, 128.1, 125.2, 120.2, 116.8, 109.1, 76.5,
75.8, 75.3, 71.4, 45.1, 41.8, 40.9, 40.6, 40.1, 23.6, 18.6, 16.6 ppm.
IR (thin film): ν = 3368, 2924, 2854, 2360, 1636, 1539, 1456, 1357,
˜
1281, 1260, 1210, 1149, 1086, 979, 889, 803, 666 cm–1. MS (ESI):
m/z = 518 [M + Na]+. HRMS (ESI): calcd. for C29H37NO6Na
518.2519; found 518.2520.
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Job/Unit: O20286
/KAP1
Date: 12-06-12 18:20:27
Pages: 11
A. K. Ghosh, X. Cheng, R. Bai, E. Hamel
FULL PAPER
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Zampanolide Diastereomer 39: Rf = 0.35 (hexanes/ethyl acetate =
1:1, UV, PMA). 1H NMR (500 MHz, CDCl3): δ = 7.66 (dd, J =
11.6, 15.0 Hz, 1 H), 7.46–7.39 (m, 1 H), 6.86–6.80 (m, 1 H), 6.46
(t, J = 11.3 Hz, 1 H), 6.35 (d, J = 7.5 Hz, 1 H), 6.12 (d, J = 11.6 Hz,
1 H), 6.06 (dd, J = 6.9, 15.0 Hz, 1 H), 6.00 (d, J = 15.0 Hz, 1 H),
5.94 (d, J = 16.3 Hz, 1 H), 5.54–5.51 (m, 1 H), 5.45 (d, J = 11.2 Hz,
1 H), 5.37–5.31 (m, 1 H), 5.24 (d, J = 8.6 Hz, 1 H), 4.73 (br. s, 2
H), 4.11 (d, J = 13.7 Hz, 1 H), 3.98–3.94 (m, 1 H), 3.60 (br., 1 H),
3.34–3.27 (m, 1 H), 3.05 (d, J = 13.7 Hz, 1 H), 2.51 (dd, J = 11.0,
13.8 Hz, 1 H), 2.39–2.23 (m, 3 H), 2.15 (d, J = 13.7 Hz, 1 H), 2.10
(d, J = 13.1 Hz, 1 H), 2.00–1.93 (m, 2 H), 1.87 (d, J = 6.6 Hz, 3
H), 1.82 (s, 3 H), 1.71 (s, 3 H) ppm.
[9]
[10]
[11]
Biological Methods: Tubulin and heat-treated microtubule-associ-
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of compound-induced microtubule assembly by turbidimetry at
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Supporting Information (see footnote on the first page of this arti-
1
cle): H and 13C NMR spectra for all new compounds and HPLC
[13]
[14]
traces for compounds 1, 16, and 39.
Acknowledgments
[15]
[16]
Financial support for this work was provided in part by the
National Institutes of Health and Purdue University. The MCF-
7, OVCAR-8, and NCI/ADR-RES human cancer cell lines were
generously provided by the National Cancer Institute drug screen-
ing group. Paclitaxel was generously provided by the Drug Chemis-
try and Synthesis Branch of the National Cancer Institute.
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Received: March 13, 2012
Published Online: ᭿
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Eur. J. Org. Chem. 0000, 0–0

Job/Unit: O20286
/KAP1
Date: 12-06-12 18:20:27
Pages: 11
Total Synthesis of Potent Antitumor Macrolide (–)-Zampanolide
Natural Product Synthesis
An enantioselective synthesis of (–)-zam-
panolide has been achieved. This rare mar-
ine natural product shows microtubule-sta-
bilizing properties. It also displays potent
activity against taxol-resistant cells. This
synthesis will provide a convenient access
to analogues for important structure–ac-
tivity relationship studies and less complex
anticancer agents related to zampanolide.
A. K. Ghosh,* X. Cheng, R. Bai,
E. Hamel ........................................ 1–11
Total Synthesis of Potent Antitumor Mac-
rolide (–)-Zampanolide: An Oxidative In-
tramolecular Cyclization-Based Strategy
Keywords: Natural products
synthesis / C–H activation / Metathesis /
Stereoselective catalysis
/
Total
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