Chemistry and biology of khafrefungin. Large-scale synthesis, design, and structure-activity relationship of khafrefungin, an antifungal agent

Article abstract of DOI:10.1039/b305818b

Large-scale synthesis, design, and structure-activity relationships of khafrefungin are reported. Khafrefungin is an antifungal agent that inhibits inositol phosphorylceramide (IPC) synthase, a enzyme involved in fungal sphingolipid biosynthesis. Unlike o

Full text of DOI:10.1039/b305818b

View Article Online / Journal Homepage / Table of Contents for this issue
Chemistry and biology of khafrefungin. Large-scale synthesis,
design, and structure–activity relationship of khafrefungin, an
antifungal agent†
Masayuki Nakamura,a Yuichiro Mori,a Kennichi Okuyama,a Kunihiro Tanikawa,a Satoshi
Yasuda,b Kentaro Hanadab and Shu¯ Kobayashi*a
a Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan
b Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases,
Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. E-mail: skobayas@mol.f.u-tokyo.ac.jp;
Fax: ϩ81-3-5684-0634
Received 27th May 2003, Accepted 31st July 2003
First published as an Advance Article on the web 5th September 2003
Large-scale synthesis, design, and structure–activity relationships of khafrefungin are reported. Khafrefungin is an
antifungal agent that inhibits inositol phosphorylceramide (IPC) synthase, an enzyme involved in fungal sphingolipid
biosynthesis. Unlike other inhibitors that inhibit the corresponding enzyme in fungi and mammals to the same
extent, khafrefungin does not impair sphingolipid synthesis in mammals. We have developed an efficient method for
large-scale synthesis of khafrefungin, and various khafrefungin derivatives were synthesized based on this method.
While most of the khafrefungin derivatives lost antifungal activity, a lactone-type derivative had almost the same
activity as khafrefungin. We also designed and synthesized derivatives which contain a five- or six-membered ring at
the central part of the structure based on NOE experiments of khafrefungin. A macrocyclic khafrefungin derivative
was also synthesized, but the antifungal activity was lost. These results suggest that the structure of khafrefungin
might be strictly recognized in fungi.
khafrefungin.3,4 However, in order to investigate the structure–
activity relationship of khafrefungin, a more efficient route to
Introduction
Khafrefungin (1), an antifungal agent isolated from the fermen-
khafrefungin which tolerates hundreds-of-milligrams to gram
tation culture MF6020 by a Merck group,1 has been shown to
scale preparation was necessary. One drawback of the previous
inhibit IPC (inositol phosphorylceramide) synthase, which
convergent route was the use of a toxic thallium reagent in the
catalyzes the fungal specific step in Saccharomyces cerevisiae
and pathogenic fungi. Different from other inhibitors that
Suzuki–Miyaura coupling reaction. Therefore, we tried to avoid
the use of the thallium reagent in a newly developed convergent
inhibit the corresponding enzyme in fungi and mammals to the
route.
same extent, khafrefungin does not impair sphingolipid syn-
Retrosynthetic analysis of khafrefungin is shown in Scheme
1. Khafrefungin (1) is divided into three fragments: alkyne 3,
thesis in mammals.2 As regards the structure of khafrefungin,
although the Merck group showed the plane structure, the
alkenyliodide 4, and protected alcohol
5 (aldonic acid
stereochemistry remained unknown until we revealed the
relative and absolute configuration of khafrefungin in 2001. We
have already completed the first total synthesis of khafrefungin
using chiral tin()-catalyzed aldol reaction, Sharpless asym-
metric epoxidation, and Keck esterification as the key steps.3
Furthermore, we have accomplished another convergent total
synthesis of khafrefungin4 using chiral zirconium()-catalyzed
aldol reaction and Suzuki–Miyaura coupling as the key steps.
In the course of our further investigations on the chemistry
and biology of khafrefungin, it was necessary to prepare
khafrefungin on a hundreds-of-milligrams to gram scale. In this
paper, we report an efficient method for large-scale synthesis
of khafrefungin and its derivatives, and the structure–activity
relationship. We designed various analogues of khafrefungin
partially based on a metabolism experiment and NOE and
HMBC experiments of khafrefungin. Especially, a macrocyclic
khafrefungin analogue was designed as an analogue to
rustmicin, which is also an IPC synthase inhibitor.2d
part). In the previous report, 3 was connected with 4 first,
and then 5 was introduced. Namely, Suzuki–Miyaura coupling
of 3 with 4 followed by esterification with 5 gave khafrefungin.
However, a problem is that synthesis of fragment 3 needs
the most multiple transformations among those of the
three fragments, and therefore, it would be more efficient if
4 was connected with 5 by esterification first, followed by
Suzuki–Miyaura coupling with 3. As mentioned above,
another drawback of the previous convergent synthesis is
the use of a thallium reagent as a base in the Suzuki–Miyaura
coupling.
First, we decided to develop a new synthetic pathway to
alcohol 10, which was a key intermediate for the preparation
of 3 (Scheme 2). Commercially available methyl (R)-(Ϫ)-3-
hydroxy-2-methylpropionate (8) was protected as its benzyl
ether, and successive reduction using lithium aluminium
hydride and tosylation afforded 9 (91%, 3 steps). Benzyl ether 9
was then alkylated, and the benzyl group was deprotected using
BCl3 to produce chiral alcohol 10. Alcohol 10 was then con-
verted to alkyne 3 according to the reported procedure.4 The
whole transformations were conducted on a multigram scale.
Since we have already synthesized units 4 and 5 on a multigram
scale, we completed the synthesis of all the three units (3–5) also
in multigram-scale quantities.5 Ester 4 was converted to carb-
oxylic acid 12, which was coupled with alcohol 5 under Keck’s
esterification conditions6 to afford the desired adduct (13)
(Scheme 3). We then examined several conditions of the Suzuki–
Results and discussion
Large-scale synthesis of khafrefungin
Recently, we have completed the first total synthesis of
khafrefungin and also developed a convergent route to
† Electronic supplementary information (ESI) available: full experi-
mental details. See http://www.rsc.org/suppdata/ob/b3/b305818b/
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 6 2 – 3 3 7 6
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
3362

View Article Online
Scheme 1 Retrosynthetic analysis for the synthesis of khafrefungin.
Scheme 2 Synthesis of chiral alcohol 10. Reagents and conditions: (a) i) BnOC(᎐NH)CCl3, cat. TfOH, Et2O, ii) LiAlH4, THF, iii) TsCl, DABCO,
᎐
CH2Cl2, 3 steps, 91%; (b) i) (C9H19)2 CuLi, Et2O, ii) BCl3, CH2Cl2, 2 steps, 84%.
Scheme 3 Synthesis of 2. Reagents and conditions: (a) DIBAL, CH2Cl2, Ϫ78 ЊC, 99%; (b) i) MnO2, CH2Cl2, ii) NaClO2 NaH2PO4, t-BuOH, H2O,
2 steps, 87%; (c) 5, DCC, DMAP, DMAPؒHCl, CH2Cl2, reflux, 79%; (d) 3, 9-BBN, THF, rt, then PdCl2(dppf ), K3PO4, H2O, 65 ЊC, 84%.
Miyaura coupling7 of 13 with alkyne 3, and the results are
summarized in Table 1. In the previous synthesis, cate-
cholborane and thallium ethoxide8 were used as reagents for
this coupling reaction; however, thallium ethoxide is not
suitable for large-scale synthesis due to its high toxicity. After
screening several reaction conditions, it was found that a
one-pot procedure with 9-borabicyclo[3.3.1]nonane (9-BBN),
PdCl2(dppf ),9 and K3PO4 gave 2 in high yield (76%, Table 1,
entry 4). Moreover, the yield was improved to 84% in a larger-
scale reaction (Scheme 3). After purification by column chro-
matography on silica gel, no regioisomer of 2 was observed
by NMR analysis. It is noted that multigram-preparation of 2
has been successfully performed according to a novel synthetic
strategy.
On the other hand, the Suzuki–Miyaura coupling of
alkenyliodide 4 with alkyne 3 also proceeded smoothly under
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 6 2 – 3 3 7 6
3363

View Article Online
Table 1 Suzuki coupling reaction
Entry
Borane
Temp.
Pd
Base
Yield (%)
1
2
3
4
catecholborane
catecholborane
catecholborane
9-BBN
50 ЊC
50 ЊC
rt
Pd(PPh3)4
Pd(PPh3)4
PdCl2(dppf )
PdCl2(dppf )
TlOEt
Na2CO3
K3PO4
K3PO4
42 (33)a
27 (53)a
50
rt
76
a Recovered 13 (%).
Scheme 4 Synthesis of 2. Reagents and conditions: (a) 3, 9-BBN, THF, rt, then PdCl2(dppf ), K3PO4, H2O, 65 ЊC, 88%; (b) DIBAL, CH2Cl2, Ϫ78 ЊC,
94%; (c) i) MnO2, CH2Cl2, ii) NaClO2, NaH2PO4, t-BuOH, H2O, 2 steps, 73%; (d) 5, DCC, DMAP, DMAPؒHCl, CH2Cl2, reflux, 69%.
Scheme 5 Synthesis of khafrefungin 1. Reagents and conditions: (a) HCl, THF, 84%; (b) i) Dess–Martin periodinane, pyridine, CH2Cl2, ii) NaClO2,
NaH2PO4, t-BuOH, H2O, 2 steps, 55%; (c) BCl3, CH2Cl2, Ϫ78 ЊC, 51%.
thallium-free conditions. Indeed, the coupling reaction of 4
with 3 proceeded smoothly to afford the desired product 14
in 88% yield (Scheme 4). After treatment with DIBAL, the
resulted alcohol was oxidized to the corresponding aldehyde
first, and then to the carboxylic acid by treatment with sodium
chlorite10 in 73% yield for two steps. Keck’s esterification
reaction of the carboxylic acid with alcohol 5 furnished the
desired ester 2 in 69%.
The final stage of the total synthesis of khafrefungin is
shown in Scheme 5. Deprotection of two silyl groups of ester 2
was conducted with 1 M aqueous hydrochloric acid in THF in
84% yield. Oxidation of the resulting diol 15 was successfully
performed using Dess–Martin periodinane11 to afford the keto-
aldehyde, which was treated with sodium chlorite to furnish the
ketocarboxylic acid 16 in good yields. Finally, four PMB groups
were removed with BCl3 to afford khafrefungin.
observed.13 We conducted the conformational analysis of
khafrefungin in detail once again using HMBC and NOE
experiments in CD3OD (Fig. 1). The analysis suggested that the
conformation of khafrefungin seemed to bend around C4 and
play an important role in the antifungal activity. To examine
the effect of this unique structure on the antifungal activity, we
designed rigid derivatives 17 and 18 and dimethyl derivative 19.
Compared to rustmicin which is also known as an IPC
synthase inhibitor, macrocyclic khafrefungin analogue 20 was
also planned to be synthesized with both antifungal activity
and synthetic interest.
Second, from the initial metabolism study of khafrefungin,
the ester bond was easily metabolized to afford carboxylic
acids 22 and 23, and oxidation of the lipophilic side chain was
observed in mouse plasma, rat hepatocytes, and liver micro-
somes from mice and humans (Fig. 2). To elucidate the function
of this linkage, we designed representative linkage derivatives,
for example, amides 24, thioester 25, ether 26, and methylene
ketone 27 (Fig. 3). To decrease the lipophilicity of khafrefungin,
we also designed anti-derivatives 28 and 29, in which their
stereochemistry would be constructed using asymmetric aldol
reactions in the presence of a chiral zirconium catalyst.
Furthermore, a derivative without two methyl groups (30) and
a derivative having a shorter alkyl chain (31) were designed.
The aldonic acid part of khafrefungin has the characteristic
structure (three stereogenic centers and three hydroxy groups).
To investigate the effect of this aldonic acid part, derivatives 22,
32–36 were designed (Fig. 4).
Thus, new synthetic routes to khafrefungin, which tolerate
large-scale preparation, have been developed. Synthetic
khafrefungin was used for the following investigations to
prepare khafrefungin-based derivatives.
Structure–activity relationship
Design. We examined the structure–activity relationship of
khafrefungin.12 Our purpose is not only enhancement of the
antifungal activity of khafrefungin but also the conformational
study of khafrefungin as well as improvement of metabolism
stability for therapeutic agents.
First, we focused on the Merck group’s suggestion that
strong NOE between H4 and H7 of khafrefungin was
Finally, as mentioned above, we had synthesized large
quantities of khafrefungin in hand. We then planned to modify
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 6 2 – 3 3 7 6
3364

View Article Online
Fig. 1 NOE experiment study of khafrefungin in CD3OD and designed derivatives from conformational analysis.
Fig. 2 Proposed metabolites of khafrefungin in mouse plasma, rat hepatocytes, and liver microsomes from mice and humans.
khafrefungin in order to investigate several functional groups
of khafrefungin.
moderate yield. According to the usual protocol, macrocyclic
khafrefungin analogue 20 was obtained.
The synthesis of the amide derivative is shown in Scheme 9.
The amino group was introduced via protection of primary
alcohol 55 with trityl chloride (TrCl), inversion of the
secondary alcohol using Mitsunobu reaction,16 further
inversion using sodium azide, and reduction using triphenyl-
phosphine and H2O. Amine 58 was converted to protected
amine 59, which was coupled with 62 using bromotris-
(pyrrolidino)phosphonium hexafluorophosphate (PyBrop) as a
coupling reagent to afford 60. The 2,4-dimethoxybenzyl (DMB)
group was removed to afford lactone 61. Compound 24 was not
observed under these conditions. In a similar way, thioester
derivative 25 was obtained as shown in Scheme 10.
The synthesis of ether derivative 26 is shown in Scheme 11.
After examination of several reaction conditions, the ether
bond was formed from 68 and 5 in moderate yield using AgOTf
in the presence of MS 4A and 2,6-dibutylpyridine. After con-
version of iodide 69 into 70, alcohol 70 was oxidized via two
steps, followed by deprotection of the PMB groups according
to the previous procedures to afford ether derivative 26.
Methylene ketone derivatives 27a and 27b (diastereomers
at C4) were synthesized according to Scheme 12. Aldehyde 71
Synthesis. The synthesis of 5-membered derivative 17 is
summarized in Scheme 6. Iodide 37, which was prepared
according to the literature,14 was converted into fragment 38
and then 39. After successive connection with alkyne 3 using
Suzuki–Miyaura coupling reaction and with alcohol 5 by
modified Keck esterification, 5-membered derivative 17 was
obtained according to the procedures shown in our total
synthesis of khafrefungin. 6-Membered derivative 18 was also
prepared as shown in Scheme 6. Dimethyl derivative 19 was
synthesized from alcohol 47 as shown in Scheme 7.
Synthesis of macrocyclic khafrefungin 20 was performed
starting from fragments 3, 4, and 5 (Scheme 8). Replacement
of the p-methoxybenzyl (PMB) group into the acetyl (Ac)
group at the C11 position was conducted first, and then one-pot
Suzuki–Miyaura coupling reaction with iodide 13 and selective
desilylation by cooled HF in an MeCN solution afforded
fully functionalized adduct 52. Seco acid 53 was furnished by
oxidation into the carboxylic acid and deprotection of the
acetyl group by DIBAL. Yamaguchi macrolactonization15
was then applied to afford the desired macrocycle 54 in
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 6 2 – 3 3 7 6
3365

View Article Online
Fig. 3 Designed derivatives for study of metabolism stability.
Fig. 4 Khafrefungin derivatives containing modified aldonic acid parts.
was readily prepared from alcohol 5 using Swern oxidation,
Wittig reaction, 1,4-reduction, and DIBAL reduction. Two
diastereomers with the ratio of 3 : 5 were separated by silica gel
column chromatography. Anti-adduct 72 was converted into 73,
and zirconation using Cp2ZrHCl17 followed by iodination gave
iodide 75. After Nozaki–Hiyama–Kishi coupling18 with the
vinyl iodide, two silyl groups were deprotected to afford triols
76ac–76bd. According to the similar protocol of the total
synthesis of khafrefungin, methylene ketones 27a and 27b were
obtained.
stereo- and enantioselectivities. Each adduct was converted to
an alkyne according to the previous synthesis of fragment 3.
After oxidation and deprotection, derivatives 28 and 29 were
obtained as shown in Scheme 13. On the other hand, derivatives
30 and 31 were synthesized based on the first synthetic route
to natural khafrefungin (1) (Schemes 14 and 15).3 Similarly,
derivatives 22, 32–36 were prepared (Schemes 16–19). In the
synthesis of the sugar parts of derivatives 34–36, the corre-
sponding carboxylic acid or monosaccharides were used as
starting materials (Scheme 19).
We have already reported that anti aldol adducts were
obtained stereoselectively using a novel chiral zirconium
catalyst.19 Thus, catalytic asymmetric aldol reactions of alde-
hydes 77 and 78 with silyl enol ether 79 using a chiral zirconium
catalyst prepared from Zr(Ot-Bu)4, (S)-3,3Ј,6,6Ј-tetraiodo-1,1Ј-
binaphthyl-2,2Ј-diol ((S)-3,3Ј,6,6Ј-I4BINOL), propanol, and
H2O proceeded smoothly to afford the corresponding aldol
adducts 80 and 81, respectively, in high yields with high dia-
From synthetic khafrefungin, which was supplied in the
large-scale synthesis, several derivatives were prepared as shown
in Scheme 20. Treatment of khafrefungin under acidic con-
ditions gave 6-membered lactone 111. It was found that the
lactone was stable during the course of purification by work-up
and silica gel chromatography. To investigate the effect of the
aldonic acid part, some other derivatives were obtained
(Scheme 20).
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 6 2 – 3 3 7 6
3366

View Article Online
Scheme 6 Synthesis of fused derivatives. Reagents and conditions: (a) KHMDS, toluene, Ϫ78 ЊC, ClCO2Et then CH3I, rt, 65%; (b) NaBH4, MeOH,
0 ЊC, 70%; (c) TPSCl, imidazole, DMF, quant.; (d) i) DIBAL, CH2Cl2, Ϫ78 ЊC, ii) (COCl)2, DMSO, Et3N, CH2Cl2, iii) PPh3᎐᎐C(CH3)CO2Et, toluene,
reflux, 3 steps, 93%; (e) i) 3, 9-BBN, THF, rt then PdCl2(dppf ), K3PO4, DMF, H2O, 65 ЊC, ii) DIBAL, CH2Cl2, Ϫ78 ЊC, 2 steps, 67%; (f ) i) (COCl)2,
DMSO, Et3N, CH2Cl2, ii) NaClO2, NaH2PO4, t-BuOH, H2O, iii) 5, EDClؒHCl, DMAP, CH2Cl2, reflux, 3 steps, 57%; (g) TBAF, AcOH, THF, 50
ЊC, 93%; (h) i) Dess–Martin periodinane, pyridine, CH2Cl2, ii) NaClO2, NaH2PO4, t-BuOH, H2O, 2 steps, 44%; (i) BCl3, CH2Cl2, Ϫ78 ЊC, 68%;
(j) HCHO aq, NaOH, dioxane, 62%; (k) (COCl)2, DMSO, Et3N, CH2Cl2, 92%; (l) PPh3᎐᎐C(CH3)CO2Et, THF, reflux, 97%; (m) (CH3)3SiI, 80 ЊC, 54%;
(n) 5, EDClؒHCl, DMAP, CH2Cl2, reflux, 44%; (o) i) 3, 9-BBN, THF, rt then PdCl2(dppf ), K3PO4, DMF, H2O, 65 ЊC, ii) HCl, THF, 2 steps, 44%;
(p) Dess–Martin periodinane, pyridine, CH2Cl2, 91%; (q) NaClO2, NaH2PO4, t-BuOH, H2O, 85%; (r) BCl3, CH2Cl2, Ϫ78 ЊC, 47%.
Scheme 7 Synthesis of 19. Reagents and conditions: (a) i) MnO2, CH2Cl2, ii) methyl isobutyrate, LDA, THF, Ϫ78 ЊC, 2 steps, 80%; (b) i) TPSCl,
imidazole, DMF, ii) DIBAL, CH2Cl2, Ϫ78 ЊC, 2 steps, 97%; (c) i) (COCl)2, DMSO, Et3N, CH2Cl2, ii) PPh3᎐᎐C(CH3)CO2Et, toluene, reflux, 2 steps,
91%; (d) i) 3, 9-BBN, THF, rt then PdCl2(dppf ), K3PO4, DMF, H2O, 65 ЊC, ii) DIBAL, CH2Cl2, Ϫ78 ЊC, 2 steps, 86%; (e) i) (COCl)2, DMSO,
Et3N, CH2Cl2, ii) NaClO2, NaH2PO4, t-BuOH, H2O, 2 steps, 86%; (f ) i) 5, EDClؒHCl, DMAP, CH2Cl2, reflux, ii) HCl, THF, 2 steps, 65%; (g) TBAF,
AcOH, THF, 50 ЊC, 89% (h) i) Dess–Martin periodinane, pyridine, CH2Cl2, ii) NaClO2, NaH2PO4, t-BuOH, H2O, 2 steps, 41%; (i) BCl3, CH2Cl2,
Ϫ78 ЊC, 50%.
Biological evaluation. We next explored the structure–activity
Discussion
relationship of khafrefungin. The MIC values of khafrefungin
on the liquid culture growth of Saccharomyces cereviciae cells
was ∼10 µM, being consistent with previous studies.4. Lactone-
type derivative 111 showed almost the same MIC (∼10 µM) as
natural khafrefungin. However, other novel derivatives syn-
thesized in the present study showed far less or no antifungal
activity.The derivatives 28, 29 and 30, in which the C10 or C12
methyl group was removed, inhibited the yeast cell growth
by 30–60% at 0.2 mM, but did not reach MIC value even at
0.2 mM, the highest concentration we examined. For other
derivatives, no inhibition of the yeast cell growth was observed
up to 0.2 mM.
In the structure of khafrefungin, the ester part is assumed to be
important because it was found to be easily metabolized in
mouse plasma, rat hepatocytes, and liver microsomes in a
metabolic stability study. The fact that carboxylic acid 22,
which lacks the aldonic acid part, does not have the antifungal
activity suggests that cleavage of the ester bond results in dis-
appearance of the activity. We synthesized various derivatives
that have other functional groups instead of the ester group. To
our surprise, in the cases of other functional groups such as
amide, ether, thioester, and methylene ketone, the activity dis-
appeared. These results suggest that the ester group is essential
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 6 2 – 3 3 7 6
3367

View Article Online
Scheme 8 Synthesis of macrocyclic khafrefungin. Reagents and conditions: (a) i) TFA, CH2Cl2, 0 ЊC, ii) Ac2O, pyridine, DMAP, CH2Cl2, 2 steps,
81%; (b) i) 13, 9-BBN, THF, rt then PdCl2(dppf ), K3PO4, DMF, H2O, 65 ЊC, ii) HF aq., CH3CN, Ϫ10 ЊC, 2 steps, 42%; (c) i) Dess–Martin
periodinane, pyridine, CH2Cl2, ii) NaClO2, NaH2PO4, t-BuOH, H2O, 2 steps, 66%; (d) DIBAL, CH2Cl2, Ϫ78 ЊC, 48%; (e) 2,4,6-trichlorobenzoyl
chloride, Et3N, THF then DMAP, toluene, reflux, 40%; (f ) HF aq., CH3CN, 0 ЊC, 54%; (g) Dess–Martin periodinane, pyridine, CH2Cl2, 93%;
(h) BCl3, CH2Cl2, Ϫ78 ЊC, 73%.
Scheme 9 Synthesis of amide derivative. Reagents and conditions: (a) TrCl, Et3N, CH2Cl2, 86%; (b) i) m-nitrobenzoic acid, DEAD, PPh3, THF, ii)
DIBAL, CH2Cl2, Ϫ78 ЊC, 2 steps, 55%; (c) i) MsCl, Et3N, CH2Cl2, ii) NaN3, DMF, 100 ЊC, 2 steps, 55%; (d) TsOH, MeOH, 62%; (e) TBSCl,
imidazole, DMF, 90%; (f ) PPh3, H2O, THF, 94%; (g) i), 2,4-(MeO)2C6H3CHO, MS 4A, toluene, ii) NaBH4, MeOH, 2 steps, 92%; (h) 62, PyBrop,
i-Pr2NEt, CH2Cl2, 45%; (i) HCl, THF, 88%; (j) Dess–Martin periodinane, pyridine, CH2Cl2, 60%; (k) NaClO2, NaH2PO4, t-BuOH, H2O; (l) TFA,
anisole, CH2Cl2, 40%.
for the activity, possibly because the ester carbonyl group might
be attacked by a nucleophile, which is an important step for the
Conclusion
In conclusion, we have developed an efficient synthetic route to
IPC synthase inhibition.
khafrefungin, and prepared three key units for the synthesis on
The derivatives in which the methyl group at the C12 position
a gram scale. We designed various derivatives of khafrefungin
and synthesized them by applying the efficient synthetic route
was removed (28, 29) showed far lower antifungal activity than
khafrefungin. Derivative 31 with a shorter alkyl chain lost the
to khafrefungin. Study of the structure–activity relationship
activity. The aldonic acid part seems to have an important
has revealed that most of the stereochemistry and functional
groups of khafrefungin are crucial for the antifungal activity. A
role in the antifungal activity. Lactone-type derivative 111 has
almost the same activity as that of khafrefungin, which suggests
lactone-type derivative has almost the same antifungal activity,
that khafrefungin and its lactone derivative may have the same
suggesting that there may be an equilibrium between natural
active species under equilibrium in the aqueous phase. The
khafrefungin and its lactone-type derivative. It was also
hydroxy groups in the aldonic acid part also seem to play an
revealed that the structure of khafrefungin was strictly dis-
important role for the activity. The antifungal activity dis-
tinguished in fungi. Further design and synthesis of khafrefun-
appears upon protecting one of the hydroxy groups as its acetyl
gin derivatives are now under investigation.
ester. The stereochemistry of the hydroxy groups is also crucial
for the biological activity.
The 5-membered derivative 17 and 6-membered derivative 18
Experimental
which were designed based on NOE data of khafrefungin
exhibited no activity. The active conformation may be different
from that of khafrefungin. The macrocyclic khafrefungin
derivative also lost the activity. The structure of khafrefungin
might be strictly recognized in fungi.
General
IR spectra were recorded on a JASCO FT/IR-610. 1H and
13C NMR spectra were recorded on a JEOL JNM-LA300,
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 6 2 – 3 3 7 6
3368

View Article Online
Scheme 10 Synthesis of thioester 25. Reagents and conditions: (a) TBDPSCl, imidazole, DMF, 81%; (b) i) m-nitrobenzoic acid, DIAD, Ph3P, THF,
ii) DIBAL, CH2Cl2, Ϫ78 ЊC, 2 steps, 57%; (c) MsCl, Et3N, CH2Cl2, 85%; (d) AcSK, toluene, DMF, 140 ЊC, 57%; (e) LiAlH4, Et2O, 88%; (f ) 67, EDClؒ
HCl, DMAP, CH2Cl2, reflux, 62%; (g) TBAF, AcOH, THF, 50 ЊC, 79%; (h) Dess–Martin periodinane, pyridine, CH2Cl2, 31%; (i) NaClO2, NaH2PO4,
t-BuOH, H2O, 59%; (j) BCl3, CH2Cl2, Ϫ78 ЊC, 30%.
Scheme 11 Synthesis of 26. Reagents and conditions: (a) CBr4, PPh3, THF, 98%; (b) 5, AgOTf, 2,6-dibutylpyridine, MS 4A, CH2Cl2, 0 ЊC, 41%;
(c) i) 3, 9-BBN, THF, rt then PdCl2(dppf ), K3PO4, DMF, H2O, 65 ЊC, ii) HCl, THF, 2 steps, 40%; (d) Dess–Martin periodinane, pyridine, CH2Cl2,
34%; (e) NaClO2, NaH2PO4, t-BuOH, H2O, 73%; (f ) BCl3, CH2Cl2, Ϫ78 ЊC, 17%.
JNM-LA400 or a JNM-LA500 spectrometer in CDCl3 unless
otherwise noted. Tetramethylsilane (TMS) served as internal
The organic layer was separated and the aqueous layer was
extracted with diethyl ether. The combined organic layer
was washed with brine, dried over Na2SO4, and evaporated. The
resulting residue was filtered through a short column of silica
gel (hexane : EtOAc = 10 : 1) to give the corresponding benzyl-
ate as a colorless oil, which was used without further purifi-
cation. To a suspension of lithium aluminium hydride (8.56 g,
226 mmol) in THF (200 mL) was added the benzylate in THF
(100 mL) at Ϫ10 ЊC slowly. After stirring at this temperature for
2 h, the reaction mixture was quenched with MeOH and then
added aqueous Rochelle salt and diethyl ether. The mixture was
warmed to room temperature and stirred vigorously for 5 h.
The organic layer was separated, and the aqueous layer was
extracted with diethyl ether. The combined organic layer was
washed with brine, dried over Na2SO4, and evaporated. The
resulting residue was filtered through a column of silica gel
(hexane : EtOAc = 3 : 1) to give the corresponding alcohol as a
colorless oil, which was used without further purification. To
a solution of the alcohol in dichloromethane (200 mL) was
added 1,4-diazabicyclo[2.2.2]octane (26.8 g, 239 mmol) and
TsCl (33.7 g, 177 mmol). After stirring at room temperature for
1.5 h, the reaction mixture was poured into saturated NaHCO3
and then extracted with diethyl ether. The combined organic
layer was washed with brine, dried over Na2SO4, and evaporated.
The resulting residue was purified by silica gel column chroma-
tography (hexane : EtOAc = 6 : 1) to give the tosylate (9) (27.4 g,
3 steps, 91%) as a colorless oil (Found: C, 64.89; H 6.47.
C18H22O4S requires C, 64.64; H, 6.63%); [α]D25.5 Ϫ4.74 (c 1.50 in
1
standard (δ 0) for H NMR, and CDCl3 was used as internal
standard (δ 77.0) for 13C NMR. When CD3OD was used,
1
CD3OD served as internal standard (δ 3.3) for H NMR, and
(δ 49.0) for 13C NMR. HPLC was carried out using a Shimadzu
C-R6A chromatopac, SPD-10A, and LC-10AT. Optical rota-
tions were recorded on a JASCO P-1010. Column chromato-
graphy was performed on Silica gel 60 (Merck). Mass spectra
were recorded on a JEOL JMS-SX102A mass spectrometer or a
micromass Quattro II and Q-Toff-2 instrument. Preparative
thin layer chromatography was performed on Wakogel B5F or
using Silica gel 60 F254 (Merck). All non-aqueous reactions
were performed under an oxygen-free atmosphere of argon
with rigid exclusion of moisture from reagents and glassware.
All solvents were purified according to standard procedures.
Growth inhibition was determined according to the pro-
cedure shown in ref. 4.
Synthesis of khafrefungin (1)
(2R)-2-Methyl-3-(benzyloxy)propyl 4-methylbenzenesulfonate
9. To a solution of (R)-(Ϫ)-3-hydroxy-2-methylpropionate
(10.0 mL, 90.2 mmol) and benzyl 2,2,2-trichloroacetimidate
(21.1 mL, 108 mmol) in diethyl ether (300 mL) was added tri-
fluoromethanesulfonic acid (1.20 mL, 12.6 mmol) dropwise.
After stirring at room temperature for 3 h, the reaction mixture
was cooled to 0 ЊC and quenched with saturated NaHCO3.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 6 2 – 3 3 7 6
3369

View Article Online
Scheme 12 Synthesis of 27. Reagents and conditions: (a) i) (COCl)2, DMSO, Et3N, CH2Cl2, ii) PPh3᎐᎐CHCO2 Et, THF, reflux, iii) NiCl2, NaBH4,
THF, EtOH, 3 steps, 77%; (b) DIBAL, CH2Cl2, 36% (71a), 57% (71b); (c) i) 3, 9-BBN, THF then PdCl2(dppf ), K3PO4, DMF, H2O, 65 ЊC, ii) DIBAL,
CH2Cl2 Ϫ78 ЊC, 2 steps, 78%; (d) i) (COCl)2, DMSO, Et3N, CH2Cl2, ii) PPh3, CBr4, CH2Cl2, 2 steps, 92%; (e) n-BuLi, THF, Ϫ78 ЊC,CH3I, rt, 91%;
(f ) Cp2ZrHCl, THF then I2, 80%; (g) i) 71a or 71b, NiCl2, CrCl2, DMSO, ii) HCl, THF, 2 steps, 13% (76ac), 20% (76ad), 15% (76bc), 10% (76bd);
(h) i) (COCl)2, DMSO, Et3N, CH2Cl2, ii) NaClO2, NaH2PO4, t-BuOH, H2O, iii) BCl3, CH2Cl2, Ϫ78 ЊC.
Scheme 13 Synthesis of 28 and 29. Reagents and conditions: (a) (S)-3,3,6,6-I4BINOL, Zr(Ot-Bu)4, PrOH, H2O, toluene, 0 ЊC; (b) i) LiAlH4, THF,
ii) PMPCH(OMe)2, p-TsOH, CH2Cl2, iii) DIBAL, CH2Cl2, 0 ЊC, iv) (COCl)2, DMSO, Et3N, CH2Cl2, v) CBr4, PPh3, CH2Cl2, 0 ЊC, vi) n-BuLi, MeI,
THF, 6 steps, 41% (82), 86% (83); (c) i) 13, 9-BBN, THF, then PdCl2(dppf )2, K3PO4, DMF, H2O, 65 ЊC, ii) HCl, THF, 2 steps, 53% (84), 58% (85);
(d) i) Dess–Martin periodinane, pyridine, CH2Cl2, ii) NaClO2, NaH2PO4, t-BuOH, H2O, iii) BCl3, CH2Cl2, Ϫ78 ЊC.
CHCl3); νmax(film)/cmϪ1 1598, 1362, 1176, 1095, 973, 812, 666;
δH (400 MHz, CDCl3) 7.78 (2 H, d, J 8.3 Hz), 7.35–7.20 (7 H,
m), 4.39 (2 H, s), 4.05 (1 H, dd, J 9.5, 5.6 Hz), 3.98 (1 H, dd,
J 9.5, 5.8 Hz), 3.55 (1 H, dd, J 9.3, 5.4 Hz), 3.52 (1 H, dd, J 9.3,
6.8 Hz), 2.41 (3 H, s), 2.17–2.05 (1 H, m), 0.94 (3 H, d, J 7.1
Hz); δC (100 MHz, CDCl3) 144.6, 138.1, 132.9, 129.7, 128.2,
127.8, 127.5, 127.3, 72.9, 72.1, 71.0, 33.6, 21.5, 13.5; m/z (ESI)
335 [M ϩ H]ϩ.
The organic layer was separated, and the aqueous layer
was extracted with diethyl ether. The combined organic layer
was washed with brine, dried over Na2SO4, and evaporated. The
resulting residue was filtered through a short column of silica
gel (hexane : EtOAc = 30 : 1) to give the corresponding adduct
as a colorless oil, which was used without further purification.
To a solution of the adduct in CH2Cl2 (300 mL) was added BCl3
(100 mL, 100 mmol, 1.0 M in heptane) at Ϫ78 ЊC. After stirring
at 0 ЊC for 30 min, the reaction mixture was quenched with
MeOH and saturated NaHCO3, and diethyl ether was added.
The organic layer was separated, and the aqueous layer was
extracted with diethyl ether. The combined organic layer
was washed with brine, dried over Na2SO4, and evaporated. The
resulting residue was purified by silica gel column chromato-
graphy (hexane : EtOAc = 10 : 1) to give the alcohol (10) (14.7 g,
2 steps, 84%) as a colorless oil (Found: C, 77.69; H 14.03.
(2S )-2-Methyl-1-dodecanol 10. To a suspension of CuI (41.7
g, 219 mmol) in diethyl ether (100 mL) was added nonyllithium
(93.4 mL, 93.4 mmol, 1.0 M in diethyl ether) at Ϫ23 ЊC slowly.
After stirring at this temperature for 10 min, the reaction mix-
ture was cooled to Ϫ78 ЊC. The tosylate (9) (29.3 g, 87.5 mmol)
in diethyl ether (100 mL) was added, and the mixture was
stirred at Ϫ23 ЊC for 30 min. The reaction mixture was
quenched with NH4Cl and then diethyl ether was added.
26
C13H28O requires C, 77.93; H, 14.09%); [α]D Ϫ10 (c 0.50
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 6 2 – 3 3 7 6
3370

View Article Online
Scheme 14 Synthesis of 30. Reagents and conditions: (a) Ph3P᎐CHCO2Et, CH2Cl2, 97%; (b) DIBAL, CH2Cl2, Ϫ78 ЊC, 88%; (c) Ti(Oi-Pr)4, ()-
᎐
DET, TBHP, CH2Cl2, Ϫ23 ЊC, 98%; (d) Red-Al, THF, 91%; (e) PMPCH(OMe)2, TsOH, CH2Cl2, 90%; (f ) DIBAL, CH2Cl2, Ϫ78 ЊC, 98%; (g)
(COCl)2, DMSO, Et3N, CH2Cl2, 84%; (h) i) Ph3P᎐C(CH3)CO2Et, THF, ii) DIBAL, CH2Cl2, Ϫ78 ЊC, 2 steps, 84%; (i) TPAP, NMO, MS 4A, CH2Cl2,
᎐
89%; (j) i) Ph3P᎐C(CH3)CO2Et, THF, i) DIBAL, CH2Cl2, Ϫ78 ЊC, 2 steps, 78%; (k) MnO2, CH2Cl2, 95%; (l) chiral Sn(OTf )2-catalyzed aldol reaction
᎐
(see ref. 3), 78%; (m) TESOTf, 2,6-lutidine, CH2Cl2, 85%; (n) DIBAL, CH2Cl2, Ϫ78 ЊC, 95%; (o) Ph3P᎐(CH3)CO2Et, CH2Cl2, 98%; (p) DIBAL,
᎐
CH2Cl2, Ϫ78 ЊC, 87%; (q) i) MnO2, CH2Cl2, ii) NaClO2, NaH2PO4, t-BuOH, H2O, 2 steps; 91%; (r) 5, DCC, DMAP, DMAPؒHCl, CH2Cl2, reflux,
65%; (s) HCl, THF, 84%; (t) i) Dess–Martin periodinane, pyridine, CH2Cl2, ii) NaClO2, NaH2PO4, t-BuOH, H2O, 2 steps, 36%; (u) BCl3, CH2Cl2,
Ϫ78 ЊC.
Scheme 15 Synthesis of 31. Reagents and conditions: (a) i) (COCl)2, DMSO, Et3N, CH2Cl2, ii) Ph3P᎐CHCO2Et, CH2Cl2, 2 steps, 70%; (b) DIBAL,
᎐
CH2Cl2, Ϫ78 ЊC, 72%; (c) Ti(Oi-Pr)4, ()-DET, TBHP, CH2Cl2, Ϫ23 ЊC, 79%; (d) MeLi, CuI, Et2O, Ϫ23 ЊC, 73%; (e) PMPCH(OMe)2, TsOH,
CH2Cl2, 87%; (f ) DIBAL, CH2Cl2, Ϫ78 ЊC, 91%; (g) i) (COCl)2, DMSO, Et3N, CH2Cl2, ii) Ph3P᎐C(CH3)CO2Et, CH2Cl2, 2 steps, 82%; (h) DIBAL,
᎐
CH2Cl2, Ϫ78 ЊC, 93%; (i) i) (COCl)2, DMSO, Et3N, CH2Cl2, ii) Ph3P᎐C(CH3)CO2Et, CH2Cl2, 2 steps, 81%; (j) i) DIBAL, CH2Cl2, Ϫ78 ЊC, ii) TPAP,
᎐
NMO, MS 4A, CH2Cl2, 2 steps, 81%; (k) chiral Sn(OTf )2-catalyzed aldol reaction (see ref. 3), 75%; (l) (i) TESOTf, lutidine, CH2Cl2, ii) DIBAL,
CH2Cl2, Ϫ78 ЊC, iii) Ph3P᎐C(CH3)CO2Et, CH2Cl2, iv) DIBAL, CH2Cl2, Ϫ78 ЊC, (v) TPAP, NMO, MS 4A, CH2Cl2, 5 steps, 57%; (m) i) NaClO2,
᎐
NaH2PO4, t-BuOH, H2O, ii) 5, DCC, DMAP, DMAPؒHCl, CH2Cl2, reflux, iii) HCl, THF, 3 steps, 39%; (n) i) Dess–Martin periodinane, pyridine,
CH2Cl2, ii) NaClO2, NaH2PO4, t-BuOH, H2O, (o) BCl3, CH2Cl2, Ϫ78 ЊC.
Scheme 16 Synthesis of 22. Reagents and conditions: (a) i) allyl bromide, Cs2CO3, DMF, ii) HCl, EtOH, 2 steps, 72%; (b) Dess–Martin periodinane,
pyridine, CH2Cl2, 70%; (c) DDQ, CH2Cl2, H2O, 71%; (d) cat. Pd(PPh3)4, morpholine, THF, 51%.
in EtOH); νmax(film)/cmϪ1 3360, 2920; δH (400 MHz, CDCl3)
3.51 (1 H, dd, J 10.5, 5.9 Hz), 3.41 (1 H, dd, J 10.5, 6.6 Hz),
1.64–1.53 (1 H, m), 1.47 (1 H, br s), 1.40–1.05 (18 H, m), 0.91
(3 H, d, J 6.6 Hz), 0.88 (3 H, t, J 6.8 Hz); δC (100 MHz, CDCl3)
68.4, 35.7, 33.1, 31.9, 29.9, 29.7, 29.6, 29.3, 27.0, 22.7, 16.6,
14.1; m/z (EI) 199 [M]ϩ.
(2E,4R,5R,6E )-2,4,6-Trimethyl-5-(triphenylsiloxy)-7-iodo-
2,6-heptadien-1-ol 11. To a solution of the ester (4) (9.00 g,
15.1 mmol) in CH2Cl2 (60 mL) at Ϫ78 ЊC was added dibutyl-
aluminium hydride (40.5 mL, 37.7 mmol, 0.93 M in hexane)
dropwise. After stirring at this temperature for 30 min, the
reaction mixture was quenched with MeOH and then aqueous
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 6 2 – 3 3 7 6
3371

View Article Online
Rochelle salt and EtOAc. The mixture was warmed to room
temperature and stirred vigorously for 5 h. The organic layer
was separated, and the aqueous layer was extracted with
EtOAc. The combined organic layer was washed with brine,
dried over Na2SO4, and evaporated. The resulting residue was
purified by silica gel column chromatography (hexane : EtOAc
= 6 : 1) to give the alcohol (11) (8.06 g, 99%) as a colorless oil
(Found: C, 60.60; H 5.67. C28H31IO2Si requires C, 60.65; H,
5.63%); [α]D26.6 ϩ33.8 (c 0.895 in CHCl3); νmax(film)/cmϪ1 3367,
3062, 2965, 1428; δH (400 MHz, CDCl3) 7.54 (6 H, dt, J 8.0, 1.5
Hz), 7.42 (3 H, tt, J 7.4, 1.5 Hz), 7.36 (6 H, dd, J 8.0, 7.4 Hz),
5.86 (1 H, s), 5.03 (1 H, dq, J 9.8, 1.0 Hz), 4.04 (1 H, d, J 8.0
Hz), 3.82 (3 H, d, J 1.0 Hz), 1.66 (3 H, d, J 1.0 Hz), 0.71 (3 H, d,
J 6.8 Hz); δC (100 MHz, CDCl3) 148.1, 135.5, 135.3, 134.1,
130.0, 128.8, 127.7, 82.8, 80.5, 68.6, 37.0, 19.5, 16.9, 14.0;
m/z (ESI) 577 [M ϩ Na]ϩ.
perature for 12 h, the reaction mixture was filtered through
Celite and evaporated to give the crude aldehyde, which was
used without further purification. To a solution of the crude
aldehyde in tert-butyl alcohol (30 mL) and 2-methyl-2-butene
(18 mL) was added a mixture of sodium chlorite (5.16 g) and
sodium dihydrogenphosphate (5.16 g) in water (30 mL). After
stirring for 20 h at room temperature, the reaction mixture was
diluted with EtOAc and 0.5 M KHSO4. The organic layer was
separated, and the aqueous layer was extracted with EtOAc.
The combined organic layer was washed with 10% NaHSO3
and brine, dried over Na2SO4, and evaporated. The resulting
residue was purified by silica gel column chromatography
(hexane : EtOAc = 5 : 1) to give the acid (12) (1.27 g, 2 steps,
87%) as a colorless oil (Found: C, 58.92; H 5.32. C28H29IO3Si
23.5
requires C, 59.15; H, 5.14%); [α]D ϩ38.8 (c 1.37 in CHCl3);
νmax(film)/cmϪ1 3441, 2972, 1683, 1645, 1426, 1271, 1068, 746,
706; δH (400 MHz, CDCl3) 7.57 (6 H, dd, J 7.8, 1.2 Hz), 7.40–
7.25 (9 H, m), 7.23 (2 H, d, J 8.6 Hz), 6.84 (2 H, d, J 8.6 Hz),
6.62 (1 H, dd, J 10.0, 1.3 Hz), 5.50 (1 H, s), 5.17 (1 H, d, J 9.8
Hz), 4.46 (1 H, d, J 10.7 Hz), 4.43 (1 H, d, J 10.7 Hz), 4.12
(2 H, q, J 7.2 Hz), 3.91 (1 H, d, J 6.6 Hz), 3.78 (3 H, s), 3.04
(1 H, dd, J 5.4, 5.1 Hz), 2.93–2.81 (1 H, m), 2.75–2.64 (1 H,
m), 1.88 (3 H, d, J 1.3 Hz), 1.68 (3 H, s), 1.55 (3 H, s), 1.42–
1.17(19 H, m), 1.22 (3 H, t, J 7.2 Hz), 0.98 (3 H, d, J 6.6 Hz),
0.94 (3 H, d, J 6.8 Hz), 0.88 (3 H, t, J 6.7 Hz), 0.78 (3 H, d, J 6.8
Hz); δC (100 MHz, CDCl3) 168.1, 158.8, 145.9, 135.6, 134.5,
133.8, 133.7, 133.2, 131.6, 131.1, 129.7, 128.9, 127.7, 127.5,
113.5, 87.2, 84.6, 74.4, 60.3, 55.2, 38.2, 36.1, 35.7, 34.2, 31.9,
30.0, 29.7, 29.7, 29.6, 29.3, 27.3, 22.7, 18.4, 16.8, 16.4, 14.7,
14.2, 14.1, 13.1, 12.8; m/z (ESI) 591 [M ϩ Na]ϩ; HRMS
(ESI) calcd for C28H33NO3SiI (M ϩ NH4)ϩ 586.1274, found
586.1273.
(2E,4R,5R,6E )-2,4,6-Trimethyl-5-(triphenylsiloxy)-7-iodo-
2,6-heptadienoic acid 12. To a suspension of manganese dioxide
(2.24 g, 25.8 mmol) in dichloromethane (13 mL) was added a
solution of the alcohol (11) (1.39 g, 2.58 mmol) in dichloro-
methane (5 mL) at room temperature. After stirring at this tem-
Scheme 17 Synthesis of 32. Reagents and conditions: (a) HCl, THF,
80%; (b) Dess–Martin periodinane, pyridine, CH2Cl2, 60%; (c) TFA,
CH2Cl2, 0 ЊC, 74%.
Scheme 18 Synthesis of 33. Reagents and conditions: (a) TBSOTf, 2,6-lutidine, CH2Cl2, 63%; (b) i) Dess–Martin periodinane, pyridine, CH2Cl2,
ii) HCl, THF, 2 steps, 58%; (c) BCl3, CH2Cl2, Ϫ78 ЊC, 46%.
Scheme 19 Synthesis of 34, 35, and 36. Reagents and conditions: (a) LiAlH4, THF, 0 ЊC, 97%; (b) TBSCl, imidazole, 70% (104), 90% (107), 77%
(110); (c) i) Allyl alcohol, H2SO4, ii) PMBCl, TBAB, KOH, THF, H2O, iii) DIBAL, NiCl2(dppp), Et2O, 20% (107), 47% (110); (d) i) EDCIؒHCl,
DMAP, CH2Cl2, reflux, ii) HCl, THF, 50 ЊC, iii) Dess–Martin periodinane, pyridine, CH2Cl2, iv) NaClO2, NaH2PO4, t-BuOH, H2O, v) BCl3, CH2Cl2,
Ϫ78 ЊC.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 6 2 – 3 3 7 6
3372

View Article Online
Scheme 20 Modification of khafrefungin. Reagents and conditions: (a) TFA, CH2Cl2, rt, 41%; (b) Ac2O, pyridine, CH2Cl2, rt, 10%; (c) TMSCHN2,
MeOH, 0 ЊC, 12% (113), 15% (114), 12% (115).
(1R,2S,3R)-1-[(4-Methoxybenzyloxy)methyl]-2,3-bis(4-meth-
oxybenzyloxy)-4-(tert-butyldimethylsiloxy)butyl(2E,4R,5R,6E )-
2,4,6-trimethyl-5-(triphenylsiloxy)-7-iodo-2,6-heptadienoate 13.
To a solution of the acid (12) (1.90 g, 3.35 mmol) and the
alcohol (5) (2.93 g, 4.68 mmol) in dichloromethane (35 mL)
was added N,N-dimethylaminopyridine (1.23 g, 10.1 mmol),
N,N-dimethylaminopyridine hydrogenchloride (1.06 g, 6.68
mmol), and dicyclohexylcarbodiimide (1.38 g, 6.69 mmol). The
reaction mixture was heated at reflux for 6 h. After cooling
to room temperature, the reaction mixture was diluted with
EtOAc. The organic layer was washed with 0.5 M KHSO4, sat-
urated NaHCO3 and brine, dried over Na2SO4, and evaporated.
The resulting residue was purified by silica gel column chroma-
tography (hexane : EtOAc = 4 : 1) to give the adduct (13) (3.10
g, 79%) as a colorless oil (Found: C, 64.56; H 6.63. C63H77-
Na2SO4, and evaporated. The resulting residue was purified by
silica gel column chromatography (hexane : EtOAc = 6 : 1) to
give the adduct (2) (2.92 g, 84%) as a colorless oil; [α]D26.0 Ϫ8.17
(c 0.655 in CHCl3); νmax(film)/cmϪ1 1710, 1615, 1510, 1462,
1249, 1088; δH (400 MHz, CDCl3) 7.56 (6 H, dd, J 7.8, 1.4 Hz),
7.38–7.23 (9 H, m), 7.22 (2 H, d, J 8.5 Hz), 7.21 (2 H, d, J 8.5
Hz), 7.16 (2 H, d, J 8.8 Hz), 7.10 (2 H, d, J 8.8 Hz), 6.84 (2 H,
d, J 8.8 Hz), 6.80 (2 H, d, J 8.8 Hz), 6.77 (2 H, d, J 8.5 Hz), 6.77
(2 H, d, J 8.5 Hz), 6.75 (1 H, m), 5.52 (1 H, s), 5.31 (1 H, m),
5.14 (1 H, d, J 9.6 Hz), 4.56–4.38 (7 H, m), 4.32 (1 H, d, J 11.7
Hz), 3.98 (1 H, d, J 8.1 Hz), 3.90 (1 H, dd, J 5.4, 3.4 Hz), 3.85
(1 H, dd, J 11.3, 3.2 Hz), 3.81–3.75 (1 H, m), 3.78 (3 H, s), 3.76
(3 H, s), 3.76 (3 H, s), 3.73 (3 H, s), 3.68–3.56 (3 H, m), 3.03
(1 H, t, J 5.3 Hz), 2.93–2.82 (1 H, m), 2.74–2.62 (1 H, m), 1.90
(3 H, s), 1.64 (3 H, s), 1.54 (3 H, s), 1.41–1.20 (19 H, m), 0.96
(3 H, d, J 6.8 Hz), 0.93 (3 H, d, J 6.8 Hz), 0.88 (9 H, s), 0.82 (3 H,
t, J 6.8 Hz), 0.81 (3H, d, J 6.8 Hz), 0.00 (6 H, s); δC (100 MHz,
CDCl3) 167.4, 159.1, 159.1, 159.0, 158.8, 146,6, 144.6, 135.6,
134.4, 133.8, 133.2, 131.6, 131.1, 130.8, 130.6, 130.4, 129.8,
129.8, 129.7, 129.1, 128.9, 127.7, 127.6, 113.6, 113.6, 113.6,
113.5, 87.1, 84.3, 79.7, 77.7, 74.4, 74.0, 73.4, 72.6, 68.4, 63.2,
55.2, 55.2, 55.2, 55.1, 38.5, 36.0, 35.7, 34.2, 31.9, 30.0, 29.7,
29.7, 29.6, 29.3, 27.3, 25.9, 22.7, 18.3, 18.3, 16.8, 16.5, 14.7,
14.1, 13.2, 13.0, Ϫ5.4, Ϫ5.5; m/z (ESI) 1459 (M ϩ Na)ϩ.
23.1
IO10Si2 requires C, 64.27; H, 6.59%); [α]D ϩ20.3 (c 0.995 in
CHCl3); νmax(film)/cmϪ1 1711, 1613, 1510, 1251, 1088, 837, 707;
δH (300 MHz, CDCl3) 7.62–7.53 (6 H, m), 7.45–7.31 (9 H, m),
7.22 (2 H, d, J 8.6 Hz), 7.18 (2 H, d, J 8.6 Hz), 7.13 (2 H, d, J 8.6
Hz), 6.82 (2 H, d, J 8.6 Hz), 6.79 (4 H, d, J 8.6 Hz), 6.73 (1 H, d,
J 10.1 Hz), 5.93 (1 H, s), 5.33 (1 H, m), 4.55 (2 H, d, J 11.0 Hz),
4.46 (2 H, d, J 11.0 Hz), 4.45 (1 H, d, J 11.7 Hz), 4.34 (1 H, d,
J 11.7 Hz), 4.20 (1 H, d, J 6.8 Hz), 3.94–3.73 (3 H, m), 3.78
(3 H, s), 3.78 (3 H, s), 3.75 (3 H, s), 3.73–3.57 (3 H, m), 2.88–
2.78 (1 H, m), 1.85 (3 H, s), 1.66 (3 H, s), 0.89 (9 H, s), 0.86
(3 H, d, J 6.8 Hz), 0.02 (6 H, s); δC (75 MHz, CDCl3)
167.2, 159.1, 159.1, 159.0, 147.4, 144.6, 135.5, 133.7, 130.7,
130.5, 130.4, 130.0, 129.8, 129.1, 129.1, 128.4, 127.8, 113.6,
113.6, 113.6, 81.8, 81.1, 79.6, 77.7, 74.0, 73.4, 73.3, 72.7, 68.3,
63.0, 55.2, 55.2, 55.2, 38.5, 25.9, 20.1, 18.2, 16.2, 12.9, Ϫ5.4,
Ϫ5.4; m/z (ESI) 1199 (M ϩ Na)ϩ; HRMS (ESI) calcd for
C63H81NO10Si2I (M ϩ NH4)ϩ 1194.4444, found 1194.4437.
Ethyl
(2E,4R,5R,6E,8E,10S,11R,12S )-2,4,6,8,10,12-hexa-
methyl-5-(triphenylsiloxy)-11-(4-methoxybenzyloxy)-2,6,8-doco-
satrienoate 14. To a solution of alkyne (3) (3.49 g, 9.04 mmol) in
THF (36 mL) was added 9-BBN (36.0 mL, 18.0 mmol, 0.5 M in
THF) at 0 ЊC. After stirring at room temperature for 12 h, a
solution of iodide (4) (4.50 g, 7.54 mmol) in DMF (36 mL),
H2O (36 mL), K3PO4 (7.64 g, 36.0 mmol), and PdCl2(dppf )
(1.23 g, 1.51 mmol) were added and the mixture was stirred at
65 ЊC for 30 min. The reaction mixture was cooled to room
temperature, diluted with EtOAc, and filtered through Celite.
The filtrate was washed with saturated NaHCO3, H2O, and
brine, dried over Na2SO4, and evaporated. The resulting residue
was purified by silica gel column chromatography (hexane :
EtOAc = 7 : 1) to give the adduct (14) (5.66 g, 88%) as a color-
less oil; [α]D23.2 Ϫ12.4 (c 0.605 in CHCl3); νmax(film)/cmϪ1 1710,
1516, 1459, 1247, 1113, 706; δH (400 MHz, CDCl3) 7.57 (6 H,
dd, J 7.8, 1.2 Hz), 7.40–7.25 (9 H, m), 7.23 (2 H, d, J 8.6 Hz),
6.84 (2 H, d, J 8.6 Hz), 6.62 (1 H, dd, J 10.0, 1.3 Hz), 5.50 (1 H,
s), 5.17 (1 H, d, J 9.8 Hz), 4.46 (1 H, d, J 10.7 Hz), 4.43 (1 H, d,
J 10.7 Hz), 4.12 (2 H, q, J 7.2 Hz), 3.91 (1 H, d, J 6.6 Hz), 3.78
(3 H, s), 3.04 (1 H, dd, J 5.4, 5.1 Hz), 2.93–2.81 (1 H, m), 2.75–
(1R,2S,3R)-1-[(4-Methoxybenzyloxy)methyl]-2,3-bis(4-meth-
oxybenzyloxy)-4-(tert-butyldimethylsiloxy)butyl (2E,4R,5R,6E,
8E,10S,11R,12S )-2,4,6,8,10,12-hexamethyl-5-(triphenylsiloxy)-
11-(4-methoxybenzyloxy)-2,6,8-docosatrienoate 2. To
a
solution of alkyne (3) (1.09 g, 2.82 mmol) in THF (12 mL) was
added 9-BBN (12.0 mL, 6.00 mmol, 0.5 M in THF) at 0 ЊC.
After stirring at room temperature for 12 h, a solution of iodide
(13) (2.76 g, 2.34 mmol) in THF (12 mL), H2O (12 mL), K3PO4
(2.50 g, 11.8 mmol), and PdCl2(dppf ) (384 mg, 0.470 mmol)
were added, and the reaction mixture was stirred at 65 ЊC for
30 min. The reaction mixture was cooled to room temperature,
diluted with EtOAc, and filtered through Celite. The filtrate was
washed with saturated NaHCO3, H2O, and brine, dried over
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 6 2 – 3 3 7 6
3373

View Article Online
2.64 (1 H, m), 1.88 (3 H, d, J 1.3 Hz), 1.68 (3 H, s), 1.55 (3 H, s),
1.42–1.17 (19 H, m), 1.22 (3 H, t, J 7.2 Hz), 0.98 (3 H, d, J 6.6
Hz), 0.94 (3 H, d, J 6.8 Hz), 0.88 (3 H, t, J 6.7 Hz), 0.78 (3 H, d,
J 6.8 Hz); δC (100 MHz, CDCl3) 168.1, 158.8, 145.9, 135.6,
134.5, 133.8, 133.7, 133.2, 131.6, 131.1, 129.7, 128.9, 127.7,
127.5, 113.5, 87.2, 84.6, 74.4, 60.3, 55.2, 38.2, 36.1, 35.7, 34.2,
31.9, 30.0. 29.7, 29.7, 29.6, 29.3, 27.3, 22.7, 18.4, 16.8, 16.4,
14.7, 14.2, 14.1, 13.1, 12.8; m/z (ESI) 879 (M ϩ Na)ϩ. HRMS
(ESI) calcd for C56H80NO5Si (M ϩ NH4)ϩ 874.5806, found
874.5797.
a solution of fragment (3) (1.45 g, 3.76 mmol) in dichloro-
methane (4.0 mL) was added trifluoroacetic acid (2.0 mL) at
0 ЊC. After stirring at this temperature for 1 h, the reaction
mixture was quenched with saturated NaHCO3. The organic
layer was separated, and the aqueous layer was extracted with
EtOAc. The combined organic layer was washed with brine,
dried over Na2SO4, and evaporated. The resulting residue was
filtered through a short column of silica gel (hexane : EtOAc =
9 : 1) to give the crude alcohol as a colorless oil, which was used
without further purification. To a solution of the crude alcohol
in dichloromethane (3.6 mL) at 0 ЊC was added pyridine
(3.6 mL) and acetic anhydride (3.6 mL). After stirring at
room temperature for 3 h, 4-dimethylaminopyridine (22 mg,
0.18 mmol) was added, and the mixture was further stirred
for 1 h. The reaction mixture was evaporated, and the residue
was diluted with EtOAc. The organic layer was washed with
saturated NH4Cl, saturated NaHCO3, and brine, dried over
Na2SO4, and evaporated. The resulting residue was purified by
silica gel column chromatography (hexane : Et2O = 40 : 1) to
give the corresponding acetate (51) (938 mg, 2 steps, 81%) as a
colorless oil. δH (300 MHz, CDCl3) 4.73 (1 H, dd, J 6.4, 5.9 Hz),
2.74 (1 H, m), 2.11 (3 H, s), 1.78 (1 H, d, J 2.4 Hz), 1.40–1.18
(19 H, m), 1.10 (3 H, d, J 7.0 Hz), 0.88 (3 H, t, J 6.7 Hz), 0.86
(3 H, d, J 6.6 Hz); δC (75 MHz, CDCl3) 170.9, 79.7, 79.0, 77.0,
34.5, 33.1, 31.9, 29.7, 29.6, 29.6, 29.6, 29.3, 28.6, 26.1, 22.7,
21.0, 17.9, 14.4, 14.1, 3.5.
(1R,2S,3R)-1-[(4-Methoxybenzyloxy)methyl]-2,3-bis(4-meth-
oxybenzyloxy)-4-hydroxybutyl
(2E,4R,5R,6E,8E,10S,11R,-
12S )-2,4,6,8,10,12-hexamethyl-5-hydroxy-11-(4-methoxybenzy-
loxy)-2,6,8-docosatrienoate 15. To a solution of the adduct (2)
(2.95 g, 2.03 mmol) in THF (70 mL) was added a 1 M aqueous
HCl solution (20 mL) at room temperature. After stirring at this
temperature for 2 days, the reaction mixture was quenched with
saturated NaHCO3, and extracted with EtOAc. The combined
organic layer was washed with brine, dried over Na2SO4, and
evaporated. The resulting residue was purified by silica gel
column chromatography (hexane : EtOAc = 2 : 1) to give the
diol (15) (1.82 g, 84%) as a colorless oil. The physical data of
the diol (15) were completely consistent with those of an
authentic sample.4
(2S,3S,4R)-2,3,5-Tris(4-methoxybenzyloxy)-4-[[(2E,4R,6E,-
8E,10S,11R,12S )-1,5-dioxo-2,4,6,8,10,12-hexamethyl-11-(4-
methoxybenzyloxy)-2,6,8-docosatrienyl]oxy]pentanoic acid 16.
To a solution of the diol (15) (1.58 g, 1.48 mmol) and pyridine
(0.60 mL, 7.42 mmol) in dichloromethane (20 mL) was added
Dess–Martin periodinane (2.51 g, 5.92 mmol) at 0 ЊC. After
stirring at room temperature for 2.5 h, the reaction mixture
was diluted with EtOAc. The organic layer was washed with
saturated NaHCO3, 10% NaHSO3, and brine, dried over
Na2SO4 and evaporated. The residue was filtered through a
short column of silica gel (hexane : EtOAc = 3 : 1) to give the
ketoaldehyde as a colorless oil, which was used without further
purification. To a solution of the ketoaldehyde in tert-butyl
alcohol (18 mL) and 2-methyl-2-butene (11 mL) was added a
mixture of sodium chlorite (2.96 g) and sodium dihydrogen-
phosphate (2.96 g) in water (18 mL). After stirring for 20 h at
room temperature, the reaction mixture was diluted with
EtOAc and 0.5 M KHSO4. The organic layer was separated,
and the aqueous layer was extracted with EtOAc. The com-
bined organic layers were washed with 10% NaHSO3 and brine,
dried over Na2SO4, and evaporated. The resulting residue was
purified by silica gel column chromatography (hexane : acetone
= 2 : 1) to give the ketoacid (16) (847 mg, 2 steps, 55%) as a
colorless oil. The physical data of the ketoacid (16) were com-
pletely consistent with those of an authentic sample.3
(1R,2S,3R)-1-[(4-Methoxybenzyloxy)methyl]-2,3-bis(4-meth-
oxybenzyloxy)-4-hydroxybutyl
(2E,4R,5R,6E,8E,10S,11R,-
12S )-2,4,6,8,10,12-hexamethyl-5-(triphenylsiloxy)-11-acetoxy-
2,6,8-docosatrienoate 52. To a solution of the alkyne (51)
(450 mg, 1.46 mmol) in THF (5.8 mL) was added 9-BBN
(5.84 mL, 2.92 mmol, 0.5 M in THF) at 0 ЊC. After stirring at
room temperature for 12 h, a solution of the iodine (13) (1.56 g,
1.33 mmol) in DMF (6.0 mL), H2O (6.0 mL), K3PO4 (1.41 g,
6.64 mmol), and PdCl2(dppf ) (217 mg, 0.266 mmol) were
added, and the mixture was stirred at 65 ЊC for 30 min. The
reaction mixture was cooled to room temperature, diluted with
EtOAc, and filtered through Celite. The filtrate was washed
with saturated NaHCO3, H2O, and brine, dried over Na2SO4,
and evaporated. The resulting residue was filtered through a
short column of silica gel (hexane : EtOAc = 7 : 1) to give the
corresponding adduct as a colorless oil, which was used without
further purification. To a solution of the adduct in acetonitrile
(70 mL) at Ϫ10 ЊC was added aqueous 46% hydrogen fluoride
(0.7 mL) dropwise. After stirring at this temperature for 3 h,
the reaction mixture was quenched with saturated NaHCO3
and extracted with EtOAc. The organic layer was washed with
brine, dried over Na2SO4, and evaporated. The resulting residue
was purified by silica gel column chromatography (hexane :
EtOAc = 2 : 1) to give the corresponding alcohol (52) (692 mg,
2 steps, 42%) as a colorless oil and diol (310 mg, 2 steps, 24%) as
a colorless oil. νmax(film)/cmϪ1 3498, 2927, 1710, 1615, 1515,
1460, 1247, 1039, 823, 707; δH (400 MHz, CDCl3) 7.56 (6 H, d,
J 7.5 Hz), 7.40–7.23 (9 H, m), 7.23–7.06 (6 H, m), 6.88–6.73
(7 H, m), 5.46 (1 H, s), 5.29 (1 H, m), 4.98 (1 H, d, J 9.5 Hz),
4.77 (1 H, dd, J 6.1, 5.4 Hz), 4.54 (1 H, J 10.5 Hz), 4.48 (1 H, d,
J 10.9 Hz), 4.46 (1 H, d, J 11.7 Hz), 4.43 (1 H d, J 11.7 Hz), 4.43
(1 H, d, J 10.5 Hz), 4.39 (1 H, d, J 10.9 Hz), 3.98 (1 H, d, J 7.3
Hz), 3.99–3.50 (6 H, m), 3.77 (3 H, s), 3.77 (3 H, s), 3.75 (3 H, s),
2.88 (1 H, m), 2.70 (1 H, m), 1.98 (3 H, s), 1.90 (3 H, s), 1.60
(3 H, s), 1.53 (3 H, s), 1.40–1.15 (18 H, m), 1.09( 1 H, m),
0.90 (3 H, d, J 6.8 Hz), 0.88 (3 H, t, J 6.8 Hz), 0.82 (3 H, d, J 6.6
Hz); δC (100 MHz, CDCl3) 170.7, 167.4, 159.2, 159.2, 159.1,
146.6, 135.6, 134.5, 134.4, 134.4, 132.5, 132.1, 131.7, 130.7,
130.3, 130.2, 129.9, 129.8, 129.6, 129.2, 127.6, 113.7, 113.7,
113.7, 84..0, 80.0, 79.1, 79.0, 74.0, 73.2, 72.8, 72.8, 68.0, 61.9,
55.2, 55.2, 55.2, 38.7, 34.5, 34.4, 33.6, 31.9, 29.9, 29.6, 29.6,
29.6, 29.3, 26.9, 22.6, 20.9, 17.5, 16.7, 16.5, 14.1, 14.0, 13.2,
12.9.
Khafrefungin (1). To a solution of the ketoacid (16) (305 mg,
0.283 mmol) in dichloromethane (24 mL) was added BCl3 (1.41
mL, 1.41 mmol, 1.0 M in heptane) at Ϫ78 ЊC. After stirring at
this temperature for 5 min, the reaction mixture was quenched
with saturated NaHCO3 and poured into aqueous citric acid
and EtOAc. The organic layer was separated, and the aqueous
layer was extracted with EtOAc. The combined organic layers
were washed with brine, dried over Na2SO4, and evaporated.
The resulting residue was purified by silica gel column chroma-
tography (CHCl3 : MeOH : H2O = 10 : 1 : 0 to 4 : 1 : 0.1) and
then freeze-dried by benzene to give khafrefungin (1) (86.1 mg,
51%) as a colorless amorphous powder. The physical data of
the synthetic khafrefungin (1) were completely consistent with
those of an authentic sample.3
Synthesis of derivative 20
(4S,5R,6S )-4,6-Dimethyl-5-acetoxy-2-hexadecyne 51. To
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 6 2 – 3 3 7 6
3374

View Article Online
(2S,3S,4R)-2,3,5-Tris(4-methoxybenzyloxy)-4-[[(2E,4R,6E,-
8E,10S,11R,12S )-1-oxo-2,4,6,8,10,12-hexamethyl-5-(triphenyl-
siloxy)-11-hydroxy-2,6,8-docosatrienyl]oxy]pentanoic acid 53.
To a solution of the alcohol (52) (692 mg, 0.556 mmol) and
pyridine (0.180 mL, 2.23 mmol) in dichloromethane (10.0 mL)
was added Dess–Martin periodinane (472 mg, 1.11 mmol) at
0 ЊC. After stirring at room temperature for 3 h, the reaction
mixture was diluted with EtOAc. The organic layer was washed
with saturated NaHCO3, 10% NaHSO3, and brine, dried over
Na2SO4, and evaporated. The residue was filtered through a
short column of silica gel (hexane : EtOAc = 4 : 1) to give
the corresponding aldehyde as a colorless oil, which was used
without further purification. To a solution of the aldehyde in
tert-butyl alcohol (5.5 mL) and 2-methyl-2-butene (3.3 mL)
was added a mixture of sodium chlorite (1.1 g) and sodium
dihydrogenphosphate (1.1 g) in water (5.5 mL). After stirring
for 20 h at room temperature, the reaction mixture was
diluted with EtOAc and 0.5 M KHSO4. The organic layer was
separated, and the aqueous layer was extracted with EtOAc.
The combined organic layer was washed with 10% NaHSO3
and brine, dried over Na2SO4, and evaporated. The resulting
residue was purified by silica gel column chromatography
(hexane : acetone = 3 : 1) to give the corresponding acid (462
mg, 2 steps, 66%) as a colorless oil. νmax(film)/cmϪ1 3497, 2927,
2857, 1712, 1614, 1515, 1248, 1037, 823, 744, 707; δH (400 MHz,
CDCl3) 7.55 (6 H, d, J 7.1 Hz), 7.38–7.25 (9 H, m), 7.16 (2 H,
d, J 8.5 Hz), 7.14 (2 H, d, J 8.5 Hz), 7.06 (2 H, d, J 8.5 Hz), 6.79
(2 H, d, J 8.5 Hz), 6.78 (2 H, d, J 8.5 Hz), 6.78 (1 H, m), 6.76
(2 H, d, J 8.5 Hz), 5.50 (1 H, s), 5.26 (1 H, ddd, J 7.6, 3.8, 3.1
Hz), 4.99 (1 H, d, J 9.5 Hz), 4.76 (1 H, dd, J 6.1, 5.9 Hz), 4.46
(1 H, d, J 10.3 Hz), 4.44 (1 H, d, J 11.4 Hz), 4.40 (1 H, d, J 10.3
Hz), 4.36–4.21 (6 H, m), 4.04–3.97 (2 H, m), 3.76 (3 H, s), 3.74
(3 H, s), 3.74 (3 H, s), 2.94–2.81 (1 H, m), 2.75–2.65 (1 H, m),
1.98 (3 H, s), 1.88 (3 H, s), 1.67 (1 H, m), 1.60 (3 H, s), 1.52 (3 H,
s), 1.37–1.17 (17 H, m), 1.09 (1 H, m), 0.90 (3 H, d, J 6.8 Hz),
0.88 (3 H, d, J 6.8 Hz), 0.88 (3 H, t, J 6.8 Hz), 0.83 (3 H, d, J 6.8
Hz): δC (100 MHz, CDCl3) 172.2, 170.8, 167.0, 159.7, 159.4,
159.2, 147.0, 135.6, 134.5, 134.4, 132.6, 132.1, 131.8, 130.3,
130.0, 129.8, 129.5, 129.3, 128.2, 127.7, 127.6, 127.5, 114.0,
113.8, 113.7, 84.3, 80.1, 78.0, 77.6, 74.5, 74.1, 72.9, 71.6, 67.7,
55.2, 55.2, 55.2, 38.7, 34.5, 34.4, 33.6, 31.9, 29.9, 29.6, 29.6,
29.6, 29.3, 27.0, 22.7, 20.9, 17.6, 16.7, 16.5, 14.1, 14.0, 13.2,
12.9.
To a solution of the acid (20.0 mg, 0.0159 mmol) in CH2Cl2
(0.6 mL) at Ϫ78 ЊC was added dibutylaluminium hydride (0.060
mL, 0.056 mmol, 0.93 M in hexane) dropwise. After stirring at
this temperature for 10 min, the reaction mixture was quenched
with MeOH and then added aqueous Rochelle salt and EtOAc.
The mixture was warmed to room temperature and stirred
vigorously for 5 h. The organic layer was separated, and the
aqueous layer was extracted with EtOAc. The combined
organic layer was washed with brine, dried over Na2SO4, and
evaporated. The resulting residue was purified by silica gel
column chromatography (hexane : acetone = 2 : 1) to give the
corresponding seco acid (53, 9.3 mg, 48%) as a colorless oil.
νmax(film)/cmϪ1 3497, 2926, 1711, 1615, 1515, 1461, 1249, 1037,
822, 744, 707; δH (400 MHz, CDCl3) 7.56 (6 H, d, J 7.6 Hz),
7.35–7.27 (9 H, m), 7.18 (2 H, d, J 8.5 Hz), 7.13 (2 H, d, J 8.5
Hz), 7.07 (2 H, d, J 8.5 Hz), 6.78 (2 H, d, J 8.5 Hz), 6.78 (2 H,
d, J 8.5 Hz), 6.77 (1 H, m), 6.76 (2 H, d, J 8.5 Hz), 5.58 (1 H,
s), 5.24 (1 H, ddd, J 8.3, 4.9, 3.4 Hz), 4.85 (1 H, d, J 10.0 Hz),
4.50–4.38 (6 H, m), 4.35–4.22 (3 H, m), 4.07–3.99 (2 H, m), 3.75
(3 H, s), 3.75 (3 H, s), 3.74 (3 H, s), 8.18 (1 H, dd, J 8.0, 3.7 Hz),
2.95–2.80 (1 H, m), 2.58–2.47 (1 H, m), 1.86 (3 H, s), 1.59 (3 H,
s), 1.55 (1 H, m), 1.35–1.18 (9 H, m), 0.88 (3 H, d, J 6.8 Hz),
0.93–0.85 (9 H, m); δC (100 MHz, CDCl3) 172.6, 167.0,
159.6, 159.3, 159.1, 146.7, 135.6, 134.6, 134.3, 133.7, 132.6,
132.4, 130.3, 130.0, 129.9, 129.4, 129.3, 128.3, 127.7, 127.7,
127.5, 113.9, 113.7, 113.6, 83.6, 78.2, 77.6, 77.5, 74.4, 73.8, 72.9,
71.6, 67.7, 55.2, 55.2, 55.2, 38.6, 36.3, 34.5, 34.4, 31.9, 29.9,
29.7, 29.7, 29.6, 29.3, 27.4, 22.7, 17.1, 17.1, 16.3, 14.1, 13.7,
12.9, 12.8.
(3S,4S,5R,8E,10R,11R,12E,14E,16S,17R)-3,4-Bis(4-meth-
oxybenzyloxy)-5-[(4-methoxybenzyloxy)methyl]-8,10,12,14,16-
pentamethyl-11-(triphenylsiloxy)-17-[(1S )-1-methylundecyl]-
1,6-dioxa-8,12,14-cycloheptadecatriene-2,7-dione 54. To
a
solution of the seco acid (53, 106.2 mg, 0.0873 mmol) and tri-
ethylamine (0.057 mL, 0.411 mmol) in THF (3.0 mL) was
added 2,4,6-trichlorobenzoyl chloride (0.035 mL, 0.23 mmol)
at room temperature. After stirring at this temperature for 2 h,
DMAP (150 mg, 1.23 mmol) in toluene (78 mL) was added,
and the mixture was stirred for further 15 h at reflux then
cooled to room temperature. The reaction mixture was washed
with 0.5 M KHSO4, saturated NaHCO3 and brine, dried over
Na2SO4, and evaporated. The resulting residue was purified by
silica gel column chromatography (hexane : EtOAc = 4 : 1)
to give the corresponding macrocycle (54, 42.0 mg, 40%) as a
23.1
colorless oil. [α]D
ϩ40.0 (c 0.25 in CHCl3); νmax(film)/cmϪ1
3503, 2926, 2856, 1748, 1710, 1614, 1513, 1248, 1096, 1036,
823, 712; δH (400 MHz, CDCl3) 7.63 (6 H, d, J 8.1 Hz), 7.45–
7.32 (9 H, m), 7.18 (2 H, d, J 8.8 Hz), 7.14 (2 H, d, J 8.8 Hz),
7.08 (2 H, d, J 8.8 Hz), 6.79 (2 H, d, J 8.8 Hz), 6.76 (2 H, d,
J 8.8 Hz), 6.73 (2 H, d, J 8.8 Hz), 6.43 (1 H, d, J 10.2 Hz), 5.58
(1 H, s), 5.17 (1 H, d, J 7.8 Hz), 4.95 (1 H, d, J 9.8 Hz), 4.85
(1 H, m), 4.72 (1 H, d, J 10.5 Hz), 4.59 (1 H, d, J 11.5 Hz), 4.45
(1 H, d, J 12.2 Hz), 4.38 (1 H, m), 4.17 (1 H, m), 4.12 (1 H, m),
3.80–3.70 (2 H, m), 3.77 (3 H, s), 3.76 (3 H, s), 3.69 (3 H, s),
2.89–2.79 (1 H, m), 2.75–2.53 (1 H, m), 1.81 (3 H, s), 1.76 (3 H,
s), 1.75–1.57 (2 H, m), 1.51 (3 H, s), 1.41 (1 H, m), 1.35–1.10 (16
H, m), 1.10 (3 H, d, J 5.8 Hz), 1.01 (3 H, d, J 6.1 Hz), 0.95 (3 H,
d, J 7.1 Hz), 0.88 (3 H, d, J 6.4 Hz); δC (100 MHz, CDCl3)
171.5, 166.3, 159.2, 159.1, 159.1, 143.9, 135.4, 134.5, 134.4,
134.3, 134.3, 134.1, 132.9, 132.6, 130.0, 130.0, 129.1, 129.0,
127.8, 127.8, 127.0, 113.7, 113.6, 113.5, 82.6, 80.1, 77.2, 73.4,
72.8, 72.6, 72.6, 71.4, 67.6, 55.2, 55.2, 55.1, 38.7, 40.2, 35.2,
34.7, 34.0, 31.9, 29.8, 29.7, 29.7, 29.6, 29.3, 27.4, 22.7, 18.8,
16.8, 16.2, 14.4, 14.2, 14.1, 12.3. m/z (ESI) 1121 (M ϩ Na)ϩ.
(3S,4S,5R,8E,10R,12E,14E,16S,17R)-3,4-Dihydroxy-5-
(hydroxymethyl)-8,10,12,14,16-pentamethyl-17-[(1S )-1-methyl-
undecyl]-1,6-dioxa-8,12,14-cycloheptadecatriene-2,7,11-trione
20. To a solution of the macrocycle (54, 62.5 mg, 0.0521 mmol)
in acetonitrile (5.0 mL) was added 46% hydrogen fluoride in
water (0.2 mL) at 0 ЊC. After stirring at this temperature for 10
h, the reaction mixture was quenched with saturated NaHCO3
and extracted with EtOAc. The organic layer was washed with
brine, dried over Na2SO4, and evaporated. The resulting residue
was purified by silica gel column chromatography (hexane :
EtOAc = 3 : 1) to give the corresponding alcohol (26.6 mg, 54%)
as a colorless oil and recovered starting material (5.8 mg,
25.1
9%). [α]D
ϩ 26.9 (c 1.33 in CHCl3); νmax(film)/cmϪ1 3506,
2927, 1745, 1712, 1613, 1515, 1465, 1248, 1093, 1038, 820, 746;
δH (400 MHz, CDCl3) 7.25 (2 H, d, J 8.5 Hz), 7.21 (2 H, d, J 8.5
Hz), 7.12 (2 H, d, J 8.5 Hz), 6.83 (2 H, d, J 8.5 Hz), 6.80 (2 H, d,
J 8.5 Hz), 6.79 (2 H, d, J 8.5 Hz), 6.54 (1 H, dq, J 9.5, 1.2 Hz),
5.84 (1 H, s), 5.27 (1 H, ddd, J 9.3, 3.4, 2.9 Hz), 5.00 (1 H, d,
J 9.8 Hz), 4.84 (1 H, t, J 5.1 Hz), 4.77 (1 H, d, J 10.5 Hz), 4.60
(1 H, d, J 10.5 Hz), 4.47 (1 H, d, J 11.7 Hz), 4.43 (2 H, d, J 11.4
Hz), 4.42 (1 H, d, J 11.4 Hz), 4.41 (1 H, d, J 11.7 Hz), 4.32 (1 H,
dd, J 9.3, 2.2 Hz), 4.05 (1 H, d, J 3.4 Hz), 3.94 (1 H, d, J 2.2 Hz),
3.78 (1 H, m), 3.78 (3 H, s), 3.77 (3 H, s), 3.77 (3 H, s), 3.73 (1 H,
dd, J 11.3 Hz), 2.91–2.78 (2 H, m), 1.84 (3 H, d, J 1.2 Hz), 1.81
(3 H, d, J 0.7 Hz), 1.73 (1 H, m), 1.68 (3 H, s), 1.63 (1 H, m),
1.43 (1 H, m), 1.36–1.08 (16 H, m), 1.10 (3 H, d, J 7.1 Hz), 0.99
(3 H, d, J 6.8 Hz), 0.93 (3 H, d, J 6.8 Hz), 0.88 (3 H, t, J 6.8 Hz);
δC (100 MHz, CDCl3) 171.3, 166.6, 159.3, 159.1, 159.1, 142.6,
135.0, 133.1, 132.3, 130.7, 130.4, 130.3, 130.2, 129.3, 129.2,
129.1, 127.7, 113.7, 113.7, 113.5, 82.9, 77.7, 77.5, 75.7, 73.5,
72.6, 72.5, 71.4, 67.8, 55.2, 55.2, 55.2, 38.3, 35.6, 34.5, 33.7,
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 6 2 – 3 3 7 6
3375

View Article Online
31.9, 29.8, 29.7, 29.7, 29.6, 29.3, 27.3, 22.7, 19.3, 16.9, 15.5,
14.9, 14.7, 14.1, 12.6.
1.21 (3 H, d, J 6.6 Hz), 1.01 (3 H, d, J 6.8 Hz), 0.91 (3 H, d, J 6.8
Hz), 0.89 (3 H, t, J 7.1 Hz); HRMS (ESI) calcd. for C33H53O8
(M Ϫ H)Ϫ 577.3741, found 577.3727.
To a solution of the alcohol (9.7 mg, 0.010 mmol) and pyridine
(0.004 mL, 0.05 mmol) in dichloromethane (0.5 mL) was added
Dess–Martin periodinane (8.7 mg, 0.021 mmol) at 0 ЊC. After
stirring at room temperature for 30 min, the reaction mixture
was diluted with EtOAc The organic layer was washed with
saturated NaHCO3, 10% NaHSO3, and brine, dried over
Na2SO4, and evaporated. The resulting residue was purified by
silica gel column chromatography (hexane : EtOAc = 3 : 1) to
give the corresponding ketone (9.0 mg, 93%) as a colorless oil.
δH (400 MHz, CDCl3) 7.25 (2 H, d, J 8.8 Hz), 7.16 (2 H, d, J 8.8
Hz), 7.10 (2 H, d, J 8.8 Hz), 6.81 (2 H, d, J 8.8 Hz), 6.81 (2 H,
d, J 8.8 Hz), 6.80 (1 H, s), 6.78 (2 H, d, J 8.8 Hz), 6.67 (1 H, dq,
J 9.3, 1.2 Hz), 5.30 (1 H, m), 4.87 (1 H, dq, J 7.0, 1.2 Hz), 4.78
(1 H, d, J 11.8 Hz), 4.53 (1 H, d, J 10.5 Hz), 4.45 (1 H, d, J 11.8
Hz), 4.43 (1 H, d, J 11.8 Hz), 4.39 (1 H, d, J 11.8 Hz), 4.36 (1 H,
d, J 10.5 Hz), 4.18 (1 H, dd, J 9.5, 1.2 Hz), 3.78 (3 H, s), 3.77
(3 H, s), 3.77 (3 H, s), 3.75 (1 H, m), 3.73 (2 H, m), 3.68 (1 H, d,
J 2.4 Hz), 3.64 (1 H, m), 2.84 (1 H, m), 1.90 (3 H, d, J 1.2 Hz),
1.83 (3 H, d, J 1.0 Hz), 1.79 (3 H, d, J 1.2 Hz), 1.44–1.10 (19 H,
m), 1.22 (3 H, d, J 6.9 Hz), 1.01 (3 H, d, J 7.1 Hz), 0.88 (3 H, d,
J 6.4 Hz), 0.87 (3 H, t, J 6.7 Hz).
Antifungal activity
Growth inhibition was determined by microtiter broth dilution
assay in Difco yeast nitrogen base medium containing 2%
glucose, 20 mg lϪ1 uracil, and 100 mg lϪ1 leucine with Saccharo-
myces cerevisiae (KA311A strain) inoculated at A600nm = 0.01.
Serial 3-fold dilutions of drugs were made from 200 µM. The
minimum inhibitory concentration (MIC) was defined as the
lowest drug concentration, which prevented visible growth after
24 hours at 30 ЊC.
Acknowledgements
The authors are grateful to Dr. Takeshi Wakabayashi, Mr.
Kouhei Mori, and Mr. Hiroki Kobayashi (The University of
Tokyo) for their contributions at the initial stage of this work.
The authors also thank Dr. Shigeru Nakajima (NMR experi-
ments), Mr. Haruki Shimokawa (metabolism experiments),
and Mr. Hirokazu Ohsawa (high resolution mass specroscopy)
(Banyu Pharmaceutical Co., Ltd.) for their technical supports.
This work was partially supported by CREST and SORST,
Japan Science and Technology Corporation (JST), and a
Grant-in-Aid for Scientific Research from Japan Society of the
Promotion of Science (JSPS).
To a solution of the ketone (14.5 mg, 0.0154 mmol) in
CH2Cl2 (2.0 mL) was added BCl3 (0.070 mL, 0.070 mmol,
1.0 M in heptane) at Ϫ78 ЊC. After stirring at this temperature
for 5 min, the reaction mixture was quenched with saturated
NaHCO3 and poured into aqueous citric acid. After addition
of EtOAc, the organic layer was separated, and the aqueous
layer was extracted with EtOAc. The combined organic layer
was washed with brine, dried over Na2SO4, and evaporated.
The resulting residue was purified by silica gel column chroma-
tography (hexane : EtOAc = 1 : 1) to give macrocyclic
References and notes
1 S. M. Mandala, R. A. Thornton, M. Rosenbach, J. Milligan,
M. Garcia-Calvo, H. G. Bull and M. B. Kurtz, J. Biol. Chem., 1997,
272, 32709–32714.
2 Reviews: (a) T. Kolter and K. Sandhoff, Angew. Chem., Int. Ed.,
1999, 38, 1532–1568; (b) see also: R. C. Dickson, Annu. Rev.
Biochem., 1998, 67, 27–48; (c) other IPC inhibitors: M. M. Nagiec,
E. E. Nagiec, J. A. Baltisberger, G. B. Wells, R. L. Lester and R. C.
Dickson, J. Biol. Chem., 1997, 272, 9809–9817; (d ) S. M. Mandala,
R. A. Thornton, J. Milligan, M. Rosenbach, M. Garcia-Calvo,
H. G. Bull, G. Harris, G. K. Abruzzo, A. M. Flattery, C. J. Gill,
K. Bartizal, S. Dreikorn and M. B. Kurtz, J. Biol. Chem., 1998, 273,
14942–14949.
3 T. Wakabayashi, K. Mori and S. Kobayashi, J. Am. Chem. Soc.,
2001, 123, 1372–1375.
4 S. Kobayashi, K. Mori, T. Wakabayashi, S. Yasuda and K. Hanada,
J. Org. Chem., 2001, 66, 5580–5584.
25.1
khafrefungin (20, 6.5 mg, 73%) as a colorless oil. [α]D ϩ84
(c 0.16 in CHCl3); νmax(film)/cmϪ1 3466, 2926, 2856, 1743, 1714,
1652, 1457, 1384, 1229, 1126, 1079, 1028, 741; δH (400 MHz,
CDCl3) 6.79 (1 H, dq, J 9.8, 1.2 Hz), 6.76 (1 H, d, J 1.2 Hz),
5.28 (1 H, d, J 10.3 Hz), 5.08 (1 H, dt, J 9.3, 3.5 Hz), 4.76 (1 H,
dq, J 7.8, 1.2 Hz), 4.17 (1 H, m), 4.10 (1 H, m), 3.98 (2 H, d,
J 3.5 Hz), 3.73 (1 H, dq, J 9.8, 6.8 Hz), 3.26 (1 H, br s), 2.97
(1 H, br s), 2.88 (1 H, dq, J 6.8, 10.3 Hz), 2.56 (1 H, br s), 1.99
(3 H, d, J 1.2 Hz), 1.93 (3 H, d, J 1.2 Hz), 1.86 (3 H, d, J 1.2 Hz),
1.56 (1 H, m), 1.67 (18 H, m), 1.23 (3 H, d, J 6.8 Hz), 1.00 (3 H,
d, J 6.8 Hz), 0.88 (3 H, d, J 6.8 Hz), 0.81 (3 H, d, J 6.8 Hz);
δC (100 MHz, CDCl3) 202.3, 172.1, 166.9, 143.6, 142.5, 142.4,
133.9, 132.2, 128.5, 85.1, 74.0, 69.8, 68.8, 62.5, 42.8, 36.0, 34.1,
33.0, 31.9, 29.9, 29.6, 29.6, 29.6, 29.3, 26.7, 22.7, 19.6, 16.1,
15.2, 14.5, 14.1, 14.0, 12.6; HRMS (ESI) calcd. for C33H53O8
(M Ϫ H)Ϫ 577.3741, found 577.3748.
5 A preliminary communication on large scale synthesis of
khafrefungin: Y. Mori, M. Nakamura, T. Wakabayashi, K. Mori
and S. Kobayashi, Synlett, 2002, 601–603.
6 E. P. Boden and G. E. Keck, J. Org. Chem., 1985, 50, 2394–2395.
7 (a) N. Miyaura and A. Suzuki, J. Chem. Soc., Chem. Commun.,
1979, 866–867; (b) review: A. Suzuki, Acc. Chem. Res., 1982, 15, 178;
(c) N. Miyaura and A. Suzuki, Chem. Rev., 1995, 95, 2457–2483.
8 J. Uenishi, J. M. Beau, R. W. Armstrong and Y. Kishi, J. Am. Chem.
Soc., 1987, 109, 4756–4758.
Synthesis of derivative 111
9 PdCl2(dppf ):
[1,1Ј-bis(diphenylphosphino)ferrocene]dichloro-
palladium(), complex with dichloromethane (1 : 1).
10 B. S. Bal, W. E. Childers Jr. and H. W. Pinnick, Tetrahedron, 1981,
37, 2091–2096.
11 D. B. Dess and J. C. Martin, J. Org. Chem., 1983, 48, 4155–4156.
12 In ref. 4, we have already reported the antifugal activity of seven
stereoisomers of khafrefungin. In all cases, the activity was lost.
13 We are grateful to Dr Guy Harris (Merck) for providing us with
valuable information.
14 Synthesis of compounds 37 and 43: E. Piers, J. R. Grierson,
C. K. Lau and I. Nagakura, Can. J. Chem., 1982, 60, 210–223.
15 J. Inanaga, K. Hirata, H. Saeki, T. Katsuki and M. Yamaguchi, Bull.
Chem. Soc. Jpn., 1979, 52, 1989–1993.
16 (a) O. Mitsunobu, M. Yamada and T. Mukaiyama, Bull. Chem. Soc.
Jpn., 1967, 40, 935–939; (b) O. Mitsunobu, Synthesis, 1981, 1–28.
17 D. W. Hart, T. F. Blackburn and J. Schwarts, J. Am. Chem. Soc.,
1975, 97, 678–680.
(3R,4S,5S )-4,5-Dihydroxy-6-oxotetrahydro-2H-pyran-3-yl
(2E,4R,6E,8E,10S,11R,12S )-5-oxo-2,4,6,8,10,12-hexamethyl-
11-hydroxy-2,6,8-docosatrienoate 111. To a solution of syn-
thetic khafrefungin (1) (50.0 mg, 0.0839 mmol) in dichloro-
methane (1.0 mL) was added trifluoroacetic acid (0.25 mL) at
0 ЊC. After stirring at this temperature for 1 h, the reaction
mixture was warmed to room temperature and further stirred
for 2 h. The mixture was concentrated under reduced pressure.
The resulting residue was purified by preparative thin layer
chromatography on silica gel (hexane : EtOAc = 2 : 3) to give the
derivative (111) (21.1 mg, 41%) as a colorless oil and recovered
khafrefungin (1) (24.7 mg, 49%). δH (400 MHz, CD3OD) 7.12
(1 H, s), 6.79 (1 H, dd, J 9.8, 1.4 Hz), 5.75 (1 H, d, J 9.8 Hz),
5.40 (1 H, ddd, J 3.2, 2.7, 2.2 Hz), 4.46 (1 H, dd, J 12.7, 2.2 Hz),
4.41 (1 H, dq, J 9.8, 6.8 Hz), 4.36 (1 H, dd, J 12.7, 2.7 Hz), 4.34
(1 H, d, J 10.0 Hz), 4.12 (1 H, dd, J 10.0, 3.2 Hz), 3.25 (1 H, dd,
J 5.9, 5.6 Hz), 2.75 (1 H, m), 1.96 (3 H, d, J 1.5 Hz), 1.94 (3 H,
s), 1.91 (3 H, d, J 1.0 Hz), 1.53 (1 H, m), 1.40–1.15 (18 H, m),
18 (a) K. Takai, K. Kimura, T. Kuroda, T. Hiyama and H. Nozaki,
Tetrahedron Lett., 1983, 24, 5281–5284; (b) H. Jin, J. Uenishi,
W. J. Christ and Y. Kishi, J. Am. Chem. Soc., 1986, 108, 5644–5646.
19 Y. Yamashita, H. Ishitani, H. Shimizu and S. Kobayashi, J. Am.
Chem. Soc., 2002, 124, 3292–3302.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 6 2 – 3 3 7 6
3376