Synthesis and biological evaluation of water soluble taxoids bearing sugar moieties

Article abstract of DOI:10.3987/COM-00-S(I)34

Synthesis and biological evaluation of a variety of water soluble taxoids with sugar moieties are described.

Full text of DOI:10.3987/COM-00-S(I)34

HETEROCYCLES, Vol. 54, No. 2, pp. 561-566, Received, 25th May, 2000
SYNTHESIS AND BIOLOGICAL EVALUATION OF
WATER SOLUBLE TAXOIDS BEARING SUGAR MOIETIES
Tadakatsu Mandai,a* Hiroshi Okumoto,a Tetsuta Oshitari,a Katsuyoshi Nakanishi,b
Katsuhiko Mikuni,b Ko-ji Hara,b Ko-zo Hara,b Wakao Iwatani,c Tetsuya Amano,c
Kosho Nakamura,c and Yoshinori Tsuchiyac
aDepartment of Chemical Technology, Kurashiki University of Science & the Arts
2640 Nishinoura, Tsurajima-cho, Kurashiki 712-8505, Japan, bBio Research Corportion of
Yokohama 13-46 Daikoku-cho, Tsurumi-ku, Yokohama 230-0053, Japan, cKaken Pharmaceutical
Co., LTD. 28-8 Honkomagome 2-chome, Bunkyo-ku, Tokyo 113-8650, Japan
Abstract- Synthesis and biological evaluation of a variety of water soluble taxoids with
sugar moieties are described.
Paclitaxel (1a) has been recognized as the most exciting anticancer agent for the treatment of ovarian and
breast cancer. However, its intrinsic poor water solubility compromises the use as a drug for clinical trials,
which stimulates intensive research activities toward the synthesis and structure-activity relationship
(SAR) studies of water soluble paclitaxel derivatives.1
Several years ago, we launched systematic SAR investigation of water soluble paclitaxel prodrugs that
exhibit superior water solubility as well as the higher level of cytotoxicity to that of 1a. On designing
such compounds that meet the above demands, sugars seemed to be the first-rate candidate as hydrophilic
functionalities to attach to the specific sites of 1a and to be expected to exert additional biological
activities of themselves. However, the conventional glycosylation protocols were not likely to be effective
because 1a might be susceptible of the core skeletal rearrangements, the oxirane cleavage, and so forth by
the inevitable use of Lewis acids. Consequently, tethering a sugar to a hydroxyl group in 1a by means of
an ester linkage seemed to be the most effective strategy.
We wish to describe here the synthesis and the biological evaluation of a new array of water soluble
taxoids (3) and (4), which feature an ester linkage to C-7 or C-10 hydroxyl group of taxoids with a variety
of w-glycosyloxycarboxylic acids (n=0-8).

O
O
O
AcO
HO
OH
7
OH
7
HO
OTES
7
O
O
10
10
10
Ph
NH
O
O
NH
O
O
O
O
Ph
O
H
Ph
O
H
HO
H
OAc
OAc
HO OBz
OAc
HO OBz
HO OBz
OH
OH
Paclitaxel (1a)
7-TES-10-Deacetyl
baccatin III (2)
Docetaxel (1b)
Glycosyl
O
O
O
Glycosyl
O
O
n
O
AcO
O
O
OH
O
O
10
10
7
7
Ph
NH
O
O
NH
O
O
O
H
Ph
O
H
Ph
O
OAc
OAc
HO OBz
HO OBz
OH
OH
3
4
In the earlier stage of our project, C-7 modified taxoids (3) bearing various types of sugars were
synthesized. Scheme 1 shows the synthetic outline of glucose-linked paclitaxel (3a). Condensation of 2’-
TES-1a with glucosyloxyacetic acid (5)2 (1.2 eq) [EDCI/DMAP, CH2Cl2, rt, 12 h] provided 6 in 99%
yield. Both the triethylsilyl and benzyl groups were successfully removed by the use of Pd black in wet
acetic acid to give 3a3 in 98% yield. Analogously, compounds (3b) (galactose), (3c) (mannose), and (3d)
(xylose) were synthesized as a diastereomeric mixture of anomers except for 3c.4
Scheme 1
OBn
O
BnO
BnO
O
OBn
O
O
BnO
AcO
O
O
BnO
BnO
AcO
OH
7
O
7
O
O
O
CO2H
BnO
10
10
5
Ph
NH
O
Ph
NH O
EDCI/DMAP/CH2Cl2
rt, 12 h, 99%
O
O
Ph
O
H
Ph
O
H
OAc
OAc
HO OBz
HO OBz
OTES
OTES
2'-TES-1a
6
OH
O
O
O
HO
HO
HO
AcO
O
O
O
H2/Pd black
10
7
Ph
Ph
NH O
wet HOAc
O
rt, 48 h, 98%
O
H
OAc
HO OBz
OH
3a
The water solubility5 of 3a-3d thus obtained ranges from 58 to 258 times of 1a as shown in Table 1.
Disappointingly their in vitro cytotoxicity (IC50)6 against P388 leukemia cells was much lower than that
of 1a.

Table 1 Water solubility and cytotoxicity of 3
3 (Glycosyl) Water solubility (µg/mL)
Cytotoxicity (IC /IC
)
50
50(1a)
Paclitaxel (1a)
3a (glucose)
0.4
1
3.3
3.8
18.3
2.7
23
68
3b (galactose)
3c (α-mannose)
3d (xylose)
103
32
Then, we turned our attention to C-10 modified taxoids (4) (n=0) whose amino group protection is
simultaneously replaced by a more hydrophilic carbamate as is seen in docetaxel (1b). The synthesis of
compound (4a) possessing a-glucose is represented in Scheme 2. Condensation of 7-TES-10-
deacetylbaccatin III (2) with a-glucosyloxyacetic acid (7) 7 (1.1 eq) [EDCI/DMAP, CH2Cl2, rt, 6 h]
proceeded in a highly regioselective manner to give 8 in 98% yield. Then, 8 was condensed with a side
chain precursor (9)8 (1.2 eq) [EDCI/DMAP, CH2Cl2, rt, 3 h] to afford 10 in 93% yield. The amino group
protection of 10 was cleaved very smoothly by the treatment with an excess of HCO2H/Et3N (5 eq/10 eq)
in the presence of Pd(OAc)2/PPh3 catalyst (2 mol%/4 mol%) (THF, rt, 15h)9 to give a free amine which,
without purification, was led to 11 in 82% after treatment with Boc2O. Finally, the all protecting groups in
11 were simultaneously removed by hydrogenation (H2/10% Pd-C/EtOH/0.5% HCl, rt, 3 days) to produce
4a10 in 71% yield.
Scheme 2
OCPh
3
OCPh
3
O
BnO
O
BnO
BnO
Ar
OCPh
BnO
O
3
O
BnO
O
BnO
O
BnO
BnO
O
O
AllocN
Ph
O
O
OTES
7
O
O
OTES
7
9
BnO
O
CO H
2
10
CO H
7
10
2
Ph
O
2
EDCI/DMAP/CH Cl
EDCI/DMAP/CH Cl
2
2
2
2
O
O
H
O
AllocN
Ar
rt, 6 h, 98%
HO
H
rt, 3 h, 93%
OAc
HO OBz
OAc
O
HO OBz
8
10
OCPh
OH
3
O
O
BnO
BnO
HO
HO
O
O
BnO
HO
O
O
1) Pd(0)/PPh
O
O
O
O
3
OTES
7
OH
O
O
H /10%Pd-C
HCOOH/Et N
2
3
10
10
7
rt, 15 h
O
NH
O
EtOH/0.5% HCl
rt, 3 days, 71%
O
NH O
2) Boc O
2
O
O
Ph
O
H
Ph
O
H
82% in 2 steps
OAc
OAc
HO OBz
HO OBz
OH
OH
11
4a

Compounds (4b) (b-glucose), (4c) (a-galactose), (4d) (b-galactose), and (4e) (a-mannose) were
synthesized by the same procedures. Both the water solubility and in vitro cytotoxicity against P388
leukemia cells of 4a-4e were listed in Table 2. The 18 to 74 fold solubility of 1b was achieved. Of
particular interest was that compounds (4a-4d) showed considerably stronger cytotoxicity than 1b.
Table 2 Water solubility and cytotoxicity of 4 (n=0)
Cytotoxicity (IC /IC
)
4 (Glycosyl)
Water solubility(µg/mL)
50
50(1b)
Docetaxel (1b)
4a (α-glucose)
4b (β-glucose)
4c (α-galactose)
4d (β-galactose)
4e (α-mannose)
14
332
253
482
301
1039
1
0.46
0.38
0.50
0.63
3.1
To investigate the effect of the tether length on the water solubility and the cytotoxicity, we prepared a
new array of taxoids (4f-4j) (n=2~4, 6, 8) by condensing w-galactosyloxy carboxylic acids (12) (n=2~4, 6,
8) followed by the same synthetic procedures of 4a as depicted in Scheme 2.
OH
HO
HO
O
HO
O
O
O
n
O
O
OH
OBn
BnO
O
NH O
O
BnO
BnO
O
Ph
O
H
OAc
O
HO OBz
OH
CO H
n
2
4f-4j
12
(n=2~4, 6, and 8)
(n=2~4, 6, and 8)
The water solubility of 4f-4j thus obtained is listed in Table 3. Interestingly, 4f (n=2), 4g (n=3), and 4h
(n=4) were found to be more soluble than 4a-4d (n=0). On the contrary to these observations, the in vitro
cytotoxicity11 of 4f-4j against B16 melanoma cells was much lower than that of 1b.

Table 3 Water solubility and cytotoxicity of 4f-4j
Water solubility(µg/mL) Cytotoxicity [IC50 (ng/mL)
]
4 (n)
<3.9
700
Docetaxel (1b)
4f (n=2)
4g (n=3)
4h (n=4)
4i (n=6)
14
712
928
525
95
700
155
360
-*
4j (n=8)
10
* Not measured.
In conclusion, we succeeded in exploiting a variety of taxoids with the superior water solubility to 1a or
1b by tethering a sugar moiety by an ester linkage at 7- or 10-position. Above all, 10-modified
compounds (4a-4d) showed the satisfactory improvement in both water solubility and cytotoxicity in
comparison with 1b. These encouraging observations have much stimulated our challenge to SAR study
of taxoids. Further investigation on their biological activity in vivo and biochemical behaviors will be
reported in due course.
ACKNOWLEDGMENTS
This work was supported by the Grant-in-Aid for Scientific Research on Priority Area No. 08245103 from
the Ministry of Education, Science, Sports and Culture, of Japanese Government.
REFERENCES AND NOTES
1. For a review: Taxol Science and Application, ed. by M. Suffness, CRC:Boca Raton, FL, 1995.
2. Compound (5) was prepared as follows:
OH
OBn
OBn
OBn
O
O
b, c
O
O
a
d
HO
HO
BnO
BnO
BnO
BnO
BnO
BnO
O
CO Et
O
CO H
2
2
HO
BnO
BnO
BnO
OMe
OMe
5
a) BnBr/NaH/DMF, rt, 12 h, 90% b) H SO /HOAc, 100 °C, 3 h, 85% c) HOCH CO Et/p-TsOH/PhH,
2
4
2
2
reflux, 3 h d) KOH, 75% in 2 steps
3. 1H NMR (500 MHz, CDCl3) d 1.14 (s, 3H, 17-CH3), 1.20 (s, 3H, CH3), 1.81 (s, 3H, CH3), 1.84 (s,
3H, CH3), 2.17 (s, 3H, CH3CO), 2.38 (s, 3H, CH3CO), 2.25-2.35 (m, 2H), 2.5-2.7 (m, 2H), 3.3-3.9
(m, 5H), 4.1-4.5 (m, 4H), 4.85 (br, 1H, H2’), 4.95 (brd, J=9.1, 1H, H5), 5.5-5.8 (m, 3H), 6.1-6.2 (m,
2H), 7.3-7.6 (m, 11H, Ar, NH), 7.6-7.7 (m, 1H, Ar), 7.7-7.8 (m, 2H, Ar), 8.1-8.2 (m, 2H, Ar).
4. Galactosyloxy-, xylosyloxy-, and mannosyloxyacetic acids were prepared, respectively, by the same
procedure depicted in reference 2. However, only the a-anomer was obtained in mannosyloxyacetic
acid synthesis.
5. The water solubility was measured as follows: After a mixture of water (0.5 mL) and taxoid (2.5
mg) being stirred at room temperature for 18 h, the solution was filtered, and the filtrate was

analyzed by HPLC (ODS column).
6. P388 murine leukemia cells (5 103 cells) were cultured in a well of a 96-well microtestplate for 24
h and further incubated in the presence of serial 2-fold dilutions of each taxoid for 48 h in
RPMI1640 medium supplemented with 10% fetal calf serum. At the termination of culture, the
number of viable cells was determined by MTT assay.10 The IC50 indicates the concentration
showing 50% of compound free control value (absorbance at 540 nm).
7. T. Mandai, H. Okumoto, T. Oshitari, K. Nakanishi, K. Mikuni, K. Hara, and K. Hara, Heterocycles,
2000, 52, 129.
8. Compound (9) was prepared according to the reported procedures.
HO
Ph
Br
N3
c, d
a
b
CO2Me
CO2Me
OH
CO2Me
OAc
CO2Me
OY
Ph
Ph
Ph
O
Y=Ac
=H
O
NH
e, f
g, h
9
CO2Me
OH
a) Sharpless AD, 70% b) 33% HBr/HOAc, rt, 4 h c) NaN /DMF, 60 °C, 20 h d) MeOH/AcCl (cat.),
Ph
3
rt, 12 h, 75% in 3 steps e) PPh /H O/THF, rt, 12 h f) ClCO CH CH=CH /Et O, NaHCO , rt, 12 h,
3
2
2
2
2
2
3
93% in 2 steps g) p-anisaldehyde dimethyl acetal/CSA, toluene, reflux, 5 h, 93% h) 6N NaOH/EtOH,
rt, 3 h, 95%
Cf: (a) Z.-M. Wang, H. C. Kolb, and K. B. Sharpless, J. Org. Chem., 1994, 59, 5104. (b) J. Deng, Y. Hamada, and T.
Shioiri, J. Am. Chem. Soc., 1995, 117, 7824. (c) I. Shiina, K. Saitoh, I. Fréchard-Ortuno, and T. Mukaiyama, Chem. Lett.,
1998, 3.
9. For a review: J. Tsuji and T. Mandai, Synthesis, 1996, 1.
10. 1H NMR spectrum (500 MHz, CDCl3/CD3OD=10/1) showed ambiguous broad peaks. However, it
was observed that the ester linkage at C-10 of 4a was gradually cleaved in whole human blood,
human serum, or rat serum to release docetaxel (1b) and a-glucosyloxyacetic acid which were
easily monitored by HPLC.
11. B16 murine melanoma cells (5 103 cells) were cultured in a well of a 96-well microtestplate for 24
h and further incubated in the presence of serial 2-fold dilutions of each taxoid for 48 h in Eagle’s
MEM medium supplemented with 10% fetal calf serum. At the termination of culture, the number
of viable cells was determined by MTT assay.12 The IC50 indicates the concentration showing 50%
of compound free control value (absorbance at 540 nm).
12. H. Tada, O. Shiho, K. Kuroshima, M. Koyama, and K. Tsukamoto, J. Immunol. Method, 1986, 93,
157.