Synthesis and antitumor activity of new D-galactose-containing derivatives of doxorubicin

Article abstract of DOI:10.1016/S0008-6215(03)00181-2

A general scheme of synthesis of antibiotic doxorubicin derivatives is based on the 13-dimethyl ketal of 14-bromodaunorubicin (4). The interaction of 4 with melibiose (5), lactose (6), 3-methoxy-4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-4- oxybenz

Full text of DOI:10.1016/S0008-6215(03)00181-2

Carbohydrate Research 338 (2003) 1359Á1367
/
www.elsevier.com/locate/carres
Synthesis and antitumor activity of new D-galactose-containing
derivatives of doxorubicin
Eugenia N. Olsufyeva,a Anna N. Tevyashova,a Ivan D. Trestchalin,a
Maria N. Preobrazhenskaya,a David Platt,b Anatole Klyosovb,*
a Gause Institute of New Antibiotics, Russian Academy of Medical Sciences, B.Pirogovskaya 11, Moscow 119021, Russia
b Pro-Pharmaceuticals, Inc., Newton, MA 02459, USA
Received 24 September 2002; accepted 5 April 2003
Abstract
A general scheme of synthesis of antibiotic doxorubicin derivatives is based on the 13-dimethyl ketal of 14-bromodaunorubicin
(4). The interaction of 4 with melibiose (5), lactose (6), 3-methoxy-4-O-(2,3,4,6-tetra-O-acetyl-b- -galactopyranosyl)-4-oxybenzal-
dehyde (12) or 4-O-(2,3,4,6-tetra-O-acetyl-b- -galactopyranosyl)-4-oxybenzaldehyde (13) by reductive alkylation followed by
hydrolysis of the corresponding intermediate bromoketals produced 3?-N-[a- -galactopyranosyl-(106)-O-1-deoxy- -glucit-1-
yl]doxorubicin (7), 3?-N-[b- -galactopyranosyl-(104)-O-1-deoxy- -glucit-1-yl]doxorubicin (8), 3?-N-[3ƒ-methoxy-4ƒ-O-(b- -ga-
lactopyranosyl)-4ƒ-oxybenzyl]doxorubicin (16), and 3?-N-[4ƒ-O-(b- -galactopyranosyl)-4ƒ-oxybenzyl]doxorubicin (17). Cytotoxic
D
D
D
/
D
D
/
D
D
D
and antitumor activity of the synthesized drug candidates compared to the parent doxorubicin was studied using various
experimental models, in particular, on mice bearing lymphocyte leukemia P-388 at single and multiple i.v. injection regimens.
# 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Doxorubicin; Reductive alkylation with mono- and disaccharides; D-Galactose; Lactose; Meliobiose
1. Introduction
connected to the antibiotic through alkyl- or acyl-type
spacers were synthesized in connection with gene-
directed enzyme prodrug therapy (GDEPT).5,6 In these
cases, the sugar moieties serve as enzyme-specific func-
tional groups of the anthracycline substrate. When a
spacer is hydrolyzed by specific enzymes in the target
tissue, the inactive prodrug is activated into the initial
drug. The goal of our research, in contrast, was to
modify the initial drug with a stable galactose residue,
aiming at reducing the drug toxicity, increasing its
efficacy, or both. The galactose residue was chosen
because of the important role that galactose-specific
receptors, galectins, play in tumor development.7,8
Here, we report the synthesis of new N-substituted
Chemical modification of anthracycline antibiotics re-
mains important for the development of compounds
that overcome natural and induced resistance to the
existing compounds. Many researchers in the field are
currently focused on the synthesis and study of hydro-
phobic derivatives of anthracycline antibiotics, some of
which seem to be poor substrates of MDR-determining
glycoproteins.1 However, the introduction of an addi-
tional sugar residue may also lead to valuable com-
pounds as a sugar may enhance the antitumor activity of
a modified antibiotic through interaction with specific
receptors in tumor cells.2Á4 In recent years, a series of
anthracycline derivatives containing sugar moieties
derivatives of doxorubicin containing
D-galactose with
hydrophilic (1-deoxyglucit-1-yl) or hydrophobic (benzyl
or 3-methoxybenzyl) spacers, and test their cytotoxic
and antitumor activity as compared to the parent
doxorubicin. Up to now no methods for the conjugation
of the 3-amino group of daunosamine with aldoses have
been published.
* Corresponding author. Tel.: ꢀ
/
1-617-5990033; fax: ꢀ1-
/
617-9283450.
E-mail address: klyosov@pro-pharmaceuticals.com (A.
Klyosov).
0008-6215/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0008-6215(03)00181-2

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/
2. Results and discussion
antibiotic through the more hydrophobic spacer, 3-
methoxy-4-O-(2,3,4,6-tetra-O-acetyl-b- -galactopyra-
nosyl)oxybenzaldehyde (12) and 4-O-(2,3,4,6-tetra-O-
acetyl-b- -galactopyranosyl)oxybenzaldehyde (13) were
obtained by the reaction of 2,3,4,6-tetra-O-acetyl-a-
galactopyranosyl bromide (11) with vanillin (9) or 4-
hydroxybenzaldehyde (10), respectively (Scheme 3).
Reductive alkylation of the 3?-amino group of 4 with
compound 12 or 13 by the use of NaBCNH3 gave the
corresponding derivatives 14 and 15 of the 13-di-
methylketal of 14-bromodaunorubicin. After deacetyla-
tion of the galactose moiety in 14 and 15 with NaOMe
in methanol followed by acid hydrolysis of the inter-
mediates 14a and 15a, the desired doxorubicin deriva-
tives 16 and 17 were obtained (Scheme 3).
D
In the first stage of our research, we studied the
possibility of 3?-N-substitution of daunorubicin (1) by
reductive alkylation of 1 using D-glucose or D-galactose
and NaBH3CN. Although the reductive N-alkylation of
anthracycline antibiotics has been widely studied neither
mono- nor disaccharides have been used in this reaction.
D
D-
By this method, 3?-(1-deoxy-
deoxy- -galactit-1-yl) derivatives of 13-dihydro-13-
(RS)-daunorubicin (2, 3) were isolated in Â20% yields
D-glucit-1-yl)- and 3?-(1-
D
/
(Scheme 1). To protect the 13-CO group of the
antibiotic from the reduction, the 13-dimethyl ketal of
14-bromodaunorubicin (4) was used as the starting
compound, obtained from daunorubicin (1) by de-
scribed methods.9,10 To introduce the
stituent, the disaccharides 6-O-a- -galactopyranosyl-
glucose (melibiose) (5) and 4-O-b- -galactopyranosyl-
-glucose (lactose) (6) were used. A modified procedure,
D-galactose sub-
3?-N-(1-Deoxy-
N-(1-deoxy-D-galact-1-yl)doxorubicin (19) (Scheme 4)
D-glucit-1-yl)doxorubicin (18) and 3?-
D
D-
D
were obtained from 4 and D-glucose or D-galactose each
in 5% yields.
D
previously described for the reductive amination of
oligosaccharides with proteins in the presence of
Thin-layer chromatography (TLC) and high-perfor-
mance liquid chromatography (HPLC) analyses showed
that compounds 7, 8, and 16 through 19 were homo-
geneous, and contain no admixed daunorubicin or
doxorubicin. Under conditions of drastic acid hydrolysis
(1 N HCl, 105 8C, 1 h) compounds 7, 8, 16, and 17
produce the aglycon adriamycinone plus galactose, as
demonstrated by TLC and paper chromatography,
using authentic compounds as standards. Compounds
2 and 3 under similar conditions produced 13-(R,S)-
dihydrodaunomycinone, and compounds 18 and 19
gave adriamycinone. NMR investigations permitted
identification of all signals in the agycon, spacers, and
carbohydrate moieties (Table 1), and mass-spectral data
showed the correct molecular weights.
NaCNBH3, was used.11 3?N-[a-
D
-Galactopyranosyl-
-glucit-1-yl]doxorubicin (7) and 3?-
-galactopyranosyl-(104)-O-1-deoxy- -glucit-1-
(10
N-[b-
/
6)-O-1-deoxy-
D
D
/
D
yl]doxorubicin (8) were obtained in 20 and 8% yields,
respectively starting from 4 and melibiose (5) or lactose
(6) with the use of NaBCNH3 after hydrolysis of the
intermediate bromoketals 7a and 8a (Scheme 2).
D-
Galactose has the a-anomeric configuration in com-
pound 7 and the b configuration in compound 8; the
polyhydroxylated hexit-1-yl spacer in compound 8 is
shorter and more branched as compared to that in
compound 7.
In the first stage of the synthesis of the conjugates 16
and 17 of doxorubicin with D-galactose linked to the
Scheme 1.

E.N. Olsufyeva et al. / Carbohydrate Research 338 (2003) 1359Á
/1367
1361
Scheme 2.
3. Antitumor activity
centration)ꢁ
mM, whereas for doxorubicin shows IC50ꢁ
In vivo studies revealed differences in the antitumor
properties of compounds 7 and 8. The maximum
/
21 mM (L1210/0) and for 8 IC50ꢁ
/
24
/
0.213 mM.
The in vitro inhibitory effects of 7 and 8 on the
proliferation of murine leukemia L1210/0 showed that
these compounds are two orders less cytotoxic than
tolerated dosages (MTD) were 40Á
/
60 mg/kg for 7, 60Á
/
doxorubicin: for
7
IC50 (50% inhibitory con-
80 mg/kg for 8, and ꢀ60 mg/kg for both 16 and 17
/

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E.N. Olsufyeva et al. / Carbohydrate Research 338 (2003) 1359Á1367
/
Scheme 3.

E.N. Olsufyeva et al. / Carbohydrate Research 338 (2003) 1359Á
/1367
1363
toxicity and better antitumor activity as compared to the
parent doxorubicin. Furthermore, this study shows that
the a-anomeric configuration of
D-galactose rather than
b-, and/or a longer length of the spacer (six carbon
atoms rather than four in this particular case of 7 and 8)
may result in a more efficacious drug candidates in this
series.
4. Experimental
4.1. General methods
Daunorubicin was purchased from the ONOPB factory
(Omutninsk, Russia). All reagents and solvents were
purchased from Aldrich, Fluka, and Merck. The pro-
gress of reactions products, column eluates, and all final
samples were analyzed by TLC. TLC was performed on
Merck G60F254 precoated plates in the following
Scheme 4.
systems: 3:1 petroleum etherÁ
CHCl3ÁMeOHÁHCO2H (B); 130:60:10:1 CHCl3Á
MeOHÁH2OÁHCO2H (C). Reaction products were
purified by column chromatography on Merck silica
gel G60 (0.040Á0.063 mm). HPLC analyses were
performed on a Shimadzu HPLC LC 10 instrument
equipped with a Diasorb C-16 column (4.0ꢂ250 mm, 7
/
EtOAc (A); 70:10:1
(single i.v. injection, BDF1 mice (C57Blꢂ
/
DBA2, males),
/
/
/
as compared to 7Á10 mg/kg for doxorubicin. The
/
/
/
antitumor activity for these compounds was studied on
mice bearing lymphocyte leukemia P-388 at single and
/
multiple (q2dꢂ3) i.v. injection regimens (Table 2).
/
/
3.1. Single injection regimen
mc, BioChem Mack, Russia) and variable wavelength
UV detector set at 254 nm with an injection volume 10
mL. Elutions were carried out at a flow-rate of 140 mL/
When 8 was i.v. injected to mice with P-388 (BDF1 mice)
24 h post i.p. implantation of the tumor, 65% ILS at the
dose of 40 mg/kg was achieved. Compound 7 was more
active than 8: at a dose of 20 mg/kg it induced 79% ILS
and at 40 mg/kg 118% ILS (without toxic effects). A
dose of 60 mg/kg showed some toxicity. The maximal
antitumor effect of doxorubicin was 70% at the MTD
dose of 7 mg/kg. Compounds 16 and 17 at doses of 40
min with an 0.01 M 65:35 H3PO4Á/MeCN eluant
mixture at 20 8C. The sample concentration was 0.05Á
/
0.2 mg/mL. 1H NMR spectra were recorded on a Varian
VXR-400 spectrometer at 400 MHz using the DQ-
COSY method. Mass spectra were determined by
electrospray ionization (ESI) on a Finnigan MAT
900S spectrometer (Germany, Bremen). For compounds
of high molecular weights the data for the predominant
monoisotope peak were obtained. All solutions were
dried over Na2SO4 and evaporated at reduced pressure
on a Buchi rotary evaporator at a temperature below
35 8C.
and 60 mg/kg induced ILS in the range 35Á
/44% (16) and
52Á59% (17), respectively.
/
3.2. Multiple injection regimen
Multiple injections of doxorubicin revealed an increased
toxicity of the drug, apparently due to its cumulative
toxic effect. Compared to a single dose regimen, when at
the dose of 7 mg/kg there was no deaths (70% ILS),
triple injection of doxorubicin with 2-day intervals
4.2. 3?-N-(1-Deoxy- -glucit-1-yl)-13-(R,S)-
dihydrodaunorubicin (2)
D
To a stirred solution of daunorubicin hydrochloride (1,
(q2dꢂ/3) at 2.3 mg/kg each dose (6.9 mg/kg total
140 mg, 0.25 mmol) in a 1:1 mixture of DMFÁ
/H2O (6
dose) resulted in four toxic deaths out of seven animals.
However, compound 7 did not show any cumulative
toxic effect at the triple dose of 40 mg/kg (120 mg/kg
total dose), and induced ILS equal to 133%. Hence,
compound 7 may be of interest for supportive therapy.
This study shows that doxorubicin derivatives con-
taining at the nitrogen atom of the daunosamine moiety
a polyhydroxylated spacer connected in turn with the
galactose moiety may afford compounds having lower
mL) D-glucose (1.8 g, 10 mmol) was added. The mixture
was stirred at 40 8C for 20 h, and NaBH3CN (32 mg, 0.5
mmol) was then added. The resulting mixture was
stirred for 3 h, an additional amount of NaBH3CN
(32 mg, 0.5 mmol) was then added and the mixture was
stirred again at 40 8C for 24 h. The reaction mixture was
then diluted with water (50 mL), washed with 10:1
CHCl3Á/MeOH (20 mL) and extracted with n-BuOH
(5ꢂ20 mL). The butanol layers were combined, washed
/

Table 1
1H NMR spectra of compounds 2, 3, 7, 8, 16Á
/19 (d ppm)
Compounds
Chemical shifts
2
3
7
8
16 b
17 b
18
19
Anthracyclinone part
1
8.08
7.72
8.02
7.78
7.46
3.98
5.37
8.05
7.73
Nd
8.02
7.70
7.39
7.85
7.60
8.05
7.72
7.90
7.64
7.90
3.98
4.95
2.15
3.43
7.87
7.62
7.90
3.97
4.94
2.15
3.43
2
3
4-OMe
7.40
3.98
7.85
3.97
7.40
3.93
7
5.42
5.45
2.84; 2.54
3.58; 3.47
5.40
2.83; 2.51
3.56; 3.43
4.93
2.18; 2.15
2.97; 2.10
5.42
2.75; 2.50
3.52; 3.38
8
10
2.99, 2.26/2.60, 2.42
3.58, 3.22/3.48, 3.22
4.10
2.86, 2.22/2.61, 2.35
3.49, 3.41/3.06, 3.06, Jgem18.2
13
14
4.15
1.58/1.56
1.65/1.63
5.45; 5.38
5.45; 5.39
4.58
5.32
4.57
4.57
Daunosamine part
1?
2?
3?
4?
5?
6?
5.84
2.68/2.63
4.25
5.82
5.81
5.78
2.43
3.74
4.13
4.64
1.47
5.32
5.82
2.78; 2.68
4.12
5.32
5.32
2.70/2.61
4.25
4.69
5.72; 5.62
4.36
4.54
1.95; 2.05
3.45
4.12
1.85; 1.97
3.39
3.55
1.97; 2.01
3.41
3.65
Nd a
4.74
Nd a
4.45
4.74
1.52/1.50
4.72
1.50
4.17
1.20
4.16
1.17
4.16
1.19
1.53/1.50
1.48
Polyole part
1ƒ
2ƒ
3ƒ
4ƒ
5ƒ
6ƒ
3.91/3.72
4.92
4.70
3.92/3.72, Jgem12.7
4.04/3.84
5.02
4.54
3.50
4.66
2.98; 2.93
3.86
3.65
3.03
3.87
3.63
3.10
3.85
3.40
4.50Á4.15
/
4.64Á
/
4.44
4.44
4.25Á/4.50
4.63
3.09
3.75
4.64Á/3.96
4.23
3.39
D
-Galactose part
1§
5.15
4.36
5.40
5.57
3.88
4.52
4.77
4.12
4.70
4.80
5.30
4.65
4.20
4.68
4.18
4.32
4.28
2§
4.64Á
/
3§
4.64Á/3.96
4§
5§
4.54
4.36
4.36
6§a
6§b
Spectra for compounds 2, 3, 7, 8, 16 were recorded in pyridine-d5ꢀ
/
CF3CO2D, rt; spectra for compounds 17Á
/
19 were recorded in Me2SO-d6.
a
Nd*not detected.
/
b
1H NMR parameters for spacers of compounds: 16 (3-methoxy-4-oxybenzyl part): 3.40 (2 H, CH2), 7.07 (1 H, H-2ƒ), 3.61 (3 H, OMe), 5ƒ (nd), 6ƒ (nd); 17 (4-oxybenzyl
part): 3.52 (2 H, CH2), 6.98 (1 H, H-2ƒ), 7.44 (1 H, H-3ƒ), 7.44 (1 H, H-5ƒ), 6.98 (1 H, H-6ƒ).

E.N. Olsufyeva et al. / Carbohydrate Research 338 (2003) 1359Á
/1367
1365
Table 2
Antitumor activity of compounds 7, 8, 17, and 18 [i.v. injection to mice (BDF1 C57 B1ꢂ
implantation of tumor] in comparison with doxorubicin
/DBA2, males) with P388, 24 h i.p. post
Compound
Dose (mg/kg)
Injection (i.v.) regimen
Toxic death
ILS (%) a
Control
Doxorubicin
0/10
0/6
2/6
0/6
0/6
1/6
0/6
1/6
0/6
0/6
0/6
0/6
4/7
0/10
0/10
0
70
7
14
20
40
60
40
80
40
60
40
60
2.3
20
40
single
single
single
single
single
single
single
single
single
single
single
Doxorubicin
7
79
118
7
7
8
65
8
16
35
44
52
59
16
17
17
Doxorubicin
q2ꢂ
q2ꢂ
q2ꢂ
/
3 b
3 b
3 b
7
7
/
79
133
/
a
Increase of lifespan
24 h post i.p. tumor implanting
b
with concd aq NaHCO3 (30 mL) and water (30 mL) and
evaporated. The addition of Et2O resulted in 150 mg of
crude 2 as a dark-red powder, which was put onto
filtrate evaporated. The resulting crude 13-dimethyl
ketal of 14-bromodaunorubicin (4) (Â0.75 g) was
used immediately without purification in the next stage.
/
column of silica gel and eluted with 80:20:1 CHCl3Á
/
Rf 0.43 (system B).
MeOHÁHCO2H (100 mL), 2:1 CHCl3ÁMeOH (100
/
/
mL) (to separate the byproduct 13-(R,S)-dihydrodau-
norubicin). Compound 2 was eluated with 13:6:1
4.5. 3?N-[-a-
D
-(Galactopyranosy
/
(10
/
6)-O-D-1-deoxy-
Á
D-glucit-1-yl]doxorubicin (7)
CHCl3Á
/
MeOHÁH2O. The resulting fractions contain-
/
ing compound 2 were combined, and evaporated to low
volume. Addition of i-PrOH (15 mL) gave a precipitated
solid which was filtered, washed with Et2O and dried to
give 2 as an amorphous red powder (70 mg, 21%), Rf
Crude 4 (Â1.5 g) was dissolved in MeOH (65 mL). A
/
solution of melibiose (5, 3.4 g, 10 mmol) in water (30
mL) was added and the reaction mixture was kept at
40 8C for 4 h, and then NaBH3CN (0.275 g, 4 mmol) in
MeOH (0.5 mL) was added. The mixture was stirred
overnight at 37 8C, an additional amount of NaCNBH3
(0.275 g, 4 mmol) in MeOH (0.5 mL) was added, and the
mixture was stirred for 24 h. This procedure was
repeated twice (16 mmol totally of NaCNBH3 was
used) with TLC control. The resulting conjugate 7a
had Rf 0.50 (system C), the starting 4 showed Rf 0.90.
Water (200 mL) was added to the reaction mixture and
the aqueous solution extracted with CHCl3 (3ꢂ
The organic layers were combined, extracted with aq
0.25 N HBr (2ꢂ50 mL). The dark-red residue that
formed between the layers was dissolved in 200 mL of
0.25 N 1:1 HBrÁMeOH mixture, and combined with the
0.42 (system C), mp 177Á
Calcd for C33H43NO15 MW 693.2633. Found: 694.2654
[MꢀH].
/
178 8C (dec). HR-ESIMS:
/
4.3. 3?-N-1-(Deoxy- -galactit-1-yl)-13-(R,S)-
D
dihydrodaunorubicin (3)
Compound 3 was obtained in a similar manner as 2,
starting from 1 (140 mg, 0.25 mmol) and
(940 mg, 5 mmol) in 20% yield, Rf 0.42 (system C), mp
186Á187 8C (dec), HR-ESIMS: Calcd for C33H43NO15
MW 693.2633. Found: 694.2664 [MꢀH].
D-galactose
/70 mL).
/
/
/
/
4.4. 13-Dimethyl ketal of 14-bromodaunorubicin (4)
extracts of the red compound in aq 0.25 N HBr. The
combined extracts were incubated during 6 h at 37 8C
(to hydrolyze 13-OMe-kethal groups), and after that a
solution of HCO2Na (1.5 g) in water (1 mL) was added
Daunorubicin hydrochloride (1) (0.7 g, 1.2 mmol) was
dissolved in a mixture of MeOH (5 mL), dioxane (2.5
mL), and ethyl orthoformate (2.5 mL) and then Br2
(0.06 mL) were added. The mixture was stirred for 1 h at
20 8C and afterwards dry K2CO3 (0.140 g) was added.
The inorganic residue was filtered off quickly and the
to the mixture (pH Â4) to hydrolyze the 14-Br group.
/
The mixture was kept at 37 8C for 24 h under TCL
control in the system C. The crude solution of 7 was
diluted with water to a volume of 500 mL and combined

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E.N. Olsufyeva et al. / Carbohydrate Research 338 (2003) 1359Á1367
/
with the sorbent XAD-2 swollen in water (Â
/
100 mL)
and 4-hydroxybenzaldehyde (10) in 50% yield, Rf 0.09
(system A), mp 121.5Á 1.18 (c 1,
122.0 8C, [a]2D0
MeOH).
1H NMR (Me2CO-d6), d, ppm: 9.98 (1 H, s, ꢀ
7.97 (2 H, d, H-2, H-6), 7.27 (2 H, d, H-3, H-5), 5.64 (1
H, d, J1?,2? 7.69 Hz, H-1?), 5.52 (1 H, dd, J4?,3? 3.48, J4?,5?
1.11 Hz, H-4?), 5.47 (1 H, dd, J2?,3? 10.39, J2?,1? 7.69 Hz,
H-2?), 5.32 (1 H, dd, J3?,2? 10.39, J3?,4? 3.48 Hz, H-3?),
4.55 (1 H, dt, J5?,6? 6.95, J5?,4? 1.11 Hz, H-5?), 4.21 (2 H,
and stirred at rt for 6 h until the red color of the solution
disappeared. The sorbent was filtered off and washed
with water (500 mL). The resulting compound 7 was
/
ꢃ
/
/CHO),
eluated by a mixture of 1:1:1 n-BuOHÁ
/
Me2COÁH2O,
/
evaporated to dryness, and purified by column chroma-
tography (in system C). The resulting fractions contain-
ing compound 7 were combined, and evaporated to low
volume. Addition of i-PrOH (15 mL) gave a precipitate
which was filtered off, washed with Et2O and dried in
vacuum to give 7 as amorphous dark-red powder (390
mg, 20%), Rf 0.29 (system C), HPLC Rt 8.62 min, mp
d, J6?,5? 6.39 Hz, 2 H-6?), 2.19, 2.06, 2.04, 1.98 (4ꢂ
/3H,
4ꢂs, 4ꢂCH3CO2).
/
/
121Á
/
123 8C (dec). HR-ESIMS: Calcd for C39H51NO21
MW 869.2954. Found: 870.2976 [Mꢀ
/
H].
4.9. 3?-N-[3ƒ-Methoxy-4ƒ-O-(b-
D-galactopyranosyl)-4-
oxybenzyl]doxorubicin (16)
4.6. 3?-N-b-
glucit-1-yl-doxorubicin (8)
D-Galactopyranosyl-(10
/
4)-O-1-deoxy-D-
To a stirred solution of 4 (Â
was added 3-methoxy-4-O-(2,3,4,6-tetra-O-acetyl-b-
/
0.75 g) in MeOH (30 mL)
D-
Compound 8 was obtained by a similar procedure,
starting from 1.3 g of 4 and lactose (6) in 8% yield. Rf
galactopyranosyl)hydroxybenzaldehyde (12, 1.5 g, 3.11
mmol). The mixture was stirred at 40 8C for 2 h, and
NaBH3CN (0.145 g, 2.3 mmol) was then added. The
mixture was stirred overnight at 20 8C, and then
additional of NaBH3CN (0.200 g, 3.2 mmol) was added
and the stirring was prolonged for 24 h. The resulting
conjugate 14 had Rf 0.55 (system B). The mixture was
evaporated at 35 8C under reduced pressure. The residue
0.31(system C), HPLC Rt 7.11 min, mp 155Á
(dec), HR-ESIMS: Calcd for C39H51NO21 MW
869.2954. Found: 870.2981 [MꢀH].
/
157 8C
/
4.7. 3-Methoxy-4-O-(2,3,4,6-tetra-O-acetyl-b-D-
galactopyranosyl)-4-oxybenzaldehyde (12)
To a stirred solution of tetra-O-acetyl-a-
D-galactopyr-
was dissolved in CHCl3 (70 mL), washed with H2O (2ꢂ
50 mL). The organic layer was dried and evaporated.
The resulted oil was dissolved in a 1:1:1 CHCl3Á
MeOHÁC6H6 (45 mL) mixture, evaporated and dried
/
anosyl bromide (11) (3.825 g, 9.31 mmol) in dry Me2CO
(30 mL) and dry DMF (20 mL) were added vanillin (9,
2.124 g, 13.96 mmol) and K2CO3 (2.61 g, 18.61 mmol).
The mixture was stirred at 20 8C overnight, and EtOAc
(100 mL) was then added. The organic layer was washed
/
/
in vacuo, dissolved in dry MeOH (30 mL) and a 0.1 N
solution of NaOCH3 (60 mL) was added at 0 8C. The
mixture was stirred for 1 h at 20 8C, the resulting
compound (14a) had Rf 0.05 (system B). Aqueous HBr
(0.25 N) was added to the mixture until pH 6 was
reached, and then 100 mL of aq 0.25 N HBr was added
additionally. The mixture was incubated at 37 8C over-
sequentially with 10% aq CH3CO2H (2ꢂ100 mL) and
/
H2O until pH 7 was reached. The organic phase was
dried and evaporated. The residue was purified by
column chromatography (in system A). Fractions con-
taining compound 12 were combined and evaporated at
35 8C under reduced pressure. The residue was dissolved
in petroleum ether (10 mL). On adding Et2O, the
precipitated solid was filtered off and dried to give 12
night and then a solution of HCO2Na (1 g, pH Â4) in
/
H2O (10 mL) was added. The mixture was kept for 24 h
at 37 8C, the desired compound 16 had Rf 0.48 (system
(2.42 g, 54%), Rf 0.11 (system A), mp 123.5Á
[a]2D0
3.58 (c 1, MeOH).
1H NMR (Me2CO-d6), d, ppm: 9.93 (1 H, s, ꢀ
/
124.0 8C,
C). The mixture was diluted with H2O to 500 mL and Â
/
ꢃ
/
100 mL of the sorbent XAD-2 swollen in water was
added. The mixture was stirred for 6 h until the red color
of the solution disappeared. The sorbent was filtered off
/CHO),
7.57 (1 H, dd, J6,5 8.24, J6,2 1.88 Hz, H-6), 7.53 (1 H, d,
J2,6 1.88 Hz, H-2), 7.43 (d, 1 H, J5,6 8.24 Hz, H-5), 5.49
(3 H, m, H-1?, H-2?, H-4?), 5.29 (1 H, m, H-3?), 4.48 (1
H, dt, J5?,6? 6.45, J5?,4? 1.14 Hz, H-5?), 4.22 (2 H, d, J6?,5
6.45 Hz, 2 H-6?), 3.93 (3 H; s, OCH3), 2.20, 2.06, 2.03,
and washed with water (Â/500 mL). Compound 16 was
eluted with 1:1:1 n-BuOHÁ
/
Me2COÁH2O mixture, the
/
eluate was evaporated. The dry residue was purified by
column chromatography (in system C). Fractions con-
taining 16 were combined and evaporated to a volume
1.97 (4ꢂ
/
3H, 4ꢂ
/
s, 4ꢂCH3CO2).
/
Â1 mL. Addition of 20 mL of i-PrOH gave a
precipitate, which was filtered off, washed with Et2O
and dried to give 16 (125 mg, 12%), HPLC Rt 8.62 min,
/
4.8. 4-O-(2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl)-
4-oxybenzaldehyde (13)
mp 173Á
/
174 8C (dec). HR-ESIMS: Calcd for
Compound 13 was obtained as described for 12 starting
from tetra-O-acetyl-a- -galactopyranosyl bromide (11)
D
C41H47NO18 MW 841.2793. Found: 842.2777 [Mꢀ
/H].

E.N. Olsufyeva et al. / Carbohydrate Research 338 (2003) 1359Á
/1367
1367
4.10. 3?-N-[4ƒ-O-(b-
oxybenzyl]doxorubicin (17)
D
-Galactopyranosyl)-4-
mmol) in 5% yield, Rf (system C) 0.44, HPLC Rt 5.25
min, mp 175Á176 8C (dec) HR-ESIMS: Calcd for
C33H41NO16 MW 707.2425. Found: 708.2460 [MꢀH].
/
/
Compound 17 was obtained as for 16 in 34% yield;
HPLC Rt 8.48 min, mp 170Á
Calcd for C40H45NO17 MW 841.2793. Found: 842.2777
[MꢀH]. HR-ESIMS: C40H45NO17 MW Calcd:
811.2687. Found: 812.2699 [MꢀH].
/
171 8C (dec), HR-ESIMS:
Acknowledgements
/
/
The authors are grateful to Professor Anatoly Ya.
Khorlin (Moscow, Russia) for valuable discussions,
Yury F. Oprunenko (Lomonosov Moscow State Uni-
versity, Moscow, Russia) for study of NMR spectra,
and to Professor Jan Balzarini (Rega Institute, Leuven,
Belgium) for the in vitro data of the doxorubicin
derivatives.
4.11. 3?-N-(D-1-Deoxyglucit-1-yl)doxorubicin (18)
Crude 4 (Â
solution of
/
0.72 g) was dissolved in MeOH (30 mL). A
-glucopyranose (4.95 g, 27.0 mmol) in
D
water (30 mL) was added and the mixture was kept at
40 8C for 2 h, and NaBH3CN (0.275 g, 4 mmol) in
MeOH (1 mL) was then added and the mixture was
stirred overnight at 37 8C. Next, NaCNBH3 (0.275 g, 4
mmol) in MeOH (1 mL) was added and the mixture was
stirred at the same temperature for 24 h. This procedure
was repeated twice (a total of 16 mmol of NaCNBH3
was added) using TLC monitoring in system C. Water
(100 mL) was added to the mixture and the aqueous
References
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T.; Priebe, W. Curr. Med. Chem. 2001, 8, 51Á64.
/
solution was extracted with CHCl3 (3ꢂ
organic layers were combined, extracted with aq 0.25 N
HBr (2ꢂ50 mL). The acidic aqueous extracts were
/70 mL). The
2. Mazerska, Z.; Woynarowska, B.; Stefanska, B.; Borowski,
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incubated for 6 h at 37 8C (to hydrolyze the 13-OMe-
ketal groups), and then a solution of NaHCO2 (1 g) in
(6), 345Á351.
/
3. Arcamone, F.; Animati, F; Bigioni, M; Capranico, G;
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water (1 mL) was added to the mixture (to pH Â4.5) to
/
hydrolyze the 14-Br group. The mixture was kept at
37 8C for 24 h under TCL monitoring. The crude
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col. 1999, 57, 1133Á
4. Zunino, F.; Prateshi, G.; Perego, P. Biochem. Pharmacol.
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combined with Â100 mL of the sorbent XAD-2 swollen
/
/
in water and stirred at rt for 6 h until the red color of the
solution disappeared. The sorbent was filtered off and
washed with water (500 mL). The resulting compound
/
6. Houba, P. H. J.; Boven, E.; Erkelens, C. A. M.; Leenders,
R.g.g.; Scheren, J. W.; Pinedo, H. M.; Haisma, H. J. Br. J.
18 was eluted with 1:1:1 n-BuOHÁ
/
Me2COÁH2O mix-
/
ture and the eluate was evaporated to dryness and
purified by column chromatography (system C). The
resulting fractions containing compound 18 were com-
bined, and evaporated to a low volume. Addition of i-
PrOH (15 mL) gave a precipitate, which was filtered off,
washed with Et2O, and dried to give 18 as an amorphous
dark-red powder (30 mg, 5%), Rf (system C) 0.24,
Cancer 1998, 78 (12), 1600Á1606.
/
7. Barondes, S. H.; Castronovo, V.; Cooper, D. N. W.;
Cummings, R. D.; Drickamer, K.; Feizi, T.; Gitt, M. A.;
Hirabayashi, J.; Hudges, C.; Kasai, K.-I.; Leffler, H.; Liu,
F.-T.; Lotan, R.; Mercurio, A. M.; Monsigny, M.; Pillai,
S.; Poirer, F.; Raz, A.; Rigby, P. W. J.; Rini, J. M.; Wang,
J. L. Cell 1994, 76, 597Á
8. Lotan, R.; Ito, H.; Yasui, W.; Yokozaki, H.; Lotan, D.;
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Olsufyeva, E. N. Russ. J. Org. Chem. 1979, 15, 1560Á
/598.
HPLC Rt 5.25 min, mp 154Á
Calcd for C33H41NO16 MW 707.2425. Found: 708.2456
[MꢀH].
/
155 8C (dec). HR-ESIMS:
/
/
/
1561 (Russ).
10. Olsufyeva, E. N.; Rosynov, B. V. Soviet J. Bioorg. Chem.
4.12. 3?-N-(1-Deoxy-D-galactit-1-yl)doxorubicin (19)
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11. Gray, G. R. Arch. Biochem. Biophys. 1974, 163 (1), 426Á
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/
481.
Compound 19 was obtained as for 18, starting from 4
(0.65 g, 1.1 mmol) and -galactopyranose (4.95 g, 27
/
D