Synthesis of 2′-paclitaxel methyl 2-glucopyranosyl succinate for specific targeted delivery to cancer cells

Article abstract of DOI:10.1016/j.bmcl.2006.11.008

A novel glucose-conjugated paclitaxel 5 was synthesized using succinic acid as linker between 2′-paclitaxel and methyl 2′-glucopyranose. 5 has not only improved the pharmaceutical properties of paclitaxel, such as solubility and stability, but also enhanc

Full text of DOI:10.1016/j.bmcl.2006.11.008

Bioorganic & Medicinal Chemistry Letters 17 (2007) 617–620
Synthesis of 20-paclitaxel methyl 2-glucopyranosyl succinate for
specific targeted delivery to cancer cells
Der-Zen Liu,a Supachok Sinchaikul,b,c Peddiahgari Vasu Govardhana Reddy,b
Meng-Yang Changd and Shui-Tein Chenb,e,
*
aGraduate Institute of Biomedical Materials, Taipei Medical University, Taipei 110, Taiwan
bInstitute of Biological Chemistry and Genomics Research Center, Academia Sinica, Taipei 11529, Taiwan
cALPS Biotech Co. Ltd, Taipei 11529, Taiwan
dDepartment of Applied Chemistry, National University of Kaohsiung, Kaohsiung 811, Taiwan
eInstitute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei 10617, Taiwan
Received 9 August 2006; revised 1 November 2006; accepted 2 November 2006
Available online 7 November 2006
Abstract—A novel glucose-conjugated paclitaxel 5 was synthesized using succinic acid as linker between 20-paclitaxel and methyl 20-
glucopyranose. 5 has not only improved the pharmaceutical properties of paclitaxel, such as solubility and stability, but also
enhanced the specific target delivery to MCF-7 cells without the cytotoxicity against normal cells. Therefore, the glucose conjugation
may be potentially used in the targeted delivery of other drugs into cells via glucose transporters (GLUTs) for cancer therapy.
Ó 2006 Elsevier Ltd. All rights reserved.
Glucose is an important energy source for living organ-
isms. There are three routes of glucose uptake in human.
First, glucose diffuses slowly through the cell membrane
of the human tissues by passive transport. Second, glu-
cose is absorbed into the intestinal and kidney epithelial
cells by utilizing specific sodium-glucose transporters,
such as SGLT-1 and SGLT-2.1 Third, glucose is trans-
ported across the human cells by the facilitative glucose
transporters (GLUTs) on cell membrane, by which this
route is the main glucose uptake across the cell mem-
brane.2–4 In general, cancer cells express higher levels
of GLUTs than do normal cells and these transporters
belong to a family of homologous proteins (GLUT-1–
7) that differ in their tissue distribution and affinity for
glucose. Accordingly, the purpose of this present inves-
tigation is to design a novel vehicle that improves the
physiological property of potent drug and facilitates
the delivery of drugs to specific carcinoma cells via the
GLUTs.
ity of paclitaxel seriously reduces its wider clinical appli-
cation. There is considerable difficulty in developing
paclitaxel to be widely used due to the hydrophobic mol-
ecule with low aqueous solubility. Although many at-
tempts have been employed to improve the solubility
and pharmacological properties of paclitaxel, such as
cosolvent systems, emulsification, micellization, lipo-
some formation, nanoparticle conjugate, and inclusion
in cyclodextrins,9–12 none of these paclitaxel derivatives
or prodrugs have been applied to use in the clinical test.
Among the most notable paclitaxel derivatives synthe-
sized so far are those in which the C-20 and C-70 hydrox-
yl groups of the molecule are engaged in a functional
group that collapses, upon in vivo activation, releasing
paclitaxel.13–16 Thus, the design and chemical synthesis
of novel derivatives of paclitaxel, in which the C-20
hydroxyl group is functionalized, is interested in pursu-
ing a program that synthesized glucose-conjugated pac-
litaxel 5 as a novel prodrug (Scheme 1) for enhancing the
solubility and specific targeted delivery of paclitaxel to
the cancer cells via GLUTs.
Paclitaxel is a potent anticancer drug used for the treat-
ment of breast, ovarian, and lung carcinomas.5–8 In spite
of its potent anticancer activity, the poor water solubil-
The synthesis of 5 was carried out according to Scheme
2. Briefly, 20-succinyl paclitaxel 2 was prepared by con-
jugation of paclitaxel 1 with succinic anhydride in the
presence of DIPEA and dry dichloromethane.16,17
2
Keywords: Paclitaxel; Glucose; Targeted delivery; Cancer cells.
*
was reacted with 3-O-benzyl-4,6-O-benzylidene methyl-
a-D-glucopyranoside 3 in the presence of DCC and
Corresponding author. Tel.: +886 2 2788 6230; fax: +886 2 2785
5981; e-mail: bcchen@gate.sinica.edu.tw
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.11.008

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D.-Z. Liu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 617–620
maintained the activity of paclitaxel upon the concentra-
tion used and could be used for the next conjugation
with 20-glucopyranose.
The solubility of 5 in water was determined to be
0.35 mg/mL which was 88-fold increased as compared
with paclitaxel that showed the solubility in water to
be <0.004 mg/mL and required the formulation of emul-
sions between alcohol and oils, such as Cremophor
EL.22,23 The 20-glucose conjugation of 5 could decrease
the lipophilicity of the drug and allowed to formulate
in aqueous solution. In addition, the stability of 5 in hu-
man serum (4 lg/mL serum) at 37 °C was examined in vi-
tro by incubating 5 in human serum, extracting 5 with
dichloromethane, and analyzing by RP-HPLC using
C18 column.24 Compound 5 was stable in the human
serum for 4 h with the stability up to 85% and slowly de-
creased until 96 h. The half-life of 5 in human serum was
approximately 66 h, which was longer than that of pac-
litaxel.25 Thus, the glucose conjugate could enhance
both the solubility and the stability of 5 that may be use-
ful for preclinical test. Moreover, the glucose conjugate
in 5 was not hydrolyzed along experimental time, by
which its molecular weight was confirmed by mass spec-
trometry. It indicated that the succinic acid serving as
linker was the strong linkage between glucose and 5.
Scheme 1. Structure of novel glucose-conjugated paclitaxel 5.
DMAP in dry THF to get the compound 4.18,19 Com-
pound 4 was hydrogenated with the excess of 10% Pd/
C in ethanol to obtain 5.20,21 The structure of synthe-
1
sized derivatives was determined by H NMR and 13C
NMR, and the molecular mass was determined by mass
spectrometer. In order to examine the effect of succinic
acid as linker on cell toxicity, we have examined the
inhibition of 20-succinyl paclitaxel 2 on MCF-7 cells
and the results showed concentration-dependent inhibi-
tion of MCF-7 cells with the 50% inhibition at 25 ng/
mL. Although the modification on 20-hydroxyl group
of paclitaxel with succinic acid appeared to reduce the
toxicity of paclitaxel on MCF-7 cell growth, this linker
The inhibitory activity of 5 against eight cancer cells
appeared to be less active than paclitaxel, except that
Scheme 2. Synthesis of glucose-conjugated paclitaxel 5. Reagents and conditions: (a) DIPEA/DCM, rt, 4 h, 98%; (b) THF, DMAP, DCC, 0 °C, 24 h,
27%; (c) Pd/C, H2, EtOH, rt, 4 h, 88%.

D.-Z. Liu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 617–620
619
MCF-7 cells showed the similar IC50 values (Table 1).
It was evident that 5 showed a great specific inhibition
with MCF-7 cells, while the other cells appeared to be
less active as opposite to paclitaxel. The cell morphol-
ogy of MCF-7 cells treated with 5 at IC50 of
0.0007 lM was also distinctly changed and the total
cell number was less than half that of control
(Fig. 1). On the other hand, 5 had not affected the
RPTEC normal cells, whereas paclitaxel strongly
inhibited its cell proliferation. The selectivity index
(SI) was determined by the proportion of the IC50
of cancer cells against that of RPTEC normal cells
and the SI values of 5 against MCF-7 and NPC-
TW01 cells were relatively low (0.0004 and 0.0046,
respectively) as compared to that of paclitaxel (0.56
and 0.33, respectively). Due to no toxicity of 5 on
RPTEC cells, it indicated that the conjugation of glu-
cose could reduce the toxicity of paclitaxel and
showed the safety on human epithelial cells. It also
enhanced the selectivity of glucose uptake and cyto-
toxicity for cancer cells. In addition, the glucose
uptake via GLUT in various cancer cells has been
investigated using [18F]fluorodeoxyglucose and the
uptake of [18F]glucose analogs was detected by
positron-emission tomography (PET).36,37 According
to cytotoxicity of 5 against various cells, it can also
support our ideal that glucose could mediate drugs
into cancer cells. Moreover, we have examined the
fluorescein-labeled octreotide-conjugated paclitaxel in
MCF-7 cells under fluorescence microscopy. It
indicated that the size of 5 was not too big to be
transported by GLUTs.38 Therefore, we suggest that
the glucose conjugate in 5 may be involved in the glu-
cose uptake or glucose transport via GLUTs and
leads to easy delivery of the paclitaxel to cytosol
through the cell membranes for selective cancer
therapy without toxicity on normal cells. Furthermore,
we will study the mechanism of drug delivery by glu-
cose transportation via GLUTs and the pharmacoki-
netics of 5 through the therapeutic application.
In summary, we successfully developed the drug deliv-
ery based on the glucose transportation via GLUTs
on cell membranes. The novel prodrug 5 showed the
improved pharmaceutical properties and the excellent
selective cytotoxicity against MCF-7 breast cancer
cells without toxicity on normal cells. Furthermore,
5 may be potentially used for therapeutic application
of breast cancer and the drug design based on glucose
conjugate may be useful for targeted delivery of other
drugs.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2006.11.008.
Table 1. Comparison of cytotoxicity between paclitaxel and 5 against various cells
a
Cells
Cell type
GLUTs
IC50 (lM)
Paclitaxel
5
1. A498
Renal carcinoma
Nasopharyngeal carcinoma
Colon carcinoma
GLUT-126
0.0610
0.0003
0.0006
0.0005
0.0054
0.0005
0.0005
0.0068
0.0009
0.2577
0.0072
0.0256
0.0007
0.0603
0.0141
0.0091
0.1140
1.5750
2. NPC-TW01
3. HCT-116
4. MCF-7
GLUT-1b,27
GLUT-128
Breast adenocarcinoma
Heptocellular carcinoma
Gastric carcinoma
GLUT-129, ꢀ229, ꢀ329, ꢀ529, ꢀ1230
5. Hep3B
GLUT-131
6. MKN45
7. MES-SA
8. NCI-H226
9. RPTEC
GLUTꢀ132, ꢀ432
GLUTꢀ1b,33
Uterine sarcoma
Non-small cell lung cancer (squamous carcinoma)
Renal tubular epithelial cells
GLUT-1b,34
GLUT-135
a Eight cancer cells (A498, NPC-TW01, HCT-116, MCF-7, Hep3B, MKN45, MES-SA, NCI-H226) and one normal cells (RPTEC) were examined.
The values of IC50 (50% inhibition concentration) represent the means of at least three independent experiments with SD less than 10%.
b Expected GLUT, according to references of GLUT in cancer cells.
(A)
(B)
Figure 1. Cell morphological changes of MCF-7 cells (A) and MCF-7 cells treated with 5 (0.0007 lM) (B) for 72 h.

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19. Hunt, D. K.; Seeberger, P. H. Org. Lett. 2002, 16, 2751.
References and notes
20. Dorn, I. T.; Neumaier, K. R.; Tampe, R. J. Am. Chem.
Soc. 1998, 120, 2753.
21. Lindberg, J.; Ekeroth, J.; Konradsson, P. J. Org. Chem.
2002, 67, 194.
22. Gelderblom, H.; Verweij, J.; Nooter, K.; Sparreboom, A.
Eur. J. Cancer 2001, 37, 1590.
23. Wrasidlo, W.; Gaedicke, G.; Guy, R. K.; Renaud, J.;
Pitsinos, E.; Nicolaou, K. C.; Reisfeld, R. A.; Lode, H. N.
Bioconjugate Chem. 2002, 13, 1093.
1. Kanai, Y.; Lee, W. S.; You, G.; Brown, D.; Hediger, M.
A. J. Clin. Invest. 2004, 93, 397.
2. Longo, N.; Elsas, L. J. Adv. Pediatr. 1998, 45, 293.
3. Kayano, T.; Burant, C. F.; Fukumoto, H.; Gould, G. W.;
Fan, Y. S.; Eddy, R. L.; Byers, M. G.; Shows, T. B.; Seino,
S.; Bell, G. I. J. Biol. Chem. 1990, 265, 13276.
4. Wheeler, T. J.; Hinkle, P. C. Annu. Rev. Physiol. 1985, 47,
503.
´
´
24. Coudore, F.; Authier, N.; Guillaume, D.; Beal, A.;
Duroux, E.; Fialip, J. J. Chromatogr., B 1999, 721, 317.
25. Rowinsky, E. K.; Cazenave, L. A.; Donehower, R. C. J.
Natl. Cancer Inst. 1990, 82, 1247.
5. Rowinsky, E. K.; Donehower, R. C. N. Engl. J. Med.
1995, 332, 1004.
6. Passardi, A.; Massa, I.; Zoli, W.; Gianni, L.; Milandri, C.;
Zumaglini, F.; Nanni, O.; Maltoni, R.; Frassineti, G. L.;
Amadori, D. BMC Cancer 2006, 6, 76.
7. Eisenhauer, E. A.; ten Bokkel Huinink, W. W.; Swener-
ton, K. D.; Gianni, L.; Myles, J.; van der Burg, M. E.;
Kerr, I.; Vermorken, J. B.; Buser, K.; Colombo, N. J. Clin.
Oncol. 1994, 12, 2654.
8. Akerley, W.; Herndon, J. E.; Egorin, M. J.; Lyss, A. P.;
Kindler, H. L.; Savarese, D. M.; Sherman, C. A.; Rosen,
D. M.; Hollis, D.; Ratain, M. J.; Green, M. R. Cancer
2003, 97, 2480.
9. Lee, J.; Lee, S. C.; Acharya, G.; Chang, C. J.; Park, K.
Pharm. Res. 2003, 20, 1022.
10. Dhanikula, A. B.; Panchagnula, R. Curr. Drug Deliv.
2005, 2, 75.
11. Schmitt-Sody, M.; Strieth, S.; Krasnici, S.; Sauer, B.;
Schulze, B.; Teifel, M.; Michaelis, U.; Naujoks, K.;
Dellian, M. Clin. Cancer Res. 2003, 9, 2335.
12. Liu, Y.; Chen, G. S.; Chen, Y.; Cao, D. X.; Ge, Z. Q.;
Yuan, Y. J. Bioorg. Med. Chem. 2004, 12, 5767.
13. Rodrigues, M. L.; Carter, P.; Wirth, C.; Mullins, S.; Lee,
A.; Blackburn, B. K. Chem. Biol. 1995, 2, 223.
14. Golik, J.; Wong, H. S. L.; Chen, S. H.; Doyle, T. W.;
Wright, J. J. K.; Knipe, J.; Rose, W. C.; Casazza, A. M.;
Vyas, D. M. Bioorg. Med. Chem. Lett. 1996, 6, 1837.
15. Lee, J. W.; Fuchs, P. L. Org. Lett. 1999, 1, 179.
16. Kirschberg, T. A.; VanDeusen, C. L.; Rothbard, J. B.;
Yang, M.; Wender, P. A. Org. Lett. 2003, 5, 3459.
17. Kleinert, M.; Rockendorf, N.; Lindhorst, T. K. Eur. J.
Org. Chem. 2004, 18, 3931.
26. Raval, R. R.; Lau, K. W.; Tran, M. G. B.; Sowter, H. M.;
Mandriota, S. J.; Li, J. L.; Pugh, C. W.; Maxwell, P. H.;
Harris, A. L.; Ratcliffe, P. J. Mol. Cell. Biol. 2005, 25,
5675.
27. Isa, A. Y.; Phil, M.; Ward, T. H.; West, C. M. L.; Slevin,
N. J.; Homer, J. J. Br. J. Radiol. 2006, 79, 791.
28. Funasaka, T.; Yanagawa, T.; Hogan, V.; Raz, A. FASEB
J. 2005, 19, 1422.
29. Grover-Mckay, M.; Walsh, S. A.; Seftor, E. A.; Thomas,
P. A.; Hendrix, M. J. C. Pathol. Oncol. Res. 1998, 4, 115.
30. Rogers, S.; Macheda, M. L.; Docherty, S. E.; Carty, M.
D.; Henderson, M. A.; Soeller, W. C.; Gibbs, E. M.;
James, D. E.; Best, J. D. Am. J. Physiol. Endocrinol.
Metab. 2002, 282, E733.
31. Gleadle, J. M.; Ratcliffe, P. J. Blood 1997, 89, 503.
32. Noguchi, Y.; Sato, S.; Marat, D.; Doi, C.; Yoshikawa, T.;
Saito, A.; Ito, T.; Tsuburaya, A.; Yanuma, S. Cancer Lett.
1999, 140, 69.
33. Pandit-Taskar, N. J. Nucl. Med. 2005, 46, 1842.
34. Ito, T.; Noguchi, Y.; Satoh, S.; Hayashi, H.; Inayama, Y.;
Kitamura, H. Mod. Pathol. 1998, 11, 437.
35. Jiang, Y.; Zhang, W.; Kondo, K.; Klco, J. M.; St Martin,
T. B.; Dufault, M. R.; Madden, S. L.; Kaelin, W. G.;
Nacht, M. Mol. Cancer Res. 2003, 1, 453.
36. Ilknur, A. K.; Stokkel, M. P. M.; Pauwels, E. K. J. J.
Cancer Res. Clin. Oncol. 2000, 126, 560.
37. Pauwels, E. K. J.; Sturm, E. J. C.; Bombardieri, E.;
Cleton, F. J.; Stokkel, M. P. M. J. Cancer Res. Clin.
Oncol. 2000, 126, 549.
18. Tinazli, A.; Tang, J.; Valiokas, R.; Picuric, S.; Lata, S.;
Piehler, J.; Liedberg, B.; Tampe, R. Chemistry 2005, 11,
5249.
38. Huang, C. M.; Wu, Y. T.; Chen, S. T. Chem. Biol. 2000, 7,
453.