Steroid-fullerene adducts from Diels-Alder reactions: Characterization and the effect on the activity of Ca2+-ATPase

Article abstract of DOI:10.1039/b007498p

Two new fullerene steroids (1 and 6) have been prepared by the Diels-Alder reaction of silyloxydienes (2 and 3) with [60]fulterene, followed by hydrolysis of the silyl enol ether under acidic conditions. The isolation, characterization, UV-Vis and CD absorption studies of the adducts are presented. A preliminary study on the effect of 1 on sarcoplasmic reticulum (SR) Ca²⁺-ATPase and survival of human lung adenocarcinoma cancer A₅₄₉ cells has also been carried out.

Full text of DOI:10.1039/b007498p

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Steroid-fullerene adducts from Diels–Alder reactions:
2+
characterization and the effect on the activity of Ca -ATPase
a
a
a
a
b
b
Lian-Sheng Li, Yun-Jin Hu, Yikang Wu, Yu-Lin Wu,* Jiachang Yue and Fuyu Yang*
1
a
State Key Laboratory of Bio-organic and Natural Products Chemistry,
Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 354 Fenglin Road,
Shanghai 200032, China. E-mail: ylwu@pub.sioc.ac.cn
b
National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica,
15 Datun Road, Beijing 100101, China
Received (in Cambridge, UK) 15th September 2000, Accepted 23rd January 2001
First published as an Advance Article on the web 23rd February 2001
Two new fullerene steroids (1 and 6) have been prepared by the Diels–Alder reaction of silyloxydienes (2 and 3) with
60]fullerene, followed by hydrolysis of the silyl enol ether under acidic conditions. The isolation, characterization,
UV-Vis and CD absorption studies of the adducts are presented. A preliminary study on the effect of 1 on
[
2ϩ
sarcoplasmic reticulum (SR) Ca -ATPase and survival of human lung adenocarcinoma cancer A cells has also
549
been carried out.
Introduction
been demonstrated to be one of the most successful methods
3c,5
for the functionalization of fullerene. Accordingly, our syn-
1
Since the discovery of [60]fullerene and its large scale prepar-
thesis of target molecule 1 used the Diels–Alder reaction of C60
2
ation, more and more attention has been paid to the introduc-
with relatively sterically hindered silyloxydienes 2 and 3 (Fig. 1)
as a key step.
tion of biologically active groups on to fullerenes because of its
interesting physical and biological properties. Steroids (sterols,
3
such as cholesterol) are the main component of biomembranes
and therefore we anticipated that coupling a fullerene with a
steroid may change the physicochemical properties of the
fullerene, improve its solubility and biocompatibility, and facili-
tate further studies on membrane–drug interactions and
membrane-related processes such as signal transduction and
solute transport etc. To the best of our knowledge, among the
multitude of fullerene derivatives that have been published
recently, only a very few examples of fullerenes with a steroid
Results and discussion
We began with examination of the cycloaddition of C60 to diene
2
3
(Scheme 1), which can be readily prepared from the available
β-acetoxypregna-5,16-dien-20-one (4) (95% isolated yield) by
treatment with tert-butyldimethylsilyl triflate (TBSOTf) and
triethylamine. The following Diels–Alder reaction with C60 was
carried out at 90 ЊC (at lower temperature the reaction did not
occur at all) in toluene under a nitrogen atmosphere. TLC [silica
gel, petroleum ether (PE)–CH Cl = 2:1, R = 0.8] analysis
4
moiety have been reported. Thus, we wish to report a synthesis
of a new steroid-linked fullerene 1 from a steroidal enol-diene
2
2
f
showed the expected cycloadduct 5 was formed quickly and the
reaction was completed within 4 h. A longer reaction time did
not improve the yield of the product. However, all attempts to
separate the adduct 5 by column chromatography on silica gel
were unsuccessful due to the undesired retro Diels–Alder reac-
(
Fig. 1) and the preliminary results of the effect of target
2+
molecule 1 on SR Ca -ATPase, and survival of human lung
adenocarcinoma cancer A549 cells.
Due to the pronounced dienophilic character of its [6–6]
electron-deficient double bond, the Diels–Alder reaction has
3c,6
tion. Therefore, the crude silyl enol ether intermediate 5 was
converted directly to the corresponding stable ketone 6 by
treatment with p-TsOH at 60 ЊC for 10 h followed by chroma-
tography, as a brown solid, in 27% yield (over two steps, or 46%
yield based on consumed C ). It was noteworthy that only one
60
stereoisomer 6 was obtained (the other stereoisomer was
formed only in trace amounts and could not be identified
clearly). The excellent diastereoselectivity was attributed to the
induction of the substrate’s chirality. The structure of product 6
was clearly established by spectroscopic means. Thus, both the
+
ESI mass spectrum, [molecular-ion peak at m/z 1077 [M+H]
for C H O ], and the MALDI-TOF spectrum of compound 6
8
3
32
3
+
[
molecular-ion peak at m/z 1077 [M+H] together with a base
peak at m/z 720 due to the fragment C ], corresponded with the
60
1
proposed structure of compound 6. In the H NMR spectrum,
owing to the effect of C , there was a significant difference in
60
the chemical shift of the two 21-CH geminal protons, which
2
appeared as doublets downfield (δ 4.69 and 4.08, J = 16.8 Hz,
H
geminal coupling) compared with the normal methylene pro-
tons. The configuration of 6 (Fig. 2) was confirmed by a large
coupling constant J16,17 = 12.2 Hz (trans coupling), and the
Fig. 1
nuclear Overhauser effects (NOE) between 16-H/18-CH , 16-H/
3
DOI: 10.1039/b007498p
J. Chem. Soc., Perkin Trans. 1, 2001, 617–621
This journal is © The Royal Society of Chemistry 2001
617

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Scheme 1
Scheme 2
Fig. 2
1
5β-H, 16-H/21β-H, 17-H/14-H and 17-H/12α-H. Further evi-
13 1 1
Fig. 3 UV-Vis absorption spectra of compound 1 and 6 in dichloro-
dence was obtained from C NMR, DEPT, H– H COSY, 2D
heteronuclear multiple quantum-filtered coherence (HMQC)
NMR, FT-IR, UV-Vis, and CD spectra (see Experimental).
In comparison with the structure of cholesterol, the hydroxy
group at C-3 in fullerene-steroid 6 is present in the protected
form. Unfortunately, all efforts to remove the protective group
under weakly basic conditions (such as KHCO –EtOH–CH Cl
methane.
Steroid 4 was readily converted into 7 in 90% yield by treat-
ment with LiOH. In a similar reaction, the desired silyloxydiene
3 was obtained in 85% isolated yield from 7 by reaction with
tert-butyldimethylsilyl triflate (2.5 eq.). As expected, the target
molecule 1 was successfully obtained in 30% yield (over two
steps, or 40% yield based on consumed C ) by reaction of C
3
2
2
and K CO –EtOH–CH Cl ) failed. Under more basic condi-
tions (LiOH–THF–H O), the product mixture was very com-
2
3
2
2
2
60
60
plicated as shown by TLC (PE–CH Cl = 1:9). We attempted
with 3 in toluene at 90 ЊC for 2 h, followed by hydrolysis of the
silyl enol ether and deprotecting of the hydroxy group at C-3
using p-TsOH in one pot. Compound 1 was fully characterized
by H NMR, H– H COSY, C NMR, 2D heteronuclear
multiple quantum-filtered coherence (HMQC) NMR, UV-Vis,
2
2
the reaction under acidic conditions (acidic ion-exchange resin
Amberlyst 15). The result was also disappointing. Therefore, we
turned to an alternative route to finish the synthesis (see
Scheme 2).
1
1
1
13
6
18
J. Chem. Soc., Perkin Trans. 1, 2001, 617–621

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2
+
Table 1 Effect of steroid-C60 (1) on the ATP hydrolysis activity of Ca -ATPase
Steroid-C60 content
0
1%
3%
5%
2
+
Ca -ATPase ATP hydrolysis activity
Activity ratio (%)
3.49 ± 0.01
100
1.47 ± 0.02
42
1.43 ± 0.04
41.0
1.12 ± 0.06
32
2
+
Fig. 5 Effect of steroid-C (1) on the Ca uptake activity of
6
0
2
+
reconstituted Ca -ATPase in soybean phospholipid liposomes (SPL):
soybean phospholipid liposomes (SPL); ꢀ SPL containing 1%
Steroid-C ; ᮀ SPL containing 3% Steroid-C .
᭿
Fig. 4 CD spectra of compound 1 (thin line) and 6 (thick line) in
60
60
Ϫ4
Ϫ3
dichloromethane (4 × 10 mol dm , 10 mm cell).
CD and FT-IR. All spectra are consistent with the proposed
structure.
The UV-Vis absorption spectra of 1 and 6 (Fig. 3) over 400–
8
00 nm showed a similar absorption pattern as those reported
4i
in the literature. There is a sharp absorption in the spectrum of
compound 6 with λmax = 432 nm, which is very characteristic for
the spectra of the mono-adducts from addition across a 6–6
7
bond of fullerene. The corresponding band in the spectrum of
compound 1 is weaker.
Recently, the chiroptical properties of chiral fullerene deriva-
8
tives have been studied by CD spectroscopy. The chiral
fullerene-steroids 1 and 6 obtained here are also CD-active,
with typical Cotton effects (Fig. 4). The CD spectra of 6 and 1
are similar in shape except for two differences: i) the Cotton
effects in 6 are stronger than those in 1, and ii) there is a weak
positive Cotton effect at 456 nm (∆ε = +0.19) in the spectrum of
Fig. 6 Effect of steroid-C60 (1) on survival of human lung
adenocarcinoma cancer A549 cells.
Ϫ1
Ϫ1
6
which is not observed for 1. According to the sector rule for
glutamate, 50 units ml
penicillin G and 50 units ml
9
C60 derivatives reported by Wilson, the sign of the Cotton
effect at 430 nm can be used to determine the absolute con-
figuration of attached groups. Clearly, the CD spectra of both
fullerene-steroids show similar negative Cotton effects near
streptomycin) at 37 ЊC in the dark, in 5% CO
. Cells were resus-
2
6
Ϫ1
pended to 1 × 10 cells ml , and 100 µL was plated into a 96-
well plate. The survival was evaluated after 48 h of incubation
by the calorimetric method using the reduction of MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to
MTT formazan, which was determined by measurement of the
absorbance at 595 nm. Results obtained clearly showed that the
survival of A549 cells following treatment with steroid-C60 (1)
was obviously decreased (Fig. 6). Eukaryotic cells are rich in
membranous structures. It is tentatively suggested that the
steroid-linked fullerene mediated changes in physical state of
membrane lipid could result in abnormality of cell functions
and hence decrease the survival of A549 cells. Generally, the
susceptibility of A549 cells to anticancer drugs is quite low. So,
the mechanism of such steroid-C60 effect deserves further
investigation. Further studies are still in progress.
4
30 nm, and the negative sign is consistent with the sector rule
9
model, from which the proposed absolute configuration of
compound 6 and 1 (see Fig. 2) can be further confirmed.
After the target molecule steroid-C60 (1) had been obtained,
the study of its cytotoxic effects both at subcellular and cellular
level was carried out. In the former case the effect on the
reconstituted sarcoplasmic reticulum membrane transmem-
2+
2+
brane Ca -ATPase (SR Ca -ATPase) in soybean phospholipid
liposomes was examined. The preliminary results in Table 1 and
Fig. 5 show that steroid-C (1) obviously decreased both the
60
2+
2+
ATP hydrolysis and Ca uptake activity of Ca -ATPase and
the inhibitions were concentration-dependent. It is well known
that cholesterol can decrease the lipid fluidity of biomem-
2+
branes. The lower activity of membrane-bound Ca -ATPase
may result from a change in the physical state of the lipid
induced by steroid-C . However, here the C moiety may
Conclusions
In summary, we have carried out the synthesis of a steroid-
linked fullerene based on the Diels–Alder reaction between C60
and silyloxydienes 2 and 3, followed by hydrolysis of the silyl
enol ether under acidic conditions. The fullerene steroids 6 and
1 were fully characterized by spectral analyses. Compound 1
with the synthetically versatile ketone and hydroxy function-
60
60
increase such an effect of cholesterol owing to steric hindrance.
Moreover, the effect of 1 on the survival of human lung adeno-
carcinoma A549 cells was also studied. The cell line was main-
tained in cultures in complete medium (Eagle’s balanced salt
solution with 10% heat-inactivated fetal bovine serum, 4 mM
J. Chem. Soc., Perkin Trans. 1, 2001, 617–621
619

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alities permits the preparation of a variety of other C -
derivatives with interesting applications. Results of a prelimin-
(dichloromethane) λmax (∆ε) 674 (+0.66), 602 (+0.82), 456
(+0.19), 432 (Ϫ0.02), 285 (+0.14) nm.
60
ary assay show that compound 1 can inhibit the reconstituted
2
+
SR Ca -ATPase in soybean phospholipid liposomes and affect
3,20-Bis(tert-butyldimethylsilyl)oxypregna-5,16,20-triene (3).
Compound 3 was obtained in 85% yield by a similar procedure
to the synthesis of compound 2, but the amount of tert-
the survival of A549 cells.
1
butyldimethylsilyl triflate was changed to 2.5 eq. H NMR
Experimental
(
CDCl ; 300 MHz): δ 6.01 (1 H, dd, J = 3.0, 1.9 Hz), 5.34 (1 H,
3
m), 4.45 (1 H, s), 4.28 (1 H, s), 3.49 (1 H, dt, J = 10.7, 4.6 Hz),
Synthesis and structure
2
0
.30–1.35 (17 H, m), 1.03 (3 H, s), 0.98 (9 H, s), 0.96 (3 H, s),
1
13
1
1
1
General. The H NMR, C NMR, H– H COSY,
H
.93 (9 H, s), 0.15 (6 H, s), 0.07 (6 H, s). EIMS (m/z, %): 542
NOESY and HMQC spectra were recorded on a Bruker AMX-
+
(
M , 4.18), 527 (30.94), 486 (35.32), 429 (6.72), 352 (10.71), 249
1
1
6
00 operating at 600 MHz for H (some H spectra were
13
(
1
6.52), 141 (11.05), 75 (100.00). IR (KBr): 2957, 2930, 1670,
615, 1472, 1253, 1093, 1043, 836, 778 cm . HR EIMS calcd.
recorded on a Bruker AMX 300) and 150 MHz for C with
TMS as the internal standard, respectively. IR spectra were
recorded on a Bruker FT instrument. Mass spectra were taken
on a VG Quattro MS/MS or an HP5989A instrument. HRMS
Ϫ1
for C H O Si 342.39754, found 342.39583.
33
58
2
2
Steroid-C60 (1). A mixture of C60 (100 mg, 0.14 mmol) and
silyloxydiene 3 (100 mg, 0.18 mmol) was dissolved in 60 mL of
toluene under nitrogen, and the solution was heated at 90 ЊC for
(
EI) spectra were obtained on a Finnigan Mat 8430 mass spec-
trometer. The UV-Vis spectra were obtained with a Shimadzu
UV-240 spectrometer. Optical rotations were measured on a
Perkin-Elmer 241 MC polarimeter. Flash column chromato-
graphy was performed on silica gel H (10–40 µm) (or Al O )
with a petroleum ether–dichloromethane or petroleum ether–
ethyl acetate system as eluant. Microanalyses were carried out
in the Microanalytical Laboratory at Shanghai Institute of
Organic Chemistry.
2
h. Then the solution was cooled to room temperature,
p-TsOH (40 mg, 1.5 eq.) was added, and the stirring was con-
tinued for 10 h at 60 ЊC. The solvent was then evaporated under
reduced pressure, and the residue was chromatographed on
silica gel (eluting with first toluene then 1:9 petroleum ether–
2
3
dichloromethane) to afford C60 (35 mg, 35%), and compound 1
1
(
43 mg, 30%, 40% based on consumed C ). H NMR (CDCl ;
60
3
3
00 MHz): δ 5.34 (1 H, d, J = 3.5 Hz), 4.70 (1 H, d, J = 17.0 Hz,
3
ꢀ-Acetoxy-20-(tert-butyldimethylsilyl)oxypregna-5,16,20-
10
2
1-H), 4.56 (1 H, dt, J = 4.2, 12.2 Hz, 16-H), 4.08 (1 H, d,
triene (2). To an ice-cooled solution of 4 (712 mg, 2 mmol) in
anhydrous ether (30 ml) was added dropwise triethylamine
332 µl, 2.4 mmol) and tert-butyldimethylsilyl triflate (0.5 ml,
.2 mmol). After stirring at 0 ЊC for 15 min and then at room
temperature for 30 min, the mixture was concentrated under
J = 17.0 Hz, 21-H), 3.58 (1 H, d, J = 12.2 Hz, 17-H), 3.52 (1 H,
m, 3-H), 2.79 (1 H, m), 2.6–1.2 (16 H, m), 1.29 (3 H, s), 1.12
(
2
13
(
3 H, s). C NMR (CDCl , 150 MHz): δ 208.73 (CO), 156.52,
3
1
1
56.36, 154.64, 151.80, 147.78, 146.64, 146.59, 146.55, 146.40,
46.37, 145.90, 145.86, 145,63, 145.48, 145.39, 145.05, 144.97,
2
reduced pressure, then passed through a pad of Al O3
144.76, 144.64, 144.59, 144.49, 143.31, 142.87, 142.72, 142.33,
42.26, 142.23, 142.16, 141.89, 141.77, 141.71, 141.64, 141.56,
141.12, 140.46, 140.28, 140.08, 139.35, 136.84, 135.56, 135.21,
(
petroleum ether as the eluant) to give compound 2 as a
white solid (1.25 g, 95% yield): H NMR (CD COCD ;
1
1
3
3
3
00 MHz): δ 6.01 (1 H, dd, J = 3.0, 2.0 Hz), 5.40 (1 H, dd,
J = 4.1, 1.3 Hz), 4.50 (1 H, m), 4.48 (1 H, s), 4.29 (1 H, s), 2.35–
.0 (17 H, m), 1.97 (3 H, s), 1.08 (3 H, s), 0.97 (3 H, s), 0.95 (9 H,
s), 0.16 (3 H, s), 0.15 (3 H, s). EIMS (m/z, %): 470 (M ), 455
55.54), 399 (100.00), 339 (42.23), 235 (3.54), 75 (56.73).
IR (KBr): 3060, 2932, 1736, 1640, 1571, 1249, 1039, 1004,
3
1
6
4
1
1
6
33.88, 121.38, 71.39 (C-3), 67.69 (sp -C for C ), 66.78 (C-17),
60
3
4.41 (sp -C for C ), 56.77, 54.45 (C-21), 49.73, 46.96 (C-16),
60
1
3.25, 38.21, 36.61, 32.34, 31.67 (2 C), 30.97, 29.69, 26.45,
9.91, 19.05 (CH ), 13.82 (CH ). IR (KBr): 3411, 2932, 1720,
+
3
3
(
Ϫ1
431, 754, 527 cm . CD spectrum (dichloromethane) λ (∆ε)
max
85 (+0.39), 616 (+0.41), 432 (Ϫ0.03), 283 (+0.05) nm.
Ϫ1
8
32 cm .
Biological studies
Preparation of SR Ca -ATPase. Rabbit SR was prepared
Steroid-C60 (6). A mixture of C60 (144 mg, 0.2 mmol) and
2
؉
silyloxydiene 2 (141 mg, 0.3 mmol) was dissolved in 70 mL of
anhydrous toluene under nitrogen, and the solution was heated
at 90 ЊC for 4 h. Then the solution was cooled to room temper-
ature, p-TsOH (50 mg, 1.5 eq.) was added, and the stirring was
continued for 10 h at 60 ЊC. The solvent was then evaporated
under reduced pressure, and the residue was chromatographed
on silica gel (eluting with first toluene then 2:1 petroleum
ether–dichloromethane) to afford C60 (60 mg, 42%), and com-
11
2+
according to MacLennan, and SR Ca -ATPase according to
12
Coll and Murphy with slight modification. The enzyme was
purified to homogeneity on SDS-PAGE.
2
+
Preparation of proteoliposomes containing SR Ca -ATPase.
Preparation of soybean phospholipid proteoliposomes was
13
based on the methods described by Gould et al. and Tu and
14
1
Yang. The lipid–protein ratio was 100:1 (µmol–µmol).
pound 6 (58 mg, 27%, 46% based on consumed C ). H NMR
60
(
CDCl ; 600 MHz): δ 5.30 (1 H, d, J = 3.6 Hz), 4.69 (1 H, d,
3
2
؉
2؉
J = 16.8 Hz, 21-H), 4.60 (1 H, m, 3-H), 4.56 (1 H, dt, J = 4.2,
2.6 Hz, 16-H), 4.08 (1 H, d, J = 16.8 Hz, 21-H), 3.58 (1 H, d,
J = 12.6 Hz, 17-H), 2.79 (1 H, m), 2.02 (3 H, s), 2.59–1.14 (16 H,
ATP hydrolysis activity and Ca uptake of Ca -ATPase.
The ATP hydrolysis activity was monitored at 30 ЊC by con-
tinuous spectrophotometry following the oxidation of NADH
1
13
15
2+
m), 1.27 (3 H, s), 1.11 (3 H, s). C NMR (CDCl ; 75 MHz):
δ 208.69 (CO), 170.44 (CH CO), 156.44, 156.29, 154.59, 151.71,
1
1
1
(
(
2
as described by Carafoli et al. Ca uptake activity was meas-
ured at 30 ЊC by following the decrease in the absorbance of
3
3
2+
47.73, 146.49, 146.34, 145.84, 145.58, 145.00, 144.61, 143.27,
42.68, 142.22, 141.87, 141.59, 140.01, 139.37, 136.81, 135.56,
arsenano III used as Ca indicator (675–685 nm) in a Hitachi
13
model 557 spectrophotometer as described by Gould et al.
3
35.18, 133.86, 121.84, 73.74 (C-3), 67.60 (sp -C for C ), 66.97
60
3
C-17), 64.36 (sp -C for C ), 56.69, 54.31 (C-21), 49.92, 46.73
Cell culture and survival assay. Cell line A549 was from Dr Jing
Gao (Institute of Biophysics, Chinese Academy of Sciences,
Beijing). The cell line was maintained in cultures in complete
medium (Eagle balanced salt solution with 10% heat-
inactivated fetal bovine serum, 4 mM glutamate penicillin G
60
C-16), 43.20, 38.06, 36.96, 36.71, 31.53 (2 C), 27.69, 26.89,
6.40, 21.39 (CH , OAc), 20.37, 19.38 (CH ), 13.59 (CH ).
3
3
3
+
ESI MS: 1077 (M + 1 for C H O ). MALDI-TOF MS:
83
32
3
+
1
2
3
077 (M + 1, 24), 720 (C60 fragment, 100). IR (KBr): 2924,
Ϫ1
Ϫ1
850, 1724, 1239, 1031, 751, 527 cm . UV-Vis: 700.0, 432.0,
and 50 units ml streptomycin) at 37 ЊC in the dark, in 5%
CO . The survival was evaluated after 48 h of incubation by the
10.0, 256, 228, 222, 218, 212, 208, 204 nm. CD spectrum
2
6
20
J. Chem. Soc., Perkin Trans. 1, 2001, 617–621

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calorimetric method using the reduction of MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to
MTT formazan, which was determined by measurement of the
Sci. Technol., 1997, 5, 977; (d) W. Sliwa, Fullerene Sci. Technol.,
997, 5, 1133; (e) G. Torres-Garcia, H. Luftmann, C. Wolf and
1
J. Mattay, J. Org. Chem., 1997, 62, 2752; (f) S. Eguchi, M. Ohno,
S. Kojima, N. Koide, A. Yashiro, Y. Shirakawa and H. Ishida,
Fullerene Sci. Technol., 1996, 4, 303; (g) M. Ohno, T. Azuma,
S. Kojima, Y. Shirakawa and S. Eguchi, Tetrahedron, 1996, 52, 4983;
(h) A. Kraus, A. Gügel, P. Belik, M. Walter and K. M. Müllen,
Tetrahedron, 1995, 51, 9927; (i) Y.-Z. An, J. L. Anderson and
Y. Robin, J. Org. Chem., 1993, 58, 4799.
U. M. Fernandez-Paniagua, B. Illescas, N. Martin and C. Seoane,
J. Org. Chem., 1997, 62, 3705.
(a) A. Puplovskis, J. Kacens and O. Neilands, Tetrahedron Lett.,
absorbance at 595 nm. The steroid-C60 was dissolved in DMSO
Ϫ1
(
dimethyl sulfoxide) and medium containing 0.15 mg ml
steroid-C .
60
Acknowledgements
6
7
We thank the Chinese Academy of Sciences (KJ952-S1-503,
KJ95-A1-504), the Chinese Natural Science Foundation
1
997, 38, 285; (b) A. Hirsch, The Chemistry of the Fullerenes, Thieme
(
29790126, 29872049, 29832020), and the Ministry of Science
and Technology of China (the Pan Deng Program No.
70211006-6, the Major State Basic Research Development
Medical Publishers, New York, 1994.
For review see: C. Thilgen and F. Diederich, Top. Curr. Chem., 1999,
199, 135.
8
9
Program No.G2000077502).
9 S. R. Wilson, Q. Lu, J. Cao, Y. Wu, C. J. Welch and D. I. Schuster,
Tetrahedron, 1996, 52, 5131; according to their sector rule, the sector
projection of compound 1 can be drawn as below and its CD
spectrum should show negative Cotton effect, because the steroid
part was located in the right-upper sector, the negative sector..
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