Total syntheses, fragmentation studies, and antitumor/antiproliferative activities of FR901464 and its low picomolar analogue

Article abstract of DOI:10.1021/ja067870m

FR901464 is a potent anticancer natural product that lowers the mRNA levels of oncogenes and tumor suppressor genes. In this article, we report a convergent enantioselective synthesis of FR901464, which was accomplished in 13 linear steps. Central to the synthetic approach was the diene-ene cross olefin metathesis reaction to generate the C6-C7 olefin without the use of protecting groups as the final step. Additional key reactions include a Zr/Ag-promoted alkynylation to set the C4 Stereocenter, a mild and chemoselective Red-Al reduction, a reagent-controlled stereoselective Mislow-Evans-type [2,3]-sigmatropic rearrangement to install the C5 Stereocenter, a Carreira asymmetric alkynylation to generate the C4′ stereocenter, and a highly efficient ring-closing metathesis-allylic oxidation sequence to form an unsaturated lactone. The decomposition pathways of FR901464's right fragment were studied under physiologically relevant conditions. Facile epoxide opening by β-elimination gave two enones, one of which could undergo dehydration via its hemiketal to form a furan. To prevent this decomposition pathway, a right fragment was rationally designed and synthesized. This analogue was 12 times more stable than the right fragment of the natural product. Using this more stable right fragment analogue, an FR901464 analogue, meayamycin, was prepared in 13 linear steps. The inhibitions of human breast cancer MCF-7 cell proliferation by synthetic FR901464 and meayamycin were studied, and the GI50 values for these compounds were determined to be 1.1 nM and 10 pM, respectively. Thus, meayamycin is among the most potent anticancer small molecules that do not bind to either DNA or microtubule.

Full text of DOI:10.1021/ja067870m

Published on Web 02/06/2007
Total Syntheses, Fragmentation Studies, and Antitumor/
Antiproliferative Activities of FR901464 and Its Low
Picomolar Analogue
Brian J. Albert, Ananthapadmanabhan Sivaramakrishnan, Tadaatsu Naka,
Nancy L. Czaicki, and Kazunori Koide*
Contribution from the Department of Chemistry, UniVersity of Pittsburgh, 219 Parkman AVenue,
Pittsburgh, PennsylVania 15260
Received November 3, 2006; E-mail: koide@pitt.edu
Abstract: FR901464 is a potent anticancer natural product that lowers the mRNA levels of oncogenes
and tumor suppressor genes. In this article, we report a convergent enantioselective synthesis of FR901464,
which was accomplished in 13 linear steps. Central to the synthetic approach was the diene-ene cross
olefin metathesis reaction to generate the C6-C7 olefin without the use of protecting groups as the final
step. Additional key reactions include a Zr/Ag-promoted alkynylation to set the C4 stereocenter, a mild and
chemoselective Red-Al reduction, a reagent-controlled stereoselective Mislow-Evans-type [2,3]-sigmatropic
rearrangement to install the C5 stereocenter, a Carreira asymmetric alkynylation to generate the C4′
stereocenter, and a highly efficient ring-closing metathesis-allylic oxidation sequence to form an unsaturated
lactone. The decomposition pathways of FR901464’s right fragment were studied under physiologically
relevant conditions. Facile epoxide opening by â-elimination gave two enones, one of which could undergo
dehydration via its hemiketal to form a furan. To prevent this decomposition pathway, a right fragment was
rationally designed and synthesized. This analogue was 12 times more stable than the right fragment of
the natural product. Using this more stable right fragment analogue, an FR901464 analogue, meayamycin,
was prepared in 13 linear steps. The inhibitions of human breast cancer MCF-7 cell proliferation by synthetic
FR901464 and meayamycin were studied, and the GI50 values for these compounds were determined to
be 1.1 nM and 10 pM, respectively. Thus, meayamycin is among the most potent anticancer small molecules
that do not bind to either DNA or microtubule.
promoter10 upstream of a reporter gene, which was stably
transfected. Through their screening efforts, structurally unique
Introduction
Controlled cellular processes such as signal transduction and
gene expression are essential for homeostasis. When these
processes become irregular, tumors may develop. Tumorigenesis
can be accounted for by approximately 200 molecular mecha-
nisms,1 many of which can in principle be targeted by drugs.
However, currently available chemotherapeutic methods are
FR901464 was isolated from the culture broth of a bacterium
of Pseudomonas sp. No. 2663, which activated the aforemen-
tioned reporter gene by 30-fold at 20 nM concentration in
MCF-7 cells.11-13 The same group revealed that FR901464
possessed antitumor activity against MCF-7 cells, human lung
adenocarcinoma A549 cells, colon cancer SW480 and HCT116
cells, and murine leukemia P388 cells with an IC50 of 0.6-3.4
nM.11 This natural product also exhibited a prominent effect at
0.056-0.18 mg/kg against human adenocarcinoma A549 and
0.18-1 mg/kg against murine Colon 38 and Meth A cells
implanted in mice, indicating that FR901464 or its structurally
related analogues have the potential for clinical application.13
limited to targeting DNA,2,3 nuclear hormone receptors,4
a
tyrosine kinase,5 a proteasome,6,7 and the microtubule.8,9 Clearly,
there is an urgent need to fill the gap between the molecular
mechanisms of tumorigenesis and available pharmacological
approaches.
In the quest for anticancer natural products possessing new
modes of action, the Nakajima group at the Fujisawa Pharma-
ceutical Company employed a reporter assay in human breast
adenocarcinoma MCF-7 cells using the simian virus 40 (SV40)
(7) Adams, J. Cancer Cell 2004, 5, 417-421.
(8) Jordan, M. A.; Wilson, L. Nat. ReV. Cancer 2004, 4, 253-265.
(9) Zhou, J.; Giannakakou, P. Curr. Med. Chem. Anti-Cancer Agents 2005, 5,
65-71.
(1) Service, R. F. Science 2005, 310, 1132-1134.
(2) Jamieson, E. R.; Lippard, S. J. Chem. ReV. 1999, 99, 2467-2498.
(3) Burger, R. M. Chem. ReV. 1998, 98, 1153-1169.
(4) Clarke, R.; Leonessa, F.; Welch, J. N.; Skaar, T. C. Pharmacol. ReV. 2001,
53, 25-71.
(5) Druker, B. J. Trends Mol. Med. 2002, 8, S14-S18.
(6) Rajkumar, S. V.; Richardson, P. G.; Hideshima, T.; Anderson, K. C.
J. Clin. Oncol. 2005, 23, 630-639.
(10) Jones, N. C.; Rigby, P. W. J.; Ziff, E. B. Genes DeV. 1988, 2, 267-281.
(11) Nakajima, H.; Sato, B.; Fujita, T.; Takase, S.; Terano, H.; Okuhara, M.
J. Antibiot. 1996, 49, 1196-1203.
(12) Nakajima, H.; Takase, S.; Terano, H.; Tanaka, H. J. Antibiot. 1997, 50,
96-99.
(13) Nakajima, H.; Hori, Y.; Terano, H.; Okuhara, M.; Manda, T.; Matsumoto,
S.; Shimomura, K. J. Antibiot. 1996, 49, 1204-1211.
9
2648
J. AM. CHEM. SOC. 2007, 129, 2648-2659
10.1021/ja067870m CCC: $37.00 © 2007 American Chemical Society

Synthesis and Antitumor Activity of FR901464
A R T I C L E S
Scheme 1. First Generation Retrosynthetic Analysis of FR901464
FR901464 induced both G1 and G2/M phase arrest in MCF-7
cells.13,14 In contrast, adriamycin and camptothecin, both DNA
synthesis inhibitors, induced S-phase arrest in the cell cycle,
and Taxol, a microtubule modulator, induced G2/M-phase
arrest.13 Since FR901464 was originally discovered as an
activator of SV40 promoter, the Nakajima group sought
endogenous genes that were upregulated by the natural product.
Their focused approach, prior to the invention of DNA mi-
croarray,15 showed that the mRNA levels of p53,16,17 p21 Cip-
1,18,19 E2F-1,20,21 and c-Myc22,23 were downregulated while a
housekeeping gene was intact. This result is both exciting and
perplexing since p53 is a well-known tumor suppressor gene
and c-Myc is an oncogene. These findings reported by the
Nakajima group and our subsequent findings strongly suggest
that the mode of action of FR901464 is different from that of
clinically used anticancer drugs. After the discovery of FR901464,
other pharmaceutical companies used the same SV40 promoter
in reporter gene systems and found that the previously known
natural product herboxidiene (aka. GEX1)24-27 and the new
natural product TMC-20528 exhibited closely related biological
activities as FR901464, although these compounds were less
potent.
The unique profile of FR901464 has intrigued many synthetic
chemists,29 which culminated in the first total synthesis of this
natural product by the Jacobsen group30,31 and the second and
third by the Kitahara group.32,33 The Jacobsen synthesis elegantly
demonstrated the power of their asymmetric hetero-Diels-Alder
reaction34 in their total synthesis, which consisted of 19 steps
in the longest linear sequence and a total of 40 steps.
revealed inefficient installation of the spiroepoxide at late stages
and the latent instability of FR901464. Clearly, a more versatile
and concise approach was desired for synthetic accessibility to
analogues of FR901464 for biological studies. Moreover,
quantitative analysis of the instability of FR901464 was needed
as part of our efforts to understand the mode of action of this
natural product. This full account describes our synthetic efforts
that eventually allowed us to develop a remarkably potent
FR901464 analogue via earlier and potentially risky installation
of the spiroepoxide and ultimate convergency, namely the cross-
coupling at the very end of the synthesis.36
The subsequent total synthesis from the Kitahara group took
advantage of the chiral pool to assemble their fragments. This
synthesis required 42 total steps with the longest linear sequence
being 24 steps.35 The third total synthesis by the same group
was the improvement of their earlier version with 22 steps in
the longest linear sequence and 41 total steps.33 These syntheses
Results and Discussion
First Generation Synthetic Studies. Scheme 1 shows our
first generation retrosynthetic analysis of FR901464. We
envisioned a Nozaki-Hiyama-Kishi (NHK) reaction37,38 of 1
and 2 as the final coupling reaction because the reaction
conditions are mild and it is generally in accordance with Felkin
selectivity for additions to R-chiral aldehydes. Vinyl iodide 1
would be prepared from acid 3 and amine 4 that were expected
to be derived from propargylic alcohol 6 and the L-threonine
derivative 7, respectively. Ketoaldehyde 2 could be prepared
from allylic alcohol 5 by oxidative cleavages of both olefins.
Finally, this alcohol would arise from epoxyalcohol 8.
(14) Nakajima, H.; Hori, Y.; Terano, H.; Okuhara, M.; Manda, T.; Matsumoto,
S.; Shimomura, K. J. Antibiot. 1996, 49, 1204-1211.
(15) Schena, M.; Shalon, D.; Davies, R. W.; Brown, P. O. Science 1995, 270,
467-470.
(16) Resnick-Silverman, L.; Manfredi, J. J. J. Cell. Biochem. 2006, 99, 679-
689.
(17) Braithwaite, A. W.; Prives, C. L. Cell Death Differ. 2006, 13, 877-880.
(18) Kumar, R.; Gururaj, A. E.; Barnes, C. J. Nat. ReV. Cancer 2006, 6, 459-
471.
(19) Gartel, A. L.; Radhakrishnan, S. K. Cancer Res. 2005, 65, 3980-3985.
(20) Stevens, C.; La Thangue, N. B. DNA Repair 2004, 3, 1071-1079.
(21) Bell, L. A.; Ryan, K. M. Cell Death Differ. 2004, 11, 137-142.
(22) Dominguez-Sola, D.; Dalla-Favera, R. Nat. Cell Biol. 2004, 6, 288-289.
(23) Janz, S. Oncogene 2005, 24, 3541-3543.
(24) Sakai, Y.; Yoshida, T.; Ochiai, K.; Uosaki, Y.; Saitoh, Y.; Tanaka, F.;
Akiyama, T.; Akinaga, S.; Mizukami, T. J. Antibiot. 2002, 55, 855-862.
(25) Isaac, B. G.; Ayer, S. W.; Elliott, R. C.; Stonard, R. J. J. Org. Chem. 1992,
57, 7220-7226.
We envisioned that the first milestone of our synthetic studies
would be the preparation of ketoaldehyde 2 (Scheme 2). The
first step toward this end, a zinc-mediated coupling of propargyl
alcohol and methallyl bromide39 afforded alcohol 9 in 93% yield.
The subsequent Sharpless asymmetric epoxidation40 proceeded
smoothly to generate epoxyalcohol 8 in 90% yield with >97:3
er. The volatile aldehyde 10 was obtained by the Dess-Martin
(26) Millerwideman, M.; Makkar, N.; Tran, M.; Isaac, B.; Biest, N.; Stonard,
R. J. Antibiot. 1992, 45, 914-921.
(27) Sakai, Y.; Tsujita, T.; Akiyama, T.; Yoshida, T.; Mizukami, T.; Akinaga,
S.; Horinouchi, S.; Yoshida, M.; Yoshida, T. J. Antibiot. 2002, 55, 863-
872.
(28) Sakurai, M.; Kohno, J.; Nishio, M.; Yamamoto, K.; Okuda, T.; Kawano,
K.; Nakanishi, N. J. Antibiot. 2001, 54, 628-634.
(29) Koide, K.; Albert, B. J. Yuki Gosei Kagaku Kyokaishi 2007, 65, 119-126.
(30) Thompson, C. F.; Jamison, T. F.; Jacobsen, E. N. J. Am. Chem. Soc. 2001,
123, 9974-9983.
(31) Thompson, C. F.; Jamison, T. F.; Jacobsen, E. N. J. Am. Chem. Soc. 2000,
122, 10482-10483.
(36) Albert, B. J.; Sivaramakrishnan, A.; Naka, T.; Koide, K. J. Am. Chem.
Soc. 2006, 128, 2792-2793.
(32) Horigome, M.; Motoyoshi, H.; Watanabe, H.; Kitahara, T. Tetrahedron
Lett. 2001, 42, 8207-8210.
(37) Haolun, J.; Uenishi, J.; Christ, W. J.; Kishi, Y. J. Am. Chem. Soc. 1986,
108, 5644-5646.
(33) Motoyoshi, H.; Horigome, M.; Watanabe, H.; Kitahara, T. Tetrahedron
2006, 62, 1378-1389.
(38) Takai, K.; Kimura, K.; Kuroda, T.; Hiyama, T.; Nozaki, H. Tetrahedron
Lett. 1983, 24, 5281-5284.
(34) Dossetter, A. G.; Jamison, T. F.; Jacobsen, E. N. Angew. Chem., Int. Ed.
1999, 38, 2398-2400.
(39) Frangin, Y.; Gaudemar, M. J. Organomet. Chem. 1977, 142, 9-22.
(40) Katsuki, T.; Sharpless, K. B. J. Am. Chem. Soc. 1980, 102, 5974-5976.
(35) Starting from the commercially available compound 32.
9
J. AM. CHEM. SOC. VOL. 129, NO. 9, 2007 2649

A R T I C L E S
Albert et al.
Scheme 2. Preparation of Ketoaldehyde 2a
a Conditions: (a) propargyl alcohol (1.1 equiv), allyl bromide (1.1 equiv), zinc dust (3.1 equiv); then methallyl bromide (1.0 equiv), THF, 23 °C, 93%;
t
(b) (+)-DIPT (9 mol %), Ti(OiPr)4 (8 mol %), BuOOH (1.9-2.2 equiv), CH2Cl2, -20 °C, 90%, er > 97:3; (c) Dess-Martin periodinane (1.5 equiv),
CH2Cl2, 0f23 °C, 81%; (d) alkyne 11 or 12 (1.3 equiv), Zn(OTf)2 (1.2 equiv), (-)-N-methylephedrine (1.3 equiv), Et3N (1.3 equiv), toluene, 23f40 °C,
22 h; (e) see Table 1; (f) 4-O2NPhCO2H (3.0 equiv), diisopropyl azodicarboxylate (2.9 equiv), Ph3P (3.0 equiv), THF, 0f23 °C, 85%; (g) K2CO3 (2.5
equiv), MeOH, 0 °C, 88%; (h) TESCl (1.1 equiv), imidazole (1.4 equiv), THF, 0 °C, quant.; (i) OsO4 (0.05 equiv), NMO (1.0 equiv), THF, 0f23 °C, 58%;
then NaIO4 (1.0), THF/H2O (1:1), 23 °C, quant.; (j) O3, CH2Cl2/MeOH (1:1), -78 °C; then Me2S (10 equiv), -78f23 °C, 54-82%.
Table 1. Step e in Scheme 2: Optimization for the Preparation of
Allylic Alcohols 5 and 4-epi-5
combined 46% yield (entry 6). Alkynylation of 10 with 18 (3.5
equiv) at -78 °C (entry 7) gave an inseparable mixture of
diastereomers in 32% yield and low stereoselectivity (dr ) 1.7
:1), and higher reaction temperatures caused Payne rearrange-
ments to occur. Partial hydrogenation of these propargylic
alcohols gave 5 and 4-epi-5 in a combined yield of 93%, which
enabled us to confirm the structures. After these and other
attempts, high levels of stereocontrol in this transformation
remained elusive.
isolated
yielda (%)
entry
conditions
solvents
5:4-epi-5b
1
2
3
4
16
THF
THF
THF
THF
THF/hexane (1:1)
THF
THF
51
71
42
42
55
46
32
1.0:1
0.3:1
1.2:1
0.8:1
2.0:1
1.2:1
1.7:1
16/ZnCl2 (2:1)
16/ZnCl2 (1:1)
16/ZnCl2 (1:2)
16/ZnCl2 (1:1)
17 + nBuLi
18
5
6
7a,c
At this point, since our priority was to examine the validity
of the final coupling, we proceeded to prepare ketoaldehyde 2
with the result shown in entry 5, Table 1. To improve the overall
yield for the formation of 5, the undesired 4-epi-5 was subjected
to Mitsunobu conditions using 4-nitrobenzoic acid to invert the
C4 stereocenter, and subsequent methanolysis of the resulting
ester 13 with K2CO3 afforded alcohol 5 in 75% yield for the
two steps. The resulting hydroxy group was protected as its
TES ether, 14, in quantitative yield. This compound was
subjected to oxidative cleavage conditions (OsO4, NMO; then
NaIO4) to afford enone 15 in 58% yield. Subsequent ozonolysis
produced the unstable ketoaldehyde 2 in 54-82% yield. It
should be mentioned that the ozonolysis of diene 14 to
ketoaldehyde 2 was low yielding (<10%). Although this
synthetic route allowed us to prepare 2 in a concise manner,
we soon realized that chromatographic separation of alcohols
5 and 4-epi-5 in a large scale was a daunting task.
a Combined yield. b Determined by 1H NMR analysis c Result after
partial hydrogenation with H2 and Lindlar’s catalyst
oxidation41 of this alcohol in 81% yield. All of these steps could
be executed in large scales (>20 g) without compromising
yields.
Our next objective was to develop a stereoselective carbon-
nucleophile addition to this aldehyde. Step d illustrates the two
failed diastereomeric alkynylation reactions of aldehyde 10 using
11 and 12 as latent nucleophiles. We presumed that Carreira’s
asymmetric alkynylation conditions42 could be sufficiently mild
to be compatible with aldehyde 10. To our disappointment, this
aldehyde did not react at ambient temperature and decomposed
at 40 °C.
In light of the above failure, we turned to substrate control
(step e; Table 1). Vinylation of aldehyde 10 using 16 in THF
(entry 1) gave a 1:1 mixture of the desired alcohol 5 and the
undesired alcohol 4-epi-5 in a combined 51% yield. The absolute
configuration of these allylic alcohols was determined after
further transformations as shown in Scheme 11. The addition
of ZnCl2 with various stoichiometry did not improve the
selectivity for the desired alcohol 5 (entries 2-4). Addition of
hexanes finally improved the stereoselectivity for the desired
alcohol (2:1) in a 55% combined yield (entry 5). Treatment of
10 with vinyllithium, prepared in situ from 17 (1.4 equiv) and
nBuLi (1.3 equiv),43 gave a 1.2:1 mixture of 5 to 4-epi-5 in a
With an established synthetic route to ketoaldehyde 2, we
still desired to improve the C4 stereoselectivity (Scheme 3).
Treatment of epoxyaldehyde 10 with methyl propiolate and
nBuLi formed alcohol 19 and its C4-epimer in a 50% combined
yield but with no diastereoselectivity (dr ) 1:1). In contrast,
the alkynylation method developed in our laboratory afforded
alcohol 19 in 84% with good stereocontrol (dr ) 6:1),44 and
the C4 stereochemistry was determined as described in our
previous communication.36 After all the struggles, the solution
was right under our noses! The partial hydrogenations of this
alcohol and its TES ether 20 using Lindlar’s catalyst failed,
(41) Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113, 7277-7287.
(42) Frantz, D. E.; Fassler, R.; Carreira, E. M. J. Am. Chem. Soc. 2000, 122,
1806-1807.
(43) Seyferth, D.; Weiner, M. A. J. Am. Chem. Soc. 1962, 84, 361-363.
(44) Shahi, S. P.; Koide, K. Angew. Chem., Int. Ed. 2004, 43, 2525-2527.
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Synthesis and Antitumor Activity of FR901464
A R T I C L E S
Scheme 3. Formation of C4 Stereocenter with Higher
Scheme 5. Alternative Methods to Prepare Acid 3a
Stereocontrola
a Conditions: (a) HCtCCH2OTHP (3.0 equiv), nBuLi (3.0 equiv), THF,
-78f0 °C, 78-93%; (b) (S)-2-methyl-CBS-oxazaborolidine (0.2 equiv),
Catecholborane (1.6 equiv), EtNO2, -78 °C; (c) Ac2O (4.6 equiv), pyridine
(excess), 23 °C, 97% (two steps); (d) Na2Cr2O7 (3.5 equiv), H2SO4, H2O,
acetone, 0f23 °C, 74%; (e) see Scheme 4.
a Conditions: (a) AgsCtCsCO2Me (1.7 equiv), Cp2ZrCl2 (1.3 equiv),
AgOTf (0.2 equiv), CH2Cl2, 23 °C, 84% (dr ) 6:1); (b) H2 (1 atm), Lindlar’s
catalyst (1.2 -2.0 mol %), quinoline (0.94-1.5 equiv), EtOH, 23 °C, 7-17
h; (c) TRSCl (1.1 equiv), imidazole (1.4 equiv), THF, 0 °C, 74%; (d) Red-
Al (2.0 equiv), THF, -72 °C, 81%; (e) TESCl (1.4 equiv), imidazole (1.5
equiv), THF, 0 °C, quant.; (f) O3, CH2Cl2/MeOH (1:1), -78 °C; then Me2S
1
(17 equiv), -78f23 °C, quant. (based on H NMR).
the er to 98:2. Unfortunately the catalytic version of the
asymmetric alkynylation reaction50 was unsuccessful. After
acetylation of propargylic alcohol 6, the resulting ester 26 was
converted to aldehyde 27 by ozonolysis in 89% yield. Ensuing
oxidation of aldehyde 27 and partial reduction of the resulting
alkyne using Lindlar’s catalyst afforded acid 3 in 75% yield
for the two steps and in six total steps from cinnamaldehyde.
Later, we converted enyne 26 to acid 28 in one step via OsO4
mediated oxidative cleavage, and subsequent partial reduction
afforded 3 in 60% yield for the two steps. Therefore, acid 3
can now be prepared in five steps from cinnamaldehyde.
Despite the successful preparation of acid 3, we wished to
further improve the synthetic efficiency and scalability. More
specifically, we deemed the use of TMSCHN2 less attractive
in large scales due to potential safety concerns. Because other
homologation methods to convert cinnamaldehyde to enyne 25
failed as described above, we decided to pursue a different
synthetic route; tetrahydro-2-(2-propynoxy)-2H-pyran was coupled
with N-acetylmorpholine to form ynone 29 in 78-93% yields
(Scheme 5). This ynone was reduced using the CBS catalyst51
in EtNO252 to afford 30, which was acetylated to provide 31 in
97% yield for the two steps. Concurrent THP removal and
oxidation using the Jones reagent afforded ynoic acid 28 in 74%
yield and 86:14 er. Subsequent partial hydrogenation again gave
acid 3. This scheme could provide acid 3 in five steps and should
be more scalable than Scheme 4.
The last building block needed to test our final coupling
strategy was intermediate 4, and our initial efforts toward this
intermediate is shown in Scheme 6. Treatment of the com-
mercially available L-threonine derivative 32 with 2-methox-
ypropene and CSA afforded oxazolidine 7 in quantitative yield.
Subsequent reduction with DIBALH, and immediate exposure
of Garner aldehyde53 to Horner-Wadsworth-Emmons olefi-
nation conditions generated 33 in 84% yield. Hydrogenation of
this unsaturated ester and lactonization afforded lactone 34 but
revealed the poor diastereoselectivity (dr ) 2:1) in the hydro-
genation of 33.
Scheme 4. Preparation of Acid 3a
a Conditions: (a) cinnamaldehyde (1.1 equiv), TMSCHN2 (1.0 equiv),
LDA (1.0 equiv), THF, -78f0 °C, 84%; (b) CH3CHO (2.3 equiv),
Zn(OTf)2 (1.0 equiv), (-)-N-methylephedrine (1.0 equiv), Et3N (1.0 equiv),
toluene, 23 °C, 41% (86:14 er); (c) Ac2O (5.0 equiv), pyridine, 23 °C, quant.;
(d) O3, CH2Cl2, -78 °C; then Me2S (10 equiv), -78f23 °C, 89%; (e)
NaClO2 (3.0 equiv), NaH2PO4 (2.0 equiv), 2-methyl-2-butene (15 equiv),
tBuOH/H2O (1:1), 23 °C; (f) OsO4 (0.7 mol %), Oxone (4.0 equiv), DMF,
23 °C; (g) H2, Lindlar’s catalyst (1 mol %), quinoline (10 mol %), EtOH,
23 °C, 75% (for steps e and g), 60% (for steps f and g).
but the reduction with Red-Al produced 23 in 81% yield chemo-
and stereoselectively.45 Subsequent TES ether formation pro-
duced 24 in quantitative yield, and ozonolyses of both of the
olefins of this compound produced ketoaldehyde 2 cleanly by
crude 1H NMR analysis. Thus, access to this ketoaldehyde was
attainable in a total of seven steps. It is noteworthy that the
ozonolysis of 24 was far more effective than that of 14.
With the B-ring fragment 2 in hand, we embarked on the
preparation of the A-ring fragment 1 as shown in Scheme 4.
We chose the styryl unit to mask an aldehyde that would
effectively suppress the volatility of otherwise low molecular
weight intermediates. The preparation of acid 3 began by the
homologation of cinnamaldehyde by the action of TMSCHN2
to give enyne 25 in 84% yield.46 The Corey-Fuchs method47
was less efficient, and the Seyforth-Ohira-Bestmann method48,49
gave 1-(1-methoxybut-3-ynyl)benzene. Subsequent Carreira
asymmetric alkynylation42 with acetaldehyde generated prop-
argylic alcohol 6 in 41% yield with 86:14 er. In this reaction,
slow addition of acetaldehyde to the reaction mixture proved
to be crucial since rapid addition resulted in the aldol condensa-
tion of acetaldehyde. Recrystallization of 6 further improved
We planned to obtain better stereocontrol in a more rigid
cyclic substrate. Oxazolidine 7 was transformed into 35 in a
one-pot procedure (DIBALH; Ph3PdCH2)54 in 77% yield.
(45) Meta, C. T.; Koide, K. Org. Lett. 2004, 6, 1785-1787.
(46) Miwa, K.; Aoyama, T.; Shioiri, T. Synlett 1994, 107-108.
(47) Corey, E. J.; Fuchs, P. L. Tetrahedron Lett. 1972, 36, 3769-3772.
(48) Ohira, S. Synth. Commun. 1989, 19, 561.
(50) Anand, N. K.; Carreira, E. M. J. Am. Chem. Soc. 2001, 123, 9687-9688.
(51) Corey, E. J.; Raman, K. B.; Shibata, S.; Chen, C.-P.; Singh, V. K. J. Am.
Chem. Soc. 1987, 109, 7925-7926.
(52) Yu, C. M.; Kim, C.; Kweon, J. H. Chem. Comm. 2004, 2494-2495.
(53) Garner, P.; Park, J. M. J. Org. Chem. 1987, 52, 2361-2364.
(54) Wei, Z. Y.; Knaus, E. E. Synthesis 1994, 1463-1466.
(49) Muller, S.; Liepold, B.; Roth, G. J.; Bestmann, H. J. Synlett 1996, 521-
522.
9
J. AM. CHEM. SOC. VOL. 129, NO. 9, 2007 2651

A R T I C L E S
Albert et al.
Scheme 6. Synthetic Approach A-Ring Formationa
a Conditions: (a) 2-methoxypropene (2.0 equiv), CSA (1 mol %), CH2Cl2, 0 °C, quant.; (b) DIBALH (2.0 equiv), CH2Cl2, -78 °C; (c)
(EtO)2P(O)CH(CH3)CO2Et (3.3 equiv), NaH (3.0 equiv), EtOH, 23 °C, 84% (two steps); (d) H2 (1 atm), PtO2 (1 mol %), EtOH, 23 °C, quant. (dr ) 2:1);
(e) AcOH, 58%; (f) DIBALH (2.0 equiv), CH2Cl2, -78 °C; then Ph3PCH3Br (2.1 equiv), KOtBu (2.0 equiv), THF, -78f48 °C, 77%; (g) CSA (10 mol %),
MeOH, 23 °C, 95%; (h) methacrylic acid (1.2 equiv), DCC (1.2 equiv), DMAP (0.1 equiv), CH2Cl2, 0 °C, 95%; (i) Ru-3 (10 mol %), ClCH2CH2Cl, 83 °C;
(j) methallyl bromide (4.0 equiv), Ag2O (1.5 equiv), DMF, 23 °C, 86%; (k) Ru-1 (1 mol %), benzene, reflux, quant.; (l) PDC (6.0 equiv), ClCH2CH2Cl,
t
reflux, 72%; (m) PCC (2.0 equiv), BuOOH (4.0 equiv), Celite, benzene, 23 °C, 67%; (n) H2, PtO2 (1 mol %), EtOH, 23 °C, quant. (dr ) 10:1); (o)
allyl-MgCl (2.0 equiv), THF, -78 °C, 96%.
Scheme 7. Preparation of 42a
Subsequent removal of the acetonide using catalytic CSA in
MeOH afforded 36 in 95% yield. Methallylation of 36 by the
action of Ag2O and methallyl bromide generated diene 37 in
86% yield, which was then subjected to ring-closing olefin
metathesis conditions using 1 mol % of Ru-155 to form
dihydropyran 38 in quantitative yield. Catalyst Ru-256 was also
able to catalyze this reaction at 1 mol %. To prepare unsaturated
lactone 39, we found PDC to be the most efficient and selective
allylic oxidant for 38 (72%). PCC-TBHP gave similar results
for the oxidation of 38 but gave peroxy-acetal 40 as a byproduct.
Unfortunately, we were unable to effectively convert peroxy-
acetal 40 to lactone 39 primarily due to epimerization at C14.
A more direct approach to form the unsaturated lactone 39 was
attempted by methacryl ester formation (step h) then ring-closing
metathesis using catalyst Ru-3 (step i),57,58 but the second step
was unsuccessful. Stereoselective hydrogenation of the unsatur-
ated lactone 39 set the C-12 stereocenter of 34 in a 10:1 dr in
quantitative yield. Allylation of this lactone afforded 41 as a
mixture of hemiketal anomers and aminal anomers.
a Conditions: (a) Et3SiH (4.0 equiv), BF3‚OEt2 (4.0 equiv), CH2Cl2, -78
°C, 42 (dr ) 10:1); (b) Et3SiH (10 equiv), BF3‚OEt2 (4.0 equiv), CF3CH2OH
(8.0 equiv), CH2Cl2, -78 °C; then Boc2O (0.5 equiv), -78f23 °C, 42
(dr ) 10:1).
tetrahydropyran 42 to 45% yield, possibly by establishing an
equilibrium between the putative oxocarbenium and N-acylimin-
ium ions. 12-epi-41 was subjected to the same reaction sequence,
which gave 12-epi-42 stereo- and chemoselectively in 85%
yield.
Prior to the discovery of the reduction of 41 in the presence
of CF3CH2OH, we explored other methods to improve the
hemiketal reduction. To thwart the production of pyrrolidine
43-type compounds, we also tested other amine-protecting
groups (Scheme 8). For example, we prepared picolinamide 44
from 38 in 82% yield. Subsequent oxidation and stereoselective
reduction were comparable to using the Boc protecting group.
Allylation of 46 was accomplished in 58% yield (not optimized),
and reduction with Et3SiH-BF3‚OEt2 afforded 50 in 44% yield
As shown in Scheme 7, subjection of 41 to reducing
conditions (Et3SiH-BF3‚OEt2) provided tetrahydropyran 42 in
20% as well as pyrrolidine 43 in 50% yield. The addition of
CF3CH2OH to the reduction conditions improved the yield of
(55) Scholl, M.; Ding, S.; Lee, C. W.; Grubbs, R. H. Org. Lett. 1999, 1, 953-
956.
(56) Kingsbury, J. S.; Harrity, J. P. A.; Bonitatebus, P. J.; Hoveyda, A. H.
J. Am. Chem. Soc. 1999, 121, 791-799.
(57) Grela, K.; Harutyunyan, S.; Michrowska, A. Angew. Chem., Int. Ed. 2002,
41, 4038-4040.
(58) Michrowska, A.; Bujok, R.; Harutyunyan, S.; Sashuk, V.; Dolgonos, G.;
Grela, K. J. Am. Chem. Soc. 2004, 126, 9318-9325.
9
2652 J. AM. CHEM. SOC. VOL. 129, NO. 9, 2007

Synthesis and Antitumor Activity of FR901464
A R T I C L E S
Scheme 8. Preparation of 41 Using Different Amine Protecting
Scheme 9. Failed Synthetic Attempts Using the NHK Strategya
Groupsa
a Conditions: (a) TFA/CH2Cl2 (1:9), 23 °C; then pivaloyl chloride (3.0
equiv), DMAP (0.01 equiv), pyridine, 0f23 °C, 82% (38f44); (b) 6 N
HCl/THF (2:1), 23 °C; then TsCl (1.3 equiv), K2CO3, 23 °C, 86% (38f45);
t
(c) PDC (4.0 equiv), BuOOH (4.0 equiv), benzene, 23 °C, 70% (25) or
65% (26); (d) H2, PtO2 (3 mol %), EtOH, 23 °C, 97% (44f46) or 99%
(45f47); (e) allyl-MgCl (3.0 equiv), THF, -98 °C, 58% (46f48), or -78
°C, 63% (47f49); (f) Et3SiH (10 equiv), BF3‚OEt2 (4.0 equiv), CH2Cl2/
MeCN (1:1), -20 °C, 44% (48f50, 61% BORSM; dr ) 8:1), or THF,
-20 °C, 68% (49f51, dr ) 12:1); (g) (from 51) sodium naphthalenide,
THF, -78 °C; then Boc2O (2.3 equiv), Et3N (4.0 equiv), DMAP (0.08
equiv), MeCN, 23 °C, 87% (51f42, two steps).
a Conditions: (a) Ru-1 (5 mol %), methacrolein (10 equiv), ClCH2CH2Cl,
40 °C, 67%; (b) CHI3 (2 equiv), CrCl2 (6 equiv), THF, 0 °C, 63%; (c)
TFA/CH2Cl2 (1:9), 23 °C; then 3 (1.7 equiv), HATU (1.7 equiv), Pr2NEt
i
(3.6 equiv), MeCN, 23 °C, 45%; (d) CrCl2 (7 equiv), NiCl2 (∼5 mol %),
(61% BORSM; dr ) 8:1). Disappointingly, we failed to find
deprotection conditions to give 50 after vigorous screening of
reaction conditions including the literature procedure.59 There-
fore, we turned our attention to other protecting groups.
The undesired pyrrolidine formation proceeds via the putative
highly electrophilic acyliminium ion, and the corresponding
sulfonyliminium ion would be less electrophilic,60 suppressing
the pyrrolidine formation. On the basis of this consideration,
we decided to test a tosyl protecting group. Deprotection of the
Boc group of 38 and subsequent protection of the resulting
amine afforded sulfonamide 45 in 86% yield. Allylic oxidation
and stereoselective hydrogenation were accomplished in 65 and
99% yields, respectively, to afford lactone 47. Allylation of 47
gave 49 in 63% yield (not optimized), which was then subjected
to reduction conditions (Et3SiH-BF3‚OEt2) to afford 51 in 68%
yield (dr ) 12:1). Deprotection proceeded smoothly using
sodium naphthalenide,61 which provided an alternative route to
prepare amine 4 or carbamate 42.
n
DMSO, 23 °C; (e) BuLi (1-2 equiv), THF, -100 f -78 °C.
Scheme 10. Julia Olefination Coupling Strategy
As shown in Scheme 9, compound 42 was converted to
unsaturated aldehyde 52 using cross-olefin metathesis conditions
employing catalytic Ru-1. Takai vinyl iodide formation62
generated iodoalkene 53 in 63% yield. Removal of the Boc
group of this compound was accomplished in 1:9 TFA/CH2-
Cl2, and subsequent amide bond formation with acid 3 was
accomplished by the action of HATU in 45% yield. Thus, access
to vinyl iodide 1 was obtained in 10 steps in the longest linear
sequence.
With both coupling partners in hand, the stage was set to
test the NHK coupling. To our disappointment, treatment of
vinyl iodide 1 and ketoaldehyde 2 under NHK conditions37,38
caused proto-deiodination of 1 and decomposition of the
ketoaldehyde. Similarly, treatment of vinyl iodide 53 and
ketoaldehyde 2 to NHK conditions gave diene 55 and decom-
position of the fragile ketoaldehyde. Therefore, we opted to
modify our strategy and attempted a lithium-halogen exchange
n
between BuLi and 53 to generate the corresponding alkenyl-
lithium but did not find any appreciable coupling to ketoalde-
hyde 2. Having realized the fragile nature of ketoaldehyde 2
and the difficulty of forming the C5-C6 bond in the late stages
of a synthesis, we began to explore other coupling possibilities.
Second Generation Synthetic Studies. We envisioned the
second retrosynthetic analysis featuring modified Julia coupling
to form the C6-C7 bond as shown in Scheme 10. Julia
olefinations63,64 are typically highly E-selective and compatible
with various functional groups. Therefore, we decided to pursue
this strategy, for which acid 3 and aldehyde 52 were already
prepared, but the preparation of the B-ring fragment 56 from
ketone 15 required new methodology.
Displayed in Scheme 11 are our efforts toward the B-ring
fragment 56. Typically, hemiketals are formed by the intramo-
(59) Koul, A. K.; Prashad, B.; Bachawat, J. M.; Ramegowda, N. S.; Mathur,
N. K. Synth. Commun. 1972, 2, 383-388.
(60) Royer, J.; Bonin, M.; Micouin, L. Chem. ReV. 2004, 104, 2311-2352.
(61) Ji, S.; Gortler, L. B.; Waring, A.; Battisti, A.; Bank, S.; Closson, W. D.
J. Am. Chem. Soc. 1967, 89, 5311-5312.
(63) Julia, M.; Paris, J.-M. Tetrahedron Lett. 1973, 14, 4833-4836.
(64) Kocienski, P. J.; Lythgoe, B.; Ruston, S. J. Chem. Soc., Perkin Trans. 1
1978, 829-834.
(62) Takai, K.; Nitta, K.; Utimoto, K. J. Am. Chem. Soc. 1986, 108, 7408-
7410.
9
J. AM. CHEM. SOC. VOL. 129, NO. 9, 2007 2653

A R T I C L E S
Albert et al.
Scheme 11. Preparation of Methyl Glycoside 58a
Scheme 12. Preparation of Thioethers 61 and 63a
a Conditions: (a) NBS (1.1 equiv), THF, H2O, 0f23 °C, 75%; (b) PPTS
(10 mol %), MeOH, CH2Cl2, 23 °C, 40%; (c) NBS (1.5 equiv), 3 Å MS,
MeOH, MeCN, 0f23 °C, 59%.
lecular attack of a hydroxy group onto a ketone, thus the
hydroxy group is the origin of the ring oxygen atom. Here we
questioned whether the ketone oxygen atom could be used as
an alternative source for the ring oxygen atom. This would allow
for the exciting possibility of trapping the putative intermediate
oxocarbenium ion with water or other nucleophiles to obtain
hemiketals and ketals, among others. To test this idea, enone
15 was treated with NBS in H2O/THF (1:8) to give 57 in 75%
yield as a single diastereomer.65 Unambiguous structural de-
termination was determined after conversion of this hemiketal
to methyl glycoside 58 in 40% yield. Similarly, we could treat
enone 15 with NBS in the presence of MeOH and obtain methyl
glycoside 58 in 59% in one step. However, the stereochemistry
at C5 was incorrect thereby making preparation of sulfone 56
too difficult from this intermediate.
a Conditions: (a) TESCl (1.2 equiv), imidazole (1.4 equiv), THF, 0 °C,
quant.; (b) OsO4 (4.5 mol %), NMO (1.0 equiv), THF, H2O, 23 °C; then
NaIO4 (1.0 equiv), Et2O, H2O, 23 °C, 60%; (c) NBS (2.0 equiv), acetone,
H2O, 23 °C, 95%; (d) PPTS (10 mol %), MeOH, CH2Cl2, 23 °C, 75%; (e)
NBS (1.5 equiv), 3 Å MS, MeOH, MeCN, 0f23 °C, 47%; (f) 4-phenyltet-
razole thiol (1.2 equiv), Et3N (2.0 equiv), MeCN, reflux, 60%; (g) TBAF
(1.1 equiv), THF, 0 °C, 80%; (h) 4-phenyltetrazole thiol (1.0 equiv), Et3N
(2.0 equiv), MeCN, 50 °C, 74%; (i) 4-O2NPhCO2H (2.1 equiv), DIAD (2.7
equiv), Ph3P (2.2 equiv), THF, 0f23 °C; then K2CO3 (1.0 equiv), MeOH,
23 °C.
We thought that using 4-epi-15 for the cyclization could
reverse the observed stereocontrol for C5 (Scheme 12). Toward
this end, 4-epi-5 was protected as its TES ether, and subsequent
regioselective oxidative cleavage (OsO4, NMO; NaIO4) gave
enone 4-epi-15 in 60% yield. Treatment of 4-epi-15 with NBS
in H2O/acetone (1:8) gave hemiketal 59 in 95% yield as a single
diastereomer. Methyl glycoside 60 could be prepared by the
treatment of 59 with catalytic PPTS in MeOH/CH2Cl2 (1:3) in
75% yield. Alternatively, the same methyl glycoside could be
formed in one step by treating 4-epi-15 with NBS in MeOH/
MeCN (1:10) in 47% yield. The absolute stereochemistry of
60 was determined by X-ray crystallographic analysis, also
revealing the diastereoselectivities for the vinylations of ep-
oxyaldehyde 10 shown in Table 1 and the alkynylation of
epoxyaldehyde 10 shown in Scheme 3.
experiment in CDCl3, which supports the notion of a “strong”
hydrogen bond.66,67
Third Generation Synthetic Studies. At this point, we had
no choice but to employ a bolder approach to unite two fully
functionalized fragments. Such retrosynthetic analysis is shown
in Scheme 13 (A), in which we continued to envisage forming
the C6-C7 olefin as the final coupling. At the onset of this
study, 1,3-diene-ene cross metatheses were poorly explored
transformations68 and had not been used in natural product
synthesis (Scheme 13, B). However, we believed that this would
be a viable approach because: (1) a ruthenium catalyst would
preferentially react with the olefin of monoene 70 rather than
the conjugated olefins of diene 69; (2) the resulting ruthenium
alkylidene 71 would preferentially react with 70 to form 67,
but this is reversible at elevated temperature and could be
minimized by adding 70 slowly to the reaction mixture; (3)
eventually, alkylidene 71 would react with the terminal olefin
of 69 to form the thermodynamically favored 72; and (4) when
ruthenium alkylidene 68 does form, this ruthenium species will
react with 70 faster than 69 to form 72 rather than 66. This
hypothesis was also corroborated by the Crimmins group in the
crucial cross-coupling step.69
To arrive at sulfone 56, the C-6 bromide needed to be
substituted with a thiol, and the C4 stereocenter needed to be
inverted. Treatment of methyl glycoside 60 with 4-phenyltet-
razole thiol under various conditions only gave the undesired
thioether 61. At this point, we were unsure whether the axial
TES ether group was shielding the C6-Br group from SN2
reactions. To make the C6 position more sterically accessible,
we removed the TES group from 60 to give 62 in 80% yield.
We then treated this compound with 4-phenyltetrazole thiol
under basic conditions but again only found epoxide opening
with the thiol to form 63. A more severe problem is that
Mitsunobu conditions failed to invert the C4 stereocenter
presumably because of steric reasons. Due to the difficulty in
inverting the C4-hyroxy group and sensitivity of the epoxide
toward thiols, we abandoned the Julia olefination strategy. It is
noteworthy that despite these synthetic problems, in 61 and 63
we observed couplings between the 3-hydroxy group and
To pursue this strategy, the first task was to prepare the right
fragment 64. With ketoaldehyde 2 already in hand, a direct
approach would be a chemoselective vinylation of this aldehyde
(66) Alkorta, I.; Rozas, I.; Elguero, J. Chem. Soc. ReV. 1998, 27, 163-170.
(67) Perrin, C. L.; Nielson, J. B. Annu. ReV. Phys. Chem. 1997, 48, 511-544.
(68) Randall, M. L.; Tallarico, J. A.; Snapper, M. L. J. Am. Chem. Soc. 1995,
117, 9610-9611.
(69) Crimmins, M. T.; Christie, H. S.; Chaudhary, K.; Long, A. J. Am. Chem.
Soc. 2005, 127, 13810-13812.
1
methylene protons adjacent to the sulfur atom in the H NMR
(65) Albert, B. J.; Koide, K. Org. Lett. 2004, 6, 3655-3658.
9
2654 J. AM. CHEM. SOC. VOL. 129, NO. 9, 2007

Synthesis and Antitumor Activity of FR901464
A R T I C L E S
Scheme 13. Diene-ene Cross Metathesis Coupling Strategy
Table 2. Optimization for the Preparation of Right Fragment 73a
reagent
additives
(equiv)
entry
(2 equiv)
% yielda
73/5-epi-73b
1
2
3
4
5
16
16
16
17
17
---
40
46
31
33
16
1.0:1
1.3:1
2.0:1
1.0:1
2.0:1
HMPA (2.5)
TMEDA (2.5)
nBuLi (1.9)
nBuLi (1.9), HMPA (2.5)
1
a Combined yield. b Determined by H NMR analysis.
with acid 3 in one pot afforded 75 in 86% yield. Stereo- and
regioselective cross metathesis of 75 with methacrolein using
Ru-3 gave aldehyde 76 in 57% yield (67% BORSM), and
subsequent Wittig reaction with ylide Ph3PdCH2 gave diene
54 in 86% yield.
With the fully functionalized intermediates 54 and 73 in hand,
the stage was set to test our coupling strategy (Scheme 15).
Treatment of diene 54 and the C6 epimeric mixture 73 gave
the metathesis adduct 77 in 22% yield. Subsequent removal of
the TES group was accomplished with HF‚pyridine to furnish
FR901464 (confirmed by HPLC analysis using the authentic
FR901464) and presumably its C5-epimer, thereby supporting
the viability of this strategy. We were very much surprised to
find that when the reaction was neutralized with a pH 7.0
phosphate buffer, the natural product decomposed. This interest-
ing observation prompted us to investigate the stability of
FR901464 under physiological conditions (see Scheme 20).
Having realized that the final steps could potentially produce
many byproducts due to instability, we strongly desired to
prepare a right fragment in higher efficiency and with better
stereocontrol of the C5 stereocenter. To test whether we could
improve the C5 stereoselectivity, we switched to the TBS ether
that might potentially favor the desired Felkin selectivity while
preventing unwanted chelation of the R-alkoxy group (see
Supporting Information). Preparation of this ketoaldehyde
followed similarly as shown with the TES derivative (Scheme
2). However, subjection of the C4-OTBS-protected ketoaldehyde
to various vinylation and alkynylation conditions formed the
desired adduct in <5% yield and lacked the desired chemose-
lectivity. Therefore, we decided to develop a different approach
to obtain the right fragment.
(Table 2). Treatment of the fragile ketoaldehyde 2 with 16 gave
an inseparable mixture of 73 and 5-epi-73 with no diastereo-
selectivity (entry 1). Addition of HMPA or TMEDA (entries 2
and 3) slightly improved the Felkin selectivity and the overall
yield of 73. Generation of vinyllithium in situ from 17 and
nBuLi and subsequent addition of 2 afforded 73 and 5-epi-73
in 33% yield but again with no stereoselectivity (entry 4).
Addition of HMPA gave better diastereoselectivity (dr ) 2:1)
but lower combined yield (16%) (entry 5). Neither switching
the solvent to CH2Cl2 or toluene nor making the organocerium
reagent from CeCl3 and 1670,71 improved this difficult trans-
formation. Additionally, ethynylation of ketoaldehyde 2 required
higher temperatures (g-20 °C), leading to decomposition. We
speculate that the low yields for these reactions are due to an
intramolecular deprotonation-elimination pathway; the alkoxide
generated after addition to the aldehyde can intramolecularly
deprotonate a hydrogen atom at C2 and induce an E1cb reaction.
Although low yielding, we obtained the right coupling fragment
73 along with its inseparable diastereomer 5-epi-73 and decided
to pursue preliminary coupling studies after preparing diene 54.
Our attempts to prepare diene 54 are shown in Scheme 14.
Aldehyde 52 was treated with ylide Ph3PdCH2 to form diene
55. Deprotection of the Boc protecting group in TFA/CH2Cl2
(1:9) and coupling with acid 3 gave 54 and 74 as an isomeric
mixture. Therefore, the Boc group removal should precede diene
formation to avoid this acid-catalyzed isomerization. Removal
of the Boc group in 42 (1:9 TFA/CH2Cl2) followed by coupling
We turned to the previously prepared enoate 24 (Scheme 3)
since it contained all of the necessary carbons and the desired
C4 stereochemistry. Up to this point, we were reluctant to pursue
the following strategy because we could not predict the
stereoselectivity of the [2,3]-sigmatropic rearrangement (Scheme
16).72 Regardless, we began to explore methods to prepare 81
from intermediate 24 (Table 3), hoping that we could somehow
control the stereoselectivity in question. The DIBALH reduction
of 24 afforded allylic alcohol 78 in 95% yield, which was
(70) Dimitrov, V.; Bratovanov, S.; Simova, S.; Kostova, K. Tetrahedron Lett.
1994, 35, 6713-6716.
(71) Dimitrov, V.; Kostova, K.; Genov, M. Tetrahedron Lett. 1996, 37, 6787-
6790.
(72) Wirth, T. Angew. Chem., Int. Ed. 2000, 39, 4189-4189.
9
J. AM. CHEM. SOC. VOL. 129, NO. 9, 2007 2655

A R T I C L E S
Albert et al.
Scheme 14. Preparation of Diene 54a
t
a Conditions: (a) Ph3PCH3Br (1.4 equiv), BuOK (1.3 equiv), THF, 0 °C, 80%; (b) TFA/CH2Cl2 (1:9), 23 °C; then 3 (1.7 equiv), HATU (1.7 equiv),
i
iPr2NEt (4.6 equiv), MeCN, 0 °C; (c) TFA/CH2Cl2 (1:9), 23 °C; then 3 (1.2 equiv), HATU (1.2 equiv), Pr2NEt (4.0 equiv), MeCN, 23 °C, 86%; (d) Ru-3
(0.05 equiv), methacrolein (20 equiv), CH2Cl2, 23 °C, 57% (67% based on recovered 75); (e) Ph3PCH3Br (1.4 equiv), KOtBu (1.2 equiv), THF, 0 °C, 86%.
Scheme 15. Our First Synthesis of FR901464a
the DMAP stoichiometry are shown in entries 6, 8, 9, and 10;
decreasing the amount of DMAP below 3 equiv eroded
selectivity presumably due to background hydrolysis reactions
of the putative selenate. Finally, the nitro group on the aromatic
ring was unnecessary for the high diastereoselectivity (entry 11).
With the stereoselective [2,3]-sigmatropic rearrangement in
hand, we proceeded to prepare right fragment 64 (Scheme 17).
Primary alcohol 78 was converted to the desired secondary
alcohol 81 this time in a one-pot procedure utilizing the selenide
formation, [2,3]-rearrangement strategy in 91% combined yield
(dr ) 7:1). Oxidative cleavage of the 1,1-disubstituted olefin
of 81 gave the previously prepared compound 73 in a diaste-
reomerically pure form but in only 27% yield. To improve the
regioselectivity of the oxidative cleavage sequence, we prepared
bis-TES ether 82 in 95% yield. Subsequent dihydroxylation and
Pb(OAc)4-promoted diol cleavage afforded enone 83 in 71%
yield. The removal of the TES ethers was found to occur best
using AcOH/H2O/THF (3:1:3) to afford 64 in 91% yield. The
diaxial nature of the C4 and C5 hydrogen atoms was confirmed
a Conditions: (a) Ru-1 (0.2 equiv), THF, 40 °C, 22%; (b) HF‚pyridine,
THF, 0 °C.
Table 3. Step c in Scheme 16: Optimization of the
[2,3]-Sigmatropic Rearrangements of 79 and 80
entry
R
base (equiv)
solvent
temp (
°
C)
% yielda 81/5-epi-81b
1
2
3
4
5
6
7
NO2 pyridine (5)
NO2 pyridine (5)
NO2 pyridine (5)
NO2 pyridine (5)
NO2 2,6-lutidine (5) THF
NO2 DMAP (5) THF
THF
EtOH
acetone -20
CH2Cl2 0f20
-20
-44f-20
94
87
96
67
73
95
84
4.0:1
4.0:1
3.5:1
3.5:1
4.0:1
7.5:1
8.1:1
-20f0
-44f23
-44f23
NO2 4-pyrrolidino- THF
pyridine (5)
8
9
NO2 DMAP (3)
NO2 DMAP (1.5)
THF
THF
THF
THF
-44f23
-44f23
-44f23
-44f23
80
87
34
91
7.8:1
5.3:1
3.3:1
7.0:1
1
by the H NMR analysis showing JH4-H5 ) 9.8 Hz, similar to
10 NO2 DMAP (0.5)
11 DMAP (5)
that found in the natural product (J ) 10 Hz).12 We now
stereoselectively and efficiently formed 64 in 11 linear steps.
Thus, the only remaining question was the cross metathesis in
the absence of any protecting groups.
H
1
a Combined yield. b Determined by H NMR analysis.
subsequently transformed to selenide 79 or 80 in quantitative
yields, setting us up to explore a Mislow-Evans-type [2,3]-
sigmatropic rearrangement despite no closely related reported
reactions.
As shown in Scheme 18, the catalyst employed in the cross-
metathesis of 54 and 64 influenced the efficiency of the reaction.
The desirable conditions found were to employ a 1:1.8 ratio of
54 to 64 in the presence of 12 mol % of Ru-3 in 1,2-
dichloroethane to afford FR901464 in 40% yield after one
recycle of unreacted starting materials (51% based on recovered
54 after one recycle). The fragile nature of 64 prevented more
forcing reaction conditions, since facile fragmentation occurred
at g47 °C in 1,2-dichloroethane. Additionally, right fragment
64 formed an unreactive homodimer under the reaction condi-
tions, as determined by preparation of the right fragment
homodimer and subjection of it to left fragment 54 and Ru-3.
Table 3 shows the optimization of the [2,3]-sigmatropic
rearrangements of 79 and 80 to form the desired alcohol 81,
whose stereochemistry was determined as shown in Scheme
17.36 While the transformation occurred rapidly in polar solvents
(entries 1-3), it was sluggish in CH2Cl2 (entry 4). The base
employed in this reaction played a critical role as shown in
entries 5-7. When 2,6-lutidine was used, the reaction was
slower as compared to pyridine presumably due to the added
steric bulk of the nucleophile, leading to no improved diaste-
reoselectivity. When DMAP was used, the reaction was very
slow at low temperatures and needed to be warmed to ambient
temperature to proceed at an adequate rate. Gratifyingly, the
diastereoselectivity of this reaction was improved to 7.5:1 in
favor of alcohol 81. The more nucleophilic base, 4-pyrrolidi-
nopyridine, gave the highest diastereoselectivity for this alcohol
(dr ) 8.1:1).73 Although DMAP showed slightly lower stereo-
selectivity, we were attracted by its lower cost. The effect of
To recapitulate our total synthesis of FR901464, we com-
pleted the synthesis in 29 total steps with the longest linear
sequence of 13 steps (Scheme 19). Highlights of the synthesis
include a diene-ene cross metathesis as the final synthetic step
in the absence of protecting groups, a Mislow-Evans-type [2,3]-
sigmatropic rearrangement of a selenoxide, an asymmetric
(73) Hassner, A.; Krepski, L. R.; Alexanian, V. Tetrahedron 1978, 34, 2069-
2076.
9
2656 J. AM. CHEM. SOC. VOL. 129, NO. 9, 2007

Synthesis and Antitumor Activity of FR901464
A R T I C L E S
Scheme 16. Preparation of Alcohols 81 and 5-epi-81a
n
a Conditions: (a) DIBALH (3.0 equiv), THF, -78 °C, 95%; (b) ArSeCN (1.2 equiv), Bu3P (1.4 equiv), THF, 0 °C, quant.
Scheme 17. Preparation of 64a
Scheme 19. Summary of the Total Synthesis of FR901464
a Conditions: (a) 2-O2NPhSeCN (1.2 equiv), Bu3P (1.4 equiv), THF,
0 °C; then H2O2 (10 equiv), DMAP (4.0 equiv), -44f23 °C, 91% (dr )
7:1); (b) OsO4 (0.02 equiv), NaIO4 (2.5 equiv), 2,6-lutidine (1.6 equiv),
dioxane/H2O (3:1), 0f23 °C, 27%; (c) TESCl (1.4 equiv), imidazole (1.6
equiv), THF, 0 °C, 95%; (d) OsO4 (0.01 equiv), NMO (0.96 equiv), THF/
H2O (10:1), 0 to 23 °C; then Pb(OAc)4 (1.2 equiv), benzene, 0f23 °C,
71% (86% based on recovered 82); (e) AcOH/H2O/THF (3:1:3), 0f23 °C,
91%.
n
Table 4. Instability of 64 in Buffers
pH
t1 2 (h)
/
Scheme 18. Final Stage of the Total Synthesis of FR901464
6
7
12
8
7.4
4
°C as shown in Table 4. Alarmingly, the half-lives of 64 are
only 8 and 4 h at pH 7 and 7.4, respectively. Consequently, we
became intrigued by the decomposition pathways and subjected
64 to pH 7.4 buffer at 37 °C for 1 day and observed >3 products
by TLC and HPLC analyses, some of which were minor and
not isolated (Scheme 20). Among the products that we were
able to partially characterize were furan 90 and enones 84 and
1
85 (a). We did not observe acrolein (by H NMR or HPLC),
91, or 92 via Kitahara’s decomposition pathway (b).74 We also
looked for acetate 93 that would be formed by a Grob
fragmentation (c)75,76 but have insufficient evidence to confirm
this product formation. Hemiketal 64 exists as a 10:1 equilibrium
mixture with linear ketoalcohol 64-open in both CD2Cl2 and
D2O, in accordance with Jacobsen’s observation.30 Therefore,
enones 84 and 85 were presumably formed by the â-elimination
via ketone 64-open, and furan 90 was formed by the dehydration
of hemiketal 88. Enone 84 can exist as its hemiketals 86 or 87,
but 86 should be thermodynamically disfavored due to its ring
strain, and therefore furan 89 is not a major byproduct of this
reaction over the time period studied.
Carreira alkynylation, formation of an unsaturated lactone by a
ring-closing metathesis-regioselective allylic oxidation se-
quence, a mild, diastereoselective alkynylation reaction, and an
E-selective Red-Al reduction. This total synthesis is the most
concise to date, allowing for facile analogue preparation and
access to each fully functionalized fragment for biological and
chemical studies.29
Tumor Specificity with pH Sensitivity. It is intriguing to
contemplate on why nature produces FR901464 with such subtle
Stability of FR901464. Similar to the Jacobsen and Kitahara
groups, we also noticed the instability of FR901464 toward
acids, even as mild as silica gel.30,74 However, the sensitivity
of FR901464 under physiologically relevant conditions remained
unreported, and therefore we decided to determine the half-life
of 64 to examine its stability in various phosphate buffers at 37
(74) Motoyoshi, H.; Horigome, M.; Ishigami, K.; Yoshida, T.; Horinouchi, S.;
Yoshida, M.; Watanabe, H.; Kitahara, T. Biosci., Biotechnol., Biochem.
2004, 68, 2178-2182.
(75) Grob, C. A. Angew. Chem., Int. Ed. Engl. 1969, 8, 535-546.
(76) Grob, C. A.; Schiess, P. W. Angew. Chem., Int. Ed. Engl. 1967, 6, 1-15.
9
J. AM. CHEM. SOC. VOL. 129, NO. 9, 2007 2657

A R T I C L E S
Albert et al.
Scheme 20. Decomposition of 64
pH sensitivity. We speculate that under pressure for survival,
the FR901464-producing organism may have had to find a way
to inhibit the growth of enemies in acidic environments. This
idea is difficult to test because the exact nature of the FR901464-
producing organism has not yet been characterized. In the
context of cancer medicine, we propose that the stability of the
â-epoxy hemiketal under an acidic environment can be exploited
for the development of cancer-specific drugs because the pH
within tumor cells is significantly lower than that of normal
cells77,78 and therefore â-epoxy hemiketal-containing anticancer
agents would decompose more rapidly in normal cells while
remaining active in tumor cells longer. Although the acidity
difference between cancer and normal cells is well-known, this
difference has not yet been fully exploited. Thus this study may
open the door for such an approach.
Scheme 21. Preparation of the Right Fragment Analogue 95a
a Conditions: (a) Hg(OAc)2 (1.1 equiv), THF, 0f23 °C; then NaBH4
(2.0 equiv), Et3B (1.0 equiv), -78 f -44 °C, 76%; (b) TBAF (1.2 equiv),
THF, 0 °C, 97%; (c) HF‚pyr, THF, 0 °C, 73%.
Although the pH-sensitivity could be exploited in therapeutic
areas, the instability of FR901464 may limit its potential as a
probe for biological study, requiring a more stable analogue.
The replacement of the C1-hydroxy group with either a
hydrogen atom30 or a methoxy group74 only marginally im-
proved the potency of FR901464. We hypothesized that such
increased stability and more desirable van der Waals interaction
with target proteins might improve the potency of FR901464.
To test this hypothesis, we prepared right fragment 95 (Scheme
21) which should be more stable based on the fragmentation
studies. Treatment of alcohol 81 with Hg(OAc)2 followed by
NaBH4 and Et3B79 afforded ether 94 in 76% yield. Deprotection
of this tetrahydropyran was accomplished by the action of TBAF
to give 95 in 97% yield. Interestingly, we found that deprotection
of 94 with HF‚pyridine caused a pinacol-like rearrangement to
yield the ring-contracted aldehyde 96. Treatment of 95 with HF‚
pyridine also caused this rearrangement to occur, indicating that
the TES group is not essential for the rearrangement.
The stability of 95 was then tested in various phosphate
buffers at 37 °C (Table 5). The t1/2 of 95 at pH 7 and 7.4 was
now 48 h, which is a dramatic increase from 64 (t1/2 ) 4-8 h).
Additionally, 95 did not appear to be significantly labile until
0.1 N H2SO4 was employed as the solvent. It should also be
mentioned that aldehyde 96 (Scheme 19) was not observed even
in the presence of H2SO4, showing the pinacol-like rearrange-
ment should not be biologically relevant. Replacement of the
C1-OH with a methyl group enhanced the stability of the right
fragment of FR901464 and, therefore, could provide a suitable
biological probe to study the unique biology of FR901464.
Synthesis of a Stable FR901464 Analogue. Encouraged by
the chemical stability of 95, we proceeded to synthesize the
more stable FR901464 analogue meayamycin (97) in 59% yield
after one recycling of recovered starting materials using the same
diene-ene metathesis strategy (Scheme 22). By having a more
stable right fragment, we were able to more efficiently produce
the desired product. Therefore, we completed the synthesis of
(77) Warburg, O. Science 1956, 123, 309-314.
(78) Simon, S. M.; Schindler, M. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 3497-
3504.
(79) Kang, S. H.; Lee, J. H.; Lee, S. B. Tetrahedron Lett. 1998, 39, 59-62.
(80) Buttke, T. M.; Mccubrey, J. A.; Owen, T. C. J. Immunol. Methods 1993,
157, 233-240.
(81) Pommier, Y.; Vogt, A.; McPherson, P.; Koide, K. Unpublished results.
9
2658 J. AM. CHEM. SOC. VOL. 129, NO. 9, 2007

Synthesis and Antitumor Activity of FR901464
A R T I C L E S
Scheme 22. Cross Metathesis of 54 and 95a
a Conditions: (a) 54 (1.0 equiv), 95 (1.5 equiv), Ru-3 (0.10 equiv),
ClCH2CH2Cl, 40 °C, 59% after one recycling of recovered 54 and 95.
Table 5. Decomposition of 95
pH
t1 2 (h)
/
Figure 1. Growth inhibition of MCF-7 cells by FR901464 (blue squares)
and meayamycin (97; red circles).
0.1 N H2SO4
3
3.5
24
4
40
5
6
7
7.4
48
40
48
48
an FR901464 analogue (meayamycin) using the more stable
right fragment in a total of 28 steps. The GI50 value of this
analogue was a remarkable 10 pM in MCF-7 cells, making it
one of the most potent anticancer agents known to date. With
the successes of FR901464 in the xenograft models13 and
impressive potency of meayamycin, we are currently working
toward preparing this analogue on a larger scale. The potency
and stability of this analogue should facilitate our efforts to
isolate cellular targets of FR901464. We are also studying how
the stability of meayamycin would affect the tumor specificity.
97 in 28 total steps with the longest linear sequence consisting
of 13 steps. This synthetic scheme enabled us to produce 30
mg of this analogue and should be scalable.
Biological Assay. We tested synthetic FR901464 and its
analogue 97 against MCF-7 breast cancer cells and measured
the cell viability using the MTS assay after 7- and 10-day
incubation periods.80 As Figure 1 shows, we observed the
inhibition of cell growth in a concentration-dependent manner
and determined the GI50 value of FR901464 to be 1.1 nM, which
is in good agreement with the literature value 1.8 nM.11 The
potency of meayamycin was better and exceeded our expecta-
tion: the GI50 value of this analogue was 10 pM, giving
approximately a 100-fold improvement in the antiproliferative
activity. We also found that the mode of action of FR901464
did not involve either DNA binding or antimitotic activity.81
Acknowledgment. B.J.A. is thankful for a Graduate Excel-
lence Fellowship. N.L.C. is a recipient of the Arnold and Mabel
Beckman Scholar Award, the Lilly Summer Research Fellow-
ship, and the Averill Scholarship. This work was supported
bythe University of Pittsburgh, the American Chemical Society
(PRF No. 38542-G1), The American Cancer Society George
Heckman Institutional Research Grant, The Competitive Medical
ResearchFund,andtheNationalCancerInstitute(1R01CA120792-
01).
Conclusion
Supporting Information Available: Experimental procedures
and spectroscopic data for all the new compounds and FR901464.
This material is available free of charge via the Internet at
http://pubs.acs.org.
We completed the most concise total synthesis of FR901464
to date, in a total of 29 steps. We then performed degradation
studies on the fully functionalized right fragment of FR901464
and used this insight to rationally design a more stable analogue.
Enabled by our total synthesis, we completed the synthesis of
JA067870M
9
J. AM. CHEM. SOC. VOL. 129, NO. 9, 2007 2659