Syntheses and biological activities of disaccharide daunorubicins

Article abstract of DOI:10.1021/jm050144u

Carbohydrate moiety is found in many anticancer nature products. To explore the carbohydrate moiety of daunorubicin in enhancing anticancer efficacy, several daunorubicin derivatives bearing disaccharide (1-8) have been synthesized. Their cytotoxicities w

Full text of DOI:10.1021/jm050144u

J. Med. Chem. 2005, 48, 5269-5278
5269
Syntheses and Biological Activities of Disaccharide Daunorubicins
Guisheng Zhang,† Lanyan Fang,‡ Lizhi Zhu,† Josephine E. Aimiuwu,‡ Jie Shen,† Hao Cheng,‡ Mark T. Muller,§
Gun Eui Lee,§ Duxin Sun,*,‡ and Peng George Wang*,†
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, Division of Pharmaceutics,
College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, and Department of Molecular Biology and
Microbiology, University of Central Florida, Orlando, Florida 32826
Received February 14, 2005
Carbohydrate moiety is found in many anticancer nature products. To explore the carbohydrate
moiety of daunorubicin in enhancing anticancer efficacy, several daunorubicin derivatives
bearing disaccharide (1-8) have been synthesized. Their cytotoxicities were tested in leukemia
K562 and colon cancer SW620 cells. Topoisomerase II (topo II) poisoning was performed with
the in vivo complex of topoisomerase bioassay. In both cell lines, compounds with various
terminal 2,6-dideoxy sugars (compounds 1, 3, 5, and 8) showed 30- to 60-fold higher anticancer
activity than compounds with 2-deoxy- or 6-deoxy sugar (compounds 6 and 7). Compounds
with an R-linkage between two sugar units (compound 3) showed 35-fold higher anticancer
activity than compounds with a â-linkage (compound 4). In addition, the anticancer activities
of these compounds correlated with their ability to target topo II mediated genomic DNA damage
in vivo. Compounds 1 and 3 with 2,6-dideoxy sugars produced more covalent topo-DNA complex
than compounds with 2-deoxy sugar (6) and 6-deoxy sugar (7). Compounds with an R-config-
uration of terminal 2,6-dideoxy sugar (compounds 1 and 3) showed higher topo II poisoning
than their counterparts with the â-configuration (compounds 2 and 4). These results indicate
that sugar moieties in daunorubicin play a significant role in its anticancer activity and topo
II inhibition. The second sugar of disaccharide daunorubicin should possess 2,6-dideoxy with
R-linkage to the first sugar to exhibit better anticancer activity.
Introduction
The anthracycline quinone antibiotics daunorubicin
(DNR) and doxorubicin (Figure 1) are potent antitumor
agents against a variety of solid tumors.1 In addition to
DNA intercalation, topoisomerase II (topo II) is consid-
ered as the primary cellular target of anthracyclines.2,3
A well-accepted mechanism of action is through interac-
tion with DNA-topo II by stabilization of a topo II-
DNA-drug ternary complex, which also triggers cell
apoptosis. It is noted that DNA binding and interaction
is necessary, but not sufficient, for topo II poisoning.4
In fact, the external (nonintercalating) sugar moieties
Figure 1.
and the cyclohexane ring A are recognized as crucial
moieties for therapeutic efficacy of anthracyclines (Fig-
ure 1). The sugar moiety of anthracyclines serves as a
minor groove binder of DNA.5,6 Indeed, modifications
on the sugar structures have led to the second genera-
tion of doxorubicin analogues as monosaccharides such
as epirubicin, valrubicin, and pirarubicin. The third
generation of anthracycline as disaccharide (MEN
10755,7-9 Figure 2) is under clinical trials. MEN 10755
possesses a 2-deoxyfucose linked to the aglycon and
daunosamine as the second sugar unit. It exhibits a
broader spectrum of antitumor activity than doxo-
rubicin. Structure-activity relationship (SAR) studies
Figure 2.
* To whom correspondence should be addressed. For D.S.: phone,
on MEN 10755 revealed the following. (a) The axial
(614) 292-4381; fax, (614) 292-7766; e-mail: sun.176@osu.edu. For
orientation R (1f4) of the second sugar daunosamine
P.G.W.: phone: (614) 292-9884; fax, (614) 688-3106; e-mail, wang.892@
is critical for the topo II poisoning ability. Indeed,
glycosides with equatorial configuration are ineffective
in topo II poisoning.10,11 (b) The presence of a 4-methoxy
group on the aglycone core dramatically decreases its
osu.edu.
† Department of Chemistry and Biochemistry, The Ohio State
University.
‡ College of Pharmacy, The Ohio State University.
§ University of Central Florida.
10.1021/jm050144u CCC: $30.25 © 2005 American Chemical Society
Published on Web 07/12/2005

5270 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16
Zhang et al.
Figure 3. Synthetic compounds.
cytotoxicity because of the reduced topo II poisoning.10,11
A crystal structure of MEN 10755 and DNA hexamer
(CGATCG) complex showed two different DNA binding
sites.12 In one binding site, the disaccharide resides in
the DNA minor groove. In the other binding site, the
second sugar protrudes from the DNA helix and is
linked to the guanine of another DNA strand through
hydrogen bonds. This peculiar behavior suggested that
the second sugar may interact with other cellular target
such as topoisomerase.12
of uncommon sugars on the second sugar moiety of
anthracycline to depict their role in anticancer activity.
In the present study, we designed novel disaccharide
daunorubicins with uncommon sugars as terminal sugar
moieties to reveal its function in anticancer activity
(Figure 3).
Results and Discussion
Chemistry. For disaccharide daunorubicins, the
optimal configuration should be an uncommon sugar
linked to the 4-position of the first sugar through R
(1f4) linkage. In our efforts to gain access to semisyn-
thetic disaccharide daunorubicins, we focused on the
glycosylation of 3′-amino protected daunorubicin (9)
with appropriate glycosyl donors. To make R-glyco-
sylation with 2,6-dideoxy sugars, we anticipate three
synthetic challenges: first, the stability of 2,6-dideoxy
sugar donors; second, R-selective formation of a glyco-
sidic bond between a ribo-configured sugar (11) and an
aglycon, and finally the known low reactivity of the axial
4-hydroxyl group in the glycosyl acceptors.22 Glycosyl
sulfide donors are believed to be the best choice for the
R-glycosylation of 2,6-dideoxy sugars in this case. Thiogly-
cosides have been introduced as glycosyl donors because
they are stable to normal protecting group manipulation
encountered in oligosaccharide synthesis.23-25 Various
activating systems can be applied, such as, MeOTf,
NBS, BSP/Tf2O/TTBP,26 IDCP, NIS/TfOH, and NIS/
TMSOTf, etc. For a 2-deoxy sugar, without the stereo-
directing ability of 2-substituents, direct and efficient
R-selective glycosylations are difficult to realize.23,27
Strategies to prepare 2-deoxyglycosides selectively in
high R- or â-anomeric forms rely heavily on indirect
sequences from glycals or latent 2-deoxysugars.28,29 As
such, these strategies require a subsequent reductive
step that would be unsuitable for our cases. Recently,
Hirama reported a direct and efficient R-selective glyco-
sylation protocol for the kedarcidin sugar and L-my-
carose using AgPF6 as a remarkable activator of 2-de-
oxythioglycosides.27 In this paper, we report a concise
promoter system AgPF6/TTBP (2,4,6-tri-tert-butylpyri-
midine) for syntheses of disaccharide daunorubicins.
The disaccharide daunorubicins were assembled using
Indeed, natural anthracycline analogues isolated from
Streptomyces sp. often contain two or more sugars
connected by axial (1f4) linkage. The anthracycline
disaccharides or oligosaccharides invariably possess
amino sugar as the first moiety attached to the agly-
cone.1 For instance, daunosaminyl daunorubicin (Figure
2), which was isolated from fermentation, contains a
disaccharide with two daunosamines R (1f4). The
structures of the terminal sugar and sugar chain length
have been shown to significantly influence DNA bind-
ing, topoisomerase poisoning, and anticancer efficacy.13
Taken together, compelling evidence has shown that
extending the monosaccharide to disaccharide with
uncommon sugars may have significant impact on
topoisomerase inhibition besides DNA binding. Surpris-
ingly, the SAR on anthracyclines has not involved a
large variation in the sugar structure. So far, only six
uncommon sugars were used in disaccharide anthra-
cycline analogues. We hypothesize that alteration of the
second sugar of anthracycline analogues with uncom-
mon sugars may provide more information for rational
design to improve the pharmacological efficacy14 and to
reduce side effects.15-17 Uncommon sugars have received
special attention because of the increased recognition
of their vital roles in the biological function of many
natural products. They add important features to the
shape and the stereoelectronic properties of a molecule
and often play an essential role in the biological activity
of many natural product drugs. Their significance has
been studied in cellular adhesion, fertilization, protein
folding, neurobiology, xenotransplantation, and target
recognition in the immune response.18-21 However, to
date, there have not been systematic studies for SAR

Disaccharide Daunorubicins
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5271
Scheme 1a
Figure 4. Cytotoxicity of compounds 1-8 on leukemia K562
cells (a) and colon cancer SW620 cells (b). The 2000-10000
cells were incubated with 0.005-5 µM tested compounds for
72 h. MTS/PMS solution was added, and cells were incubated
for another 1-4 h. Then the absorbance was recorded at
490 nM. The survival rate was expressed as a percentage of
control group.
a Reagents and conditions: (i) (CF3CO)2O/pyridine, -20 °C,
15 min; (ii) Ac2O/pyridine, room temp, 24 h; (iii) PhSH, BF3‚Et2O/
CH2Cl2, 0 °C, 2 h, (iv) K2CO3, CuSO4, TfN3 solution, then Ac2O/
pyridine.
thiophenyl 2,6-dideoxyglycosides as glycosyl donors.
This protocol overcame the low reactivity of the axial
4′-hydroxy group and produced the desired R-glycosidic
bond with good yields.
Condensation of the glycosyl acceptor 9 with donors
24-27 in the presence of TTBP and AgPF6 exclusively
formed the R-products 32, 33, 34, and 35 with yields of
81%, 40%, 28%, and 82%, respectively. Under the same
reaction conditions, however, the reactions of 9 with 22
and 23 gave anomeric mixtures (R:â ) 2:1) in yields of
83% and 60%, respectively (Scheme 2). Mild deprotec-
tion of 28-35 with 0.1 M NaOH in THF afforded the
final products 1, 2, 3, 4, 5, 6, 7, and 8 in good yields
(88%, 81%, 76%, 75%, 85%, 62%, 67%, and 66%,
respectively).
Biology. Cytotoxicity. The cytotoxicity of these
compounds was examined against leukemia cell line
K562 cells (Figure 4a) and colon cancer cell line SW620
cells (Figure 4b) with MTS assay as described.37-39 The
2000-10000 cells were incubated with 0.001-5 µM
DNR and its derivatives for 72 h. Then 20 µL of MTS/
PMS assay solution was added to each well and the
absorbance was recorded. The cell survival was calcu-
lated as the percentage of cell control group without
treatment. In both cell lines, compounds 1, 3, 5, and 8
with various terminal 2,6-dideoxy sugars showed very
potent cytotoxicity with IC50 of 20-60 nM. Compounds
6 (with 2-deoxy terminal sugar) and 7 (with 6-deoxy
terminal sugar), when compared to other compounds,
showed much worse activity (30- to 60-fold less) than
compounds 1, 3, and 5 (with 2,6-dideoxy terminal
sugar). This suggests that the presence of 2-OH or 6-OH
groups in a terminal sugar (compounds 6 and 7)
decreases cytotoxicity, while compound 3 with 3-OH and
compound 5 with 3-OMe in the terminal 2,6-dideoxy-
Treatment of DNR with trifluroacetic anhydride in
pyridine gave the glycosyl acceptor 9 with a yield of
95%.30 The acetyl group was used for protecting the
hydroxyl functions present in sugar molecule. Acetyl
and trifluoroacetyl groups were cleavable under 0.1 M
NaOH in THF, which allowed the acid and strong base
sensitive aglycon moiety in the anthracycline molecule
to be unaffected in the final deprotection. According to
the literature procedures, isopropyl glycosides 10-12
were obtained through acidic cleavage of the natural
products erythromycin, tylosin, and avermectin in iso-
propyl alcohol instead of water.31,32 Treatment of 10-
12 and free sugars 13 and 14 with acetyl anhydride in
dry pyridine at room temperature overnight produced
the corresponding acetates 16, 17, 18, 19, and 20 with
yields of 90%, 79%, 92%, 87%, and 89%, respectively. It
was of interest to note that in the acetylation of 11 3-OH
was intact. However, the acetylation of free mycarose
gave 1,3,4-tri-O-acetylmycarose in 83% of yield. There-
fore, it was suggested that the hindrance of the bulky
isopropyl group allowed for 3-OH survival in the acety-
lation of 11. The azido sugar 21 could be prepared
readily from 15 in an overall yield of 80% according to
C.-H. Wong’s method.33 Treatment of 16-21 with
thiophenol in the presence of boron trifluoride diethyl
etherate gave thioglycosides 22, 23, 24, 25, 26, and 27
in 90%, 96%, 96%, 85%, 90%, and 80% of yields,
respectively (Scheme 1).34-36

5272 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16
Zhang et al.
Scheme 2a
a Reagents and conditions: (i) TTBP, AgPF6/CH2Cl2, 0 °C, 2 h; (ii) 0.1 M NaOH/THF, 0 °C, 6 h.
Table 1. Anticancer Activity (IC50) of Synthesized Compounds in Two Cancer Cell Lines
compounds
1
2
3
4
5
6
7
8
DNR
IC50 in K562 (nM)
IC50 in SW620 (nM)
39.5
18.2
45.8
38.9
44.3
21.7
1531.6
>1000
21.0
11.4
1378.3
>1000
>4000
>1000
31.3
60.3
15.6
8.6
sugar showed very similar activity. Thus, 2,6-dideoxy
sugars are necessary for cytotoxicity, while modification
of other sites does not affect its activity significantly.
Very interestingly, compound 3 with an R-configuration
of the terminal 2,6-dideoxy sugar moiety showed 35-
fold higher cytotoxicity than compound 4 with the
â-configuration. The IC50 values of all these compounds
against these two cell lines are summarized in Table 1.
It was demonstrated that these compounds have similar
IC50 range and comparable efficacy in K562 and SW620
cells. Altogether, these data suggest that modification
on the terminal sugar of daunorubicin can significantly
change its cytotoxicity against cancer cells. The second
sugar of disaccharide daunorubicin should possess a
2,6-dideoxy withan R-linkage to the first sugar to exhibit
better activity.
Inhibition of Topoisomerase II. Compounds 1-8
were also tested for their ability to mediate topo II
cleavage (or covalent DNA) complexes in vivo using the
ICT (in vivo complex of topoisomerase) bioassay in HeLa
cells. Formation of the covalent topo-DNA complex is
essential for the cytotoxic action of the topoisomerase
poisons. The unique nature of topoisomerase-DNA
interaction (formation of the covalent intermediate) can
be exploited to quantify topoisomerase-mediated DNA
damage in the intact cell. In the presence of topo II
poisons, the topo-mediated DNA breaks are stabilized
and a covalent topo-DNA complex can be trapped upon

Disaccharide Daunorubicins
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5273
cer activity of these compounds correlated with their
ability to target topo II mediated genomic DNA damage
in vivo. Compounds with the R-configuration of the
terminal 2,6-dideoxy sugar showed higher topo II tar-
getability than their counterparts with the â-configu-
ration, while compounds with 6-deoxy or 2-deoxy ter-
minal sugar do not show any topo II inhibition. These
results indicate that the structures and conformations
of sugar moieties are important for their anticancer
activity and topo II targeting. The second sugar
2,6-dideoxy with R-linkage to the first sugar of dauno-
rubicin disaccharide is preferred to exhibit anticancer
activity.
Figure 5. Inhibition of topoisomerase II by compounds 1-8.
Exponentially growing HeLa cells were incubated with 50 µM
etoposide or tested compounds for 30 min at 37 °C. DNA and
denatured proteins were fractionated by CsCl gradient cen-
trifugation. The bottom fraction was collected. Each fraction
(50 µL) was slotted onto a Hybond ECL membrane, and the
blot was probed with anti-topo-II antibody. The immunoblot
signals were visualized by chemiluminescence.
Experimental Section
Chemistry. All solvents were dried with a solvent-purifica-
tion system from Innovative Technology, Inc. All reagents were
used as obtained from commercial sources. Analytical TLC was
carried out on E. Merck silica gel 60 F254 aluminum-backed
plates. The 230-400 mesh size of the same absorbent was
utilized for all chromatographic purifications. 1H and 13C NMR
spectra are recorded at the indicated field strengths. The high-
resolution mass spectra were collected at the Campus Chemi-
cal Instrumentation Center of The Ohio State University.
N-(Trifluoroacetyl)daunorubicin (9). Daunorubicin hy-
drochloride (16.0 g, 28.4 mmol) was stirred in dry pyridine
(16 mL) at -20 °C for 0.5 h. Trifluoroacetic anhydride (28 mL)
in anhydrous ether (200 mL) was added dropwise over a 1.5 h
period. After 0.5 h, water (250 mL) was added and stirring
was continued for 0.5 h. The reaction mixture was extracted
with ethyl acetate, and the extracts were washed with water.
After the mixture was dried over Na2SO4, the solvent was
filtered and concentrated and the residue was heated at reflux
with MeOH (50 mL) for 0.5 h. MeOH was removed on the
rotary evaporator, and the residue was precipitated from
chloroform-pentane to afford 9 as a deep-red solid (16.8 g,
95%). HRMS (M + Na)+ (ESI+) calcd for C29H28F3NO11Na+
646.1507, found 646.1493. 1H NMR (500 MHz, CDCl3) δ 13.98
(1H, s, OH-6), 13.25 (1H, s, OH-11), 8.02 (1H, d, J ) 7.8 Hz,
H-1), 7.68 (1H, t, J ) 8.1 Hz, H-2), 7.37 (1H, d, J ) 8.2 Hz,
H-3), 6.63 (1H, d, J ) 8.5 Hz, NHCOCF3), 5.50 (1H, d, J )
3.9 Hz, H-1′), 5.25 (1H, dd, J ) 1.9, 4.0 Hz, H-7), 4.24 (2H, m,
H-3′, H-5′), 4.06 (3H, s, OMe-4), 3.65 (1H, d, J ) 2.2 Hz, H-4′),
3.25 (1H, dd, J ) 1.9, 18.8 Hz, H-10), 2.92 (1H, d, J ) 18.8
Hz, H-10), 2.39 (3H, s, H-14), 2.29 (1H, m, Ha-8), 2.14 (1H, m,
Hb-8), 1.94 (1H, m, Ha-2′), 1.80 (1H, m, Hb-2′), 1.28 (3H, d,
J ) 6.6 Hz, H-6′).
addition of protein denaturants. DNA is then purified
(without proteases) by CsCl gradient centrifugation,
which completely dissociates noncovalent protein-DNA
interactions and allows quantitative separation and
recovery of DNA. An antibody is then used to quantify
topo II levels in the DNA peak fraction from the
gradient as shown in Figure 5. The anticancer activities
of these compounds correlated with their ability to
target topo II mediated genomic DNA damage in vivo
(Figure 5). Clearly, compound 1 produced more covalent
topo-DNA complex than compounds 2-8. Among these
compounds, compounds 1 and 3 with an R-configuration
of the terminal 2,6-dideoxy sugar showed high topo II
targetability than their counterparts with â-configura-
tion (compounds 2 and 4). By comparison of compounds
3 and 5, 3-OH in the terminal sugar seems to play a
more important role than 3-OMe in poisoning topo II.
However, compound 1 with only an equatorial 3-OMe
showed higher topo II poison than compounds 3 and 5
with both axial 3-OMe (or OH) and equatorial 3-C-Me
groups. Compounds 6 and 7 with 6-deoxy or 2-deoxy
terminal sugar did not show any topo II inhibition.
These results indicate that sugar moieties in dauno-
rubincin play a significant role in topo II inhibition, in
which 2,6-dideoxy sugars with an R-linkage are required
for maintaining anthracycline cytotoxicity against can-
cer cells.
General Procedure A for the Preparation of Isopropyl
Glycosides (16-20). A mixture of substrate (12.3 mmol),
acetyl anhydride (30 mL), and dry pyridine (36 mL) was stirred
overnight at room temperature. Then methanol (10 mL) was
added and the mixture was stirred for 0.5 h. The reaction
mixture was diluted with EtOAc (200 mL) and washed with
ice-water, saturated aqueous NaHCO3 solution, and brine.
Concentration at the rotary evaporator and chromatography
on silica gel gave the protected glycosides.
Conclusion
In summary, we have developed an effective method
to prepare disaccharide daunorubicins from DNR using
AgPF6/TTBP as a mild activating system for simple
2,6-dideoxythioglycosides. The biological activities of
these compounds in two cancer cell lines show that
compounds 1, 3, 5, and 8 with various terminal
2,6-dideoxy sugars display 30- to 60-fold better cytotox-
icity than compounds 6 and 7, which contain either a
2- or 6-deoxy sugar. Meanwhile, structural modification
on other sites in the second sugar moiety (such as the
3-position, -OH or -OMe) does not affect the activity
significantly. This suggests that 2,6-dideoxy sugars are
required for anticancer activity of daunorubicin and its
derivatives. In addition, the results show that a com-
pound bearing an R-configuration of the terminal
2,6-dideoxy sugar moiety is 35- to 50-fold more potent
than compounds with the â-configuration. The antican-
Isopropyl 4-O-Acetyl-L-oleandroside (16). Compound 16
was prepared from glycoside 10 according to general procedure
A with 90% yield (purified on a column of silica gel using
EtOAc/hexanes 1:9). HRMS (M + Na)+ (ESI+) calcd for
1
C12H22O5Na+ 269.1359, found 269.1359. H NMR (250 MHz,
CDCl3), â-anomer, δ 4.59 (1H, m), 4.49 (1H, d, J ) 8.2 Hz,
H-1), 3.93 (1H, m), 3.29 (5H, m), 2.25 (1H, m), 2.06 (3H, s),
1.59 (1H, m), 1.15 (9H, m); R-anomer, δ 4.59 (1H, m), 4.49 (1H,
d, J ) 8.2 Hz, H-1), 3.93 (1H, m), 3.29 (5H, m), 2.25 (1H, m),
2.06 (3H, s), 1.59 (1H, m), 1.15 (9H, m).
Isopropyl 4-O-Acetyl-L-mycaroside (17). Compound 17
was prepared from glycoside 11 according to general procedure
A with 79% yield (purified on a column of silica gel using
EtOAc/hexane 1:4). HRMS (M + Na)+ (ESI+) calcd for C12H22O5-
Na+ 269.1359, found 269.1362. 1H NMR (250 MHz, CDCl3),
â-anomer, δ 4.89 (1H, dd, J ) 2.2 Hz, 9.6 Hz, H-1), 4.60 (1H,

5274 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16
Zhang et al.
d, J ) 9.7 Hz, H-4), 3.96 (1H, quint, J ) 6.2 Hz), 3.81 (1H, m),
2.11 (3H, s, OAc), 1.92 (1H, m), 1.76 (1H, br, OH), 1.21 (3H, d,
J ) 6.2 Hz, H-6), 1.14 (9H, m).
gel, EtOAc/hexane 1:4) as a colorless oil (85%, R:â ) 1.5:1).
HRMS (M + Na)+ (ESI+) calcd for C18H22O7SNa+ 405.0978,
found 405.0970. 1H NMR (500 MHz, CDCl3), R-anomer, δ 7.45
(2H, m, Ar), 7.29 (3H, m, Ar), 5.47 (1H, d, J ) 5.5 Hz, H-1),
5.28 (1H, m, H-3), 4.99 (1H, m, H-4), 4.51 (1H, m, H-5), 4.30
(1H, dd, J ) 5.3 Hz, J ) 12.3 Hz, Ha-6), 4.01 (1H, dd, J )
2.2 Hz, J ) 12.3 Hz, Hb-6), 2.43 (1H. m, H-2), 2.23 (1H, m,
H-2), 2.05 (3H, s, OAc), 2.01 (3H, s, OAc), 2.00 (3H, s, OAc);
â -anomer, 7.49 (2H, m, Ar), 7.29 (3H, m, Ar), 4.98 (1H, m,
H-3), 4.96 (1H, t, J ) 9.6 Hz, H-4), 4.78 (1H, dd, J ) 1.9 Hz,
11.9 Hz, H-1), 4.22 (1H, m, Ha-6), 4.13 (1H, m, Hb-6), 3.62
(1H, m, H-5), 2.45 (1H. m, H-2), 1.84 (1H, m, H-2), 2.06 (3H,
s, OAc), 2.02 (3H, s, OAc), 1.99 (3H, s, OAc).
Thiophenyl 2,3,4-Tri-O-acetyl-L-rhamnopyranoside (26).
Compound 26 was prepared from glycoside 20 according to
general procedure B (purified on a column of silica gel, EtOAc/
hexane 1:4) as a pale-yellow solid (90%, R-anomer only). HRMS
(M + Na)+ (ESI+) calcd for C18H22O7SNa+ 405.0978, found
405.0972. 1H NMR (500 MHz, CDCl3) δ 7.44 (2H, m, Ar), 7.29
(3H, m, Ar), 5.47 (1H, dd, J ) 1.6 Hz, J ) 3.3 Hz, H-2), 5.38
(1H, d, J ) 1.4 Hz, H-1), 5.27 (1H, dd, J ) 3.4 Hz, J ) 10.1
Hz, H-3), 5.12 (1H, t, J ) 9.8 Hz, H-4), 4.34 (1H, m, H-5), 2.12
(3H, s, OAc), 2.05 (3H, s, OAc), 1.98 (3H, s, OAc), 1.22 (3H, d,
J ) 6.3 Hz, H-6).
Isopropyl 4-O-Acetyl-L-cladinoside (18). Compound 18
was prepared from glycoside 12 according to general procedure
A with 92% yield (purified on a column of silica gel using
EtOAc/hexanes 1:9). HRMS (M + Na)+ (ESI+) calcd for
1
C13H24O5Na+ 283.1516, found 283.1529. H NMR (250 MHz,
CDCl3) δ 4.73 (2H, m), 3.93 (2H, m), 3.25 (3H, s, OMe-3), 2.12
(4H, m), 1.46 (1H, m), 1.59 (12H, m).
2-Deoxy-1,3,4,6-tetra-O-acetyl-D-glucopyranose (19).
Compound 19 was prepared from 2-deoxyglucopyranose 13
according to general procedure A with 87% yield (purified on
a column of silica gel using EtOAc/hexanes 1:4).
1,2,3,4-Tetra-O-acetyl-L-rhamnopyranose (20). Com-
pound 20 was prepared from rhamnopyranose 14 according
to general procedure A with 89% yield (purified on a column
of silica gel using EtOAc/hexane 1:4).
3-Azido-1,4-di-O-acetyl-2,3,6-trideoxy-L-lyxo-hexopyra-
nose (21). Daunosamine hydrochloride 15 (184.0 mg,
1.0 mmol) was dissolved in water (3 mL) and treated with
potassium carbonate (207.0 mg, 1.5 mmol) and CuSO4‚5H2O
(1.5 mg, 0.01 mmol). To the sugar solution was added MeOH
(6 mL) and the TfN3 solution made using 2 equiv of Tf2O.33
Then more MeOH was added to obtain homogeneity. The
reaction mixture was allowed to be stirred at room tempera-
ture for 18 h, and the solvent was removed. The residue was
acetylated using Ac2O (3 mL) and pyridine (5 mL) with
catalytic DMAP. It was worked up by removal of solvent and
extraction with water from EtOAc. Column chromatography
of the organic phase on silica gel using EtOAc/hexanes (1:4)
afforded the product 21 as a yellow oil (205 mg, 80%).
General Procedure B for the Preparation of Thiophe-
nyl Glycosides (22-27). A solution of substrate (6.2 mmol)
and thiophenol (678 mg, 6.2 mmol) in toluene (10 mL) was
cooled to 0 °C (ice bath), and then boron trifluoride ethyl
etherate (678 µL) was added. The mixture was warmed to room
temperature and aged for 2 h. After the mixture was cooled
to 0 °C by an ice bath, aqueous NaOH (5%, 15 mL) was added.
The organic phase was separated, and the aqueous phase was
extracted with toluene (50 mL). The combined organic phases
were washed with saturated NaCl and concentrated under
vacuum to give the crude products.
Thiophenyl 4-O-Acetyl-L-oleandroside (22). Compound
22 was prepared from glycoside 16 according to general
procedure B (purified on a column of silica gel, EtOAc/benzene
1:19) as a pale-yellow solid (90%, R:â ) 1.7:1). HRMS (M +
Na)+ (ESI+) calcd for C15H20O4SNa+ 319.0974, found 319.0970.
1H NMR (500 MHz, CDCl3) δ 7.48 (m, Ar), 7.31 (m, Ar), 5.65
(d, J ) 5.6 Hz, H-1R), 4.71 (m), 4.29 (m), 3.65 (m), 3.41 (m),
3.39 (s, H-OMeR), 3.37 (s, H-OMeâ), 2.48(m), 2.14 (s, H-OAcR),
2.11 (s, H-OAcâ), 2.07 (m), 1.26 (d, J ) 6.2 Hz, H-6â), 1.20
(d, J ) 6.3 Hz, H-6R).
Thiophenyl 4-O-Acetyl-3-azido-2,3,6-trideoxy-L-lyxo-
hexopyranoside (27). Compound 27 was prepared from
glycoside 21 according to general procedure B (purified on a
column of silica gel, 5% Et2O in hexane) as a pale-yellow oil
(80%, R-anomer). HRMS (M + Na)+ (ESI+) calcd for C14H17N3O3-
1
SNa+ 330.0883, found 330.0885. H NMR (500 MHz, CDCl3)
δ 7.44 (2H, m, Ar), 7.29 (3H, m, Ar), 5.71 (1H, d, J ) 5.6 Hz,
H-1), 5.19 (1H, d, J ) 2.4 Hz, H-4), 4.45 (1H, q, J ) 6.5 Hz,
H-5), 3.18 (1H, m, H-3), 2.44, 2.12 (2H, m, H-2), 2.16 (3H, s,
OAc), 1.12 (3H, d, H-6).
General Procedure C for the Preparation of the
Protected Disaccharide Daunorubicins (28-35). A mix-
ture of 2,6-dideoxythioglycoside (4.8 mmol), compound 9
(7.2 mmol), TTBP (17.3 mmol), and molecular sieves (4 Å, <5
µm, freshly activated) in CH2Cl2 (50 mL) was stirred at room
temperature for 2 h under N2, then cooled to 0 °C (ice bath).
Powdered AgPF6 (17.3 mmol) was added, and the mixture was
stirred for 3 h. Subsequently, pyridine (23 mL) was added, and
the mixture was stirred for a further 0.5 h. Filtration (through
a Celite pad), concentration, and chromatographic purification
provided the products.
7-[4-O-(4-O-Acetyl-r-L-oleandrosyl)-2,3,6-trideoxy-3-tri-
fluoroacetoamido-r-L-lyxo-hexopyranosyl]daunorubici-
none (28) and 7-[4-O-(4-O-Acetyl-â-L-oleandrosyl)-
2,3,6-trideoxy-3-trifluoroacetoamido-r-L-lyxo-hexopyra-
nosyl]daunorubicinone (29). The glycoside 22 was reacted
according to general procedure C to give R-anomer 28 and
â-anomer 29 (separated on a column of silica gel, MeOH/
CH2Cl2 1:100) as red solids.
28. Yield, 55%. HRMS (M + Na)+ (ESI+) calcd for C38H42F3-
NO15Na+ 832.2398, found 832.2387. 1H NMR (500 MHz,
CDCl3) δ 13.89 (1H, s, HO-6), 13.15 (1H, s, HO-11), 8.11 (1H,
d, J ) 8.3 Hz, NHCOCF3), 7.94 (1H, d, J ) 7.7 Hz, H-1), 7.71
(1H, t, J ) 8.2 Hz, H-2), 7.34 (1H, d, J ) 8.1 Hz, H-3), 5.51
(1H, d, J ) 3.3 Hz, H-1′), 5.23 (1H, d, J ) 1.8 Hz, H-7), 4.89
(1H, br, H-1′′), 4.71 (1H, m, H-4′′), 4.35 (1H, br, OH-9), 4.21
(2H, m, H-3′, H-5′), 4.20 (3H, s, OMe-4), 3.95 (1H, m, H-5′′),
3.65 (1H, m, H-3′′), 3.53 (1H, br, H-4′), 3.36 (3H, s, OMe-3′′),
3.11 (1H, d, J ) 18.7 Hz, Ha-10), 2.85 (1H, d, J ) 18.7 Hz,
Hb-10), 2.37 (3H, s, H-14), 2.25 (2H, m, Ha-2′′, Ha-8), 2.09 (4H,
m, OAc-4′′, Hb-8), 1.83 (3H, m, Hb-2′′, H-2′), 1.27 (3H, d, J )
6.4 Hz, H-6′), 1.20 (3H, d, J ) 6.7 Hz, H-6′′). 13C NMR (125
MHz, CDCl3) δ 211.9, 186.9, 186.6, 170.1, 160.9, 156.7, 156.3,
155.7, 135.6, 135.4, 134.3, 133.9, 120.8, 119.8, 118.4, 116.9,
111.5, 111.3, 100.4, 99.3, 80.1, 76.7, 75.4, 73.3, 69.9, 69.4, 67.4,
57.1, 56.6, 45.5, 35.1, 34.1, 33.4, 30.6, 24.1, 21.0, 17.2, 17.0.
Thiophenyl 4-O-Acetyl-L-mycaroside (23). Compound 23
was prepared from glycoside 17 according to general procedure
B (purified on a column of silica gel, EtOAc/benzene 1:11) as
a pale-yellow solid (96%, R:â ) 1:3). HRMS (M + Na)+ (ESI+)
calcd for C15H20O4SNa+ 319.0974, found 319.0976. 1H NMR
(500 MHz, CDCl3) δ 7.48 (m, Ar), 7.26 (m, Ar), 5.48 (dd, J )
3.0 Hz, J ) 4.6 Hz, H-1R), 5.15 (dd, J ) 2.2 Hz, J ) 11.9 Hz,
H-1â), 4.65 (m), 3.87 (m), 2.36 (s), 2.13 (m), 1.82 (m).
Thiophenyl 4-O-Acetyl-L-cladinoside (24). Compound 24
was prepared from glycoside 18 according to general procedure
B to give 24 (purified on a column of silica gel, EtOAc/benzene
1:19) as a pale-yellow solid (96%, R:â ) 1.8:1). HRMS (M +
Na)+ (ESI+) calcd for C15H20O4SNa+ 283.1516, found 283.1529.
1H NMR (500 MHz, CDCl3) δ 7.48 (m, Ar), 7.30 (m, Ar), 5.65
(d, J ) 6.3 Hz, H-1R), 5.04 (dd, J ) 1.8 Hz, J ) 11.8 Hz, H-1â),
4.63 (m), 4.01 (m), 3.34 (s, H-OMeR), 3.24 (s, H-OMeâ), 2.48-
(d, J ) 15.0 Hz), 2.26 (dd, J ) 1.8 Hz, J ) 14.3 Hz), 2.14
(s, H-OAcR), 2.11 (s, H-OAcâ), 2.03 (m), 1.66 (m), 1.14 (m).
Thiophenyl 2-Deoxy-3,4,6-tri-O-acetyl-D-glucopyrano-
side (25). Compound 25 was prepared from glycoside 19
according to general procedure B (purified on a column of silica
29. Yield, 27%. HRMS (M + Na)+ (ESI+) calcd for C38H42F3-
NO15Na+ 832.2398, found 832.2381. 1H NMR (500 MHz,
CDCl3) δ 13.87 (1H, s, HO-6), 13.14 (1H, s, HO-11), 7.94 (1H,
d, J ) 7.6 Hz, H-1), 7.72 (1H, t, J ) 8.4 Hz, H-2), 7.33 (1H, d,

Disaccharide Daunorubicins
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5275
J ) 8.5 Hz, H-3), 6.89 (1H, d, J ) 7.6 Hz, NHCOCF3), 5.48
(1H, d, J ) 3.3 Hz, H-1′), 5.19 (1H, d, J ) 1.9 Hz, H-7), 4.58
(2H, m, H-1′′, H-4′′), 4.31 (1H, s, OH-9), 4.17 (2H, m, H-3′,
H-5′), 4.02 (3H, s, OMe-4), 3.89 (1H, br, H-4′), 3.35 (5H, m,
H-5′′, H-3′′, OMe-3′′), 3.11 (1H, d, J ) 18.7 Hz, Ha-10), 2.85
(1H, d, J ) 18.7 Hz, Hb-10), 2.37 (3H, s, H-14), 2.27 (2H, m,
Ha-2′′, Ha-8), 2.09 (4H, m, OAc-4′′, Hb-8), 1.89 (2H, m, H-2′),
1.65 (1H, m, H-2′′), 1.27 (3H, d, J ) 6.6 Hz, H-6′), 1.15 (3H, d,
J ) 6.2 Hz, H-6′′). 13C NMR (125 MHz, CDCl3) δ 211.9, 186.9,
186.5, 170.1, 160.9, 156.3, 156.2, 155.7, 135.5, 135.4, 134.3,
133.9, 120.8, 119.7, 118.4, 116.8, 111.4, 111.2, 100.2, 99.5, 77.9,
76.6, 75.0, 73.2, 70.8, 70.0, 66.8, 56.8, 56.6, 46.1, 35.5, 35.0,
33.3, 29.8, 24.8, 20.8, 17.5, 17.3.
100.5, 98.2, 77.7, 76.9, 76.7, 72.9, 69.9, 67.8, 63.9, 56.7, 49.8,
45.6, 35.6, 35.1, 33.4, 30.7, 24.8, 21.1, 20.8, 17.8, 17.1.
7-[4-O-(2-Deoxy-3,4,6-tri-O-acetyl-r-D-glucopyranosyl)-
2,3,6-trideoxy-3-trifluoroacetoamido-r-L-lyxo-hexopyra-
nosyl]daunorubicinone (33). Compound 33 was prepared
from glycoside 25 according to general procedure C (purified
on a column of silica gel, MeOH/CH2Cl2 1:75) as a red solid
(40%). HRMS (M + Na)+ (ESI+) calcd for C41H44F3NO18Na+
918.2403, found 918.2387. 1H NMR (125 MHz, CDCl3) δ 13.95
(1H, s, HO-6), 13.18 (1H, s, HO-11), 7.97 (1H, d, J ) 7.6 Hz,
H-1), 7.74 (1H, m, H-2), 7.34 (1H, d, J ) 8.5 Hz, H-3), 6.69
(1H, d, J ) 7.1 Hz, NHCOCF3), 5.56 (1H, d, J ) 3.5 Hz, H-1′),
5.28 (1H, m, H-3′′), 5.18 (1H, d, J ) 2.2 Hz, H-7), 5.04 (1H, t,
J ) 9.7 Hz, H-4′′), 4.88 (1H, d, J ) 1.6 Hz, H-1′′), 4.28-4.06
(7H, m, H-3′, H-3′′, H-5′, H-5′′, H-6′′, OH-9), 4.03 (3H, s, MeO-
4), 3.84 (1H, br, H-4′), 3.14 (1H, d, J ) 18.7 Hz, Ha-10), 2.85
(1H, d, J ) 18.7 Hz, Hb-10), 2.38 (3H, s, H-14), 2.28 (2H, m,
Ha-2′′, Ha-8), 2.06 (2H, m, Ha-2′, Hb-8), 2.05, 2.03, 2.02 (9H,
3s, OAc), 1.83 (2H, m, Hb-2′, Hb-2′′), 1.31 (3H, d, J ) 6.6 Hz,
H-6′). 13C NMR (125 MHz, CDCl3) δ 211.8, 187.0, 186.6, 170.7,
170.7, 169.7, 161.0, 156.4, 156.3, 155.7, 135.7, 135.5, 134.4,
133.8, 120.8, 119.8, 118.4, 116.7, 111.5, 111.3, 100.4, 100.3,
78.6, 76.5, 70.4, 69.3, 69.1, 68.8, 66.4, 62.4, 56.6, 46.9, 35.4,
35.1, 33.3, 29.6, 24.8, 20.9, 20.7, 20.6, 17.3.
7-[4-O-(2,3,4-Tri-O-acetyl-r-L-rhamnopyranosyl)-2,3,6-
trideoxy-3-trifluoroacetoamido-r-L-lyxo-hexopyranosyl]-
daunorubicinone (34). Compound 34 was prepared from
glycoside 26 according to general procedure C (purified on a
column of silica gel, MeOH/CH2Cl2 1:75) as a red solid (28%).
HRMS (M + Na)+ (ESI+) calcd for C41H44F3NO18Na+ 918.2403,
found 918.2411. 1H NMR (500 MHz, CDCl3) δ 13.95 (1H, s,
HO-6), 13.24 (1H, s, HO-11), 8.02 (1H, d, J ) 7.7 Hz, H-1),
7.76 (1H, t, J ) 8.3 Hz, H-2), 7.52 (1H, d, J ) 8.7 Hz,
NHCOCF3), 7.37 (1H, d, J ) 8.5 Hz, H-3), 5.53 (1H, d, J ) 3.2
Hz, H-1′), 5.36 (1H, m, H-3′′), 5.29 (1H, br, H-2′′), 5.25 (1H,
br, H-7), 5.10 (1H, t, H-4′′), 4.78 (1H, d, J ) 2.3 Hz, H-1′′),
4.31 (1H, s, OH-9), 4.25 (1H, m, H-3′), 4.23 (1H, m, H-5′), 4.05
(3H, s, MeO-4), 4.04 (1H, m, H-5′′), 3.64 (1H, br, H-4′), 3.20
(1H, d, J ) 18.8 Hz, Ha-10), 2.91 (1H, d, J ) 18.8 Hz, Hb-10),
2.38 (3H, s, H-14), 2.27 (1H, m, Ha-8), 2.16 (1H, m, Hb-8), 2.14,
2.07, 2.01 (9H, 3s, OAc), 1.87 (2H, m, H-2′), 1.33 (3H, d, J )
6.5 Hz, H-6′), 1.24 (3H, d, J ) 6.3 Hz, H-6′′). 13C NMR
(125 MHz, CDCl3) δ 211.8, 187.1, 186.7, 170.1, 169.9, 169.8,
161.1, 156.4, 156.3, 155.8, 135.7, 135.6, 134.4, 133.8, 121.0,
119.8, 118.4, 116.7, 111.6, 111.4, 100.3, 100.2, 80.9, 76.7, 70.5,
70.2, 69.5, 68.6, 68.5, 67.2, 56.7, 45.5, 35.1, 33.4, 30.6, 24.8,
20.9, 20.8, 20.7, 17.2, 17.1.
7-[4-O-(4-O-Acetyl-3-azido-r-L-lyxo-hexopyranosyl)-2,3,6-
trideoxy-3-trifluoroacetoamido-r-L-lyxo-hexopyranosyl]-
daunorubicinone (35). Compound 35 was prepared from
glycoside 27 according to general procedure C (purified on a
column of silica gel, MeOH/CH2Cl2 1:150) as a red solid (82%).
HRMS (M + Na)+ (ESI+) calcd for C37H39F3N4O14Na+ 843.2307,
found 843.2331. 1H NMR (500 MHz, CDCl3) δ 13.94 (1H, s,
HO-6), 13.21 (1H, s, HO-11), 8.60 (1H, d, J ) 4.3 Hz,
NHCOCF3), 7.99 (1H, d, J ) 7.9 Hz, H-1), 7.73 (1H, m, H-2),
7.33 (1H, m, H-3), 5.50 (1H, d, J ) 3.4 Hz, H-1′), 5.24 (1H, br,
H-7), 5.21 (1H, br, H-4′′), 4.99 (1H, br, H-1′′), 4.20 (3H, m, H-3′,
H-5′, H-5′′), 4.04 (3H, s, MeO-4), 3.92 (1H, m, H-3′′), 3.58 (1H,
br, H-4′), 3.18 (1H, d, J ) 18.8 Hz, Ha-10), 2.90 (1H, d, J )
18.8 Hz, Hb-10), 2.38 (3H, s, H-14), 2.17 (1H, m, Ha-8), 2.17
(3H, s, OAc), 2.13(2H, m, Ha-2′′, Hb-8), 2.02 (1H, m, Hb-2′′),
1.86 (1H, m, Ha-2′), 1.77 (1H, m, Hb-2′), 1.27 (3H, d, J )
6.5 Hz, H-6′), 1.14 (3H, d, J ) 6.6 Hz, H-6′′). 13C NMR
(125 MHz, CDCl3) δ 211.8, 187.1, 186.7, 170.3, 161.0, 156.3,
155.8, 150.4, 135.7, 135.5, 134.3, 133.8, 124.3, 120.9, 119.8,
118.4, 111.5, 111.4, 100.4, 100.2, 80.9, 76.6, 69.9, 69.7, 67.4,
67.3, 56.7, 53.9, 45.6, 35.1, 33.5, 30.8, 30.1, 24.8, 20.7, 17.3,
16.4.
7-[4-O-(4-O-Acetyl-r-L-mycarosyl)-2,3,6-trideoxy-3-tri-
fluoroacetoamido-r-L-lyxo-hexopyranosyl]daunorubici-
none
(30)
and
7-[4-O-(4-O-acetyl-â-L-mycarosyl)-
2,3,6-trideoxy-3-trifluoroacetoamido-r-L-lyxo-hexopyra-
nosyl]daunorubicinone (31). The glycoside 23 was reacted
according to general procedure C to give R-anomer 30 and
â-anomer 31 (separated on a column of silica gel, MeOH/
CH2Cl2 1:75) as red solids.
30. Yield, 40%. HRMS (M + Na)+ (ESI+) calcd for C38H42F3-
NO15Na+ 832.2398, found 832.2438. 1H NMR (500 MHz,
CDCl3) δ 13.87 (1H, s, HO-6), 13.83 (1H, s, HO-11), 7.99 (1H,
d, J ) 8.8 Hz, NHCOCF3), 7.87 (1H, d, J ) 7.7 Hz, H-1), 7.66
(1H, t, J ) 8.0 Hz, H-2), 7.30 (1H, d, J ) 7.6 Hz, H-3), 5.44
(1H, d, J ) 3.3 Hz, H-1′), 5.14 (1H, s, H-7), 4.97 (1H, br, J )
3.5 Hz, H-1′′), 4.65 (1H, d, J ) 9.9 Hz, H-4′′), 4.19 (4H, m,
H-3′, H-5′, H-5′′, H-4′), 3.99 (3H, s, OMe-4), 3.61 (1H, s,
OH-9), 3.09 (1H, d, J ) 18.9 Hz, Ha-10), 2.80 (1H, d, J ) 18.9
Hz, Hb-10), 2.35 (3H, s, H-14), 2.10, 1.91, (9H, m, H-8, H-2′,
H-2′′, OAc), 1.27 (3H, d, J ) 6.2 Hz, H-6′), 1.45 (6H, m, CH3-
3′′, H-6′′). 13C NMR (125 MHz, CDCl3) δ 211.7, 186.7, 186.5,
170.4, 160.9, 156.7, 156.3, 155.6, 135.6, 135.3, 134.1, 133.8,
120.7, 119.7, 118.4, 116.9, 111.5, 111.2, 100.4, 99.9, 79.7, 76.6,
70.2, 69.4, 67.2, 64.6, 56.6, 45.5, 41.28, 35.1, 33.3, 30.7, 26.0,
24.8, 20.7, 17.5, 17.1.
31. Yield, 20%. HRMS (M + Na)+ (ESI+) calcd for C38H42F3-
NO15Na+ 832.2398, found 832.2368. 1H NMR (500 MHz,
CDCl3) δ 14.06 (1H, s, HO-6), 13.34 (1H, s, HO-11), 8.01 (1H,
d, J ) 7.6 Hz, H-1), 7.77 (1H, t, J ) 8.1 Hz, H-2), 7.35 (1H, d,
J ) 8.3 Hz, H-3), 6.48 (1H, d, J ) 8.5 Hz, NHCOCF3), 5.48
(1H, br, H-1′), 4.97 (1H, d, J ) 5.5 Hz, H-1′′), 4.88 (1H, br,
H-1′′), 4.75 (1H, m, H-3′), 4.55 (1H, m, H-5′), 4.38 (1H, d, J )
10.0 Hz, H-4′′), 4.07 (3H, s, OMe-4), 3.67 (3H, m, Ha-10, H-5′′,
H-4′), 2.96 (1H, d, J ) 18.9 Hz, Hb-10), 2.55 (1H, m, Ha-8),
2.31 (3H, s, H-14), 2.1-1.8 (8H, m, H-2′′, Hb-8, H-2′, OAc),
1.29 (3H, d, J ) 6.6 Hz, H-6′), 1.05 (3H, s, CH3-3′′), 0.46 (3H,
d, J ) 6.2 Hz, H-6′′). 13C NMR (125 MHz, CDCl3) δ 211.9,
186.9, 186.5, 170.1, 160.9, 156.3, 156.2, 155.7, 135.5, 135.4,
134.3, 133.9, 120.8, 119.7, 118.4, 116.8, 111.4, 111.2, 100.2,
99.5, 77.9, 76.6, 75.0, 73.2, 70.8, 70.0, 66.8, 56.8, 56.6, 46.1,
35.5, 35.0, 33.3, 29.8, 24.8, 20.8, 17.5, 17.3.
7-[4-O-(4-O-Acetyl-r-L-cladinosyl)-2,3,6-trideoxy-3-tri-
fluoroacetoamido-r-L-lyxo-hexopyranosyl]daunorubici-
none (32). Compound 32 was prepared from glycoside 24
according to general procedure C (purified on a column of silica
gel, MeOH/CH2Cl2 1:100) as a red solid (81%). HRMS (M +
Na)+ (ESI+) calcd for C39H44F3NO15Na+ 846.2555, found
1
846.2545. H NMR (500 MHz, CDCl3) δ 13.90 (1H, s, HO-6),
13.20 (1H, s, HO-11), 7.98 (1H, d, J ) 7.7 Hz, H-1), 7.73 (2H,
m, H-2, NHCOCF3), 7.34 (1H, d, J ) 8.5 Hz, H-3), 5.48 (1H, d,
J ) 3.5 Hz, H-1′), 5.24 (1H, d, J ) 1.6 Hz, H-7), 4.92 (1H, d,
J ) 4.3 Hz, H-1′′), 4.68 (1H, d, J ) 9.7 Hz, H-4′′), 4.38 (1H, s,
OH-9), 4.26 (2H, m, H-3′, H-5′′), 4.18 (1H, m, H-5′), 4.04 (3H,
s, OMe-4), 3.61 (1H, br, H-4′), 3.30 (3H, s, OMe-3′′), 3.17 (1H,
d, J ) 18.8 Hz, Ha-10), 2.85 (1H, d, J ) 18.8 Hz, Hb-10), 2.46
(1H, m, Ha-2′′), 2.37 (3H, s, H-14), 2.25 (1H, m, Ha-8), 2.12
(4H, m, OAc-4′′, Hb-8), 1.83 (2H, m, H-2′), 1.65 (1H, m,
Hb-2′′), 1.29 (3H, d, J ) 6.6 Hz, H-6′), 1.14 (3H, s, CH3-3′′),
1.08 (3H, d, J ) 6.3 Hz, H-6′′). 13C NMR (125 MHz, CDCl3)
δ 211.9, 187.0, 186.6, 170.6, 161.0, 156.7, 156.4, 155.8, 135.6,
135.5, 134.3, 133.9, 120.9, 119.8, 118.4, 116.9, 111.5, 111.3,
General Procedure D for the Preparation of the
Deprotected Disaccharide Daunorubicins (1-8). A solu-
tion of protected disaccharide daunorubicin (100 mg) in THF
(5 mL) was cooled in an ice bath to 0 °C, and then 0.1 M NaOH
aqueous solution (70 mL), which was cooled in advance in ice

5276 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16
Zhang et al.
bath, was added. After being stirred for 6-8 h, the reaction
mixture was neutralized with 0.1 M citric acid and extracted
with CHCl3. The extractions were washed with saturated
aqueous NaHCO3 solution. Concentration and chromato-
graphic purification provided the products.
Hb-2′′, H-2′), 1.32 (3H, d, J ) 6.6 Hz, H-6′), 1.10 (3H, s,
CH3-3′′), 0.72 (3H, d, J ) 6.1 Hz, H-6′′). 13C NMR (125 MHz,
CDCl3) δ 208.3, 187.1, 186.6, 161.1, 157.3, 155.4, 135.6, 135.5,
135.0, 134.9, 121.1, 119.8, 118.5, 111.1, 111.0, 101.8, 95.5, 82.4,
76.3, 71.0, 69.9, 69.1, 66.9, 66.8, 56.7, 41.9, 37.9, 34.3, 29.5,
27.3, 25.7, 24.3, 17.5, 17.1.
7-[3-Amino-4-O-(r-L-oleandrosyl)-2,3,6-trideoxy-r-L-lyxo-
hexopyranosyl]daunorubicinone (1). Compound 1 was
prepared from protected glycoside 28 according to the general
procedure (purified on a column of silica gel, MeOH/CH2Cl2
1:25 to 1:15) as a red solids (88%). HRMS (M + Na)+ (ESI+)
7-[3-Amino-4-O-(r-L-cladinosyl)-2,3,6-trideoxy-r-L-lyxo-
hexopyranosyl]daunorubicinone (5). Compound 5 was
prepared from the protected glycoside 32 according to general
procedure D (purified on a column of silica gel, MeOH/CH2Cl2
1:30-1:15) as a deep-red solid (85%). HRMS (M + Na)+ (ESI+)
1
calcd for C34H41NO13Na+ 694.2470, found 694.2483. H NMR
1
(500 MHz, CDCl3) δ 7.94 (1H, d, J ) 7.6 Hz, H-1), 7.71 (1H, t,
J ) 8.0 Hz, H-2), 7.32 (1H, d, J ) 8.5 Hz, H-3), 5.45 (1H, d,
J ) 2.9 Hz, H-1′), 5.20 (1H, br, H-7), 4.92 (1H, d, J ) 2.5 Hz,
H-1′′), 4.05 (4H, m, H-5′, OMe-4), 3.85 (1H, m, H-5′′), 3.53 (2H,
m, H-3′, H-4′), 3.39 (3H, s, OMe-3′′), 3.12 (2H, m, H-4′′,
Ha-10), 2.95 (1H, m, H-3′′), 2.83 (1H, d, J ) 10.8 Hz, Hb-10),
2.37 (4H, m, Ha-8, H-14), 2.26 (1H, m, Ha-2′′), 2.07 (1H, m,
Hb-8), 1.74 (2H, m, H-2′), 1.53 (1H, m, Hb-2′′), 1.23 (6H, m,
H-6′, H-6′′). 13C NMR (125 MHz, CDCl3) δ 211.9, 186.8, 186.5,
160.9, 156.4, 155.7, 135.6, 135.4, 134.3, 134.2, 120.8, 119.7,
118.4, 111.3, 111.2, 101.1, 100.3, 81.9, 77.9, 75.9, 69.4, 68.8,
68.2, 56.6, 56.5, 46.7, 34.8, 34.3, 33.4, 29.7, 25.1, 24.7, 17.8,
17.6.
calcd for C35H43NO13Na+ 708.2626, found 708.2601. H NMR
(500 MHz, CDCl3) δ 7.95 (1H, d, J ) 7.8 Hz, H-1), 7.71 (1H, t,
J ) 8.2 Hz, H-2), 7.32 (1H, d, J ) 8.1 Hz, H-3), 5.43 (1H, d,
J ) 3.1 Hz, H-1′), 5.21 (1H, br, H-7), 4.79 (1H, d, J ) 4.3 Hz,
H-1′′), 4.01 (5H, m, H-5′, H-5′′, OMe-4), 3.48 (1H, br, H-4′),
3.29 (3H, s, OMe-3′′), 3.14 (1H, d, J ) 18.7 Hz, Ha-10), 2.98
(2H, m, H-3′, H-4′′), 2.84 (1H, d, J ) 18.7 Hz, Hb-10), 2.43
(1H, m, Ha-2′′), 2.36 (3H, s, H-14), 2.25 (1H, m, Ha-8), 2.05
(1H, m, Hb-8), 1.73 (2H, m, H-2′), 1.55 (1H, m, Hb-2′′), 1.21
(9H, m, H-6′, CH3-3′′, H-6′′). 13C NMR (125 MHz, CDCl3)
δ 212.0, 186.7, 186.5, 160.9, 156.4, 155.8, 135.6, 135.5, 134.4,
134.4, 120.9, 119.7, 118.4, 111.3, 111.2, 101.3, 98.1, 79.9, 77.9,
76.8, 72.8, 69.4, 68.3, 65.7, 56.6, 49.5, 46.6, 35.5, 34.8, 34.3,
33.4, 24.8, 21.5, 17.9, 17.8.
7-[3-Amino-4-O-(â-L-oleandrosyl)-2,3,6-trideoxy-r-L-lyxo-
hexopyranosyl]daunorubicinone (2). Compound 2 was
prepared from protected glycoside 29 according to general
procedure D (purified on a column of silica gel, MeOH/CH2Cl2
1:10) as a red solids (81%). HRMS (M + Na)+ (ESI+) calcd for
C34H41NO13Na+ 694.2470, found 694.2473. 1H NMR (500 MHz,
CDCl3) δ 7.94 (1H, d, J ) 7.7 Hz, H-1), 7.73 (1H, t, J ) 8.1
Hz, H-2), 7.32 (1H, d, J ) 8.3 Hz, H-3), 5.47 (1H, d, J ) 3.1
Hz, H-1′), 5.22 (1H, br, H-7), 4.64 (1H, d, J ) 8.6 Hz, H-1′′),
4.06 (4H, m, H-5′, OMe-4), 3.69 (1H, br, H-4′), 3.78 (H, m,
OMe-3′′, H-4′′), 3.17 (4H, m, H-3′′, Ha-10, H-3′, H-5′′), 2.91 (1H,
d, J ) 18.8 Hz, Hb-10), 2.39 (5H, m, Ha-8, H-14, Ha-2′′), 2.04
(1H, m, Hb-8), 1.74 (2H, m, H-2′), 1.51 (1H, m, Hb-2′′), 1.30
(6H, m, H-6′, H-6′′). 13C NMR (125 MHz, CDCl3) δ 211.9, 186.9,
186.6, 161.0, 156.4, 155.8, 135.6, 135.4, 134.5, 134.4, 120.9,
119.7, 118.4, 111.3, 111.2, 100.9, 100.6, 80.7, 77.2, 75.5, 71.5,
69.5, 67.8, 56.6, 56.2, 47.0, 35.1, 34.8, 33.3, 29.7, 24.9, 24.7,
17.8, 17.5.
7-[3-Amino-4-O-(2-deoxy-l-r-D-glucopyranosyl)-2,3,6-
trideoxy-r-L-lyxo-hexopyranosyl]daunorubicinone (6).
Compound 6 was prepared from glycoside 33 according to the
general procedure (purified on a column of silica gel, MeOH/
CH2Cl2 1:15) as a red solid (62%). HRMS (M + Na)+ (ESI+)
1
calcd for C33H39NO14Na+ 696.2263, found 696.2245. H NMR
(500 MHz, MeOH-d4) δ 7.55 (2H, br, H-1, H-2), 7.25 (1H, br,
H-3), 5.36 (1H, br, H-1′), 4.91 (1H, d, J ) 3.2 Hz, H-7), 4.84
(1H, br, H-1′′), 4.34 (1H, m, H-5′), 4.24 (1H, m, H-3′), 3.95 (1H,
br, H-4′), 3.93 (1H, m, H-5′′), 3.84 (3H, s, MeO-4), 3.81 (2H,
m, H-3′′, Ha-6′′), 3.74 (1H, m, Hb-6′′), 3.28 (1H, t, H-4′′), 2.85
(1H, m, Ha-10), 2.73 (1H, m, Hb-10), 2.36 (3H, s, H-14), 2.30
(1H, m, Ha-8), 2.23 (2H, m, Ha-2′′, Hb-8), 2.05 (1H, m,
Hb-2′′), 1.80 (1H, m, Ha-2′), 1.60 (1H, m, Hb-2′), 1.32 (3H, d,
J ) 6.5 Hz, H-6′). 13C NMR (125 MHz, MeOH-d4) δ 212.3,
186.5, 186.3, 160.7, 157.4, 155.8, 135.5, 134.4, 134.3, 134.1,
118.9, 118.5, 117.1, 110.6, 110.4, 100.4, 99.6, 75.8, 75.0, 73.5,
71.8, 70.2, 68.3, 66.7, 61.3, 55.5, 48.3, 37.4, 35.5, 31.9, 28.7,
23.3, 17.0.
7-[3-Amino-4-O-(r-L-rhamnopyranosyl)-2,3,6-trideoxy-
r-L-lyxo-hexopyranosyl]daunorubicinone (7). Compound
16 was prepared from glycoside 34 according to general
procedure D (purified on a column of silica gel, MeOH/CH2Cl2
1:6) as a red solid (67%). HRMS (M + Na)+ (ESI+) calcd for
C33H39NO14Na+ 696.2263, found 696.2265. 1H NMR (500 MHz,
MeOH-d4) δ 7.68 (2H, m, H-1, H-2), 7.39 (1H, d, J ) 7.8 Hz,
H-3), 5.37 (1H, d, J ) 2.0 Hz, H-1′), 4.94 (1H, br, H-7), 4.82
(1H, br, H-1′′), 4.28 (1H, m, H-5′), 4.23 (1H, m, H-2′′), 3.93
(3H, s, MeO-4), 3.79 (2H, m, H-3′′, H-5′′), 3.67 (1H, br, H-4′),
3.48 (1H, t, J ) 9.1 Hz, H-4′′), 3.19 (1H, m, H-3′), 2.91 (1H, d,
J ) 18.2 Hz, Ha-10), 2.77 (1H, d, J ) 18.2 Hz, Hb-10), 2.28
(1H, m, Ha-8), 2.06 (1H, m, Hb-8), 1.83 (2H, m, H-2′), 1.32
(6H, m, H-6′, H-6′′). 13C NMR (125 MHz, MeOH-d4) δ 212.3,
186.7, 186.5, 161.1, 156.0, 154.9, 135.7, 135.1, 134.6, 134.3,
120.3, 119.1, 118.9, 111.1, 110.8, 103.0, 100.5, 80.8, 76.0, 72.2,
70.9, 70.7, 70.2.3, 70.0, 67.5, 55.7, 48.4, 46.5, 35.5, 32.3, 31.9,
16.6, 16.3.
7-[3-Amino-4-O-(r-L-mycarosyl)-2,3,6-trideoxy-r-L-lyxo-
hexopyranosyl]daunorubicinone (3). Compound 3 was
prepared from protected glycoside 30 according to general
procedure D (purified on a column of silica gel, MeOH/CH2Cl2
1:25-1:10) as a deep red solid (76%). HRMS (M + Na)+ (ESI+)
1
calcd for C34H41NO13Na+ 694.2470, found 694.2454. H NMR
(500 MHz, CDCl3) δ 7.89 (1H, d, J ) 7.6 Hz, H-1), 7.71 (1H, t,
J ) 8.0 Hz, H-2), 7.30 (1H, d, J ) 8.4 Hz, H-3), 5.41 (1H, d,
J ) 3.0 Hz, H-1′), 5.13 (1H, br, H-7), 4.95 (1H, J ) 2.6 Hz,
H-1′′), 4.07 (1H, m, H-5′), 3.98 (3H, s, OMe-4), 3.87 (1H, m,
H-5′′), 3.53 (1H, br, H-4′), 3.07 (2H, m, Ha-10, H-3′), 2.94 (1H,
d, J ) 79.6 Hz, H-4′′), 2.79 (1H, d, J ) 18.7 Hz, Hb-10), 2.34
(3H, s, H-14), 2.15 (2H, m, Ha-2′′, Ha-8), 2.01 (1H, m, Hb-8),
1.81 (1H, m, Hb-2′′), 1.68 (2H, m, H-2′), 1.24 (9H, m, H-6′, H-6′′,
CH3-3′′). 13C NMR (125 MHz, CDCl3) δ 211.7, 186.6, 186.4,
160.9, 156.3, 155.6, 135.6, 135.3, 134.2, 134.1, 120.7, 119.6,
118.4, 111.2, 111.1, 100.9, 99.8, 81.6, 76.7, 76.5, 69.7, 69.5, 67.8,
66.7, 56.6, 46.5, 41.0, 34.8, 34.3, 33.2, 25.4, 24.7, 17.9, 17.7
7-[3-Amino-4-O-(â-L-mycarosyl)-2,3,6-trideoxy-r-L-lyxo-
hexopyranosyl]daunorubicinone (4). Compound 4 was
prepared from protected glycoside 31 according to general
procedure D (purified on a column of silica gel, MeOH/CH2Cl2
1:10-1:6) as a deep red solids (75%). HRMS (M + Na)+ (ESI+)
7-[3-Amino-4-O-(3-azido-2,3,6-trideoxy-r-L-lyxo-hexopy-
ranosyl)-2,3,6-trideoxy-r-L-lyxo-hexopyranosyl]daunoru-
bicinone (8). Compound 8 was prepared from glycoside 35
according to general procedure D (purified on a column of silica
gel, MeOH/CH2Cl2 1:35) as a red solid (66%). HRMS (M + Na)+
(ESI+) calcd for C33H38N4O12Na+ 705.2378, found 705.2375.
1H NMR (500 MHz, MeOH-d4) δ 7.65 (2H, m, H-1, H-2), 7.37
(1H, m, H-3), 5.34 (1H, br, H-1′), 5.04 (1H, d, J ) 2.4 Hz, H-7),
4.93 (1H, br, H-1′′), 4.24 (1H, m, H-5′), 4.08 (1H, m, H-5′′),
3.91 (3H, s, MeO-4), 3.76 (2H, m, H-3′, H-4′), 3.62 (1H, br,
H-4′′), 3.16 (1H, m, H-3′′), 2.87 (1H, d, J ) 18.3 Hz, Ha-10),
2.76 (1H, d, J ) 18.3 Hz, Hb-10), 2.33, 2.20, 2.00, 1.80 (6H, m,
1
calcd for C34H41NO13Na+ 694.2470, found 694.2462. H NMR
(500 MHz, CDCl3) δ 7.99 (1H, d, J ) 7.7 Hz, H-1), 7.77 (1H, t,
J ) 8.1 Hz, H-2), 7.37 (1H, d, J ) 8.3 Hz, H-3), 5.51 (1H, d,
J ) 3.9 Hz, H-1′), 4.98 (1H, d, J ) 7.3 Hz, H-1′′), 4.85 (1H,
J ) 3.5 Hz, H-7), 4.05 (4H, m, H-5′, OMe-4), 3.76 (1H, d, J )
2.5 Hz, J ) 18.8 Hz, Ha-10), 3.55 (1H, m, H-3′), 3.45 (1H, br,
H-4′), 2.94 (1H, m, H-5′′), 2.75 (1H, d, J ) 18.8 Hz, Hb-10),
2.69 (1H, d, J ) 9.7 Hz, H-4′′), 2.43 (1H, m, Ha-8), 2.29 (3H,
s, H-14), 2.03 (1H, m, Ha-2′′), 1.91 (1H, m, Hb-8), 1.74 (3H, m,

Disaccharide Daunorubicins
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5277
(10) Arcamone, F.; Animati, F.; Bigioni, M.; Capranico, G.; Caserini,
C.; Cipollone, A.; De Cesare, M.; Ettorre, A.; Guano, F.; Manzini,
S.; Monteagudo, E.; Pratesi, G.; Salvatore, C.; Supino, R.; Zunino,
F. Configurational requirements of the sugar moiety for the
pharmacological activity of anthracycline disaccharides. Bio-
chem. Pharmacol. 1999, 57, 1133-1139.
(11) Cipollone, A.; Berettoni, M.; Bigioni, M.; Binaschi, M.; Cermele,
C.; Monteagudo, E.; Olivieri, L.; Palomba, D.; Animati, F.; Goso,
C.; Maggi, C. A. Novel anthracycline oligosaccharides: influence
of chemical modifications of the carbohydrate moiety on biologi-
cal activity. Bioorg. Med. Chem. 2002, 10, 1459-1470.
(12) Temperini, C.; Messori, L.; Orioli, P.; Di Bugno, C.; Animati, F.;
Ughetto, G. The crystal structure of the complex between a
disaccharide anthracycline and the DNA hexamer d(CGATCG)
reveals two different binding sites involving two DNA duplexes.
Nucleic Acids Res. 2003, 31, 1464-1469.
(13) DuVernay, V. H. Molecular pharmacology of anthracycline
antitumor antibiotics. Cancer Chemother. 1981, 3, 233-271.
(14) Bos, A. M.; de Vries, E. G.; Dombernovsky, P.; Aamdal, S.; Uges,
D. R.; Schrijvers, D.; Wanders, J.; Roelvink, M. W.; Hanauske,
A. R.; Bortini, S.; Capriati, A.; Crea, A. E.; Vermorken, J. B.
Pharmacokinetics of MEN-10755, a novel anthracycline disac-
charide analogue, in two phase I studies in adults with advanced
solid tumours. Cancer Chemother. Pharmacol. 2001, 48, 361-
369.
H-8, H-2′, H-2′′), 1.28 (3H, d, J ) 6.4 Hz, H-6′), 1.21 (3H, d,
J ) 6.2 Hz, H-6′′). 13C NMR (125 MHz, MeOH-d4) δ 212.3,
186.1, 185.9, 160.9, 155.9, 154.7, 135.6, 134.6, 134.4, 134.2,
119.8, 118.9, 118.7, 110.7, 110.5, 101.0, 99.9, 81.0, 77.5, 76.0,
72.0, 69.8, 69.5, 67.7, 55.7, 55.6, 46.4, 32.8, 31.9, 29.5, 28.4,
16.5, 15.9.
Cell Culture. Cell lines K562 and SW620 were cultured
in RPMI 1640 supplemented with 10% fetal bovine serum
(FBS), 1% nonessential amino acid, penicillin (100 units/mL),
and streptomycin (100 µg/mL) in a humidified atmosphere of
5% CO2 and 95% air at 37 °C. The culture mediums were
changed every 2-3 days.
Cytotoxicity of Synthesized Compounds (Daunorubi-
cin (DNR), 1-8) (MTS Assay). Leukemia K562 cells and
colon cancer SW620 cells (2000-10000) were seeded in
96-well plates in RPMI-1640 and incubated for 24 h. The
exponentially growing cancer cells were incubated with various
concentrations of compounds for 72 h at 37 °C (5% CO2, 95%
humidity). After 72 h of incubation, tetrazolium[3-(4,5-dim-
ethythiazol-2-yl)]-5-(3-carboxymethoxyphenyl)-2-(4-sulfophe-
nyl)-2H-tetrazolium, inner salt (MTS, 2 mg/mL) and phenazine
methosulfate (PMS, 25 µM) were mixed and added directly to
the cells. After incubation for 3 h at 37 °C, the absorbance of
formazan (the metabolite of MTS by viable cells) was measured
at 490 nm. The IC50 values of the carbohydrate-drug conju-
gates for cytotoxicity were calculated by WinNonlin software
from the dose-response curves of the percentage of cell growth
vs control (no compound added).
Inhibition of Topoisomerase II with ICT Bioassay. ICT
bioassay was performed according to previously described
procedures.40-42 Briefly, HeLa cells were incubated with or
without 50 µM positive control etoposide or tested compounds
for 30 min during exponential growth at 37 °C. Following
detergent lyses with sarkosyl, DNA and denatured proteins
were fractionated by CsCl gradient centrifugation. The fraction
was collected from the bottom. The DNA and covalently bonded
proteins are associated with fractions, while free proteins and
debris are at the top of the gradient. Each fraction (50 µL)
was slotted onto a Hybond ECL membrane (Amersham Bio-
sciences), and the blot was probed with anti-topo-I antibody.
The immunoblot signals were visualized by chemiluminescence
(Amersham Biosciences).
(15) Zucchi, R.; Danesi, R. Cardiac toxicity of antineoplastic anthra-
cyclines. Curr. Med. Chem.: Anti-Cancer Agents 2003, 3, 151-
171.
(16) Pratesi, G.; De Cesare, M.; Caserini, C.; Perego, P.; Dal Bo, L.;
Polizzi, D.; Supino, R.; Bigioni, M.; Manzini, S.; Iafrate, E.;
Salvatore, C.; Casazza, A.; Arcamone, F.; Zunino, F. Improved
efficacy and enlarged spectrum of activity of a novel anthra-
cycline disaccharide analogue of doxorubicin against human
tumor xenografts. Clin. Cancer Res. 1998, 4, 2833-2839.
(17) Arcamone, F.; Animati, F.; Berettoni, M.; Bigioni, M.; Capranico,
G.; Casazza, A. M.; Caserini, C.; Cipollone, A.; De Cesare, M.;
Franciotti, M.; Lombardi, P.; Madami, A.; Manzini, S.; Mon-
teagudo, E.; Polizzi, D.; Pratesi, G.; Righetti, S. C.; Salvatore,
C.; Supino, R.; Zunino, F. Doxorubicin disaccharide analogue:
apoptosis-related improvement of efficacy in vivo. J. Natl. Cancer
Inst. 1997, 89, 1217-1223.
(18) Coderre, J. A.; Button, T. M.; Micca, P. L.; Fisher, C. D.;
Nawrocky, M. M.; Liu, H. B. Neutron capture therapy of the 9L
rat gliosarcoma using the p-boronophenylalanine-fructose com-
plex. Int. J. Radiat. Oncol., Biol., Phys. 1994, 30, 643-652.
(19) Hallis, T. M.; Liu, H.-w. Learning nature’s strategies for making
deoxy sugars: pathways, mechanisms, and combinatorial ap-
plications. Acc. Chem. Res. 1999, 32, 579-588.
(20) Kirschning, A.; Bechthold, A. F. W.; Rohr, J. Chemical and
biochemical aspects of deoxysugars and deoxysugar oligosac-
charides. Top. Curr. Chem. 1997, 188, 1-84.
(21) Trefzer, A.; Bechthold, A.; Salas, J. A. Genes and enzymes
involved in deoxysugar biosynthesis in bacteria. Nat. Prod. Rep.
1999, 16, 283-299.
(22) Thiem, J.; Klaffke, W. Facile stereospecific synthesis of deoxy-
fucosyl disaccharide units of anthracyclines. J. Org. Chem. 1989,
54, 2006-2009.
(23) Nicolaou, K. C.; Seitz, S. P.; Papahatjis, D. P. A mild and general
method for the synthesis of O-glycosides. J. Am. Chem. Soc.
1983, 105, 2430-2434.
(24) Veeneman, G. H.; Van Leeuwen, S. H.; Van Boom, J. H.
Iodonium ion-promoted reactions at the anomeric center. II. An
efficient thioglycoside-mediated approach toward the formation
of 1,2-trans-linked glycosides and glycosidic esters. Tetrahedron
Lett. 1990, 31, 1331-1334.
(25) Veeneman, G. H.; Van Boom, J. H. An efficient thioglycoside-
mediated formation of R-glycosidic linkages promoted by iodo-
nium dicollidine perchlorate. Tetrahedron Lett. 1990, 31, 275-
278.
(26) Crich, D.; Smith, M. 1-Benzenesulfinyl piperidine/trifluoro-
methanesulfonic anhydride: A potent combination of shelf-stable
reagents for the low-temperature conversion of thioglycosides
to glycosyl triflates and for the formation of diverse glycosidic
linkages. J. Am. Chem. Soc. 2001, 123, 9015-9020.
(27) Lear, M. J.; Yoshimura, F.; Hirama, M. A direct and efficient
a-selective glycosylation protocol for the kedarcidin sugar,
L-mycarose: AgPF6 as a remarkable activator of 2-deoxythiogly-
cosides. Angew. Chem., Int. Ed. 2001, 40, 946-949.
Acknowledgment. This work was supported by
NSF (Grant CH-0316806) to P.G.W. and partially sup-
ported by ACS Grant IRG-98-278-04, a Research Starter
Grant from PhRMA Foundation to D.S.
References
(1) Arcamone, F. Doxorubicin: Anticancer Antibiotics; Medinal
Chemistry, a Series of Monographs; Academic Press: New York,
1981; Vol. 17, pp 1-369.
(2) Wang, J. C. DNA topoisomerases. Annu. Rev. Biochem 1996, 65,
635-692.
(3) Gewirtz, D. A. A critical evaluation of the mechanisms of action
proposed for the antitumor effects of the anthracycline antibiot-
ics adriamycin and daunorubicin. Biochem. Pharmacol. 1999,
57, 727-741.
(4) Zunino, F.; Capranico, G. DNA topoisomerase II as the primary
target of anti-tumor anthracyclines. Anti-Cancer Drug Des. 1990,
5, 307-317.
(5) Capranico, G.; Palumbo, M.; Tinelli, S.; Mabilia, M.; Pozzan, A.;
Zunino, F. Conformational drug determinants of the sequence
specificity of drug-stimulated topoisomerase II DNA cleavage.
J. Mol. Biol. 1994, 235, 1218-1230.
(6) Capranico, G.; Butelli, E.; Zunino, F. Change of the sequence
specificity of daunorubicin-stimulated topoisomerase II DNA
cleavage by epimerization of the amino group of the sugar
moiety. Cancer Res. 1995, 55, 312-317.
(7) Arcamone, F.; Animati, F.; Capranico, G.; Lombardi, P.; Pratesi,
G.; Manzini, S.; Supino, R.; Zunino, F. New developments in
antitumor anthracyclines. Pharmacol. Ther. 1997, 76, 117-124.
(8) Arcamone, F. M. From the pigments of the actinomycetes to third
generation antitumor anthracyclines. Biochimie 1998, 80, 201-
206.
(28) Marzabadi, C. H.; Franck, R. W. The synthesis of 2-deoxygly-
cosides: 1988-1999. Tetrahedron 2000, 56, 8385-8417.
(29) Toshima, K.; Tatsuta, K. Recent progress in O-glycosylation
methods and its application to natural products synthesis. Chem.
Rev. 1993, 93, 1503-1531.
(30) Israel, M.; Taube, D.; Seshadri, R.; Idriss, J. M. Adriamycin
analogues. Preparation and biological evaluation of some N-(tri-
fluoroacetyl)-14-O-[N-acetylamino)acyl]adriamycin derivatives.
J. Med. Chem. 1986, 29, 1273-1276.
(9) Binaschi, M.; Bigioni, M.; Cipollone, A.; Rossi, C.; Goso, C.;
Maggi, C. A.; Capranico, G.; Animati, F. Anthracyclines: selected
new developments. Curr. Med. Chem.: Anti-Cancer Agents 2001,
1, 113-130.

5278 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16
Zhang et al.
(31) Flynn, E. H.; Sigal, M. V., Jr.; Wiley, P. F.; Gerzon, K.
Erythromycin. I. Properties and degradation studies. J. Am.
Chem. Soc. 1954, 76, 3121-3131.
(32) Hamill, R. L.; Haney, M. E., Jr.; Stamper, M.; Wiley, P. F.
Tylosin, a new antibiotic. II. Isolation, properties, and prepara-
tion of desmycosin, a microbiologically active degradation prod-
uct. Antibiotics Chemother. 1961, 11, 328-334.
(33) Alper, P. B.; Hung, S.-C.; Wong, C.-H. Metal catalyzed diazo
transfer for the synthesis of azides from amines. Tetrahedron
Lett. 1996, 37, 6029-6032.
(34) Fukunaga, K.; Yoshida, M.; Nakajima, F.; Uematsu, R.; Hara,
M.; Inoue, S.; Kondo, H.; Nishimura, S.-I. Design, synthesis, and
evaluation of â-galactosylceramide mimics promoting b-gluco-
cerebrosidase activity in keratinocytes. Bioorg. Med. Chem. Lett.
2003, 13, 813-815.
(35) Janczuk, A. J.; Zhang, W.; Andreana, P. R.; Warrick, J.; Wang,
P. G. The synthesis of deoxy-R-Gal epitope derivatives for the
evaluation of an anti-R-Gal antibody binding. Carbohydr. Res.
2002, 337, 1247-1259.
(36) Ohlsson, J.; Magnusson, G. Galabiosyl donors; efficient synthesis
from 1,2,3,4,6-penta-O-acetyl-â-D-galactopyranose. Carbohydr.
Res. 2000, 329, 49-55.
(37) Qian, J.; Zhou, C. H.; Qian, Z.; Nan, F. J.; Ye, Q. Z. Development
of a K562 cell-based assay for screening anticancer agents. Acta
Pharmacol. Sin. 2001, 22, 821-826.
(38) Chen, X. S.; Fang, T. L.; Cai, R. B.; Guo, G. L. Development of
MTS/pms colorimetric assay in the proliferation of leukemic cells.
Zhongguo Shiyan Xueyexue Zazhi 2002, 10, 438-440.
(39) Wang, M. J.; Liu, Y. X.; Li, X. L.; Shi, J.; Liu, S. L.; Zheng, D. X.
Chemotherapeutic drugs enhanced rsTRAIL tumoricidal activ-
ity. Zhongguo Yixue Kexueyuan Xuebao 2004, 26, 524-528.
(40) Muller, M. T.; Mehta, V. B. DNase I hypersensitivity is inde-
pendent of endogenous topoisomerase II activity during chicken
erythrocyte differentiation. Mol. Cell. Biol. 1988, 8, 3661-3669.
(41) Subramanian, D.; Kraut, E.; Staubus, A.; Young, D. C.; Muller,
M. T. Analysis of topoisomerase I/DNA complexes in patients
administered topotecan. Cancer Res. 1995, 55, 2097-2103.
(42) Subramanian, D.; Furbee, C. S.; Muller, M. T. ICE bioassay.
Isolating in vivo complexes of enzyme to DNA. Methods Mol.
Biol. 2001, 95, 137-147.
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