Total synthesis and structural elucidation of khafrefungin

Article abstract of DOI:10.1021/ja0057272

Total synthesis and structural elucidation of khafrefungin, a novel antifungal agent isolated from the fermentation culture MF6020, have been achieved. Unlike other inhibitors that inhibit the corresponding enzyme in fungi and mammals to the same extent,

Full text of DOI:10.1021/ja0057272

1372
J. Am. Chem. Soc. 2001, 123, 1372-1375
Total Synthesis and Structural Elucidation of Khafrefungin
Takeshi Wakabayashi, Kouhei Mori, and Shuj Kobayashi*
Contribution from the Graduate School of Pharmaceutical Sciences, The UniVersity of Tokyo,
Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
ReceiVed October 25, 2000
Abstract: Total synthesis and structural elucidation of khafrefungin, a novel antifungal agent isolated from
the fermentation culture MF6020, have been achieved. Unlike other inhibitors that inhibit the corresponding
enzyme in fungi and mammals to the same extent, khafrefungin does not impair sphingolipid synthesis of
mammals. The basic strategy for the structural elucidation is to prepare all stereoisomers of the structurally
simplified khafrefungin mimics 1 and 2 that were designed for the elucidation of C10,11,12 and C2′,3′,4′
relative stereochemistry, respectively. The comparison of their spectra with those of natural khafrefungin would
result in the identification of eight possible stereoisomers, and the analytical details of these eight stereoisomers
have led to the complete stereochemical assignment. On the basis of the structural elucidation, the total synthesis
of khafrefungin has been accomplished by using tin(II)-catalyzed asymmetric aldol reactions as key steps.
Khafrefungin is a novel antifungal agent isolated from the
fermentation culture MF6020 by a Merck group in 1997.1 It
has been shown to inhibit the biosynthesis of inositol phosphor-
ylceramide (IPC) in Sacchromyces cereVisiae and pathogenic
fungi such as Candida albicans and Cryptococcus neoformans
in picomolar and nanomolar concentrations and causes ceramide
accumulation. Unlike other inhibitors that inhibit the corre-
sponding enzyme in fungi and mammals to the same extent,
khafrefungin does not impair sphingolipid synthesis of mam-
mals.2 Khafrefungin is composed of C5 aldonic acid esterified
at the C4 hydroxy group with C22 linear polyketide acid
including four chiral centers, and its absolute and relative
configuration until now had remained unknown due to both the
limited availability from nature and the complexity of the
chemical degradation. In this paper, we report the complete
stereochemical assignment and the first asymmetric total
synthesis of khafrefungin.
Figure 1. Khafrefungin and its mimics 1 and 2.
aluminum hydride (LAH) followed by Swern oxidation gave
Our basic strategy is to prepare all stereoisomers of the
structurally simplified khafrefungin mimics 1 and 2 that were
designed for the elucidation of C10,11,12 and C2′,3′,4′ relative
stereochemistry, respectively (Figure 1). The comparison of their
spectra with those of natural khafrefungin would result in the
identification of eight possible stereoisomers, so that the
analytical details of these eight stereoisomers would lead to the
complete stereochemical assignment as well as the total
synthesis of khafrefungin.
The synthesis of 1 was performed according to Scheme 1.
The catalytic asymmetric aldol reaction of decanal with the silyl
enol ether derived from S-ethyl propanethioate (3) by using 0.2
equiv of a tin(II)-chiral diamine complex afforded the corre-
sponding aldol adduct (4) in 83% yield with 94% ee.3 The
deoxygenation of the aldol adduct was performed by thiocar-
bonylation of the hydroxy group of 4 followed by treatment
with tributyltin hydride in the presence of a catalytic amount
of AIBN.4 Reduction of the resulting thioester by lithium
the key aldehyde (5). The aldol reaction of 5 with silyl enol
ether 3 with 0.1 equiv of Sc(OTf)3 proceeded smoothly in
5
propionitrile to afford four stereoisomers of the adduct 6 in 92%
yield (6a:6b:6c:6d ) 22:38:9:31).6 The easily separated 6d was
reduced by using lithium borohydride, and the secondary
hydroxy group of the resulting diol 7d was protected selectively
as its p-methoxybenzyl (PMB) ether via hydride addition of
the corresponding p-methoxybenzylidene acetal to give the
primary alcohol, which was converted to the aldehyde 8d by
Swern oxidation. The Wittig reaction of 8d with (carbethoxy-
ethylidene)triphenylphosphorane afforded the corresponding
ester stereoselectively (E/Z ) >95/5), which was reduced with
(3) (a) Kobayashi, S.; Uchiro, H.; Fujishita, Y.; Shiina, I.; Mukaiyama,
T. J. Am. Chem. Soc. 1991, 113, 4247. (b) Kobayashi, S.; Uchiro, H.; Shiina,
I.; Mukaiyama, T. Tetrahedron 1993, 49, 1761. (c) Kobayashi, S.; Kawasuji,
T.; Mori, N. Chem. Lett. 1994, 217. (d) Kobayashi, S.; Horibe, M. Chem.
Eur. J. 1997, 3, 1472. This reaction was already utilized in our total
synthesis20 of sphingofungin B and F that was shown to inhibit serine
palmitoyltransferase (SPT) in the biosynthesis of sphingolipid.21
(4) (a) Barton, D. H. R.; McCombie, S. W. J. Chem. Soc., Perkin Trans.
1 1975, 1574. (b) Robins, M. J.; Wilson, J. S. J. Am. Chem. Soc. 1983,
105, 4059.
(1) Mandala, S. M.; Thornton, R. A.; Rosenbach, M.; Milligan, J.; Garcia-
Calvo, M.; Bull, H. G.; Kurtz, M. B. J. Biol. Chem. 1997, 272, 32709.
(2) Reviews: (a) Kolter, T.; Sandhoff, K. Angew. Chem., Int. Ed. Engl.
1999, 38, 1532. See also: (b) Dickson, R. C. Annu. ReV. Biochem. 1998,
67, 27.
(5) (a) Kobayashi, S.; Hachiya, I.; Ishitani, H.; Araki, M. Synlett 1993,
472. (b) Kobayashi, S. Eur. J. Org. Chem. 1999, 15.
(6) For stereochemical assignment of 6a-d, see Supporting Information.
10.1021/ja0057272 CCC: $20.00 © 2001 American Chemical Society
Published on Web 01/27/2001

Synthesis and Structural Elucidation of Khafrefungin
J. Am. Chem. Soc., Vol. 123, No. 7, 2001 1373
Scheme 2. Synthesis of 2a
Scheme 1. Synthesis of 1d
Scheme 3. Spectral Data of 2b, 2c, and 2d
derivative 2a (Scheme 2). The anomeric hydroxy group of
D-ribose was protected as its allyl ether, and the three remaining
hydroxy groups were subsequently protected as their PMB ethers
to give compound 14. Deprotection of the allyl group of 14
and successive reduction of the resulting hemiacetal were carried
out in the same pot with DIBAL in the presence of NiCl2(dppp)9
to afford the corresponding diol, which was protected selectively
as its triphenylmethyl (Tr) ether to give secondary alcohol 15a.
DCC-mediated esterification of 15a with trans-2-methyl-2-
pentadecenoic acid followed by deprotection of the Tr group
afforded alcohol 16a. TPAP-catalyzed oxidation7 followed by
treatment with sodium chlorite gave the corresponding car-
boxylic acid. After the carboxylic acid was converted to methyl
ester 17a, deprotection of the three PMB groups of 17a was
successfully performed with BBr3 to afford 2a.
diisobutylaluminum hydride (DIBAL). The resulting alcohol was
oxidized with a catalytic amount of TPAP in the presence of
NMO7 to give aldehyde 9d. A similar transformation from
aldehyde 9d to dienal 10d was performed. The asymmetric aldol
reaction of dienal 10d with silyl enol ether 3 by using a tin-
(II)-chiral diamine complex proceeded smoothly to afford the
corresponding aldol adduct (11d) in 90% yield with >98% ds.3,8
After the hydroxy group of 11d was protected as its triethylsilyl
(TES) ether, reduction of the thioester group with DIBAL gave
aldehyde 12d. A propionate unit was installed to the aldehyde
12d by using Wittig reaction to give the corresponding ester
stereoslectively (E/Z ) >95/5), which was treated with aqueous
HCl in ethanol to afford secondary alcohol 13d. Finally, TPAP-
catalyzed oxidation7 of 13d followed by deprotection of the
PMB group with DDQ furnished 1d.
Three other stereoisomers, 1a, 1b, and 1c, were prepared from
the corresponding aldol adducts 6a, 6b, and 6c, respectively,
in the same way. According to the NMR spectra of the four
synthesized compounds and natural khafrefungin (H11: 5.7 Hz
(t); C11: δ 79.8), it is reasonable to assume that natural
khafrefungin would bear 10,11-anti and 11,12-syn stereochem-
ical relationships.
Arabinonic acid derivative 2b was also prepared from alcohol
15b derived from D-arabinose in the same way. On the other
hand, xylonic acid derivative 2c and lyxonic acid derivative 2d
were prepared from alcohols 15c and 15d, respectively, which
were derived via Mitsunobu reaction10 of alcohol 15b and 15a,
1
respectively. According to H and 13C NMR analyses of the
four synthesized compounds 2a, 2b, 2c, 2d, and natural
khafrefungin (1H NMR: J2′3′ ) 1.9, J3′4′ ) 9.1, J4′5′ ) 2.8, 4.2
Hz; 13C NMR: δ 61.5, 71.3, 71.8, 75.5), the spectral data of
2b were the most similar to those of khafrefungin, suggesting
(8) Stereochemical assignment of newly created asymmetric centers was
made based on the chirality of the chiral ligand.
(9) Taniguchi, T.; Ogasawara, T. Angew. Chem., Int. Ed. Engl. 1998,
37, 1136.
Our attempt to elucidate the C2′-C4′ relative stereochemistry
of khafrefungin was initiated by preparation of ribonic acid
(7) Griffith, W. P.; Ley, S. V. Aldrichim. Acta 1990, 23, 13.
(10) Review: Mitsunobu, O. Synthesis 1981, 1.

1374 J. Am. Chem. Soc., Vol. 123, No. 7, 2001
Wakabayashi et al.
Scheme 5. Synthesis of 18a
Figure 2. Possible stereoisomers of khafrefungin.
Scheme 4. Stereoselective Synthesis of 7b
yield. Sharpless epoxidation14 of 20 with D-tartaric acid diethyl
ester followed by treatment of the resulting epoxide 21 with
Me2CuLi gave the desired diol (7b) in good yield with high
stereoselectivity (>90% ds).
We next undertook the synthesis of compound 18a having a
fully functionalized khafrefungin structure (Scheme 5). Diol 7b
was converted to ester 22ab according to similar procedures in
Scheme 1. Reduction of the ester 22ab was conducted with
DIBAL, and successive TPAP-catalyzed oxidation7 gave alde-
hyde 23ab, which was treated with sodium chlorite to provide
carboxylic acid 24ab in high yield. The DCC-mediated esteri-
fication reaction of 1 equiv of carboxylic acid 24ab with a slight
excess amount of alcohol 15e, which was prepared from
D-arabinose by a similar procedure shown in Scheme 2, in the
presence of (dimethylamino)pyridine (DMAP) and DMAP‚HCl
(Keck’s conditions)15 proceeded smoothly to afford the corre-
sponding ester 25a. It was found that the addition of DMAP‚
HCl was essential to obtain high yield. Both TBS and TES
groups of ester 25a were removed by exposure to aqueous HCl
in THF to give diol 26a in 71% yield for two steps. Oxidation
of diol 26a was successfully performed by Dess-Martin
periodinane16 to afford the keto aldehyde without any epimerized
product at the C4 position, which was treated with sodium
chlorite to furnish keto carboxylic acid 27a in 77% yield for
two steps. Finally, four PMB groups were removed with BCl3
to afford 18a in 57% yield.
that khafrefungin would be an arabinonic acid derivative
(Scheme 3).
At this stage, it was assumed that natural khafrefungin would
bear 10,11-anti, 11,12-syn, 2′,3′-syn, and 3′,4′-syn stereochem-
ical relationships, which led to the identification of eight possible
stereoisomers of natural khafreungin (Figure 2). Our next task
is to synthesize four of the eight stereoisomers. Comparison of
physical data of these synthesized compounds with those of
natural khafrefungin would reveal the real structure of khafre-
fungin.
Initially, we undertook the stereoselective synthesis of diol
7b including three contiguous chiral centers. The Sc(OTf)3-
catalyzed aldol reaction shown in Scheme 1 proceeded in high
yield,5 albeit the desired stereochemical outcome that yielded
the R,â-anti, â,γ-syn aldol adduct was low (38% ds). At this
stage, it was necessary to develop a new method for highly
stereoselective synthesis of diol 7b. We attempted several types
of aldol reactions. The TiCl4-promoted system gave the desired
aldol adduct in moderate stereoselectivity (56% ds). The
selectivity was slightly improved by the reaction of the lithium
enolate generated from S-ethyl propanethioate with aldehyde 5
(66% ds). These results encouraged us to examine some chiral
ligands;11 however, all chiral catalyst-controlled aldol reactions,
including chiral tin(II)-catalyzed reactions,12 failed for the
preparation of the desired R,â-anti, â,γ-syn adduct. Thus, the
Kishi protocol,13 including Sharpless asymmetric epoxidation
of a chiral allylic alcohol, was then employed instead of the
aldol process for stereoselective synthesis of diol 7b (Scheme
4). Wittig reaction of aldehyde 5 with (carbethoxymethylidene)-
triphenylphosphorane gave the corresponding ester (19) stereo-
selectively (E/Z ) >95/5), which was reduced by diisobutyl-
aluminum hydride (DIBAL) to afford allylic alcohol 20 in high
The other three stereoisomers 18b, 18c, and 18d were also
prepared via esterification reactions of the corresponding
carboxylic acids with the corresponding alcohols in the same
way. Among the four stereoisomers, the spectra of 1H and 13
C
NMR, Rf value of TLC, the retention time of RP HPLC, and
the optical rotation of 18a were completely consistent with those
of natural khafrefungin (18a: [R]D -27 (c 0.095, MeOH);
natural: [R]D -27 (c 0.29, MeOH).17,18 In addition, among the
(14) Katsuki, T.; Sharpless, K. B. J. Am. Chem. Soc. 1980, 102, 5974.
(15) Boden, E.; Keck, G. E. J. Org. Chem. 1985, 50, 2394.
(16) Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48, 4155.
(17) While the optical rotation of natural khafrefungin is [R]D -27
(c 0.29 MeOH), the optical rotations of all four synthesized compounds
(18a-d) show negative values indicating that ent 18a-d are not natural
khafrefungin. 18b: [R]D -3.6; 18c: [R]D -6.1; 18d: [R]D -34 (MeOH).
(18) The concentration for the measurement of optical rotations of 18a-d
was determined by using the extinction coefficient at 286 nm (14300) based
on the original procedure. See Supporting Information.
(11) Reviews: (a) Carreira, E. M. In ComprehensiVe Asymmetric
Catalysis; Jacobsen, E. N., Pfaltz, A., Yamamoto, H., Eds.; Springer-
Verlag: Berlin, 1999; p 997. (b) Mahrwald, R. Chem. ReV. 1999, 99, 1095.
(12) Kobayashi, S.; Ohtsubo, A.; Mukaiyama, T. Chem. Lett. 1991, 831.
(13) Nagaoka, H.; Kishi, Y. Tetrahedron 1981, 37, 3873.

Synthesis and Structural Elucidation of Khafrefungin
J. Am. Chem. Soc., Vol. 123, No. 7, 2001 1375
four stereoisomers, only 18a has been shown to inhibit IPC
synthase to almost the same extent as natural khafrefungin.19
These analyses have revealed the stereochemistry, including the
absolute configuration, of khafrefungin to be 18a, and at the
same time, total synthesis of khafrefungin has been completed.
completion of the total synthesis, we also found that khafre-
fungin exhibits a strict stereochemical requirement for antifungal
activity, and biological details, including structure-activity
relationships and structural modification, are now under inves-
tigation.
In summary, complete stereochemical assignment and total
synthesis of khafrefungin have been achieved by using highly
stereocontrolled reactions. Several possible stereoisomers have
been synthesized, and comparison of their physical data as well
as biological activity with those of an authentic sample have
revealed the true structure of khafrefungin. For the total
synthesis, the tin(II)-catalyzed asymmetric aldol reaction has
been shown to be a powerful synthetic tool for the construction
of acyclic compounds containing several chiral centers. After
Acknowledgment. The authors are grateful to Dr. Guy
Harris (Merck) for providing us with an authentic sample of
khafrefungin, spectral data, and other valuable information. Drs.
Kentaro Hanada and Satoshi Yasuda (National Institute of
Infectious Diseases) are also acknowledged for measurement
of the biological activity of khafrefungin. The authors also thank
Mr. Hiroki Kobayashi for his technical support. This work was
partially supported by CREST, Japan Science and Technology
Corporation (JST), and a Grant-in-Aid for Scientific Research
from the Ministry of Education, Science, Sport, and Culture,
Japane. T.W. thanks the JSPS fellowship for Japanese Junior
Scientists.
(19) Yasuda, S.; Hanada, K. Unpublished. Details will be reported in
due course.
(20) (a) Kobayashi, S.; Furuta, T.; Hayashi, T.; Nishijima, M.; Hanada,
K. J. Am. Chem. Soc. 1998, 120, 908. (b) Kobayashi, S.; Furuta, T.
Tetrahedron 1998, 54, 10275.
(21) (a) VanMiddlesworth, F.; Gracobbe, R. A.; Lopez, M.; Garrity, G.;
Bland, J. A.; Bartizal, K.; Fromtling, R. A.; Polishook, J.; Zweerink, M.;
Edison, A. M.; Rozdilsky, W.; Wilson, K. E.; Monaghan, R. L. J. Antibiot.
1992, 45, 861. (b) Zweerink, M. M.; Edison, A. M.; Well, G. B.; Pinto,
W.; Lester, R. L. J. Biol. Chem. 1992, 267, 25032.
Supporting Information Available: Experimental details
and 1H and 13C NMR spectra (PDF). This material is available
free of charge via the Internet at http://pubs.acs.org.
JA0057272