Enhanced efficacy of 7-hydroxy-3-methoxycadalene via glycosylation in in vivo xenograft study

Article abstract of DOI:10.1016/j.bmcl.2007.08.071

7-Hydroxy-3-methoxycadalene, isolated from Zelkova serrata Makino, was confirmed as a biologically active natural compound. In this study, the efficacy of cadalene as an anticancer agent was tested. In order to address the poor physicochemical properties of cadalene, we designed and synthesized glycosylated cadalene derivatives for improved solubility and efficient drug delivery as a potential prodrug. In vitro cell viability assays confirmed that glycosylated cadalenes were less toxic and more soluble than cadalene. In an in vivo xenograft study in mice, the oral administration of glycosylated cadalenes caused a significant reduction in tumor size.

Full text of DOI:10.1016/j.bmcl.2007.08.071

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6335–6339
Enhanced efficacy of 7-hydroxy-3-methoxycadalene
via glycosylation in in vivo xenograft study
Hyang Yeon Lee,a,ꢀ Jung-Taek Kwon,b,ꢀ Minseob Koh,a
Myung-Haing Chob,* and Seung Bum Parka,*
aDepartment of Chemistry, Seoul National University, Seoul 151-747, Republic of Korea
bCollege of Veterinary Medicine, Seoul National University, Seoul 151-742, Republic of Korea
Received 13 July 2007; revised 28 August 2007; accepted 29 August 2007
Available online 1 September 2007
Abstract—7-Hydroxy-3-methoxycadalene, isolated from Zelkova serrata Makino, was confirmed as a biologically active natural
compound. In this study, the efficacy of cadalene as an anticancer agent was tested. In order to address the poor physicochemical
properties of cadalene, we designed and synthesized glycosylated cadalene derivatives for improved solubility and efficient drug
delivery as a potential prodrug. In vitro cell viability assays confirmed that glycosylated cadalenes were less toxic and more soluble
than cadalene. In an in vivo xenograft study in mice, the oral administration of glycosylated cadalenes caused a significant reduction
in tumor size.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Cadalene (7-hydroxy-3-methoxycadalene, 1), a small
molecular secondary metabolite classified as a plant-de-
rived flavonoid,1 was isolated from Zelkova serrata
Makino and made available in large quantities from
Zelkova wood powder by extraction with ethanol and
purification with flash silica column chromatography.
In our previous study, cadalene demonstrated various
biological activities such as chemopreventive effects
and antioxidative activities in murine lung tumorigenesis
induced by 4-(methylinitrosamino)-1-(3-pyridyl)-1-buta-
none (NNK); NNK is the major causative factor of lung
cancer.2,3 When A/J mice with oxidative damages in-
duced by NNK were orally treated with cadalene on a
dosage of 100 mg/kg, the glutathione (GSH) level was
maintained and the oxidative stress was reduced. With
regard to its chemopreventive efficacy, cadalene-treated
A/J mice (oral dosage of 100 mg/kg) demonstrated a sig-
nificant reduction in the incidence of lung adenomas
from 45% to 10%.2b The results supported that cadalene
is an antioxidant and potent chemopreventive agent
against NNK-induced murine lung tumorigenesis.2
However, the efficacy of cadalene as an anticancer agent
has not been studied. Furthermore, there are potential
problems in the clinical application of cadalene due to
its low solubility in water.
In order to address these issues, we performed the chem-
ical modification of cadalene. Glycosylation is one of the
major approaches for increasing water solubility and
cellular uptake.4–9 There are many literature precedents
for the enhancement in the solubility and efficacy of nat-
ural products such as quercetin,6 kaempferitrin,7 and
chrysoeriol.8 Therefore, it is expected that glycosylated
cadalenes will have enhanced water solubility and im-
proved cellular uptake.5,7 Due to the fast proliferation
of cancer cells, glucose uptake in them is significantly
9
faster than that in normal cells. We also considered
the importance of the stereochemistries of glycolytic
linkage at anomeric positions because we observed the
differences in the cellular uptakes of fluorescent glucose
bioprobes in a-anomer and b-anomer.9
In this study, we focused on the enhancement of the
water solubility and efficient delivery of cadalene via gly-
cosylation. The transient modification of cadalene might
facilitate its targeted delivery into cancer cells in the
form of an inactive prodrug, which can be unmasked
by the cellular enzymatic reaction of glycosidase or sim-
ple hydrolysis.10 The antitumor efficacies of cadalene 1
Keywords: Cadalene; Glycosylation; Prodrug; Drug delivery;
Anticancer.
*
Corresponding authors. Tel.: +82 2 880 2376; fax: +82 2 873 1268
(M.-H.C.); tel.: +82 2 880 9090; fax: +82 2 884 4025 (S.B.P.); e-mail
addresses: mchotox@snu.ac.kr; sbpark@snu.ac.kr
ꢀ
These authors contributed equally to this work.
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.08.071

6336
H. Y. Lee et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6335–6339
and glycosylated cadalenes (2 and 3) were examined by
an in vitro cell viability assay and confirmed with a hu-
man nonsmall cell lung cancer xenograft model by oral
administration.
OAc
O
OH
O
i, ii
AcO
AcO
HO
HO
OH
AcO
Br
OH
O
6
OH
As shown in Figure 1, we designed and synthesized two
derivatives of glycosylated cadalene; one derivative was
obtained by direct b-glycosylation on cadalene (b-Glc-
O-cadalene, 2), and the other was obtained by a-glyco-
sylation on cadalene via an ethoxy linker (a-Glc-
OCH2CH2-O-cadalene, 3). The key features of these
two glycosylated cadalenes are two different stereochem-
istry of the hydrolytic linkage at anomeric positions,
namely, a- and b-anomers. The synthesis of the glycos-
ylated cadalene derivatives (2 and 3) is shown in
Schemes 1 and 2. The synthetic scheme was initiated
with the global acetylation of hydroxyl groups on glu-
cose. Subsequently, the hydroxyl group at the anomeric
position on compound 4 was selectively deprotected
with ethylenediamine, followed by activation with tri-
chloroacetonitrile in the presence of DBU.11,12a Glyco-
syl trichloroacetimidate 5 was a thermodynamically
favored a-anomeric product, which was confirmed by
1H NMR; therefore, the treatment of cadalene with gly-
cosyl trichloroacetimidate led to the desired b-glycosyl-
ated cadalene in high yield with the assistance of
BF3•OEt2.12 The other derivative was a-glycosylated
cadalene with an ethoxy linker, 3. The stereochemical
enrichment of the a-anomer of glycosylated cadalene
O
HO
HO
iii, iv
O
HO
O
O
3
Scheme 2. Synthesis of a-Glc-OCH2CH2-O-cadalene (3). Reagents
and conditions: (i) 2-bromoethanol, Dowex 50wx8-400 ion exchange
resin, 70 ꢁC, 82%; (ii) glacial acetic anhydride, pyridine, DMAP, 65%;
(iii) 1, Cs2CO3, anhyd DMF, 60 ꢁC, 70%; (iv) NaOMe, MeOH,
quantitative yield.
was achieved by the following pathway. The treatment
of glucose with 2-bromoethanol in the presence of acidic
ion exchange resin yielded an a-anomer compound as
the major product due to its anomeric effect.9 After glo-
bal acetylation, the a-anomer of glycosyl bromide 6 was
purified by flash silica column chromatography and
treated with cadalene 1 in the presence of Cs2CO3.13
The deacetylation of the resulting substitution products
yielded the desired a-Glc-OCH2CH2-O-cadalene, 3.
To test the in vitro cytotoxicity of cadalene and glycos-
ylated cadalene, the cell viability assay was performed.
Cadalene (1), b-Glc-O-cadalene (2), and a-Glc-
OCH2CH2-O-cadalene (3) at indicated concentrations
were treated on A549 cells (human lung carcinoma)
and HeLa cells (human cervical carcinoma). The
in vitro cell viability of individual compounds was mea-
sured after 24 h of treatment using a cell counting kit
(Dojindo Laboratories, Kumamoto, Japan), which
counts live cells with 2-(2-methoxy-4-nitrophenyl)-3-
(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium
(WST-8) and 1-methoxyphenazine methosulfate (1-
methoxy-PMS).14 The transformation of WST-8 to for-
mazan by metabolically active cells was quantified by
the ELISA plate reader (ELx800, BioTek, USA) at a
wavelength of 450 nm due to the enhanced absorbance
by formazan. As shown in Figure 2, the IC50 values of
cadalene 1, b-Glc-O-cadalene 2, and a-Glc-OCH2CH2-
O-cadalene 3 on A549 cells were 34.9, 193.7, and
129.4 lM, respectively. In the case of HeLa cells, the
IC50 values of 1, 2, and 3 were 28.3, 230.9, and
160.6 lM, respectively. As expected, we observed a sig-
nificant reduction in the cytotoxicity of the cells and
the improved solubility of glycosylated cadalene. The
cell viability data on two different cancer cell lines show
similar patterns of cytotoxicity reduction.
OH
HO
O
HO
HO
O
OH
O
O
O
OH
2
1
O
HO
HO
OH
O
O
3
Figure 1. Structures of cadalene and glycosylated derivatives: (1) 7-
hydroxy-3-methoxycadalene, (2) b-Glc-O-cadalene, and (3) a-Glc-
OCH2CH2-O-cadalene.
OAc
O
OH
O
i, ii
iii
AcO
AcO
HO
HO
OH
OH
OH
OAc
OAc
4
OH
O
O
AcO
AcO
iv, v
HO
HO
O
From the in vitro cell viability assay data, we could con-
firm that glycosylated cadalenes (2 and 3) have lower
cytotoxicity than cadalene (1). In our previous research,
we confirmed the chemopreventive efficacy of cadalene
against the NNK-induced pulmonary tumorigenesis in
mice.2b However, the antitumor activity of cadalene
was not tested. Therefore, we performed an in vivo
tumor xenograft study and tested two glycosylated
AcO
OH
NH
CCl3
O
O
5
2
Scheme 1. Synthesis of b-Glc-O-cadalene (2). Reagents: (i) acetic
anhydride, pyridine, DMAP; (ii) ethylenediamine, glacial acetic acid,
DCM, 80% from D-glucose; (iii) Cl3CCN, DBU, DCM, 72%; (iv) 1,
BF3ÆOEt2, anhyd DMF, 89%; (v) NaOMe, MeOH, quantitative yield.

H. Y. Lee et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6335–6339
6337
Figure 2. Cell viability data at various dosages of cadalene (1) and glycosylated cadalenes (2 and 3): (a) A549 human lung carcinoma cell line; (b)
HeLa human cervical carcinoma cell line.
1600
1200
800
400
0
Con
Cadalene
β-Glc-O-Cadalene
α-Glc-O-CH2CH2-O-Cadalene
cadalene as prodrugs in order to achieve better solubility
and enhanced drug delivery toward cancer cells (see
Fig. 3). Athymic (BALB/c, nu/nu, 5 weeks old) male
nude mice were obtained from SLC Inc. (Hamamatsu,
Japan). The mice were housed in autoclaved plastic fil-
ter-top cages, maintained in a laboratory animal facility
(23 2 ꢁC, 50 20% relative humidity, 12-h light/dark
cycle), and acclimatized for at least 1 week prior to the
beginning of the study. All animal experiments were per-
formed in accordance with the guidelines of Seoul Na-
tional University for the care and use of animals.
Xenografts were established from the A549 cell lines
by the subcutaneous implantation of 1 · 107 cells in ser-
um-free media in the flank of the animals.15 The tumor
volume was calculated by using the mean diameter mea-
sured with calipers using the formula [v = 0.5 · a · b2],
1
5
9
13
17
21
25
29
Time (day)
Figure 3. Xenograft data of tumor volume for 29 days.

6338
H. Y. Lee et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6335–6339
where a and b are the largest and smallest tumor diam-
eters, respectively.16 Cadalene and glycosylated cada-
lenes were administered after the tumors grew to a size
of approximately 160 mm3 (approximately 4 weeks after
implantation). The vehicle control and chemicals were
administered at 100 mg/kg via oral gavage (in a mixture
of 20:1 vegetable oil/DMSO) once every 28 days. After
the experimental period (28 days), the tumor volume
of the control increased sevenfold. Interestingly, the
treatment of cadalene 1 did not affect the tumor volume.
However, the oral treatment of both glycosylated cada-
lene derivatives (2 and 3) caused a significant reduction
in the tumor volume. As shown in Figure 3, there was a
50% reduction in the tumor size as compared to the tu-
mor volume of the control by the treatment of b-Glc-O-
cadalene (2). In the case of a-Glc-OCH2CH2-O-cadalene
(3), there was a 25% reduction in the tumor volume,
which is consistent with the in vitro cell viability study.
Therefore, we could conclude that the hydrolytic glyco-
sylation of bioactive cadalene significantly enhances the
efficacy of the anticancer agent through efficient drug
delivery and improved water solubility.
for the Seoul Science Fellowship award. H.Y. Lee, J.T.
Kwon, and M. Koh are grateful for the BK21 fellowship
award.
Supplementary data
Supplementary data, including experimental procedures
and analytical data, associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2007.08.071.
References and notes
1. (a) Hayashi, Y.; Sakurai, K.; Takahashi, T. Mokuzai
Gakkaishi 1976, 22, 202; (b) Tu, T. T. N.; Derenne, S.;
Largeau, C.; Mariotti, A.; Bocherens, H.; Pons, D. Org.
Geochem. 2000, 31, 1743; (c) Lee, S. S.; Lee, H. J.; Kang,
H. Y.; Choi, D. H. FRI J. Forest Sci. 1999, 61, 82; (d) Lee,
S. S.; Lee, H. J.; Kang, H. Y.; Choi, D. H. Mokchae
Konghak 2000, 28, 32.
2. (a) Kim, J. H.; Lee, H. J.; Yeon, S. C.; Choi, D. H.; Lee, S.
S.; Kang, J. K.; Chae, C. H.; Paik, N. W.; Lee, K. H.;
Cho, M.-H. Phytother. Res. 2004, 18, 425; (b) Kim, J. H.;
Lee, H. J.; Kim, G. S.; Choi, D. H.; Lee, S. S.; Kang, J. K.;
Chae, C.; Paik, N. W.; Cho, M.-H. Cancer Lett. 2004, 213,
139.
3. Kohno, H.; Taima, M.; Sumida, T. Cancer Lett. 2001,
174, 141.
4. For targeted delivery: Hashida, M.; Hirabayashi, H.;
Nishikawa, M.; Takakura, Y. J. Control. Release 1997, 46,
129.
5. For increasing water solubility Lowery, J. J.; Yeomans, L.;
Keyari, C. M.; Davis, P.; Porreca, F.; Knapp, B. I.;
Bidlack, J. M.; Bilsky, E. J.; Polt, R. Chem. Biol. Drug
Des. 2007, 69, 41.
6. Bernard, F-X.; Sable, S.; Cameron, B.; Provost, J.;
Desnottes, J.-F.; Crouzet, J.; Blanche, F. Antimicrob.
Agents Chemother. 1997, 41, 992.
7. Jorge, A. P.; Horst, H.; de Sousa, E.; Pizzolatti, M. G.;
Silva, F. R. M. B. Chem.-Biol. Interact. 2004, 149, 89.
8. Mishra, B.; Priyadarsini, K. I.; Kumar, M. S.; Unnikrish-
nan, M. K.; Mohan, H. Bioorg. Med. Chem. 2003, 11, 2677.
9. Park, J.; Lee, H. Y.; Cho, M.-H.; Park, S. B. Angew.
Chem., Int. Ed. 2007, 46, 2018.
10. (a) Tietze, L. F.; Herzig, T.; Fecher, A.; Haunert, F.;
Schuberth, I. Chembiochem 2001, 2, 758; (b) Wang, S.;
Liu, D.; Zhang, X.; Li, S.; Yongxu, S.; Li, J.; Zhou, Y.;
Zhang, L. Carbohydr. Res. 2007, 342, 1254; (c) Mahmood,
I.; Green, M. D. Clin. Pharmacokinet. 2005, 44, 331.
11. (a) Chiara, J. L.; Encinas, L.; Diaz, B. Tetrahedron Lett.
2005, 46, 2445; (b) Read, J. A.; Ahmed, R. A.; Morrison,
J. P.; Coleman, W. C., Jr.; Tanner, M. E. J. Am. Chem.
Soc. 2004, 126, 8878.
12. (a) Lindhorst, T. K. Essentials of Carbohydrate Chemistry
and Biochemistry; Wiley-VCH: Weinhein, Germany, 2000,
pp 50–89; (b) Uhrig, R. K.; Picard, M. A.; Beyreuther, K.;
Wiessler, M. Carbohydr. Res. 2000, 325, 72; (c) Shen, G.;
Yu, X. M.; Blagg, B. S. J. Bioorg. Med. Chem. Lett. 2004,
14, 5903; (d) Wang, J.; Li, J.; Tuttle, D.; Takemoto, J. Y.;
Chang, C.-W. T. Org. Lett. 2002, 4, 3997; (e) Tietze, L. F.;
Feuerstein, T.; Fecher, A.; Haunert, F.; Panknin, O.;
Borchers, U.; Schuberth, I.; Alves, F. Angew. Chem., Int.
Ed. 2002, 41, 759; (f) Jeselnik, M.; Plavec, J.; Polanc, S.;
Kocevar, M. Carbohydr. Res. 2000, 328, 591.
In summary, cadalene is a biologically active natural
product isolated from Z. serrata Makino. In our previ-
ous study, the biological activities of cadalene were con-
firmed in chemopreventive effects and antioxidative
activities in NNK-induced lung tumorigenesis in mice.
In this study, cadalene 1 was tested as an anticancer
agent and the chemical modification of cadalene was
performed in order to address the limitations of cada-
lene as a therapeutic agent due to its poor solubility.
Glycosylation is one of the general approaches for
achieving enhanced water solubility and targeted drug
delivery toward cancer cells; therefore, the transient gly-
cosylation of cadalene might facilitate its targeted deliv-
ery into the cancer cells in the form of an inactive
prodrug, which can be unmasked by the cellular enzy-
matic reaction of glycosidase or simple hydrolysis. In
this study, we synthesized two types of glycosylated
cadalene (2 and 3) with two distinct stereochemistry of
the hydrolytic linkage at anomeric positions, i.e., a-
and b-anomers. The in vitro cell viability test of glycos-
ylated cadalenes showed a significant reduction in cyto-
toxicity via two different cancer cell lines. The efficacy of
these compounds was confirmed by an in vivo xenograft
study. The oral administration of b-Glc-O-cadalene (2)
induced a 50% reduction in the tumor size. Therefore,
we concluded that the glycosylation of bioactive small
molecules with undesirable physical properties is an effi-
cient approach to enhance cell permeability, water solu-
bility, and targeted drug delivery.
Acknowledgments
This work is supported by the Korea Science and Engi-
neering Foundation (KOSEF); Molecular and Cellular
BioDiscovery Research Program of the Ministry of Sci-
ence and Technology (MOST); and MarineBio21,
Ministry of Maritime Affairs and Fisheries, Korea
(MOMAF). M.H.C. is supported by KOSEF
(M20704000010-07M0400-01010). H.Y. Lee is grateful
13. Luo, J.; Zheng, Q.-Y.; Chem, C.-F.; Huang, Z.-T.
Tetrahedron 2005, 61, 8517.

H. Y. Lee et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6335–6339
6339
14. Ishiyama, M.; Tominaga, H.; Shiga, M.; Sasamoto, K.;
Ohkura, Y.; Ueno, K. Biol. Pharm. Bull. 1996, 19, 1518.
15. Guichard, S. M.; Else, R.; Reid, E.; Zeitlin, B.; Aird, R.;
Muir, M.; Dodds, M.; Fiebig, H.; Sadler, P. J.; Jodrell, D.
I. Biochem. Pharmacol. 2006, 71, 408.
16. Putt, K. S.; Chen, G. W.; Pearson, J. M.; Sandhorst, J.
S.; Hoagland, M. S.; Kwon, J.-T.; Hwang, S-K.; Jin, H.;
Churchwell, M. I.; Cho, M.-H.; Doerge, D. R.; Helfe-
rich, W. G.; Hergenrother, P. J. Nat. Chem. Biol. 2006,
2, 543.