Facilely accessible multidrug resistance modulator derived from sucrose.

Article abstract of DOI:10.1016/S0960-894X(02)00702-3

Exploration for new MDR-modulators utilizing atractysucroses as scaffolds disclosed 2,3,4,3',4'-O-pentaisovalerylsucrose (9) as a readily accessible medicinal lead. This lead was prepared from sucrose in 65% total yield for three steps. In addition, compound 9 exhibited more potent MDR modulating activity than verapamil, a representative modulator of MDR mediated by P-gp.

Full text of DOI:10.1016/S0960-894X(02)00702-3

Bioorganic & Medicinal Chemistry Letters 12 (2002) 3267–3270
Facilely Accessible Multidrug Resistance Modulator Derived
from Sucrose
Nobutoshi Murakami,a Satoru Tamura,a Etsuko Iwata,a Shunji Aoki,a
Shin-ichi Akiyamab and Motomasa Kobayashia,*
aGraduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan
bInstitute for Cancer Research, Faculty of Medicine, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima, 890-8520, Japan
Received 25 July 2002; accepted 27 August 2002
Abstract—Exploration for new MDR-modulators utilizing atractysucroses as scaffolds disclosed 2,3,4,30,40-O-pentaisovaleryl-
sucrose (9) as a readily accessible medicinal lead. This lead was prepared from sucrose in 65% total yield for three steps. In addi-
tion, compound 9 exhibited more potent MDR modulating activity than verapamil, a representative modulator of MDR mediated
by P-gp.
# 2002 Elsevier Science Ltd. All rights reserved.
Drug resistance has been a major unsolved problem in
chemotherapy for cancer patients. Especially, multidrug
resistance (MDR) is a highly complicated phenom-
enon.1 In many cases, MDR tumor cells acquire the
resistance to many structurally and functionally unre-
lated anti-tumor drugs by the overexpression of P-gly-
coprotein (P-gp). This specific protein acts as an energy-
dependent extrusion pump, which efficiently transports
lipophilic chemotherapeutic agents outside tumor
cells.2,3 MDR-modulators are able to restore the inher-
ent potency of anti-tumor agents through inhibition of
the function of P-gp.4 Thereby, exploration for MDR-
modulators has been a challenging topic for anti-cancer
therapeutic intervention. In this regard, we have been
engaged in a search for new MDR-modulators inhibit-
ing the transport activity of membrane proteins such as
P-gp and multidrug-resistance-associated protein 1
(MRP1) from natural compounds.5,6 During the course
of this research program, we characterized new MDR-
modulating oligoacylated sucroses, atractysucroses-I
(1), II (2), and III (3), from Atractylodis Lanceae Rhi-
zoma (So-jutsu in Japanese).7 Analysis for contribution
of the acyl residues to the MDR-modulating activity of
atractysucroses brought about 2,3,4,30,40-penta-O-iso-
valerylsucrose (9). This paper communicates the readily
accessible MDR-modulator 9 derived from sucrose.
Atractysucroses-I (1), II (2), and III (3) showed growth
inhibition against MDR tumor cells overexpressing
P-gp (KB-C2)8 in the presence of 0.1 mg/mL of colchi-
cine, a typical cytotoxic agent. Among the three con-
geners, 1 and 2 with five acyl residues showed similar
activity, whereas 3 with four acyl residues had less
potent activity than 1 and 2. This difference led us to
presume that the number and/or the location of acyl
residues are crucial for the MDR-modulating activity.
Thus, participation of the number and/or location of
acyl residues in MDR-modulating activity was exam-
ined by derivatization of atractysucrose-I (1) as illu-
strated in Scheme 1. The condensation between 1 and
0.3 equiv of isovaleric acid by using 3-(3-dimethylami-
.
nopropyl)-1-ethylcarbodiimide hydrochloride (EDCI HCl)
and 4-dimethylaminopyridine (DMAP) furnished 6-O-
isovaleryl (4) and 4-O-isovalerylatractysucrose-I (5) in
34% and 21% yields, respectively.9 On the other hand,
1 was treated with 3.3 equiv of isovaleric acid in the
.
presence of EDCI HCl and DMAP to give completely
acylated analogue 6 in 81% yield. Since direct intro-
duction of an isovaleryl group to the 3-hydroxyl residue
in 1 could be accomplished but only poorly, esterifica-
tion at the 3-OH group was carried out after protection
of the 4-OH and 6-OH groups as a benzylidene acetal.
Namely, treatment of 1 with benzaldehyde dimethyl
acetal in the presence of p-toluenesulfonic acid mono-
hydrate provided a benzylidene acetal 7 in 94% yield.
Condensation of isovaleric acid with 7 under the same
conditions as the preparation for 6 followed by removal
*Corresponding author. Tel.: +81-6-6879-8215; fax: +81-6-6879-
8219; e-mail: kobayasi@phs.osaka-u.ac.jp
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00702-3

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N. Murakami et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3267–3270
.
Scheme 1. Synthesis of derivatives of atractysucrose-I (1). Reagents and conditions: (a) isovaleric acid (0.3 equiv to 1), EDCI HCl, DMAP, CH2Cl2,
.
34% for 4, 21% for 5; (b) isovaleric acid (3.3 equiv to 1), EDCI HCl, DMAP, CH2Cl2, 81%; (c) benzaldehyde dimethyl acetal, p-toluenesulfonic
.
acid monohydrate, DMF, 94%; (d) isovaleric acid, EDCI HCl, DMAP, CH2Cl2; (e) H2, Pd/C, MeOH–AcOH (10:1), 92% two steps.
of the protecting group with Pd/C under hydrogen
atmosphere successfully furnished 3-O-isovaleryl con-
gener 8 in 92% yield for two steps.
modulating activity as 1, whereas 6-O-isovaleryl (4) and
3-O-isovaleryl congeners (8) showed slightly weaker
activity.
On
the
other
hand,
per-iso-
valerylatractysucrose-I (6) showed little activity. This
result suggested that the three hydroxyl groups in 1
would not be necessarily requested for MDR-modulat-
ing activity. In addition, enhancement of lipophilicity by
introduction of the three isovaleryl residues was shown
to diminish biological potency fairly. These findings led
us to assume that a pentaisovalerylsucrose with similar
lipophilicity to atractysucrose-I (1) was anticipated as a
potent MDR-modulator. According to this assumption,
we designed a readily accessible candidate 9, of which
only the primary hydroxyl groups are free from iso-
valeryl functions.
Assessment of MDR-modulating activity of all deriva-
tives was conducted by comparison of growth inhibition
against KB 3–1 cells with that against KB-C2cells in the
presence of colchicine.10 The biological outcome is
summarized in Table 1. At the concentration of 3 mg/
mL or less, each monoisovalerylatractysucrose-I (4, 5,
8) reversed MDR in KB-C2cells. Among them, 4- O-
isovalerylatractysucrose-I (5) displayed as potent MDR-
Table 1. Reversal of MDR in KB-C2cells by atractysucroses-I ( 1)
and its derivatives (4, 5, 6, 8, and 9)
This pentaisovaleryl candidate 9 was prepared from
sucrose in three steps as shown in Scheme 2. Protection
of all primary hydroxyl groups in sucrose was con-
ducted by using tert-butyldiphenylsilyl chloride
(TBDPSCl) in the presence of Et3N and DMAP to give
a tri-TBDPS ether in 68% yield. Coupling of isovaleric
acid and the tri-TBDPS ether in the same manner as the
preparation for 8, and subsequent deprotection of the
TBDPS groups by nBu4NF in acidic medium (THF-
AcOH=4:1)11 provided 2,3,4,30,40-O-pentaisovaler-
ylsucrose (9)12 in 95% yield for two steps. As a result of
4
Compd
Dose
Growth inhibition (%)
(mg/mL)
KB-3–1a
KB-C2b
1
4
5
6
8
9
10
3
1
10
3
1
10
3
1
10
3
1
10
3
1
10
3
1
92ꢀ6
93ꢀ1
76ꢀ4
40ꢀ9
ꢀ3
2 ꢀ9
1
10ꢀ16
93ꢀ292
16ꢀ16
18ꢀ11
40ꢀ5
60ꢀ10
23ꢀ8
88ꢀ3
79ꢀ8
39ꢀ11
2 ꢀ12
12ꢀ11
4ꢀ11
94ꢀ1
53ꢀ11
18ꢀ7
97ꢀ1
92ꢀ1
ꢀ18
10ꢀ19
2ꢀ11
25ꢀ6
15ꢀ15
10ꢀ13
66ꢀ13
16ꢀ9
6ꢀ11
98ꢀ0
8ꢀ11
9ꢀ1265
Each value presents meanꢀS.D. Colchicine shows no cytotoxicity
against KB-C2cells at 0.1 mg/mL.
Scheme 2. Synthesis of 2,3,4,30,40-O-pentaisovalerylsucrose (9).
Reagents and conditions: (a) TBDPSCl, Et3N, DMAP, DMF, 68%;
.
(b) isovaleric acid, EDCI HCl, DMAP, CH2Cl2; (c) nBu4NF, THF–
AcOH (4:1), 95% two steps.
aCytotoxicity of each compound.
bGrowth inhibition in the presence of colchicine (0.1 mg/mL).

N. Murakami et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3267–3270
3269
Science, Sports, and Culture of Japan. The authors are
also grateful to the Naito Foundation, the Hoh-ansha
Foundation, and the San-Ei Gen Foundation for Food
Chemical Research for financial support.
References and Notes
1. Mayer, L. D.; Shabbits, J. A. Cancer Metastasis. Rev. 2001,
20, 87.
2. Gottesman, M. M.; Pastan, I.; Ambudkar, S. V. Curr.
Opin. Gen. Devel. 1996, 6, 610.
3. Bellamy, W. T. Annu. Rev. Pharmacol. Toxicol. 1996, 36,
161.
4. Ford, J. M. Eur. J. Cancer. 1996, 32A, 991.
5. 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.
6. Aoki, S.; Okano, M.; Matsui, K.; Itoh, T.; Satari, R.;
Akiyama, S.; Kobayashi, M. Tetrahedron 2001, 57, 8951.
7. Murakami, N.; Iwata, E.; Tamura, S.; Akiyama, S.;
Kobayashi, M. Bioorg. Med. Chem. Lett. 2000, 10, 2629.
8. Akiyama, S.; Fojo, A.; Hanover, J. A.; Pastan, I.; Gottes-
man, M. M. Somat. Cell. Mol. Genet. 1985, 11, 117.
9. Dhaon, M. K.; Olsen, R. K.; Ramasamy, K. J. Org. Chem.
1982, 47, 1962.
10. Human epidermoid carcinoma KB cells (KB-3–1) were
used as the parental cell line for the present study. KB-3–1
cells were cultured in RPMI 1640 medium with 0.44 mg/mL of
glutamine, 50 mg/mL of kanamycin sulfate, supplemented with
10% newborn calf serum. Multidrug resistant (MDR) KB-C2
cells were selected and maintained from KB-3–1 in the med-
ium containing 2 mg/mL of colchicine. Reversing activity and
cytotoxicity were measured by means of MTT colorimetric
assay performed in 96-well plates. Equal numbers of cells
(10,000 cells) were inoculated into each well with 100 mL of the
culture medium. After 24 h preincubation (37 ꢃC, 5% CO2), 50
mL solution of the anticancer agent (colchicine) and the testing
sample was added to each well. Then, the whole mixture was
further incubated for 48 h under the same condition as pre-
incubation. The cytotoxic activity of the testing sample was
also examined by MTT assay using parental KB-3–1 cells.
Thereafter, 25 mL of MTT solution (2mg/mL in PBS) was
added to each well and incubated for further 3 h. After
removing the medium by aspiration, the resulting formazan
was extracted with 200 mL of dimethylsulfoxide. The percen-
tage of cell growth inhibition was calculated from the absor-
bance at 540 nm.
Figure 1. Reversal of MDR by 2,3,4,30,40-O-pentaisovalerylsucrose (9)
and verapamil under various concentrations of colchicine.
evaluation for MDR-modulating activity, compound 9
showed more potent efficacy than atractysucrose-I (1) as
shown in Table 1.
This pronounced biological potency directed us to
compare activity between 2,3,4,30,40-O-pentaisovaleryl-
sucrose (9) and verapamil,13 a representative modulator
of MDR mediated by P-gp. Thus, under the two con-
centrations of 9 (3 and 1 mg/mL), proliferation of KB-
C2cells resistant to colchicine was monitored. In both
concentrations, compound 9 restored cytotoxicity of
colchicine against KB-C2cells and completely reversed
colchicine-resistance at the concentration of 3 mg/mL as
depicted in Figure 1. 2,3,4,30,40-O-Pentaisovaler-
ylsucrose (9) restored the sensitivity of KB-C2cells
against colchicine in the lower concentration (3.9ꢁ10ꢂ6
M) than verapamil (1.0ꢁ10ꢂ5 M).14
11. Smith, A. B., III; Ott, G. R. J. Am. Chem. Soc. 1996, 118,
13095.
12. 9: Colorless oil, [a]D22 +19.8ꢃ(c 1.08 in CHCl3). IR (KBr):
3472, 1748, 1296, 1254 cmꢂ1 1H NMR (500 MHz, CDCl3)
.
Sugar moiety d: 5.66 (1H, d, J=3.7 Hz, 1-H), 5.61 (1H, dd,
J=7.3, 7.9 Hz, 40-H), 5.49 (1H, d, J=7.9 Hz, 30-H), 5.48 (1H,
dd, J=9.8, 10.4 Hz, 3-H), 4.97 (1H, dd, J=9.8, 9.8 Hz, 4-H),
4.90 (1H, dd, J=3.7, 10.4 Hz, 2-H), 4.17 (1H, ddd, J=2.4, 5.5,
9.8 Hz, 5-H), 4.00 (1H, ddd, J=3.1, 4.3, 7.3 Hz, 50-H), 3.85
(1H, dd, J=3.1, 12.8 Hz, 60-Ha), 3.70 (1H, dd, J=2.4, 12.8
Hz, 6-Ha), 3.65 (1H, dd, J=4.3, 12.8 Hz, 60-Hb), 3.63 (1H, d,
J=12.8 Hz, 10-Ha), 3.60 (1H, dd, J=5.5, 12.8 Hz, 6-Hb), 3.52
(1H, d, J=12.8 Hz, 10-Hb). Isovaleryl moiety d: 2.39 (1H, dd,
J=6.9, 15.1 Hz, COCH2CH(CH3)2), 2.32 (1H, dd, J=7.5, 14.9
Hz, COCH2CH(CH3)2), 2.09–2.21 (m, COCH2CH(CH3)2), 1.94–
2.07 (m, COCH2CH(CH3)2), 0.99–1.01 (m, COCH2CH(CH3)2),
0.89–0.91 (m, COCH2CH(CH3)2). 13C NMR (125 MHz,
In summary, we have disclosed a new readily accessible
MDR-modulator, 2,3,4,30,40-O-pentaisovalerylsucrose
(9), utilizing atractysucroses as scaffolds. It should be
noted that 2,3,4,30,40-O-pentaisovalerylsucrose (9) was
synthesized from cheaply available sucrose in 65% total
yield for 3 steps.
Acknowledgements
0
CDCl3) Sugar moiety dc: 104.4 (C-20), 89.7 (C-1), 81.2(C-5 ),
76.0 (C-30), 72.6 (C-40), 71.8 (C-5), 70.2(C-2), 68.8 (C-3), 68.5
This study was financially supported by Grants-in-Aid
for Scientific Research from the Ministry of Education,
(C-4), 64.1 (C-10), 61.5 (C-6), 60.7 (C-60). Isovaleryl moiety dc:

3270
N. Murakami et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3267–3270
171.5–173.0 (COCH2CH(CH3)2), 42.7–43.0 (COCH2CH(CH3)2),
25.5–25.8 (COCH2CH(CH3)2), 22.2–22.7 (COCH2CH(CH3)2).
FABMS m/z: 785 [M+Na]+. FABHRMS m/z: calcd for
C37H62O16+Na: 785.3935: Found 785.3936.
13. Tsuruo, T.; Iida, H.; Tsukagoshi, S.; Sakurai, Y. Cancer
Res. 1981, 41, 1967.
14. As shown in Figure 1, the incubation with 10 mg/mL of 9 in
the presence of low concentration of colchicine (1ꢁ10ꢂ3 mg/
mL) exhibited no cytotoxicity against KB C-2. However, the 10
mg/mL concentration of 9 was cytotoxic against parental KB-
3–1 cells (Table 1). These findings might be ascribable to selec-
tive cytotoxic behavior of 9 against KB 3–1 cells similar to 1.