Total synthesis of the cyclodepsipeptide apratoxin A and its analogues and assessment of their biological activities

Article abstract of DOI:10.1002/chem.200600599

A novel total synthesis of apratoxin A is described, with key steps including the assembly of its ketide segment through a D-proline-catalyzed direct aldol reaction and Oppolzer's anti aldol reaction and the preparation of its thiazoline unit in a biomimetic synthesis. An oxazoline analogue of apratoxin A has also been elaborated by a similar approach. This compound has a potency against HeLa cell proliferation only slightly lower than that of apratoxin A, whilst a C(40)-demethylated oxazoline analogue of apratoxin A displays a much lower cytotoxicity and the C(37)-epimer and C(37) demethylation prod uct of this new analogue are inactive. These results suggest that the two methyl groups at C(37) and C(40) and the stereochemistry at C(37) are essential for the potent cellular activity of the oxazoline analogue of apratoxin A. Further biological analysis revealed that both synthetic apratoxin A and its oxazoline analogue inhibited cell proliferation by causing cell cycle arrest in the G1 phase.

Full text of DOI:10.1002/chem.200600599

FULL PAPER
DOI: 10.1002/chem.200600599
Total Synthesis of the Cyclodepsipeptide Apratoxin A and Its Analogues and
Assessment of TheirBiological Activities
Dawei Ma,*[a] Bin Zou,[a] Guorong Cai,[b] Xiaoyi Hu,[c] and Jun O. Liu*[c]
Abstract: A novel total synthesis of
apratoxin A is described, with key
steps including the assembly of its
ketide segment through a d-proline-
catalyzed direct aldol reaction and Op-
polzerꢀs anti aldol reaction and the
preparation of its thiazoline unit in a
biomimetic synthesis. An oxazoline an-
alogue of apratoxin A has also been
elaborated by a similar approach. This
compound has a potency against HeLa
cell proliferation only slightly lower
than that of apratoxin A, whilst
C(40)-demethylated oxazoline ana-
logue of apratoxin A displays a much
lower cytotoxicity and the C(37)-
epimer and C(37) demethylation prod-
uct of this new analogue are inactive.
These results suggest that the two
methyl groups at C(37) and C(40) and
the stereochemistry at C(37) are essen-
tial for the potent cellular activity of
the oxazoline analogue of apratoxin A.
Further biological analysis revealed
that both synthetic apratoxin A and its
oxazoline analogue inhibited cell pro-
liferation by causing cell cycle arrest in
the G1 phase.
a
Keywords: aldol reaction
cycle cyclic depsipeptides
cytotoxicity · total synthesis
·
cell
·
·
Introduction
from the marine cyanobacterium Lyngbya majuscula by
Moore and co-workers in 2001.[2] This compound was shown
to be cytotoxic against the LoVo and KB cancer cell lines,
exhibiting in vitro IC50 values ranging from 0.36 to 0.52 nm
and being the most cytotoxic among several cyclodepsipepti-
des discovered in the marine cyanobacterium.[2] Preliminary
biological studies have indicated that apratoxin A affects
neither microtubule polymerization dynamics nor topoiso-
merase I, but further investigation of its mode of action has
been hampered by the scarcity of the natural material. In
addition, apratoxin A was poorly tolerated in mice, probably
due to its intrinsic toxicity to normal cells and lack of selec-
tivity for tumor cell lines.[2] One possible solution to this
problem is to carry out structure–activity relationship
(SAR) studies through synthesis and testing of new ana-
logues of apratoxin A. In fact, the different cytotoxicity pat-
terns displayed by its new family members, apratoxins B (2)
and C (3), suggest that the cytotoxicities of different mem-
bers of this family of natural products are highly dependent
on their structures.[3] It was reported that 3 exhibits in vitro
IC50 cytotoxicity values almost identical to those of 1 whilst
2 is significantly less cytotoxic than 1. These observations,
together with the intriguing structure of apratoxin A, have
attracted considerable synthetic attention, which culminated
in the first total synthesis of apratoxin A by Forsyth and
Chen.[4a,b] Here we report a novel synthetic route to apratox-
in A, which also enabled the synthesis of a number of ana-
logues for preliminary SAR and mechanistic studies.[5,6]
Natural products and analogues showing potent cytotoxic ef-
fects are important sources of new anticancer drugs.[1] Since
resistance often occurs, and not all tumor cells are equally
sensitive to a given drug, discovery of new compounds with
potent cytotoxicity and novel modes of action is crucial.
Apratoxin A (1, Scheme 1) is a cyclodepsipeptide isolated
[a] Prof. Dr. D. Ma, Dr. B. Zou
State Key Laboratory of Bioorganic and Natural Products Chemistry
Shanghai Institute of Organic Chemistry, Chinese Academy of Scien-
ces
354 Fenglin Lu, Shanghai 200032 (China)
Fax : (+86)21-6416-6128
E-mail: madw@mail.sioc.ac.cn
[b] G. Cai
Department of Chemistry, Fudan University
Shanghai 200433 (China)
[c] Dr. X. Hu, Prof. Dr. J. O. Liu
Department of Pharmacology and Department of Neuroscience
Johns Hopkins University School of Medicine
725 North Wolfe St., Baltimore, MD 21205 (USA)
Fax : (+1)410-955-4520
E-mail: joliu@jhu.edu
Supporting information for this article (experimental procedures for
preparation of the intermediates for assembling 39–41, and copies of
1H NMR spectra for compounds 1, 4, and 39–41) is available on the
WWW under http://www.chemeurj.org/ or from the author.
Chem. Eur. J. 2006, 12, 7615 – 7626
ꢁ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
7615

Scheme 2. Synthesis of the acid 5a. a) TBSCl, imidazole; b) NaBH4,
MeOH; c) MsCl, Et3N; d) tBuOK, 71% yield over four steps; e) O3,
Me2S; f) LiCH2CO2Et; g) 40% HF; h) MsCl, Et3N, 45% yield over four
steps; i) CH2=CHCOCl, Et3N, 90%; j) 10 mol% Grubbs’ catalyst, 80%;
k) Me2(CuCN)Li2, 94%; l) LiAlH4, THF; m) AcCl, pyridine, then
N
K2CO3, MeOH; n) Dess–Martin oxidation, 69% yield over three steps;
o) N-Propionylsultam, Et2BOTf, iPr2NEt, then TiCl4, 90%; p) LiAlH4,
THF, then DMP, PPTs, 67%; q) Fmoc-l-proline, 2,4,6-trichlorobenzoyl
chloride, DMAP, 90%; r) TsOH, MeOH; s) TEMPO, NaClO;
t) NaH2PO4, NaClO2, 81% yield over three steps. TBS = tert-butyldime-
thylsilyl. DMAP = 4-dimethylaminopyridine; TEMPO = 2,2,6,6-tetra-
methylpiperidinyl-1-oxyl.
Scheme 1. Structures of apratoxins and an oxazoline analogue and their
retrosynthetic analysis.
Results and Discussion
As outlined in Scheme 1, our synthetic strategy was based
on the retrosynthetic degradation of apratoxin A into a tri-
peptide part (A) and a thiazoline unit (B). Further bond dis-
connection of B gave a cysteine-derived a,b-unsaturated
ester (D) and an l-proline-based carboxylic acid (5a). One
challenge for the assembly of intermediate B was the instal-
lation of its b-hydroxy-2,4-disubstituted thiazoline unit, as
most existing methods for thiazoline ring elaboration are
forbidden to this unstable moiety. Indeed, Forsyth and
Chen’s success in the total synthesis of apratoxin A was
highly dependent on their newly developed methodology for
preparing thiazoline rings.[4c] This problem prompted us to
consider replacing the thiazoline ring with a more conven-
iently accessible oxazoline ring in further SAR studies.[7,8]
Thus, if the modified compound 4 still retained potent cyto-
toxicity, not only would we have determined the importance
of the thiazoline moiety for the cytotoxicity of apratoxin A,
but we would also have facilitated future SAR studies.
acetone.[9] Protection of 6 with TBSCl, followed by reduc-
tion of the ketone moiety with NaBH4, produced an alcohol,
which was subjected to elimination through its mesylate to
afford allyl ether 7. Ozonolysis of 7 provided an aldehyde,
which was condensed with LiCH2CO2Et at À788C, followed
by cyclization and elimination, to give the a,b-unsaturated
lactone 8. We should note that this lactone was initially pre-
pared by a shorter route, which employed asymmetric allyl-
boration[10] of trimethylacetaldehyde and ring-closing meta-
thesis as the key steps,[11] but that in the asymmetric allylbo-
ration step it was found that the desired product 9 was diffi-
cult to separate from the resulting isopinocampheol either
by distillation or by column chromatography. This drawback,
together with the high cost of Grubbs’ first-generation cata-
lyst for conversion of 10 into 8, forced us to abandon this al-
ternative route to 8.
The synthesis of apratoxin A (1) and its oxazoline ana-
logue 4 began with the construction of their common pro-
line-ketide ester 5a as outlined in Scheme 2. As the starting
material we chose b-hydroxy ketone 6 (>99% ee), a d-pro-
line-catalyzed direct aldol reaction product that could be
prepared on a large scale from trimethylacetaldehyde and
Methylcupration[12] of 8 with Me2Cu(CN)Li2 gave 11 in
94% yield as a single diastereomer as determined by
1H NMR spectroscopy. Reduction of the lactone 11 with
LAH, followed by protection of the resulting diol with
acetic chloride, afforded a diester, which was treated with
1.2 equivalents of K2CO3 in methanol to liberate the pri-
7616
www.chemeurj.org
ꢁ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 7615 – 7626

Total Synthesis of Apratoxin A
FULL PAPER
mary alcohol selectively. The alcohol was then subjected to
Dess–Martin oxidation to yield aldehyde 12. For further
carbon chain elongation, Oppolzerꢀs methodology[13] for pre-
paring anti-diols from bornanesultam-derived boryl enolates
appeared particularly attractive. To this end, treatment of
N-propionylsultam with Et2BOTf resulted in a Z-enolate,
which was treated with the aldehyde 12 in the presence of
TiCl4 to afford “anti”-aldol 13 in 90% yield as the only de-
tectable diastereomer. It is noteworthy that use of eight
equivalents of TiCl4 was necessary to ensure the high yield.
Next, removal of both the chiral auxiliary and the acyl
group by LAH reduction gave a triol, which was protected
with DMP to furnish alcohol 14 in 67% yield. Esterification
of 14 with N-Fmoc-l-proline by Yamaguchiꢀs procedure[14]
produced 15 in 90% yield. Finally, treatment of 15 with
TsOH in methanol to liberate its diol moiety, and subse-
quent selective oxidation of the resulting primary alcohol
with the hindered chloro oxammonium salt generated from
TEMPO/NaClO, afforded an aldehyde, which was subse-
quently oxidized with NaClO2 to provide the desired acid
5a.
subsequent installation of a thiazoline or oxazoline ring
bearing an a,b-unsaturated ester side chain. Treatment of 21
with trifluoroacetic acid/water (3:1) liberated the amine,
which was coupled with the acid 5a in the presence of
HATU and DIPEA to provide amide 22a in 90% yield
(Scheme 4). Cyclodehydration[16] of 15 with 2.5 equivalents
For installation of the modified cysteine or serine units in
1 or 4, a,b-unsaturated esters 19 and 21 were prepared as
depicted in Scheme 3. Weinreb amide 17 was synthesized
Scheme 4. Attempts to construct thioamide 24a. a) CF3CO2H, H2O,
CH2Cl2; b) 5a, HATU, iPr2NEt, 90% yield over two steps; c) DAST,
CH2Cl2, À788C, 86%; d) H2S, Et3N, MeOH. DAST = (diethylamino)sul-
fur trifluoride.
of DAST in methylene chloride at À788C proceeded
smoothly, delivering oxazoline 23 in 86% yield. Formation
of the required thiazoline intermediate, however, proved
challenging as expected. Many existing methods were tried
without success for this special case: Wipfꢀs thiolysis/cyclo-
dehydration strategy,[17] for example, failed to give thioa-
mide 24a because of Michael addition of H2S to the a,b-un-
saturated ester moiety. To solve this problem, we also tried
to prepare 24a through in situ generation of active thiono
ester by a procedure reported by Hoeg-Jensen and co-work-
ers.[18] Unfortunately, although this method did give some
24b, the coupling yield was low, with the major product
being amide 22b (Scheme 5).
Scheme 3. Assembly of the a,b-unsaturated esters 19 and 21.
a) HNMeOMe, HATU, iPr2NEt, 78%; b) LiAlH4, THF; c) 18, n-BuLi,
then 17, 30% yield over two steps; d) Ph3P=C(Me)CO2Et, 100%; e) aq.
NaOH; f) allyl alcohol, EDCI, DMAP, 74% yield over two steps. HATU
=
N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-
A
methylmethanaminium hexafluorophosphate N-oxide, EDCI = 1-(3-di-
methylaminopropyl)-3-ethylcarbodiimide hydrochloride.
from protected d-cysteine, and reduction of 17 with LAH
afforded an aldehyde, which was directly subjected to a
Wittig–Horner reaction to provide 19. Ethyl ester 20, on the
other hand, was obtained in quantitative yield from (R)-
Garner aldehyde. Hydrolysis of 20 and esterification with
allyl alcohol yielded 21, in which the allyl ester is removable
under neutral conditions.[15]
Scheme 5. Synthesis of thioamide 22b. a) CF3CO2H, H2O, CH2Cl2;
b) TBSCl, iPr2NEt, 90% yield over two steps; c) C2F5OH, EDCI,
CH2Cl2; d) H2S, iPr2NEt, MeOH, 56% yield over two steps; e) PyBOP,
iPr2NEt, 30%. PyBOP = benzotriazol-1-yloxy)tripyrrolidinophosphoni-
um hexafluorophosphate.
With the a,b-unsaturated esters 19 and 21 in hand, our
next tasks were to connect them with the acid 5a, and the
Chem. Eur. J. 2006, 12, 7615 – 7626
ꢁ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
7617

D. Ma, J. O. Liu et al.
Having failed in the above attempts, we turned our atten-
tion to the assembly of the thiazoline intermediate by a
tandem deprotection/cyclodehydration strategy reported by
Kelly and co-workers.[19] Deprotection of 19 liberated
amines, which were then condensed with 5a in the presence
of HATU to afford amides 27 (Scheme 6). Initially, five
Scheme 7. Assembly of the thiazoline intermediate 31: a) AcCl, DMAP;
b) Ph3PO, Tf2O, 0 8C.
Scheme 6. Elaboration of the thiazoline intermediate 28: a) Et2NH,
MeCN; b) 5a, HATU, iPr2NEt; c) 1.5 equiv TiCl4, 15%; d) CF3CO2H,
Et3SiH, CH2Cl2; e) 1.5 equiv TiCl4, 68%.
equivalents of TiCl4 were used to induce deprotection/cyclo-
dehydration, but this gave a complex mixture as determined
by TLC, with no desired product 28. We then tried the use
of varying amounts of TiCl4 and found that when 1.5 equiva-
lents of TiCl4 were used, thiazoline 28 could be isolated in
less than 15% yield, together with the deprotection product
29 in about 30% yield. As a result, we decided to run the
deprotection reaction first, followed by cyclodehydration.
Treatment of 27a with TFA thus produced thiol 29, which
was treated with 1.5 equivalents of TiCl4 to provide 28 in
68% yield. Unfortunately, this route to the key thiazoline
intermediate was found to be suitable only for small-scale
preparations, and much lower yields were obtained if this re-
action was run on scales over 100 mg. The underlying cause
of this difference remains unclear; attempts to switch the
Lewis acids from TiCl4 to PCl5 and F3B·OEt2 failed to give
any desired product 28.
At that point Kellyꢀs new report on biomimetic synthesis
of thiazolines appeared, offering a potential solution to the
problem,[20] and we were pleased to find that treatment of
30, the acylation products of 27, with Ph3PO/Tf2O provided
thiazoline intermediates 31 in excellent yields (Scheme 7).
For the synthesis of the highly methylated tripeptide frag-
ment, 2-bromo-1-ethylpyridinium tetrafluoroborate (BEP),
an efficient coupling reagent for hindered peptide synthesis
developed by Xu and Li,[21] was employed. As shown in
Scheme 8, BEP-mediated coupling of 32 with N-Fmoc-O-
methyl-l-tyrosine (33) gave dipeptide 34 in 55% yield. Pd/
C-catalyzed hydrogenolysis of 34 afforded an acid, which
Scheme 8. Preparation of the tripeptide 36. a) BEP, iPr2NEt, 55%; b) Pd/
C, H2, EtOAc; c) Et2NH, MeCN; d) BEP, iPr2NEt, 60% yield from 34.
BEP = 2-bromo-1-ethyl-pyridinium tetrafluoroborate.
was connected with a liberated amine from 35 to furnish tri-
peptide 36 in 60% yield.
With the required coupling components in hand, we fol-
lowed our strategy to create the macrocycle. As outlined in
Scheme 9, the allyl protecting group in 23 was removed with
the aid of [Pd(PPh3)4] catalysis in the presence of N-methyl-
A
aniline to give an acid,[15] which was coupled with a liberated
amine from 36 to provide 37b in 59% yield. The utilization
of N-methylaniline was essential, as other bases employed
(such as morphine) caused the simultaneous cleavage of the
N-Fmoc protecting group during the deprotection reaction.
This procedure was unsuccessful for deallylation of thiazo-
line 31a, however, so 31b, a prenyl-protected intermedi-
ate,[22] was selected for further conversion. To our delight,
treatment of 31b with TMSOTf produced a good yield of
the desired deprotection product, which was connected with
the liberated amine from 36 to afford amide 37a. Cleavage
of the Fmoc and TMSE protecting groups in 37 was accom-
plished by treatment with TBAF/THF to give cyclization
precursors. Macrocyclization of masked amino acids 37a
and 37b with HATU/DIPEA in dilute methyl chloride af-
forded 38 and 4, respectively, and removal of the acyl group
in 38 with KCN in ethanol afforded apratoxin A.
7618
www.chemeurj.org
ꢁ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 7615 – 7626

Total Synthesis of Apratoxin A
FULL PAPER
work by specifically interfering with the function of a target
required for cell cycle progression of HeLa and probably
other cells.[23]
These results also indicated that the replacement of the
thiazoline ring of apratoxin A with an oxazoline ring had
only a marginal effect on potency, which prompted us to
design and synthesize more simplified analogues of apratoxi-
n A containing the more accessible oxazoline ring for a pre-
liminary structure–activity relationship study. To this end,
the apratoxin C oxazoline analogue 39, its C(37)-epimer 40,
and the C(37)-demethylated analogue 41 (Scheme 10),
became our new synthetic targets. These could be assembled
from more conveniently available aldehydes 42, by the same
process as used for elaborating 4.
As shown in Scheme 11, we synthesized the requisite alde-
hydes 42a and 42b from ketone 43, another product of a d-
proline-catalyzed direct aldol reaction,[9] as the starting ma-
terial. Protection of 43 with TBSCl, followed by a Peterson
reaction, provided olefin 44 as an inseparable mixture of cis
and trans isomers.[24] Treatment of 44 with 40% HF in
MeCN afforded trans olefin 45 and lactone 46, and hydroge-
nation and subsequent LAH reduction of 45 gave a separa-
ble mixture of alcohol 47a and its epimer. The latter com-
pound was also obtained from 46 as a single product by the
same reaction sequence. Protection of 47 with acetyl chlor-
ide produced diesters, which were selectively hydrolyzed,
and the resulting primary alcohols were then oxidized to fur-
nish 42 in 81% yield. For construction of aldehyde 42c,
ring-opening of epoxide 48 with a lithium reagent generated
from 1-benzyloxyprop-2-yne with the assistance of boron tri-
fluoride diethyl etherate was conducted to give alcohol 49.
After 49 had been masked with an acetyl group, hydrogena-
tion was carried out to provide alcohol 50, and Dess–Martin
oxidation of 50 furnished 42c in 93% yield. By the same
procedure used for preparing 4 from aldehyde 12, the three
aldehydes 42a–c were converted into 39–41, respectively.
We next determined the biological activities of analogues
39, 40, and 41 in the HeLa cell proliferation assay. Surpris-
ingly, 39 was almost 100 times less active than its structurally
closely related analogue 4 (see Table 1), indicating that the
methyl group at C(40) is important for the biological activity
in oxazoline analogues of apratoxin A. This result is distinct
from what has been observed in the natural apratoxin
family, because apratoxins A and C have almost identical
potencies.[3] This difference might be the result of a confor-
mation change caused by some subtle structural variation,
whilst it is noteworthy that in Wipfꢀs report, replacement of
thiazoline with oxazoline resulted in a significant decrease
in cytotoxicity in SAR studies of lissoclinamide 7.[25] In addi-
tion, when the stereochemistry at C(37) was inverted, the re-
sulting analogue 40 was inactive even at 10000 nm, whilst a
similar loss of activity was seen when the C(37) methyl
group was removed in analogue 41. Together, these results
suggest that the two methyl groups at C(37) and C(40), as
well as the stereochemistry at C(37), are essential for the
cellular activity of the oxazoline analogue of apratoxin A,
probably because of their intimate involvement in binding
Scheme 9. Assembly of apratoxin A and its oxazoline analogue. a) [Pd-
A
from 31b, 59% yield for 37b from 23; e) TBAF, THF, then HATU,
iPr2NEt, CH2Cl2 (0.002m); f) KCN, EtOH, 508C. TBAF = tetrabutylam-
monium fluoride.
The synthetic apratoxin A (1) and its oxazoline analogue
4 were tested for cytotoxicity against human cervical cancer
cell line HeLa. Both compounds exhibited potent inhibitory
effects on the proliferation of HeLa, with IC50 values of 2.2
and 9.7 nm, respectively (Table 1). Further analysis revealed
Table 1. Apratoxin A and its analogues inhibit HeLa cell proliferation.
Compound
Potency (IC50, nm)
1
4
39
40
41
2.2Æ0.2
9.7Æ1.4
920Æ18
>10000
>10000
that both synthetic apratoxin A and its oxazoline analogue
inhibited cell proliferation by causing cell cycle arrest in the
G1 phase (Figure 1), so the cell cycle distribution of HeLa
cells was followed by fluorescence-activated cell sorting
(FACS) after staining of cells with the DNA-intercalating
dye propidium iodide. Upon treatment with either com-
pound for 24 h, more HeLa cells accumulate at the G1
phase of the cell cycle, with corresponding decreases in cells
at the S or G2/M phases of the cell cycle (Table 2 and Figur-
e 1B, C, vs. A). Prolonged treatment of HeLa cells with the
compounds for 48 h resulted in more pronounced cell cycle
arrest in G1 (Figure 1E, F, vs. D). The cell cycle effect of
apratoxin A and its analogue suggests that they are likely to
Chem. Eur. J. 2006, 12, 7615 – 7626
ꢁ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
7619

D. Ma, J. O. Liu et al.
Figure 1. Cell cycle effects of apratoxin A and its oxazoline analogue. A representative HeLa cell cycle profile is shown with 24 h treatment of vehicle
control (A). Significant increases in G1 cell populations were observed for 4 (B) and 1 (C) with corresponding decreases in both S phase and G2/M
phase cell populations. When cells were incubated for 48 h, the profile of vehicle remains the same (D), whereas only G1 cell populations remained for 4
(E) and 1 (F) treatments. 50 nm final concentrations of either compound were used.
Table 2. Percentages of cell populations in G1, S, and G2/M phases when
treated with compounds 1 and 4.[a]
Compound
G1
S
G2/M
DMSO
1
4
54.11 (57.88)
71.70 (75.16)
75.45 (76.81)
18.83 (17.46)
10.46 (10.09)
10.88 (8.68)
27.27 (24.11)
17.22 (12.74)
13.35 (11.82)
[a] Treatment with 50 nm compounds for 24 h. The data in parentheses
were obtained by treatment of these compounds for 48 h.
to the molecular target responsible for cell cycle inhibition
by apratoxin A.
Scheme 10. Structures of apratoxin C analogues and their retrosynthetic
analysis.
Conclusion
the analogue 4 inhibit cell proliferation by blocking the pro-
gression of cell cycle at the G1 phase. Since the oxazoline
analogue 4 could be more easily assembled, it may serve as
a more expedient probe with which to explore the biological
function of apratoxin A. Moreover, the use of the synthetic
analogues of apratoxin A identified essential structural ele-
ments that are important for the biological activity, which
should guide future design and synthesis of bioprobes of
apratoxin A for the identification of its molecular target and
for the improvement of its biological activity.
In summary, we have devised a new synthetic route for the
total synthesis of apratoxin A. Novel elements of the synthe-
sis include the concise assembly of its polyketide acid seg-
ment through two asymmetric aldol reactions and the elabo-
ration of its thiazoline unit by Kellyꢀs biomimetic method.
Several oxazoline analogues were elaborated by a similar
approach, and compound 4 was found to be nearly as potent
as apratoxin A in inhibiting proliferation of HeLa cells. Pre-
liminary investigations revealed that both apratoxin A and
7620
www.chemeurj.org
ꢁ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 7615 – 7626

Total Synthesis of Apratoxin A
FULL PAPER
centrated in vacuo. The residual oil was purified by chromatography (elu-
tion with pure petroleum ether) to give 7 (10 g, 71% yield from 6) as a
pale yellow oil. [a]2D0 = +3.8 (c = 12.2 in CHCl3); 1H NMR (300 MHz,
CDCl3): d = 5.90 (m, 1H), 5.03 (m, 2H), 3.35 (m, 1H), 2.41 (m, 1H),
2.20 (m, 1H), 0.92 (s, 9H), 0.88 (s, 9H), 0.08 (s, 3H), 0.06 (s, 3H) ppm;
IR (film): n˜max
= ; EI-MS: m/z: 227
2958, 2931, 1394, 1363 cmÀ1
[MÀCH3]+, 185 [MÀt-Bu]+; elemental analysis calcd (%) for C14H30SiO:
C 69.35, H 12.47; found: C 68.88, H 12.21.
Compound 8:
A solution of 7 (6 g, 25 mmol) in dichloromethane
(100 mL) was cooled to À788C and treated with ozone until the solution
became blue in color. The excess ozone was removed in a stream of N2,
the resulting mixture was allowed to warm to room temperature, and
then Me2S (4 mL) was added. The solution was kept at 408C for 24 h and
was then cooled and washed with water. The separated organic layer was
dried over Na2SO4 and concentrated in vacuo to give the aldehyde as a
yellow oil, which was used in the next step without further purification.
n-Butyllithium (1.6m in hexane, 15.6 mL, 25 mmol) was added dropwise
at 08C under N2 to a stirred solution of diisopropylamine (3.5 mL,
25 mmol) in dry THF (80 mL). The solution was stirred for 15 min, the
temperature was lowered to À788C, and dry ethyl acetate (2.5 mL,
25.5 mmol) was added dropwise. The solution was stirred for 20 min and
a solution of above crude aldehyde in THF was added slowly. One hour
later, the solution was allowed to warm to room temperature and
quenched with saturated NH4Cl solution. The separated organic layer
was washed with brine and water, dried over Na2SO4, and concentrated
to give the crude alcohol, which was dissolved in acetonitrile containing a
suitable amount (5–30%) of aqueous HF (40%). The resulting solution
was stirred at room temperature until the starting material had disap-
peared, as monitored by TLC. The solution was concentrated in vacuo,
diluted with ethyl acetate, and washed with saturated NaHCO3 and
water. The organic layer was dried over Na2SO4 and concentrated to give
the crude hydroxy lactone.
Et3N (7 mL, 50 mmol), followed by MsCl (4.9 mL, 50 mmol), were added
to a solution of the above lactone in dichloromethane (50 mL). The re-
sulting solution was stirred for 2 h and was then quenched with water
and separated. The aqueous layer was extracted three times with CH2Cl2,
and the combined organic layers were dried over Na2SO4 and concentrat-
ed in vacuo. The resulting oil was purified by chromatography (elution
with ethyl acetate/petroleum ether 1:4) to give 8 (1.75 g, 45% yield from
7). [a]2D0 = À129 (c = 0.51 in CHCl3); 1H NMR (300 MHz, CDCl3): d =
6.95 (m, 1H), 6.05 (m, 1H), 4.11 (m, 1H), 2.40 (m, 2H), 1.03 (m,
9H) ppm; 13C NMR (75 MHz, CDCl3): d = 164.77, 145.44, 121.09, 85.22,
Scheme 11. Synthesis of oxazoline analogues 39–41. a) TBSCl, imidazole;
b) TMSCH2CO2Et, LDA, À788C, 90% yield over two steps; c) 40% HF,
MeCN; d) Pd/C, H2, then LiAlH4; e) AcCl, pyridine; f) Aq. K2CO3,
MeOH; g) Dess–Martin oxidation, 80% yield over three steps; h) nBuLi,
3-benzyloxyprop-1-yne, then F3B·OEt2, 96%; i) AcCl, pyridine, then Pd/
C, H2, 86%; j) Dess–Martin oxidation, 93%.
Experimental Section
33.78, 25.42, 24.53 ppm; IR (film): n˜max = 3474, 1719, 1482, 1383 cmÀ1
;
EI-MS: m/z: 153 [MÀH]+, 149; HRMS: m/z: calcd for C9H14O2:
Compound 7: Imidazole (12.2 g, 179 mmol) and tert-butyldimethylsilyl
chloride (14.8 g, 91 mmol) were added to a stirred solution of 6 (8.5 g,
59 mmol) in DMF (12 mL). The resulting solution was stirred for 24 h,
and was then quenched with methanol (25 mL) and water (200 mL). The
mixture was extracted with ethyl acetate, the combined organic layers
were dried over Na2SO4 and concentrated in vacuo, and the resulting oil
was purified by chromatography (elution with ethyl acetate/petroleum
ether 1:10) to give 15 g of silyl ether 7.
154.0994; found 154.1006 [M]+.
Compound 11: Methyllithium (1.6m solution in diethyl ether, 33 mL,
52.8 mmol) was added under nitrogen at À788C to a suspension of CuCN
(2.4 g, 26.4 mmol) in dry ether (100 mL). The mixture was stirred for 2 h
at this temperature, after which a solution of 8 (2.7 g, 17.8 mol) in ether
was added slowly. The resulting mixture was stirred for 1 h and was then
quenched with aqueous FeCl3 solution, the organic layer was washed
with brine and water, dried over Na2SO4, and concentrated in vacuo, and
the residual oil was purified by chromatography (elution with ethyl ace-
tate/petroleum ether 1:10) to give 11 (2.84 g, 94%). [a]2D0 = +46.5 (c =
0.3 in CHCl3); 1H NMR (300 MHz, CDCl3): d = 4.00 (dd, J = 11.7,
3.9 Hz, 1H), 2.55 (m, 1H), 2.20 (m, 2H), 1.80 (m, 1H), 1.53 (m, 1H),
1.10 (d, J = 6.0 Hz, 3H), 1.00 (s, 9H) ppm; 13C NMR (75 MHz, CDCl3):
d = 173.07, 83.57, 36.89, 33.84, 29.70, 25.33, 23.95, 21.11 ppm; IR (film):
n˜max = 1733, 1481, 1398, 1367, 1073 cmÀ1; EI-MS: m/z: 170 [M]+, 155,
127, 43; HRMS: m/z: calcd for C10H18O2: 170.1307; found 170.1309 [M]+.
NaBH4 (4.4 g, 116 mmol) was added at 08C to a solution of the above
silyl ether (15 g, 58 mmol) in methanol (150 mL) and the resulting solu-
tion was allowed to stir for 0.5 h. The solution was concentrated and then
diluted with water (200 mL), the mixture was extracted with ethyl ace-
tate, and the organic layers were washed with brine and water, dried over
Na2SO4, and concentrated in vacuo to give the crude alcohol. This was
dissolved in dry CH2Cl2 (200 mL), after which Et3N (16 mL, 116 mmol)
and MsCl (9 mL, 116 mmol) were added successively at 08C under nitro-
gen atmosphere. Two hours later the reaction was quenched with brine
and the organic layer was washed with water, dried over Na2SO4, and
concentrated to give the crude mesylate.
Compound 12: A solution of the lactone 11 (2.6 g, 15.3 mmol) in dry
THF (10 mL) was added dropwise under nitrogen to a boiling THF
(70 mL) suspension of LiAlH4 (1.0 g, 30 mmol). After having been
heated at reflux for 1 h, the reaction mixture was allowed to cool to
room temperature and quenched by sequential slow addition of water
(1 mL), aqueous NaOH solution (15%, 1 mL), and water (1 mL). After
stirring for 2 h, the white solid was filtered and the filtrate was concen-
trated to give the crude diol, which was dissolved in dichloromethane
tBuOK (21.3 g, 189 mmol) was added to a solution of the above mesylate
in toluene (200 mL), and the resulting mixture was heated at reflux for
1 h and then allowed to cool to room temperature. After the reaction
had been quenched with water, the organic layer was separated, the
aqueous layer was extracted with n-heptane, and the combined organic
layers were washed with brine and water, dried over Na2SO4, and con-
Chem. Eur. J. 2006, 12, 7615 – 7626
ꢁ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
7621

D. Ma, J. O. Liu et al.
(50 mL). Pyridine (5 mL, 62 mmol) was added at 08C to this solution, fol-
lowed by AcCl (4.4 mL, 62 mmol). The solution was allowed to stir over-
night and was then quenched with water and separated, the aqueous
layer was extracted three times with CH2Cl2, the combined organic layers
were dried over Na2SO4 and concentrated, and the residue was dissolved
in methanol (40 mL) and water (10 mL), after which K2CO3 (2.2 g,
15.9 mmol) was added. The resulting mixture was stirred for 5 h and then
concentrated in vacuo to remove the methanol, and the resulting mixture
was diluted with ethyl acetate, washed with brine, dried over Na2SO4,
and concentrated to give the monoalcohol.
2871, 1464 cmÀ1; EI-MS: m/z: 257 [MÀCH3]+, 215, 157, 59; HRMS: m/z:
calcd for C15H29O3: 257.2117; found 257.2134 [MÀCH3]+.
Compound 15: 2,4,6-Trichlorobenzoyl chloride (0.17 mL, 1.08 mmol) was
added under nitrogen to a suspension of N-Fmoc-l-proline (250 mg,
0.74 mmol) in benzene (1 mL), followed by DIPEA (0.18 mL,
1.08 mmol). A solution of 14 (200 mg, 0.74 mmol) in benzene (0.5 mL)
was added to the resulting solution, after which DMAP (180 mg,
1.5 mmol) was added in one portion. The mixture was stirred overnight,
diluted with ethyl acetate, and washed with water, the organic layer was
dried over Na2SO4 and concentrated in vacuo, and the residual oil was
purified by chromatography (elution with ethyl acetate/petroleum ether
1:10) to give 15 as a white foam (391 mg, 90%). [a]2D0 = À71 (c = 1.07 in
CHCl3); 1H NMR (300 MHz, CDCl3): d = 7.77 (m, 2H), 7.61 (m, 2H),
7.45–7.25 (m, 4H), 4.92 (m, 1H), 4.58–4.48 (m, 1H), 4.44–4.25 (m, 2H),
4.20 (m, 1H), 3.80–3.41 (m, 6H), 2.41–2.15 (m, 2H), 2.12–1.91 (m, 2H),
1.80–1.53 (m, 2H), 1.41 (s, 3H), 1.38–1.32 (m, 3H), 1.25 (s, 3H), 1.23–
1.10 (m, 1H), 1.00–0.95 (m, 1H), 0.87 (s, 9H), 0.83–0.67 (m, 4H) ppm;
13C NMR (75 MHz, CDCl3): d = 172.0, 154.3, 144.1, 143.9, 141.2, 127.6,
127.0, 125.1, 119.8, 97.8, 79.5, 72.4, 72.2, 67.6, 67.3, 66.1, 59.4, 47.1, 46.8,
46.2, 38.8, 37.8, 34.9, 34.4, 31.0, 29.6, 25.8, 24.2, 23.2, 20.2, 18.9, 12.6 ppm;
IR (film): n˜max = 1710, 1417, 1198 cmÀ1; ESI-MS: m/z: 592 [M+H]+, 609
[M+NH4]+; HRMS: m/z: calcd for C36H49NO6Na: 614.3452; found
614.3457 [M+Na]+.
Dess–Martin reagent (7.1 g, 16.8 mmol) was added at 08C to a solution
of the above alcohol in dry CH2Cl2 (60 mL). The mixture was stirred for
3 h and quenched with sat. Na2S2O3, the organic layer was washed with
brine, dried over Na2SO4, and concentrated in vacuo, and the resulting
oil was purified by chromatography (elution with ethyl acetate/petroleum
ether 1:10) to give 12 (2.3 g, 69% yield from 8). [a]2D0 = À16 (c = 1.0 in
CHCl3); 1H NMR (300 MHz, CDCl3): d = 9.7 (t, J = 1.2 Hz, 1H), 4.79
(dd, J = 7.8, 3.6 Hz 1H), 2.67 (dd, J = 16.2, 3.6 Hz, 1H), 2.21–2.10 (m,
1H), 2.08 (s, 3H), 2.01–1.92 (m, 1H), 1.51 (m, 2H), 0.97 (d, J = 6.6 Hz,
3H), 0.89 (s, 9H) ppm; 13C NMR (75 MHz, CDCl3): d = 202.5, 171.2,
78.2, 49.4, 36.6, 34.6, 25.9, 25.1, 21.2, 21.0 ppm; IR (film): n˜max = 2877,
1733, 1710, 1481, 1373 cmÀ1; EI-MS: m/z: 171 [MÀAc]+, 153, 113, 43;
HRMS: m/z: calcd for C10H19O2: 171.1385; found 171.1385 [MÀAc]+.
Compound 5a: TsOH (129 mg, 0.676 mmol) was added to a solution of
15 (200 mg, 0.338 mmol) in methanol (5 mL). The resulting solution was
stirred overnight and concentrated to remove the solvent, and the residue
was diluted with ethyl acetate and washed sequentially with sat.
NaHCO3, brine, and water. The organic layer was dried over Na2SO4 and
concentrated to give the crude diol.
Compound 13: CF3SO3H (1.3 mL, 15 mmol) was added at room temper-
ature under nitrogen to Et3B (1.0m solution in hexane, 15 mL, 15 mmol)
and the mixture was stirred at 408C until gas evolution had ceased. A
solution of N-propionylsultam (1.7 g, 6.2 mmol) in CH2Cl2 (10 mL) and
DIPEA (2.7 mL, 15.9 mmol) were added successively at À108C and the
stirring was continued for 30 min. The resulting boryl enolate solution
was cooled to À788C, and a precooled (À788C) solution of the aldehyde
12 (2.66 g, 12.4 mmol) and TiCl4 (5.5 mL, 50 mmol) in CH2Cl2 (80 mL)
was added by cannula. The resulting mixture was stirred for 2 h and
quenched with saturated NH4Cl. After the water phase had been extract-
ed with CH2Cl2, the combined organic layers were dried over Na2SO4
and concentrated in vacuo, and the residue was purified by chromatogra-
phy (elution with ethyl acetate/petroleum ether 1:5) to give 13 as a white
2,2,6,6-Tetramethylpiperidinyloxyl (10 mg) and KBr (10 mg) were added
at 08C to a two-phase mixture of the above crude diol product in CH2Cl2
(4 mL) and saturated NaHCO3 (2 mL). The mixture was stirred vigorous-
ly, and NaClO (0.1m solution in water, 4 mL, 0.40 mmol) was added. Stir-
ring was continued for 1 h and the reaction mixture was quenched with
saturated Na2S2O3 and extracted with CHCl3. The combined organic
layers were dried over Na2SO4 and concentrated to give the crude alde-
hyde.
solid (2.8 g, 90%). [a]D20
=
À70.5 (c
=
1.1 in CHCl3); 1H NMR
A
solution of NaClO2 (123 mg, 1.35 mmol) and NaH2PO4 (81 mg,
(300 MHz, CDCl3): d = 4.78 (dd, J = 10.8, 1.8 Hz, 1H), 3.92 (dd, J =
7.8, 5.1 Hz, 1H), 3.71 (m, 1H), 3.52 (q, J = 13.8 Hz, 2H), 3.15 (t, J =
6.6 Hz, 1H), 2.33 (d, J = 10.2 Hz), 2.18 (m, 1H), 2.12 (t, J = 6.9 Hz,
1H), 2.05 (s, 3H), 1.91 (m, 3H), 1.65 (s, 3H), 1.51–1.30 (m, 4H), 1.24 (s,
3H), 1.20 (d, J = 6.9 Hz, 3H), 0.98 (s, 3H), 0.92 (d, J = 6.6 Hz, 3H),
0.88 (s, 9H) ppm; 13C NMR (75 MHz, CDCl3): d = 175.1, 171.6, 78.1,
73.1, 65.4, 53.2, 48.3, 47.8, 46.5, 44.8, 40.8, 38.4, 37.5, 34.3, 33.0, 26.5, 26.0,
25.2, 21.1, 20.9, 20.6, 19.9, 13.9 ppm; IR (KBr): n˜max = 3528, 2962, 1727,
1696, 1481 cmÀ1; ESI-MS: m/z: 486.3 [M+H]+; elemental analysis calcd
(%) for C25H43NO6S: C 61.82, H 8.92, N 2.88; found: C 61.94, H 9.20, N
2.62.
0.676 mmol) in H2O (1.5 mL) was added to a solution of the above crude
aldehyde in t-BuOH (3 mL) and 2-methylbutene (1.5 mL). The mixture
was stirred for 1 h and extracted with ethyl acetate, the organic layer was
washed with brine, dried over Na2SO4, and concentrated in vacuo, and
the residue was purified by chromatography (elution with ethyl acetate/
petroleum ether 1:1) to give 5a as a colorless solid (155 mg, 81% yield
from 15). [a]2D0
=
À49.5 (c
=
1.15 in CHCl3); 1H NMR (300 MHz,
CDCl3): d = 7.77 (d, J = 7.2 Hz, 2H), 7.62 (dd, J = 10.8, 7.2 Hz, 2H),
7.41 (t, J = 7.5 Hz, 2H), 7.33 (t, J = 7.5 Hz, 2H), 4.92 (d, J = 10.5 Hz,
1H), 4.44 (dd, J = 9.9, 6.6 Hz, 1H), 4.38–4.31 (m, 2H), 4.29 (d, J =
6.6 Hz, 1H), 3.82 (m, 1H), 3.71–3.61 (m, 1H), 3.57–3.49 (m, 1H), 2.45 (t,
J = 7.2 Hz, 1H), 2.35–2.25 (m, 1H), 2.11–1.91 (m, 4H), 1.72 (m, 2H),
1.35–1.25 (m, 3H), 1.19 (d, J = 9.0 Hz, 3H), 0.99 (d, J = 6.3 Hz, 3H),
Compound 14: A solution of 13 (2.8 g, 5.8 mmol) in dry THF (5 mL) was
added under nitrogen to a suspension of LiAlH4 (0.5 g, 15 mmol) in THF
(30 mL) at reflux. After having been heated at reflux for 0.5 h, the reac-
tion mixture was allowed to cool to room temperature and quenched by
slow sequential addition of water (0.5 mL), aqueous NaOH (15%,
0.5 mL), and water (0.5 mL). The white solid was filtered and the filtrate
was concentrated to give the crude triol.
0.91 (s, 9H) ppm; 13C NMR (75 MHz, CDCl3): d
= 178.77, 172.53,
155.15, 144.03, 143.74, 141.31, 142.28, 127.67, 127.15, 127.10, 125.24,
125.14, 119.91, 78.34, 70.56, 67.77, 59.49, 47.12, 46.65, 46.56, 38.84, 37.38,
34.72, 29.91, 29.72, 26.01, 25.96, 24.80. 24.50, 20.32, 20.18, 13.41 ppm; IR
(KBr): n˜max = 3477 (br), 2967, 1741, 1687, 1453 cmÀ1; ESI-MS: m/z: 566
[M+H]+; HRMS: m/z: calcd for C33H43NO7Na: 588.2932; found 588.2908
[M+Na]+.
PPTs (200 mg) was added to a solution of the above triol in 2,2-dime-
thoxypropane (30 mL). The resulting mixture was stirred for 24 h and
was then diluted with ether and washed with water. The organic layer
was dried over Na2SO4 and concentrated in vacuo, and the residual oil
was purified by chromatography (elution with ethyl acetate/petroleum
ether 1:8) to give 14 (1.05 g, 67% yield from 13). [a]2D0 = À60 (c = 0.88
in CHCl3); 1H NMR (300 MHz, CDCl3): d = 3.79 (dd, J = 12.0, 5.1 Hz,
1H), 3.50 (t, J = 11.4 Hz, 2H), 3.35 (d, J = 9.0 Hz, 1H), 2.41 (m, 1H),
1.83 (m, 1H), 1.51 (m, 1H), 1.45 (s, 3H), 1.40 (s, 3H), 1.39–1.28 (m, 3H),
0.98 (d, J = 6.9 Hz, 3H), 0.90 (s, 9H), 0.75 (d, J = 6.6 Hz, 3H) ppm;
13C NMR (75 MHz, CDCl3): d = 98.2, 75.8, 73.9, 66.0, 38.7, 38.2, 34.6,
34.4, 29.5, 26.1, 25.7, 21.5, 19.0, 12.7 ppm; IR (film): n˜max = 3497, 2955,
Compound 17: Weinreb amine (1.35 g, 13.8 mmol), EDCI (2.91 g,
15.2 mmol), HOBt (2.05 g, 15.2 mmol), and DIPEA (4.7 g, 27.6 mmol)
were added successively to a solution of acid 16 (8.08 g, 13.8 mmol) in
dry CH2Cl2 (20 mL). The solution was stirred overnight and was then
washed with brine, the organic layers were dried over Na2SO4 and con-
centrated, and the residue was purified by chromatography (elution with
ethyl acetate/petroleum ether 1:5) to give 17 (6.76 g, 78%). [a]D20
=
+13.1 (c = 2.0 in CHCl3); 1H NMR (300 MHz, CDCl3): d = 7.78 (d, J
= 6.9 Hz, 2H), 7.62 (m, 2H), 7.40 (m, 9H), 7.33–7.18 (m, 10H), 5.40 (d,
J = 9.0 Hz, 1H), 4.80 (m, 1H), 4.19 (m, 2H), 4.10 (m, 1H), 3.65 (s, 3H),
7622
www.chemeurj.org
ꢁ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 7615 – 7626

Total Synthesis of Apratoxin A
FULL PAPER
3.19 (s, 3H), 2.62 (dd, J = 4.5, 12.3 Hz, 1H), 2.46 (dd, J = 7.8, 12.0 Hz,
1702, 1388, 1366 cmÀ1; ESI-MS: m/z: 326 [M+H]+; HRMS: m/z: calcd
for C17H27NO5Na: 348.1781; found 348.1789 [M+Na]+.
1H) ppm; 13C NMR (75 MHz, CDCl3):
d = 155.72, 144.36, 143.83,
143.71, 141.21, 141.18, 129.52, 127.88, 127.62, 127.03, 126.99, 126.72,
125.14, 125.12, 119.86, 67.02, 66.84, 61.49, 50.15, 47.04, 33.95 ppm; IR
(KBr): n˜max = 3303, 3058, 2939, 1723, 1660, 1446 cmÀ1; ESI-MS: m/z: 651
[M+Na]+; HRMS: m/z: calcd for C39H36N2O4SNa: 651.2288; found
651.2284 [M+Na]+.
Compound 22: The acid 21 (300 mg, 0.92 mmol) was dissolved in
CF3COOH (0.6 mL) and water (0.2 mL) and the resulting mixture was
stirred for 1 h and diluted with toluene (2 mL). The mixture was concen-
trated to dryness, and the residue was then redissolved in toluene (2 mL)
and concentrated to dryness again.
Compound 19b: A solution of 17 (0.7 g, 1.1 mmol) in dry THF (5 mL)
was cooled to À788C under nitrogen, and LiAlH4 (0.08 g, 2.1 mmol) was
then added. After the solution had been stirred for 2 h, water (0.1 mL)
was added carefully at À788C. After the reaction mixture had been al-
lowed to warm to room temperature, it was quenched by sequential slow
addition of aqueous NaOH (15%, 0.1 mL) and water (0.1 mL). The
white solid was filtered off and the filtrate was concentrated to give the
crude aldehyde.
The acid 5a (430 mg, 0.76 mmol), HATU (525 mg, 1.38 mmol), and
DIPEA (0.4 mL, 2.4 mmol) were sequentially added to a solution of the
above residue in dry CH2Cl2 (3 mL). The resulting mixture was stirred
for 24 h, and the mixture was then concentrated in vacuo and the residue
was purified by chromatography (elution with ethyl acetate/petroleum
ether 1:1) to give 22 as a colorless solid (505 mg, 90% yield). [a]D20
=
1
À16.1 (c = 0.66 in CHCl3); H NMR (300 MHz, CDCl3): d = 7.78 (d, J
= 7.5 Hz, 2H), 7.62 (d, J = 7.2 Hz, 2H), 7.41 (t, J = 7.5 Hz, 2H), 7.3 (t,
J = 7.2 Hz, 2H), 6.60 (m, 2H), 5.90–5.81 (m, 1H), 5.31–5.15 (m, 2H),
4.92 (d, J = 11.1 Hz, 1H), 4.75 (m, 1H), 4.61–4.45 (m, 3H), 4.38–4.25
(m, 3H), 3.62 (m, 3H), 3.51–3.42 (m, 1H), 3.34 (dd, J = 11.4, 6.3 Hz,
1H), 2.31–2.22 (m, 1H), 2.15–2.05 (m, 1H), 2.01–1.92 (m, 3H), 1.89 (s,
3H), 1.71–1.52 (m, 3H), 1.43–1.21 (m, 3H), 1.12 (d, J = 7.2 Hz, 3H),
0.95 (d, J = 6.3 Hz, 3H), 0.91 (s, 9H) ppm; 13C NMR (75 MHz, CDCl3):
d = 176.00, 172.26, 167.05, 155.40, 143.91, 143.71, 141.22, 137.66, 132.20,
130.71, 127.71, 127.65, 127.10, 127.03, 125.10, 119.92, 119.87, 118.06, 78.63,
71.49, 67.67, 65.35, 64.10, 59.39, 50.34, 47.56, 47.16, 46.54, 40.17, 38.56,
37.15, 34.70, 29.87, 25.88, 24.84. 24.24, 20.27, 14.54, 12.94 ppm; ESI-MS:
m/z: 733 [M+H]+; HRMS: m/z: calcd for C42H56N2O9Na: 755.3878;
found 755.3895 [M+Na]+.
n-Butyllithium (1.6m in hexane, 0.66 mL, 1.06 mmol) was added dropwise
at À788C under N2 to a stirred solution of 18 (293 mg, 1.05 mmol) in dry
toluene (2 mL). The resulting solution was stirred for 0.5 h, after which a
solution of the above crude aldehyde in toluene (2 mL) was added. After
it had been stirred at À788C for 1 h, the solution was allowed to warm to
room temperature and was then quenched with saturated NH4Cl solu-
tion. The separated organic layer was washed with brine and water, dried
over Na2SO4, and concentrated in vacuo, and the residue was purified by
chromatography (elution with ethyl acetate/petroleum ether 1:10) to give
19b (220 mg, 30% yield from 17). [a]2D0 = À5.9 (c = 0.8 in CHCl3);
1H NMR (300 MHz, CDCl3): d = 7.70 (m, 2H), 7.58 (m, 2H), 7.45–7.20
(m, 19H), 6.4 (m, 1H), 5.38 (m, 1H), 4.80–4.60 (m, 3H), 4.40 (m, 3H),
2.45 (m, 2H), 1.78 (s, 3H), 1.72 (s, 3H), 1.25 (s, 3H) ppm; 13C NMR
Compound 23: A solution of amido alcohol 22 (150 mg, 0.2 mmol) in
CH2Cl2 (3 mL) was cooled at À788C under nitrogen and DAST (25 mL,
0.2 mmol) was slowly added. One hour later, further DAST (25 mL,
0.2 mmol) was added. The solution was stirred for another one hour, and
then DAST (12 mL, 0.1 mmol) was again added. The solution was stirred
for a further 1 h and quenched with NH4OH (4m, 1.0 mL) at À788C. The
aqueous layer was then extracted with CHCl3, the combined organic
layers were dried over Na2SO4 and concentrated, and the residue was pu-
rified by chromatography (elution with ethyl acetate/petroleum ether
2:1) to give the oxazoline product 23 (130 mg, 90% yield). [a]2D0 = À79.0
(75 MHz, CDCl3): d
= 167.47, 155.34, 144.39, 143.76, 141.23, 139.09,
138.72, 129.51, 127.94, 127.62, 126.98, 126.83, 124.94, 119.89, 118.68, 67.17,
66.62, 64.72, 61.78, 47.16, 36.12, 31.52, 25.70, 22.59, 19.17, 18.01, 14.06,
12.83 ppm; IR (film): n˜max = 2934, 1706, 1450 cmÀ1; ESI-MS: m/z: 716
[M+Na]+; HRMS: m/z: calcd for C45H43NO4SNa: 716.2805; found
716.2799 [M+Na]+.
Compound 20: A solution of (R)-Garner aldehyde (1.15 g, 5.0 mmol) in
dry CH2Cl2 (10 mL) was slowly added at 08C to a solution of 2-triphenyl-
phosphoranylidene-propionic acid ethyl ester (2.0 g, 5.5 mmol) in dry
CH2Cl2 (25 mL). The resulting solution was allowed to warm to room
temperature and stirred for 12 h, and the solution was concentrated and
then purified by chromatography (elution with ethyl acetate/petroleum
(c = 1.1 in CHCl3); 1H NMR (300 MHz, CDCl3): d = 7.75 (d, J
=
7.2 Hz, 2H), 7.65 (d, J = 6.1 Hz, 2H), 7.43–7.25 (m, 4H), 6.68 (d, J =
8.4 Hz, 1H), 5.90 (m, 1H), 5.20 (q, J = 13.5 Hz, 2H), 4.90 (m, 2H), 4.62
(dd, J = 6.0, 15.0 Hz, 1H), 4.56–4.40 (m, 1H), 4.37 (dd, J = 3.0, 8.4 Hz,
3H), 4.30–4.20 (m, 2H), 3.83 (t, J = 8.1 Hz, 1H), 3.78–3.70 (m, 1H),
3.69–3.60 (m, 2H), 3.58–3.48 (m, 2H), 2.50 (t, J = 6.3 Hz, 1H), 2.30–2.20
(m, 1H), 2.10–1.92 (m, 3H), 1.90 (s, 3H), 1.50–1.40 (m, 1H), 1.30–1.20
(m, 3H), 0.97 (d, J = 6.3 Hz, 3H), 0.88 (s, 9H), 0.80 (t, J = 6.6 Hz,
3H) ppm; IR (KBr): n˜max = 2959, 2929, 1712, 1452, 1421 cmÀ1; ESI-MS:
m/z: 715 [M+H]+; HRMS: m/z: calcd for C42H55N2O8: 715.3953; found
715.3920 [M+H]+.
ether 1:10) to give 20 (1.72 g, 100%): [a]2D0 +14.4 (c = 1.1 in CH2Cl2);
=
1H NMR (300 MHz, CDCl3): d = 6.64 (d, J = 7.9 Hz, 1H), 4.62 (m,
1H), 4.21 (m, 2H), 4.16–4.11 (m, 1H), 3.70 (dd, J = 3.6, 8.9 Hz, 1H),
1.89 (s, 3H), 1.63 (s, 3H), 1.54 (s, 3H), 1.41 (s, 9H), 1.30 (t, J = 7.1 Hz,
3H) ppm; IR (film): n˜max
=
1741, 1698, 1657 cmÀ1; EI-MS: m/z: 298
[MÀCH3]+, 199, 198, 156, 126, 110, 109, 57, 41; elemental analysis calcd
(%) for C16H27NO5: C 61.31, H 8.68, N 4.47; found: C 61.04, H 8.37, N
4.70.
Compound 21: Sodium hydroxide (0.1 g, 2.5 mmol) was added to a solu-
tion of 20 (0.4 g, 1.3 mmol) in methanol (4 mL) and water (1 mL). The
resulting solution was warmed to 508C, stirred for 4 h, and concentrated
in vacuo before addition of water and acidification to pH 2 with HCl
(1n). The aqueous layer was extracted with ethyl acetate, the combined
organic layers were dried over Na2SO4 and concentrated, and the residue
was purified by chromatography (elution with ethyl acetate/petroleum
ether 1:1) to give the acid (0.36 g).
Compound 34: Fmoc-N-Me-Ala-OBn (32, 0.7 g, 1.7 mmol) was dissolved
in MeCN (2 mL) and Et2NH (1 mL). The resulting solution was stirred
for 15 min and concentrated to dryness, the residue was dissolved in dry
CH2Cl2 (3 mL), and the acid 33 (0.7 g, 1.7 mmol) and BEP (0.8 g,
2.9 mmol) were then added successively. DIPEA (0.5 mL, 2.9 mmol) was
added to this solution at 08C, the resulting mixture was stirred for 24 h
and concentrated in vacuo, and the residue was purified by chromatogra-
phy (elution with ethyl acetate/petroleum ether 1:4) to give the dipeptide
product 34 (60% yield from 32). [a]2D0 = À20.8 (c = 3.5 in CHCl3);
1H NMR (300 MHz, CDCl3): d = 7.77 (d, J = 7.5 Hz, 2H), 7.57 (t, J =
5.7 Hz, 2H), 7.45–7.25 (m, 9H), 7.08 (d, J = 8.7 Hz, 2H), 6.76 (d, J =
8.4 Hz, 2H), 5.61 (d, J = 8.7 Hz, 1H), 5.18 (m, 2H), 4.85 (m, 1H), 4.40–
4.10 (m, 4H), 3.75 (s, 3H), 3.00 (m, 1H), 2.88 (s, 3H), 2.80 (m, 1H), 1.42
This acid was dissolved in dry CH2Cl2 (3 mL), and allyl alcohol (89 mg,
1.5 mmol), DMAP (24 mg, 0.2 mmol), and EDCI (0.37 mg, 1.9 mmol)
were then added successively. The solution was stirred for 2 h and was
then diluted with CH2Cl2 and washed with brine, the organic layers were
dried over Na2SO4 and concentrated, and the residue was purified by
chromatography (elution with ethyl acetate/petroleum ether 1:20) to give
(d, J
= = 171.50,
7.2 Hz, 3H) ppm; 13C NMR (75 MHz, CDCl3): d
1
21 (308 mg, 74%). [a]2D0 = +22.5 (c = 1.4, CHCl3); H NMR (300 MHz,
170.99, 158.42, 155.52, 143.76, 143.72, 141.14, 135.40, 130.51, 128.47,
128.29, 128.09, 127.81, 127.69, 127.54, 126.90, 125.06, 124.99, 119.81,
113.71, 66.82, 55.00, 52.82, 52.08, 47.03, 37.72, 31.43, 14.02 ppm; IR (film):
n˜max = 3298, 3065, 2945, 2837, 1739, 1718, 1647, 1513, 1451 cmÀ1; ESI-
MS: m/z: 593 [M+H]+; HRMS: m/z: calcd for C36H37N2O6: 593.2646;
found 593.2649 [M+H]+.
CDCl3): d = 6.70 (d, J = 5.1 Hz, 1H), 6.00 (m, 1H), 5.30 (m, 2H), 4.80–
4.60 (m, 3H), 4.15 (t, J = 7.5 Hz, 1H), 3.70 (dd, J = 3.3, 8.7 Hz, 1H),
1.95 (d, J = 14.4 Hz, 3H), 1.65 (s, 3H), 1.55 (s, 3H), 1.45 (s, 9H) ppm;
13C NMR (75 MHz, CDCl3): d = 166.9, 140.9, 132.1, 117.6, 94.2, 80.0,
67.4, 65.0, 55.1, 28.1, 27.1, 26.0, 24.8, 23.9, 12.3 ppm; IR (film): n˜max
=
Chem. Eur. J. 2006, 12, 7615 – 7626
ꢁ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
7623

D. Ma, J. O. Liu et al.
Compound 36: A solution of the above dipeptide 34 in ethyl acetate was
hydrogenated over 10% palladium on charcoal at room temperature and
1 atm for 12 h. After the mixture had been filtered through Celite, the fil-
trate was concentrated to give the crude acid.
2H), 6.50 (dd, J = 12.1, 8.1 Hz, 1H), 6.11 (m, 1H), 5.43 (dd, J = 7.0,
2.0 Hz, 1H), 5.15 (d, J = 11.1 Hz, 1H), 5.05 (m, 1H), 4.94 (m, 1H), 4.84
(dd, J = 8.5, 2.9 Hz, 1H), 4.54 (dd, J = 8.3, 3.5 Hz, 1H), 4.35 (dt, J =
8.6, 3.0 Hz, 1H), 4.22 (t, J = 6.0 Hz, 1H), 4.02 (m, 1H), 3.77 (s, 3H),
3.70 (m, 1H), 3.66 (m, 1H), 3.61 (m, 1H), 3.06 (m, 1H), 3.01 (s, 3H),
2.91 (m, 1H), 2.85 (m, 1H), 2.81 (s, 3H), 2.41 (m, 1H), 2.30 (m, 1H),
2.25 (m, 1H), 2.12 (m, 1H), 2.05 (m, 1H), 1.95 (m, 1H), 1.87 (m, 1H),
1.86 (s, 3H), 1.81 (m, 1H), 1.69 (m, 1H), 1.58 (m, 1H), 1.37 (m, 1H),
1.22 (m, 1H), 1.16 (d, J = 7.1 Hz, 3H), 1.09 (d, J = 7.2 Hz, 3H), 0.97 (d,
J = 6.6 Hz, 3H), 0.93 (m, 1H), 0.91 (m, 3H), 0.86 (m, 6H), 0.90 (m,
3H) ppm; ESI-MS: m/z: 810.3 [M+H]+; HRMS: m/z: calcd for
C44H68N5O9: 810.5012; found 810.5017 [M+H]+.
Fmoc-N-Me-Ile-OTMSE (35, 0.1 g, 0.2 mmol) was dissolved in MeCN
(2 mL) and Et2NH (1 mL). The solution was stirred for 15 min and con-
centrated to dryness, and the residue was dissolved in dry CH2Cl2 (3 mL),
after which the above acid (0.1 g, 0.2 mmol) and BEP (0.12 g, 0.44 mmol)
were added successively. DIPEA (0.1 mL, 0.6 mmol) was added at 08C to
this resulting solution, the resulting mixture was stirred for 24 h and con-
centrated in vacuo, and the residue was purified by chromatography (elu-
tion with ethyl acetate/petroleum ether 1:4) to give 36 as a pale yellow
solid (90 mg, 60% overall yield). [a]D20
=
À36 (c
= 1.23 in CHCl3);
Compound 40: [a]2D0 = À74.7 (c = 0.5 in CHCl3); 1H NMR (500 MHz,
1H NMR (300 MHz, CDCl3): d = 7.81 (m, 2H), 7.62 (m, 2H), 7.51–7.32
(m, 4H), 7.15 (t, J = 8.4 Hz, 2H), 6.81 (d, J = 7.8 Hz, 2H), 5.50 (m,
1H), 4.95 (m, 2H), 4.51–4.15 (m, 5H), 3.82 (s, 3H), 3.40 (m, 1H), 3.05
(m, 1H), 3.01 (s, 3H), 2.93–2.82 (m, 1H), 2.82 (s, 3H), 2.01 (m, 1H), 1.31
(m, 4H), 1.01–0.81 (m, 9H), 0.05 (s, 9H) ppm; IR (KBr): n˜max = 2950,
2940, 1729, 1512 cmÀ1; ESI-MS: m/z: 730 [M+H]+.
CDCl3): d
= 7.15 (m, 2H), 6.80 (d, J = 8.1 Hz, 2H), 6.40 (d, J =
10.0 Hz, 1H), 6.25 (d, J = 8.1 Hz, 1H), 5.25 (m, 1H), 5.20–5.10 (m, 3H),
5.00 (d, J = 11.2 Hz, 1H), 4.80 (m, 1H), 4.77 (s, 1H), 4.32 (m, 1H), 4.00
(m, 1H), 3.75 (s, 3H), 3.70–3.60 (m, 3H), 3.50 (m, 1H), 3.15 (d, J =
13.3 Hz, 1H), 3.10 (d, J = 8.6 Hz, 1H), 3.00 (m, 1H), 2.75 (s, 3H), 2.67
(d, J = 8.1 Hz, 1H), 2.58 (s, 3H), 2.56 (m, 1H), 2.25–2.05 (m, 3H), 1.93
(s, 3H), 1.90–1.75 (m, 3H), 1.45 (m, 1H), 1.28–1.13 (m, 4H), 1.10–0.75
(m, 20H) ppm; ESI-MS: m/z: 810 [M+H]+; HRMS: m/z: calcd for
C44H68N5O9: 810.5012; found 810.5019 [M+H]+.
Compound 41: [a]2D0 = À39.4 (c = 0.11 in CHCl3); 1H NMR (500 MHz,
CDCl3): d = 7.12 (d, J = 8.2 Hz, 2H), 6.80 (d, J = 8.2 Hz, 2H), 6.49
(dd, J = 15.3, 8.6 Hz, 1H), 6.12 (d, J = 8.6 Hz, 2H), 5.43 (dd, J = 8.6,
2.0 Hz, 1H), 5.16 (d, J = 10.9 Hz, 1H), 4.94 (m, 1H), 4.86 (dd, J = 8.5,
2.9 Hz, 1H), 4.82 (d, J = 8.2 Hz, 1H), 4.52 (dd, J = 6.8, 3.5 Hz, 1H),
4.38 (t, J = 8.0 Hz, 1H), 4.02 (m, 1H), 3.90 (m, 1H), 3.81 (s, 3H), 3.77
(m, 1H), 3.68 (m, 1H), 3.55 (m, 1H), 3.40 (m, 1H), 3.06 (m, 1H), 3.01 (s,
3H), 2.90 (m, 1H), 2.86 (m, 1H), 2.81 (s, 3H), 2.40 (m, 1H), 2.34 (m,
1H), 2.20 (m, 1H), 2.09 (m, 1H), 1.94 (m, 1H), 1.88 (m, 1H), 1.87 (s,
3H), 1.82 (m, 1H), 1.62 (m, 1H), 1.54 (m, 1H), 1.47 (m, 1H), 1.45 (m,
1H), 1.39 (m, 1H), 1.20 (m, 1H), 1.13 (d, J = 7.1 Hz, 3H), 1.10 (d, J =
7.5 Hz, 3H), 0.96 (d, J = 6.2 Hz, 3H), 0.92 (m, 1H), 0.88 (m, 3H), 0.86
(m, 6H) ppm; ESI-MS: m/z: 796.5 [M+H]+; HRMS: m/z: calcd for
C43H66N5O9: 796.4855; found 796.4862 [M+H]+.
Compound 37b: [Pd(PPh3)4] (20 mg, 0.017 mmol) was added under nitro-
N
gen to a solution of the oxazoline 23 (120 mg, 0.17 mmol) in dry THF
(3 mL), followed by N-methylaniline (40 mL, 0.37 mmol). The resulting
solution was stirred for 2 h and concentrated, and the residue was puri-
fied by chromatography (elution with ethyl acetate/petroleum ether 2:3)
to give the crude acid.
Tripeptide 36 (130 mg, 0.18 mmol) was dissolved in MeCN (2 mL) and
Et2NH (1 mL). The solution was stirred for 15 min and concentrated to
dryness. After the residue had been dissolved in dry CH2Cl2 (3 mL), the
above acid (95 mg, 0.14 mmol), HATU (80 mg, 0.21 mmol), and DIPEA
(0.07 mL, 0.41 mmol) were added successively, the resulting mixture was
stirred for 48 h at room temperature and concentrated in vacuo, and the
residue was purified by chromatography (elution with ethyl acetate/pe-
troleum ether 2:3) to give 37b as a pale yellow solid (96 mg, 59% yield).
[a]2D0 = À63.3 (c = 1.38 in CHCl3); 1H NMR (300 MHz, CDCl3): d =
7.75 (d, J = 7.8 Hz, 2H), 7.58 (d, J = 7.5 Hz, 2H), 7.41–7.32 (m, 4H),
7.07 (m, 2H), 6.78 (m, 2H), 6.51 (d, J = 7.8 Hz, 1H), 6.15 (d, J =
8.7 Hz, 1H), 5.42 (m, 1H), 5.21 (m, 1H), 4.95–4.81 (m, 3H), 4.52–4.11
(m, 7H), 3.75 (s, 3H), 3.71–3.52 (m, 3H), 3.15 (m, 1H), 3.03–2.96 (m,
1H), 2.95 (s, 3H), 2.91–2.82 (m, 2H), 2.71 (s, 3H), 2.52 (m, 1H), 2.11–
1.91 (m, 4H), 1.82 (s, 3H), 1.81–1.62 (m, 3H), 1.45 (m, 1H), 1.31–1.22
(m, 5H), 1.05–0.92 (m, 12H), 0.90 (s, 9H), 0.86–0.78 (m, 4H), 0.06 (s,
9H) ppm; ESI-MS: m/z: 1164 [M+H]+; HRMS: m/z: calcd for
C65H94N5O12Si: 1164.6663; found 1164.6687 [M+H]+.
Compound 27b: A solution of 19b (200 mg, 0.29 mmol) in MeCN (2 mL)
and Et2NH (1 mL) was stirred for 15 min and was then concentrated to
dryness. After the residue had been dissolved in dry CH2Cl2 (3 mL), the
acid 5a (160 mg, 0.29 mmol), HATU (164 mg, 0.43 mmol), and DIPEA
(0.10 mL, 0.58 mmol) were added successively. The resulting mixture was
stirred for 12 h at room temperature and concentrated in vacuo, and the
residue was purified by chromatography (elution with ethyl acetate/pe-
troleum ether 1:3) to give 27b (237 mg, 81% yield). [a]2D0 = À22.7 (c =
0.3 in CHCl3); 1H NMR (300 MHz, CDCl3): d = 7.77 (d, J = 6.6 Hz,
2H), 7.60 (t, J = 6.9 Hz, 2H), 7.50–7.15 (m, 19H), 7.00 (d, J = 13.5 Hz,
1H), 6.41 (d, J = 9.3 Hz, 1H), 5.30 (m, 1H), 4.90 (d, J = 9.6 Hz, 1H),
4.70–4.40 (m, 5H), 4.30–4.20 (m, 2H), 3.75–3.45 (m, 4H), 2.45 (m, 1H),
2.35–2.05 (m, 5H), 1.95 (m, 3H), 1.71 (s, 3H), 1.66 (s, 3H), 1.61 (s, 3H),
The oxazoline analogue of apratoxin A (4): TBAF (1.0m solution in
THF, 50 mL, 50 mmol) was added to a solution of 37b (20 mg, 17.2 mmol)
in dry THF (1 mL). The resulting solution was stirred overnight and con-
centrated to dryness, and the residue was dissolved in toluene (2 mL) and
concentrated to dryness. This residue was dissolved in dry CH2Cl2 (9 mL,
0.002m), and HATU (20 mg, 52.6 mmol) and DIPEA (25 mL, 143 mmol)
were then added successively. The resulting mixture was stirred for three
days at room temperature and concentrated in vacuo, and the residue
was purified by chromatography (elution with pure ethyl acetate) to give
4 as a colorless solid (7 mg, 45% yield). [a]2D0 = À117.5 (c = 0.2 in
1.50–1.30 (m, 3H), 1.15 (d,
J = 6.9 Hz, 3H), 0.9 (m, 12H) ppm;
13C NMR (75 MHz, CDCl3): d = 175.26, 172.09, 167.51, 155.12, 144.56,
144.50, 144.06, 143.68, 141.22, 139.35, 130.19, 129.55, 127.92, 127.84,
127.63, 127.09, 127.01, 126.76, 126.64, 125.10, 125.03, 119.89, 118.86, 78.52,
71.05, 67.68, 61.58, 59.52, 47.19, 47.10, 46.39, 46.18, 40.42, 37.17, 36.03,
34.85, 34.79, 29.83, 29.80, 25.85, 25.68, 25.65, 25.40, 24.19, 19.99, 17.97,
1
CHCl3); H NMR (500 MHz, CDCl3): d = 7.15 (d, J = 8.7 Hz, 2H), 6.80
(d, J = 8.2 Hz, 2H), 6.21 (d, J = 9.3 Hz, 1H), 6.01 (d, J = 9.3 Hz, 1H),
5.25 (d, J = 11.5 Hz, 1H), 5.05 (td, J = 10.2, 5.6 Hz, 1H), 4.97 (d, J =
11.6 Hz, 1H), 4.81 (m, 1H), 4.70 (d, J = 10.6 Hz, 1H), 4.38 (m, 1H),
4.20 (m, 1H), 3.78 (s, 3H), 3.70–3.55 (m, 3H), 3.28 (brq, J = 6.7 Hz,
1H), 3.15 (d, J = 3.8 Hz, 1H), 3.10 (d, J = 11.7 Hz, 1H), 2.85 (dd, J =
12.7, 4.6 Hz, 1H), 2.81 (s, 3H), 2.75 (s, 3H), 2.65 (d, J = 13.5 Hz, 1H),
2.33 (m, 1H), 2.25 (m, 1H), 2.15 (m, 1H), 2.05 (m, 1H), 1.92 (s, 3H),
1.90–1.75 (m, 3H), 1.45 (m, 1H), 1.31 (m, 1H), 1.26 (m, 1H), 1.25 (s,
3H), 1.10 (m, 1H), 1.07 (d, J = 6.9 Hz, 3H), 1.01 (t, J = 7.0 Hz, 3H),
0.99 (d, J = 6.4 Hz, 3H), 0.96 (m, 1H), 0.95 (d, J = 7.6 Hz, 3H), 0.87
(m, 9H) ppm; ESI-MS: m/z: 824 [M+H]+; HRMS: m/z: calcd for
C45H70N5O9: 824.5168; found 824.5160 [M+H]+.
15.10, 12.93 ppm; IR (film): n˜max
= 3322, 2962, 2928, 1712, 1684,
1450 cmÀ1
;
ESI-MS: m/z: 1041 [M+Na]+; HRMS: m/z: calcd for
C63H74N2O8SNa: 1041.5058; found 1041.5061 [M+Na]+.
Compound 30b: DIPEA (40 mL, 0.24 mmol) was added at 08C to a solu-
tion of 27b (120 mg, 0.18 mmol) in dichloromethane (2 mL), followed by
AcCl (15 mL, 0.21 mmol). After the solution had been stirred for 0.5 h,
DIPEA (40 mL, 0.24 mmol) and AcCl (15 mL, 0.21 mmol) were again
added. The solution was stirred for 1 h and then concentrated in vacuo,
and the residue was purified by chromatography (elution with ethyl ace-
tate/petroleum ether 1:3) to give 30b (100 mg, 81% yield). [a]2D0 = À40.3
(c = 0.7 in CHCl3); 1H NMR (300 MHz, CDCl3): d = 7.78 (d, J
=
Compound 39: [a]D20
(500 MHz, CDCl3): d = 7.08 (d, J = 7.5 Hz, 2H), 6.79 (d, J = 7.5 Hz,
=
À113.3 (c
=
0.055 in CHCl3); 1H NMR
6.6 Hz, 2H), 7.64 (m, 2H), 7.55–7.15 (m, 19H), 6.39 (m, 1H), 5.35 (m,
1H), 5.05 (m, 1H), 4.90–4.10 (m, 9H), 3.70–3.50 (m, 2H), 2.55–2.30 (m,
7624
www.chemeurj.org
ꢁ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 7615 – 7626

Total Synthesis of Apratoxin A
FULL PAPER
4H), 2.10 (s, 3H), 1.98 (s, 3H), 1.85–1.65 (m, 9H), 1.50–1.25 (m, 5H),
1.10 (d, J = 4.5 Hz, 3H), 0.90 (s, 9H), 0.85 (d, J = 5.4 Hz, 3H) ppm;
13C NMR (75 MHz, CDCl3): d = 172.19, 172.00, 167.41, 154.59, 154.12,
144.41, 144.31, 143.80, 143.51, 141.17, 138.98, 129.47, 127.83, 127.56,
126.96, 126.92, 126.85, 126.72, 126.69, 124.99, 124.95, 119.83, 119.76,
118.70, 79.66, 79.09, 72.90, 67.31, 61.64, 60.23, 59.57, 59.23, 47.16, 47.04,
46.18, 44.88, 34.71, 29.87, 25.98, 25.74, 25.72, 25.62, 24.12, 23.21, 20.90,
20.78, 20.33, 17.92, 14.09, 13.30, 12.85 ppm; IR (film): n˜max = 3300, 1711,
1421 cmÀ1; ESI-MS: m/z: 1061 [M+H]+, 1083 [M+Na]+; HRMS: m/z:
calcd for C65H76N2O9SNa: 1083.5164; found 1083.5166 [M+Na]+.
1H), 4.19 (t, J = 7.3 Hz, 1H), 3.78 (s, 3H), 3.66 (m, 1H), 3.54 (m, 1H),
3.45 (t, J = 6.9 Hz, 1H), 3.25 (m, 1H), 3.14 (dd, J = 3.8 Hz, 1H), 3.10
(dd, J = 11.7 Hz, 1H), 2.83 (dd, J = 12.7, 4.6 Hz, 1H), 2.79 (s, 3H), 2.72
(s, 3H), 2.60 (m, 1H), 2.19 (m, 3H), 2.10 (m, 1H), 1.95 (s, 3H), 1.85 (m,
2H), 1.80 (m, 1H), 1.55 (m, 1H), 1.22 (m, 1H), 1.20 (m, 4H), 1.10 (m,
1H), 1.05 (d, J = 6.9 Hz, 3H), 0.96 (m, 1H), 0.95 (m, 6H), 0.91 (m, 3H),
0.87 (s, 9H) ppm; ESI-MS: m/z: 862 [M+Na]+; HRMS: m/z: calcd for
C45H70N5O8S: 840.4940; found 840.4931 [M+H]+.
Compound
31b:
Trifluoromethanesulfonic
anhydride
(120 mL,
0.73 mmol) was added at 08C under nitrogen to a solution of triphenyl-
phosphine oxide (410 mg, 1.47 mmol) in dry dichloromethane (5 mL).
After the mixture had been stirred for 0.5 h, a solution of 30b (195 mg,
0.18 mmol) in dry dichloromethane (2 mL) was added, the resulting solu-
tion was stirred for 1 h, and then sodium hydrogen carbonate solution
(5%) was added. The aqueous layer was extracted with CHCl3, the com-
bined organic layers were dried over Na2SO4 and concentrated, and the
residue was purified by chromatography (elution with ethyl acetate/pe-
troleum ether 1:3) to give 31b (135 mg, 92% yield). [a]2D0 = À56.1 (c =
0.4 in CHCl3); 1H NMR (300 MHz, CDCl3): d = 7.77 (d, J = 6.6 Hz,
2H), 7.64 (m, 2H), 7.40 (t, J = 7.5 Hz, 2H), 7.32 (m, 2H), 6.72 (m, 1H),
5.35 (m, 1H), 5.15 (m, 2H), 4.80 (d, J = 8.4 Hz, 1H), 4.63 (d, J =
7.2 Hz, 2H), 4.56–4.20 (m, 5H), 3.70–3.38 (m, 4H), 3.00 (m, 2H), 2.09 (s,
3H), 2.05 (s, 3H), 1.75 (s, 3H), 1.71 (s, 3H), 1.45–1.25 (m, 10H), 0.90 (m,
12H) ppm; IR (film): n˜max = 2961, 1737, 1711, 1618, 1452 cmÀ1; ESI-MS:
m/z: 801 [M+H]+, 823 [M+Na]+; HRMS: m/z: calcd for C46H61N2O8S:
801.4143; found 801.4145 [M+H]+.
Acknowledgements
The authors are grateful to the Chinese Academy of Sciences, the Na-
tional Natural Science Foundation of China (grant 20321202), and the
Science and Technology Commission of Shanghai Municipality (grants
02 JC14032 & 03XD14001) for their financial support.
[1] For recent reviews, see: a) M. S. Butler, J. Nat. Prod. 2004, 67, 2141–
2153; b) D. J. Newman, G. M. Cragg, K. M. Snader, J. Nat. Prod.
2003, 66, 1022–1037.
[2] H. Leusch, W. Y. Yoshida, R. E. Moore, V. J. Paul, T. H. Corbett, J.
Am. Chem. Soc. 2001, 123, 5418–5423.
[3] H. Leusch, W. Y. Yoshida, R. E. Moore, V. J. Paul, Bioorg. Med.
Chem. 2002, 10, 1973–1978.
[4] a) J. Chen, C. J. Forsyth, J. Am. Chem. Soc. 2003, 125, 8734–8735;
b) J. Chen, C. J. Forsyth, Proc. Natl. Acad. Sci. USA 2004, 101,
12067–12072; c) J. Chen, C. J. Forsyth, Org. Lett. 2003, 5, 1281–
1283; d) Z. Xu, Z. Chen, T. Ye, Tetrahedron: Asymmetry 2004, 15,
355–363.
Compound 37a: TMSOTf (13 mL, 62.5 mmol) was added under nitrogen
to a solution of 31b (50 mg, 62.5 mmol) in dry dichloromethane (2 mL).
The resulting solution was stirred for 1 h and concentrated to give the
crude acid.
[5] Part of this work has been published in a preliminary communica-
tion, see: B. Zou, J. Wei, G. Cai, D. Ma, Org. Lett. 2003, 5, 3503–
3506.
[6] During the preparation of this manuscript, Doi and co-workers re-
ported their total synthesis of apratoxin A, see: T. Doi, Y. Numajiri,
A. Munakata, T. Takahashi, Org. Lett. 2006, 8, 531–534.
[7] For reviews, see: a) P. Wipf, Chem. Rev. 1995, 95, 2115–2134; b) P.
Wipf, S. Venkatraman, Synlett 1997, 1–10.
[8] For recent examples on the synthesis of thiazoline- and oxazoline-
containing cyclopeptides, see: a) S. Yu, X. Pan, X. Lin, D. Ma,
Angew. Chem. 2005, 117, 137–140; Angew. Chem. Int. Ed. 2005, 44,
135–138; b) K. C. Nicolaou, D. W. Kim, D. Schlawe, D. E. Lizos,
R. G. Noronha, D. A. Longbottom, Angew. Chem. 2005, 117, 5005–
5009; Angew. Chem. Int. Ed. 2005, 44, 4925–4929; c) K. C. Nicolaou,
D. Schlawe, D. W. Kim, D. A. Longbottom, R. G. Noronha, D. E.
Lizos, R. Rao Mannam, D. J. Faulkner, Chem. Eur. J. 2005, 11,
6197–6211; d) B. McKeever, G. Pattenden, Tetrahedron 2003, 59,
2713–2727; e) B. McKeever, G. Pattenden, Tetrahedron 2003, 59,
2701–2712; f) K. C. Nicolaou, M. Nevalainen, M. Zak, S. Bulat, M.
Bella, B. S. Safina, Angew. Chem. 2003, 115, 3540–3543; Angew.
Chem. Int. Ed. 2003, 42, 3418–3421; g) J. M. Caba, I. M. Rodriguez,
I. Manzanares, E. Giralt, F. Albericio, J. Org. Chem. 2001, 66, 7568;
h) P. Wipf, Y. Uto, J. Org. Chem. 2000, 65, 1037.
[9] a) B. List, Synlett 2001, 1675–1685; b) B. List, P. Pojarliev, C. Castel-
lo, C. Org. Lett. 2001, 3, 573–575.
[10] a) U. S. Racherla, H. C. Brown, J. Org. Chem. 1991, 56, 401–401;
b) P. K. Jadhav, K. S. Bhat, P. T. Perumal, H. C. Brown, J. Org.
Chem. 1986, 51, 432–439.
[11] R. H. Grubbs, S. Chang, Tetrahedron 1998, 54, 4413–4450.
[12] W. H. Pirkle, P. E. Adams, J. Org. Chem. 1980, 45, 4117–4121.
[13] W. Oppolzer, P. Lienard, Tetrahedron Lett. 1993, 34, 4321–4324.
[14] a) J. Inanaga, K. Hirata, H. Saeki, T. Katsuki, M. Yamaguchi, Bull.
Chem. Soc. Jpn. 1979, 52, 1989–1993; b) M. Hikota, Y. Sakurai, K.
Horita, O. Yonemitsu, Tetrahedron Lett. 1990, 31, 6367–6370.
[15] M. Ciommer, H. Kunz, Synlett 1991, 593–595.
Tripeptide 36 (57 mg, 78 mmol) was dissolved in MeCN (1 mL) and
Et2NH (0.5 mL), and the solution was stirred for 15 min and concentrat-
ed to dryness. After the residue had been dissolved in dry CH2Cl2
(2 mL), HATU (80 mg, 0.21 mmol), a solution of the above acid in dry
CH2Cl2 (1 mL), and DIPEA (27 mL, 156 mmol) were added successively,
the resulting mixture was stirred overnight at room temperature and con-
centrated in vacuo, and the residue was purified by chromatography (elu-
tion with ethyl acetate/petroleum ether 1:1) to give 37a (38 mg, 50%
yield). [a]2D0 = À100.9 (c = 1.0 in CHCl3); 1H NMR (300 MHz, CDCl3):
d = 7.78 (m, 2H), 7.66 (m, 2H), 7.43 (t, J = 7.2 Hz, 2H), 7.34 (t, J =
7.5 Hz, 2H), 7.12 (d, J = 7.5 Hz, 2H), 6.81 (d, J = 8.7 Hz, 2H), 6.58 (m,
1H), 6.32 (m, 1H), 5.43 (m, 1H), 5.30–5.05 (m, 3H), 4.94 (d, J
=
13.2 Hz, 1H), 4.82 (m, 1H), 4.60–4.05 (m, 7H), 3.80 (s, 3H), 3.62 (m,
2H), 3.42 (m, 1H), 3.10 (m, 1H), 3.01 (s, 3H), 2.90 (m, 3H), 2.75 (s, 3H),
2.30 (m, 1H), 2.12 (s, 3H), 2.07 (s, 3H), 2.00–1.90 (m, 6H), 1.50–1.20 (m,
5H), 1.10–0.70 (m, 25H), 0.07 (s, 9H) ppm; ESI-MS: m/z: 1244
[M+Na]+; HRMS: m/z: calcd for C67H95N5O12SSiNa: 1244.6359; found
1244.6348 [M+Na]+.
Apratoxin A (1): TBAF (1.0m solution in THF, 130 mL, 130 mmol) was
added to a solution of 37a (40 mg, 32.8 mmol) in dry THF (2 mL). The re-
sulting solution was stirred overnight and concentrated to dryness, the
residue was dissolved in toluene (2 mL), was again concentrated to dry-
ness, and was redissolved in dry CH2Cl2 (16 mL, 0.002m), and HATU
(25 mg, 65.5 mmol) and DIPEA (28 mL, 164 mmol) were then added suc-
cessively. The resulting mixture was stirred for 3 days at room tempera-
ture and concentrated in vacuo, and the residue was purified by chroma-
tography (elution with ethyl acetate/petroleum ether 4:1) to give 38
(10 mg).
This product was dissolved in ethanol (95%, 2 mL), and KCN (0.7 mg,
11 mmol) was added. The solution was warmed to 508C, stirred overnight,
and then concentrated in vacuo and purified by chromatography (elution
with pure ethyl acetate) to give 1 (7 mg, 26% yield from 37a). [a]D20
=
1
À159 (c = 0.45 in MeOH); H NMR (500 MHz, CDCl3): d = 7.20 (d, J
= 8.3 Hz, 2H), 6.80 (d, J = 8.2 Hz, 2H), 6.40 (d, J = 9.3 Hz, 1H), 6.05
(d, J = 9.3 Hz, 1H), 5.28 (m, 1H), 5.21 (d, J = 11.5 Hz, 1H), 5.05 (m,
1H), 5.00 (dd, J = 10.2, 5.6 Hz, 1H), 4.69 (d, J = 10.6 Hz, 1H), 4.23 (m,
[16] P. Lafargue, P. Guenot, J.-P. Lellouche, Heterocycles 1995, 41, 947–
957.
Chem. Eur. J. 2006, 12, 7615 – 7626
ꢁ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
7625

D. Ma, J. O. Liu et al.
[17] a) P. Wipf, C. P. Miller, S. Venkatraman, P. C. Fritch, Tetrahedron
Lett. 1995, 36, 6395–6398; b) P. Wipf, P. C. Fritch, J. Am. Chem. Soc.
1996, 118, 12358–12367.
[18] T. Hoeg-Jensen, A. Holm, H. Sorensen, Synthesis 1996, 383–387.
[19] P. Raman, H. Razavi, J. W. Kelly, Org. Lett. 2000, 2, 3289–3292.
[20] S. You, H. Razavi, J. W. Kelly, Angew. Chem. 2003, 115, 87–89;
Angew. Chem. Int. Ed. 2003, 42, 83–85.
[21] P. Li, J.-C. Xu, Tetrahedron 2000, 56, 8119–8131.
[22] M. Nishizawa, H. Yamamoto, K. Seo, H. Imagawa, T. Sugihara, Org.
Lett. 2002, 4, 1947–1949.
[23] During the submission of this manuscript, a report regarding the bi-
ological mode of action of apratoxin A appeared, see: H. Luesch,
S. K. Chanda, R. M. Raya, P. D. DeJesus, A. P. Orth, J. R. Walker,
J. C. I. Belmonte, P. G. Schultz, Nature: Chem. Biol. 2006, 2, 158.
[24] K. Shimoji, H. Taguchi, K. Oshima, H. Yamamoto, H. Nozaki, J.
Am. Chem. Soc. 1974, 96, 1620.
[25] P. Wipf, P. C. Fritch, S. J. Geib, A. M. Sefler, J. Am. Chem. Soc.
1998, 120, 4105–4112.
Received: April 29, 2006
Published online: July 10, 2006
7626
www.chemeurj.org
ꢁ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 7615 – 7626