Syntheses and biological activity of bisdaunorubicins

Article abstract of DOI:10.1016/j.bmc.2005.08.014

To study the length and flexibility of the linkers between two monomers of bisdaunorubicins for their activity against cancer cells, seven bisdaunorubicins were rationally designed and synthesized through click chemistry. Their cytotoxicity was tested in

Full text of DOI:10.1016/j.bmc.2005.08.014

Bioorganic & Medicinal Chemistry 14 (2006) 426–434
Syntheses and biological activity of bisdaunorubicins
Guisheng Zhang,a Lanyan Fang,b Lizhi Zhu,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 11 July 2005; revised 8 August 2005; accepted 9 August 2005
Available online 22 September 2005
Abstract—To study the length and flexibility of the linkers between two monomers of bisdaunorubicins for their activity against
cancer cells, seven bisdaunorubicins were rationally designed and synthesized through click chemistry. Their cytotoxicity was tested
in leukemia cells with MTS assay. The results showed that the compounds with short linkers exhibited higher activity than the com-
pounds with long linkers, while the flexibility of the linker also contributed to their activity. These results indicated that the length
and flexibility of the linkers between two monomers in bisdaunorubicins are very critical to maintain their activity against cancer
cells.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
MEN 10755 (a disaccharide anthracycline) has a differ-
ent DNA binding behavior. The crystal structure of the
complex between MEN 10755 and DNA hexamer
(CGATCG)9 showed two different DNA binding sites.
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
guanine of another DNA molecule through hydrogen
bonds. This peculiar behavior suggests that the sugar
structure may interact with other cellular targets if
appropriate modifications are being made.
Many anticancer compounds in clinical use are DNA
intercalating agents, which can bind to DNA.1 These
agents bind to specific DNA sequences, form topoiso-
merase–DNA complex, and cause double strand DNA
breaks.2,3 Drug affinity to DNA is one of the dominant
factors in these interactions.2–4 Therefore, small mole-
cules that show high DNA binding and intercalation
capability are expected to provide better anticancer
activity.
The anthracycline antibiotics, daunorubicin (daunomy-
cin, DNR) and doxorubicin (adriamycin), are among
the most potent anticancer drugs in cancer chemothera-
py.5 The mechanism of action includes DNA intercala-
tion, topoisomerase inhibition, and production of free
radicals to attack DNA.6 However, the clinical effective-
ness of anthracycline is limited by their side effects and
development of multidrug resistance (MDR).7,8 Previ-
ous studies have been performed to modify anthracy-
cline glycoside antibiotics for generating analogs with
high anticancer activity and low toxicity. However, these
efforts have only achieved limited success. For instance,
To increase the DNA binding affinity of anthracycline,
the bisintercalator, which is the dimer of anthracyclines,
may provide better opportunity to develop a new series
of anticancer analogs. The potential advantages of a bis-
intercalator over its monomeric counterpart include: (1)
DNA binding affinity should be greatly enhanced. The
binding constant for the bisintercalator is roughly the
square of that for the monomer.10 (2) The interaction
area between the bisintercalator and DNA is increased
compared to the monomer. In fact, four types of
bisanthracyclines have been reported10–13 (Fig. 1). BA1
links two monomers of daunorubicin through either
C13 or C14 positions and exhibits 1.2- to 7.5-fold higher
cytotoxicity than daunorubicin.10 Doxform links two
monomers of doxorubicin through 30-NH2 group and
shows more activity than doxorubicin.12 Daunorubicin
dimer WP631 even overcomes multidrug resistance in
cultured cells.13 However, most of the dimers through
squaric acid amide show less activity than monomer.11
Keywords: Bisdaunorubicin; Click chemistry; Structure–activity
relationship; Anticancer.
*
Corresponding authors. Tel.: +1 614 292 4381; fax: +1 614 292 7766
(D.S.); tel.: +1 614 292 9884; fax: +1 614 688 3106 (P.G.W.); e-mail
addresses: sun.176@osu.edu; wang.892@osu.edu
0968-0896/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.08.014

G. Zhang et al. / Bioorg. Med. Chem. 14 (2006) 426–434
427
O
O
OH
O
O
OH
O
O
O
OH
O
O
O
OH
O
NNH
OH
OH
OH
OH
OH
CO
MeO
OH O
O
MeO
OH
OH O
O
(CH2)n
MeO
OH O
MeO
OH O
O
NH
O
N
NH2
CO
NH
HO
HO
O
NH
HO
O
(CH2)m
O
CO
O
N
O
NH
O
NH
NH
O
OH O OMe
(CH2)n
(CH2)6
O
OH O OMe
CO
O
O
HO
O
HN
OH
O
O
OH
HO
HO
O
NNH
OH
OH O
O
OH O
NH
MeO
OH O
O
O
OH O OMe
WP631
Doxform
HO
O
NH2
HO
OH O
BA1
Dimer with squaric
acid amide
Figure 1. Structures of known bisdaunorubicins.
In addition, the linkers between the two monomers are
not well characterized to maintain the anticancer activi-
ty of the bisintercalator.
two amino groups of daunorubicin to form a bisanthra-
cycline with the potential of bisintercalating into DNA.
These bisdaunorubicins were synthesized via click chem-
istry through the linkers with various lengths and flexi-
bility to test their activities against cancer cells.
Huisgen 1,3-dipolar cycloaddition reaction, click chem-
istry, has proven to be a powerful tool in biomedical re-
search and drug discovery.15 This reaction has many
advantages: (1) the azido group and alkyne are tolerable
to most reaction manipulations (no protection and
deprotection are necessary); (2) the product can be easily
isolated after the reaction. The transformation is espe-
cially useful for drug discovery because of its reliability
as a linking reaction and the favorable physicochemical
properties of triazoles. The triazole products are often
associated with biological targets through hydrogen
bonding and dipole interactions.16 In this paper, we re-
port the synthetic method of bisdaunorubicins with var-
ious linkers through click chemistry and their biological
activity.
In the present research, we intend to study the length
and the flexibility of the linkers between two monomers
of bisdaunorubicins for their anticancer activity. The
information of anthracyclines binding to DNA provides
a foundation for our rational design.6,14 It was found
that the non-covalent complexes of anthracyclines
bound to DNA are in a 2:1 stoichiometry of drug to
DNA. In such a complex, each drug intercalated at
either end of the hexanucleotide, with the daunosamine
moieties of the two drug molecules lining the minor
groove and pointing toward each other (Fig. 2).13 The
˚
distance between the two sugars is around 6A. Based
on this discovery, we rationally designed to link the
2. Results and discussion
2.1. Chemistry
O
We designed and synthesized two types of bisdaunoru-
bicins 1–7 (Fig. 3) through click reaction. The synthetic
routes of compounds 1–7 are depicted in Schemes 1 and
2. The synthetic procedures first focused on the interme-
diates 8, 9, 14, 15, 17, and 19.
o
6 A
O
Daunorubicin hydrochloride was readily reacted with
azido acetic acid in the presence of DCC at rt overnight
to produce the intermediate 8 as a red solid in 63% yield.
A similar procedure was adopted to give compound 9 in
66% yield. The 30-azido daunorubicin 15 was prepared
from daunorubicin by treatment with TfN3 solution
(made by using 2 equiv of Tf2O17) in 70% yield.
Figure 2. A schematic of the structure of monomeric anthracycline
molecules bound to a DNA hexanucleotide as revealed by high-
resolution crystallographic structures.

428
G. Zhang et al. / Bioorg. Med. Chem. 14 (2006) 426–434
studies on MEN 10755 reveal that the a-orientation of
MeO
O
O
OH
OH
OH
the second sugar is critical for the topo II poisoning abil-
ity.19,20 To make a-glycosylation with 2,6-dideoxy sug-
ars, we anticipate three synthetic challenges: first, the
stability of 2,6-dideoxy sugar donor; second, a-selective
formation of the glycosidic bond; and finally the known
low reactivity of an axial-4-hydroxyl group in the glyco-
syl acceptors.21 Thioglycosides are stable to normal pro-
tecting manipulation in oligosaccharide synthesis.22,23
Therefore, thioglycoside 12 was chosen as the glycosyl
donor. Recently, Hirama reported a direct and efficient
a-selective glycosylation protocol for the kedarcidin su-
gar and L-mycarose using AgPF6 as a activator of
2-deoxythioglycosides.24 In our research, we found that
AgPF6/TTBP (2,4,6-tert-butylpyrimidine) is the better
promoter system for the synthesis of 14. Condensation
between the thioglycosides 12 and the glycosyl acceptor
13 in the presence of AgPF6/TTBP produced exclusively
a-glycoside 14 with 82% yield. It is worth noting that the
present method is an efficient method for the synthesis
of disaccharide anthracyclines. This protocol overcame
the low reactivity of the axial-40-hydroxy group and
produced the desired a-glycosidic bond with good yield.
O
O
HO
O
O
N
HO
O
OH
O
4
HO
O
OH
N
N
N
O
N
OH
N
O
HO
HO
O
OMe
1
MeO
O
O
HO
O
O
O
H
O
O
N
N
OH
HO
O
OH
O
HN
4
O
N
N
N
O
N
OH
N
O
O
HO
O
OMe
2
O
O
O
NH2
O
HO
N
N
MeO
O
HO
O
OH
O
N
N
N
N
O
O
O
O
O
O
4
HO
OH
OH
OMe
H2N
O
3
O
O
OH
HO
O
O
MeO
O
N
N
O
N
O
HO
O
O
NH
HO
O
OH
O
HO
HN
OH
OH
N
N
Hydrolysis of daunorubicin with 0.2 M HCl at 90 °C for
1 h gave daunosamine hydrochloride 10 in 90% yield,25
which was readily converted to the 3-azido sugar 11 in
overall yield of 80% according to C.-H. Wongꢀs meth-
od.17 Treatment of 11 with thiophenol in the presence
of boron trifluoride diethyl etherate afforded thioglyco-
side 12 in 80% yield. Trifluoroacetylation of daunorubi-
cin in pyridine gave the glycosyl acceptor 13 in 95%
yield.26 Thus, condensation of 13 with 12 in the presence
of AgPF6/TTBP in CH2Cl2 at 0 °C for 3 h gave 14 with
82% yield. The spectra of 1H NMR (4.99 ppm, br H-100)
O
N
OMe
O
4
HO
O
MeO
O
O
O
OH
HO
O
HO
O
OH
O
HN
O
O
O
NH
N
HO
O
OH
O
N
OH
N
HO
O
OMe
5
1
and H-1H COSY indicate the newly formed glycosidic
O
OH
MeO
O
O
bond is a-linkage.
HO
O
O
NH
OH
O
O
N
HO
O
OH
N
O
N
The bisfunctional intermediates 17 and 19 were readily
prepared from corresponding tetraethylene glycol 16
and 1,4-bis(chloromethyl)benzene 18. Treatment of 16
with propargyl bromide in the presence of NaH in
THF at 0 °C for 6 h formed 17 with 72% yield. Starting
with commercially available 18, after treatment with
sodium azide in the presence of Et4NBr (4% weight) at
110 °C overnight, compound 19 was obtained in a quan-
titative yield.
HO
O
OH
H2N
O
O
6
OMe
HO
O
MeO
O
O
OH
O
O
HO
O
HO
OH
O
N
N
NH
O
N
HO
OH
O
O
OH
O
HO
O
7
OMe
To prepare bisdaunorubicins 1–7 through click chemis-
try, three catalytic systems were explored: CuI/DIPEA
(diisopropylethylamine),27 CuSO4Æ5H2O/sodium ascor-
bate,28 and (EtO)3PCuI/DIPEA.29 However, only
(EtO)3PCuI/DIPEA gave a satisfactory yield. Thus,
all the bisdaunorubicins were assembled through click
chemistry using this catalytic system in THF at rt.
Compounds 1, 2, 4, 5, and 7 were obtained by a click
reaction between the corresponding intermediates with
azido group and alkyne in yields of 72, 52, 70, 54, and
54%, respectively. After condensation of 14 with 17 or
9, the coupling products were deprotected by treatment
with 0.1 M NaOH. Thus, compounds 3 or 6 were
obtained in the overall yields of 32%, and 37%,
respectively.
Figure 3. Synthetic bisdaunorubicins (1–7).
To produce bisdaunorubicins containing disaccharide
monomer, we designed a disaccharide daunorubicin
14. The second sugar was produced from daunosamine
and linked to the first sugar through an a-glycosidic
bond. The rational design of 14 was based on the follow-
ing considerations: (1) natural disaccharide anthracy-
cline analogs isolated from Streptomyces sp. often
contain two or more sugars connected through an a
(1 ! 4) linkage and invariably possess an amino sugar
as the first sugar moiety attached to the aglycone.5,18
(2) intensive structure–activity relationship (SAR)

G. Zhang et al. / Bioorg. Med. Chem. 14 (2006) 426–434
429
O
OH
O
O
OH
O
SPh
h
O
+
OH
OH
82%
N3
AcO
12
OMe O
OH
O
OMe O
OH
O
g
80%
O
O
O
NHCOCF3
NHCOCF3
OAc
OH
HO
13
O
N3
AcO
80%
11
14
O
d
95%
f
N3
AcO
O
OH
O
O
OH
O
O
e
c
NH3Cl
HO
OH
90%
OH
70%
10
OMe O
OH O
OMe O
OH
O
O
O
a
b
NH3Cl
N3
HO
HO
Daunorubicin
63%
O
66%
15
O
OH
O
OH
O
OH
OH
OMe O
OH O
OMe O
OH O
O
O
NH
HO
NH
HO
O
O
8
N3
9
Scheme 1. Reagents and conditions: (a) N3CH2CO2H, DCC, CH2Cl2, overnight; (b) CHꢀCCH2CO2H, DCC, CH2Cl2, overnight; (c) K2CO3,
CuSO4, TfN3 solution; (d) (CF3CO)2O/pyridine, ꢁ20 °C, 15 min; (e) 0.2 M HCl, 90 °C, 1 h; (f) K2CO3, CuSO4, TfN3 solution; then Ac2O/pyridine;
(g) PhSH, BF3ÆEt2O, CH2Cl2, 0 °C, 2 h; (h) AgPF6, TTBP, CH2Cl2, 0 °C, 2 h.
H
between the two monomers. Compound 1 killed about
35% of K562 cells at the concentration of 25 lM, while
compounds 2 and 3 had no cytotoxicity at all at 25 lM
concentration (Fig. 4). On the other hand, compounds
HO
c
O
a
17
17
+
15
8
1
2
4
72%
c
16
72%
+
52%
O
O
c, d
4
17
19
+
+
14
3
32%
17
140
8
c
9
120
70%
1
Cl
100
2
c
8
+
+
+
3
Cl
80
54%
18
60
40
20
0
c, d
14
15
9
9
6
7
b
100%
37%
N3
c
N3
54%
19
0
0.5
5
10
25
Drug Concentration(uM)
Scheme 2. Reagents and conditions: (a) CHꢀCCH2Br, NaH, THF,
6 h; (b) NaN3, Et4NBr (cat), overnight; (c) (EtO)3PCuI, DIPEA, THF,
overnight; (d) 0.1 M NaOH, THF, 0 °C.
140
120
100
80
8
9
4
5
6
7
2.2. Cytotoxicity
The cytotoxicities of these bisdaunorubicins (1–7) and
two intermediates (8 and 9) were examined in leukemia
cell line K562 cells with MTS assay as described.30–32
2000–10,000 cells were incubated with 0.001–5 lM of
each compound 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 per-
centage of cell control group without treatment.
60
40
20
0
0
0.5
5
10
25
Drug Concentration(uM)
Figure 4. Cytotoxicity of compounds 1–9 on leukemia K562 cells by
MTS assay (cell survival is compared to control group without
treatment of any drugs. The data are average (X SD) of six
experiments).
Compounds 1, 2, and 3 belong to a group with long flex-
ible chains, but compounds 2 and 3 have longer linkers

430
G. Zhang et al. / Bioorg. Med. Chem. 14 (2006) 426–434
4–7 belong to another group carrying similar relative
rigid chains. Compound 7 showed the highest activity
with the IC50 value of 20 lM, while compounds 4–6
(with longer linkers) completely lost their activity even
at highest concentration (25 lM) (Fig. 4). These results
indicated that the length of the linker between the two
monomers in the bisdaunorubicins is very critical for
their activity against cancer cells.
Intermediate 9 was obtained by condensation of dauno-
rubicin hydrochloride with propiolic acid as the same
procedure with a yield of 66%.
3.1.3. Procedure for the synthesis of sugar donor 12 from
daunorubicin. The daunorubicin hydrochloride (10.0 g)
was stirred in 0.2 M hydrochloric acid (800 mL) at
90 °C for 1 h. After cooling, the orange precipitate was
collected and dried to give daunorubicinone (6.4 g,
91% yield). The filtrate was evaporated to dryness to
give 10 (2.9 g, 90%) as a white solid, which was used
in the next reaction without further purification.
Comparing compounds 1, 4–6, the linker in compound 1
has higher flexibility than those in compounds 4–6.
Interestingly, although the linker in compound 1 is even
longer than those in compounds 4–6, compound 1
showed significant better activity while compounds 4–6
completely lost their activity. This result suggested that
the flexibility of the linker may also play a critical role
to determine the activity of these bisdaunorubicins. It
was also observed that the bisdaunorubicin 7 and mono-
mers (8 and 9) exhibited similar activity, but lower than
that of the parent compound daunorubicin. This indi-
cated that conversion of the 30-amino group to an amide
is not favored for developing more potent drug candi-
dates. The reservation of the basicity of the amino group
is required in the future drug design.
Daunosamine hydrochloride 10 (1.84 g, 10 mmol) was
dissolved in water (30 mL) and treated with potassium
carbonate (2.07 g, 15 mmol) and CuSO4Æ5H2O (15 mg,
0.1 mmol). To the mixture were added MeOH (60 mL)
and the TfN3 solution (made by using 2 equiv of
Tf2O17). Then, more MeOH was added to homogeneity.
The reaction mixture was stirred for 18 h. After the sol-
vent was removed, the residue was acetylated using
Ac2O (30 mL) and pyridine (40 mL) with catalytic
DMAP. The solvent was evaporated under vacuum.
The residue was dissolved in EtOAc and washed with
water. After removal of the solvent, column chromatog-
raphy of the crude product on a silica gel using EtOAc/
hexane (1:4) afforded the product 11 as a yellow oil
(2.05 g, 80%).
In summary, seven bisdaunorubicins were effectively
constructed using the click reactions with (EtO)3PCuI
as catalyst in organic solvent. These bisdaunorubicins
have various lengths and flexibility of the linkers be-
tween two monomers. The cytotoxicity studies indicated
that the compound with shorter linker displays the high-
er activity against cancer cells, while the flexibility of the
linker also contributes to their activity.
A solution of 11 (1.9 g, 6.2 mmol), thiophenol (678 mg,
6.2 mmol) in toluene (10 mL) was cooled to 0 °C (ice
bath), and then boron trifluoride ethyl etherate
(678 lL) was added. The mixture was warmed to rt
and stirred for 2 h. After cooling to 0 °C by 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 evaporat-
ed under vacuum to give the crude product. Purification
through a column of silica gel (5% Et2O in hexane) gave
the pure compound 12 as a pale yellow oil (80%,
a-anomer).
3. Experimental section
3.1. Chemistry
3.1.1. General information. All solvents were dried with
solvent-purification system from Innovative Technolo-
gy, Inc. All reagents were obtained from commercial
sources and used without further purification. Analyti-
cal TLC was carried out on silica gel 60 F254 alumi-
num-backed plates (E. Merck). The 230–400 mesh size
of the same absorbent was utilized for all chromato-
3.1.4. Procedure for the synthesis of sugar acceptor 13
from daunorubicin. Daunorubicin hydrochloride (16.0 g,
2.6 mmol) was stirred in dry pyridine (16 mL) at ꢁ20 °C
for 0.5 h. Trifluoroacetic anhydride (28 mL) in anhy-
drous ether (200 mL) was added dropwise over a 1.5 h
period. After 0.5 h, water (250 mL) was added and stir-
ring was continued for additional 0.5 h. The reaction
mixture was extracted with ethyl acetate and the extracts
were washed with water. After drying over Na2SO4, the
solvent was evaporated, and the residue was heated at
reflux with MeOH (50 mL) for 0.5 h. MeOH was dis-
tilled off on the rotary evaporator, and the residue was
precipitated from chloroform–pentane to afford 13 as
a deep red solid (16.8 g, 95%).
1
graphic purifications. H and 13C NMR spectra were
recorded at the indicated field strengths. The high-reso-
lution mass spectra were collected at The Ohio State
University Campus Chemical Instrumentation Center.
3.1.2. Procedure for preparation of intermediates 8 and 9.
A solution of daunorubicin hydrochloride (1.65 g,
2.93 mmol), azidoacetic acid (0.28 g, 2.77 mmol), and
DMAP (0.35 g, 2.86 mmol) in CH2Cl2 (100 mL) was
stirred at 0 °C for 1 h, and then DCC (0.88 g, 4.3 mmol)
was added to the solution. The reaction mixture was
then stirred at rt overnight. The sediment was removed
and the filtrate was washed with saturated NH4Cl aque-
ous solution, water, and dried over Na2SO4. After the
solvent was evaporated, the crude product was purified
on a column of silica gel (MeOH/CH2Cl2 1:40) to give
compound 8 as a red solid (1.2 g, 63%).
3.1.5. Procedure for preparation of intermediate 14. A
mixture of 12 (0.35 g, 1.1 mmol), 13 (1.2 g, 1.9 mmol),
˚
TTBP (1.46 g, 5.9 mmol), and molecular sieves (4 A,
<5 microns, freshly activated, 1.8 g) in CH2Cl2
(15 mL) was stirred at rt for 2 h under N2 and then

G. Zhang et al. / Bioorg. Med. Chem. 14 (2006) 426–434
431
cooled to 0 °C (ice bath). Powdered AgPF6 (1 g,
3.9 mmol) was added and stirred for 3 h. Subsequently,
pyridine (7 mL) was added and stirred for a further
0.5 h. Filtration (through a Celite pad), concentration,
and chromatographic purification (MeOH/CH2Cl2
1:150) provided the products 14 as a red solid (2 g, 82%).
0.074 mmol) in THF (2 mL) were added (EtO)3PCuI
(0.2 equiv) and diisopropylethylamine (0.3 equiv). The
reaction mixture was stirred at rt for 48 h. Water
(2 mL) and (NH4)2S solution (22%, 2 mL) were added,
and the resultant mixture was stirred for additional
2 h. The product was extracted with CH2Cl2. The com-
bined organic layers were washed with saturated NaH-
CO3 solution and dried over Na2SO4. Concentration
and purification on a column of silica gel (CH2Cl2/
MeOH 30:1) provided a red solid.
3.1.6. Procedure for preparation of intermediate 15. Dau-
norubicin hydrochloride (5.24 g, 9.3 mmol) was dis-
solved in water (30 mL) and treated with potassium
carbonate (1.92 g, 13.9 mmol) and CuSO4Æ5H2O
(14 mg, 88 lmol). MeOH (60 mL) was added to the
solution and the TfN3 solution (made using 2 equiv of
Tf2O17). Then, adequate MeOH was added to homoge-
neity. The reaction mixture was allowed to be stirred
overnight. The mixture was diluted with H2O
(100 mL) and extracted with CH2Cl2. The combined
extractions were dried over anhydrous Na2SO4. After
removal of the solvent, the residue was purified through
a column of silica gel using MeOH/CH2Cl2 (1:100–50) to
afford 15 as a red solid (70%).
A solution of the solid obtained above in THF (5 mL)
was cooled in ice bath to 0 °C, and then 0.1 M NaOH
aqueous solution (70 mL), which was cooled in advance
in ice bath, was added. After stirring 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 and dried
over Na2SO4. Concentration and chromatographic puri-
fication on a column of silica gel (CH2Cl2/MeOH 4:1)
provided 3 as a dark red solid (45.6 mg, 32% of two
steps).
3.1.7. Procedure for preparation of intermediate 17. To a
solution of tetraethylene glycol (16) (1 g, 5.15 mmol) in
THF (30 mL) was added NaH (0.62 g, of 60% suspen-
sion in oil, 15.5 mmol) at 0 °C. The resulting mixture
was stirred at 0 °C for 0.5 h and then at rt for 0.5 h fol-
lowed by the addition of propargyl bromide (1.84 g,
15.5 mmol) under N2 atmosphere. After 6 h, the resul-
tant solution was quenched with water (150 mL) at
0 °C and extracted with EtOAc (500 mL). The organic
layer was washed with water and dried over MgSO4.
The crude product was purified on a column of silica
gel (EtOAc/hexane 1:3) to give 17 as a yellow oil (1 g,
72%).
3.1.11. Procedure for synthesis of bisdaunorubicin 4. To a
solution of 8 (116 mg, 0.20 mmol) and 19 (10 mg,
0.05 mmol) in THF (2 mL) were added (EtO)3PCuI
(0.2 equiv) and diisopropylethylamine (0.3 equiv). The
reaction mixture was stirred at rt for 48 h. Water
(2 mL) and (NH4)2S solution (22%, 2 mL) were added,
and the resultant mixture was stirred for additional
2 h. The product was extracted with CH2Cl2. The
combined organic layers were washed with saturated
NaHCO3 solution and dried over Na2SO4. Concentra-
tion and purification on a column of silica gel
(CH2Cl2/MeOH 20:1–10:1) provided 4 as a red solid
(50 mg, 70%).
3.1.8. Procedure for preparation of intermediate 19. The
(5.0 g,
mixture
of
1,4-bis(chloromethyl)benzene
3.1.12. Procedure for synthesis of bisdaunorubicin 5 and
7. To a solution of 8 (61 mg, 0.10 mmol) and 9 (58 mg,
0.1 mmol) in THF (2 mL) were added (EtO)3PCuI (0.2
equiv) and diisopropylethylamine (0.3 equiv). The reac-
tion mixture was stirred at rt for 48 h. Water (2 mL) and
(NH4)2S solution (22%, 2 mL) were added, and the
resultant mixture was stirred for additional 2 h. The
product was extracted with CH2Cl2. The combined
organic layers were washed with saturated NaHCO3
solution and dried over Na2SO4. Concentration and
purification on a column of silica gel (CH2Cl2/MeOH
20:1–10:1) provided 5 as a red solid (64 mg, 54%).
28 mmol), sodium azide (3 equiv), and Et4NBr (4%
weight) was heated to 110 °C and stirred overnight.
After cooling, ether (100 mL) was added. The sediment
was removed and the filtrate was evaporated to give 19
as a yellow oil in quantitative yield.
3.1.9. Procedure for preparation of bisdaunorubicin 1 and
2. To a solution of 15 (110 mg, 0.20 mmol) and 17
(13.5 mg, 0.05 mmol) in THF (1.5 mL) were added
(EtO)3PCuI (0.2 equiv) and diisopropylethylamine (0.3
equiv). The reaction mixture was stirred at rt for 48 h.
Water (2 mL) and (NH4)2S solution (22%, 2 mL) were
added, and the resultant mixture was stirred for addi-
tional 2 h. The product was extracted with CH2Cl2.
The combined organic layers were washed with saturat-
ed NaHCO3 solution and dried over Na2SO4. Concen-
tration and purification on a column of silica gel
(CH2Cl2/MeOH 20:1–10:1) provided 1 as a dark red
solid (49.7 mg, 72%).
Compound 7 was obtained by coupling of 15 with 9 as
the same procedure in a yield of 54%.
3.1.13. Procedure for synthesis of bisdaunorubicin 6. To a
solution of 14 (82 mg, 0.1 mmol) and 9 (58 mg,
0.1 mmol) in THF (2 mL) were added (EtO)3PCuI (0.2
equiv) and diisopropylethylamine (0.3 equiv). The reac-
tion mixture was stirred at rt for 48 h. Water (2 mL) and
(NH4)2S solution (22%, 2 mL) were added, and the
resultant mixture was stirred for additional 2 h. The
product was extracted with CH2Cl2. The combined
organic layers were washed with saturated NaHCO3
solution and dried over Na2SO4. Concentration and
Compound 2 was obtained by condensation of 8 with 17
as the same procedure in a yield of 52%.
3.1.10. Procedure for synthesis of bisdaunorubicin 3. To a
solution of 14 (188 mg, 0.22 mmol) and 17 (20 mg,

432
G. Zhang et al. / Bioorg. Med. Chem. 14 (2006) 426–434
purification on column of silica gel (CH2Cl2/MeOH
25:1) provided a red solid.
3.94 (3H, s, MeO-4), 3.54 (1H, br, H-400), 3.29 (1H, s,
CCH), 3.28 (1H, d, J = 18.5 Hz, Ha-10), 2.74 (1H, d,
J = 18.5 Hz, Hb-10), 2.31 (3H, s, H-14), 2.24 (1H, m,
Ha-8), 2.00 (1H, m, Hb-8), 1.87 (1H, m, Ha-20), 1.68
(1H, m, Hb-20), 1.21 (3H, d, J = 6.5 Hz, H-60). 13C
NMR (125 MHz, CD3OD + CDCl3) 212.5, 186.3,
186.2, 166.5, 160.7, 156.1, 154.5, 136.1, 135.5, 134.5,
134.4, 119.9, 119.6, 118.9, 110.6, 110.5, 100.2, 75.1,
70.1, 67.8, 66.6, 56.5, 54.9, 50.5, 45.3, 36.1, 31.5, 29.6,
24.0, 16.9.
A solution of the solid obtained above in THF (5 mL)
was cooled in ice bath to 0 °C, and then 0.1 M NaOH
aqueous solution (70 mL), which was cooled in advance
in ice bath, was added. After stirring for 6–8 h, the
reaction mixture was neutralized with 0.1 M citric acid
and extracted with CHCl3. The extracts were washed
with saturated aqueous NaHCO3 solution and dried
over Na2SO4. Concentration and chromatographic
purification on a column of silica gel (CH2Cl2/MeOH
10:1) provided 6 (46.7 mg, 37% of two steps).
3.3.3. Thiophenyl 4-O-acetyl-3-azido-L-lyxo-hexopyrano-
side (12). HRMS (M+Na)+ (ESI+) calcd for
1
C14H17N3O3NSa+ 330.0883, found 330.0885; H NMR
3.2. Biology
(500 MHz, CDCl3) 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, 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,
J = 6.3 Hz, H-6).
3.2.1. Cell culture. Cell line K562 was cultured in RPMI
1640 supplemented with 10% fetal bovine serum (FBS),
1% non-essential amino acids, and penicillin (100 U/mL)
streptomycin (100 lg/mL) in a humidified atmosphere of
5% CO2 and 95% air at 37 °C. The culture media were
changed every 2–3 days.
3.3.4. N-(Trifluoroacetyl)daunorubicin (13). HRMS
(M+Na)+ (ESI+) calcd for C29H28F3NO11Na+
646.1507, found 646.1493. 1H NMR (500 MHz, CDCl3)
13.98 (1H, s, OH-6), 13.25 (1H, s, OH-11), 8.02 (1H,
d, J = 7.6 Hz, H-1), 7.68 (1H, t, J = 8.2 Hz, H-3), 7.37
(1H, d, J = 8.2 Hz, H-3), 6.63 (1H, d, J = 8.4 Hz,
NHCOCF3), 5.50 (1H, d, J = 3.9 Hz, H-10), 5.25 (1H,
dd, J = 1.9 Hz, , J = 3.9 Hz, H-7), 4.24 (2H, m, H-30,
H-50), 4.06 (3H, s, OMe-4), 3.65 (1H, d, J = 2.2 Hz, H-
40), 3.25 (1H, d, J = 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-20), 1.80
(1H, m, Hb-20), 1.28 (3H, d, J = 6.6 Hz, H-60).
3.2.2. Cytotoxicity of synthesized compounds (MTS
assay). K562 leukemia cells (2000–10,000) were seeded
in 96-well plates in RPMI-1640 and incubated for
24 h. The exponentially growing cancer cells were incu-
bated with various concentrations of each compound
for 72 h at 37 °C (5% CO2, 95% humidity). After 72 h
incubation, tetrazolium[3-(4,5-dimethythiazol-2-yl)]-5-
(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetra-
zolium, inner salt (MTS, 2 mg/mL), and phenazine
methosulfate (PMS, 25 lM) were mixed and added
directly to the cells. After incubated for 3 h at 37 °C,
the absorbance of formazan (the metabolite of MTS
by viable cells) was measured at 490 nm.
3.3.5.
7-[4-O-(4-O-Acetyl-3-azido-L-lyxo-hexopyrano-
syl)-2,3,6-trideoxy-3-trifluoroacetoamido-a-L-lyxo-hexo-
pyranosyl]-daunorubicinone (14). HRMS (M+Na)+
(ESI+) calcd for C37H39F3N4O14Na+ 843.2307, found
3.3. Spectroscopic data
1
843.2331. H NMR (500 MHz, CDCl3) 13.94 (1H, s,
3.3.1. N-Azidoacetyl daunorubicin (8). HRMS (M+Na)+
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-10), 5.24 (1H, br, H-7), 5.21 (1H, br, H-400), 4.99
(1H, br, H-100), 4.20 (3H, m, H-30, H-50, H-500), 4.04
(3H, s, MeO-4), 3.92 (1H, m, H-300), 3.58 (1H, br, H-
40), 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-200, Hb-8),
2.02 (1H, m, Hb-200), 1.86 (1H, m, Ha-20), 1.77 (1H,
m, Hb-20), 1.27 (3H, d, J = 6.4 Hz, H-60), 1.14 (3 H, d,
J = 6.6 Hz, H-600). 13C NMR (125 MHz, CDCl3) 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.
(ESI+) calcd for C29H30N4O11Na+ 633.1803, found
633.1787. H NMR (500 MHz, DMSO-d6) 13.95 (1H,
1
s, HO-6), 13.20 (1H, s, HO-11), 7.84 (3H, m, H-1, H-
2, NHCO), 7.56 (1H, m, H-3), 5.48 (1H, s, OH-8),
5.22 (1H, d, J = 3.3 Hz, H-10), 4.89 (1H, m, H-7), 4.83
(1H, d, J = 6.1 Hz, OH-40), 4.19 (1H, q, J = 6.7 Hz, H-
50), 4.00 (1H, m, H-30), 3.94 (3H, s, MeO-4), 3.77 (1H,
d, J = 15.5 Hz, Ha-10), 3.75 (1H, d, J = 15.5 Hz, Hb-
10), 3.42 (1H, m, H-40), 2.89 (2H, s, CH2N3), 2.27
(3H, s, H-14), 2.21 (1H, m, Ha-8), 2.06 (1H, m, Hb-8),
1.84 (1H, m, Ha-20), 1.47 (1H, m, Hb-20), 1.14 (3H, d,
J = 6.5 Hz, H-60). 13C NMR (125 MHz, DMSO-d6)
211.7, 186.4, 186.0, 160.5, 155.6, 154.7, 151.9, 134.7,
133.8, 133.7, 120.1, 110.9, 110.6, 100.2, 76.5, 76.0,
73.9, 69.5, 67.9, 66.8, 55.9, 49.2, 48.9, 46.5, 45.7, 34.9,
32.4, 28.7, 24.1, 16.1.
3.3.2. N-Propiolyl daunorubicin (9). HRMS (M+Na)+
3.3.6.
7-(3-Azido-2,3,6-trideoxy-a-L-lyxo-hexopyrano-
(ESI+) calcd for C30H29NO11Na+ 602.1633, found
602.1630. H NMR (500 MHz, CD3OD + CDCl3) 7.79
syl)-daunorubicinone (15). HRMS (M+Na)+ (ESI+)
calcd for C27H27N3O10Na+ 576.1589, found 576.1612.
1H NMR (500 MHz, CDCl3) 13.95 (1H, s, HO-6),
13.19 (1H, s, HO-11), 7.98 (1H, d, J = 7.4 Hz, H-1),
7.74 (1H, t, J = 8.2 Hz, H-2), 7.36 (1H, d, J = 8.4 Hz,
1
(1H, d, J = 7.5 Hz, H-1), 7.64 (1H, t, J = 8.0 Hz, H-2),
7.30 (1H, d, J = 8.4 Hz, H-3), 5.37 (1H, d, J = 3.2 Hz,
H-10), 5.06 (1H, br, H-7), 4.08 (2H, m, H-30, H-50),

G. Zhang et al. / Bioorg. Med. Chem. 14 (2006) 426–434
433
1
H-3), 5.54 (1H, d, J = 3.6 Hz, H-10), 5.23 (1H, d,
J = 1.9 Hz, H-7), 4.37 (1H, s, HO-9), 4.10 (1H, m, H-
50), 4.05 (3H, s, MeO-4), 3.69 (1H, br, H-40), 3.60 (1H,
m, H-30), 3.15 (1H, dd, J = 1.6 Hz, J = 18.8 Hz, Ha-
10), 2.87 (1H, d, J = 18.8 Hz, Hb-10), 2.38 (3H, s, H-
14), 2.28 (1H, m, Ha-8), 2.09 (2H, m, Hb-8, Ha-20),
1.91 (1H, m, Hb-20), 1.30 (3H, d, J = 6.6 Hz, H-60).
13C NMR (125 MHz, CDCl3) 211.5, 186.9, 186.7,
161.1, 156.3, 155.7, 135.7, 135.5, 134.2, 133.9, 120.8,
119.8, 118.5, 111.5, 111.3, 100.6, 76.7, 70.1, 69.5, 67.1,
56.8, 56.7, 34.9, 33.3, 28.5, 24.7, 16.8.
for C80H98N8O29Na+ 1657.6332, found 1657.6334. H
NMR (400 MHz, DMSO-d6) 8.07 (2H, s, H-triazole),
7.80 (4H, m, H-1, H-2), 7.55 (2H, d, J = 8.1 Hz, H-
3), 5.37 (2H, br, H-10), 5.23 (2H, br, H-100), 5.16 (2H,
d, J = 4.3 Hz, H-7), 5.10 (4H, m, HO-9, H-30), 4.85
(2H, br, HO-400), 4.55 (4H, s, OCH2-triazole), 4.34
(2H, q, J = 6.6 Hz, H-50), 4.15 (2H, q, J = 6.5 Hz, H-
500), 3.94 (4H, s, MeO-4), 3.73 (2H, br, H-40), 3.59
(18H, m, H-400, OCH2CH2O), 3.20 (4H, m, NH2-30),
2.97 (2H, m, H-300), 2.52 (2H, m, Ha-8), 2.27 (6H, s,
H-14), 2.14 (2H, m, Hb-8), 1.99 (4H, m, H-200), 1.71
(2H, m, Ha-20), 1.15 (2H, m, Hb-20), 1.17 (6H, d,
J = 6.3 Hz, H-60), 1.09 (6H, d, J = 6.3 Hz, H-600). 13C
NMR (100 MHz, DMSO-d6) 212.0, 186.5, 186.4,
161.2, 161.1, 156.6, 154.9, 143.9, 136.5, 136.4, 136.0,
135.2, 135.1, 123.2, 123.1, 120.3, 119.3, 111.1, 110.9,
100.7, 98.7, 75.7, 70.3, 70.2, 70.1, 69.5, 69.0, 67.7,
67.4, 64.2, 64.1, 56.9, 56.7, 48.6, 36.4, 29.8, 29.7,
24.5, 17.8, 17.3.
1
3.3.7. 7,10,13,16-Pentaoxanonadeca-1,18-diyne (17). H
NMR (250 MHz, CDCl3) 4.21 (4H, s, CꢀCCH2O),
3.60 (16H, m, OCH2CH2O), 2.35 (2H, s, Cꢀ CH).
3.3.8. 1,4-Bis(azidomethyl)benzene (19). 1H NMR
(250 MHz, CDCl3) 7.33 (4H, s, Ar-H), 4.34 (4H, s, CH2).
3.3.9. 1,15-Bis(1-(daunorubicin-30-yl)-[1,2,3]triazol-4- yl)-
2,5,8,11-tetraoxapentadecane (1). HRMS (M+Na)+
(ESI+) calcd for C68H76N6O25Na+ 1399.4752, found
3.3.12.
1,4-Bis-(4-(daunorubicin-30-N-yl-carbonyl)-
[1,2,3]triazol-1-yl-methyl)benzene (4). HRMS (M+Na)+
1
1399.4684. H NMR (500 MHz, CDCl3) 13.89 (2H, s,
(ESI+) calcd for C68H66N8O22Na+ 1369.4184, found
1369.4224. H NMR (400 MHz, DMSO-d6) 13.91 (2H,
1
HO-6), 13.09 (2H, s, HO-11), 7.91 (2H, d, J = 7.6 Hz,
H-1), 7.69 (2H, t, J = 8.2 Hz, H-2), 7.62 (2H, s, H-tria-
zole), 7.29 (2H, d, J = 8.5 Hz, H-3), 5.61 (2H, d,
J = 3.2 Hz, H-10), 5.21 (1H, br, H-7), 4.86 (2H, m, H-
30), 4.64 (2H, d, J = 6.1 Hz, HO-40), 4.50 (2H, d,
J = 12.3 Hz, OCH2-triazole), 4.44 (2H, d, J = 12.3 Hz,
OCH2-triazole), 4.37 (2H, s, HO-9), 4.30 (2H, q,
J = 6.5 Hz, H-50), 4.04 (2H, d, J = 5.6 Hz, H-40), 3.98
(6H, s, MeO-4), 3.58 (16H, m, –OCH2CH2O–), 3.14
(2H, d, J = 18.5 Hz, Ha-10), 2.84 (2H, d, J = 18.5 Hz,
Hb-10), 2.51 (2H, m, Ha-8), 2.39 (6H, s, H-14), 2.35
(2H, m, Hb-8), 2.10 (2H, m, Ha-20), 1.98 (2H, m, Hb-
20), 1.35 (6H, d, H-60). 13C NMR (125 MHz, CDCl3)
211.9, 186.8, 186.4, 160.9, 156.3, 155.6, 144.0, 135.7,
135.3, 134.4, 133.9, 121.9, 120.7, 119.7, 118.4, 111.3,
111.2, 100.3, 76.6, 70.6, 70.5, 70.0, 69.8, 68.8, 67.7,
64.4, 57.1, 56.6, 35.1, 33.2, 29.2, 24.8, 16.9.
s, HO-6), 13.17 (2H, s, HO-11), 8.54 (2H, s, H-triazole),
7.81 (4H, m, H-1, H-2), 7.65 (2H, d, J = 6.8 Hz,
NHCO), 7.55 (2H, d, J = 6.4 Hz, H-3), 7.29 (4H, s, H-
Ph), 5.57 (4H, s, CH2-Ph), 5.46 (2H, s, HO-9), 5.25
(2H, d, J = 1.8 Hz, H-10), 4.97 (2H, d, J = 4.9 Hz, HO-
40), 4.93 (2H, br, H-7), 4.21 (4H, m, H-30, H-50), 3.92
(6H, s, MeO-4), 3.47 (2H, d, J = 3.2 Hz, H-40), 2.92
(4H, s, H-10), 2.27 (6H, s, H-14), 2.22 (2H, m, Ha-8),
2.12 (2H, m, Hb-8), 1.91 (2H, m, Ha-20), 1.62 (2H, m,
Hb-20), 1.15 (6H, d, J = 6.1 Hz, H-60). 13C NMR
(100 MHz, DMSO-d6) 212.1, 186.8, 186.7, 161.2,
159.0, 156.6, 154.9, 143.2, 136.5, 136.1, 136.0, 135.0,
134.9, 128.9, 126.9, 120.4, 120.1, 119.3, 111.1, 111.0,
100.6, 75.7, 70.5, 68.5, 67.2, 56.9, 55.3, 53.2, 45.3, 36.7,
32.1, 30.4, 24.5, 17.3.
3.3.13. 1-(Daunorubicin-30-N-yl-carbonylmethyl)-4-(dau-
norubicin-30-N-yl-carbonyl)[1,2,3]triazole (5). HRMS
3.3.10. 1,15-Bis(1-(daunorubicin-30-N-yl-carbonylmeth-
yl)-[1,2,3]triazol-4-yl)-2,5,8,11-tetraoxapentadecane (2).
HRMS (M+Na)+ (ESI+) calcd for C72H82N8O27Na+
1513.5182, found 1513.5233. 1H NMR (400 MHz,
DMSO-d6) 8.14 (2H, d, J = 8.0 Hz, H-1), 7.92 (2H, s, H-
triazole), 7.76 (4H, m, H-2, NHCO), 7.49 (2H, m, H-3),
5.45 (2H, s, HO-9), 5.25 (2H, br, H-10), 5.05 (2H, br, H-
7), 4.91 (2H, m, H-30), 4.87 (2H, d, J = 6.1 Hz, HO-40),
4.47 (4H, s, OCH2-triazole), 4.19 (2H, q, J = 6.5 Hz, H-
50), 4.03 (2H, m, H-40), 3.91 (6H, s, MeO-4), 3.43 (20H,
m, –OCH2CH2O–, H-10), 2.89 (4H, s, NCH2CO), 2.32
(8H, m, H-14, Ha-8), 2.21 (2H, m, Hb-8), 1.93 (2H, m,
Ha-20), 1.55 (2H, m, Hb-20), 1.16 (6H, d, J = 6.4 Hz, H-
60). 13C NMR (100 MHz, DMSO-d6) 212.1, 212.0,
186.7, 186.6, 165.1, 161.1, 156.6, 154.9, 144.1, 136.4,
135.9, 134.9, 134.8, 125.6, 120.3, 119.9, 119.2, 111.1,
110.9, 100.7, 75.7, 70.1, 70.0, 69.3, 68.3, 67.1, 63.9, 58.9,
52.0, 46.0, 36.7, 36.6, 32.0, 30.1, 24.5, 17.4.
(M+Na)+
(ESI+) calcd
for
C59H59N5O22Na+
1212.3544, found 1212.3579. 1H NMR (500 MHz,
DMSO-d6) 13.91 (2H, s), 13.17 (2H, d), 8.36 (1H, s),
8.13 (1H, d, J = 8.1 Hz), 7.81 (4H, m), 7.64 (1H, d,
J = 8.6 Hz), 7.54 (2H, m), 5.46 (1H, s), 5.41 (1H, s),
5.23 (2H, br), 5.08 (2H, s), 4.99 (1H, d, J = 6.1 Hz),
4.91 (2H, br), 4.82 (1H, s), 4.17 (3H, m), 3.93 (1H, m),
3.92 (6H, 2s), 3.42 (2H, m), 2.89 (4H, m), 2.26 (3H, s),
2.25 (3H, s), 2.24 (1H, m), 2.08 (2H, m), 1.89 (2H, m),
1.55 (1H, m), 1.45 (1H, m), 1.21 (1H, m), 1.12 (6H,
m). 13C NMR (100 MHz, DMSO-d6) 212.1, 186.8,
186.7, 161.2, 159.0, 156.6, 154.9, 142.7, 136.5, 136.0,
135.1, 134.9, 128.2, 120.5, 120.1, 119.4, 111.2, 111.1,
100.6, 75.7, 75.6, 70.6, 70.4 68.5, 65.5, 57.0, 55.3, 55.3,
52.2, 46.0, 45.3, 36.9, 32.5, 30.9, 30.3, 24.5, 24.4, 17.4.
3.3.14.
1-(1-(Daunorubicin-40-O-yl)-2,3,6-trideoxy-L-
lyxo-hexopyranos-3-yl)-4-(daunorubicin-30-N-yl-carbon-
yl) [1,2,3]triazole (6). HRMS (M+Na)+ (ESI+) calcd for
C63H67N5O23Na+ 1284.4119, found 1284.4154. 1H
NMR (400 MHz, DMSO-d6) 8.46 (1H, s), 7.75 (4H,
3.3.11. 1,15-Bis(1-(1-(daunorubicin-40-O-yl)-2,3,6-tride-
oxy-L-lyxo-hexopyranos-3-yl)[1,2,3]triazol-4-yl)-2,5,8,11-
tetraoxapentadecane (3). HRMS (M+Na)+ (ESI+) calcd

434
G. Zhang et al. / Bioorg. Med. Chem. 14 (2006) 426–434
6. Chaires, J. B. Advances in DNA Sequence Specific Agents
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m), 7.53 (2H, d, J = 7.6 Hz), 5.47 (1H, s), 5.35 (1H, s),
5.26 (1H, s), 5.22 (1H, s), 5.13 (4H, m), 4.85 (1H, br),
4.53 (1H, br), 4.29 (3H, m), 4.11 (1H, m), 3.92 (6H,
2s), 3.71 (1H, br), 3.51 (2H, m), 2.95 (5H, m), 2.49
(1H, m), 2.28 (3H, s), 2.24 (3H, s), 1.97 (5H, m), 1.65
(2H, m), 1.51 (1H, m), 1.18 (3H, d, J = 6.2 Hz), 1.14
(3H, d, J = 6.2 Hz), 1.05 (3H, d, J = 6.2 Hz). 13C
NMR (100 MHz, DMSO-d6) 212.2, 212.0, 186.6,
186.54, 186.5, 161.2, 159.3, 156.6, 154.9, 154.7, 142.6,
142.5, 136.5, 136.49, 136.47, 135.9, 135.0, 134.9,
134.85, 134.83, 120.3, 120.2, 119.6, 119.3, 110.9,
110.85, 100.7, 100.6, 98.6, 75.7, 70.6, 68.6, 68.5, 67.1,
57.0, 56.9, 45.2, 36.5, 36.4, 32.1, 32.0, 30.5, 24.5, 24.4,
17.8, 17.4, 17.2.
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3.3.15. 1-(Daunorubicin-30-yl)-4-(daunorubicin-30-N-yl-
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calcd for C57H56N4O21Na+ 1155.3329, found
1
1155.3352. H NMR (500 MHz, DMSO-d6) 13.54 (1H,
m), 13.45 (1H, m), 12.91 (1H, br), 12.78 (1H, br), 8.51
(1H, s), 7.66 (1H, d, J = 7.1 Hz), 7.30 (4H, m), 6.91
(2H, m), 5.40 (1H, s), 5.33 (1H, s), 5.18 (2H, s), 5.10
(1H, d), 5.00 (1H, m), 4.84 (1H, d, J = 5.5 Hz), 4.70
(1H, br), 4.36 (1H, d, J = 6.3 Hz), 4.19 (2H, d,
J = 6.1 Hz), 3.78 (1H, d, J = 4.6 Hz), 3.55 (1H, br),
3.35 (1H, m), 3.25 (6H, s), 2.80 (4H, m), 2.53 (1H, m),
2.28 (3H, s), 2.25 (3H, s), 2.11 (5H, m), 1.84 (1H, m),
1.58 (1H, m), 1.18 (3H, d, J = 6.5 Hz), 1.16 (3H, d,
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156.1, 154.4, 142.6, 135.8, 135.7, 135.6, 135.1, 134.2,
134.1, 134.0, 125.5, 119.4, 117.9, 117.7, 110.7, 110.6,
110.5, 100.7, 99.6, 75.7, 75.6, 79.3, 70.7, 69.4, 69.3,
68.4, 67.1, 67.0, 57.4, 56.4, 56.3, 45.6, 36.3, 31.9, 31.8,
29.7, 28.9, 24.5, 24.4, 17.5, 17.2.
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This work was supported by NSF (CH-0316806) to P. G.
Wang and was partially supported by ACS Grant IRG-
98-278-04, Research Starter Grant from PhRMA Foun-
dation, and NIP from AACP to D. Sun. The authors
thank the Ohio State University for providing research
resources, analysis instruments, and funding support.
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