Syntheses and biological activities of daunorubicin analogs with uncommon sugars

Article abstract of DOI:10.1016/j.bmc.2005.06.053

To study the effects of the sugar structure on the activity of anthracycline against cancer cells, six daunorubicin analogs containing different uncommon sugars were synthesized. Their cytotoxicities were tested against colon cancer cells by MTS assay. Th

Full text of DOI:10.1016/j.bmc.2005.06.053

Bioorganic & Medicinal Chemistry 13 (2005) 6381–6387
Syntheses and biological activities of daunorubicin analogs
with uncommon sugars
Lizhi Zhu,a Xianhua Cao,b Wenlan Chen,a Guisheng Zhang,a
Duxin Sunb,* and Peng George Wanga,*
aDepartment of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA
bDivision of Pharmaceutics, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA
Received 7 June 2005; accepted 24 June 2005
Available online 1 August 2005
Abstract—To study the effects of the sugar structure on the activity of anthracycline against cancer cells, six daunorubicin analogs
containing different uncommon sugars were synthesized. Their cytotoxicities were tested against colon cancer cells by MTS assay.
The results showed that the aglycon without sugar moiety has 70–100-fold lower activity against cancer cells than daunorubicin
derivatives with various uncommon sugars. It suggests that the sugar structure in daunorubicin plays a critical role in determining
its anticancer activity. In the compounds with various sugars, the 40-OH of the sugar is an important determinant for their activity,
while the axial-30-substituent in the sugar interferes with the binding of daunorubicins to DNA. Therefore, 2,6-dideoxy sugars are a
better choice for generating biologically active daunorubicin analogs than 6-deoxysugars, 2,3,6-trideoxysugars, or 2,3,4,6-
tetradeoxysugars.
ꢀ 2005 Elsevier Ltd. All rights reserved.
O
O
OH
O
1. Introduction
O
C
OH
B
O
13
1
10
A
12
11
9
R
2
3
OH
OH
D
The anthracycline quinone antibiotics daunorubicin and
doxorubicin (adriamycin) have been used clinically since
the 1970s.1 So far anthracyclines are among the most
effective drugs against a variety of human solid tumors
(Fig. 1).2 The primary cellular target for anthracyclines
is DNA topoisomerase II (topo II).3 The well-accepted
mechanism of action is through interaction with DNA
topo II by stabilization of the topo II–DNA–drug terna-
ry complex. It is noted that DNA binding and interac-
tion are necessary, but not sufficient, for topo II
poisoning.4 In fact, the external (non-intercalating) moi-
eties involving the sugar structure and the cyclohexane
ring A (Fig. 1) are recognized as crucial moieties for
therapeutic efficacy of anthracyclines. The sugar moiety
of anthracyclines lies in the minor groove of the DNA
and contacts base pairs.5,6 This mechanisms of anthra-
cycline to intercalate DNA causes DNA breakage, pro-
duces oxidative stress, and triggers cell apoptosis.7 The
8
4
5
O
6
7
MeO
OH OH
MeO
OH O
O
NH2HCl
HO
R = H, Daunorubicin
R = OH, Doxorubicin
Aglycon of daunorubicin
Figure 1. Daunorubicin, doxorubicin, and aglycon of daunorubicin.
major problems associated with anthracycline drugs
are cardiotoxicity and drug resistance mediated by the
multidrug resistance gene (MDR).8,9 Many researchers
have explored ways to modify the structure of anthracy-
cline to generate various analogs to reduce the side
effects and reverse multidrug resistance; however, these
efforts only have had limited success.10,11
In fact, a wealth of data has been published on the struc-
ture–activity relationship (SAR) of DOXꢀs aglycon por-
tion and carbohydrate moiety over the past 30 years.
The results show that the carbohydrates moiety is a crit-
ical component of anthracycline anticancer activity. For
instance, the aglycon itself is nearly inactive when tested
Keywords: Daunorubicin; Glycosylation; Structure–activity relation-
ship (SAR); Anticancer.
*
Corresponding authors. Tel.: +1 614 292 9884; fax: +1 614 688 3106
(P.G.W.); tel.: +1 614 292 4381; fax: +1 614 292 7766 (D.S.); e-mail
addresses: sun.176@osu.edu; wang.892@osu.edu
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.06.053

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L. Zhu et al. / Bioorg. Med. Chem. 13 (2005) 6381–6387
as an anticancer agent. It is well established that the su-
gar plays a vital role in the binding of anthracycline with
the DNA topo II complex and the orientation of sugar is
critical for the binding. a-Glycoside is assumed as a
common motif for anthracyclines with high anticancer
activities, while the b-anomer is even less active than
the aglycon itself.12,13 Chemical and configurational
modifications on the sugar moiety, which mainly focus
on the 30- and 40-positions, markedly determine the dif-
ference in anticancer activity, toxicity, the sequence
specificity of the DNA break sites, and other cellular
process/pathway besides topo II.14
daunorubicin analogs. The monosaccharide daunoru-
bicins were synthesized using thiophenyl glycoside of
2,6-dideoxysugar donors in the presence of AgPF6 and
TTBP. This protocol produced the desired a-glycosidic
bond with good yields. Taking advantage of our uncom-
mon sugar library, we synthesized six daunorubicin
analogs (Fig. 2) using this glycosylation method. The
biological activities of these compounds have been
tested with colon cancer cell line SW620.
The syntheses of daunorubicin analogs started with the
preparation of the aglycon. The aglycon (DNR-A) was
readily obtained from the hydrolysis of daunorubicin
hydrochloride with dilute HCl at 90 ꢁC for 1 h. DNR-
A was obtained after filtration as a red powder (Scheme
1) and was used directly for further glycosylations. The
corresponding sugar donors were prepared from their
corresponding precursors (compounds 1–6). Acetyl
group was used for protecting the hydroxyl groups pres-
ent in the sugar molecule, because they were cleavable
under 0.1 M NaOH in THF, which allowed the acid
and strong base-sensitive aglycon moiety in the anthra-
cycline not to be affected in the final deprotection proce-
dures. As shown in Scheme 1, after treatment with
phenylthiol in the presence of BF3ÆEt2O at 0 ꢁC for
4 h, the desired sugar donors (compounds 7–12) were
obtained in excellent yields (>94%). The thiolglycosides
were obtained as a mixture of a- and b-isomers. Since
both the isomers are able to be used for the glycosyla-
tion to produce the desired a-linked daunorubicin deriv-
atives, separation of them is not necessary. Previous
research22 found that the tertiary hydroxyl group would
not affect further glycosylation, thus compound 2 was
directly converted to the sugar donor 8. With the agly-
con and the sugar donors in hand, the glycosylation
was performed subsequently.
Although the chemical modifications of anthracycline so
far support the hypothesis that the sugar structure is a
very important determinant for its anticancer activity,
the diversity of sugar structure in that study is rather
limited by the unavailability of the various uncommon
sugars. In our research, we have generated an uncom-
mon sugar library, which provides us a powerful tool
in the systematic SAR investigation of sugar structures
for anticancer activity. We synthesized six daunorubicin
analogs, with different uncommon sugars attached, to
explore the structure–activity relation of the uncommon
sugar moiety of daunorubicin analogs. Their activity
against colon cancer cells SW 620 was measured by
MTS assay (tetrazolium[3-(4,5-dimethy-thiazol-2-yl)]-5-
(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetra-
zolium, inner salt). Indeed, different uncommon sugars
in daunorubicin analogs show distinct cytotoxicity
against cancer cells.
2. Results and discussion
2.1. Chemistry
Previous research has indicated that the linkage between
the sugar moiety and the aglycon is very important.
Only the a-linked daunorubicin monosaccharide ana-
logs are biologically active. Therefore, the uncommon
sugar has to be introduced stereoselectively. Glycosyl
sulfide donors are believed to be the best choice for
the a-glycosylation of 2,6-dideoxy sugars in this case.
Thiolglycosides have been introduced as glycosyl donors
because they are stable to normal protecting group
manipulation encountered in oligosaccharide synthe-
ses.15–17 Various activating systems can be applied, such
as, MeOTf, NBS, BSP/Tf2O/TTBP (2,4,6-tri-tert-butyl-
pyrimidine),18 IDCP, NIS/TfOH, and NIS/TMSOTf.
For 2-deoxy sugars, without the stereodirecting ability
of 2-substituents, strategies to prepare 2-deoxyglyco-
sides selectively in high a- or b-anomeric forms rely
heavily on indirect sequences from glycols or latent 2-
deoxysugars.19,20 In addition, direct and efficient a-selec-
tive glycosylations are difficult to realize.15,21 Recently,
Hirama and co-workers reported a direct and efficient
a-selective glycosylation protocol for the kedarcidin
sugar and L-mycarose using AgPF6 as a remarkable
activator of 2-deoxythioglycosides.21
O
OH
O
O
OH
O
OH
OH
MeO
MeO
MeO
O
OH
O
MeO
O
OH
OH
O
O
O
HO
HO
HO
DNR-1
OH
DNR-2
O
OH
O
O
OH
O
OH
OH
MeO
O
OH
O
O
O
OH O
OMe
O
HO
HO
MeO
DNR-3
DNR-4
O
OH
O
O
O
OH
O
OH
OH
O
OH
O
MeO
OH
O
O
O
HO
N3
DNR-5
Figure 2. Synthetic daunorubicin derivatives.
DNR-6
In this paper, we report a concise promoter system
AgPF6/TTBP for syntheses of a-linked monosaccharide

L. Zhu et al. / Bioorg. Med. Chem. 13 (2005) 6381–6387
6383
O
OH
O
(a:b = 3:1). In addition, compound 14 was unable to
be separated from the unreacted aglycon (same Rf value
using 100:1 CH2Cl2/MeOH as eluent). Finally, the pure
DNR-2 was obtained after the deprotection step in 39%
overall yield for two steps. Compounds 13–17 were
treated with 0.1 M NaOH for 6 h, and the acetyl groups
were successfully removed to allow the isolation of the
desired DNR-1–DNR-5 in 65–75% yields.
O
O
OH
OH
O
a
OH
OH
92%
MeO
O
OH OH
MeO
O
O
DNR-A
NH2HCl
HO
b
OAc
SPh
SPh
O
O
AcO
AcO
AcO
AcO
94%
2.2. Cytotoxicity
OAc
OAc
1
7
OH
O
OH
O
b
OPri
The cytotoxicity of these compounds was examined in
colon cancer cell line SW620 cells (Fig. 3) with MTS as-
say. Five thousand cells were incubated with 0.001–
10 lM DNR and its derivatives for 72 h. Then 20 lL
MTS/PMS assay solution was added to each well and
the absorbance was recorded. The cell survival was
calculated as a percentage of the cell control group
(without treatment with drug). The IC50 of all these
compounds against colon cancer cell line SW620 are
summarized in Table 1. The IC50 values were calculated
by WinNonlin 4.1 (Pharsight) from the dose–response
curves of percentage of cell growth with the model:
E = Emax ꢀ (Emax ꢀ E0) · [C/(C + EC50)].
AcO
AcO
98%
2
8
OMe
O
OMe
O
b
OPri
OPri
SPh
SPh
AcO
AcO
AcO
AcO
97%
3
9
b
O
O
96%
b
MeO
MeO
4
10
OAc
OPri
SPh
SPh
O
5
O
11
O
AcO
AcO
93%
b
O
96%
N3
N3
12
6
Scheme 1. Reagents and conditions: (a) 0.2 M HCl, 90 ꢁC, 1 h; (b)
PhSH, BF3ÆEt2O/CH2Cl2, 0 ꢁC, 4 h.
The results showed that the aglycon, DNR-A, exhibited
70–100-fold lower cytotoxicity than daunorubicin deriv-
atives with various uncommon sugars. This suggests
that the sugar structure in daunorubicin plays a critical
role in determining its anticancer activity. Indeed, this
finding has also been confirmed by other reports.20,21
The mixture of aglycon (DNR-A) and sugar donors 7–
˚
12, in the presence of TTBP and 4 A molecular sieves,
was treated with AgPF6 at 0 ꢁC for 4 h to give the gly-
cosylated products 13–17 and DNR-6 in around 60%
yields (Scheme 2). The 1H NMR data indicated that
the desired a-linkage was formed predominantly
(a:b > 9:1). However, glycosylation of DNR-A with
sugar donor 10 produced a mixture of both isomers
Compound DNR-4 with a 30-OMe terminal 2,6-dideoxy-
sugar showed very potent cytotoxicity with IC50 of
104 nM (Fig. 3A). Importantly, compared to com-
pounds DNR-2 and DNR-3 (with an axial-30-OMe or
˚
Scheme 2. Reagents and conditions: (a) TTBP, AgPF6/CH2Cl2, 4 A MS, 0 ꢁC, 4 h; (b) 0.1 M NaOH/THF, 0 ꢁC, 6 h.

6384
L. Zhu et al. / Bioorg. Med. Chem. 13 (2005) 6381–6387
A
140
simply lying in the minor groove of the DNA, the func-
tional group of sugar rather interact with the base pairs
for the biological activity of daunorubicin analogs.
DNR
120
100
80
60
40
20
0
DNR-A
DNR-2
DNR-3
DNR-4
3. Conclusions
In summary, six daunorubicin analogs with different
uncommon sugars were synthesized. Their biological
activities were tested against colon cancer cell line
SW620 cells. The results showed that the aglycon of
anthracycline without sugar moiety decreased its activity
by 70–100-fold. This suggests that the sugar moiety in
these analogs plays a crucial role in determining the
cytotoxicities of these compounds. In addition, different
modifications in sugar structures of anthracylcine indi-
cate that the axial-30-substituent in sugar (such as in
compounds DNR-2 and DNR-3) interferes with the
binding of daunorubicin to DNA, while the 40-OH of
sugar is a critical determinant for the activity of dauno-
rubicin. Therefore, increasing the hindrance of the a-
face of the sugar leads to decreased cytotoxicity against
cancer cells. The 2,6-dideoxy sugars are the best choice
to modify daunorubicin compared with 6-deoxysugar,
2,3,6-trideoxysugars, and 2,3,4,6-tetradeoxysugars.
1
10
100
1000
10000
Concentration (nM)
140
120
100
80
B
DNR
DNR-A
DNR-1
DNR-5
DNR-6
60
40
20
0
1
10
100
1000
10000
Concentration (nM)
Figure 3. Cytotoxicity of compounds DNR, DNR-A, DNR-2–4 (A)
and compounds DNR, DNR-A, DNR-1, DNR-5–6 (B) against colon
cancer SW620 cells. Five thousand cells were incubated with 0.001–
10 lM tested compounds for 72 h. MTS/PMS solution was added and
further incubated with cells for 2.5–3 h. Then the absorbance was
recorded at 490 nM. The survival rate was expressed as percentage of
control group.
4. Experimental
4.1. Chemistry
axial-30-OH group), DNR-4 (with an equatorial-30-OMe
group) showed 10–20-fold higher anticancer activity.
This suggested that the axial-30-substituent in the sugar
(such as in compounds DNR-2 and DNR-3) may inter-
fere daunorubicin binding to DNA.
4.1.1. General information. All the solvents were dried
with solvent-purification system from Innovative Tech-
nology, Inc. All the reagents were purchased from com-
mercial sources and used without further purification.
Analytical TLC was carried out on silica gel 60 F254 alu-
minum-backed plates (E. Merck). The preparation TLC
was carried out on silica gel 60 F254 plates (EMD Chem-
icals, Inc. 20 · 20 cm, 1 mm). The 230–400 mesh size of
the same absorbent was utilized for all chromatographic
purifications. 1H and 13C NMR spectra were recorded at
the indicated field strengths with the indicated solvent.
The high-resolution mass spectra were obtained at The
Ohio State University Campus Chemical Instrumenta-
tion Center.
Compounds DNR-1 and DNR-5 with an equatorial-40-
OH showed similar activity, while DNR-6 (substituted
40-OH with an axial-40-N3 in the sugar moiety), which
is similar to the aglycon DNR-A, lost its cytotoxicity
(Fig. 3B). This suggests that the 40-OH in the sugar
may also be important for sugar containing anthracy-
cline as an anticancer agent. Further SAR studies are re-
quired to clarify this finding.
From the published results,4 it is found that the a-face of
the sugar is lying toward the DNA base pairs when bind-
ing to DNA. Therefore, increasing the size of the a-face
will decrease the binding ability of the sugar moiety to
DNA, thus decreasing cytotoxicity decreases. The IC50
values of DNR-1, DNR-4, DNR-5, and DNR-6 showed
that compound DNR-4 had the best activity followed by
DNR-1, DNR-5, and DNR-6. This tendency suggests that
the 2,6-dideoxy sugars are better choices for making bio-
logically active daunorubicin analogs than 6-deoxysu-
gars, 2,3,6-trideoxysugars, or 2,3,4,6-tetradeoxysugars.
These data also indicate that the sugar moiety is not just
4.1.2. General procedure for preparation of the sugar
donor. To a stirred solution of acetated sugar 3 (0.2 g,
0.77 mmol) in CH2Cl2 (5.0 mL) at 0 ꢁC, phenylthiol
(0.1 mL, 0.98 mmol) and BF3ÆEt2O (0.11 mL,
0.87 mmol) were added. The reaction mixture was al-
lowed to warm slowly to room temperature. After stir-
ring for 4 h, the reaction mixture was quenched with
aqueous NaOH (0.1 N, 10 mL). Then the reaction mix-
ture was washed with water. The aqueous layer was
extracted with CH2Cl2, and the combined organic layers
were dried over MgSO4 and concentrated under
vacuum. The crude product was purified by flash
Table 1. Cytotoxicity (IC50) of synthesized compounds against colon cancer SW620 cells
Compounds
DNR-1
DNR-2
DNR-3
DNR-4
DNR-5
DNR-6
DNR-A
DNR
IC50 in SW620 (nM)
264.6
>1000
>1000
104
350
>1000
>2000
33.4

L. Zhu et al. / Bioorg. Med. Chem. 13 (2005) 6381–6387
6385
column chromatography (50:1 petroleum ether–EtOAc
as eluent) and provided donor 9 as syrup (0.23 g, 97%
yield).
J = 7.4 Hz, 1H), 5.62 (d, J = 5.4 Hz, 1H), 4.49 (m,
1H), 3.53 (s, 1H), 2.37 (m, 1H), 2.14 (tt, J = 14.1,
3.8 Hz, 1H), 2.04 (dd, J = 14.3, 3.4 Hz, 1H), 1.82 (dt,
J = 14.2, 3.2 Hz, 1H), 1.23 (d, J = 6.5 Hz, 3H); 13C
NMR (125.69 MHz, CDCl3) d 135.2, 130.9 (2C), 128.9
(2C), 126.9, 84.5, 66.4, 60.0, 25.3, 24.6, 17.8. The parent
ion of this compound was not observed by HRMS, due
to the decomposition of this compound under the anal-
ysis conditions.
4.1.3. General procedure for glycosylation. The mixture
of donor
9 (0.17 g, 0.55 mmol), aglycon (0.29 g,
˚
0.75 mmol), 4 A MS, and TTBP (0.55 g, 2.2 mmol) in
CH2Cl2 (5.0 mL) was stirred (avoid light) at room tem-
perature for 1.5 h. Then the mixture was cooled to 0 ꢁC
and AgPF6 (0.43 g, 1.7 mmol) was added. After stirring
for 6 h at 0 ꢁC, pyridine (2 mL) was added, and the reac-
tion mixture was stirred for a further 0.5 h at 0 ꢁC. The
mixture was filtered through Celite, and the residue was
thoroughly washed with CH2Cl2. The crude product was
purified by flash column chromatography (100:1 dichlo-
romethane–methanol as eluent) to give compound 15
(220 mg, 68% yield) as a red powder.
4.3.2. 7-(2,3,4-O-Triacetyl-a-rhamnosyl)daunorubicinone
1
(13). H NMR (500 MHz, CDCl3) d 3.86 (s, 1H), 13.14
(s, 1H), 7.93 (d, J = 7.7 Hz, 1H), 7.72 (t, J = 8.2 Hz, 1H),
7.34 (d, J = 8.5 Hz, 1H), 5.33 (d, J = 1.5 Hz, 1H), 5.31
(m, 1H), 5.26 (d, J = 2.1 Hz, 1H), 5.10 (s, 1H), 5.08 (s,
1H), 4.07 (m, 1H), 4.03 (s, 3H), 3.19 (dd, J = 18.5,
1.0 Hz, 1H), 2.85 (d, J = 18.7 Hz, 1H), 2.41 (s, 3H),
2.32 (d, J = 15.0 Hz, 1H), 2.16 (dd, J = 14.8, 4.3 Hz,
1H), 2.15 (s, 3H), 2.04 (m, 1H), 2.02 (s, 3H), 1.91 (s,
3H), 1.27 (d, J = 6.3 Hz, 3H); 13C NMR (125.69 MHz,
CDCl3) d 211.6, 186.7, 186.6, 170.0, 169.7, 169.6,
161.0, 156.1, 155.4, 137.3, 135.3, 134.3, 132.9, 120.7,
119.7, 118.5, 111.5 (2C), 100.5, 76.4, 70.8, 70.4, 69.4,
69.1, 67.7, 56.6, 35.1, 33.3, 24.8, 20.9, 20.8, 20.6, 17.4;
HRMS (ESI) m/z calcd for C33H34O15Na (M+Na+)
693.1790, found 693.1768.
4.1.4. General procedure for deprotection. To a solution
of compound 15 (180 mg, 0.3 mmol) in THF (5 mL) at
0 ꢁC, aqueous NaOH (0.1 M, 70 mL) was added. After
stirring at 0 ꢁC for 6 h, the reaction mixture was neutral-
ized with citric acid (10 wt %). The aqueous layer was
extracted with CH2Cl2, and the combined organic layers
were washed with saturated NaHCO3 and water. After
drying with MgSO4 and concentrating under vacuum,
the crude product was purified by flash column chroma-
tography (80:1 dichloromethane–methanol) to give
DNR-3 (110 mg, 66% yield) as a red powder.
4.3.3. 7-(4-O-Acetyl-a-cladinosyl)daunorubicinone (15).
1H NMR (500 MHz, CDCl3) d 13.83 (s, 1H), 13.11 (s,
1H), 7.92 (d, J = 7.6 Hz, 1H), 7.71 (t, J = 8.1 Hz, 1H),
7.33 (d, J = 8.5 Hz, 1H), 5.35 (d, J = 4.5 Hz, 1H), 5.27
(s, 1H), 5.15 (m, 1H), 4.73 (d, J = 10.0 Hz, 1H), 4.43
(m, 1H), 4.04 (s, 3H), 3.09 (d, J = 20.0 Hz, 1H), 3.09
(s, 3H), 2.78 (d, J = 19.0 Hz, 1H), 2.41 (s, 3H), 2.31 (d,
J = 14.5 Hz, 1H), 2.19 (d, J = 15.1 Hz, 1H), 2.13 (s,
3H), 2.03 (m, 1H), 1.63 (m, 1H), 1.17 (d, J = 6.3 Hz,
3H), 1.06 (s, 3H); 13C NMR (125.69 MHz, CDCl3) d
213.0, 186.9, 186.3, 170.6, 160.9, 156.3, 155.9, 135.5,
135.4, 134.8, 134.6, 120.8, 119.6, 118.2, 111.1, 110.9,
99.7, 78.4, 76.4, 73.3, 69.5, 63.3, 56.6, 50.0, 37.1, 34.8,
33.3, 24.9, 20.9, 20.7, 17.2; HRMS (ESI) m/z calcd for
C31H34O12Na 621.1942 (M+Na+), found 621.1913.
4.2. Biology
4.2.1. Cell culture. Colon cancer cell line SW620 was cul-
tured in RPMI 1640 supplemented with 10% fetal
bovine serum (FBS), 1% non-essential amino acid and
Penicillin (100 units/mL) Streptomycin (100 lg/mL) in
a humidified atmosphere of 5% CO2 and 95% air at
37 ꢁC. The culture mediums were changed every
2–3 days.
4.2.2. Cytotoxicity of synthesized compounds against
cancer cells by MTS assay. Colon cancer SW620 cells
(5000) 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 com-
pounds for 72 h at 37 ꢁC (5% CO2, 95% humidity). After
72 h incubation, tetrazolium[3-(4,5-dimethy-thiazol-2-
yl)]-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-
tetrazolium, inner salt (MTS, 2 mg/mL), and phenazine
methosulfate (PMS, 25 lM) 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 val-
ues of the carbohydrate–drug conjugates for cytotoxic-
ity were calculated by WinNonlin 4.1 (Pharsight) from
the dose–response curves of percentage of cell growth
versus control (no compound added).
4.3.4. 7-(4-O-Acetyl-a-oleaudrosyl)daunorubicinone (16).
1H NMR (500 MHz, CDCl3) d 13.92 (s, 1H), 13.16 (s,
1H), 7.96 (d, J = 7.7 Hz, 1H), 7.74 (t, J = 8.3 Hz, 1H),
7.35 (d, J = 8.2 Hz, 1H), 5.49 (d, J = 3.1 Hz, 1H), 5.20
(d, J = 2.2 Hz, 1H), 4.67 (t, J = 9.3 Hz, 1H), 4.52 (s,
1H), 4.04 (s, 3H), 4.93 (m, 1H), 3.40 (m, 1H), 3.24 (s,
3H), 3.15 (dd, J = 18.8, 1.8 Hz, 1H), 2.84 (d,
J = 18.7 Hz, 1H), 2.40 (s, 3H), 2.31 (d, J = 14.8 Hz,
1H), 2.25 (m, 1H), 2.09 (m, 1H), 2.07 (s, 3H), 1.66 (m,
1H), 1.19 (d, J = 6.5 Hz, 3H); 13C NMR (125.69 MHz,
CDCl3) d 211.7, 186.9, 186.6, 170.2, 161.0, 156.4,
155.7, 135.7, 135.4, 134.4, 134.0, 120.8, 119.8, 118.4,
111.4, 111.3, 101.0, 76.7, 75.8, 75.5, 70.2, 67.2, 56.9,
56.7, 35.0, 34.7, 33.3, 24.8, 21.1, 17.5; HRMS (ESI)
m/z calcd for C30H32O12Na 607.1786 (M+Na+), found
607.1799.
4.3. Spectroscopic data
4.3.1. Phenylthio-4-azido-2,3,4,6-tetradeoxy-a-L-erythro-
hexopyranoside (12). 1H NMR (500 MHz, CDCl3) d 7.45
(d, J = 7.7 Hz, 2H), 7.29 (t, J = 7.3 Hz, 2H), 7.22 (t,
4.3.5. 7-(4-O-Acetyl-a-amicetosyl)daunorubicinone (17).
1H NMR (500 MHz, CDCl3) d 13.97 (s, 1H), 13.29 (s,
1H), 8.02 (d, J = 7.7 Hz, 1H), 7.76 (t, J = 8.1 Hz, 1H),

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L. Zhu et al. / Bioorg. Med. Chem. 13 (2005) 6381–6387
7.37 (d, J = 8.5 Hz, 1H), 5.39 (m, 1H), 5.32 (m, 1H), 4.81
(s, 1H), 4.52 (m, 1H), 4.07 (s, 3H), 3.96 (m, 1H), 3.24
(dd, J = 18.9, 1.9 Hz, 1H), 2.97 (d, J = 18.8 Hz, 1H),
2.43 (s, 3H), 2.38 (m, 1H), 2.10 (dd, J = 14.8, 4.1 Hz,
1H), 2.04 (s, 3H), 1.90 (m, 1H), 1.82 (m, 2H), 1.59 (m,
1H), 1.21 (d, J = 6.3 Hz, 3H); 13C NMR (125.69 MHz,
CDCl3) d 212.0, 187.2, 186.8, 170.3, 161.1, 156.5,
156.0, 135.7, 135.6, 134.6, 134.3, 121.1, 119.8, 118.4,
111.5, 111.4, 99.8, 76.9, 73.2, 69.5, 68.0, 56.7, 35.0,
33.5, 29.7, 28.9, 24.8, 21.2, 17.8; HRMS (ESI) m/z calcd
for C29H30O11Na 577.1680 (M+Na+), found 577.1688.
(m, 1H), 3.27 (m, 1H), 3.26 (s, 3H), 3.18 (t, J = 9.1 Hz,
1H), 3.13 (dd, J = 18.8, 1.9 Hz, 1H), 2.80 (d,
J = 18.7 Hz, 1H), 2.39 (s, 3H), 2.32 (d, J = 14.8 Hz,
1H), 2.25 (dd, J = 13.1, 4.5 Hz, 1H), 2.08 (dd, J = 14.8,
4.2 Hz, 1H), 1.51 (m, 1H), 1.33 (d, J = 6.2 Hz, 3H);
13C NMR (125.69 MHz, CDCl3) d 212.1, 186.9, 186.5,
160.9, 156.4, 155.8, 135.6, 135.4, 134.6, 134.1, 120.8,
119.7, 118.4, 111.3, 111.2, 101.3, 78.0, 75.8, 70.1, 68.9,
56.6, 56.4, 35.0, 33.9, 33.3, 24.8 (2C), 17.7; HRMS
(ESI) m/z calcd for C28H30O11Na 565.1680 (M+Na+),
found 565.1706.
4.3.6. 7-(a-Rhamnosyl)daunorubicinone (DNR-1). 1H
NMR (500 MHz, DMSO-d6) d 14.03 (s, 1H), 13.24 (s,
1H), 7.90 (m, 2H), 7.64 (m, 1H), 5.45 (s, 1H), 5.02 (s,
1H), 4.95 (s, 1H), 4.70 (dd, J = 13.4, 5.1 Hz, 2H), 4.41
(d, J = 5.6 Hz, 1H), 3.98 (s, 3H), 3.77 (m, 1H), 3.58 (s,
1H), 3.33 (m, 1H), 3.22 (m, 1H), 2.99 (d, J = 17.7 Hz,
1H), 2.87 (d, J = 17.9 Hz, 1H), 2.55 (s, 1H), 2.24 (s,
3H), 2.16 (m, 1H), 1.18 (d, J = 6.2 Hz, 3H); 13C NMR
(125.69 MHz, DMSO-d6) d 212.0, 187.0, 186.9, 161.3,
156.6, 150.1, 136.7, 136.6, 136.0, 135.2, 135.1, 120.5,
120.2, 119.5, 111.3, 111.2, 104.2, 75.8, 72.3, 71.1, 71.0,
69.6, 57.1, 37.1, 32.1, 24.4, 18.2; HRMS (ESI) m/z calcd
for C27H28O12Na 567.1743 (M+Na+), found 567.1465.
4.3.10. 7-(a-Amicetosyl)daunorubicinone (DNR-5). 1H
NMR (500 MHz, CDCl3) d 13.95 (s, 1H), 13.24 (s,
1H), 8.00 (d, J = 7.7 Hz, 1H), 7.76 (t, J = 8.1 Hz, 1H),
7.37 (d, J = 8.4 Hz, 1H), 5.38 (d, J = 3.0 Hz, 1H), 5.30
(m, 1H), 4.85 (s, 1H), 4.07 (s, 3H), 3.77 (m, 1H), 3.32
(br, 1H), 3.20 (dd, J = 18.9, 1.9 Hz, 1H), 2.92 (d,
J = 18.8 Hz, 1H), 2.42 (s, 3 H), 2.38 (dt, J = 14.5,
2.1 Hz, 1H), 2.09 (dd, J = 14.8, 4.1 Hz, 1H), 1.82 (m,
3H), 1.55 (m, 2H), 1.32 (d, J = 6.2 Hz, 3H); 13C NMR
(125.69 MHz, CDCl3) d 212.3, 187.0, 186.6, 161.0,
156.5, 155.9, 135.6, 135.5, 134.7, 134.4, 120.9, 119.8,
118.3, 111.4, 111.2, 99.7, 76.9, 71.8, 70.9, 69.3, 56.6,
34.9, 33.5, 29.4, 27.6, 24.9, 17.8; HRMS (ESI) m/z calcd
for C27H28O10Na 535.1575 (M+Na+), found 535.1566.
4.3.7. 7-(a-Mycarosyl)daunorubicinone (DNR-2). 1H
NMR (500 MHz, CDCl3) d 13.87 (s, 1H), 13.18 (s,
1H), 7.95 (d, J = 7.6 Hz, 1H), 7.71 (t, J = 8.1 Hz, 1H),
7.32 (d, J = 8.5 Hz, 1H), 5.40 (d, J = 3.5 Hz, 1H), 5.15
(s, 1H), 4.50 (s, 1H), 4.03 (s, 3H), 3.93 (m, 1H), 3.70
(s, 1H), 3.01 (m, 3H), 2.37 (s, 3H), 2.36 (s, 1H), 2.29
(d, J = 14.5 Hz, 1H), 2.09 (dd, J = 14.6, 4.1 Hz, 1H),
1.99 (d, J = 14.6 Hz, 1H), 1.75 (dd, J = 14.6, 4.1 Hz,
1H), 1.32 (d, J = 6.2 Hz, 3H), 1.17 (s, 3H); 13C NMR
(125.69 MHz, CDCl3) d 211.2, 186.9, 186.5, 161.0,
156.4, 155.6, 135.6, 135.5, 134.9, 134.0, 120.9, 119.8,
118.4, 111.4, 111.1, 99.8, 76.7, 76.0, 69.6, 68.0, 66.4,
56.7, 41.2, 35.1, 32.8, 26.1, 24.2, 17.9; HRMS (ESI)
m/z calcd for C28H30O11Na 565.1680 (M+Na+), found
565.1664.
4.3.11. 7-(4-Azido-2,3,4,6-tetradeoxy-a-L-erythro-hexo-
pyranosyl) daunorubicinone (DNR-6). 1H NMR
(500 MHz, CDCl3) d 13.94 (s, 1H), 13.26 (s, 1H), 8.01
(d, J = 7.7 Hz, 1H), 7.77 (t, J = 8.2 Hz, 1H), 7.38 (d,
J = 8.4 Hz, 1H), 5.45 (m, 1H), 5.29 (d, J = 5.3 Hz,
1H), 4.75 (s, 1H), 4.19 (m, 1H), 4.08 (s, 3H), 3.52 (m,
1H), 3.21 (d, J = 19.3 Hz, 1H), 2.94 (d, J = 18.8 Hz,
1H), 2.40 (s, 3H), 2.33 (d, J = 14.5 Hz, 1H), 2.10 (dd,
J = 14.8, 3.9 Hz, 1H), 1.98–1.88 (m, 3H), 1.60–1.55 (m,
1H), 1.27 (d, J = 6.5 Hz, 3H); 13C NMR (125.69 MHz,
CDCl3) d 211.7, 187.1, 186.7, 161.1, 156.4, 155.9,
135.7, 135.6, 134.4, 134.3, 121.0, 119.8, 118.4, 111.5,
111.3, 100.4, 76.9, 69.6, 66.5, 59.7, 56.7, 34.9, 33.5,
24.7, 23.9, 22.9, 17.9; HRMS (ESI) m/z calcd for
C27H27N3O9Na 560.1640 (M+Na+), found 560.1651.
4.3.8. 7-(a-Cladinosyl)daunorubicinone (DNR-3). 1H
NMR (500 MHz, CDCl3) d 13.84 (s, 1H), 13.14 (s,
1H), 7.93 (d, J = 7.6 Hz, 1H), 7.71 (t, J = 8.1 Hz, 1H),
7.34 (d, J = 8.3 Hz, 1H), 5.28 (d, J = 4.3 Hz, 1H), 5.20
(s, 1H), 5.12 (s, 1H), 4.05 (m, 1H), 4.04 (s, 3H), 3.05
(m, 2H), 3.00 (s, 3H), 2.81 (d, J = 18.8 Hz, 1H), 2.39
(s, 3H), 2.32 (m, 1H), 2.25 (d, J = 11.1 Hz, 1H), 2.19
(d, J = 15.4 Hz, 1H), 1.99 (dd, J = 14.7, 3.8 Hz, 1H),
1.50 (dd, J = 15.4, 4.6 Hz, 1H), 1.30 (d, J = 6.3 Hz,
3H), 1.16 (s, 3H); 13C NMR (125.69 MHz, CDCl3) d
212.7, 186.9, 186.4, 160.9, 156.3, 156.0, 135.6, 135.5,
134.9, 134.7, 120.9, 119.7, 118.3, 111.1, 111.0, 99.8,
77.8, 76.6, 73.2, 69.8, 65.9, 56.6, 49.3, 35.4, 34.8, 33.2,
24.8, 21.4, 17.9; HRMS (ESI) m/z calcd for
C29H32O11Na 579.1839 (M+Na+), found 579.1856.
Acknowledgments
This work was supported by NSF (CH-0316806) and a
fund from Michigan Life Science Corridor Fund
(1632) to P. G. Wang. This work was partially support-
ed by ACS grant IRG-98-278-04, Research Starter
Grant from PhRMA Foundation, and NIP from AACP
to D. Sun. The authors thank The Ohio State University
for providing research resources, analysis instruments,
and funding support.
References and notes
4.3.9. 7-(a-Oleaudrosyl)daunorubicinone (DNR-4). 1H
NMR (500 MHz, CDCl3) d 13.92 (s, 1H), 13.15 (s,
1H), 7.96 (d, J = 7.7 Hz, 1H), 7.73 (t, J = 8.0 Hz, 1H),
7.35 (d, J = 8.5 Hz, 1H), 5.50 (d, J = 3.7 Hz, 1H), 5.27
(s, 1H), 5.20 (m, 1H), 4.54 (s, 1H), 4.05 (s, 3H), 3.85
1. Weiss, R. B.; Sarosy, G.; Clagett-Carr, K.; Russo, M.
Cancer Chemother. Pharmacol. 1986, 18, 185–197.
2. Arcamone, F. In Doxorubicin, Anticancer antibiotics In
Medicinal Chemistry Series; Academic Press: New York,
1981; 17, pp 1–369.

L. Zhu et al. / Bioorg. Med. Chem. 13 (2005) 6381–6387
6387
3. Wang, J. C. Ann. Rev. Biochem. 1996, 65, 635–692.
4. Zunino, F.; Capranico, G. Anti-Cancer Drug Des. 1990, 5,
307–317.
14. Capranico, G.; Supino, R.; Binaschi, M.; Capolongo, L.;
Grandi, M.; Suarato, A.; Zunino, F. Mol. Pharmacol.
1994, 45, 908–915.
5. Capranico, G.; Palumbo, M.; Tinelli, S.; Mabilia, M.;
Pozzan, A.; Zunino, F. J. Mol. Biol. 1994, 235, 1218–1230.
6. Capranico, G.; Butelli, E.; Zunino, F. Cancer Res. 1995,
55, 312–317.
15. Nicolaou, K. C.; Seitz, S. P.; Papahatjis, D. P. J. Am.
Chem. Soc. 1983, 105, 2430–2434.
16. Veeneman, G. H.; Van Leeuwen, S. H.; Van Boom, J. H.
Tetrahedron Lett. 1990, 31, 1331–1334.
7. Aligiannis, N.; Pouli, N.; Marakos, P.; Skaltsounis, A. L.;
Florent, J. C.; Perchellet, E. M.; Sperfslage, B. J.; McIlvain,
C. J.; Perchellet, J. P. J. Antibiot. 2002, 55, 181–190.
8. Gottesman, M. M. Cancer Res. 1993, 53, 747–754.
9. Kaye, S. B. Br. J. Cancer 1988, 58, 691–694.
10. Garnier-Suillerot, A.; Marbeuf-Gueye, C.; Salerno, M.;
Loetchutinat, C.; Fokt, I.; Krawczyk, M.; Kowalczyk, T.;
Priebe, W. Curr. Med. Chem. 2001, 8, 51–64.
11. Lehne, G. Cur. Drug Targets 2000, 1, 85–99.
12. Roche, C. J.; Berkowitz, D.; Sulikowski, G. A.; Danishef-
sky, S. J.; Crothers, D. M. Biochemistry 1994, 33, 936–942.
13. Israel, M.; Murray, R. J. J. Med. Chem. 1982, 25, 24–28.
17. Veeneman, G. H.; Van Boom, J. H. Tetrahedron Lett.
1990, 31, 275–278.
18. Crich, D.; Smith, M. J. Am. Chem. Soc. 2001, 123, 9015–
9020.
19. Marzabadi, C. H.; Franck, R. W. Tetrahedron 2000, 56,
8385–8417.
20. Toshima, K.; Tatsuta, K. Chem. Rev. 1993, 93, 1503–1531.
21. Lear, M. J.; Yoshimura, F.; Hirama, M. Angew. Chem.,
Int. Ed. 2001, 40, 946–949.
22. Zhang, G; Fang, L.; Zhu, L.; Aimiuwu, J. E.; Shen, J.;
Cheng, H.; Muller, M. T.; Lee, G. E.; Sun, D.; Wang, P.
G. J. Med. Chem. 2005, 48, in press.