Evolution of the total syntheses of ustiloxin natural products and their analogues

Article abstract of DOI:10.1021/ja710363p

Ustiloxins A-F are antimitotic heterodetic cyclopeptides containing a 13-membered cyclic core structure with a synthetically challenging chiral tertiary alkyl-aryl ether linkage. The first total synthesis of ustiloxin D was achieved in 31 linear steps using an SNAr reaction. An NOE study of this synthetic product showed that ustiloxin D existed as a single atropisomer. Subsequently, highly concise and convergent syntheses of ustiloxins D and F were developed by utilizing a newly discovered ethynyl aziridine ring-opening reaction in a longest linear sequence of 15 steps. The approach was further optimized to achieve a better macrolactamization strategy. Ustiloxins D, F, and eight analogues (14-MeO-ustiloxin D, four analogues with different amino acid residues at the C-6 position, and three (9R,10S)-epi-ustiloxin analogues) were prepared via the second-generation route. Evaluation of these compounds as inhibitors of tubulin polymerization demonstrated that variation at the C-6 position is tolerated to a certain extent. In contrast, the S configuration of the C-9 methylamino group and a free phenolic hydroxyl group are essential for inhibition of tubulin polymerization.

Full text of DOI:10.1021/ja710363p

Published on Web 01/30/2008
Evolution of the Total Syntheses of Ustiloxin Natural Products
and Their Analogues
Pixu Li,†,§ Cory D. Evans,†,| Yongzhong Wu,† Bin Cao,†, Ernest Hamel,‡ and
Madeleine M. Joullie´*,†
Department of Chemistry, UniVersity of PennsylVania, 231 South 34th Street, Philadelphia,
PennsylVania 19104, Toxicology and Pharmacology Branch, DeVelopmental Therapeutics
Program, DiVision of Cancer Treatment and Diagnosis, National Cancer Institute at Frederick,
National Institutes of Health, Frederick, Maryland 21702
Received November 15, 2007; E-mail: mjoullie@sas.upenn.edu
Abstract: Ustiloxins A-F are antimitotic heterodetic cyclopeptides containing a 13-membered cyclic core
structure with a synthetically challenging chiral tertiary alkyl-aryl ether linkage. The first total synthesis of
ustiloxin D was achieved in 31 linear steps using an SNAr reaction. An NOE study of this synthetic product
showed that ustiloxin D existed as a single atropisomer. Subsequently, highly concise and convergent
syntheses of ustiloxins D and F were developed by utilizing a newly discovered ethynyl aziridine ring-
opening reaction in a longest linear sequence of 15 steps. The approach was further optimized to achieve
a better macrolactamization strategy. Ustiloxins D, F, and eight analogues (14-MeO-ustiloxin D, four
analogues with different amino acid residues at the C-6 position, and three (9R,10S)-epi-ustiloxin analogues)
were prepared via the second-generation route. Evaluation of these compounds as inhibitors of tubulin
polymerization demonstrated that variation at the C-6 position is tolerated to a certain extent. In contrast,
the S configuration of the C-9 methylamino group and a free phenolic hydroxyl group are essential for
inhibition of tubulin polymerization.
Introduction
therapy is a very important goal being extensively investigated,
the mechanism of action of the naturally occurring peptides that
Antimitotic Agents. Antimitotic agents have long been of
interest to scientists and physicians, even before their precise
mechanism of action could be articulated. Most of these
compounds interfere with microtubule assembly and functions,
thus resulting in mitotic arrest of eukaryotic cells. There are a
number of natural and synthetic compounds that interfere with
tubulin function to inhibit the formation of microtubules.1 Such
antimitotic agents exhibit a broad range of biological activities
and can be used for medicinal and agrochemical purposes, acting
as anticancer, antifungal, or anthelmintic agents. Moreover,
tubulin-binding compounds are valuable for understanding basic
mechanisms involved in the dynamics of the microtubule
network.2 Mitotic arrest is of importance in cancer chemo-
therapy, since tubulin is an established chemotherapeutic target.
In addition to the widely used vinca alkaloids and taxoids, many
other antimitotic agents are currently in clinical or preclinical
development.3 Newer potential targets that may cause mitotic
arrest are in development as well. Although cancer chemo-
inhibit tubulin polymerization remains a challenging problem.
Attempts to establish a relationship among the tubulin binding
peptides have had only limited success.4-8 As a result, the
mechanism of action of peptidic antimitotic agents is not
completely elucidated and deserves further investigation.
Ustiloxins. The first peptide shown to interact with tubulin
was phomopsin A (Figure 1).9-13 Since then, a number of
peptides and depsipeptides were found to disrupt cellular
microtubules. These compounds universally inhibit the assembly
of the R,â-tubulin dimer into its polymer and, in the cases
investigated, strongly suppress microtubule dynamics at low
(4) Hamel, E. Biopolymers 2002, 66, 142.
(5) Hamel, E.; Covell, D. G. Curr. Med. Chem.: Anti-Cancer Agents 2002, 2,
19.
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Chem. 1998, 41, 1524.
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1998, 8, 385.
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Frahn, J. L.; Jago, M. V.; Lanigan, G. W.; Payne, A. L.; Peterson, J. E.;
Petterson, D. S.; Smith, L. W.; White, R. R. Aust. J. Biol. Sci. 1977, 30,
269.
(10) Culvenor, C. C. J.; Cockrum, P. A.; Edgar, J. A.; Frahn, J. L.; Gorst-Allman,
C. P.; Jones, A. J.; Marasas, W. F. O.; Murray, K. E.; Smith, L. W.; Steyn,
P. S.; Vleggaar, R.; Wessels, P. L. Chem. Commun. 1983, 1259.
(11) Culvenor, C. C. J.; Edgar, J. A.; Mackay, M. F.; Gorst-Allman, C. P.;
Marasas, W. O. F.; Steyn, P. S.; Vleggar, R.; Wessels, P. L. Tetrahedron
1989, 45, 2351.
† University of Pennsylvania.
‡ National Cancer Institute at Frederick, National Institutes of Health.
§ Present address: Wyeth Research, 401 N. Middletown Road, Pearl
River, NY 10965.
| Present address: U.S. Food and Drug Administration, CVM/ONADE/
DMT HFV-142, 7500 Standish Place, Rockville, MD 20855.
Present address: Sanofi-Aventis, 1041 Route 202-206, Bridgewater,
NJ 08807.
(12) Mackay, M. F.; Van Donkelaar, A. V.; Culvenor, C. C. J. J. Chem. Soc.,
Chem. Commun. 1986, 1219.
(13) Tonsing, E. M.; Steyn, P. S.; Osborn, M.; Weber, K. Eur. J. Cell. Biol.
1984, 35, 156.
(1) Downing, K. H. Annu. ReV. Cell. DeV. Biol. 2000, 16, 89.
(2) Walczak, C. E. Curr. Opin. Cell Biol. 2000, 12, 52.
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9
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J. AM. CHEM. SOC. 2008, 130, 2351-2364
2351

A R T I C L E S
Li et al.
Figure 1. Structures of ustiloxins and phomopsin A.
concentrations. Among these compounds are ustiloxins A-F
(1-5) (Figure 1), which were isolated from the water extracts
of false smut balls, caused by the parasitic fungus Ustilaginoidea
Virens, found on the panicles of the rice plant.14-16
Ustiloxins are structurally similar to phomopsin A, and their
structures were elucidated by amino acid analyses, X-ray
analysis of ustiloxin A,16 and other spectroscopic data. Ustiloxins
cause mycotoxicosis and inhibit the polymerization of brain
tubulin at micromolar concentrations but have no growth
inhibitory effect on bacteria or fungi.17 Interestingly, the
ustiloxins and phomopsin A are relatively weak cytotoxins
compared with other antimitotic agents, which are lethal to cells
at subnanomolar concentrations.5
The structures of the ustiloxin congeners contain a 13-
membered heterodetic peptide macrocycle that includes a rare
chiral tertiary alkyl-aryl ether linkage. The ustiloxin congeners
(1-5) are composed of permutations of two separate sites: the
side chain attached to the macrocyclic aromatic ring para to
the ether linkage (see R1 in Figure 1) and the presence of either
alanine or valine in the macrocycle (see R2 in Figure 1). Both
ustiloxins A (1) and B (2) have a sulfinyl norvaline-based amino
acid attached to the aromatic ring at the R1 position. Ustiloxins
A and B differ only in the side chain of a single macrocyclic
amino acid. Ustiloxin C (3) contains a hydroxyethanesulfinyl
group at the R1 position and the alanine variation of the
macrocycle. Ustiloxins D (4) and F (5) have no sulfinyl
appendage on the aromatic ring; they are differentiated from
each other only by the presence of valine or alanine in the
macrocycle. A major difference between the phomopsins12 and
the ustiloxins16 is that, although both have a hydroxyl substituent
at C-10, they are of opposite configurations.
It is important to determine the mechanism of action of the
naturally occurring peptides that inhibit tubulin polymerization
so that they may be more efficiently applied in cancer
chemotherapy. In the literature, descriptions of potential binding
sites of antimitotic peptides and depsipeptides to tubulin are
somewhat contradictory.6,18-20 The ustiloxins appeared to
provide fertile ground for studying antimitotic peptide natural
products and their inhibitory effects on tubulin polymerization.
Synthesis of ustiloxins could provide access to a variety of
analogues of the natural product for SAR studies, which would
elucidate the interaction between tubulin and the ustiloxin
ligands. Ultimately, these analogues would provide information
that could lead to a better understanding of the underlying
mechanism of microtubule assembly inhibition, as well as the
dynamics and impact of modulating these processes on cancer
chemotherapy. Given our longstanding interest in the synthesis
of cyclodepsipeptides, we present two generations of total
syntheses of ustiloxins D and F and a full account of recent
progress in the total synthesis of several analogues.
Synthetic Strategy and Retrosynthetic Analysis
Ustiloxin D was chosen as the lead compound because it was
one of the simplest congeners of the ustiloxin family and it
possessed high biological activity. The total synthesis of
ustiloxin D provided a scaffold for the syntheses of other
congeners and analogues. From a synthetic perspective, ustiloxin
D is composed of four amino acid residues (Figure 2). Two of
them are naturally occurring glycine and valine. The other two
(14) Koiso, Y.; Li, Y.; Iwasaki, S.; Hanaoka, K.; Kobayashi, T.; Sonoda, R.;
Fujita, Y.; Yaegashi, H.; Sato, Z. J. Antibiot. 1994, 47, 765.
(15) Koiso, Y.; Morisaki, N.; Yamashita, Y.; Mitsui, Y.; Shirai, R.; Hashimoto,
Y.; Iwasaki, S. J. Antibiot. 1998, 51, 418.
(16) Koiso, Y.; Natori, M.; Iwasaki, S.; Sato, S.; Sonoda, R.; Fujita, Y.; Yaegashi,
H.; Sato, Z. Tetrahedron Lett. 1992, 33, 4157.
(17) Iwasaki, S. Med. Res. ReV. 1993, 13, 183.
(18) Bai, R.; Cowell, D. G.; Taylor, G. F.; Kepler, J. A.; Copeland, T. D.;
Nguyen, N. Y.; Pettit, G. R.; Hamel, E. J. Biol. Chem. 2004, 279, 30731.
(19) Barbier, P.; Gregoire, C.; Sarrazin, M.; Peyrot, V. Biochemistry 2001, 40,
13510.
(20) Ravi, M.; Zask, A.; Rush, T. S., III. Biochemistry 2005, 44, 15871.
9
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Total Syntheses of Ustiloxin and Analogues
A R T I C L E S
Scheme 1. Ether Formation Study Using the Mitsunobu Reaction
Figure 2. Amino acid components of ustiloxin D.
Since the project began in our laboratory, extensive investiga-
tions were carried out to identify a method to form the
challenging tertiary alkyl-aryl ether bond. It was envisioned that
a mild reaction tolerant of a broad range of functionalities would
lead to an expedient total synthesis. The Mitsunobu reaction
was the first choice. We had successfully synthesized a number
of cyclopeptide alkaloids bearing alkyl-aryl ether linkages using
the Mitsunobu approach.34,35 Model studies used phenol deriva-
tive 8, which was synthesized in six steps from tyrosine. Among
all the Mitsunobu reaction conditions tested, the best result was
obtained by treatment of 8 with methyl-2-hydroxyisobutyrate
(9) with PPh3 and DEAD in THF, which provided the desired
ether 10 in 35% yield (Scheme 1a).
are non-proteinogenic amino acid residues, namely â-hydroxyi-
soleucine (6) and N-methyl-â-hydroxytyrosine derivative 7. A
chiral tertiary alkyl-aryl ether linkage exists between compounds
6 and 7. The main challenges of the total synthesis are the
construction of 6 and 7 by short and stereoselective approaches
and the formation of a chiral tertiary alkyl-aryl ether between
these two amino acid residues without affecting other functional
groups and the existing stereochemistry. The latter specification
was especially challenging, given the very limited methods that
were available for the transformation and the highly function-
alized structure of the ustiloxins.
The Challenges
However, when using a more complex tertiary alcohol 12,
the desired ether 13 was not produced under Mitsunobu
conditions (PPh3, DEAD). A newly developed Mitsunobu
reagent, cyanomethylenetrimethylphosphorane (CMMP),36 was
also used but failed to give the desired ether (Scheme 1b). When
propargyl tertiary alcohol 15 was treated with isovanillin (14)
under a Mitsunobu protocol, tertiary alkyl-aryl ether 16 was
obtained in a very low yield (Scheme 1c). Although the
propargylic alcohol 15 is a more reactive Mitsunobu substrate,
no further improvement to this reaction was achieved. Our
unsuccessful model studies indicated that the scope of substrates
in the Mitsunobu reaction for the tertiary ether formation was
limited.37,38 As a result, we investigated the SNAr reaction as
an alternative approach toward stereoselective alkyl-aryl syn-
thesis.
An alkyl-aryl ether may be retrosynthetically disconnected
in two ways, one providing an alkyl alcohol and an aromatic
compound with a leaving group. The C-O bond may be formed
using a nucleophilic aromatic substitution (SNAr) reaction or a
transition metal-catalyzed coupling reaction between the same
reagents. The other disconnection would provide a phenol
derivative and an aliphatic compound with a leaving group. In
this approach, the most common methods are nucleophilic
substitution of an alkyl halide by a phenol or a Mitsunobu
reaction between a phenol derivative and an alkyl alcohol.
Although these methods are available, the formation of a tertiary
alkyl-aryl ether remains a challenging transformation. Stereo-
selective formation of this functionality is even more difficult.
In the first disconnection, the alcohol substrate needs to be a
tertiary alcohol. Tertiary alkoxides are more often employed as
non-nucleophilic bases, but rarely used as nucleophiles because
of steric bulkiness and low nucleophilicity. The second discon-
nection is not a good alternative since tertiary alkyl species with
a leaving group tend to form tertiary alkyl cations readily, which
would destroy the stereochemistry of the tertiary carbon center.
The competing elimination reaction would inhibit the effective-
ness of the nucleophilic substitution reaction. As a result, very
few reactions have been reported to form tertiary alkyl-aryl
ethers stereoselectively. To the best of our knowledge, only four
types of reactions have been reported to construct such an ether
motif, namely, SNAr reactions,21-23 Pd-catalyzed intermolecular
cross-coupling of aryl halides with alcohols,24-26 Mitsunobu-
type reactions27-29 and Pd-catalyzed asymmetric allylic O-
alkylation (AAA) reactions.30-33
The advantage of the SNAr reaction for generation of alkyl-
aryl ethers is retention of configuration of the chiral alcohol
(21) Coutts, I. G. C.; Southcott, M. R. J. Chem. Soc., Perkin Trans. 1 1990,
767.
(22) Masada, H.; Oishi, Y. Chem. Lett. 1978, 57.
(23) Woiwode, T. F.; Rose, C.; Wandless, T. J. J. Org. Chem. 1998, 63, 9594.
(24) Mann, G.; Hartwig, J. F. J. Am. Chem. Soc. 1996, 118, 13109.
(25) Mann, G.; Hartwig, J. F. J. Org. Chem. 1997, 62, 5413.
(26) Palucki, M.; Wolfe, J. P.; Buchwald, S. L. J. Am. Chem. Soc. 1997, 119,
3395.
(27) Hughes, D. L. Org. React. 1992, 42, 355.
(28) Hughes, D. L. Org. Prep. Proced. Int. 1996, 28, 127.
(29) Mitsunobu, O. Synthesis 1981, 1.
(30) Trost, B. M. Chem. Pharm. Bull. 2002, 50, 1.
(31) Trost, B. M. J. Org. Chem. 2004, 69, 5813.
(32) Trost, B. M.; Crawley, M. L. Chem. ReV. 2003, 103, 2921.
(33) Trost, B. M.; Tang, W. J. Am. Chem. Soc. 2002, 124, 14542.
(34) Heffner, R. J.; Jiang, J. J.; Joullie´, M. M. J. Am. Chem. Soc. 1992, 114,
10181.
(35) Heffner, R. J.; Joullie´, M. M. Tetrahedron Lett. 1989, 7021.
(36) Tsunoda, T.; Nagino, C.; Oguri, M.; Itoˆ, S. Tetrahedron Lett. 1996, 2459.
9
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A R T I C L E S
Li et al.
Scheme 2. Synthesis of Chiral Tertiary Alkyl-Aryl Ether 24 via
SNAr Reaction
ethylmagnesium bromide to the ketone proceeded with very
good diastereoselectivity, giving rise to the anti-Felkin diaste-
reomer of tertiary alcohol 19 as the major product. The absolute
configuration of the newly generated stereocenter was confirmed
by X-ray crystallography.
Treatment of 19 with p-toluenesulfonic acid in methanol gave
diol 20, and the primary hydroxyl group was selectively
protected as its MOM ether 21. Subsequent removal of the Boc
group with trifluoroacetic acid (TFA) afforded amino alcohol
22. Treatment of 22 with aryl fluoride 23 and 1.1 equiv of
KHMDS in THF23 successfully constructed the ether linkage
in 24 in 78% yield. A number of problems were encountered
during this SNAr reaction. Two of these are shown in Scheme
3: (a) when 22 and 23 were treated with 2.0 equiv of KHMDS,
two products were observed with the undesired aryl amine 25
as the major product due to a faster reaction between the in
situ generated amide and aryl fluoride 23; (b) when the amino
group was protected as its N-carbamate 21, an undesired
oxazolidinone 26 was produced via intramolecular cyclization.
The SNAr reaction is highly sensitive to the functional groups
on the aromatic ring as well. When the cyano group was
replaced by a nitro group, no reaction occurred under the same
reaction conditions.38
substrates, as no new stereogenic centers are formed in the
transformation. For many years, our group has focused on
building the ether linkage in cyclopeptide alkaloids using SNAr
reactions. These efforts resulted in the total syntheses of
sanjoinine A39,40 and sanjoinine G1.41 We used 4-fluoroben-
zonitrile as an effective reagent for the arylation of alcohols,42
and several SNAr reactions on activated aryl halides have been
reported.23,43,44 Wandless and co-workers developed an efficient
method for the preparation of hindered alkyl-aryl ethers using
potassium bis(trimethylsilyl)amide (KHMDS) to generate the
alkoxide.23 The enantiopure tertiary alcohols were prepared prior
to this challenging bond formation, and no selectivity issues
were noted during the reaction. However, functional group
compatibility remained an issue as the reaction uses strong basic
conditions. Nonetheless, Zhu and co-workers applied the
intramolecular SNAr reaction to synthesize a cyclic depsipeptide
using tetrabutylammonium fluoride (TBAF) as the base.44
Scheme 3. Problems Observed during the SNAr Reaction
Optimization
The First Total Synthesis of Ustiloxin D
On the basis of the reported success of SNAr reactions, this
approach was used in our first generation total synthesis of
ustiloxin D. The key aspects of our synthetic strategy were the
installation of the ether linkage at an early stage to circumvent
potential problems at the later stages and the stereoselective
generation of the â-hydroxytyrosine moiety in a single step.
The first total synthesis of ustiloxin D began with the preparation
of the protected amino alcohol 19 (Scheme 2). This alcohol
was synthesized from D-serine in five steps according to a
literature procedure.45 Treatment of D-serine with di-tert-butyl
dicarbonate and NaHCO3 protected the amino group as its Boc
carbamate, and the carboxylic acid was then coupled with N,O-
dimethylhydroxylamine to afford Weinreb amide 17 using
1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDCI‚HCl) as the coupling reagent. The amino and hydroxyl
groups of 17 were protected as the N-tert-butoxycarbonyl-N,O-
isopropylidene derivative. Treatment with methyllithium con-
verted the Weinreb amide to methyl ketone 18. Addition of
With ether 24 in hand, the amino group was protected as its
Boc carbamate 27 (Scheme 4). Later we found that the three-
step sequence from compound 21 to 27 could be performed
without purification of crude intermediates to provide better
overall yield (78% for three steps). Raney nickel reduction with
NaH2PO2 in aqueous buffer solution converted nitrile 27 to
aldehyde 28. Treatment of 28 with 30% H2O2 and catalytic di-
(o-nitrophenyl)diselenide via Dakin oxidation afforded the
corresponding formate, and subsequent hydrolysis with KOH
in methanol gave phenol 29, which was protected as a benzyl
ether 30. Treatment of 30 with ethyl acrylate, Pd(OAc)2, and
(o-tolyl)3P under Heck conditions afforded cinnamate 31 in 96%
yield.
With cinnamate 31 in hand, the regioreversed Sharpless
asymmetric aminohydroxylation (AA) protocol46 was success-
fully applied to install two stereocenters in a single step, giving
the desired (2S,3R)-â-hydroxy-R-amino ester 32 in 58% yield
(Figure 3). To determine the regioselectivity and diastereose-
lectivity of this reaction, the diastereomer 33 and the regioisomer
(37) Cao, B. Ph.D. Thesis. University of Pennsylvania, Philadelphia, PA, 2002.
(38) Li, P. Ph.D. Thesis. University of Pennsylvania, Philadelphia, PA, 2005.
(39) East, S. P.; Joullie´, M. M. Tetrahedron Lett. 1998, 39, 7211.
(40) Xiao, D.; East, S. P.; Joullie´, M. M. Tetrahedron Lett. 1998, 39, 9631.
(41) East, S. P.; Shao, F.; Williams, L.; Joullie´, M. M. Tetrahedron 1998, 54,
13371.
(42) Williams, L.; Zhang, Z.; Shao, F.; Carroll, P. J.; Joullie´, M. M. Tetrahedron
1996, 52, 11673.
(43) La¨ıb, T.; Zhu, J. Synlett 2000, 9, 1363.
(44) La¨ıb, T.; Zhu, J. Tetrahedron Lett. 1999, 83.
(45) Ageno, G.; Banfi, L.; Cascio, G.; Guanti, G.; Manghisi, E.; Riva, R.; Rocca,
V. Tetrahedron 1995, 51, 8121.
(46) Park, H.; Cao, B.; Joullie´, M. M. J. Org. Chem. 2001, 66, 7223.
9
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Total Syntheses of Ustiloxin and Analogues
A R T I C L E S
Scheme 4. Synthesis of Intermediate 31 for Use in the Sharpless
Aminohydroxylation
With the amino ester 32 in hand, the Boc group was removed
by TFA to afford an amine, which was then coupled with
N-Boc-L-valine using the DEPBT reagent to give 35 (Scheme
5). Treatment of 35 with TBSOTf protected the secondary
hydroxyl group as its silyl ether, as well as transformed the
tert-butyl carbamate to the corresponding tert-butyldimethylsilyl
carbamate. Basic hydrolysis with LiOH in tert-BuOH-H2O
afforded amino acid 36, which was then cyclized via macro-
lactamization to afford macrocycle 37 in 78% yield using
EDCI-HOBt in a mixed solvent DMF-CH2Cl2 (1:5) as the
optimized conditions for macrocyclization. The MOM group
was removed using B-bromocatecholborane at -78 °C to afford
alcohol 38 in 88% yield (Scheme 5).
Scheme 5. Synthesis of Protected Macrocycle 40
34 of compound 32 were synthesized using different chiral
ligands. With (DHQD)2AQN or (DHQ)2AQN as the ligand, iso-
mer 32 or the diastereomeric (2R,3S)-â-hydroxy-R-amino ester
33 were the major products, respectively. When (DHQD)2PHAL
In order to differentiate the nitrogen of the tyrosine R-amino
group from the two backbone nitrogens in the macrocycle and
to achieve methylation later in the synthesis, conversion of the
N-carbamate to N-sulfonamide was necessary to decrease the
pKa of the nitrogen. The benzyl carbamate was selectively
removed by catalytic hydrogenolysis in the presence of the
benzyl ether, using 2,2′-dipyridyl as a catalyst poison (Scheme
5). The intermediate amine was subsequently protected as the
2-nitrobenzenesulfonamide 39 in 85% yield for two steps.
Sequential oxidation of the primary hydroxyl group in 39 with
the Dess-Martin reagent and then with NaClO2 afforded a
carboxylic acid, which was coupled with glycine benzyl ester
to give 40. Selective deprotonation of sulfonamide 40 was
achieved with the base MTBD (1,3,4,6,7,8-hexahydro-1-methyl-
2H-pyrimido[1,2-a]-pyrimidine),47 and subsequent methylation
with methyl 4-nitrobenzenesulfonate afforded 41 (Scheme 6).
Treatment of compound 41 with n-Bu4NF-HOAc deprotected
the silyl ether to give alcohol 42. Removal of the 2-nitroben-
zenesulfonyl group with â-mercaptoethanol and DBU (1,8-
diazabicyclo[5.4.0]undec-7-ene) gave secondary amine 43.48,49
Figure 3. Synthesis of â-hydroxy-R-amino ester via regioreversed Sharpless
AA reaction.
was used as the ligand, the regioisomeric (2S,3R)-R-hydroxy-
â-amino ester 34 was the major product. Using isomers 33 and
34 as internal references, regioselectivity (5:1) and diastereo-
selectivity (91:9) were determined. The desired compound 32
was separated from other isomers by flash column chromatog-
raphy on aluminum oxide.
(47) Miller, S. C.; Scanlan, T. S. J. Am. Chem. Soc. 1997, 119, 2301.
(48) Fukuyama, T.; Jow, C.-K.; Cheung, M. Tetrahedron Lett. 1995, 36, 6373.
(49) Kan, T.; Fukuyama, T. Chem. Commun. 2004, 353.
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A R T I C L E S
Li et al.
Scheme 6. Completion of the Synthesis of Ustiloxin D
The final step involved hydrogenolysis to remove both the
benzyl ester and the benzyl ether. Direct hydrogenolysis with
10% Pd/C, as well as transfer hydrogenolysis with 10% Pd/C
and 1,4-cyclohexadiene, removed the benzyl ester but not the
benzyl ether. A more active catalyst, palladium black, was
needed to remove the protecting groups and afford ustiloxin D
(4) in 85% yield. The 1H NMR, 13C NMR, HRMS, and optical
rotation of synthetic ustiloxin D were identical to those of the
natural product.
It was clear that a new method to construct the chiral tertiary
alkyl-aryl ether linkage was necessary for a more efficient and
convergent synthesis. The new reaction should ideally be
performed after â-hydroxyisoleucine 6 and N-methyl-â-hy-
droxytyrosine derivative 7 were built. The conditions for ether
formation had to be mild to tolerate all the functionalities of
these substrates and not affect any existing chiral centers.
Furthermore, as the main difference between the macrocyclic
core structures of ustiloxins and phomopsins12,16 is the stereo-
chemistry of the C-10 hydroxyl group of the â-hydroxytyrosine
moiety, adoption of a single method to access all the diaster-
eomers of N-methyl-â-hydroxytyrosine was preferred as it would
be easily modifiable for the synthesis of phomopsin natural
products.
Atropisomerism
Like many other macrocyclic natural products, such as
vancomycins, ustiloxin D may potentially exist as two atropi-
somers. To clarify the atropisomerism issue, a two-dimensional
1H-1H NOESY experiment was carried out. Ustiloxin D
exhibited diagnostic NOE cross-peaks between protons on C-16
and C-10, C-12 and C-9, and C-16 and C-21 (Figure 4). No
NOE cross-peaks were observed between protons on C-12 and
C-10, C-16 and C-9, or C-12 and C-21. This observation
suggested that ustiloxin D was present as a single atropisomer
instead of as a mixture of two atropisomers. The three-
dimensional structure in Figure 4 was generated based on the
information from NOE signals.
The first total synthesis of ustiloxin D (4) was accomplished
from readily available D-serine and aryl fluoride starting
materials. The longest linear sequence of the synthetic route
was 31 steps, and the average yield was 82% for each step.50
We next initiated efforts to improve the efficiency of the
synthesis. The strategy was based on an intramolecular SNAr
reaction at a late stage to make the synthesis more convergent.
Although the linear precursor was synthesized very efficiently,
many attempts using different SNAr reaction conditions failed
to afford the desired macrocycle.51
Investigation of AAA Reaction
The Pd-catalyzed AAA reaction has been widely used to
construct chiral alkyl-aryl ethers in many natural product
syntheses.31,32 However, applications of the AAA reaction to
form a tertiary alkyl-aryl ether stereoselectively are rare because
of steric effects.52 Nonetheless, the mild reaction conditions and
broad functional group tolerance would be beneficial for the
late-stage synthesis of highly functionalized natural products
such as ustiloxins with a proper allylic methyl carbonate. To
investigate this approach, allylic carbonate 44, bearing a
nitrogen-containing chiral carbon center and an adjacent hy-
droxyl group, was subjected to standard AAA reaction condi-
tions with isovanillin (14, Scheme 7a).
No reaction was observed at room temperature, but a small
amount of product 46 was isolated when the reaction was heated
to 100 °C in a microwave reactor. The initial Pd-catalyzed allylic
O-alkylation presumably formed the desired tertiary alkyl-aryl
ether 45, but it was subsequently converted to the isolated
product via a Claisen rearrangement because of the high
temperature. To increase the reactivity of the allylic carbonate
and allow for lower reaction temperatures, allylic carbonate 47
was synthesized38 and reacted with isovanillin to provide
products 48 and 49 in a 4:5 ratio. Product 48 was shown by 1H
NMR to be a diastereomeric mixture in a 6:1 ratio. Although
introduction of this cyclic constraint improved the reactivity with
isovanillin, carbonate 47 failed to participate in the AAA
(50) Cao, B.; Park, H.; Joullie, M. M. J. Am. Chem. Soc. 2002, 124, 520.
(51) Li, P.; Evans, C. D.; Forbeck, E. M.; Park, H.; Bai, R.; Hamel, E.; Joullie´,
M. M. Bioorg. Med. Chem. Lett. 2006, 16, 4804.
1
Figure 4. Assignment of atropstereochemistry of ustiloxin D by H-1H
NOE study.
(52) Trost, B. M.; Toste, F. D. J. Am. Chem. Soc. 1998, 120, 9074.
9
2356 J. AM. CHEM. SOC. VOL. 130, NO. 7, 2008

Total Syntheses of Ustiloxin and Analogues
A R T I C L E S
Scheme 7. Studies of the AAA Reaction
Figure 5. Attempted Lewis acid-promoted ring-opening of aziridine by
phenol derivatives.
because of steric effects. The size and nucleophilicity of the
phenol were also significant factors.
The formation of aryl 1,1-dimethylpropargyl ethers mediated
by copper catalysts appeared to be worthy of investigation.61,62
The reaction conditions were mild, and the yields were excellent.
Although the method had not been used to form stereogenic
centers, we felt that a stereochemical induction from a vicinal
stereocenter might provide useful levels of selectivity. The chiral
amino acid carbon center next to the C-2 center could potentially
induce diastereoselectivity. Subjecting propargylic carbonate 54
and isovanillin (14) to the conditions of the copper-catalyzed
aryl 1,1-dimethylpropargyl etherification afforded the desired
tertiary alkyl-aryl ether 55 in 37% yield (Figure 6a). The more
highly functionalized â-hydroxytyrosine derivative 56 also
exhibited similar reactivity to isovanillin, albeit in poor yield
(Figure 6b).
reaction with dipeptide 50. Attempts to favor the AAA pathway
by either adding a base to increase the concentration of
phenoxide nucleophile or using the naphthoyl-based ligand did
not provide any desired product (51). A low conversion was
observed in toluene and THF, but the major product was the
undesired regioisomer. Using an iridium phosphoramidite
catalyst53 also did not yield any product.
On the basis of these results, we concluded that the more
substituted allylic carbon center was too sterically hindered or
that the nucleophile was too bulky. At that time, Wandless and
co-workers reported a total synthesis of ustiloxin D utilizing
an AAA reaction.54,55 However, a 2:1 ratio of diastereoselectivity
was obtained with a relatively simple allylic carbonate, and this
inseparable mixture was carried through all subsequent ma-
nipulations to the end of the total synthesis.54,55 On the basis of
these results and our model studies, we decided to develop a
new stereoselective method to construct tertiary alkyl-aryl ethers.
Figure 6. Diastereoselective copper-catalyzed tertiary alkyl-aryl ether
formation.
Although these results were very promising, reaction opti-
mization and determination of the stereochemistry of the coupled
product were problematic. The reactive propargylic carbonate
54 participated in various nonproductive reaction pathways, and
a less reactive leaving group was desired.
Aziridine-2-carboxylate 52, prepared in the previous study
of the Lewis acid promoted aziridine ring-opening, had shown
low reactivity where the strained N-C bond behaved as a
leaving group. Therefore, we investigated the reaction between
isovanillin (14) and 52 and found that it afforded a tertiary alkyl-
aryl ether 58 as a single product in modest yield (Figure 7a).
An advantage of this aziridine ring-opening reaction was that
Discovery of an Aziridine Ring-Opening Reaction by
Phenol Derivatives
We next investigated methods used for the formation of
tertiary alkyl-aryl ethers with the aim to extend these to a
stereoselective method. The aziridine ring-opening reaction by
phenol derivatives was very attractive for this purpose.56-60
Having learned from the results of our investigations of the
Mitsunobu reaction that the propargylic carbon center was more
reactive toward nucleophilic attack than a normal tertiary carbon
center, ethynyl aziridine 52 was used for the study of tertiary
alkyl-aryl ether formation by aziridine ring-opening. Investiga-
tion of a trisubstituted aziridine ring-opening by phenol deriva-
tives with Lewis acids51 or phosphines57 did not provide 53 in
acceptable yield (Figure 5). Our results suggested that aziridine
ring-opening at the tertiary carbon center was very difficult
(53) Lopez, F.; Ohmura, T.; Hartwig, J. F. J. Am. Chem. Soc. 2003, 125, 3426.
(54) Sawayama, A. M.; Tanaka, H.; Wandless, T. J. J. Org. Chem. 2004, 69,
8810.
(55) Tanaka, H.; Sawayama, A. M.; Wandless, T. J. J. Am. Chem. Soc. 2003,
125, 6864.
(56) Fan, R.-H.; Hou, X.-L. J. Org. Chem. 2003, 68, 726.
(57) Hou, X.-L.; Fan, R.-H.; Dai, L.-X. J. Org. Chem. 2002, 67, 5295.
(58) Palais, L.; Chemla, F.; Ferreira, F. Synlett 2006, 1039.
(59) Pineschi, M.; Bertolini, F.; Haak, R. M.; Crotti, P.; Macchia, F. Chem.
Commun. 2005, 1426.
(60) Prasad, B. A. B.; Sanghi, R.; Singh, V. K. Tetrahedron 2002, 58, 7355.
Figure 7. Ethynyl aziridine ring-opening reactions.
9
J. AM. CHEM. SOC. VOL. 130, NO. 7, 2008 2357

A R T I C L E S
Li et al.
Figure 8. Convergent approach to ustiloxin D.
Scheme 8. Synthesis of â-Hydroxytyrosine Fragment 62
the product stereochemistry was easily controlled since it was
stereoselective and yielded a single diastereomer. Should the
C-2 configuration prove to be opposite of what we proposed,
the desired product could be obtained from a diastereomer of
aziridine 52. The reaction between 52 and â-hydroxytyrosine
derivative 56 afforded the tertiary alkyl-aryl ether 59 as well
(Figure 7b).
A number of conditions, including different copper species,
bases, solvents, and temperatures were screened to optimize the
reaction shown in Figure 7a. Toluene was the solvent of choice.
During the screening of different copper species, CuOAc was
found to provide the best reaction when combined with DBU
as the base. Using these optimized conditions, the desired tertiary
alkyl-aryl ether product 58 was isolated in 60% yield after 24
h at room temperature. Moreover, X-ray structure analysis
showed that the stereochemical configuration of the product
matched that of ustiloxin D.63
and 62) were necessary for the construction of the tertiary alkyl-
aryl ether via ethynyl aziridine ring-opening reaction (Fig-
ure 8).
First Convergent Approach toward Ustiloxin. The syn-
thesis of fragment 62 started from 5-methoxy-2-(4-methoxy-
phenyl)-oxazole) 65 (Scheme 8). The oxazole 65 was originally
obtained by dehydration of (4-methoxybenzoylamino)acetic acid
methyl ester using P2O5.64 However, the reaction was low
yielding and not easy to handle, especially under humid
conditions. Furthermore, replacing the 5-methoxy group with
other alkoxy groups, such as a benzyloxy group, failed to give
the desired oxazoles. The problem was solved by using the
cyclodehydration conditions developed by Wipf,65 oxazole 65
was obtained in good yield along with 65a-b. The condensation
of p-anisoyl chloride with glycine derivatives smoothly afforded
66a-c in excellent yield. Treatment of 66a-c with tri-
phenylphosphine, iodine, and triethylamine presumably formed
a carbene that underwent a formal cyclodehydration to yield
the oxazoles 65 and 65a-b.
Second-Generation Total Synthesis of
Ustiloxins D and F
A salen-aluminum complex (67)-catalyzed aldol-type reac-
tion between oxazole 65 and orthogonally protected 3,4-
dihydroxybenzaldehyde 64 afforded the desired oxazoline-2-
Given the mild reaction conditions and broad functional group
tolerance, the new regio- and stereoselective ethynyl aziri-
dine ring-opening reaction provided an ideal solution for a
convergent ustiloxin total synthesis. Two key intermediates (60
(63) Li, P.; Forbeck, E. M.; Evans, C. D.; Joullie´, M. M. Org. Lett. 2006, 8,
5105.
(61) Bell, D.; Davies, M. R.; Geen, G. R.; Mann, I. S. Synthesis 1995, 707.
(62) Godfrey, J. D., Jr.; Mueller, R. H.; Sedergran, T. C.; Soundararajan, N.;
Colandrea, V. J. Tetrahedron Lett. 1994, 35, 6405.
(64) Karrer, P.; Miyamichi, E.; Storm, H. C.; Widmer, R. HelV. Chim. Acta
1925, 8, 205.
(65) Wipf, P.; Miller, C. P. J. Org. Chem. 1993, 58, 3604.
9
2358 J. AM. CHEM. SOC. VOL. 130, NO. 7, 2008

Total Syntheses of Ustiloxin and Analogues
A R T I C L E S
carboxylate 68.66 Electron-rich aldehydes undergo slower
turnover, so catalyst loading was increased to 10% to ensure
full conversion of aldehyde 64 to 68. Other investigators
reported nearly identical results in this aldol-type reaction.54 The
diimine ligand was easily recovered by flash column chroma-
tography, and the catalyst could be regenerated without loss of
activity and stereoselectivity. The enantiopure cis-oxazoline 68
was obtained in 85% yield after a single recrystallization.
However, when oxazole 65a was subjected to the aluminum-
salen-catalyzed aldol-type reaction, only a small amount of the
cis product was formed even with high catalyst loadings. No
cis product was observed when oxazole 65b was used in the
aldol-type reaction. It appeared that the bulky aluminum-salen
catalyst was sensitive to the increased size of the benzyl or
p-methoxybenzyl substituents, and it could not effectively
promote the aldol-type reactions of oxazoles 65a and 65b. The
electronic properties of the aromatic ring are also critical. No
reaction occurred when the acetyl group was replaced by a more
electron-donating silyl ether.
DBU-mediated isomerization of 68 afforded the thermody-
namically more stable trans-oxazoline 69,66 with the correct
absolute stereochemistry for the construction of ustiloxins. At
this stage, it was decided to install the requisite N-methyl group
by formation of an oxazolinium ion with methyl triflate,
followed by in situ reduction.67 This protocol would replace
the four-step selective N-methylation sequence employed in our
first total synthesis.
higher yield. Since diastereomers were formed upon reduction,
the p-methoxybenzylidene 70 was cleaved by 10% aqueous HCl,
and the resulting secondary amine was protected as Boc-
carbamate 71 in one pot. Simultaneous hydrolysis of the acetate
and methyl ester of compound 71 was next performed using
lithium hydroxide, followed by coupling with valine benzyl ester
to afford dipeptide 62 in a sequence of eight steps (Scheme 8).
The ethynyl aziridines were synthesized utilizing the methyl
ketone intermediate 18 of the first total synthesis of ustiloxin
D. The synthesis of ketone 18 was improved68 via a four-step
sequence without column chromatography with an overall 60%
yield. Grignard addition of ethynylmagnesium bromide to 18
afforded tertiary alcohol 15 in 80% yield with an 11:1
diastereomeric ratio. The amino alcohol was liberated by acidic
full deprotection of intermediate 15, followed by protection of
the primary amino group as its Cbz carbamate (61) or nosyl
sulfonamide (74) in one pot. A one-step TEMPO-catalyzed
oxidation protocol directly oxidized the primary hydroxyl group
of diols 61 and 74 to carboxylic acids 75a-b, which were
coupled with glycine esters to provide the dipeptides without
affecting the tertiary hydroxyl group. The Mitsunobu reaction
converted the amino alcohols 76-78 to the ethynyl aziridines
60, 79, and 80 (Scheme 9).63
Scheme 9. Trisubstituted Ethynyl Aziridine Synthesis
During the in situ reduction of the oxazolinium ion, we
encountered the same over-reduction problem that Wandless
and co-workers had observed.54 While applying the commonly
used conditions of NaBH4 in a 1:4 mixture of methanol and
THF at 0 °C to the methoxy phenol analogue of 69, the desired
N,O-acetal was obtained without any difficulty. However, when
69 was subjected to the same protocol, the C-O bond was
cleaved to afford 72 as the major product (Table 1). The relative
stereochemical configuration of the adjacent two carbon centers
was confirmed by an X-ray crystallographic analysis of 72.
The aziridine ring-opening reaction between 60 and 62
successfully generated the desired tertiary alkyl-aryl ether 81.
A Pd-catalyzed transfer hydrogenolysis simultaneously removed
the Cbz carbamate, benzyl ester, and benzyl ether and reduced
the ethynyl group to an ethyl group to provide the linear
precursor 82 (Scheme 10).
Table 1. Investigation of Iminium Ion Reduction
Scheme 10. First Attempt to Ustiloxin D via Aziridine
Ring-Opening
reductant
solvents
temp (
°
C)
producta
yieldb (%)
NaBH4
L-Selectride
NaBH4
MeOH/THF (1:4)
CH2Cl2
H2O/THF (1:4)
EtOH
0
0
0
72
73
73
70
64
67
63
85
NaBH4
-78
a Major product. b Yield of major product
L-Selectride or NaBH4 in a mixture of water and THF as the
reductants provided compound 73 as the major product. Wand-
less and co-workers developed a biphasic system which suc-
cessfully prevented over-reduction from occurring,54 but we
found that using ethanol as the solvent and lowering the reaction
temperature to -78 °C provided the desired oxazolidine 70 in
To our disappointment, macrolactamization of 82 was unsuc-
cessful with various coupling reagents, bases, and solvents.
Wandless and co-workers also experienced difficulties in
(66) Evans, D. A.; Janey, J. M.; Magomedov, N.; Tedrow, J. S. Angew. Chem.,
Int. Ed. 2001, 40, 1884.
9
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A R T I C L E S
Li et al.
Scheme 11. Synthesis of Ustiloxins D and F and C-6 Analogues
macrolactamization with a linear ustiloxin precursor at this
position. Although only macrolactamization and simultaneous
deprotection of the tert-butyl ester and the Boc carbamate were
needed to finish the total synthesis of ustiloxin D in 14 steps,
no further progress was achieved.
Total Synthesis of Ustiloxins D and F, and Three C-6
Analogues. On the basis of these results, it was decided that
the macrolactamization between the N-terminus of â-hydroxyi-
soleucine and the C-terminus of valine was not viable. Another
choice was to attempt macrocyclization between the N-terminus
of valine and the C-terminus of â-hydroxytyrosine, as both
previous total syntheses of ustiloxin D closed the macrocycle
at this position.
To improve the protecting group strategy, acid 63 was
selectively protected as its benzyl ester 84 (Scheme 11). The
ethynyl aziridine ring-opening reaction of 84 with 79 success-
fully provided the desired chiral tertiary alkyl-aryl ether 85 in
90% yield. The o-nosyl group was removed to afford the
primary amine 86 in 78% yield.69 The amine was coupled with
N-Cbz-valine and N-Cbz-alanine using EDCI and HOBt to
install all the required amino acid components for ustiloxin D
and F respectively. Palladium black catalyzed hydrogenolysis
of 87a-b simultaneously removed the benzyl carbamate, the
benzyl ester, and the benzyl ether and reduced the ethynyl group
to the requisite ethyl group to afford the linear precursors, which
underwent macrolactamization to provide macrocycles 88a-b
in moderate yield in two steps. Cleavage of the Boc carbamate
and tert-butyl ester by TFA in the presence of triethylsilane70
completed the total syntheses of ustiloxin D (4)71 and ustiloxin
F (5).51 The longest linear sequence of this second-generation
approach was 15 steps. Since the amino acid residue at the C-6
position is one of the two variable positions in the ustiloxin
family, it was valuable to investigate the structural requirements
at this position. As the new approach introduces the C-6 amino
acid residue at the late stage, three analogues (6-Tle-ustiloxin,
6-Leu-ustiloxin, and 6-Phe-ustiloxin, 89a-c) were synthesized
using the same strategy.
An Improved Total Synthesis Strategy. Although the new
total syntheses of ustiloxins D and F were convergent, concise,
and versatile, the approach suffered from a low-yielding
macrocyclization step. To facilitate the synthesis of other
analogues, the macrolactamization needed to be improved. The
yields of macrocyclization were consistently in the range of 10-
20%, and coupling reagents seemed to have little effect on the
yield. However, in a nonpolar solvent, such as methylene
chloride, no product was observed, whereas in a polar solvent,
such as DMF, macrolactamization occurred with various
coupling reagents. This observation suggested that the solution
conformation of the linear precursor, likely due to intramolecular
H-bonding, could be preventing macrocyclization. A comparison
of the linear precursors of the new route with those of the
previous two routes50,54,55 showed a major difference between
the precursors: the phenolic hydroxyl group ortho to the tertiary
alkyl-aryl ether linkage was not protected in the new linear
precursor, while the other two were both protected as their
benzyl ethers in the macrocyclization reaction. A study of the
unusual effects remote substituents may have on macrocycliza-
tion reactions was reported by Boger and Yohannes.72 The
formation of 17-membered cyclic tripeptides bearing a diaryl
ether was thoroughly examined using a full range of substrates.
The study showed that remote substituents have substantial,
conformationally derived effects on the rate of macrocyclization
reactions. Interestingly, in contrast with our results, substrates
bearing a free phenol were found to undergo macrocyclization
more productively.73
The observation led us to hypothesize that intramolecular
H-bonding between the free phenolic hydroxyl group and other
(67) Nordin, I. C. J. Heterocycl. Chem. 1966, 3, 531.
(68) Campbell, A. D.; Raynham, T. M.; Taylor, R. J. K. Synthesis 1998, 1707.
(69) Nihei, K.; Kato, M. J.; Yamane, T.; Palma, M. S.; Konno, K. Synlett 2001,
1167.
(70) Mehta, A.; Jaouhari, R.; Benson, T. J.; Douglas, K. T. Tetrahedron Lett.
1992, 33, 5441.
(71) Li, P.; Evans, C. D.; Joullie´, M. M. Org. Lett. 2005, 7, 5325.
(72) Boger, L. D.; Yohannes, D. Tetrahedron Lett. 1989, 30, 5061.
(73) Boger, L. D.; Yohannes, D. J. Org. Chem. 1990, 55, 6000.
9
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Total Syntheses of Ustiloxin and Analogues
A R T I C L E S
Scheme 12. Synthesis of 14-MeO-ustiloxin D
oxygen or nitrogen atoms in the linear precursor was the
problem for the macrocyclization reaction. Computational results
supported the hypothesis, as some of the lowest-energy con-
formations of the linear precursor calculated by MacroModel
indicated hydrogen bonding between the phenolic hydroxyl
group and other oxygen and nitrogen atoms was causing the
distance between the N-terminus and C-terminus to be too large
for amide bond formation.
benzyl ester and reduced the ethynyl group to the ethyl group
to provide the 14-MeO-ustiloxin D analogue 95 (Scheme 12).51
Final Optimization of the New Total Synthesis Route. With
the results from the model study, a slight modification of the
second-generation synthesis was pursued. In this new plan,
â-hydroxytyrosine 98 was reacted with the ethynyl aziridine
80. (Scheme 13). The synthesis of â-hydroxytyrosine derivative
98 started from trans-oxazolidine 70. Acidic deprotection of
the N,O-acetal, followed by Cbz carbamate formation of the
free amino group, afforded product 96 that, unlike the Boc
carbamate 71, easily formed an oxazolidinone through an
intramolecular cyclization under basic conditions. The C-9
benzylic hydroxyl was masked as its TBS ether 97. Following
hydrolysis of acetate and methyl ester, the free acid was
selectively protected as the PMB ester to give compound 98.
Model Study: Synthesis of 14-MeO-Ustiloxin. To achieve
a better macrocyclization reaction, a linear precursor with a
protected phenolic hydroxyl group was needed. At that time,
aryl methyl ether 90 was available. This intermediate not only
was ideal for the model study but also could provide an analogue
to test the importance of the free phenolic functionality for
biological activity.
It was realized that if the model study went as proposed and
the benzyl ether was present for the macrocyclization, this
approach would require avoiding hydrogenolysis in the linear
precursor formation as in the total synthesis route. The protecting
strategy was changed accordingly to employ the acid labile
p-methoxybenzyl (PMB) ester and Boc carbamate to protect
the termini of the linear precursor. Thus, the linear precursor
would be generated by treatment with acid without affecting
the phenolic benzyl ether. After macrolactamization, protecting
group removal by hydrogenolytic cleavage would provide the
natural product analogue.
The ethynyl aziridine ring-opening reaction between the
â-hydroxytyrosine derivative 98 and aziridine 80 successfully
constructed the desired tertiary alkyl-aryl ether 99. The free
amino group of 100, liberated from the thiol-assisted nosyl
sulfonamide cleavage, was coupled with various Boc-protected
amino acids. The resulting products 101a-g were then subjected
to TFA with triethylsilane as a cation scavenger. These acidic
conditions simultaneously cleaved the Boc carbamate, the PMB
ester, and the TBS ether to afford the linear precursors.
Macrolactamization successfully provided the macrocycles
102a-e. The two-step yields (102a-d) were significantly better
than those obtained in Scheme 11. Unfortunately, proline- and
D-valine-based linear precursors 101f-g did not undergo
successful macrolactamization to provide ustiloxin analogues.
The (6R)-ustiloxin D would have provided data on the role of
configuration at C-6 for biological activity. Simultaneous
reduction of the ethynyl group to the ethyl group and depro-
tection of Cbz carbamate, benzyl ester, and benzyl ether by
hydrogenolysis afforded ustiloxin D (4), ustiloxin F (5), 6-Tle-
ustiloxin (89a), 6-Leu-ustiloxin (89b), and 6-Ile-ustiloxin (103).
A mixture of THF-H2O instead of ethanol was the solvent of
choice for the hydrogenolysis, because it was observed that the
carboxylic acid of the glycine side chain was easily esterified
The ethynyl aziridine ring-opening reaction of 80 with
â-hydroxytyrosine derivative 90 successfully formed the tertiary
alkyl-aryl ether 91. Deprotection of the o-nosyl group and
coupling of the resulting free amine 92 and N-Boc-valine
provided compound 93. Treatment of 93 with TFA liberated
the carboxylic acid and free amino group and deprotected the
TBS ether simultaneously. The linear precursor was cyclized
to give 94 in 38% yield for the two steps, which was twice the
yield that was obtained when the linear precursor had a free
phenolic hydroxyl group. This result confirmed the hypothesis
that the free phenol made macrocyclization more difficult. The
final hydrogenolysis step deprotected the Cbz carbamate and
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A R T I C L E S
Li et al.
Scheme 13. Improved Synthesis of Ustiloxins
Scheme 14. Synthesis of Three (9R,10S)-epi-Ustiloxin Analogues
under acidic conditions, during both the first total synthesis of
ustiloxin D and the synthesis of ustiloxin F.
suggested that the C-10 hydroxyl group may not play a role in
the interaction of these peptides with tubulin. Since it is known
that tubulin polymerization activity is sensitive to dimethyla-
tion of the â-hydroxytyrosine nitrogen,74 we felt that investiga-
tion of the stereochemical configuration of the 9-amino group
was important. As we had developed a robust route to all
diastereomers of the â-hydroxytyrosine moiety using Evans’
Syntheses of (9R,10S)-epi-Ustiloxin Analogues. Another
goal of the research program was to prepare (9R,10S)-epi-
ustiloxin analogues to evaluate their ability to inhibit tubulin
polymerization. Comparison of the ustiloxin and phomopsin12,16
core structures shows that the configuration of the C-10 hydroxyl
group is not critical for tubulin binding, because the two natural
products have opposite configurations at C-10. This observation
(74) Morisaki, N.; Mitsui, Y.; Yamashita, Y.; Koiso, Y.; Shirai, R.; Hashimoto,
Y.; Iwasaki, S. J. Antibiot. 1998, 51, 423.
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Total Syntheses of Ustiloxin and Analogues
A R T I C L E S
aldol-type chemistry, we chose to prepare (9R,10S)-epi-ustiloxin
analogues, and their synthesis is shown in Scheme 14.
â-Hydroxytyrosine 104 was successfully prepared by the
same approach used for its enantiomer (98) using R-salen-
aluminum catalyst. At this stage, our investigations showed that
the use of TBD (105) as a base gave better results than DBU
and CuOAc combined and also minimized side reactions in the
ethynyl aziridine ring-opening reaction.75 Indeed, the reaction
between 104 and 80 in the presence of TBD gave superior
results and afforded the chiral tertiary alkyl-aryl ether 106 in
78% yield. An additional advantage was that a 1:1 ratio of 104
and 80 was enabled by the use of TBD versus a 1.5:1 ratio
when DBU was employed.76 This result greatly improved the
material throughput for the synthesis of analogues, and this
efficient joining of the complex â-hydroxytyrosine and aziridine
components highlights the convergence of our synthetic route.
The N-nosyl group was cleaved by treatment with benzenethiol
to provide the free amine 107 in quantitative yield. The amine
was used as a branching point to incorporate three amino acid
residues, specifically valine, alanine, and isoleucine. The amino
acids valine and alanine were chosen because these residues
are found in ustiloxin D (4) and F (5), respectively. In previous
investigations, an active 6-Ile-ustiloxin analogue had been
prepared, so we chose to introduce isoleucine to provide a
comparable (9R,10S)-epi-6-Ile-ustiloxin analogue.
Figure 9. Evaluation of C-6 substitution on inhibition of tubulin polym-
erization.
C-6 (6-Ile-ustiloxin 103), a slight decrease in activity was
observed. The isoleucine residue possesses the same degree of
branching as the valine residue in 4 but is extended by an
additional methylene unit, which may cause less effective
binding of 103 relative to 4. When the branching element at
the C-6 position was moved further from the macrocycle or
removed, as in 6-Leu-ustiloxin (89b) or ustiloxin F (5),
respectively, inhibition of tubulin polymerization was further
decreased (IC50 ) 8.2 µM). An aromatic residue, such as
phenylalanine (6-Phe-ustiloxin 89c), resulted in a still less active
compound (IC50 ) 25 µM). These results suggest that a
relatively compact, but highly branched aliphatic residue at
the C-6 position is optimal for inhibition of tubulin polymeri-
zation.
Biological evaluation of 14-MeO-ustiloxin D (95) showed
no inhibition of tubulin polymerization (IC50 > 40 µM),
demonstrating that the phenol moiety of the ustiloxin family is
essential for binding to the R,â-tubulin dimer. Iwasaki also
reported loss of activity upon methylation of the phenol in a
semisynthetic ustiloxin analogue.74 Similarly, (9R,10S)-epi-
ustiloxin D (110a), (9R,10S)-epi-ustiloxin F (110b), and (9R,-
10S)-epi-6-Ile ustiloxin (110c) yielded IC50 values >40 µM
(Figure 10). Since the phomopsin peptides13 have an S config-
uration at C-10, these results imply that the naturally occurring
The coupling of amine 107 with valine, alanine, or isoleucine
provided the desired protected linear precursors 108a-c,
respectively, in excellent yields. Using the improved macro-
lactamization and same end-game strategy, (9R,10S)-epi-
ustiloxin analogues 110a-c were prepared efficiently. These
three (9R,10S)-epi-ustiloxin analogues are the first analogues
synthesized to probe the structural basis for the interaction of
the â-hydroxytyrosine region of the ustiloxin family with
tubulin.
Bioactivity
Disturbance of the dynamic equilibrium of microtubules has
critical consequences for cell survival. Since disruption of the
mitotic pathway represents a challenging and significant target
for oncology, the investigation of ligands that interact with
tubulin is essential to better understand their mechanisms of
action and biological activity. The course of our investigations
led to the preparation of suitable analogues to initiate structure
activity relationship studies in the ustiloxin-phomopsin class
of antimitotic peptides. The analogues were designed to probe
the importance of different structural regions of these peptides
for biological activity. Thus far, modifications at the valine
residue (C-6), the â-hydroxytyrosine residue (C-9 and C-10),
and the phenolic hydroxyl have been investigated.
The biological property we examined was the effect of these
compounds on the polymerization of purified tubulin. Tubulin
showed a tolerance for structural variation at the C-6 macro-
cyclic position of the ustiloxins (Figure 9). The most active
analogues had the highest degree of branching at the C-6
position, with the synthetic 6-Tle-ustiloxin (89a) and ustiloxin
D (4) displaying comparable activities (IC50 ) 1.2 and 1.5 µM,
respectively). When an isoleucine residue was incorporated at
Figure 10. Inactivity of 14-MeO-ustiloxin D and (9R,10S)-epi-ustiloxin
analogues.
9S-methylamino group is critical for binding to the R,â-tubulin
heterodimer. Structural changes that alter the macrocyclic
conformation, such as stereochemical modifications, likely place
the 9R-methylamino group at a distance too great to make
contact with the tubulin protein and results in the observed loss
(75) Forbeck, E. M.; Evans, C. D.; Gilleran, J. A.; Li, P.; Joullie´, M. M. J. Am.
Chem. Soc 2007, 129, 14463.
(76) Evans, C. D. Ph.D. Thesis. University of Pennsylvania, Philadelphia, PA,
2007.
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A R T I C L E S
Li et al.
of activity. Future studies to investigate other regions of the
ustiloxin/phomopsin structure should reveal other critical points
for binding to tubulin and advance our understanding of the
interaction of tubulin with these heterodetic peptides.
were prepared using this highly versatile strategy. Studies on
the inhibition of tubulin polymerization revealed that limited
variation at the C-6 position is tolerated and that the free
phenolic hydroxyl group and the S configuration of the C-9
methylamino group are essential for bioactivity. Investigation
of other analogues of the ustiloxin family is ongoing.
Conclusion
Two total syntheses of ustiloxin natural products were
achieved. The first total synthesis of ustiloxin D featured
application of an SNAr reaction to construct the tertiary alkyl-
aryl ether and regioreversed Sharpless aminohydroxylation to
install the two chiral centers of the â-hydroxytyrosine moiety
in one step. The synthesis provided a scaffold for further
development. The second-generation synthesis took advantage
of a newly developed trisubstituted aziridine ring-opening by
phenol derivatives and resulted in a concise and convergent
synthesis. Ustiloxin D, ustiloxin F, and eight ustiloxin analogues
Acknowledgment. We thank NIH (CA-40081), NSF Grant
0515443, Wyeth Research, and the University of Pennsylvania
Research Foundation for financial support.
Supporting Information Available: Experimental detail and
characterization for all new compounds in the synthesis of
ustiloxin D, ustiloxin F, and eight analogues. This material is
available free of charge via the Internet at http://pubs.acs.org.
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