Fluorophore-appended steroidal saponin (dioscin and polyphyllin D) derivatives

Article abstract of DOI:10.1021/ol0475616

(Chemical Equation Presented) The synthesis of three fluorophore-appended derivatives of dioscin and polyphyllin D is reported herein. Starting from trillin, dansyl derivatives A-C were prepared in overall yields of 7-12% over 7-10 steps. A study of their behavior in a variety of polar solvents suggests that dansyl derivatives A-C are capable of micellar self-assembly and can maintain cytotoxicities (IC₅₀ = 15-18 μM) against the HeLa carcinoma cell line evaluated by standard MTT assay.

Full text of DOI:10.1021/ol0475616

ORGANIC
LETTERS
2005
Vol. 7, No. 4
669-672
Fluorophore-Appended Steroidal
Saponin (Dioscin and Polyphyllin D)
Derivatives
Zhiqi Yang,†,‡ Ella Lai-Ming Wong,‡ Tina Yuen-Ting Shum,‡ Chi-Ming Che,*,†,‡ and
Yongzheng Hui*,†
Shanghai-Hong Kong Joint Laboratory on Chemical Synthesis, Shanghai Institute of
Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China, and
Department of Chemistry and Open Laboratory of Chemical Biology of the Institute of
Molecular Technology for Drug DiscoVery and Synthesis,
The UniVersity of Hong Kong, Pokfulam Road, Hong Kong SAR, China
cmche@hkucc.hku.hk; huiyongzheng@yahoo.com
Received November 28, 2004
ABSTRACT
The synthesis of three fluorophore-appended derivatives of dioscin and polyphyllin D is reported herein. Starting from trillin, dansyl derivatives
C were prepared in overall yields of 7 12% over 7 10 steps. A study of their behavior in a variety of polar solvents suggests that dansyl
derivatives A C are capable of micellar self-assembly and can maintain cytotoxicities (IC50 15 18 M) against the HeLa carcinoma cell line
A−
−
−
−
)
−
µ
evaluated by standard MTT assay.
Saponins, a class of glycoconjugates, are widely found in
terrestrial plants and some marine organisms and are reported
to be active constituents of many well-known traditional
chinese medicines.1 Structurally, saponins contain two
distinct components, a saccharide (or polysaccharide) group
and a sapogenin moiety, with the latter being usually a steroid
or triterpene unit. Both components are important to the
bioactivities exhibited by saponins.2 The molecular structures
of two typical steroidal saponins, dioscin (diosgenyl 2,4-di-
O-R-L-rhamnopyrano-syl-â-D-glucopyranoside) and poly-
phyllin D (diosgenyl R-L-rhamno-pyranosyl-(1f2)-[R-L-
arabinofuranosyl-(1f4)]-â-D-glucopyranoside), are depicted
in Figure 1. Dioscin exhibits moderate to good cardiovas-
cular, cytotoxic, and antifungal activities.3,4 Polyphyllin D
(2) (a) Gu, G.; Du, Y.; Linhardt, R. J. J. Org. Chem. 2004, 69, 5497. (b)
Ikeda, T.; Tsumagari, H.; Honbu, T.; Nohara, T. Biol. Pharm. Bull. 2003,
26, 1198. (c) Ma, X.; Yu, B.; Hui, Y.; Miao, Z.; Ding, J. Bioorg. Med.
Chem. Lett. 2001, 11, 2153. (d) Chang, L.-C.; Tsai, T.-R.; Wang, J.-J.;
Lin, C.-N.; Kuo, K.-W. Biochem. Biophys. Res. Commun. 1998, 242, 21.
(3) (a) Nakano, K.; Murakami, K.; Takaishi, Y.; Tomimatsu, T.; Nohara,
T. Chem. Pharm. Bull. 1989, 37, 116. (b) Hufford, C. D.; Liu, S.; Clark,
A. M. J. Nat. Prod. 1988, 51, 94. (c) Hirai, Y.; Sanada, S.; Ida, Y.; Shoji,
J. Chem. Pharm. Bull. 1984, 32, 295. (d) Liu, C.; Chen, Y. Acta Pharm.
Sin. 1984, 19, 799. (e) Fang, Y.; Zhao, J.; Ho, Y.; Li, B.; Xu, C. Acta
Pharm. Sin. 1982, 17, 388.
(4) (a) Liu, M.-J.; Wang, Z.; Ju, Y.; Zhou, J.-B.; Wang, Y.; Wong, R.
N.-S. Biol. Pharm. Bull. 2004, 27, 1059. (b) Ikeda, T.; Tsumagari, H.;
Honbu, T.; Nohara, T. Biol. Pharm. Bull. 2003, 26, 1198. (c) Cai, J.; Liu,
M.; Wang, Z.; Ju, Y. Biol. Pharm. Bull. 2002, 25, 193. (d) Wang, Z.; Zhou,
J.; Ju, Y.; Zhang, H.; Liu, M.; Li, X. Biol. Pharm. Bull. 2001, 24, 159.
† Shanghai Institute of Organic Chemistry.
‡ The University of Hong Kong.
(1) (a) Hostettmann, K.; Marston, A. Saponins; Cambridge University
Press: New York, 1995. (b) Waller, G. R.; Yamasaki, K. Saponins Used
in Traditional and Modern Medicine; Plenum Press: New York, 1996.
10.1021/ol0475616 CCC: $30.25
© 2005 American Chemical Society
Published on Web 01/27/2005

previous work6b could be obtained by carrying out the
reaction at a lower temperature (-3 °C) and pyridine
concentration (50%). TMSOTf-promoted glycosylation of
diol 2 with 2,3,4-tri-O-benzoyl-R-L-rhamnopyranosyl tri-
chloroacetimidate (3)9 (1.2 equiv) gave the 2-O-glycosylated
and 2,4-di-O-glycosylated products 4 and 5 in 36 and 42%
yields, respectively. Subsequent base-mediated deprotection
of 5 gave dioscin in 90% yield. As shown in Scheme 3,
Figure 1. Structures of dioscin and polyphyllin D.
Scheme 3
has been shown to have promising cardiovascular, antitumor,
hemostatic, and immunomodulating effects.5 The synthesis
of these two saponins has been reported previously.6
To examine the pharmacological mechanisms of these two
diosgenyl saponins at the cellular level, we have prepared
three fluorescent derivatives of dioscin and polyphyllin D
(A-C). In this work, the fluorescent dansyl group was
chosen because its emission properties are sensitive to the
microenvironment.7
Lewis-acid (BF3‚OEt2)-catalyzed coupling of 4 with 2,3,5-
tri-O-acytyl-R-L-arabinofuranosyl trichloroacetimidate 6 gave
7 in 87% yield.6a,b Deprotection of 7 under basic conditions
with NaOH (12 equiv) afforded polyphyllin D in 91% yield.
The OH groups of dioscin and polyphyllin D were
benzylated with BnBr and NaH to give 8 and 9 in 90 and
91% yields, respectively (Scheme 4). Treatment of 8 with
Scheme 1
Scheme 4
A modified synthetic route to dioscin and polyphyllin D
has been developed in this work. As shown in Scheme 2,
the synthesis was started from trillin (diosgenyl â-D-
glucopyranoside) 1.6a,b,8 Treatment of 1 with pivaloyl chloride
(PivCl) in CH2Cl2/pyridine (3:1) solution afforded the 3,6-
di-Piv-protected product 2 in 85% yield. We found a higher
product yield and regioselectivity than that reported in
Scheme 2
DIBALH10 afforded the dihydro (22R)-derivative 10 in 59%
yield. Similarly, 13 was obtained in 61% yield from the
reaction of 9 with DIBALH. The 26-hydroxyl primary
alcohols 10 and 13 were readily converted to their phthal-
(5) (a) Nohara, T.; Yabuta, H.; Suenobu, M.; Hida, R.; Miyahara, K.;
Kawasaki, T. Chem. Pharm. Bull. 1973, 21, 1240. (b) Ravikumar, P. R.;
Hammesfahr, P.; Sih, C. J. J. Pharm. Sci. 1979, 68, 900. (c) Ma, J. C. N.;
Lau, F. W. Phytochemistry 1985, 24, 1561. (d) Nambu, T.; Huang, X.;
Shu, Y.; Huang, S.; Hattori, M.; Kakiuchi, N.; Wang, Q.; Xu, G.-J. Planta
Med. 1989, 55, 501. (e) Zhou, J. Pure Appl. Chem. 1989, 61, 457. (f) Chiang,
H. C.; Wang, J. J.; Wu, R. T. Anticancer Res. 1992, 12, 949.
(6) (a) Deng, S.; Yu, B.; Hui, Y.; Yu, H.; Han, X. Carbohydr. Res. 1999,
317, 53. (b) Li, B.; Yu, B.; Hui, Y.; Li, M.; Han, X.; Fung, K.-P. Carbohydr.
Res. 2001, 331, 1. (c) Yu, B.; Tao, H. J. Org. Chem. 2002, 67, 9099. (d)
Du, Y.; Gu, G.; Wei, G.; Hua, Y.; Linhardt, R. J. Org. Lett. 2003, 5, 3627.
670
Org. Lett., Vol. 7, No. 4, 2005

Scheme 5
imide derivatives by the Mitsunobu-Gabriel reaction11 to
at 514 nm [quantum yield ) 0.4,19 λ(excitation) ) 339 nm]
and two absorption bands at 262 (ꢀ ) 11 590 dm3 mol-1
cm-1) and 339 nm (ꢀ ) 3900 dm3 mol-1 cm-1). The dansyl
derivative B emits at 518 nm [quantum yield ) 0.39, λ
(excitation) ) 339 nm] and shows absorption maxima at 261
(ꢀ ) 12 800 dm3 mol-1 cm-1) and 339 nm (ꢀ ) 4600 dm3
mol-1 cm-1).
It has been reported that the dansyl fluorescence maximum
λmax increases with increasing polarity of the medium.7 In
this work, we studied the solvent effect on the emission
properties of A and B in H2O/DMSO (for C, the solvent
effect and the emission properties are the same as those for
A) and obtained some interesting findings.
As depicted in Figure 2, an increase in solvent polarity
by changing the H2O/DMSO ratio from 0 to 1:1 results in a
red shift of λmax of the dansyl fluorescence of A and B
(∆λmax(A) ) 17 nm, ∆λmax(B) ) 15 nm).
An interesting phenomenon was observed when the H2O
content was further increased. As depicted in Figure 2, the
give 11 (92% yield) and 14 (90% yield), respectively.
Subsequent removal of the N-protecting group12 with hy-
drazine hydrate and reduction of the benzyl groups13 with
lithium in liquid ammonia furnished the respective precursors
16 and 17 in 77 and 78% yields, respectively, over two steps.
Reaction of 16 with dansyl chloride14 (DsCl) and KHCO3
afforded A in 77% yield. Under similar conditions, reaction
of 17 with DsCl gave dansyl derivative C in 69% yield.
The synthesis of B is depicted in Scheme 5. First, 18 was
prepared in 66% yield by treating trillin with triphenylmethyl
chloride (TrCl) and (dimethylamino)pyridine in pyridine at
85 °C.15 Reaction of 18 with PivCl in CH2Cl2/pyridine (3:1)
solution afforded the 3-Piv product 19 in 82% yield.16
Glycosylation of diol 19 with 3 (2.5 equiv) catalyzed by
TMSOTf gave the 2,4-di-O-glycosylated product 20 in 95%
yield. The triphenylmethyl (Tr) protecting group was re-
moved to give 21 in 80% yield.17 Under the reaction
conditions of I2/PPh3/imidazole,18 the primary alcohol was
converted to the iodide 22 in 86% yield. The ethylenediamine
derivative 23 was prepared in 53% yield by refluxing 22
with 1,2-diaminoethane in THF and removing the Piv
protecting group with NaOH. Condensation of 23 with DsCl
and KHCO3 gave B in 66% yield.
Dansyl derivatives A-C are strongly emissive. The
absorption and emission spectra of A and C are essentially
the same in DMSO solution, featuring an emission with λmax
(7) (a) Abel, E.; Maguire, G. E. M.; Murillo, O.; Suzuki, I.; De Wall, S.
L.; Gokel, G. W. J. Am. Chem. Soc. 1999, 121, 9043. (b) Doyle, E. L.;
Hunter, C. A.; Phillips, H. C.; Webb, S. J.; Williams, N. H. J. Am. Chem.
Soc. 2003, 125, 4593.
(8) Deng, S.; Yu, B.; Lou, Y.; Hui, Y. J. Org. Chem. 1999, 64, 202.
(9) Li, C.; Yu, B.; Hui, Y. J. Carbohydr. Chem. 1999, 18, 1107.
(10) Oikawa, M.; Oikawa, H.; Ichihara, A. Tetrahedron 1995, 51, 6237.
(11) Ku¨hn, C.; Lindeberg, G.; Gogoll, A.; Hallberg, A.; Schmidt, B.
Tetrahedron 1997, 53, 12497.
(12) Ohtake, H.; Ichiba, N.; Ikegami, S. J. Org. Chem. 2000, 65, 8171.
(13) Birch A. J. J. Chem. Soc. 1944, 430.
(14) Zhou, X.-T.; Forestier, C.; Goff, R. D.; Li, C.; Teyton, L.; Bendelac,
A.; Savage, P. B. Org. Lett. 2002, 4, 1267.
(15) Du, Y.; Zhang, M.; Kong, F. Org. Lett. 2000, 2, 3797.
(16) Jiang, L.; Chan, T.-H. J. Org. Chem. 1998, 63, 6035.
(17) Deprotection of 21 under basic conditions with NaOH (12 equiv)
could afford dioscin. Thus, we could indirectly confirm the structure of 19.
(18) Classon, B.; Garegg, P. J.; Samuelsson, B. Can. J. Chem. 1981,
59, 339.
Figure 2. Change of the emission maximum of the dansyl deriva-
tives A and B with changes in the solvent ratio of DMSO:H2O.
Org. Lett., Vol. 7, No. 4, 2005
671

fluorescence λmax of A and B blue shifts abruptly once the
H2O:DMSO ratio is larger than 1:1. The λmax of A was found
to shift from 531 to 492 nm as the ratio of H2O:DMSO
increased from 1:1 to 2:1. A further increase in the H2O:
DMSO ratio did not have a significant effect as the ratio
was gradually increased to more than 20:1, and the λmax of
A eventually stabilized at 489 nm. For B, its λmax was found
to blue shift from 533 to 492 nm as the ratio of H2O:DMSO
increased from 1:1 to 10:1. However, the change in the λmax
of B was dissimilar to that observed for A when the H2O:
DMSO ratio was altered from 10:1 to 3000:1. An increase
of the H2O:DMSO ratio from 2:1 to 500:1 was found to
slightly increase the fluorescence λmax of B from 492 to 498
nm. A further increase in the H2O:DMSO ratio from 500:1
to 3000:1 did not have a significant effect, and the λmax of B
remained at 498 nm.
arrangement would permit a response to changes in the
solvent environment even after the micelles completely
formed.
The cytotoxicity of dioscin, polyphyllin D, and the dansyl
derivatives A-C against the HeLa cell line has been
evaluated by standard MTT assay,20 and the results are
depicted in Table 1.
Table 1. Cytotoxicities (IC50) of Dioscin, Polyphyllin D, and
Their Dansyl Derivatives against HeLa Cellsa
compound
IC50 (µM) in 72 h
dioscin
polyphyllin D
A
B
3.8 ( 0.3
1.1 ( 0.02
15.7 ( 0.6
15.3 ( 0.2
17.6 ( 0.5
7.4 ( 0.1
C
This phenomenon is unusual, as it is different from that
reported in the literature.7 A decrease in λmax suggests that
the dansyl group is located in a less polar environment. On
this premise, we propose that A and B form dispersed
micelles when the H2O:DMSO ratio is larger than 1:1; the
outer part of the micelle consists of hydrophilic sugar
moieties, and the hydrophobic aglycone and dansyl groups
form the inner core of the micelle. Thus, the dansyl groups
become surrounded by a nonpolar environment. Two possible
micellar models are depicted in Figure 3.
cisplatin
a HeLa: cervix epitheloid carcinoma.
The cytotoxic activities of dansyl derivatives A (IC50
)
15.7 ( 0.6 µM) and C (IC50 )17.6 ( 0.5 µM) are lower
than those of their parent compounds (dioscin, IC50 ) 3.8
( 0.3 µM; polyphyllin D, IC50 ) 1.1 ( 0.02 µM) on the
basis of IC50 values. This suggests that the F ring of the
steroid and spirostanol structural unit plays an important role
in the cytotoxicities toward HeLa cells. When the spirostanol
structural unit was lost, the two saponins lost considerable
efficiency. Similar findings had been reported for steroidal
saponins.4b,d
The cytotoxic activity of dansyl derivative B (IC50 ) 15.3
( 0.2 µM) is also less toxic than its parent compound,
dioscin. We conceive that attachment of a hydrophobic
dansyl group at the sugar terminal of dioscin might lower
its hydrophilic character, which results in a higher IC50 value.
In conclusion, we have synthesized three fluorophore-
appended steroidal saponin derivatives of dioscin and poly-
phyllin D and examined their micellar properties and
bioactivities. Efforts are currently under way to examine the
intercellular target and actions of the two saponins by in vitro
and in vivo studies.
Figure 3. Possible micellar models of A-C. Abbreviation: F )
Acknowledgment. This work is supported by the Area
of Excellence Scheme (AoE/P-10-01) established under the
University Grants Committee, HKSAR, the Hong Kong
Research Grants Council (HKU7039/03P), HKSAR, and The
University of Hong Kong (University Development Fund).
fluorophore moiety.
In the case of A, as the ratio of H2O:DMSO increases
above 20:1, micellar self-assembly occurs in a manner where
the dansyl groups are positioned at the center of the micelle,
presumably to minimize further changes in solvent polarity.
As depicted in Figure 3, the dansyl groups of B are proposed
to be located on the outer rim of the micellar structure. This
Supporting Information Available: Experimental details
for the synthesis of A-C as well as their cytotoxicity tests
and analytical data for new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
OL0475616
(19) Fluorescence quantum yields (Ff) of A-C were determined by using
quinine sulfate in 0.1 N H2SO4 as the reference standard (Ff ) 0.546).
(20) Mosmann, T. J. Immunol. Methods 1983, 65, 55.
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Org. Lett., Vol. 7, No. 4, 2005