Solid-phase total synthesis of (-)-apratoxin a and its analogues and their biological evaluation

Article abstract of DOI:10.1002/asia.201000549

Two approaches for the solid-phase total synthesis of apratoxinA and its derivatives were accomplished. In synthetic route A, the peptide was prepared by the sequential coupling of the corresponding amino acids on trityl chloride SynPhase Lanterns. After cleavage from the polymer-support, macrolactamization of 10, followed by thiazoline formation, provided apratoxinA. This approach, however, resulted in low yield because the chemoselectivity was not sufficient for the formation of the thiazoline ring though its analogue 33 was obtained. However, in synthetic route B, a cyclization precursor was prepared by solid-phase peptide synthesis by using amino acids 13-15 and 18. The final macrolactamization was performed in solution to provide apratoxin A in high overall yield. This method was then successfully applied to the synthesis of apratoxin analogues. The cytotoxic activity of the synthetic derivatives was then evaluated. The epimer 34 was as potent as apratoxinA, and O-methyl tyrosine can be replaced by 7-azidoheptyl tyrosine without loss of activity. The 1,3-dipolar cycloaddition of 38 with phenylacetylene was performed in the presence of a copper catalyst without affecting the thiazoline ring. Solid Approach: Two routes for the solid-phase total synthesis of apratoxinA have been described. On the basis of this method, its analogues were synthesized and their biological activity has been evaluated. Copyright

Full text of DOI:10.1002/asia.201000549

FULL PAPERS
DOI: 10.1002/asia.201000549
Solid-phase Total Synthesis of (ꢀ)-Apratoxin A and Its Analogues and Their
Biological Evaluation
Takayuki Doi,*[a, b] Yoshitaka Numajiri,[b] Takashi Takahashi,[b] Motoki Takagi,[c] and
Kazuo Shin-ya[d]
Abstract: Two approaches for the
solid-phase total synthesis of apratoxi-
n A and its derivatives were accom-
plished. In synthetic route A, the pep-
tide was prepared by the sequential
coupling of the corresponding amino
acids on trityl chloride SynPhase Lan-
terns. After cleavage from the poly-
mer-support, macrolactamization of 10,
followed by thiazoline formation, pro-
vided apratoxin A. This approach, how-
ever, resulted in low yield because the
chemoselectivity was not sufficient for
the formation of the thiazoline ring
though its analogue 33 was obtained.
However, in synthetic route B, a cycli-
zation precursor was prepared by solid-
phase peptide synthesis by using amino
acids 13–15 and 18. The final macrolac-
tamization was performed in solution
to provide apratoxin A in high overall
yield. This method was then successful-
ly applied to the synthesis of apratoxin
analogues. The cytotoxic activity of the
synthetic derivatives was then evaluat-
ed. The epimer 34 was as potent as
apratoxin A, and O-methyl tyrosine
can be replaced by 7-azidoheptyl tyro-
sine without loss of activity. The 1,3-di-
polar cycloaddition of 38 with phenyl-
acetylene was performed in the pres-
ence of a copper catalyst without af-
fecting the thiazoline ring.
Keywords: cytotoxicity · peptides ·
solid-phase synthesis
·
structure–
total
activity
relationships
·
synthesis
Introduction
(N-methylisoleucine, N-methylalanine, and O-methyltyro-
sine), a,b-unsaturated modified cysteine residue (moCys), a
dihydroxylated fatty acid moiety, and 3,7-dihydroxy-2,5,8,8-
tetramethylnonanoic acid (Dtena) (Figure 1). Apratoxin A
has a thiazoline ring in its macrocycle and epimerization at
the a position of the thiazoline at the C34 position as well
as b-elimination of the hydroxy group at the C35 position
were observed under weakly acidic conditions, such as in
CDCl3 and the resulting dehydroapratoxin A (2), lost the cy-
totoxicity.[2] Apratoxin B–G (3–8), which are structurally re-
lated to apratoxin A, were also isolated and their biological
activity was investigated.[2–5] After Forsyth and Chen report-
ed the first total synthesis of apratoxin A (1),[6] two groups,
including our group, have achieved the total synthesis of 1
and its related derivatives.[7a,8a] Oxoapratoxin A (9), an oxa-
zoline analogue, is more accessible and has been synthesized
by three groups.[7b,8b,9] In particular, Cavelier et al. recently
reported the solid-phase total synthesis of 9, which enabled
the synthesis of various analogues of oxoapratoxin deriva-
tives.[9] They prepared two linear precursors on a polymer
support, and macrolactamization was successfully performed
between O-MeTyr and a modified serine, and also between
the modified serine and Dtena. The oxazoline was finally
obtained from the modified serine moiety using dehydrocyc-
lization. They also mentioned that the formation of oxazo-
Apratoxin A (1), isolated from the marine cyanobacterium
Lyngbya majuscula, exhibits potent cytotoxic activity against
KB and LoVo cancer cells with IC50 values of 0.52 and
0.36 nm, respectively.[1] This 25-membered cyclic depsipep-
tide is comprised of proline, three methylated amino acids
[a] Prof. Dr. T. Doi
Graduate School of Pharmaceutical Sciences
Tohoku University
6-3 Aza-Aoba, Aramaki, Aoba, Sendai 980-8578 (Japan)
Fax : (+81)22-795-6864
E-mail: doi_taka@mail.pharm.tohoku.ac.jp
[b] Prof. Dr. T. Doi, Dr. Y. Numajiri, Prof. Dr. T. Takahashi
Department of Applied Chemistry
Tokyo Institute of Technology
2-12-1 Ookayama, Meguro, Tokyo 152-8552 (Japan)
[c] Dr. M. Takagi
Japan Biological Informatics Consortium (JBIC)
2-4-7 Aomi, Koto-ku, Tokyo 135-0064 (Japan)
[d] Dr. K. Shin-ya
National Institute of Advanced Industrial Science and Technology
(AIST)
2-4-7 Aomi, Koto-ku, Tokyo 135-0064 (Japan)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201000549.
180
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Chem. Asian J. 2011, 6, 180 – 188

action of apratoxin A by func-
tional genomics, where apratox-
in A induces pronounced G1
cell cycle arrest and apoptosis
by the inhibition of phosphory-
lation of the signal transducer
and the activator of transcrip-
tion (STAT) 3.[10a] Upstream
events were recently reported
in which apratoxin A inhibits
Janus kinase (JAK)/STAT sig-
naling through rapid down-reg-
ulation of interleukin 6 signal
transducer (gp130), and also de-
pletes cancer cells of several
cancer-associated receptor tyro-
sine kinases by preventing their
N-glycosylation, which led to
their rapid proteasomal degra-
dation.[10b] On the other hand,
Shen et al. have proposed that
oxoapratoxin A (9) modulates
degradation of Hsp90 client
proteins through the chaper-
one-mediated
autophagy
(CMA) pathway.[11] This feature
was proposed on the basis of af-
finity pull down assays by using
a biotin probe of an oxoapra-
toxin derivative. In view of our
independent approach to eluci-
date the biological mechanism
of apratoxin A, we report the
solid-phase total synthesis of
Figure 1. Structure of apratoxin A–G and oxoapratoxin A.
apratoxin A (1) and its ana-
line prior to macrocyclization led to 9 without any adverse
affect.
logues, evaluation of their biological activity, and structure–
activity relationships.
As apratoxin A (1) exhibits potent cytotoxicity of cancer
cells, elucidation of its biological mode of action would be
an interesting development and could lead to a new anti-
cancer agent. In 2006, Luesch et al. proposed the mode of
Results and Discussion
Retrosynthetic Analysis
Our synthetic strategy has been illustrated in Scheme 1. We
planned two synthetic routes using solid-phase peptide syn-
thesis (SPPS). For route A, thiazoline formation was per-
formed in the final step of the synthesis after macrolactami-
zation of 10, because the C34 position easily epimerizes and
dehydration occurs under weakly acidic conditions. The for-
mation of a thiazoline ring from the modified cysteine
moiety must be chemoselective in the presence of the five
amide bonds. For route B, the thiazoline ring was formed
prior to the peptide coupling reaction, as previously report-
ed in the solution phase synthesis. Macrolactamization will
be the final step in this synthetic route. The thiazoline
formed should be stable under the peptide coupling/depro-
tection conditions on a polymer-support.
Abstract in Japanese:
Chem. Asian J. 2011, 6, 180 – 188
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181

T. Doi et al.
FULL PAPERS
(UV 214 nm) after acid cleav-
age. PyBroP is known as a good
condensation reagent for N-
methylamino acids, and it ac-
tually gave better results than
other reagents, such as diisopro-
pylcarbodiimide/1-hydroxyben-
zotriazole
HATU.[14] After Fmoc cleavage,
acylation with Fmoc-Tyr(OMe)-
(DIC/HOBt)
or
ACHTUNGTRENNUNG
OH (15) was also repeatedly
performed by using PyBroP.
Surprisingly, acid cleavage with
either 0.5% TFA/CH2Cl2 or
30% hexafluoroisopropyl alco-
[15]
hol (HFIPA)/CH2Cl2 did not
provide the desired tripeptide
21, but rather provided diketo-
piperadine 22 in quantitative
yield. As we did not observe
acid free dipeptide cleaved be-
tween
two
N-methylamino
acids, MeAla-Melle,[16] it is
thought that the diketopipera-
dine would be formed, thereby
releasing H-Melle-OH as
a
leaving group, only after acid
cleavage from the polymer sup-
port. Therefore, we continued
the next acylation with Fmoc-
moCys-OH (16), after removal
of the Fmoc group on the poly-
mer support. The condensation
reaction was performed by
using DIC/HOBt to afford
polymer-supported tetrapeptide
23 in 93% yield with 91%
purity (UV 214 nm) determined
by cleavage with 30% HFIPA/
CH2Cl2. After removal of the
Scheme 1. The two synthetic routes toward solid-phase synthesis of apratoxin A (1).
Synthetic Approach of Route
Fmoc group, peptide coupling with Fmoc-Pro-Dtena-
A To synthesize various derivatives, SPPS was performed by
using the 9-fluorenylmethoxycarbonyl (Fmoc) strategy con-
nected by a trityl linker. The cyclization precursors can be
cleaved under mild conditions, such as using hexafluoroiso-
propyl alcohol (HFIPA). The attachment of Fmoc-Melle-
OH (13) to trityl chloride SynPhase Lanterns, 12 preformed
from commercially available trityl alcohol SynPhase Lan-
terns (0.037 mmol/lantern) with AcCl, was performed by
using diisopropylethylamine (DIEA)/CH2Cl2 (Scheme 2).[12]
After cleavage of 13 from the polymer-supported 19 with
0.5% TFA/CH2Cl2, the loading amount of 13 was deter-
mined to be 0.033 mmol per lantern. After removal of the
Fmoc group with 20% piperidine/DMF, acylation with
Fmoc-MeAla-OH (14) was repeatedly performed by using
PyBroP/DIEA.[13] The purity was determined to be 93% by
high-performance liquid chromatography (HPLC) analysis
(OTroc)-OH (17)[7b] was performed by using HATU as a
condensation reagent, the reaction was completed in 24 h, as
monitored by the Kaiser test.[17] We were then able to
reduce the amount of 17 to 2 equivalents. To prepare the
cyclization precursor 10, the polymer-supported peptide 25
was treated with 20% piperidine/DMF, followed by 30%
HFIPA/CH2Cl2. However, the product obtained was not the
desired compound 10, but rather a piperidine adduct to the
N-trichloroethoxycarbonyl (Troc) group. To avoid this prob-
lem, 2,6-dimethylpiperidine was utilized instead of piperi-
dine.[18] The desired compound 10 was then obtained in
93% crude yield with purity of 82%, as detected by HPLC
(UV 214 nm), and was then used for macrocyclic formation
without purification.
Macrolactamization of 10 was achieved by using a solu-
tion of HATU/DIEA prior to the formation of a thiazoline
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Chem. Asian J. 2011, 6, 180 – 188

Total Synthesis of (ꢀ)-Apratox
investigated by Kelly and co-workers.[19] Treatment of 26
with trifluoromethanesulfonic anhydride/triphenylphosphine
oxide (Tf2O/Ph3PO), followed by cleavage of the Troc group
with Zn/NH4OAc provided a complex mixture. Repeatedly
separated by preparative thin-layer chromatography (TLC),
apratoxin A (1) was isolated in 10% overall yield over two
steps. Treatment with an excess amount of Tf2O/Ph3PO de-
creased the yield because an undesired reaction with other
amido groups also occurred. Although apratoxin A (1) was
synthesized by using a solid-phase method, route A was not
suitable for the synthesis of various derivatives of 1. There-
fore, we investigated the synthetic route B.
Synthetic Approach of Route B
Coupling of polymer-supported tetrapeptide 23 with thiazo-
line-containing 18 (2.5 equiv) was investigated as we previ-
ously reported, in solution-phase synthesis (Scheme 4).[7]
PyAOP[20] was an effective condensation reagent to com-
plete the reaction in DMF/CH2Cl2 in 24 h. After removal of
the Fmoc group with 20% piperidine/DMF, the cyclization
precursor 11 was cleaved from the polymer support by using
30% HFIPA/CH2Cl2 in 90% crude yield and 80% purity
(UV 214 nm). This product was used in the next reaction
without purification. Macrolactamization was performed by
using HATU/DIEA as previously reported. After simple pu-
rification by silica gel chromatography, a diastereomeric
mixture of apratoxin A (1) and 34-epi-apratoxin A was pro-
vided in 53% combined yield from 19 in a ratio of 85:15.
The diastereomers were easily separated by preparative
TLC, and their spectral data were identical to those previ-
ously reported.[7b] As previously reported, the epimerization
at position 34 does not occur in the solution-phase synthesis.
This partial epimerization could take place during removal
of the Fmoc group in the treatment with 20% piperidine on
the polymer support (Scheme 5).
Scheme 2. Solid-phase synthesis of cyclization precursor 10 (route A).
ring (Scheme 3). After purification by silica gel chromatog-
raphy, the cyclic peptide 26 was isolated in 60% overall
yield from 19. The crucial thiazoline ring formation was
Figure 2. Synthetic fragments for the synthesis of apratoxin analogues.
Chem. Asian J. 2011, 6, 180 – 188
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T. Doi et al.
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Scheme 3. Thiazoline formation performed after macrolactamization in the total synthesis of apratoxin A (1) through route A.
also investigated. On the basis
of the Huisgen 1,3-dipolar cy-
cloaddition[21] or “click chemis-
try”,[22] an azide is a suitable
functional group for the attach-
ment of a detection tag in activ-
ity-based
small-molecular
probes.[23] When azido-contain-
ing amino acids 28–31 were uti-
lized in place of MeAla, Tyr-
AHCTUNGTERG(NNUN OMe) residue through route B,
the desired products 36–39
were provided in 31–47% over-
all yields in diastereomeric
ratios of 90:10–70:30 (Table 1,
entries 8–11). The major diaste-
reomers were isolated by prep-
arative TLC or preparative re-
versed-phase HPLC. In the at-
tempted synthesis of 34 in
which Melle was replaced by 28
(entry 6), no product was de-
tected because the linear pep-
tide had been cleaved off from
the polymer support; this led to
the formation of diketopipera-
dine.
The
compound
35
(Figure 3) was obtained in a
small amount, which was de-
tected by LC-MS when 29 was
used in place of Melle (entry 7).
Scheme 4. Solid-phase total synthesis of apratoxin A (1) by route B.
Synthesis of Apratoxin Analogues
Because we have established the solid-phase total synthesis
of apratoxin A, various analogues can now be prepared and
applied to the precursor of a molecular probe to elucidate
the structure–activity relationships (Figures 2 and 3). Silyla-
tion of 1 with triethylsilyl triflate provided triethylsilyl ether
32 in 50% yield, whereas acylation of the hydroxy group in
1 did not proceed. According to route A, simple amino acid
27, instead of 17, was utilized in the synthesis of 33. Synthe-
ses of azido-containing apratoxin derivatives 34–39 were
Cytotoxic Activity of Apratoxin Analogues
We have now synthesized several apratoxin analogues and
the cytotoxic activity of these against HeLa cells was evalu-
ated, and the results are depicted in Table 1. Synthetic apra-
toxin A (1) was potent with an IC50 value of 0.19 mm
(entry 1).[24] The 34-epimer was, interestingly, as potent as
apratoxin A (entry 2). The stereochemistry of the methyl
group at position 34 does not affect the potency. Oxoapra-
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Chem. Asian J. 2011, 6, 180 – 188

Total Synthesis of (ꢀ)-Apratox
replaced by azido-containing MeAla and MeLys derivatives,
respectively, decreased to one-tenth of that of 1 (entries 8
and 9). Substitution of the O-methyl group in a tyrosine res-
idue with a 7-azidoheptyl group resulted in potent activity
(38, entry 10). Thus, 38 could be a good precursor in the
preparation of a molecular probe, which can be utilized for
identification of the target molecule of apratoxin A.[25]
Coupling Reaction of an Azido-containing Apratoxin
Analogue with Phenylacetylene
To study a molecular probe synthesis, we performed cop-
per(I)-catalyzed 1,3-dipolar cycloaddition of 38 with phenyl
acetylene (Scheme 5).[26,27] The reaction proceeded smoothly
at room temperature in the presence of CuSO4 and sodium
ascorbate in tBuOH/H2O, thereby leading to triazole 40 in
65% yield without affecting the thiazoline ring. Compound
40 retained potent cytotoxic activity (IC50 0.03 mm; Table 1,
entry 12). As the triazole formed did not affect the biologi-
cal activity, 38 could be a good precursor for the preparation
of an activity-based molecular probe having a detection tag.
Scheme 5. Copper-catalyzed 1,3-dipolar cycloaddition of 38 with phenyl-
acetylene.
toxin (9) was slightly less active than apratoxin A, and the
result is in good agreement with that previously reported by
Ma et al. (entry 3).[8] When the hydroxy group at position 35
was protected by a triethylsilyl group (32, entry 4), and
there was no substituent on the fatty acid moiety (33,
entry 5), there was an evident lack of cytotoxic activity.
Thus, the substituents on the fatty acid moiety are important
in inducing potent activity. Azido-containing analogues 35,
38, and 39 retained the activity (entries 7, 10, and 11),
whereas the potency of 36 and 37 in which the MeAla was
Conclusions
We have demonstrated two routes for the solid-phase syn-
thesis of apratoxin A analogues. In the synthetic route A,
the peptide 25 was prepared by sequential coupling of the
Figure 3. Synthetic apratoxin analogues 32–39.
Chem. Asian J. 2011, 6, 180 – 188
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Table 1. Synthetic analogues of apratoxin A and their cytotoxic activity.
ODS-3, 3 mm, 4.6ꢁ75 mm) with a
linear gradient of solvent I=0.1%
HCOOH in H2O and solvent II=
0.1% HCOOH in CH3CN with a flow
rate of 1.0 mLminꢀ1. Method A: 10%
of solvent II in solvent I (0–1 min),
10–100% of solvent II in solvent I (1–
10 min), 100% of solvent II (10–
12 min) or Method B: 50% of solvent
II in solvent I (0–1 min), 50–100% of
Entry
Compound
Yield[a] [%]
Ratio of diastereomers
Cytotoxicity[b]
IC50 [mM]
1
2
3
4
5
6
7
8
1
53
85:15
0.19
0.19
0.38
34
28
–
0.45
2.1
1.7
0.08
0.35
0.03
34-epi-1[c]
9[c]
32
33
34
35
36
37
38
39
40
25[d]
0[e]
<3[f]
31
47
36
70:30
90:10
70:30
90:10
80:20
solvent II in solvent
I (1–10 min),
100% of solvent II (10–12 min).
9
General procedure for loading of N-
Fmoc protected amino acids to the
trityl SynPhase Lanterns: Trityl alco-
hol SynPhase Lanterns (d-series,
10
11
12
42
0.037 mmolunitꢀ1
) were treated with
[a] Overall yield from polymer-supported 19 through method B. [b] Against HeLa cells. [c] Previously synthe-
sized in our group (Ref. [7b]). [d] Through route A. [e] The desired cyclization precursor was not obtained
after cleavage from the polymer-support. [f] Detected by LC-MS.
10% AcCl in CH2Cl2 at room temper-
ature for 3 h.[12] After completion of
the reaction, the lanterns were washed
with CH2Cl2 (2 minꢁ5). A solution of
Fmoc amino acid (0.074 mmolunitꢀ1
)
amino acids 13–17 on trityl chloride SynPhase Lanterns
(12). After cleavage from the polymer support, macrolac-
tamization of 10 followed by thiazoline formation, provided
apratoxin A (1). Its analogue 33 was similarly synthesized.
This approach, however, resulted in low yield because che-
moselectivity was insufficient in the formation of a thiazo-
line ring. In contrast, in the synthetic route B, the cyclization
precursor 11 was prepared by solid-phase peptide synthesis
by using 13–15, and 18. The final macrolactamization was
performed in solution to provide apratoxin A in high overall
yield. This method was successfully applied to the synthesis
of apratoxin analogues 35–39. The cytotoxic activity of the
synthetic derivatives was then evaluated. The 34-epimer was
as potent as apratoxin A and O-methyl tyrosine could be re-
placed by O-7-azidoheptyl tyrosine with no loss of activity.
The 1,3-dipolar cycloaddition of 38 with phenylacetylene
was performed in the presence of a copper-catalyst without
the thiazoline ring being affected. This method could be ap-
plied to the synthesis of a molecular probe to identify the
target molecule of apratoxin A. Further studies are in prog-
ress in our laboratories.
and DIEA (44 mLunitꢀ1) in CH2Cl2 (1 mLunitꢀ1) at room temperature
was added to the lanterns. After being left for 12 h, the lanterns were
washed with DMF (3 minꢁ3), CH2Cl2 (3 minꢁ3), THF/H2O=4:1
(3 minꢁ3), MeOH (3 minꢁ2), and ether (3 minꢁ2), and then dried in
vacuo to give Fmoc amino acid-supported lanterns.
General procedure for cleavage of polymer-supported peptidic acids: The
peptide supported lantern (1 unit) was treated with HFIPA/CH2Cl2 (3:7).
After being left for 30 min, the lantern was removed and the reaction
mixture was concentrated in vacuo to give the corresponding acid.
General procedure for removal of the N-Fmoc group on the polymer
support: The N-Fmoc protected peptide supported lanterns were treated
with a solution of 20% piperidine or 2,6-dimethylpiperidine in case of 25
in DMF (10 minꢁ3). After the reaction was complete, the lanterns were
washed with DMF (3 minꢁ5) and were then used for the next reaction.
General procedure for acylation with 14–15 and 28–31 on the polymer
support: The amino acid-supported lanterns (0.033 mmolunitꢀ1) were
treated with a solution of Fmoc amino acid 14–15 or 28–31 (0.148 mmo-
lunitꢀ1), PyBroP (0.148 mmolunitꢀ1), and DIEA (0.296 mmolunitꢀ1) in
CH2Cl2/DMF=4:1 (1 mLunitꢀ1). After being left for 12 h, the lanterns
were washed with DMF (3 minꢁ3), and the above coupling reaction was
repeated. After the reaction was complete, the lanterns were washed
with DMF (3 minꢁ3), CH2Cl2 (3 minꢁ3), THF/H2O=4:1 (3 minꢁ3),
MeOH (3 minꢁ2), and ether (3 minꢁ2).
General procedure for acylation with 16 on the polymer support: The
amino acid-supported lanterns (0.033 mmolunitꢀ1) were treated with a
solution of Fmoc-MoCys-OH (16) (0.10 mmolunitꢀ1), DIC (15.5 mL,
0.10 mmolunitꢀ1), HOBt (13.5 mg, 0.10 mmolunitꢀ1), and DIEA
(0.043 mL, 0.25 mmolunitꢀ1) in CH2Cl2/DMF=4:1 (1 mLunitꢀ1). After
being left for 12 h, the lanterns were washed with DMF (3 minꢁ3),
CH2Cl2 (3 minꢁ3), THF/H2O=4:1 (3 minꢁ3), MeOH (3 minꢁ2), and
ether (3 minꢁ2).
Experimental Section
General procedure: NMR spectra were recorded on a JEOL Model
ECP-400 (400 MHz for 1H, 100 MHz for 13C) instrument in the indicated
solvent. The spectra were referenced internally according to residual sol-
vent signals of CD2Cl2 (1H NMR, d 5.3 ppm; 13C NMR, d 53.8 ppm).
Data for 1H NMR were recorded as follows: chemical shift (d, ppm),
multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet,
br.=broad), coupling constant (Hz), and integration. Data for 13C NMR
were recorded in terms of chemical shift (d, ppm). Optical rotations were
measured with a JASCO P-1020 polarimeter. All reactions were moni-
tored by thin-layer chromatography carried out on 0.2 mm E. Merck
silica gel plates (60F-254) with UV light, visualized by p-anisaldehyde so-
lution, ceric sulfate, or 10% ethanolic phosphomolybdic acid. Merck
silica gel was used for column chromatography. ESI-TOF mass spectra
were measured with a Waters LCT PremierTM XE spectrometer. HRMS
(ESI-TOF) were calibrated with leucine enkephalin (Sigma) as an exter-
nal standard. HPLC analysis was performed on a Waters 2695 Separation
Module equipped with Waters 2996 Photodiode Array detector (Inertsil
General procedure for acylation with 17 or 27 on a polymer support
(route A): The amino acid-supported lanterns (0.033 mmolunitꢀ1) were
treated with a solution of Fmoc amino acid 17 or 27 (0.066 mmolunitꢀ1),
HATU (32.0 mg, 0.10 mmolunitꢀ1), and DIEA (0.043 mL, 0.25 mmol
unitꢀ1) in CH2Cl2/DMF=4:1 (1 mLunitꢀ1). After being left for 24 h, the
lanterns were washed with DMF (3 minꢁ3) and CH2Cl2 (3 minꢁ3).
General procedure for acylation with 18 on the polymer support (route
B): The tetrapeptide supported lanterns (0.033 mmolunitꢀ1) were treated
with
a
solution of carboxylic acid 18 (0.0825 mmolunitꢀ1), PyAOP
(0.0825 mmolunitꢀ1), and DIEA (0.165 mmolunitꢀ1) in CH2Cl2/DMF=
1:1 (0.8 mLunitꢀ1). After being left for 24 h, the lanterns were washed
with DMF (3 minꢁ3) and CH2Cl2 (3 minꢁ3).
General procedure for macrolactamization: To a solution of the crude
cyclization precursor in CH2Cl2 (25 mL), DIEA (0.15 mmol) and HATU
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Chem. Asian J. 2011, 6, 180 – 188

Total Synthesis of (ꢀ)-Apratox
(0.050 mmol) were added at 08C under argon. After being stirred at 08C
to room temperature for 24 h, the reaction mixture was concentrated in
vacuo. The residue was used in the next reaction without purification.
J=13.2, 9.3 Hz, 1H), 2.86 (dd, J=13.2, 5.8 Hz, 1H), 2.84 (dd, J=10.7,
10.7 Hz, 1H), 2.79 (s, 3H), 2.77 (m, 1H), 2.57 (s, 3H), 2.20 (m, 1H), 2.00
(m, 1H), 1.91 (d, J=1.4 Hz, 1H), 1.79–1.89 (m, 3H), 1.69 (m, 1H), 1.49
(m, 1H), 1.24–1.38 (m, 4H), 1.19 (d, J=7.3 Hz, 3H), 0.99 (m, 1H), 0.98
(d, J=6.3 Hz, 3H), 0.93 (t, J=7.8 Hz, 9H), 0.92 (d, J=6.8 Hz, 3H), 0.84
(s, 9H), 0.81 (t, J=7.8 Hz, 3H), 0.66 (d, J=6.8 Hz, 3H), 0.58 ppm (q, J=
7.8 Hz, 6H); HRMS (ESI-TOF): m/z (%) calcd for C51H83N5O8SSi:
954.5810 [M+H+]; found: 954.5811.
26: 1H NMR (400 MHz, CD2Cl2: d=7.17–7.38 (m, 15H, Trt), 7.14 (d, J=
8.8 Hz, 2H), 6.91 (d, J=8.3 Hz, 1H), 6.79 (d, J=8.8 Hz, 2H), 6.33 (dq,
J=9.3, 1.0 Hz, 1H), 6.04 (d, J=10.2 Hz, 1H), 5.25 (m, 1H), 5.03 (d, J=
11.2 Hz, 1H), 4.92 (d, J=12.2 Hz, 1H), 4.80–4.89 (m, 3H), 4.65 (q, J=
6.8 Hz, 1H), 4.57 (d, J=12.2 Hz, 1H), 4.48 (dd, J=8.8, 3.9 Hz, 1H), 3.91
(m, 1H), 3.74 (s, 3H), 3.68 (m, 1H), 3.19 (dd, J=13.2, 8.8 Hz, 1H), 2.87
(dd, J=13.2, 6.4 Hz, 1H), 2.73 (dq, J=6.8, 3.4 Hz, 1H), 2.60 (s, 3H), 2.57
(s, 3H), 2.45 (dd, J=11.7, 7.3 Hz, 1H), 2.27 (dd, J=11.7, 5.4 Hz, 1H),
2.22 (m, 1H), 2.05 (m, 1H), 1.76–2.00 (m, 4H), 1.71 (d, J=1.0 Hz, 3H),
1.70 (m, 1H), 1.23–1.55 (m, 4H), 1.06 (d, J=6.8 Hz, 3H), 1.01 (d, J=
6.8 Hz, 3H), 0.97 (d, J=6.8 Hz, 3H), 0.96 (m, 1H), 0.86 (d, J=6.8 Hz,
3H), 0.80 (t, J=7.4 Hz, 3H), 0.78 ppm (s, 9H); HRMS (ESI-TOF): m/z
(%) calcd for C67H86N5O11SCl3: 1274.5188 [M+H+]; found: 1274.5182.
Synthesis of apratoxin A (1), 34-epi-1, and 35–39 (route B): After macro-
lactamization of the corresponding cyclization precursor obtained by the
above procedure, the residue was purified by column chromatography on
silica gel (MeOH in CHCl3) to provide the corresponding macrolactams
as a diastereomeric mixture. When necessary, further purification was
performed by preparative TLC on silica gel (ethyl acetate in n-hexane),
or reversed phase preparative HPLC (CH3CN-H2O) to isolate the diaste-
reomers in pure form.
35 major diastereomer: tR 29.3 min (Inertsil C18 ODS, 3 mm, 4.6ꢁ250 mm,
1.0 mLminꢀ1, UV detection at 220 nm, an isocratic system of 65% aque-
ous CH3CN); HRMS (ESI-TOF): m/z (%) calcd for C45H68N8O8S:
881.4959 [M+H+]; found: 881.4958.
Synthesis of apratoxin A (1) and 33 (route A): To a solution of 26 or a re-
lated compound composed of 27 instead of 17 (0.010 mmol) and triphe-
nylphosphine oxide (16.5 mg, 0.060 mmol) in CH2Cl2 (0.4 mL), Tf2O
(5.0 mL, 0.030 mmol) was added dropwise at 08C under argon. After
being stirred at the same temperature for 2 h, the reaction mixture was
quenched with saturated aqueous NaHCO3 at 08C, and the aqueous
layer was extracted with ethyl acetate. The organic layers were combined
and washed with brine, then dried over Na2SO4, and concentrated in
vacuo. The residue was used for the next reaction without further purifi-
cation. To the solution of crude thiazoline in THF (0.4 mL) and aqueous
NH4OAc (1.0m, 0.1 mL) zinc dust (10 mg, 0.15 mmol) was added at room
temperature. After being stirred at room temperature for 30 min, the re-
action mixture was partitioned between ethyl acetate and brine. The solu-
tion was extracted five times with ethyl acetate. The organic layers were
combined, dried over Na2SO4, and concentrated in vacuo. The residue
was purified by column chromatography with silica gel (1 to 4% MeOH
in CHCl3) and preparative TLC on silica gel (90% ethyl acetate in
hexane) to give 1 (0.9 mg, 0.001 mmol, 10% in 2 steps). Spectral data of
1 were identical to those previously reported. 33 was obtained according
to the above procedure but without treatment with Zn/NH4OAc. 33:
½aꢁ2D2 =ꢀ159 (c=0.705, MeOH); IR (neat): n˜ =3431, 2932, 1736, 1639,
36 major diastereomer: tR 9.13 min (method B); ½aꢁ2D3 =ꢀ169 (c=0.130,
MeOH); 1H NMR (400 MHz, CD2Cl2): d=7.14 (d, J=8.8 Hz, 2H), 6.83
(d, J=8.8 Hz, 2H), 6.23 (dq, J=9.3, 1.0 Hz, 1H), 5.93 (d, J=9.2 Hz, 1H),
5.22 (m, 1H), 5.11 (d, J=11.7 Hz, 1H), 5.07 (m, 1H), 4.93 (dd, J=13.7,
2.9 Hz, 1H), 4.46 (d, J=11.2 Hz, 1H), 4.16 (t, J=7.8 Hz, 1H), 4.10 (m,
1H), 3.97 (dd, J=12.2, 5.4 Hz, 1H), 3.75 (s, 3H), 3.44–3.61 (m, 2H), 3.42
(dd, J=10.7, 8.8 Hz, 1H), 3.20–3.36 (m, 2H), 3.02–3.08 (m, 2H), 2.98 (s,
3H), 2.89 (dd, J=13.2, 5.9 Hz, 1H), 2.65 (s, 3H), 2.57 (m, 1H), 2.00–2.26
(m, 4H), 1.91 (d, J=1.0 Hz, 3H), 1.72–1.90 (m, 3H), 1.50 (m, 1H), 1.22–
1.32 (m, 2H), 1.09 (m, 1H), 1.03 (d, J=6.8 Hz, 3H), 0.93 (d, J=6.8 Hz,
3H), 0.90 (m, 1H), 0.85 (d, J=6.8 Hz, 3H), 0.85 (s, 9H), 0.81 ppm (t, J=
6.8 Hz, 3H); HRMS (ESI-TOF): m/z (%) calcd for C45H68N8O8S:
881.4959 [M+H+]; found: 881.4965.
37 major diastereomer: tR 9.53 min (method B); ½aꢁ2D3 =ꢀ150 (c 0.10,
MeOH); 1H NMR (400 MHz, CD2Cl2): d=7.16 (d, J=8.8 Hz, 2H), 6.80
(d, J=8.8 Hz, 2H), 6.22 (dq, J=9.8, 1.0 Hz, 1H), 5.93 (d, J=9.3 Hz, 1H),
5.21 (m, 1H), 5.12 (d, J=11.7 Hz, 1H), 5.07 (m, 1H), 4.93 (dd, J=12.2,
1.5 Hz, 1H), 4.48 (d, J=11.2 Hz, 1H), 4.15 (t, J=7.8 Hz, 1H), 4.12 (m,
1H), 3.75 (s, 3H), 3.43–3.63 (m, 3H), 3.43 (dd, J=10.7, 8.3 Hz, 1H),
2.98–3.20 (m, 4H), 2.96 (s, 3H), 2.89 (dd, J=13.2, 5.4 Hz, 1H), 2.64 (s,
3H), 2.57 (m, 1H), 2.00–2.28 (m, 4H), 1.92 (d, J=1.0 Hz, 3H), 0.82–1.90
(m, 14H), 1.03 (d, J=6.8 Hz, 3H), 0.94 (d, J=6.3 Hz, 3H), 0.86 (d, J=
6.8 Hz, 3H), 0.85 (s, 9H), 0.81 ppm (t, J=6.8 Hz, 3H); HRMS (ESI-
TOF): m/z (%) calcd for C48H74N8O8S: 923.5429 [M+H+]; found:
923.5422.
38 major diastereomer: tR 11.0 min (method A); ½aꢁ2D5 =ꢀ162 (c=0.150,
MeOH); 1H NMR (400 MHz, CD2Cl2): d=7.11 (d, J=8.8 Hz, 2H), 6.78
(d, J=8.8 Hz, 2H), 6.21 (dq, J=9.8, 1.0 Hz, 1H), 5.98 (d, J=8.3 Hz, 1H),
5.23 (m, 1H), 5.12 (d, J=11.7 Hz, 1H), 5.00 (m, 1H), 4.93 (dd, J=12.2,
1.5 Hz, 1H), 4.49 (d, J=11.2 Hz, 1H), 4.11–4.16 (m, 2H), 3.90 (t, J=
6.8 Hz, 2H), 3.42–3.62 (m, 3H), 3.26 (m, 1H), 3.24 (t, J=6.8 Hz, 2H),
2.99–3.10 (m, 2H), 2.86 (dd, J=13.2, 5.4 Hz, 1H), 2.79 (s, 3H), 2.62 (s,
3H), 2.56 (m, 1H), 1.97–2.28 (m, 4H), 1.92 (d, J=1.0 Hz, 3H), 1.70–1.90
(m, 5H), 1.57 (m, 2H), 1.32–1.62 (m, 7H), 1.19–1.29 (m, 2H), 1.11 (d, J=
6.8 Hz, 3H), 1.05 (m, 1H), 1.03 (d, J=6.8 Hz, 3H), 0.94 (d, J=6.8 Hz,
3H), 0.89 (m, 1H), 0.86 (d, J=6.8 Hz, 3H), 0.85 (s, 9H), 0.82 ppm (t, J=
7.3 Hz, 3H); HRMS (ESI-TOF): m/z (%) calcd for C51H80N8O8S:
965.5898 [M+H+]; found: 965.5900.
39 major diastereomer: tR 10.8 min (method A); ½aꢁ2D4 =ꢀ134 (c=0.110,
MeOH); 1H NMR (400 MHz, CD2Cl2): d=7.13 (d, J=8.8 Hz, 2H), 6.81
(d, J=8.8 Hz, 2H), 6.20 (dq, J=8.8, 1.0 Hz, 1H), 5.98 (d, J=9.8 Hz, 1H),
5.21 (m, 1H), 5.12 (d, J=11.7 Hz, 1H), 5.01 (m, 1H), 4.93 (dd, J=12.2,
2.0 Hz, 1H), 4.48 (d, J=11.2 Hz, 1H), 4.12–4.16 (m, 2H), 4.05 (brt, J=
4.6 Hz, 2H), 3.79 (brt, J=4.6 Hz, 2H), 3.58–3.66 (m, 7H), 3.48 (m, 1H),
3.42 (dd, J=11.2, 3.9 Hz, 1H), 3.35 (t, J=5.4 Hz, 2H), 3.25 (brs, 1H),
3.07 (dd, J=11.2, 3.9 Hz, 1H), 3.04 (dd, J=12.2, 11.2 Hz, 1H), 2.86 (dd,
J=12.2, 4.9 Hz, 1H), 2.79 (s, 3H), 2.62 (s, 3H), 2.60 (m, 1H), 2.00–2.24
(m, 4H), 1.92 (d, J=1.0 Hz, 3H), 1.75–1.91 (m, 3H), 1.50 (m, 1H), 1.23–
1512, 1442, 1247, 1177 cmꢀ1 1H NMR (400 MHz, CD2Cl2, major rota-
;
mer): d=7.13 (d, J=8.8 Hz, 2H), 6.81 (d, J=8.8 Hz, 2H), 6.47 (dq, J=
9.8 Hz, 1.0 Hz, 1H), 6.35 (d, J=9.8 Hz, 1H), 5.36 (ddd, J=9.8, 9.3,
5.4 Hz, 1H), 5.25 (ddd, J=9.8, 8.8, 4.9 Hz, 1H), 4.92 (d, J=11.7 Hz, 1H),
4.81 (q, J=6.3 Hz, 1H), 4.34 (m, 1H), 4.22 (m, 1H), 4.04 (m, 1H), 3.93
(m, 1H), 3.75 (s, 3H), 3.59 (m, 1H), 3.47 (dd, J=11.2, 8.8 Hz, 1H), 3.15
(dd, J=12.7, 9.3 Hz, 1H), 3.10 (dd, J=11.2, 4.9 Hz, 1H), 2.89 (dd, J=
12.7, 5.4 Hz, 1H), 2.77 (s, 3H), 2.59 (s, 3H), 2.54 (ddd, J=15.6, 9.8,
5.9 Hz, 1H), 2.41 (ddd, J=15.6, 8.8, 4.9 Hz, 1H), 2.18 (m, 1H), 1.84–2.09
(m, 4H), 1.94 (d, J=1.0 Hz, 3H), 1.26–1.71 (m, 9H), 0.98 (m, 1H), 0.95
(d, J=6.8 Hz, 3H), 0.83 (t, J=7.8 Hz, 3H), 0.71 ppm (d, J=6.3 Hz, 3H);
13C NMR (100 MHz, CD2Cl2, major rotamer): d=172.3, 172.2, 171.9,
170.0, 169.7, 166.6, 158.8, 137.6, 130.6, 128.9, 128.7, 114.0, 73.4, 64.3, 59.6,
57.7, 55.3, 54.6, 50.0, 47.5, 39.5, 38.7, 34.1, 29.8, 29.6, 29.4, 28.8, 28.5, 27.7,
25.5, 25.4, 25.3, 25.1, 15.6, 14.1, 12.7, 9.9 ppm; HRMS (ESI-TOF): m/z
(%) calcd for C39H57N5O7S: 740.4057 [M+H+]; found: 740.4061.
35-O-Triethylsilyl apratoxin A (32): To a solution of apratoxin A (1)
(2.3 mg, 2.7 mmol) in CH2Cl2 (0.4 mL), 2,6-lutidine (3.2 mL, 27.4 mmol)
and TESOTf (3.1 mL, 13.7 mmol) were added at ꢀ508C under argon.
After being stirred at the same temperature for 30 min, the reaction mix-
ture was quenched with methanol at ꢀ508C, and diluted with ethyl ace-
tate. The organic layers were combined and washed first with saturated
aqueous NaHCO3, then with brine, and then dried over Na2SO4, and con-
centrated in vacuo. The residue was purified twice by preparative TLC
on silica gel (50% ethyl acetate in hexane) to give 35-O-triethylsilyl apra-
toxin A (32) (1.3 mg, 1.4 mmol, 50%). 1H NMR (400 MHz, CD2Cl2): d=
7.11 (d, J=8.3 Hz, 2H), 6.80 (d, J=8.3 Hz, 2H), 6.63 (dq, J=9.3, 1.4 Hz,
1H), 6.34 (d, J=9.8 Hz, 1H), 5.39 (m, 1H), 5.06 (ddd, J=10.7, 9.3,
8.8 Hz, 1H), 4.88 (d, J=11.2 Hz, 1H), 4.87 (q, J=6.8 Hz, 1H), 4.71 (dd,
J=6.4, 3.9 Hz, 1H), 4.18 (m, 1H), 4.11 (dd, J=8.3, 7.8 Hz, 1H), 3.99 (m,
1H), 3.74 (s, 3H), 3.51 (m, 1H), 3.33 (dd, J=10.7, 8.8 Hz, 1H), 3.13 (dd,
Chem. Asian J. 2011, 6, 180 – 188
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
187

T. Doi et al.
FULL PAPERS
1.29 (m, 2H), 1.12 (d, J=6.4 Hz, 3H), 1.03 (m, 1H), 1.02 (d, J=7.3 Hz,
3H), 0.94 (d, J=6.8 Hz, 3H), 0.86 (d, J=6.8 Hz, 3H), 0.85 (s, 9H), 0.84
(m, 1H), 0.82 ppm (t, J=7.3 Hz, 3H); HRMS (ESI-TOF): m/z (%) calcd
for C50H78N8O10S: 983.5640 [M+H+]; found: 983.5640.
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Formation of triazole 40 by 1,3-dipolar cycloaddition of 38 with phenyl
acetylene: To a solution of apratoxin A derivative 38 (0.7 mg, 0.7 mmol)
and phenylacetylene (1.0 mL, 3.6 mmol) in 2-methyl-2-propanol (0.1 mL)
and H2O (0.1 mL), 1m sodium ascorbate (1.0 mL, 1.0 mmol) and 0.5m
CuSO4 (1.0 mL, 0.5 mmol) was added, respectively, at room temperature
under argon. After being stirred at room temperature for 3 h, the reac-
tion mixture was quenched with saturated aqueous NaHCO3, and the
aqueous layer was extracted with ethyl acetate. The organic layers were
combined and washed with brine, then dried over Na2SO4, and concen-
trated in vacuo. The residue was purified twice by preparative TLC on
silica gel (90% EtOAc in hexane) to afford triazole 40 (0.5 mg,
0.46 mmol, 65%). tR 10.2 min (method B); ½aꢁ2D4 =ꢀ145 (c=0.070,
MeOH); 1H NMR (400 MHz, CD2Cl2): d=7.80 (dd, J=6.8, 1.5 Hz, 2H),
7.77 (s, 1H), 7.41 (dd, J=6.8, 6.8 Hz, 2H), 7.31 (m, 1H), 7.11 (d, J=
8.8 Hz, 2H), 6.77 (d, J=8.8 Hz, 2H), 6.20 (dq, J=9.3, 1.0 Hz, 1H), 5.97
(d, J=9.8 Hz, 1H), 5.21 (m, 1H), 5.12 (d, J=11.2 Hz, 1H), 5.00 (m, 1H),
4.93 (dd, J=12.2, 2.0 Hz, 1H), 4.48 (d, J=10.8 Hz, 1H), 4.37 (t, J=
7.3 Hz, 2H), 4.12–4.16 (m, 2H), 3.89 (t, J=6.4 Hz, 2H), 3.43–3.62 (m,
2H), 3.42 (dd, J=11.2, 8.8 Hz, 1H), 3.25 (brs, 1H), 3.06 (dd, J=11.2,
3.9 Hz, 1H), 3.02 (dd, J=13.6, 11.2 Hz, 1H), 2.85 (dd, J=13.6, 5.4 Hz,
1H), 2.78 (s, 3H), 2.62 (s, 3H), 2.57 (m, 1H), 1.98–2.25 (m, 4H), 1.92 (m,
2H), 1.91 (d, J=1.0 Hz, 3H), 1.80–1.90 (m, 2H), 1.68–1.77 (m, 3H),
1.20–1.52 (m, 9H), 1.11 (d, J=6.8 Hz, 3H), 1.04 (m, 1H), 1.03 (d, J=
6.8 Hz, 3H), 0.94 (d, J=6.4 Hz, 3H), 0.87 (m, 1H), 0.86 (d, J=6.8 Hz,
3H), 0.85 (s, 9H), 0.82 ppm (t, J=7.4 Hz, 3H).
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thoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoli-
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well plates were seeded with aliquots of a 20 mL medium containing 1.0ꢁ
103 cells per well and were incubated overnight before being treated with
compounds at various concentrations for 48 h. Plates were incubated for
1 h at 378C after the addition of 2 mL WST-8 reagent solution (Cell
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formazan dye was measured at 450 nm. The vehicle solvent (DMSO) was
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Acknowledgements
[24] Based on the above result, the IC50 value of synthetic apratoxin A
measured in our hands was not as small as that previously reported.
In our collaborative study with Professor Gerwickꢃs group, however,
it was found that synthetic apratoxin A exhibited the same potency
as the natural product in vitro. Furthermore, the synthetic one had a
significant in vivo antitumor effect in conventional mice bearing syn-
geneic solid tumors. See Ref. 5.
This work was supported by a Grant-in-Aid from the Ministry of Educa-
tion, Culture, Sports, Science and Technology, Japan (No.14103013), and
the Naito foundation. We thank the JSPS Research Fellowships for
Young Scientists (Y. N.).
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Received: August 9, 2010
Published online: November 15, 2010
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