N. Murakami et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2521±2524
2523
LiAlH4 reduction to furnish a diene alcohol, which
was further treated with t-butyldimethylsilyl tri¯uoro-
methanesulfonate (TBSOTf) and 2,6-lutidine to give a
TBS ether 6.
10-CH3. Selective cleavage of the MOM group in 15 was
achieved using TMSBr to give a monoalcohol in 96%
yield. The monoalcohol was further subjected to suc-
cessive acetylation using Ac2O/pyridine and removal of
the TBS group with HF±pyridine to furnish 4-deace-
toxyagosterol A (2)12 in 7.3% of total yield for 17 steps.
With a view to our exploration for more straightfor-
ward lead compounds, the chemical transformation
from 12 into a 22-deoxyanalogue (17)13 of 2 was also
carried out by the same procedure (Scheme 1).
10
Dehydrogenation of 6 with Hg(OAc)2 followed by
selective protection of the homoannular 5,7-diene by 4-
phenyl-1,2,4-triazoline-3,5-dione gave 9 in 71% yield for
two steps. Epoxidation of 9 with m-chloroperbenzoic
acid in CHCl3 predominantly aorded the 9a,11a-
epoxide 11,11 which was further subjected to reductive
cleavage of the epoxy ring and diene-deprotection by
use of LiAlH4 to give the desired 11a-alcohol 13 in
unsatisfactory yield (48%). For the purpose of over-
coming this undesirable outcome, we examined carefully
this reaction condition utilizing 12 as a model substrate.
The epoxide 12 was prepared from 7,8-didehydro-
cholesterol (8) through the same three-step transforma-
tion after protection of the 3-OH group as a MOM ether.
Intensive investigation of the reaction conditions led us to
®nd a facile Et2AlCl-catalyzed LiAlH4-reductive epoxy
cleavage method, which proceeded with concomitant
removal of the triazolidine group to furnish a 11a-
hydroxy-5,7-diene (14) from 12 in 90% yield. Namely,
after pre-treatment of 12 with Et2AlCl in THF under
re¯ux, the reaction mixture was re¯uxed with LiAlH4 to
furnish 14. Application of this method to 11 resulted in
generation of 13 in nearly the same yield (84%). The ste-
reochemistry at C-9 in 13 was con®rmed to be R by the
coupling constant (11 Hz) between 9-H and 11-H.
Assessment of the MDR-modulating activity was made
from the ability to potentiate the respective cytotoxicity
of colchicine against KB-C2 cells14 and that of vincris-
tine against KB CV-60 cells3 as compared with parental
human epidermoid carcinoma KB-3-1 cells. The former
MDR cell line was shown to overexpress P-gp, while the
latter to overexpress MRP. Table 1 summarizes the
MDR-modulating potency for agosterol A (1), 4-deace-
toxy- (2), and 4-deacetoxy-22-deoxy-congener (17). In a
previous study of SAR,5 we found that both the three
acetoxyl groups in ring AB and the two hydroxyl groups
are crucial for the MDR-modulating activity. Com-
pound 17 lacking both 4-acetoxy and 22-hydroxyl
groups showed not only serious reduction of MDR
reversal properties but also enhancement of cytotoxicity
against parental KB-3-1 cells. On the other hand, 4-
deacetoxyagosterol A (2) mostly preserves the MDR-
modulating potency of agosterol A (1). On the basis of
these ®ndings, the following SAR of 1 is assumed: (1) a
22-hydroxyl group is an important structural function
for the MDR-modulating activity of 1; (2) the MDR-
modulating activity of 1 is minimally aected by an
acetoxyl group on C-4. So far, there are few MDR-
modulators inhibiting the function of MRP.15,16 In this
context, it should be noted that the activity of 4-deacet-
oxyagosterol A (2) to restore the sensitivity of KB CV-
60 cells against vincristine is nearly as potent as that of
agosterol A (1).
The 11a-hydroxyl group in 13 was protected as a TBS
ether; then, the resulting diene with fully protected
hydroxyl functions was submitted to hydroboration by
a BH3±Me2S complex, followed by oxidation with H2O2
to aord an allyl alcohol with 6a-hydroxy-7-ene struc-
ture. Usual acetylation of the allyl alcohol provided 15
in 65% yield for two steps. The con®guration at C-5
and C-6 was elucidated by the following spectral fea-
tures: (1) in the 1H NMR spectrum of 15, the signal due
to 6-H appeared at 5.02 ppm as a broad doublet with
a coupling constant of 10 Hz; (2) the NOESY spectrum
of 15 showed a distinct cross-peak between 6-H and
Acknowledgements
The authors are grateful to the Naito Foundation, the
Houansha Foundation, and the Ministry of Education,
Science, Sports, and Culture of Japan for ®nancial
support.
Table 1. Reversal of MDR in KB-C2 and KB CV-60 cells by
agosterol A and its derivatives
Growth inhibition (%)
Compound
Dose (mg/mL)
KB-3-1a
KB-C2b
KB CV-60c
1
10
3
1
10
3
1
10
3
1
13 Æ 7
84 Æ 2
82 Æ 0
75 Æ 4
87 Æ 1
76 Æ 3
44 Æ 1
88 Æ 1
69 Æ 1
37 Æ 0
76 Æ 2
73 Æ 0
66 Æ 0
69 Æ 2
64 Æ 1
59 Æ 2
42 Æ 7
25 Æ 4
15 Æ 5
References and Notes
1. Young, R. C. Drug Resistance in Cancer Therapy; Ozols, R.
F., Ed.; Kluwer Academic Press: Boston, 1989; pp 1±12.
2. Gerlach, J. H.; Endicott, J. A.; Juranka, P. F.; Henderson,
G.; Sarangi, F.; Deuchars, K. L.; Ling, V. Nature 1986, 324,
485.
3. Nagayama, S.; Chen, Z.-S.; Kitazono, M.; Takebayashi,
Y.; Niwa, K.; Yamada, K.; Tani, A.; Haraguchi, M.; Sumi-
zawa, T.; Furukawa, T.; Aikou, T.; Akiyama, S. Cancer Lett.
1998, 130, 175.
2
19 Æ 5
13 Æ 4
17
39 Æ 7
5 Æ 10
aCytotoxicity of each compound.
bGrowth inhibition in the presence of colchicine (0.1 mg/mL).
cGrowth inhibition in the presence of vincristine (0.1 mg/mL).
Each value presents mean Æ S.D. Colchicine and vincristine show
no cytotoxicity against KB-C2 and KB CV-60 at 0.1 mg/mL dose,
respectively.
4. Aoki, S.; Yoshioka, Y.; Miyamoto, Y.; Higuchi, K.; Setia-
wan, A.; Murakami, N.; Chen, Z.-S.; Sumizawa, T.; Akiyama,
S.; Kobayashi, M. Tetrahedron Lett. 1998, 39, 6303.
Murakami, Nobutoshi
