Development of an enantioselective synthetic route to neocarzinostatin chromophore and its use for multiple radioisotopic incorporation

Article abstract of DOI:10.1021/ja012487x

A convergent, enantioselective synthetic route to the natural product neocarzinostatin chromophore (1) is described. Synthesis of the chromophore aglycon (2) was targeted initially. Chemistry previously developed for the synthesis of a neocarzinostatin core model (4) failed in the requisite 1,3-transposition of an allylic silyl ether when applied toward the preparation of 2 with use of the more highly oxygenated substrates 27 and 54. An alternative synthetic plan was therefore developed, based upon a proposed reduction of the epoxy alcohol 58 to form the aglycon 2, a transformation that was achieved in a novel manner, using a combination of the reagents triphenylphosphine, iodine, and imidazole. The successful route to 1 and 2 began with the convergent coupling of the epoxydiyne 15, obtained in 9 steps (43% overall yield) from D-glyceraldehyde acetonide, and the cyclopentenone (+)-14, prepared in one step (75-85% yield) from the prostaglandin intermediate (+)-16, affording the alcohol 22 in 80% yield and with 20:1 diastereoselectivity. The alcohol 22 was then converted into the epoxy alcohol 58 in 17 steps with an average yield of 92% and an overall yield of 22%. Key features of this sequence include the diastereoselective Sharpless asymmetric epoxidation of allylic alcohol 81 (98% yield); intramolecular acetylide addition within the epoxy aldehyde 82, using Masamune's lithium diphenyltetramethyldisilazide base (85% yield); selective esterification of the diol 84 with the naphthoic acid 13 followed by selective cleavage of the chloroacetate protective group in situ to furnish the naphthoic acid ester 85 in 80% yield; and elimination of the tertiary hydroxyl group within intermediate 88 using the Martin sulfurane reagent (79% yield). Reductive transposition of the product epoxy alcohol (58) then formed neocarzinostatin chromophore aglycon (2, 71% yield). Studies directed toward the glycosylation of 2 focused initially on the preparation of the N-methylamino → hydroxyl replacement analogue 3, an α-D-fucose derivative of neocarzinostatin chromophore, formed in 42% yield by a two-step Schmidt glycosylation-deprotection sequence. For the synthesis of 1, an extensive search for a suitable 2′-N- methylfucosamine glycosyl donor led to the discovery that the reaction of 2 with the trichloroacetimidate 108, containing a free N-methylamino group, formed the α-glycoside 114 selectively in the presence of boron trifluoride diethyl etherate. Subsequent deprotection of 114 under mildly acidic conditions then furnished the labile chromophore (1). The synthetic route was readily modified for the preparation of singly and doubly ³H- and ¹⁴C -labeled 1, compounds unavailable by other means, for studies of the mechanism of action of neocarzinostatin in vivo.

Full text of DOI:10.1021/ja012487x

Published on Web 04/19/2002
Development of an Enantioselective Synthetic Route to
Neocarzinostatin Chromophore and Its Use for Multiple
Radioisotopic Incorporation
Andrew G. Myers,* Ralf Glatthar, Marlys Hammond, Philip M. Harrington,
Elaine Y. Kuo, Jun Liang, Scott E. Schaus, Yusheng Wu, and Jia-Ning Xiang
Contribution from the Department of Chemistry and Chemical Biology, HarVard UniVersity,
Cambridge, Massachusetts 02138
Received November 6, 2001
Abstract: A convergent, enantioselective synthetic route to the natural product neocarzinostatin chro-
mophore (1) is described. Synthesis of the chromophore aglycon (2) was targeted initially. Chemistry
previously developed for the synthesis of a neocarzinostatin core model (4) failed in the requisite 1,3-
transposition of an allylic silyl ether when applied toward the preparation of 2 with use of the more highly
oxygenated substrates 27 and 54. An alternative synthetic plan was therefore developed, based upon a
proposed reduction of the epoxy alcohol 58 to form the aglycon 2, a transformation that was achieved in
a novel manner, using a combination of the reagents triphenylphosphine, iodine, and imidazole. The
successful route to 1 and 2 began with the convergent coupling of the epoxydiyne 15, obtained in 9 steps
(43% overall yield) from D-glyceraldehyde acetonide, and the cyclopentenone (+)-14, prepared in one step
(75-85% yield) from the prostaglandin intermediate (+)-16, affording the alcohol 22 in 80% yield and with
g20:1 diastereoselectivity. The alcohol 22 was then converted into the epoxy alcohol 58 in 17 steps with
an average yield of 92% and an overall yield of 22%. Key features of this sequence include the
diastereoselective Sharpless asymmetric epoxidation of allylic alcohol 81 (98% yield); intramolecular acetylide
addition within the epoxy aldehyde 82, using Masamune’s lithium diphenyltetramethyldisilazide base (85%
yield); selective esterification of the diol 84 with the naphthoic acid 13 followed by selective cleavage of the
chloroacetate protective group in situ to furnish the naphthoic acid ester 85 in 80% yield; and elimination
of the tertiary hydroxyl group within intermediate 88 using the Martin sulfurane reagent (79% yield).
Reductive transposition of the product epoxy alcohol (58) then formed neocarzinostatin chromophore aglycon
(2, 71% yield). Studies directed toward the glycosylation of 2 focused initially on the preparation of the
N-methylamino f hydroxyl replacement analogue 3, an R-D-fucose derivative of neocarzinostatin
chromophore, formed in 42% yield by a two-step Schmidt glycosylation-deprotection sequence. For the
synthesis of 1, an extensive search for a suitable 2′-N-methylfucosamine glycosyl donor led to the discovery
that the reaction of 2 with the trichloroacetimidate 108, containing a free N-methylamino group, formed the
R-glycoside 114 selectively in the presence of boron trifluoride diethyl etherate. Subsequent deprotection
of 114 under mildly acidic conditions then furnished the labile chromophore (1). The synthetic route was
readily modified for the preparation of singly and doubly 3H- and 14C-labeled 1, compounds unavailable by
other means, for studies of the mechanism of action of neocarzinostatin in vivo.
Introduction and Background
After the discovery of neocarzinostatin, several other members
of the newly defined enediyne antibiotic family were identified.4
Among these are some of the most potent antiproliferative agents
known. The family can be broadly divided into a chromoprotein
subclass that includes neocarzinostatin, kedarcidin,5 and C-1027,6
Neocarzinostatin (NCS) was the first “enediyne” antitumor
antibiotic identified and is the prototypical member of the
chromoprotein class.1 Isolated from the culture filtrate of
Streptomyces carzinostaticus, neocarzinostatin was found to
possess broad-spectrum antibiotic activity and showed potent
antiproliferative effects in a wide variety of tumor cell lines
both in vitro and in vivo.2 A polymer-conjugated version of
the drug (conjugated via its binding protein, vide infra) has been
approved for the treatment of cancers of the liver and brain, as
well as leukemia, in Japan.3
(2) (a) Kinami, Y.; Miyazaki, H.; Koyama, F.; Nogichi, M.; Konishi, K.;
Furukawa, N.; Nishita, Y. J. Jpn. Soc. Cancer Ther. 1975, 10, 502. (b)
Maeda, H.; Sakamoto, S.; Ogata, J. Anti-micro. Agents Chemother. 1977,
11, 941. (c) Satake, I.; Tari, K.; Yamamoto, M.; Nishimura, H. J. Urol.
1985, 133, 87.
(3) (a) Maeda, H.; Takeshita, J.; Kanamaru, R. Int. J. Pept. Protein Res. 1979,
14, 81. (b) Maeda, H.; Matsumoto, T.; Konno, T.; Iwai, K.; Ueda, M. J.
Protein Chem. 1984, 3, 181. (c) Maeda, H.; Ueda, M.; Morinaga, T.;
Matsumoto, T. J. Med. Chem. 1985, 28, 455.
(1) (a) Ishida, N. J.; Miyazaki, M.; Kumagai, K.; Rikimaru, M. J. Antibiot.
1965, 18, 68. (b) Maeda, H.; Kumagai, K.; Ishida, N. J. Antibiot. 1966,
19, 253. (c) Maeda, H.; Edo, K.; Ishida, N. Neocarzinostatin; Springer:
Tokyo, 1997. (d) Kumagai, K. J. Antibiotics 1962, A15, 53.
(4) Reviews: (a) Smith, A. L.; Nicolaou, K. C. J. Med. Chem. 1996, 39, 2103.
(b) Grissom, J. W.; Gunawardena, G. U.; Klingberg, D.; Huang, D.
Tetrahedron 1996, 52, 6453. (c) Zhen, X.; Goldberg, I. H. Compr. Nat.
Prod. Chem. 1999, 7, 553.
9
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Synthesis of Neocarzinostatin Chromophore
A R T I C L E S
and a nonproteinaceous group, exemplified by the molecules
dynemicin A,7 the calicheamicins,8 and the esperamicins.9
Members of the chromoprotein class are composed of a 1:1
complex of a “small molecule” chromophore component and a
binding protein, ∼11 kD (113 amino acids) in the case of NCS
apoprotein. The distinction is critical from the synthetic
chemist’s perspective, for the chromophoric components of the
chromoprotein agents identified thus far are exceedingly unstable
in isolation and therefore represent much more challenging
targets.10
crystallography. To date, this is the only chromoprotein
antibiotic (and, for that matter, the only unmodified enediyne
agent) for which such detailed structural information is avail-
able.11 Each π face of the nine-membered epoxy diyne ring
contacts the edge of the arene ring of a phenylalanine residue
(Phe52 and Phe78) in the binding pocket.11 The chromophore
component (1) is readily extracted into organic solvents and is
stable under mildly acidic conditions at -70 °C.12
The biological activity of NCS is believed to derive from
the ability of the chromophore component (1) to cleave DNA,
a process shown by Goldberg and co-workers to be accelerated
g1000-fold in the presence of thiols.13 This acceleration is
proposed to arise by a nucleophilic activation process involving
addition of thiols to C-12 of the chromophore with concomitant
epoxide opening to form a cumulene intermediate that subse-
quently undergoes biradical-forming cycloaromatization.14 This
proposal is now supported by a large body of evidence, including
the direct observation of an unstable cumulene intermediate
(methylthioglycolate adduct) and its low-temperature (-38 °C)
transformation by cycloaromatization.15 In the presence of
double-stranded DNA, the biradical is proposed to abstract
hydrogen atoms from deoxyribose residues of double-stranded
DNA, producing mainly single- and a small number of double-
stranded DNA cleavage products. There is little sequence
selectivity in this process, but it exhibits pronounced base
selectivity, with breaks occurring mainly at thymine and adenine
residues (T > A . G > C).16
The structure of NCS chromophore is highly unusual,
characterized most notably by the unsaturated epoxy bicyclo-
[7.3.0]dodecadienediyne core.17 The central nine-membered ring
of the core is presumably strained, more so than the 10-
membered diacetylene rings of, e.g., calicheamicin and dyne-
micin. Deformation within the nine-membered ring is evident
from the crystal structure of NCS;11 the average C-CtC bond
angle (161.5 ( 1.2°) is significantly distorted from linearity.
This distortion presumably contributes to the lability of the
chromophore in solution. Once separated from its carrier protein
(apo NCS), the chromophore is highly unstable, particularly in
the basic pH region, exhibiting a half-life of ∼30 s at pH 8.0
(0 °C).1c The chromophore is more stable in weakly acidic media
and in organic solvents. The half-life of the chromophore in a
1:1 mixture of acetic acid and water is ∼5 h at 23 °C. More
strongly acidic conditions lead to decomposition of the chro-
mophore.18 In addition, the chromophore is known to be light-
The structures of the NCS protein-chromophore complex
and the NCS apoprotein have been determined by X-ray
(5) Leet, J. E.; Schroeder, D. R.; Langley, D. R.; Coloson, K. L.; Huang, S.;
Klohr, S. E.; Lee, M. S.; Golik, J.; Hofstead, S. J.; Doyle, T. W.; Matson,
J. A. J. Am. Chem. Soc. 1993, 115, 8432. Kawata, S.; Ashiyawa, S.; Hirama,
M. J. Am. Chem. Soc. 1997, 119, 12012.
(6) (a) Hu, J.; Xue, Y.-C.; Xie, M.; Zhang, R.; Otani, T.; Minami, Y.; Yamada,
Y.; Marunaka, T. J. Antibiot. 1988, 41, 1575. (b) Otani, T.; Minami, Y.;
Marunaka, T.; Zhang, R.; Xie, M. J. Antibiot. 1988, 41, 1580.
(7) Konishi, M.; Ohkuma, H.; Matsumoto, K.; Tsuno, T.; Kamei, H.; Miyaki,
T.; Oki, T.; Kawaguchi, H.; Vanduyne, G. D.; Clardy, J. J. Antibiot. 1989,
42, 1449.
(8) (a) Golik, J.; Clardy, J.; Dubby, G.; Groenewold, G.; Kawaguchi, H.;
Konishi, M.; Krishnan, B.; Ohkuma, H.; Saitoh, K.; Doyle, T. W. J. Am.
Chem. Soc. 1987, 109, 3461 and 3462. (b) Lee, M. D.; Dunne, T. S.; Siegel,
M. M.; Chang, C. C.; Morton, G. O.; Borders, D. B. J. Am. Chem. Soc.
1987, 109, 3464 and 3466.
(9) (a) Konishi, M.; Ohkuma, H.; Saitoh, K.; Kawaguchi, H.; Golik, J.; Dubay,
G.; Groenewold, G.; Krishnan, B.; Doyle, T. W. J. Antibiot. 1985, 38, 1605.
(b) Golik, J.; Clardy, J.; Dubay, G.; Groenewold, G.; Kawaguchi, H.;
Konishi, M.; Krishnan, B.; Ohkuma, H.; Saitoh, K.; Doyle, T. W. J. Am.
Chem. Soc. 1987, 109, 3461.
(10) (a) Synthesis of calicheamicin: Nicoloau, K. C.; Hummel, C. W.; Pitsinos,
E. N.; Nakada, M.; Smith, A. L.; Shibayama, K.; Saimoto, H. J. Am. Chem.
Soc. 1992, 114, 10082. (b) Hitchcock, S. A.; Boyer, S. H.; Chu-Moyer, M.
Y.; Olson, S. H.; Danishefsky, S. J. Angew. Chem., Int. Ed. Engl. 1994,
33, 858. (c) Synthesis of dynemicin A and its analogues: Taunton, J.; Wood,
J. L.; Schreiber, S. L. J. Am. Chem. Soc. 1993, 115, 10378. (d) Myers, A.
G.; Fraley, M. E.; Tom, N. J.; Cohen, S. B.; Madar, D. J. Chem. Biol.
1995, 2, 33. (e) Shair, M. D.; Yoon, T.-Y.; Danishefsky, S. J. Angew. Chem.,
Int. Ed. Engl. 1995, 34, 1721.
(11) Kim, K.-H.; Kwon, B.-M.; Myers, A. G.; Rees, D. C. Science 1993, 262,
1042.
(12) Isolation of 1: Napier, M. A.; Holmquist, B.; Strydom, D. J.; Goldberg, I.
H. Biochem. Biophys. Res. Commun. 1979, 89, 635. (b) Koide, Y.; Ishii,
F.; Hasuda, K.; Koyama, Y.; Edo, K.; Katamine, S.; Kitame, F.; Ishida, N.
J. Antibiot. 1980, 33, 342.
(13) (a) Beerman, T. A.; Goldberg, I. H. Biochem. Biophys. Res. Commun. 1974,
59, 1254. (b) Beerman, T. A.; Poon, R.; Goldberg, I. H. Biochim. Biophys.
Acta 1977, 475, 294. (c) Kappen, L. S.; Goldberg, I. H. Nucleic Acids Res.
1978, 5, 2959.
(14) Myers, A. G. Tetrahedron Lett. 1987, 28, 4493.
(15) (a) Myers, A. G.; Proteau, P. J. J. Am. Chem. Soc. 1989, 111, 1146. (b)
Myers, A. G.; Cohen, S. B.; Kwon, B.-M. J. Am. Chem. Soc. 1994, 116,
1670.
(16) Hatayama, T.; Goldberg, I. H.; Takeshira, M.; Grollman, A. P. Proc. Natl.
Acad. Sci. U.S.A. 1978, 75, 3603. See also ref 9b.
(17) (a) Chromophore structure: Edo, K.; Mizugaki, M.; Koide, Y.; Seto, H.;
Furihata, K.; Otake, N.; Ishida, N. Tetrahedron Lett. 1985, 26, 331. (b)
Carbohydrate stereochemistry: Edo, K.; Akiyama, Y.; Saito, K.; Mizugaki,
M.; Koide, Y.; Ishida, N. J. Antibiot. 1986, 39, 1615. (c) Chromophore
stereochemistry: Myers, A. G.; Proteau, P. J.; Handel, T. M. J. Am. Chem.
Soc. 1988, 110, 7212.
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A R T I C L E S
Myers et al.
Scheme 1. Known Reactions of Neocarzinostatin Chromophore (1)
sensitive,19 and is especially sensitive toward decomposition in
neat form, presumably by free-radical-mediated oligomerization.
Most of the known reaction pathways involving 1 are sum-
marized in Scheme 1.1b,14,20-22
because the aglycon (2) was anticipated to be even less stable
than the chromophore (1), a prediction that was later borne out.
Efforts to form the aglycon by hydrolysis of the 2-aminosugar
of the natural product failed, not surprisingly, using acidic
conditions or any of several commercial glycosidases. Neverthe-
less, efforts directed toward the laboratory synthesis of this target
of undefined but certainly modest stability were pursued in light
of the convergence inherent in a route to the synthesis of 1 that
would involve the aglycon 2, as well as the opportunity to study
the aglycon mechanistically and to use it for the synthesis of
analogues of 1 with modified carbohydrate residues.
The significant biological activity, complex structure, and high
reactivity of NCS chromophore define it as a compelling, highly
challenging synthetic target. The reactivity of the chromophore
core in particular complicates the development of a synthetic
route to 1. Although notable achievements in this area have
been reported,23 model studies providing structures containing
the nine-membered ring have been few, and these have typically
lacked other critical structural elements of 1.
Some time ago we began a program to synthesize NCS
chromophore and study its mechanism of action. Our initial goal
was to develop methodology to synthesize the core functionality
and then to apply this chemistry to prepare the chromophore
aglycon (2). The aglycon (2) was unknown at the outset of our
studies, and remained so until it was synthesized in our
laboratory several years later. As a consequence, its stability
profile was unknown, a point of some concern, particularly
(18) For example, the half-life of the chromophore is ∼25 min in 50% aqueous
acetic acid containing trifluoroacetic acid. Myers, A. G.; Harrington, P.
M. Unpublished results. See also ref 54.
(19) (a) Uesawa, Y.; Kuwahara, J.; Sugiura, Y. Biochem. Biophys. Res. Commun.
1989, 164, 903. (b) Gomibuchi, T.; Fujiwara, K.; Nehira, T.; Hirama, M.
Tetrahedron Lett. 1993, 36, 5753. (c) Gomibuchi, T.; Hirama, M. J.
Antibiotics 1995, 48, 738.
(20) (a) Povirk, L. F.; Goldberg, I. H. Biochemistry 1980, 19, 4773. (b)
Strassinopoulos, A.; Goldberg, I. H. Biochemistry 1995, 34, 15359.
(21) (a) Sugiyama, H.; Yamashita, K.; Nishi, M.; Saito, I. Tetrahedron Lett.
1992, 33, 515. (b) Myers, A. G.; Arvedson, S. P.; Lee, W. W. J. Am. Chem.
Soc. 1996, 118, 4725.
(22) (a) Tanaka, T.; Fujiwara, K.; Hirama, M. Tetrahedron Lett. 1990, 31, 5947.
(b) Fujiwara, K.; Sakai, H.; Tanaka, T.; Hirama, M. Chem. Lett. 1994,
457.
Here, we describe in detail the chemistry leading to an
enantioselective synthesis of neocarzinostatin chromophore
aglycon (2) and then of the chromophore itself,24 to date, the
only successful synthesis of any of the chromoprotein chro-
mophore structures. We have adapted our synthetic route to
prepare two different radiolabeled forms of 1 and the R-D-
fucosyl NCS chromophore analogue 3, compounds of impor-
tance in mechanistic studies of NCS.25
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Synthesis of Neocarzinostatin Chromophore
A R T I C L E S
Scheme 2. Retrosynthetic Analysis of a Core Model Featuring an Intramolecular Acetylide Addition Strategy
Early Model Studies
vinylidene carbene intermediate into the C-H bond of a C-7
terminal alkyne failed (insertion into the solvent, THF, occurred
preferentially), our focus turned toward the formation of this
bond by a more conventional method, involving the intramo-
lecular addition of a C-7 localized acetylide anion to the
carbonyl carbon (C-8) of an aldehyde group.
In the initial planning of a synthetic route to the aglycon 2,
it was anticipated that the formation of the reactive and strained
nine-membered carbocyclic ring would be a formidable problem.
We therefore undertook as a model study the synthesis of a
simplified epoxy bicyclo[7.3.0]dienediyne, structure 4 (Scheme
2).26,27 The C-7-C-8 σ-bond of 4 was identified as a strategic
bond in our initial retrosynthetic analysis (more recently, we
have been successful in a strategy that targets the transannular
C-1-C-9 σ-bond).23m,23p When our initial efforts to form the
C-7-C-8 bond by the intramolecular insertion of a C-8
To minimize the number of synthetic transformations post
ring-closure, it was desirable to conduct the proposed intramo-
lecular acetylide addition in the presence of the epoxide ring.
Although we initially had concerns about the feasibility of an
epoxy acetylide intermediate (3-chloroacetylides form vinylidene
carbenes at low temperature),28 this aspect of the chemistry did
not prove to be problematic. The proposed acetylide-aldehyde
addition reaction was not straightforward, however. Now, with
the benefit of experience in attempts with many such closures
in diverse substrates, it is evident that this is a highly
idiosyncratic reaction, whose success depends on favorable
kinetics for closure relative to intermolecular acetylide-
aldehyde addition (dimerization), as well as other competing
reactions, such as the addition of the acetylide-forming base to
the aldehyde. Not surprisingly, these additions can be highly
(23) (a) Wender, P. A.; Harmata, M.; Jeffrey, D.; Mukai, C.; Suffert, J.
Tetrahedron Lett. 1988, 29, 909. (b) Hirama, M.; Fujiwara, K.; Shigematu,
K.; Fukazawa, Y. J. Am. Chem. Soc. 1989, 111, 4120. (c) Suffert, J.
Tetrahedron Lett. 1990, 31, 7437. (d) Doi, T.; Takahashi, T. J. Org. Chem.
1991, 56, 3456. (e) Hirama, M.; Gomibuchi, T.; Fujiwara, K. J. Am. Chem.
Soc. 1991, 113, 9851. (f) Nakatani, K.; Arai, K.; Terashima, S. Tetrahedron
1993, 49, 1901. (g) Takahashi, T.; Tanaka, H.; Hirai, Y.; Doi, T.; Yamada,
H.; Shiraki, T.; Sugiura, Y. Angew. Chem., Int. Ed. Engl. 1993, 32, 1657.
(h) Matsumoto, Y.; Kuwatani, Y.; Ueda, I. Tetrahedron Lett. 1995, 36,
3197. (i) Toshima, K.; Ohta, K.; Yanagawa, K.; Kano, T.; Nakata, M.;
Kinoshita, M.; Matsumura, S. J. Am. Chem. Soc. 1995, 117, 10825. (j)
Magnus, P.; Carter, R.; Davies, M.; Elliott, J.; Pitterna, T. Tetrahedron
1996, 52, 6283. (k) Dai, W.-M.; Fong, K. C. Tetrahedron Lett. 1996, 37,
8413. (l) Caddick, S.; Delisser, V. M. Tetrahedron Lett. 1997, 38, 2355.
(m) Myers, A. G.; Goldberg, S. D. Tetrahedron Lett. 1998, 39, 9633. (n)
Toyama, I. S.; Kikuchi, T.; Hirama, M. Synlett 1998, 1308. (o) Bru¨ckner,
R.; Suffert, J. Synlett 1999, 6, 657. (p) Myers, A. G.; Goldberg, S. D. Angew.
Chem., Int. Ed. Engl. 2000, 39, 2732. (q) Toyama, K.; Iguchi, S.; Sakazaki,
H.; Oishi, T.; Hirama, M. Bull. Chem. Soc. Jpn. 2001, 74, 997.
(24) (a) Myers, A. G.; Hammond, M.; Wu, Y.; Xiang, J.-N.; Harrington, P. M.;
Kuo, E. Y. J. Am. Chem. Soc. 1996, 118, 10006. (b) Hammond, M., Ph.D.
Thesis, California Institute of Technology, 1996. (c) Myers, A. G.; Liang,
J.; Hammond, M.; Harrington, P. M.; Wu, Y.; Kuo, E. Y. J. Am. Chem.
Soc. 1998, 120, 5319. (d) Liang, J., Ph.D. Thesis, Harvard University, 2000.
(25) Myers, A. G.; Liang, J.; Hammond, M. Tetrahedron Lett. 1999, 40, 5129.
(26) (a) Myers, A. G.; Harrington, P. M.; Kuo, E. Y. J. Am. Chem. Soc. 1991,
113, 694. (b) Myers, A. G.; Harrington, P. M.; Kwon, B.-M. J. Am. Chem.
Soc. 1992, 114, 1086.
(27) The conditions (lithium hexamethyldisilazide in the presence of cerium-
(III) chloride) have been found to be successful in other intramolecular
acetylide-aldehyde cyclizations, see: (a) Iida, K.; Hirama, M. J. Am. Chem.
Soc. 1994, 116, 10310. (b) Sato, I.; Akahori, Y.; Iida, K.; Hirama, M.
Tetrahedron Lett. 1996, 37, 5135. (c) Kawata, S.; Yoshimura, F.; Irie, J.;
Ehara, H.; Hirama, M. Synlett 1997, 250. (d) Kobayashi, S.; Reddy, R. S.;
Sugiura, Y.; Sasaki, D.; Miyagawa, N.; Hirama, M.; Iida, K.; Hirama, M.
J. Am. Chem. Soc. 2001, 123, 2778.
(28) (a) Hennion, G. F.; Maloney, D. E. J. Am. Chem. Soc. 1951, 73, 4735. (b)
Hartzler, H. D. J. Am. Chem. Soc. 1959, 81, 2024. (c) Hennion, G. F.;
Biosselle, A. P. J. Am. Chem. Soc. 1961, 83, 2677. (d) Battioni, J.-P.;
Chodkiewicz, W. Bull. Soc. Chim. Fr. 1969, 911.
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Scheme 3. Synthesis of the Core Functionality of Neocarzinostatin Chromophore
sensitive to the reaction conditions. As exemplified in the
discussion that follows, the use of Lewis-acid additives and
hindered amide bases can prove critical for successful cyclization
to occur.
4. As expected, the model dienediyne 4 was highly unstable
and, in fact, proved to be less stable than the natural product
(1). Although we were successful in developing a 1,3-transposi-
tion-elimination sequence for the synthesis of the model
compound 4, later, more advanced substrates with C-11-
oxygenation did not undergo the solvolytic transposition reac-
tion, or were diverted by internal trapping to produce compounds
that could not be used as intermediates for the synthesis of 1
(vide infra). The evolution of the successful pathway to 1 from
these initial studies is described in the discussion that follows.
In the case at hand, the most direct way to apply an
intramolecular acetylide-aldehyde addition strategy to synthe-
size 4 was to hydrate the diene group retrosynthetically by 1,4-
addition (structure 5) and then disconnect the C-7-C-8 σ-bond.
Unfortunately, all attempts to bring about the closure reaction
within such substrates (e.g., 6) and, later, more highly oxygen-
ated analogues failed, although macrocyclic dimeric products
could be isolated. One possible explanation for the failure of
substrate 6 to undergo ring closure involved trajectory analysis,
where it was recognized that the planar form of the R,â-
unsaturated aldehyde was poorly disposed for intramolecular
addition of the acetylide anion (Scheme 2). We reasoned that
if C-1 were sp3-hybridized, rather than sp2-hybridized, the
trajectory for intramolecular acetylide addition would be much
more favorable. The proposed hybridization change could be
brought about retrosynthetically by 1,4-hydration of the diene
of 4 as initially proposed (structure 5), followed by 1,3-
transposition of the resulting allylic silyl ether within 5.
Disconnection of the C-7-C-8 σ-bond after transposition affords
a cyclization substrate such as 7. When implemented, this
alternative strategy for closure of the nine-membered ring proved
to be successful, but brought with it a 1,3-transposition problem
that proved to be the most challenging problem that we were
to face in the synthetic route. The alternative cyclization
substrate 7 was prepared as shown (Scheme 3), and was found
to undergo efficient ring-closure at -78 °C upon treatment with
lithium hexamethyldisilazide in the presence of cerium(III)
chloride,26,27 providing the propargylic alcohols 10 and 11 in
87% yield. We were now confronted with the challenge of
effecting 1,3-allylic transposition and then 1,4-elimination to
form the diene of the target 4. After much experimentation, a
transposition scheme was developed involving exposure of the
bis(trimethylsilyl) ether derived from diastereomer 1129 to
trifluoroacetic acid (0.2 M in dichloromethane, 5 equiv) at 0
°C, producing the trifluoroacetate 12 in 49% yield. Subsequent
functional group manipulation provided the allylic alcohol 5,
which underwent 1,4-elimination to afford the epoxydienediyne
Initial Approaches to the Synthesis of Neocarzinostatin
Chromophore Aglycon
Mapping a strategy for the synthesis of the aglycon 2 onto a
template suggested by our route to 4, we envisioned the
convergent assembly of 2 from three components: the naphthoic
acid 13, the cyclopentenone 14, and the epoxydiyne 15.
The naphthoic acid component 13 was prepared in six steps
in 40-48% yield from 4-bromo-3-methylanisole.30 The cyclo-
(29) Use of the diastereomer 11 leads to the desired C-10 S stereoisomer 12, by
(30) Myers, A. G.; Subramanian, V.; Hammond, M. Tetrahedron Lett. 1996,
37, 587.
a suprafacial transposition process.
9
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Synthesis of Neocarzinostatin Chromophore
A R T I C L E S
Scheme 4
pentenone (+)-14 was prepared from the well-established
prostaglandin intermediate (+)-16 (>90% ee)31 in one step by
a carefully optimized application of the method of Noyori et
al.,32 involving the sequential addition of trimethylsilylphenyl
selenide, trimethyl orthoformate, and hydrogen peroxide-
pyridine to (+)-16. The timing of the addition of hydrogen
peroxide-pyridine to the intermediate â-phenylselenenyl dim-
ethyl acetal, in particular, was critical. If the addition of the
oxidant was delayed, decomposition occurred, and if the addition
was conducted too rapidly (before the trimethylsilyl enol ether
intermediate had reacted completely with added trimethyl
orthoformate), then incomplete conversion of the starting
cyclopentenone ((+)-16) was observed. The enantiomeric purity
of (+)-14 was determined to be g90% ee by a sequence
involving 1,2-reduction with DIBAL, isolation of the major (cis)
1
diastereomer, Mosher esterification, and H NMR analysis.
The synthesis of the epoxydiyne component 15 is shown in
Scheme 4. Addition of lithium (trimethylsilyl)acetylide to
D-glyceraldehyde acetonide33 produced a diastereomeric mixture
of propargylic alcohols (ratio 1.3:1, 81% yield). Oxidation of
the secondary alcohol gave the corresponding propargylic ketone
17, which was added directly (in solution) to the ylide 18,
affording a mixture of the olefins 19 (∼3:1), in which the desired
trans isomer (with the acetylenic substituents cis) predominated,
in 88% yield. The isomers were not separated at this stage, but
instead the trimethylsilyl and acetonide protective groups were
removed by sequential treatment with potassium carbonate in
methanol followed by hydrochloric acid (2 N, aqueous tetrahy-
drofuran). The desired diol (20) was then isolated in isomerically
pure form by flash column chromatography (72% yield).
Attempts to use the diol 20 as a substrate in the Sharpless
asymmetric epoxidation reaction (SAE)34 provided further
evidence that the stereochemical outcome in SAE of allylic diols
is difficult to predict.35 No reaction was observed when 20 was
treated under catalytic or stoichiometric SAE conditions36
employing (-)-diethyl D-tartrate, while the epoxidation with
(+)-diethyl L-tartrate as ligand (stoichiometric conditions)
afforded the â-epoxide in quantitative yield. This epoxide was
not useful for the synthesis of 1. This problem was easily
circumvented by the selective silylation of the primary alcohol
of 20 with tert-butyldiphenylsilyl chloride (TDSCl, quantitative
yield); using the resulting silyl ether as substrate, SAE with
(-)-diethyl tartrate as ligand (stoichiometric conditions) pro-
vided the required R-epoxide (21) in 94% yield. Cleavage of
the silyl ether occurred upon exposure of 21 to triethylamine
trihydrofluoride, affording the corresponding diol epoxide as a
pale yellow solid in 98% yield (g95% ee). Treatment of this
diol with 2-methoxypropene in the presence of catalytic amounts
of p-toluenesulfonic acid then afforded the acetonide 15 as a
white solid.
With components 13-15 in hand, studies directed toward
the synthesis of neocarzinostatin chromophore aglycon (2) along
the lines established with the core model structure 4 were
initiated (Scheme 5). Deprotonation of the epoxydiyne 15 with
lithium hexamethyldisilazide (LHMDS) followed by addition
of the enone 14 to the resulting anion afforded the adduct 22 in
80% yield and with g20:1 selectivity for the diastereomer shown
(attack opposite the tert-butyldimethylsilyl ether substituent).
Treatment of the adduct (22) with tetrabutylammonium fluoride
(TBAF) cleanly removed both silyl groups, providing the
corresponding diol in quantitative yield. Selective esterification
of the secondary alcohol with the naphthoic acid 13 in the
presence of 1,3-dicyclohexylcarbodiimide (DCC) and 4-pyrro-
lidinopyridine or 4-(dimethylamino)pyridine (DMAP) as catalyst
provided the monoester 23 in 70% yield. The acetonide
protective group was then cleaved in the presence of concen-
trated hydrochloric acid in methanol, producing the correspond-
ing tetraol in 79% yield, and the ethylene carbonate group was
introduced by using carbonyldiimidazole (CDI) in tetrahydro-
furan. Exposure of the crude reaction mixture to (()-CSA
cleaved acylimidazolide intermediates that had formed, as well
(31) Myers, A. G.; Hammond, M.; Wu, Y. Tetrahedron Lett. 1996, 37, 3083.
(32) Suzuki, M.; Kawagishi, T.; Noyori, R. Tetrahedron Lett. 1981, 22, 1809.
(33) Schmid, C. R.; Bryant, J. D.; Dowlatzedah, M.; Philips, J. L.; Prather, D.
E.; Schantz, R. D.; Sear, N. L.; Vianco, C. S. J. Org. Chem. 1991, 56,
4056.
(34) Katsuki, T.; Sharpless, K. B. J. Am. Chem. Soc. 1980, 102, 5974.
(35) (a) Takano, S.; Iwabuchi, Y.; Ogasawara, K. Synlett 1991, 2, 548. (b)
Takano, S.; Setoh, M.; Takahashi, M.; Ogasawara, K. Tetrahedron Lett.
1992, 33, 5365.
(36) Gao, Y.; Hanson, R. M.; Klunder, J. M.; Ko, S. Y.; Masamune, H.;
Sharpless, K. B. J. Am. Chem. Soc. 1987, 109, 5765.
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A R T I C L E S
Myers et al.
Scheme 5
as the dimethyl acetal group, affording the R,â-unsaturated
aldehyde 24 in 80% yield. Preparatory to ring closure, the
tertiary hydroxyl group of 24 was protected by using trimeth-
ylsilyl triflate and 2,6-lutidine to provide the corresponding
trimethylsilyl ether 25 in 91% yield.
cyclization, as the corresponding methoxyethoxymethyl (MEM)
ether 29 (Scheme 6, 94% yield). Addition of LHMDS to a
suspension of cerium(III) chloride and 29 in THF at -78 °C
provided the corresponding cyclic product 30 in 73% yield.26,27
Treatment of 30 with triflic anhydride and pyridine afforded a
product that was exceedingly unstable, decomposing within
minutes in neat form and within hours in solution (benzene).
1H NMR analysis of the chromatographically purified product
indicated that allylic transposition had indeed occurred; however,
the C-10 hydroxyl group of the product (32) was determined
to have S stereochemistry (cis to the C-11 naphthoic acid ester),
and not the R stereochemistry (trans to the C-11 naphthoic acid
ester, see 33) found in 1 and 2. The stereochemical outcome of
this transposition was rationalized as arising from neighboring-
group participation of the C-11 naphthoic acid ester. Addition
of water to the proposed dioxolenium ion intermediate 31
(Scheme 6) and selective expulsion of the C-10 hydroxyl group
(or expulsion of the C-11 hydroxyl group followed by acyl
transfer) would then afford the transposed product 32. Minor
Conditions developed for the closure of the nine-membered
ring in the synthesis of the model compound 4 were successfully
applied to the more elaborate substrate 25. Thus, treatment of
a suspension of anhydrous cerium(III) chloride (3 equiv) and
the intermediate 25 in tetrahydrofuran at -78 °C with LHMDS
(1 equiv) provided the ring-closure product 26 in 50-65%
yield.26,27 The lower yield of cyclized product (26) relative to
the earlier model study was attributed to competitive base-
induced cleavage of the ethylene carbonate in both the product
and the starting material. As expected, the cyclic product (26)
was a somewhat sensitive material, particularly in neat form,
although not to the degree exhibited by 1. The alcohol 26 could
be concentrated and held in neat form for brief periods of time
without significant decomposition, and could be stored for
several days in a frozen benzene matrix at -20 °C.
1
signals attributed to the C-10 ester were observed in the H
Treatment of the alcohol 26 with excess trimethylsilyl chloride
and triethylamine in dichloromethane furnished the bis-silyl
ether 27, which was isolated after aqueous workup and then
exposed to trifluoroacetic acid in dichloromethane at 0 °C in
an effort to effect allylic transposition, as before. However, the
only products observed under these conditions were those
resulting from cleavage of the silyl ether bonds. Consequently,
alternative means to bring about the necessary 1,3-allylic
transposition were explored.
NMR spectrum of the product 32.
Although the necessary 1,3-transposition reaction had oc-
curred, a stereochemical inversion (at C-10) was now necessary
to make use of this product for the synthesis of 1. Several
experiments were conducted in an effort to trap the intermediate
31 at C-10 with an external, oxygen-centered nucleophile such
as acetate or trifluoroacetate, without success. Efforts to invert
the C-10 stereocenter of the rearranged product 32 were also
unsuccessful. The instability of 32 greatly complicated both
strategies and, ultimately, led us to conclude that this series of
compounds was simply too unstable to complete the synthesis
of 2 and 1. We then shifted our focus to efforts to transpose the
One alternative that was investigated involved activation of
the C-8 hydroxyl group toward solvolysis by triflation. To
accomplish this the naphthol group was protected first, prior to
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Synthesis of Neocarzinostatin Chromophore
A R T I C L E S
Scheme 6
Scheme 7
C-1 hydroxyl group rather than the C-8 hydroxyl group, with
the expectation that the products in this series would be more
easily handled as a consequence of the presumed greater stability
of the tetrasubstituted olefin (Scheme 7).
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A R T I C L E S
Myers et al.
Scheme 8
Scheme 9
To activate the C-1 hydroxyl group for transposition, the C-8
hydroxyl group was first protected as the corresponding tert-
butyldimethylsilyl ether (34, 93% yield, Scheme 7); selective
cleavage of the trimethylsilyl ether was then effected by
exposure of 34 to triethylamine trihydrofluoride, affording the
tertiary alcohol 35 in 87% yield. Treatment of 35 with triflic
anhydride in the presence of 2,6-lutidine afforded the transposed
product 36, with cis stereochemistry again, presumably as a
consequence of neighboring-group participation. Unfortunately,
the transposed alcohol 36 was not significantly more stable than
the alcohol 32, undergoing rapid decomposition both in neat
form and in solution. As before, efforts to invert the C-10
hydroxyl group, now within intermediate 36, or to trap the
presumed intermediate dioxolenium ion by external nucleophilic
additions, were not successful.
36, and seemed to be an ideal candidate for direct nucleophilic
inversion; however, efforts to accomplish this under a broad
spectrum of conditions led only to decomposition.
To examine the role of the naphthoic acid ester in the
rearrangement chemistry, we prepared the substrate 38 with a
tert-butyldimethylsilyl ether at C-11 in place of the naphthoic
acid ester (Scheme 8). Not unexpectedly, the behavior of the
substrate 38 toward triflic anhydride was markedly different than
that of substrate 35, bearing a C-11 naphthoic acid ester.
Treatment of 38 with triflic anhydride and pyridine resulted in
rapid and nonspecific decomposition of the starting material.
The reactivity of 38 toward methanesulfonyl chloride, on the
other hand, paralleled that of substrate 35, affording the
(unstable) rearranged mesylate 39. Attempted invertive displace-
ment of the mesylate 39 was unsuccessful with use of several
different nucleophiles; only decomposition of the substrate was
observed. Allylic transposition of the tertiary allylic alcohols
38 (with a C-11 silyl ether) and 41 (with a C-11 naphthoic acid
ester) was also attempted under Mitsunobu conditions (Scheme
Interestingly, treatment of alcohol 35 with methanesulfonyl
chloride and triethylamine led to direct transposition to form
the allylic mesylate 37, also with cis stereochemistry. The
mesylate was slightly more stable than the rearranged alcohol
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Synthesis of Neocarzinostatin Chromophore
A R T I C L E S
Scheme 10
Scheme 11
9), although little precedent exists for the application of the
Mitsunobu reaction for the inversion of tertiary alcohols or for
1,3-transposition of allylic alcohols.37 Reaction of alcohol 38
with chloroacetic acid, triphenylphosphine, and diethyl azodi-
carboxylate (DEAD) afforded the rearranged diester 40 in 16-
18% yield, whereas the more functionalized substrate 41
afforded the product of direct invertive displacement of the
tertiary hydroxyl group (43), under substantially modified
Mitsunobu conditions (methyldiphenylphosphine, DEAD, γ-hy-
droxybutyric acid o-nitrobenzyl ether 42); neither transformation
was of apparent use for our purpose.
In considering alternative strategies to achieve the necessary
allylic transposition reaction, we returned to our earlier model
structure (11) that had rearranged successfully (after trimeth-
ylsilylation of the secondary alcohol) in the presence of
trifluoroacetic acid (11 f 12, Scheme 3). As described,
attempted extension of this strategy to the more complex
substrate 27 had failed, presumably due to C-11 oxygenation
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A R T I C L E S
Myers et al.
within this substrate. It occurred to us that an additional
distinction between two substrates was the stereochemical
variation at C-1 and C-8. This led us to consider the rearrange-
ment of the diastereomeric substrate 44, whose retrosynthesis
is outlined in Scheme 10.
Many possible means of accomplishing the necessary reduction
of 58 to form 2 were envisioned, although that which ultimately
proved successful was discovered serendipitously (vide infra).
It was expected that intramolecular acetylide addition within
the C-1 epimeric epoxy aldehyde substrate 45 would occur in
analogy to prior closures in the diastereomeric ring series, via
the s-trans conformation of the R,â-unsaturated aldehyde (45
f 44, Scheme 10). We planned to introduce the C-11 naphthoic
acid ester of 45 by Mitsunobu inversion of the secondary alcohol
derived from intermediate 46.37 Intermediate 46 would be
accessed by diastereoselective addition of the lithium acetylide
derived from 15 to (-)-14, the enantiomer of the substrate used
for the preparation of the previous diastereomeric series. The
basic elements of this plan were successfully pursued to obtain
the diastereomeric cyclization product 53 and its differentially
silylated forms 54 and 55, as shown in Scheme 11. Interestingly,
closure of the nine-membered ring required a large excess of
LHMDS (9 equiv) to proceed to completion, and the product
(53) was found to be much less stable than the diastereomeric
ring-closure products 27 or 30, decomposing within a few days
even when stored in a solid benzene matrix at -20 °C.
The instability of intermediates 53 f 55 in this series greatly
complicated their manipulation and purification, and conse-
quently the yields for intermediates beyond 53 were difficult
to determine accurately.
In contrast to the behavior of the diastereomeric alcohol 35,
the alcohol 55 underwent nonspecific decomposition upon
treatment with triflic anhydride. Interestingly, the reaction of
alcohol 55 with methanesulfonyl chloride or methanesulfonic
anhydride afforded products whose 1H NMR spectra were
consistent with structures assigned as the chloride 56 and the
mesylate 57, respectively. Although these products apparently
possessed the requisite trans stereochemistry between the C-10
and C-11 centers, we were unable to develop a means for the
conversion of either of these compounds to intermediates useful
for the synthesis of 2. More practically, the marked instability
of this diastereomeric series convinced us to return to the initial
diastereomeric series for the continuation of our studies.
The most direct means by which to access the intermediate
58 involved the epoxidation of substrates in hand, and this was
investigated first. A number of reagents were screened for the
epoxidation of the substrate 59, for example, m-CPBA, di-
methyldioxirane, and methyl(trifluoromethyl)dioxirane,38 and the
peroxycarboximidic acid derived from the combination of 90%
hydrogen peroxide and acetonitrile,39 none of which was
successful. Only buffered trifluoroperoxyacetic acid afforded
the desired epoxide (60). The 0-Hz coupling constant between
the protons at C-10 and C-11 was diagnostic for a trans
relationship, as expected if epoxidation had occurred from the
less hindered face of the olefin within 59. However, the low
yield of the epoxide product (significant decomposition of the
substrate and/or product occurred) prompted us to seek an
improved method to introduce the epoxide.
Enantioselective Synthesis of Neocarzinostatin
Chromophore Aglycon
Having exhausted many potential routes for the preparation
of the substituted epoxy dienediyne core of 1 by the transposition
of the allylic alcohols discussed in the previous section, attention
was turned to a new strategy involving the bis-epoxide 58 as a
potential precursor to neocarzinostatin chromophore aglycon (2).
(37) (a) Mitsunobu, O. Synthesis 1981, 1. (b) Hughes, R. L. Org. React. 1992,
42, 335.
(38) (a) Murry, R. W.; Jeyaraman, R. J. Org. Chem. 1985, 50, 2847. (b) Mello,
R.; Fiorentino, M.; Sciacovelli, O.; Curci, R. J. Org. Chem. 1988, 53, 3980.
(c) Adam, W.; Bialis, J.; Hadjiarapolou, L. Chem. Ber. 1991, 124, 2377.
(39) Gagieu, C. H.; Grouiller, A. V. J. Chem. Soc., Perkin Trans. 1 1982, 1009.
9
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Synthesis of Neocarzinostatin Chromophore
A R T I C L E S
We were able to direct the epoxidation reaction by using the
hydroxyl group at C-8 in the diastereomeric series of intermedi-
ates (in which the C-1 and C-8 centers were inverted); for
example, alcohol 53 was epoxidized under SAE conditions with
use of (+)-DET to afford the epoxide 61.36 The purification of
led to an improvement in the yield of the coupling reaction (12-
Crown-4 as an additive, 70%), the diastereoselectivity remained
unacceptably low (∼3:1).
61 from residual (+)-DET was difficult, and the instability of
the precursors made material throughput a challenge. Conse-
quently, introduction of the epoxide prior to closure of the nine-
membered ring was pursued, although there were concerns about
the influence the epoxide would have upon the closure reaction;
the outcome was not easily predicted (see 62 w 63).
Our first approach to the synthesis of epoxy aldehyde
intermediates having the general structure 63 involved the
coupling of the epoxy diyne component 15 with the epoxy
ketone 64, prepared from the reaction of (+)-14 with alkaline
hydrogen peroxide (91% yield, g95% de, g85% ee). Addition
of the acetylide anion derived from 15 and LHMDS to the
ketone 64 provided a 1:1 mixture of the diastereomers 65 in
10-15% yield. Although modification of the reaction conditions
Diastereoselective introduction of the second epoxy group
within 63 was best achieved by application of the SAE, as shown
in Scheme 12, beginning with the prior intermediate 66.
Selective silylation of 66 with triisopropylsilyl triflate provided
the secondary silyl ether 67 in 89% yield. Hydrolysis of the
dimethyl acetal group within this product with p-toluenesulfonic
acid (p-TSA) in acetone afforded the corresponding aldehyde.
Protection of the tertiary alcohol with trimethylsilyl triflate then
furnished the aldehyde 68 in 93% yield from 67. The aldehyde
68 underwent 1,2-reduction upon treatment with DIBAL,
Scheme 12
9
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A R T I C L E S
Myers et al.
affording the corresponding allylic alcohol in 96% yield. This
product was epoxidized diastereoselectively under SAE condi-
tions with use of (+)-DET, providing the corresponding bis-
epoxy alcohol in 89% yield; oxidation with Dess-Martin
periodinane40 buffered with pyridine then afforded the bis-epoxy
aldehyde 69 in 94% yield.
75 in 93% yield (Scheme 13). This product and all similar
compounds containing the cyclic ene-yne core were notably
more sensitive intermediates, darkening within minutes upon
concentration. These compounds could be held in neat form
only for brief periods, but were stable to storage at -20 °C in
a frozen benzene matrix for several days.
Closure of the nine-membered ring could be brought about
by treatment of epoxy aldehyde 69 with LHMDS in the presence
of cerium(III) chloride, but higher conversions and cleaner
reactions were observed with this substrate when anhydrous
lithium chloride was used in lieu of cerium(III) chloride. Thus,
addition of LHMDS to a suspension of the aldehyde 69 and
lithium chloride (50 equiv) in THF at -78 °C furnished the
cyclic alcohol 70 in 62% yield. An unexpected, but highly
noteworthy feature of the product 70 was its stability to
chromatography, concentration, and routine manipulations. This
stands in marked contrast to the “olefinic” series of cyclization
products which, as mentioned, was found to be exceedingly
difficult to handle. The stereochemistry of the addition reaction
was confirmed to be as shown (70) by the preparation of a cyclic
disiloxane derivative linking the C-1 and C-8 hydroxyl groups.
Exploratory studies to effect the reductive fragmentation step,
using the epoxy alcohol 70 or a closely related substrate,
suggested that the C-4-C-5 epoxide was more labile to
reductive cleavage than the C-9-C-10 epoxide. For example,
1H NMR analysis of the product mixture from attempted Barton
deoxygenation, using the thiocarbonylimidazolide derived from
70,41 provided evidence that the C-4-C-5 epoxide had opened,
whereas the C-9-C-10 epoxide remained intact. Similar results
were obtained by using the one-electron reductant Cp2TiCl of
RajanBabu and Nugent42 (the alcohol 74, a single diastereomer
of undetermined stereochemistry at C-4, was isolated).
Reexamination of the Cp2TiCl reagent for the reductive
fragmentation, now within the substrate 75, once again led to
selective opening of the C-4-C-5 epoxide, as well as cleavage
of the cyclic carbonate, forming the triol 76. Efforts to transform
77 into a halohydrin intermediate (as a substrate for subsequent
reduction) by selective cleavage of the allylic C-O bond were
also not successful; again, the major reaction path involved
opening of the C-4-C-5 epoxide. A thorough study of ionic
hydrogenation conditions44 led only to the realization that both
epoxy groups within 75 were unexpectedly resilient under acidic
conditions; exposure of 75 to trifluoroacetic acid-triethylsilane,
for example, provided only the alcohol 77, by acidic cleavage
of the triethylsilyl ether. This transformation was ultimately used
productively, albeit with more conventional reagents ((()-CSA
in aqueous acetonitrile, 92%).
With a slightly altered focus, we attempted to transform the
alcohol 77 into the corresponding secondary iodide, intending
to effect reductive epoxide opening by subsequent lithium-
halogen exchange. Accordingly, 77 was treated with tri-
phenylphosphine, iodine, and imidazole in dichloromethane at
-10 °C (Scheme 14).45 The product of this reaction proved to
be highly unstable. After much experimentation, a suitable
procedure for isolation of the product was found, a key element
being the direct loading of the reaction mixture onto a
pre-slurried column of silica gel immediately upon completion
of the reaction (TLC analysis). The isolated product was found
not to be the expected epoxy iodide, but instead the epoxydi-
enediyne 78.46 Further support for this assignment was obtained
1
by H NMR analysis of the corresponding triethylsilyl ether
(79), a derivative that provided well-resolved, fully assignable
proton resonances. Although the secondary iodide expected from
the reaction may be an intermediate in the formation of the
epoxy dienediyne 78, it was not observed. The transformation
of epoxy iodides into allylic alcohols under nucleophilic
conditions is known, but generally requires more forcing
conditions than those employed here.
With the somewhat serendipitous discovery of a viable
protocol to prepare the epoxy dienediyne functionality of
neocarzinostatin, the product 78 representing the first fully
oxygenated neocarzinostatin core structure to be synthesized,
we sought to incorporate the naphthoic acid ester and thus
complete the synthesis of the aglycon (2). The observed
instability of the intermediate 78 suggested that the introduction
of the ester at this stage or beyond would not be feasible. To
allow for naphthoic acid ester formation prior to reductive
transposition, small adjustments in the hydroxyl group protection
scheme were introduced (Scheme 15), the primary modification
being the substitution of tert-butyldimethylsilyl for triisopro-
pylsilyl (69 f 82). This permitted masking of the C-8 hydroxyl
We therefore elected to introduce the C-1-C-12 double bond
prior to reduction to favor cleavage of the desired epoxide; the
ethylene carbonate group was also introduced concurrently
(Schemes 12 and 13). Thus, treatment of the alcohol 70 with
methanolic p-TSA provided the tetraol 71 in 62% yield, which
afforded the carbonate 72 upon reaction with CDI in THF at
0-23 °C (72% yield). The secondary alcohol was protected as
the corresponding triethylsilyl ether (97% yield) without
competing silylation of the tertiary hydroxyl group.
Among many conditions examined for elimination with the
tertiary alcohol 73, optimal results were obtained by using the
Martin sulfurane as the dehydrating agent,43 producing the olefin
(40) (a) Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113, 7277. (b) Ireland,
R. E.; Liu, L. J. Org. Chem. 1993, 58, 2899. (c) Meyer, S. D.; Schreiber,
S. L. J. Org. Chem. 1994, 59, 7549.
(41) Barton, D. H. R.; Motherwell, R. S. H.; Motherwell, W. B. J. Chem. Soc.,
Perkin Trans. 1 1981, 2363.
(42) (a) RajanBabu, T. V.; Nugent, W. A.; Beattie, M. S. J. Am. Chem. Soc.
1990, 112, 6408. (b) Rajanbabu, T. V.; Nugent, W. A. J. Am. Chem. Soc.
1994, 116, 986.
(43) (a) Martin, J. C.; Arhart, R. J. J. Am. Chem. Soc. 1971, 93, 4327. (b) Arhart,
R. J.; Martin, J. C. J. Am. Chem. Soc. 1972, 94, 4997.
(44) For a review of ionic dehydrogenations in organic synthesis, see: Kursamov,
D. V.; Parnes, Z. N.; Loim, N. M. Synthesis 1974, 634.
(45) Garegg, P. J.; Samuelsson, B. J. J. Chem. Soc., Chem. Commun. 1979,
978.
(46) More recently, this reductive deoxygenation procedure was reported
independently by another research group: Dorta, R. L.; Rodr´ıguez, M. S.;
Salazar, J. A.; Sua´rez, E. Tetrahedron Lett. 1997, 38, 4675.
9
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Synthesis of Neocarzinostatin Chromophore
A R T I C L E S
Scheme 13
Scheme 14
Scheme 15
group as a chloroacetate ester, an orthogonal protective group
that was stable to conditions necessary for the cleavage of the
C-11 silyl ether, yet was removable under mild conditions. The
only other significant modification of prior chemistry within
Scheme 15 is the use of Masamune’s hindered base, lithium
bis(dimethylphenylsilyl)azide,47 for the ring-closure reaction (82
f 83), a modification that was necessitated by the poor
conversions observed with use of LHMDS as base. This subtle
yet critical variation in the cyclization protocol serves to
highlight the delicate interplay of relative reaction rates that
can determine the success or failure in these reactions (here,
presumably the rate of deprotonation by the base versus the
rate of its 1,2-addition to the aldehyde).
With an efficient route to the diol 84, selective acylation of
the secondary hydroxyl group with the naphthoic acid 13 was
examined. Optimum results were obtained upon combination
of 84 and 13 (3 equiv) with DCC (5 equiv) in THF at -10 °C
followed by the addition of n-propylamine to the crude reaction
(47) (a) Zhinkin, D. Y.; Malnova, G. N.; Gorislavskaya, Zh. V. Zh. Obshch.
Khim. 1968, 38, 2800. (b) Masamune, S.; Ellingboe, J. W.; Choy, W. J.
Am. Chem. Soc. 1982, 104, 5526.
9
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A R T I C L E S
Myers et al.
Scheme 16
mixture to bring about cleavage of the chloroacetate ester in
situ (Scheme 16). Under these conditions, the ester 85 was
obtained in 80% yield. Considerable experimentation was
required for the development of this procedure, for the naphthoic
acid 13 was prone to self-coupling. Competing acylation of the
tertiary hydroxyl group of 84 was also observed. The selection
of the reaction solvent and the coupling reagent proved to be
essential elements for the success of this reaction, with DCC in
THF emerging as a uniquely successful combination.
To complete the synthesis of the aglycon 2, the acetonide
protective group of 85 was cleaved in the presence of methanolic
p-TSA to provide the pentaol 86 in 81% yield; the ethylene
carbonate group was then introduced as before, by reaction with
CDI in THF, affording the cyclic carbonate 87 in 89% yield
(Scheme 16). The naphthol and secondary hydroxyl groups were
protected simultaneously with use of triethylsilyl triflate-2,6-
lutidine, (quantative yield) and the resulting bis(triethylsilyl)
ether (88) was treated with the Martin sulfurane reagent to
induce elimination of the tertiary hydroxyl group, furnishing
the olefinic product 89 in 79% yield. Removal of the triethylsilyl
groups within the product 89 in the presence of triethylamine
trihydrofluoride then provided the epoxy alcohol 58 in 99%
yield.
Treatment of the epoxy alcohol 58 with a mixture of
triphenylphosphine, iodine, and imidazole led directly to the
highly unstable aglycon 2. Close monitoring of the reaction by
TLC showed that the transformation was complete within 5 min
at -10 °C. As before, no intermediates were detectable in the
reaction. Workup involved quenching excess triphenylphos-
phine-iodine with methanol (50 equiv), followed by the
addition of pentane to precipitate triphenylphosphine oxide and
imidazolium iodide. Flash column chromatography was con-
ducted at 0 °C (cold room) and the pooled aglycon fractions
were concentrated at 0 °C by using a rotary evaporator with an
ice bath. All manipulations (chromatography, venting after
concentration, etc.) were conducted under argon. Also, Kishi’s
free-radical inhibitor (5-tert-butyl-4-hydroxy-2-methylphenyl
sulfide)48 was added to all solutions, to suppress decomposition.
Typically, test tubes used for fraction collection during aglycon
purification contained a solution of Kishi inhibitor in deoxy-
9
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Synthesis of Neocarzinostatin Chromophore
A R T I C L E S
genated benzene (50 mL, 2.5 mg/mL). With these procedures
for synthesis and purification, the optically pure aglycon (2)
was obtained in up to 71% yield. Typically, 2-3 mg batches
of neocarzinostatin chromophore aglycon (2) were prepared by
this protocol.
analysis). Groups that were labile under the latter conditions
were therefore targeted for protection of the carbohydrate
residue.
To begin our glycosylation studies, we elected to pursue the
synthesis of the R-D-fucose analogue 3 prior to addressing the
more difficult problem of 1 itself. Compounds 3 and 1 differ
only in the nature of the 2′-substituent of the carbohydrate
residue, a distinction that was of interest from mechanistic as
well as synthetic perspectives. The 2′-methylamino substituent
of 1 was presumed to make it a more challenging target than 3
(with a 2′-hydroxyl group), a prediction that was later confirmed.
The aglycon (2) was observed to degrade much more rapidly
than neocarzinostatin chromophore (1), both in solution and in
neat form. Synthetic 2 provided good quality 1H NMR and CD
spectra, but its half-life in solution was too short to obtain a
suitable 13C NMR spectrum. All efforts to obtain a mass
spectrum of 2 in isolation with a variety of soft-ionization
techniques also failed, almost certainly due to the instability of
2. We were successful in obtaining mass spectral data by
precomplexation of the synthetic aglycon (2) with purified
neocarzinostatin apoprotein in water (pH 7); peaks consistent
with the protein-complexed aglycon were observed. For further
confirmation of the structure, the aglycon (2) was treated with
methyl thioglycolate (75 equiv), triethylamine (75 equiv), and
1,4-cyclohexadiene (150 equiv) in THF at 23 °C, affording the
thiol adduct 90 in analogy to experiments with neocarzinostatin
chromophore (1). Triethylamine was required in the latter
experiment, providing support for the proposed participation of
the amino group of the carbohydrate residue as an internal base
in the thiol activation of 1.25,49
The critical issues to be resolved in any glycosylation reaction
are the method of carbohydrate activation, selectivity in the
glycosidic bond formation (R versus â), and the nature of the
protective groups as they pertain to the latter issue and, in the
case of 1 and 3, to conditions for deprotection vis-a`-vis product
stability. The Schmidt trichloroacetimidate glycosyl activation
method was recognized at the outset to be nearly ideal in the
context of our synthetic problem, as it has been in so many
complex carbohydrate syntheses, largely for the mildly acidic
conditions that are used to promote the coupling reaction, as
well as the opportunities to alter R/â selectivity by modification
of the trichloroacetimidate stereochemistry and/or the coupling
conditions.51
Three differently protected trichloroacetimidate-activated
D-fucose donors were prepared (91, 92, and 93, see Supporting
Information) and examined as substrates for the synthesis of
protected fucosyl glycosides to select an optimum substrate for
the synthesis of 3. The cyclopentenol 94 was used in lieu of
the aglycon 2 in initial studies; results of glycosylation reactions
with this substrate, as well as subsequent deprotection experi-
ments, are depicted in Scheme 17. Briefly, we learned that silyl
ethers, and triethylsilyl ethers in particular, were well suited
for protection of the hydroxyl groups of the carbohydrate
coupling partner. Ketals (acetonide, cyclopentylidene) were too
robust to liberate 1 or 3 intact, as was the disiloxane group within
substrate 92. Also, the â-selectivity observed in coupling with
the latter substrate suggested that bulky substituents in the 2′-
position were to be avoided if R-selectivity was desired, a
proposal that has proven to be somewhat general throughout
this work. Among the three substrates, the tris(triethylsilyl) ether
93 was clearly superior in terms of R-selectivity in product
formation (further improved upon use of 3 equiv of 93 and
trimethylsilyl triflate as catalyst) and the mildness of the
Glycosylation Studies
To complete the synthesis of neocarzinostatin chromophore
(1), the problem of stereoselective glycosylation of the aglycon
(2) was addressed. Direct glycosylation of 2 in unprotected form
had been planned as part of our initial retrosynthetic analysis.
The instability of the aglycon (2) limited the options available
for glycosylation conditions, however, and the instability of the
final product (1) restricted the selection of protective groups
for the amino and hydroxyl groups of the carbohydrate coupling
partner. It was known that the chromophore (1) was particularly
tolerant of mildly acidic conditions; for example, solutions of
1 in neat acetic acid showed no evidence of decomposition
within 1 d at 23 °C.50 Mildly acidic solutions of fluoride ion
were deemed to be particularly worthy of consideration for final
deprotection. These were defined more precisely in control
experiments with 1. Exposure of 1 to 10% (v/v) aqueous
hydrofluoric acid in acetonitrile at 23 °C led to rapid decom-
position (t1/2 e 30 min), but the milder reagent hydrogen
fluoride-pyridine complex in THF was well tolerated (no
evidence of decomposition of 1 after 2 h at 23 °C, rp-HPLC
(51) Reviews: (a) Schmidt, R. R. Angew. Chem., Int. Ed. Engl. 1986, 25, 212.
(b) Schmidt, R. R. In ComprehensiVe Organic Synthesis; Trost, B. M.,
Ed.; Pergamon Press: Oxford, 1991; Vol. 6, p 33. (c) Schmidt, R. R.; Kinzy,
W. AdV. Carbohydr. Chem. Biochem. 1994, 50, 21. (d) Schmidt, R. R.;
Jung, K.-H. In PreparatiVe Carbohydrate Chemistry; Hanessian, S., Ed.;
Marcel Dekker: New York, 1997; Chapter 12.
(48) Kishi, Y.; Aratani, M.; Tanino, H.; Fukuyama, T.; Goto, T.; Inoue, S.;
Sugiura, S.; Kakoi, H. J. Chem. Soc., Chem. Commun. 1972, 64.
(49) (a) Myers, A. G.; Kort, M. E.; Hammond, M. J. Am Chem. Soc. 1997,
119, 2965. See also ref 24.
(50) Myers, A. G.; Harrington, P. M. Unpublished results. See also ref 54.
9
J. AM. CHEM. SOC. VOL. 124, NO. 19, 2002 5395

A R T I C L E S
Myers et al.
Scheme 17
subsequent deprotection conditions and so was selected for
coupling with 2.
conducted with the exclusion of light. Synthetic 3 provided
spectral and analytical data fully consistent with expectations.
For further confirmation of structure, the product of nucleo-
philic activation with methyl thioglycolate (102, 47% yield,
Scheme 19) was prepared. As in prior work with the aglycon
itself (2 f 90), the latter reaction required the presence of an
exogenous base (triethylamine, 150 equiv), this providing the
most compelling evidence to date for the role of the meth-
ylamino group in thiol activation.25,49
Having established the suitability of 2 as a substrate for direct
glycosylation, efforts turned toward the more challenging
problem of introduction of the 2′-amino sugar of neocarzinosta-
tin chromophore (1). Despite the importance of 2′-amino sugars
in biology, reports of the stereocontrolled introduction of
R-glycosidic bonds in 2′-amino sugars are few.52 The key
unresolved issue here was the nature of the protective group
for the 2′-methylamino group. Although 2′-azido sugars (trichlo-
roacetimidate activation) have been employed in R-selective
glycosylation reactions,53 conditions necessary for subsequent
reduction of the 2′-azido substituent and N-methylation of the
resultant 2′-amino group would almost certainly be incompatible
with our target (1). Ideally, the group selected for protection of
Operationally, synthesis of the R-fucosyl analogue of neo-
carzinostatin chromophore (3) proceeded by straightforward
extension of our model studies with 94. A solution of the freshly
purified aglycon 2 and the tris(triethylsilyl)-protected fucosyl
R-trichloroacetimidate 93 (10 equiv) in deoxygenated benzene
containing the Kishi free-radical inhibitor (2.5 mg/mL) was very
briefly concentrated, and the residue was dissolved in ether.
Addition of 3 Å molecular sieves and stirring at 23 °C (10 min)
served to dry the solution; cooling to -30 °C and addition of
trimethylsilyl triflate (0.1 equiv) then led to smooth R-fucosy-
lation (63% yield, Scheme 18). Despite the instability of 2, and
the fact that some decomposition invariably occurred during
its manipulation, with practice the procedure was reproducible
and decomposition was minimal. It is interesting to note that
the glycosylation reaction was apparently unaffected by the
presence of the Kishi free-radical inhibitor,48 which, however,
helped to preserve the aglycon. The product (101) was more
stable than the starting aglycon, but did readily decompose,
especially when neat. Deprotection of the triethylsilyl ethers of
101 proceeded cleanly in 5% (v/v) hydrofluoric acid in
acetonitrile at -30 °C, but did require a longer time for
completion (1-1.5 h) than the model substrate (97). D-Fucosyl
neocarzinostatin chromophore (3) was obtained in pure form
after flash column chromatography (67% yield) and was found
to be relatively stable toward concentration for brief periods.
Like 1, 3 was light-sensitive, and all operations handling it were
(52) (a) Toshima, K.; Tatsuta, K. Chem. ReV. 1993, 93, 1503. (b) Danishefsky,
S. J.; Bilodeau, M. T. Angew. Chem., Int. Ed. Engl. 1996, 35, 1380.
(53) (a) Schmidt, R. R.; Grundler, G. Angew. Chem., Int. Ed. Engl. 1982, 21,
781. (b) Grundler, G.; Schmidt, R. R. Liebigs Ann. Chem. 1984, 1826. (c)
Kinzy, W.; Schmidt, R. R. Carbohydr. Res. 1989, 193, 33. (d) Boons, G.
J. P. H.; Overhand, M.; van der Marel, G. A.; van Boom, J.-H. Angew.
Chem., Int. Ed. Engl. 1989, 28, 1504. (e) Kaneko, T.; Takahashi, K.;
Hirama, M. Heterocycles 1998, 47, 91.
9
5396 J. AM. CHEM. SOC. VOL. 124, NO. 19, 2002

Synthesis of Neocarzinostatin Chromophore
A R T I C L E S
Scheme 18
Scheme 19
Table 1
conditions
result
10% HF, CH3CN
**HF‚pyr, THF
TBAF, AcOH
Et3N‚3HF, THF
AcOH
30 min
50 min
8 h
no reaction
no reaction
nominally basic nitrogen atom was a viable glycosyl donor in
the Schmidt coupling protocol, however, was revealing, and led
us to question whether it was necessary to protect the 2′-
methylamino group at all. To consider this question, the
trichloroacetimidate 108, with a free 2′-methylamino group and
triethylsilyl ether protection of the two hydroxyl groups, was
prepared and glycosylation of the model substrate 94 was
studied.24c,55 Coupling with 108 (Scheme 22) did require larger
amounts of acid activator (up to 0.8 equiv, introduced portion-
wise) and longer reaction periods than substrates in which the
2′-methylamino group was protected, but 108 was nevertheless
an excellent glycosyl donor. Optimum results were obtained
by using triflic acid as activator (Scheme 22); under these
conditions the coupled product (109) was obtained in 82% yield,
with g20:1 R:â selectivity.
the 2′-methylamino group would be labile under the conditions
of mildly acidic fluoride ion, identified as compatible with 1.
Acid-labile carbamates were reasonable to consider, but with
them arose problematic issues in the glycosylation reaction.
These are best illustrated by the results of our experiments with
the t-Boc-protected 2′-deoxy 2′-methylamino R-fucosyl trichlo-
roacetimidate 103 (Scheme 20). Neighboring-group participation
of the carbamate during the glycosylation was evident from the
â-selectivity of the reaction, and certainly was responsible for
the formation of the oxazolidinone byproduct, a common
liability of glycosyl donors with cation-stabilizing 2′-carbamate
groups.54
In a move away from carbamate protection of the 2′-
methylamino group and toward the ultimately successful solu-
tion to the problem, we investigated the use of an acid-labile
2′-N-alkyl group, such as di-p-methoxyphenylmethyl (106,
Scheme 21). In keeping with our earlier observations concerning
bulky 2′-substituents, glycosylation with this substrate was found
to be largely â-selective. The fact that a substrate with a
Mechanistically, this transformation is viewed to proceed via
the 2′-methylamino-substituted oxocarbenium ion intermediate
(55) Glycosidic coupling reactions with free 2-hydroxyl groups are well
precedented. For leading references, see: (a) Hanessian, S.; Bacquet, C.;
Lehong, N. Carbohydr. Res. 1980, 80, C17. (b) Hanessian, S. In PreparatiVe
Carbohydrate Chemistry; Hanessian, S., Ed.; Marcel Dekker: New York,
1997; Chapter 16.
(54) Kuo, E. Y., Ph.D. Thesis, California Institute of Technology, 1993.
9
J. AM. CHEM. SOC. VOL. 124, NO. 19, 2002 5397

A R T I C L E S
Myers et al.
Scheme 20
Scheme 21
Scheme 22
Scheme 23
111, as outlined in Scheme 23. It is interesting to note that
reaction via an N-methylaziridinium ion intermediate (112)
would be expected to produce the isomeric â-product. Coupling
under a wide variety of conditions was observed to be
R-selective;56 use of triflic acid as promoter, however, was
optimal.
pyridine complex provided a mild reagent for clean and rapid
deprotection of 109.
Extension of our model studies to the synthesis of 1 required
the development of a slightly modified coupling protocol. After
extensive experimentation, we found that the glycosylation of
2 with 108 (6.6 equiv) was best achieved by using boron fluoride
etherate (3.0 equiv) as the Lewis acid promoter and toluene as
Conditions for the deprotection of the triethylsilyl ethers
within the coupled product 109 were investigated (Table 1).
We found that the presence of the free 2′-N-methylamino group
in the substrate markedly slowed the rate of deprotection of
the silyl ethers, but not to the extent that the synthesis of 1 by
such a route became infeasible. In particular, hydrogen fluoride-
(56) Hirama’s group has described studies of the neocarzinostatin chromophore
glycosylation problem in which they employed a 2′-methylamino group in
unprotected form. Their work involved thioglycoside activation with
stoichiometric triflic acid, however, and gave rise to â-selective coupling.
It is likely that the N-methylamino group was protonated in the transition
state for this glycosylation reaction. Takahashi, K.; Tanaka, T.; Suzuki,
T.; Hirama, M. Tetrahedron 1994, 50, 1327.
9
5398 J. AM. CHEM. SOC. VOL. 124, NO. 19, 2002

Synthesis of Neocarzinostatin Chromophore
A R T I C L E S
Scheme 24
Scheme 25
the solvent (Scheme 24). Under these conditions, the glycosy-
lation was complete within 1 h at -30 °C, affording the coupled
product (114) in 51% yield after flash column chromatography
on silica gel. The desired R-anomer was formed exclusively,
as determined by 1H NMR analysis. Because 114 was observed
to decompose when stored (-80 °C, deoxygenated benzene
matrix), deprotection was typically conducted immediately. Use
of hydrogen fluoride-pyridine complex in THF cleanly removed
both triethylsilyl ether groups within 1 h at 23 °C to afford
synthetic neocarzinostatin chromophore (1) in unprotected form.
Purification was achieved by direct loading of the deprotection
reaction solution onto Sephadex LH-20 resin (attempted prior
neutralization of the reaction medium induced rapid decomposi-
tion of 1). With use of a mixed solvent system (dichloromethane,
methanol, acetic acid; 94:5:1), pure 1 eluted from the column,
while the lower molecular weight hydrogen fluoride-pyridine
complex was retained. Synthetic 1 provided 1H NMR, CD, and
IR spectra that were identical with spectra obtained from a
sample of natural chromophore, and both samples showed
identical mobility in rp-HPLC analysis. We were also successful
in obtaining a high-resolution mass spectrum of 1 using an
external ion source Fourier transform ion cyclotron resonance
(FT-ICR)57 mass spectrometer equipped with a nanoelectro-
spray58 ionization source (calcd for C35H34NO12 ([M + H]+),
660.2082; found, 660.2090). The exceedingly mild nature of
this soft ionization technique was evident in the fact that the
molecular ion peak was also the base peak; essentially no
fragment-ion peaks were observed (<2% of base-peak intensity).
more radiolabels within the molecule. The goal would then be
to incubate a specific cell line with the labeled compound to
determine the fate of the drug in vivo, certainly an unresolved
issue at present. Here, we describe briefly only the chemical
synthesis component of the problem and its solution as applied
to the preparation of tritiated and 14C-labeled 1, both separately
and in combination. For reasons of safety and economy it was
desirable to incorporate the radiolabel as late in the synthetic
route as possible. It was logical then that our first consideration
was to label the glycosyl donor 108; this was accomplished by
using tritiated methyl iodide, as shown in Scheme 25. In the
introduction of the radiolabel, the N-Cbz derivative 115 (used
in excess) was deprotonated with sodium hydride in the presence
of hexamethylphosphoramide (HMPA) and alkylated with 3H-
methyl iodide to afford the N-methylated product 116 in 93%
yield (specific activity ∼140 mCi/mmol). Thereafter, the
protocol for the synthesis of tritiated 1 followed established lines
(Schemes 25 and 26). After purification by chromatography with
3
Sephadex LH-20, the yield of synthetic H-1 was quantitated
by measurement of the UV absorbance of the column fractions
at 302 nm (ꢀ ) 5830 M-1cm-1); in a typical preparation, ∼0.4
3
mg H-1 was obtained, with a specific activity of ∼30 mCi/
mmol.
As in vivo mechanistic studies with 3H-1 progressed (detailed
elsewhere), it became necessary to prepare 1 with an additional
but different radiolabel, in a different part of the molecule. The
14C-labeled aglycon 2 was therefore synthesized, as shown in
Scheme 27. This route employed 14C-labeled carbonyldiimida-
zole for introduction of the first radiolabel (86 f 87), and
tritiated methyl iodide, as before, for the second label. In this
manner, ∼0.4 mg 14C-3H-1 was synthesized, with a specific
Synthesis of Singly and Doubly Radiolabeled
Neocarzinostatin chromophore
The development of an efficient synthetic pathway to
neocarzinostatin chromophore (1) provided a valuable op-
portunity to conduct a unique series of mechanistic studies,
provided that the route could be modified to introduce one or
(57) Rodgers, M. T.; Campbell, S.; Marzluff, E. M.; Beauchamp, J. L. Int. J.
Mass Spectrom. Ion Processes 1994, 137, 121.
(58) Wilm, M.; Mann, M. Anal. Chem. 1996, 68, 1.
9
J. AM. CHEM. SOC. VOL. 124, NO. 19, 2002 5399

A R T I C L E S
Myers et al.
Scheme 26
Scheme 27
14C-radioactivity of ∼30 mCi/mmol and a specific 3H-
radioactivity of ∼30 mCi/mmol (Scheme 28).
Summary
A convergent, enantioselective synthesis of neocarzinostatin
chromophore (1) was developed. The previously unknown, and
highly unstable aglycon (2) served as our key intermediate,
synthesized in 19 steps (average yield per step 89%) from the
three precursors (13, 14, and 15). It is noteworthy that the final
step in the preparation (58 f 2, Scheme 16) was not a known
transformation at the outset of our studies. This transformation
allowed us to transition from a series of olefinic synthetic
precursors to the corresponding epoxides, which proved to be
markedly more stable and therefore more practical intermediates.
Neither this stabilizing feature nor the success of the final
The ability to synthesize 3H- and 14C-3H-labeled 1 by simple
modification of our synthetic route is unique to that approach;
it would have been difficult, if not impossible, to have prepared
the same derivatives biosynthetically, for example.59 The
availability of these materials should enable the rather precise
determination of the reaction pathways 1 follows in many
different settings, in particular in vivo studies with different
cancer cell lines.
(59) (a) Iwasaki, S. Compr. Nat. Prod. Chem. 1999, 1, 557. (b) Thorson, J. S.;
Shen, B.; Whitwam, R. E.; Liu, W.; Li, Y.; Ahlert, J. Bioorg. Chem. 1999,
27, 172.
9
5400 J. AM. CHEM. SOC. VOL. 124, NO. 19, 2002

Synthesis of Neocarzinostatin Chromophore
A R T I C L E S
Scheme 28
transformation (58 f 2) could have been foreseen or predicted
with any certainty.
tive transposition of 2,3-epoxy alcohols, glycosylation with
2-amino sugar donors), we believe that these studies will allow
us to determine the fundamental chemistry of 1 that occurs in
vivo.
Aglycon 2 was shown to be a viable substrate for glycosy-
lation, despite its instability. The fucosyl (2′-methylaminof2′-
hydroxyl) analogue 3 was thus prepared, using the Schmidt
trichloroacetimidate protocol. This strategy was then extended
to the preparation of 1, with the key recognition that the 2′-
methylamino group was best introduced without a protective
group. Both glycosylation reactions were highly selective for
the desired R-anomeric glycosides. Efficient procedures for the
final deprotection and purification of synthetic 3 and 1 were
also developed. To date, the route described has been executed
successfully and independently by three different researchers
for the preparation of milligram quantities of 1 and, as described,
radiolabeled 1. In addition to expanding our understanding of,
and modes of implementation of, various basic chemical
transformations (e.g., intramolecular acetylide additions, reduc-
Acknowledgment. Generous financial support from the
National Institutes of Health and Pfizer, Inc., is gratefully
acknowledged. R.G. thanks the Deutsche Akademie der Natur-
forscher Leopoldina for a postdoctoral fellowship (BMBF-LPD
9901/8-36).
Supporting Information Available: Experimental procedures,
schemes for synthesis of compounds 13, 14, 91, 92, and 93,
and characterization data for all compounds (PDF). This material
is available free of charge via the Internet at http://pubs.acs.org.
JA012487X
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J. AM. CHEM. SOC. VOL. 124, NO. 19, 2002 5401