Syntheses and biological activities of rebeccamycin analogues with uncommon sugars

Article abstract of DOI:10.1021/jm0493764

Rebeccamycin analogues containing uncommon sugars and substitutions on the imide nitrogen have been synthesized. Their cytotoxicities were tested in colon cancer and leukemia cells. Their ability to target topoisomerase I was examined using the in vivo co

Full text of DOI:10.1021/jm0493764

2600
J. Med. Chem. 2005, 48, 2600-2611
Syntheses and Biological Activities of Rebeccamycin Analogues with
Uncommon Sugars
Guisheng Zhang,† Jie Shen,† Hao Cheng,‡ Lizhi Zhu,† Lanyan Fang,‡ Sanzhong Luo,† Mark T. Muller,|
Gun Eui Lee,| Lijun Wei,# Yuguo Du,§ 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, Department of Molecular Biology and Microbiology,
University of Central Florida, Orlando, Florida 32826, School of Chemistry and Material Engineering, Yunnan University,
China, and Research center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, China
Received August 3, 2004
Rebeccamycin analogues containing uncommon sugars and substitutions on the imide nitrogen
have been synthesized. Their cytotoxicities were tested in colon cancer and leukemia cells.
Their ability to target topoisomerase I was examined using the in vivo complex of the
topoisomerase bioassay in Hela cells. Compared with aglycon 1, the modified compounds with
various sugar moieties showed more potent cytotoxicities and topo I targeting ability. In
addition, the rebeccamycin analogues with various uncommon sugars showed distinct cyto-
toxicities and topo I targeting activities. The activity of compounds with 2-deoxyglucose (8 and
9) > compounds with 2,6-deoxyglucose (5 and 6) > compounds with 2,3,6-deoxyglucose (10).
Furthermore, the anticancer activity of compounds correlated with their ability to target
endogenous topo I. These results suggest that the sugar moiety, especially the 2-OH and 6-OH
group of the sugar, rather than the modifications in imide structure on the indolocarbazole
ring, is a key element for its activity.
Introduction
A few papers in recent years reported the synthesis and
activity of indolocabazoles with a modified sugar
moiety.14-16 These studies suggest that altering glyco-
sylation patterns on indolocarbazole has a high potential
for generating novel chemotherapeutics. For instance,
alterations from glucose to other sugars such as galac-
tose, mannose, and fucose do not change the biological
profile significantly.15 In sharp contrast, however, the
introduction of an amino group on the glucose sugar
moiety greatly enhance the binding affinity to DNA,
leading to enhanced cytotoxicity. While the 2-amino
group does not prevent indolocarbazoles from inhibiting
topo Ι,16 the 6-amino group almost abolishes topo-
isomerase inhibition activity of the compound.14 To date,
there are no systematic studies to depict the biological
effect of the functionalities on the sugar moiety.
In our present study, we intend to investigate a series
of rebeccamycin analogues for which various uncommon
sugars are attached to one of the indole nitrogens via
an R or â-N-glycosidic linkage for their cytotoxicities and
inhibition of topoisomerase I. Uncommon sugars have
received special attention, due to the increased recogni-
tion 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. Areas in which their
significance has been well-established include cellular
adhesion and cell-cell recognition, fertilization, protein
folding, neurobiology, xenotransplantation, and target
recognition in the immune response.17-20 In addition, a
methyl group is introduced on the imide nitrogen of the
indolocarbazole chromophore. Methyl-containing indolo-
carbazoles may provide a different mechanism for the
Rebeccamycin and BE-13793C (Figure 1) were iso-
lated from fermentation cultures of Saccharotrix aero-
colonigenes (ATCC 39243)1 and Streptoverticillium
mobaraense (FERM P-10489).2 Their structures, which
were elucidated in 19853,4 and 1991,2 share a common
indolocarbazole pharmacophore. Since the potent anti-
cancer activities were discovered for these compounds,
the indolocarbazole compounds have been extensively
studied. Structure-activity relationships studies have
led to the development of several analogues. For in-
stance, NB-506,5-7 a glycosylated derivative of BE-
13793C, the rebeccamycin derivative NCS655649,8-10
and J-10708811-13 are currently in clinical trials.
Generally, there are three modification approaches to
synthesize indolocarbazole derivatives for new antitu-
mor drug candidates with improved pharmaceutical
properties: (i) replacement of the hydrogen on the imide
functionality with other groups, especially a hydrophilic
group; (ii) substitution on the fused aromatic ring or a
change of the benzene ring to another hetero-aromatic
ring, and (iii) replacement of the â-glycoside with other
sugar moieties. More than 300 rebeccamycin derivatives
were synthesized and reported to date. Most of the
current modifications through the first two approaches
are to improve the water solubility of the compounds.
* To whom correspondence should be addressed. G.P.W.: Phone
(614) 292-9884. Fax (614) 688-3106. E-mail wang.892@osu.edu. D.S.:
Phone (614) 292-4381. Fax (614) 292-7766. E-mail sun.176@osu.edu.
† Department of Chemistry and Biochemistry, The Ohio State
University.
‡ Division of Pharmaceutics, College of Pharmacy, The Ohio State
University.
| University of Central Florida.
# Yunnan University.
§ Chinese Academy of Sciences.
10.1021/jm0493764 CCC: $30.25 © 2005 American Chemical Society
Published on Web 02/18/2005

Rebeccamycin Analogues with Uncommon Sugars
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2601
Figure 1. Structures of rebeccamycin and its analogues.
anhydride analogues (11, 12). The cytotoxicities and
topoisomerase I inhibition of these compounds were
tested in cancer cells in comparison with their parent
compounds, synthetic compounds 1 and 2, methylated
at the imide nitrogen.
Results and Discussion
Chemistry. The synthesis of compounds 1, 2 and 13
bearing a methyl group at the imide nitrogen is outlined
in Scheme 2. The coupling reaction of indolylmagnesium
bromide with N-methyldichloromaleimide provided the
bisindolylmaleimide intermediate.24 Treatment of the
intermediate with 2 equiv of tert-butyl dicarbonate
followed by the selective mono-deprotection gave com-
pound 13,25 which was employed as the glycosylation
aglycon in the following synthetic routes. Compound 1
was prepared via oxidative photocyclization of the
bisindolylmaleimide intermediate.26 N-Glycosylation of
compound 1 with tetra-O-benzyl glucopyranosyl chloride
was performed in the presence of potassium hydroxide
according to the methods described by Ohkubo et
al.,27-29 and the hydrogenation debenzylation led to
compound 2. Various methods of N-glycosylations of
indole or indolocarbazole moieties were described in the
literature:4,9,15,27-31 (i) coupling of indolocarbazoles to an
R-D-glucopyranosyl bromide via the Koenigs-Knorr
method provided essentially R-N-glycosylated com-
pounds; (ii) coupling of an anhydrosugar to an indole
or bis-indole moiety favored the â-N-glycosylated prod-
uct; (iii) reaction in heterogeneous basic medium with
an indolocarbazole and R-tetra-O-benzyl-glucopyranosyl
chloride gave the major â-N-glycosylated compounds.
Figure 2. Favorable conformation of compound 2 with
bifurcated intramolecular hydrogen bond.
development of antitumor drugs since two rebeccamycin
analogues (AT2433-A1 and AT2433-B1) with a methyl
group on the imide nitrogen exhibited significant anti-
tumor properties via an inhibition of topoisomerase I.9,21
Furthermore, it has been reported that the closed
conformation involving a hydrogen bond between the
pyranose oxygen and the indole NH of rebeccamycin is
essential for the activity to access DNA binding sites
and inhibit topoisomerase I10,22,23 (Figure 2). Since both
6-OH and 1-oxygen can form a hydrogen bond with
indole NH thus keeping the closed conformation, the
importance of all OH groups in the sugar moiety needs
to be further investigated.
To explore the importance of OH group at the 2, 3, 6
positions in the sugar and the impact of an uncommon
aminosugar, three uncommon sugars 25 (2-deoxy sugar),
17 (2,6-dideoxy sugar), and 34 (2,3,6-trideoxy amino-
sugar) were selected as glycosyl donors. Currently, we
have prepared a series of rebeccamycin analogues
(Scheme 1) with various uncommon sugars and substi-
tution on the imide nitrogen with a methyl group (5, 6,
8, 9, 10) or amino group (3, 4, 7), and the corresponding
Scheme 1. Synthesized Compounds

2602 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Zhang et al.
Scheme 2a
a Reagents and conditions: (a) Et2O/toluene, 88 °C, 24 h (78%); (b) Boc2O, DMAP, THF, rt, 1 h; (c) TBAF, THF, reflux, 8 h (75%, two
steps); (d) hv, air, I2, THF/MeCN, 15 h (80%); (e) NaOH, Na2SO4, MeCN, r.t, 24 h (92%); (f) Pd/C, H2 45 psi, 10 h (87%); (g) EtOH/toluene
(1:3), 48% KOH, rt, overnight; then 10% citric acid (80%); (h) 1-(3-aminopropyl)-imidazole, 60 °C, 24 h (80%); (i) Pd/C, H2, 45 psi, 1 M
HCl, THF/EtOH, 24 h (80%).
This reaction is highly stereoselective; (iv) a Mitsunobu
reaction for the coupling of a bis-indole or an indole with
R-2,3,4,6-tetra-O-benzyl-glucose afforded the â-N-gly-
cosylated compound with an electron-withdrawing group
on the substrate.
odology lies in the glycosylation reaction of mannose and
erythrose whose halosugars are not stable.15
The Mitsunobu reaction was first performed with
compound 1 using 2,6-dideoxy-3,4-di-O-acetyl-L-rham-
nopyranose (17) according to the method described by
Sabesan et al.35 in the presence of diethyl azodicarbox-
ylate (DEAD) and triphenylphosphine. However, no
reaction was observed due to the particularly insolubil-
ity of 1. Using bis(indolyl)maleimide derivative 13
instead of 1 as the glycosylated acceptor, an inseparable
anomeric mixture 18 was produced with 32% yield. The
1H NMR spectrum of the mixture indicates the presence
of R,â-anomers in the ratio 1:2.5. Treatment of the
mixture 18 with formic acid at room temperature
afforded â-N-glycoside 19 and R-N-glycoside 20 with
yield of 91%. Their stereochemistry was assigned from
the coupling constant J1′,2′. The values (10.3 Hz of 19,
and 4.4 Hz of 20) are in agreement with axial-axial,
axial-equatorial coupling, respectively. Oxidative cy-
clization of 19 and 20 by irradiation in the presence of
iodine yielded the corresponding 21 and 22, and the
deacetylation of these compounds gave the required
rebeccamycin analogues 5 and 6 with high yields
(Scheme 3). The conversion of 5 to anhydride 23 was
accomplished by treatment with aqueous KOH in
toluene/EtOH overnight at room temperature. The
reaction initially generates a mixture of regioisomeric
amic acid intermediates, which upon adjustment to pH
6-7 with aqueous citric acid undergoes dehydration to
form the anhydride 23 over the course of 3 h at room
temperature. The coupling of hydrazine with anhydride
23 was run in THF at 50 °C produced analogue 7 with
80% yield from 5.
To synthesize the â-glycoside of rebeccamycin, meth-
ods for stereospecifically forming â-glycoside of rebec-
camycin derivatives are mostly desired. In a convergent
design, it is favorable to form the indoloacarbazole core
before the glycosylation. However, the indolocarbazole
acceptor is a weaker nucleophile than the bis(indoly1)-
maleimide or indole acceptor, which limits the direct
application of many established glycosylation method-
ologies to the indolocarbazole aglycones. This challenge
was overcome by Ohkubo28 in 1997 when armed chloride
donor was used in heterogeneous basic media for the
synthesis of NB-506. This attractive methodology af-
forded both excellent yield and high stereoselectiv-
ity.15,28,32-34 In the case of the â-N-glycosylation of
2-deoxysugar, however, this method is unsuitable. The
glycosylation reaction of 3,4,6-tri-O-benzyl-2-deoxy glu-
copypyranosyl chloride with an indolocarbazole only
produced R-isomer.15 Currently, there is no general
methodology to directly couple 2-deoxy uncommon sugar
with an indolocarbazole core with â-selectivity. As an
alternative approach, glycosylation can be executed
before the indolocarbazole core formation. For instance,
the indole or bis(indolyl)maleimide derivative is used
as the glycosylation acceptor.15 Mitsunobu reaction and
1,2-anhydrosugar donors30,31 are the two most fre-
quently employed methods, both of which stereoselec-
tively generate the â-isomers. A particular superiority
of the Mitsunobu approach to the chloride donor meth-

Rebeccamycin Analogues with Uncommon Sugars
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2603
Scheme 3a
a Reagents and conditions (a) LiBr‚H2O, AG 50W-X2 resin, CH3CN, rt, 15 min (72%); (b) Ph3P, DEAD, THF, -78 °C, 15 h (32%, R:â )
1:2.5); (c) 88% HCO2H, rt, 6 h (91%); (d) hv, air, I2, benzene, 8 h (21: 82%, 22: 57%); (e) TBAF, THF, reflux, 15 h (5: 90%, 6: 70%);
(f) EtOH/toluene (1:3), 48% KOH, rt, overnight; then 10% citric acid; (g) N2H4‚H2O (80% from 5).
Scheme 4a
a Reagents and conditions: (a) LiBr‚H2O, AG 50W-X2 resin, CH3CN, rt, 1 h (71%); (b) Ph3P, DEAD, THF, -78 °C, 15 h (35%, R:â )
1:2.2); (c) 88% HCO2H, rt, 6 h (88%); (d) hv, air, I2, benzene, 8h (29: 82%, 30: 68%); (e) TBAF, THF, reflux, 15 h (8: 91%, 9: 92%).
For the synthesis of 8 and 9 from 13 and 2-deoxy-
3,4,6-tri-O-acetyl-D-glucopyranose (25),35 a synthetic
route similar to that presented in Scheme 3 was
investigated. The desired rebeccamycin analogues were
obtained and the synthetic procedure is outlined in
Scheme 4.
10.8 Hz, H-1′). Oxidative cyclization of compound 36 in
the presence of iodine led to compound 37 (85% yield),
which was treated with tetrabutylammonium fluoride
(TBAF) or sodium methoxide in methanol, to yield
analogues 10 in yields of 89% and 91%, respectively
(Scheme 5). Treatment of 10 according to the similar
procedure as described for 23 yielded the anhydride
analogues 11 (80% yield). The conversion of 37 to 11
and 12 was performed in two steps: treatment of 37
with powdered NaOH in 2-methoxyethanol under reflux
followed by acidification with 10% aqueous citric acid
to produce anhydride analogues 11 and 12, in yields of
40% and 42%, respectively.
To study the effect of base group at imide nitrogen
on the bioactivity of rebeccamicin analogues, the deriva-
tives 4, 3-imidazol-1-yl-propyl connected to imide nitro-
gen, was prepared from 14. The conversion of 14 to 15
was performed according to the method described for
23 yielded 15 in 80% yield. Treatment of 15 with
3-(amino propyl) imidazole and the hydrogenation de-
Aqueous acidic hydrolysis of hydrochloride of dauno-
rubicin with 0.2 M hydrochloric acid at 90 °C gave
daunorubicinone and hydrochloride of daunosamine.36
To get a pure hydochloride of daunosamine, we tried to
purify the crude product by recrystallization with etha-
nol. The ethyl glycoside 32 was obtained in 83% yield
instead of hydrochloride of daunosamine. A glycosyla-
tion occurred in the recrystallization process. Benzox-
ylation of 32 with benzoxy anhydride followed by acidic
hydrolysis with 3 M hydrogen chloride produced the
glycosylated donor 34. N-Glycosylation of 13 with 34 in
THF and subsequent removal of the Boc protecting
group using formic acid led to 36 as a single â-isomer
(1H NMR: δ 5.81 ppm 1H, dd, J1′,2eq′ ) 2.1 Hz, J1′,2ax′
)

2604 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Zhang et al.
Scheme 5a
a Reagents and conditions: (a) 0.2 M HCl, 90 °C, 1 h; (b) EtOH, reflux (83% from dunorubicin); (c) Bz2O, Pyridine, rt, overnight (74%);
(d) 3 M HCl, THF, 60 °C, 15 h (80%); (e) Ph3P, DEAD, THF, -78 °C, 18 h (34%); (f) 88% HCO2H, rt, 22 h (81%); (g) hv, air, I2, benzene,
15 h (85%); (h) NaOH, MeOCH2CH2OH, reflux, overnight, then 10% citric acid (11:40%, 12: 42%). (i) TBAF, THF, reflux, 14 h (89%); or
NaOMe/MeOH, rt, overnight (91%); (j) EtOH/toluene (1:3), 48% KOH, reflux, overnight; then 10% citric acid (80%).
Table 1. Anticancer Activity (IC50) of Synthesized Compounds in Two Cancer Cell Lines (µM)
1
2
3
4
5
6
7
8
9
10
11
12
Reb
SW620
K562
>100
5
3
>50
>50
2
4
24
26
34
19
>100
>100
13
12
32
12
>100
76
53
>100
4
6
40
34
55
benzylation led to compound 4 with 64% overal yield
3-OH, and 6-OH groups in sugar moieties played a very
important role in maintaining rebeccamycin anticancer
activity. The 6-OH group may have a more significant
role than other OH groups in the sugar moiety.
(Scheme 2).
Biology
The cytotoxicity of rebeccamycin analogues with
substitution at the N6 position of the imide was also
compared in SW620 and K562 cells. Despite the imid-
azole substitution at N6 of compound 4 (IC50 2-4 µM),
it showed identical activity to that of Reb and 2, which
indicates that the sugar moiety, rather than the N6
substitution, is the activity determinant. This mecha-
nism was confirmed by the activity of compound 3, the
protected sugar moiety derivative of 4. When the sugar
moiety was protected with a benzyl group (compound
3), it lost the anticancer activity (IC50 > 50 µM). The
observation is further verified by the activity of com-
pound 7, where NH2 attached at N6 of the imide did
not rescue the loss of activity by sugar modification with
2,6-deoxyglucose.
Cytotoxicity of compound 10, 11, and 12, differing in
substitution at the N6 or O6 position, was also deter-
mined in SW620 and K562 cells. All three compounds
showed low activity similar to that of aglycon 1 without
sugar moieties (IC50 > 50-100 µM). Comparing com-
pound 10 and 11, the anhydride function does not confer
higher activities compared to the amide function. In
comparison of compound 11 and 12, deprotected NH2
at the C3′ of the sugar moiety did not rescue its
anticancer activity, which indicates either 3-OH in the
sugar moiety is critical or the polarity of this position
Cytotoxicity. The cytotoxicities of these conjugates
were tested with MTS assay in two cancer cell lines,
including colorectal carcinoma cells (SW620) and leu-
kemia cells (K562) (Table 1).
The rebeccamycin (Reb) and its analogues 2, 5, 6, 8,
9 with different sugar moieties are much more active
(IC50 3-34 µM) than the corresponding aglycon 1
(IC50 > 100 µM), which lacking the sugar moiety.
Compared with rebeccamycin (Reb, IC50 4-6 µM),
compound 2 (IC50 3-5 µM) displayed similar activity,
despite the substitution of chlorine atoms at the C1, C11
positions and methyl substitution at the N6 position.
These results suggest that the sugar moiety on the
indolocarbazole ring is a key element for its cytotoxicity.
Comparing compounds with a â-linkage (8, 5) (IC50 12-
26 µM) to compounds with a R-linkage (9, 6) (IC50 12-
34 µM), no activity difference was observed between
â-configuration and R-configuration. In addition, it
seems that compounds with 2-deoxyglucose (8 and 9)
are slightly more active than compounds with 2,6-
dideoxyglucose (5 and 6). Interestingly, when the sugar
moiety was changed to 2,3,6-trideoxy-3-benzylamino-
glucose in rebeccamycin anlogues (compound 10), it
showed the least cytotoxicity (IC50 > 100 µM), which is
similar to that of the aglycon 1 without sugar moieties.
Thus, all the above results indicate that the 2-OH,

Rebeccamycin Analogues with Uncommon Sugars
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2605
This is surprising because the N6 region of rebeccamy-
cin was thought to be involved in topoisomerase inhibi-
tion while the sugar moiety is involved in DNA binding.
Our results clearly demonstrated that the sugar moiety
also modulates the topoisomerase I activity, which is
worthy of further investigation with various modifica-
tions in sugar moiety.
Conclusion
In summary, several rebeccamycin analogues were
synthesized, which contain uncommon sugars, and
substitutions on the imide nitrogen such as a methyl
group or amino group, as well as two corresponding
anhydride analogues. The results suggest that the sugar
moiety on the indolocarbazole ring rather than N6
substitution is a key element for its anticancer activity
and topoisomerase inhibition. In addition, among the
compounds tested in this study, the rebeccamycin
analogues with various uncommon sugars showed dis-
tinct cytotoxicities and topoisomerase inhibition. These
compounds with glucose (2, 4, and Reb) are more active
than compounds with 2-deoxyglucose (8 and 9) or with
2,6-deoxyglucose (5 and 6). Compounds with 2,3,6-
deoxyglucose (compound 10) showed the worst activity.
These data indicate that the 2-OH, 3-OH, and 6-OH
groups in sugar moieties, rather than the modifications
in the imide structure, play a very important role in
maintaining their anticancer activity. The better activi-
ties of compound 2, 8, and 4 imply that the 6-OH group
may have a more significant role than other OH groups
in the sugar moiety. The 6-OH group on the carbohy-
drate residue may form a hydrogen bond with the indole
NH, which may maintain the carbohydrate in a fixed
conformation for optimal activity and interaction with
the topoisomerase I-DNA complex. In addition, the
cytotoxicities of these compounds correlated with the
inhibition of topoisomerase I, where compounds with
various sugar moieties show more potent topoisomerase
inhibition compared to the aglycon 1 without a sugar
moiety. Compounds with 2-deoxyglucose showed better
topoisomerase I inhibition than the compounds with 2,6-
deoxyglucose. This indicates that the 2-OH and 6-OH
of the sugar moiety also modulate the topoisomerase I
activity in cancer cells, which is worthy of further
investigation with various modifications in the sugar
moiety. Therefore, the modifications of rebeccamycin
with uncommon sugars may provide a new class of
anticancer compounds.
Figure 3. Standard ICT assay in HeLa cells comparing
camptothecin (CPT) with Rebeccamycin-sugar derivatives 2,
5, and 8. Exponentially growing HeLa cells were incubated
with the compounds indicated to the right of the blot for 30
min at 37 °C, followed by sarkosyl lysis and a standard ICT
assay (CsCl gradient separation of DNA, recovery of DNA and
spotting from 0.01 to 5 µg of DNA on the blot). The blot was
probed with anti-topo I antibody. Signals were developed using
ECL and a short (1 min) exposure.
is not a key element for its activity. Further validation
and analysis with different compounds need to be
performed for further clarification.
Inhibition of Topoisomerase I. The compounds
were also tested for their inhibition of topoisomerase I
with in vivo complex of topoisomerase (ICT) bioassay
in HeLa cells. Formation of the covalent complex
(topoisomerase/DNA) is essential for the cytotoxic action
of the topoisomerase poisons. The unique nature of
topoisomerase/DNA interaction (viz., formation of the
covalent intermediate) can be exploited to quantify
topoisomerase-mediated DNA damage in the intact cell.
In the presence of topo I poisons, the DNA breaks are
stabilized and a covalent topo-DNA complex can be
trapped upon addition of protein denaturants. The
Standard ICT assay was performed on HeLa cells
comparing camptothecin (CPT) with three rebeccamycin
analogues (5, 8, and 2) that differ only in their sugar
side chains where compound 2 was linked with glucose,
compound 8 with 2-deoxyglucose, and compound 5 with
2,6-deoxyglucose. To ensure uniformity between six
different treatments, a range of DNA concentrations
was probed from 0.01 to 5 µg; thus, the signals can be
directly compared between each culture. Very surpris-
ingly, their topoisomerase inhibition is in agreement
with the anticancer activity (Figure 3). Compound 2
with glucose moiety showed similar strong topoisom-
erase inhibition as that of CPT (positive control) at the
same molar concentration, which was indicated by the
formation of topo-DNA complex in the immunoblot.
However, compound 8 with 2-deoxy-D-glucose moiety
gave a very weak (but detectable on longer exposure)
signal while compound 5 with 2,6-dideoxy glucose
moiety was inactive. Clearly, the 2-OH and 6-OH of the
sugar moiety in rebeccamycin analogues strongly modu-
late drug activity in topoisomerase I inhibition in vivo.
Experimental Section
Chemistry. All solvent was 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. Preparative TLC was carried out on EMD Chemicals,
Inc. silica gel 60 F254 plates (20 × 20 cm, 1 mm). The 230-
400 mesh size of the same absorbent was utilized for all
1
chromatographic purifications. H and 13C NMR spectra are
at the indicated field strengths. The high-resolution mass
spectra were recorded at The Ohio State University Campus
Chemical Instrumentation Center.
2-Deoxy-3,4-di-O-acetyl-L-rhamnopyranose (17).35 AG
50W-X2 resin (0.3 g) and water (0.6 mL) were added to a
solution of 3,4-di-O-acetyl-L-rhamnal (0.4 g) and lithium
bromide hydrate (0.5 g) in acetonitrile (15 mL). The mixture
was stirred at room temperature for 15 min. The reaction

2606 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Zhang et al.
mixture was filtered, neutralized with triethylamine, and
evaporated to dryness. The residue was dissolved in dichlo-
romethane and washed with water, ice-cold 1 M hydrochloric
acid, and saturated sodium bicarbonate solution. The colorless
solid (R:â ) 2:1) of compound 17 (310 mg, 72% yield) was
obtained after purification on a column of silica gel using ethyl
acetate-hexane (1:3) as eluant: 1H NMR (250 MHz, CDCl3)
5.24 (d, J ) 3.4 Hz, H-1R), 5.20 (m, H-3R), 4.88 (m, H-3â), 4.80
(dd, J ) 1.9 Hz, J ) 9.4 Hz, H-1â), 4.63 (t, J ) 9.6 Hz, H-4),
4.00 (m, H-5R), 3.43 (m, H-5â), 2.26 (m, H-2âeq), 2.13 (m,
H-2Req), 1.70 (m, H-2âax), 1.60 (m, H-2Rax), 1.11 (d, J ) 6.2
Hz, H-6â), 1.05 (d, J ) 6.2 Hz, H-6R).
2,5-Dihydro-3-[1-(tert-butyloxycarbonyl)-1H-indol-3-
yl]-4-[1-(2-deoxy-3,4-di-O-acetyl-L-rhamnopyranosyl)-1H-
indol-3-yl]-1H-pyrrole-2,5-dione (18). To a solution of 13
(1.50 g, 3.40 mmol) in THF (100 mL) were added 17 (1.95 g,
8.41 mmol) and triphenylphosphine (2.20 g, 8.41 mmol). The
mixture was cooled to -78 °C, and then DEAD (1.33 mL, 8.41
mmol) was added dropwise. The mixture was allowed to reach
room temperature and stirred at room temperature for 15 h,
and then water was added. After extraction with ethyl acetate,
the combined organic phase was dried over Na2SO4. The
solvent was removed, and the residue was purified through
column chromatography on silica gel using benzene-ethyl
acetate (9:1) to give unreacted substrate 13 (1.0 g) and the
anomeric mixture 18 (0.7 g, 32%, R:â ) 1:2.5) as an orange
solid: 1H NMR (250 MHz, CDCl3) 5.24 (dd, J ) 1.75 Hz, J )
11 Hz, H-1′â), 5.96 (d, J ) 4.7 Hz, H-1′R).
2,5-Dihydro-3-(1H-indol-3-yl)-4-[1-(2-deoxy-3,4-di-O-
acetyl-â-L-rhamnopyranosyl)-1H-indol-3-yl]-1H-pyrrole-
2,5-dione(19) and 2,5-Dihydro-3-(1H-indol-3-yl)-4-[1-(2-
deoxy-3,4-di-O-acetyl-R-L-rhamnopyranosyl)-1H-indol-3-
yl]-1H-pyrrole-2,5-dione (20). A solution of 18 (310 mg, 0.47
mmol) in 88% formic acid (40 mL) was stirred at room
temperature and monitored by TLC hourly. After 6 h, the
reaction was completed. Trimethylamine was added dropwise
until neutralization. Saturated aqueous NaOAc was added.
After extraction with ethyl acetate, the combined organic phase
was dried over Na2SO4. The solvent was removed, and the
residue was purified by prep-TLC (benzene-ethyl acetate, 8:1)
to afford 19 (red solid, 170 mg) and 20 (red solid, 68 mg) with
91% yield.
19: HRMS (M + Na)+ (ESI+) calcd for C31H29N3O7Na+
578.1898, found 578.1896. 1H NMR (400 MHz, CDCl3) 8.52
(1H, s, NH), 7.72 (1H, s), 7.67 (1H, s), 7.34 (1H, d, J ) 8.2
Hz), 7.30 (1H, d, J ) 8.2 Hz), 7.11 (1H, t, J ) 7.4 Hz), 7.06
(1H, t, J ) 7.4 Hz), 6.97 (2H, d, J ) 8.8 Hz), 6.77 (2H, dd, J )
7.3 Hz, J ) 12.6 Hz), 5.65 (1H, d, J ) 10.3 Hz, H-1′), 5.14
(1H, m, H-3′), 4.87 (1H, t, J ) 9.6 Hz, H-4′), 3.75 (1H, m, H-5′),
3.17 (3H, s, NCH3), 2.47 (1H, m, H-2eq), 2.23 (1H, m, H-2ax),
2.08 (3H, s, COCH3), 2.04 (3H, s, COCH3), 1.24 (3H, d, J )
6.3 Hz, H-6′). 13C NMR (400 MHz, CDCl3) 18.2 (C6′), 21.2, 21.3
(CH3CO), 24.6 (NCH3), 36.1 (C2′), 71.4, 73.3, 74.1, 81.4 (C1′,
C3′, C4′, C5′), 110.5, 111.6, 120.9, 121.4, 122.5, 122.9, 123.1,
123.2, 128.4, 128.8 (C tert arom), 107.6, 107.9, 125.7, 126.9,
127.5, 128.7, 136.1, 136.3 (C quat arom), 170.4, 170.7, 172.6,
172.7 (CdO). 20: HRMS (M + Na)+ (ESI+) calcd for C31H29N3O7-
Na+ 578.1897, found 578.1895. 1H NMR (400 MHz, CDCl3) 8.59
(1H, s, NH), 7.88 (1H, s), 7.80 (1H, d, J ) 2.7 Hz), 7.58 (1H, d,
J ) 8.3 Hz), 7.29 (1H, d, J ) 8.2 Hz), 7.20 (1H, d, J ) 8.0 Hz),
7.11 (1H, t, J ) 8.2 Hz), 7.03 (1H, t, J ) 8.1 Hz), 6.88 (2H, t,
J ) 8.1 Hz), 6.72 (1H, t, J ) 8.0 Hz), 5.99 (1H, d, J ) 4.4 Hz,
H-1′), 4.87 (2H, m, H-4′, H-3′), 3.31 (1H, m, H-5′), 3.18 (3H, s,
NCH3), 2.75 (1H, m, H-2eq), 2.17 (1H, m, H-2ax), 2.03 (3H, s,
COCH3), 2.02 (3H, s, COCH3), 1.12 (3H, d, J ) 6.3 Hz, H-6′).
13C NMR (400 MHz, CDCl3) 17.6 (C6′), 21.2, 21.3 (CH3CO), 24.6
(NCH3), 32.7 (C2′), 67.9, 69.9, 73.6, 80.5 (C1′, C3′, C4′, C5′), 111.5,
112.4, 120.6, 121.6, 122.4, 122.6, 123.1, 123.4, 129.1, 129.6 (C
tert arom), 107.5, 108.0, 125.4, 127.4, 127.6, 128.9 136.5, 137.0
(C quat arom), 170.4, 170.8, 172.6, 172.7 (CdO).
Air was continuously bubbled through the reaction while was
irradiated with a medium-pressure mercury lamp equipped
with a Vycor filter for 8 h. Benzene was added to the reaction
every 2 h to keep the solvent volume constant. The solvent
was removed, and the residue was dissolved in EtOAc and
washed with aqueous Na2S2O3 and brine. The organic phase
was dried over Na2SO4. After removal of the solvent, the
residue was purified by Prep-TLC using EtOAc-hexane (1:9)
as developing solvent to give the product 21 (41 mg, 82% yield)
as a yellow solid: HRMS (M + Na)+ (ESI+) calcd for C31H27N3O7-
Na+ 576.1741, found 576.1751. 1H NMR (400 MHz, CDCl3)
10.15 (1H, s, NH), 9.25 (1H, d, J ) 7.8 Hz), 9.15 (1H, d, J )
7.9 Hz), 7.59-7.40 (6H, m), 6.16 (1H, dd, J ) 2.9 Hz, J ) 10.7
Hz, H-1′), 5.29 (2H, m, H-3′, H-4′), 4.15 (1H, m, H-5′), 3.21
(3H, s, NCH3), 2.25 (2H, m, H-2), 2.17 (3H, s, COCH3), 1.90
(3H, s, COCH3), 1.67 (3H, d, J ) 6.2 Hz, H-6′). 13C NMR (400
MHz, CDCl3) 19.1 (C6′), 21.1, 21.2 (CH3CO), 24.1 (NCH3), 36.1
(C2′), 70.7, 73.9, 75.2, 82.7 (C1′, C3′, C4′, C5′), 109.7, 111.5, 121.7,
122.5, 126.1, 126.5, 127.8, 127.9, (C tert arom), 118.9, 119.7,
120.1, 121.8, 122.7, 123.3, 128.5, 130.0, 140.6, 140.7 (C quat
arom), 170.2, 170.3, 170.3, 170.4 (CdO).
6-Methyl-12-(2-deoxy-3,4-di-O-acetyl-R-L-rhamnopy-
ranosyl)-6,7,12,13-tetrahydroindolo[2,3-a]pyrrolo[3,4-c]car-
bazole-5,7-dione (22). Following the same procedure de-
scribed for 21, compound 22 was obtained from 20 as a yellow
solid (57% yield): HRMS (M + Na)+ (ESI+) calcd for C31H27N3O7-
Na+ 576.1741, found 576.1756. 1H NMR (500 MHz, CDCl3)
10.46 (1H, s, NH), 9.36 (1H, d, J ) 7.6 Hz), 9.26 (1H, d, J )
8.2 Hz), 7.61-7.39 (6H, m), 6.16 (1H, d, J ) 9.2 Hz, H-1′), 5.25
(1H, br, H-4′), 5.02 (1H, br, H-3′), 4.87 (1H, m, H-5′), 3.32 (3H,
s, NCH3), 2.60, 1.83 (2H, 2m, H-2), 2.32 (3H, s, COCH3), 2.24
(3H, s, COCH3), 1.86 (3H, d, J ) 7.3 Hz, H-6′). 13C NMR (500
MHz, CDCl3) 16.0 (C6′), 21.5, 21.8 (CH3CO), 24.1 (NCH3), 32.1
(C2′), 68.9, 68.9, 74.8, 75.8, (C1′, C3′, C4′, C5′), 109.7, 110.9, 121.2,
122.2, 126.3, 126.6, 127.5, 127.6, (C tert arom), 118.5, 120.0,
120.1, 121.3, 122.7, 123.2, 128.4, 129.7, 140.4, 140.5 (C quat
arom), 169.1, 169.6, 170.2 (CdO).
6-Methyl-12-(2-deoxy-â-L-rhamnopyranosyl)-6,7,12,13-
tetrahydroindolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-di-
one (5). A solution of tetrabutylammonium fluoride (4 mL,
1.0 M in THF) was added to a solution of 21 (70 mg, 0.07 mmol)
in THF (10 mL). The mixture was refluxed for 15 h, diluted
with EtOAc (80 mL), washed with water, and dried over Na2-
SO4. The solvent was removed, and the residue was purified
on a column of silica gel using MeOH-CH2Cl2 (1:50) as eluent
to give the product 5 (63 mg, 90% yield) as a yellow solid:
HRMS (M + Na)+ (ESI+) calcd for C27H23N3O5Na+ 492.1530,
found 492.1529. 1H NMR (500 MHz, DMSO) 11.35 (1H, s, NH),
9.09 (1H, d, J ) 7.9 Hz), 8.97 (1H, d, J ) 8.0 Hz), 8.02 (1H, d,
J ) 8.4 Hz), 7.73 (1H, d, J ) 8.2 Hz), 7.55 (2H, m), 7.33 (2H,
m), 6.61 (1H, dd, J ) 2.4 Hz, J ) 11.1 Hz, H-1′), 5.41 (1H, br,
H-4′), 5.09 (1H, br, HO-3′), 3.94 (1H, m, H-5′), 3.34 (2H, m,
H-3′, HO-4′), 3.03(3H, s, NCH3), 3.11, 2.25 (2H, m, H-2), 1.49
(3H, d, J ) 6.1 Hz, H-6′). 13C NMR (500 MHz, DMSO) 19.3
(C6′), 24.4 (NCH3), 39.1 (C2′), 71.1, 76.1, 77.5, 83.0 (C1′, C3′, C4′,
C5′), 112.6, 113.7, 121.4, 121.9, 125.2, 125.5, 127.8, 128.1, (C
tert arom), 118.1, 118.2, 119.5, 120.7, 121.4, 122.9, 128.8,
129.1, 140.5, 141.3 (C quat arom), 170.1, 170.2 (CdO).
6-Methyl-12-(2-deoxy-R-L-rhamnopyranosyl)-6,7,12,13-
tetrahydroindolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-di-
one (6). Followed the same procedure described for 5, 6 was
obtained from 22 as a yellow solid (70% yield): HRMS (M +
Na)+ (ESI+) calcd for C27H23N3O5Na+ 492.1530, found 492.1523.
1H NMR (400 MHz, DMSO) 12.06 (1H, s, NH), 9.20 (1H, d,
J ) 8.1 Hz), 9.09 (1H, d, J ) 8.0 Hz), 7.79 (1H, d, J ) 8.4 Hz),
7.60 (3H, m), 7.38 (2H, m), 6.69 (2H, m, H-1′, 4′), 5.56 (1H, d,
J ) 3.0 Hz, HO-3′), 4.53 (1H, m, H-3′), 4.19 (1H, br, H-5′), 3.80
(1H, br, HO-4′), 3.(3H, s, NCH3), 2.73, 1.83 (2H,m, H-2), 1.47
(3H, d, J ) 6.1 Hz, H-6′). 13C NMR (500 MHz, DMSO) 16.0
(C6′), 24.5 (NCH3), 34.5 (C2′), 68.6, 69.2, 73.9, 77.9 (C1′, C3′, C4′,
C5′), 110.4, 112.2, 121.1, 121.7, 125.3, 125.6, 127.8, 128.1, (C
tert arom), 117.4, 118.1, 119.1, 120.9, 121.5, 121.8, 128.5,
129.4, 140.3, 141.2 (C quat arom), 170.43, 170.45 (CdO).
6-Methyl-12-(2-deoxy-3,4-di-O-acetyl-â-L-rhamnopy-
ranosyl)-6,7,12,13-tetrahydroindolo[2,3-a]pyrrolo[3,4-c]car-
bazole-5,7-dione (21). A red solution of 19 (50 mg, 0.09
mmol) in benzene (150 mL) was treated with iodine (1 mg).

Rebeccamycin Analogues with Uncommon Sugars
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2607
6-Amino-12-(2-deoxy-â-L-rhamnopyranosyl)-6,7,12,13-
tetrahydroindolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-di-
one (7). A mixture of 5 (30 mg, 0.064 mmol), 48% KOH
aqueous (1 g), EtOH (3.0 mL) and toluene (1.0 mL) was stirred
at room temperature for overnight. The color of the reaction
solution was changed from deep red to pale yellow. The
solution was acidified with 10% citric acid to pH 6, and stirred
for additional 3 h at room temperature, then extracted with
EtOAc. After removal of the solvent, the crude compound 23
was obtained (HRMS (M + Na)+ (ESI+) calcd for C26H20N2O6-
Na+ 479.1213, found 479.1207). THF (2.0 mL) and hydrazine
hydrate (1.0 mL) was added to the crude compound 23. The
mixture was stirred at 50 °C for 3 h, and then poured into
brine (20 mL). The resulted mixture was stirred at room-
temperature overnight. The solid was collected with EtOAc
and dried over Na2SO4. The solvent was removed, and the
residue was purified on a prepTLC using MeOH-CH2Cl2 (1:
25) as developing solvent to give the product 7 (24 mg, 80%
yield from 5) as a yellow solid: HRMS (M + Na)+ (ESI+) calcd
Hz, H-4′), 4.27 (1H, dd, J ) 5.2 Hz, J ) 12.4 Hz, H-6′), 4.12
(1H, dd, J ) 2.9 Hz, J ) 12.4 Hz, H-6′), 3.85 (1H, m, H-5′),
3.20 (3H, s, NCH3), 2.47 (1H, m, H-2eq), 2.26 (1H, m, H-2ax),
2.08 (3H, s, COCH3), 2.05 (3H, s, COCH3), 2.04 (3H, s, COCH3).
13C NMR (500 MHz, CDCl3) 21.1, 21.2, 21.3 (CH3CO), 24.6
(NCH3), 35.7 (C2′), 62.7, 69.0, 71.4, 74.9, 81.7 (C1′, C3′, C4′, C5′,
C6′), 110.5, 111.6, 120.9, 121.5, 122.5, 122.9, 123.1, 123.3, 128.3,
129.0 (C tert arom), 107.5, 108.1, 125.6, 125.9, 126.9, 127.3,
136.2, 136.3 (C quat arom), 170.2, 170.6, 171.1, 172.6, 172.7
(CdO). 28: HRMS (M + Na)+ (ESI+) calcd for C33H31N3O9-
Na+ 636.1952, found 636.1948. 1H NMR (500 MHz, CDCl3) 8.63
(1H, s, NH), 7.86 (1H, s), 7.82 (1H, d, J ) 2.7 Hz), 7.59 (1H, d,
J ) 8.3 Hz), 7.29 (1H, d, J ) 8.3 Hz), 7.22 (1H, t, J ) 8.1 Hz),
7.12 (1H, t, J ) 7.4 Hz), 7.04 (1H, t, J ) 7.3 Hz), 6.89 (1H, t,
J ) 7.1 Hz), 6.83 (1H, d, J ) 8.2 Hz), 6.69 (1H, t, J ) 8.1 Hz),
6.07 (1H, d, J ) 4.9 Hz, H-1′), 5.10 (1H, t, J ) 9.6 Hz, H-4′),
4.99 (1H, m, H-3′), 4.26 (1H, dd, J ) 5.2 Hz, J ) 12.5 Hz, H-6′),
4.12 (1H, dd, J ) 2.8 Hz, J ) 12.5 Hz, H-6′), 3.40 (1H, m, H-5′),
3.19 (3H, s, NCH3), 2.75 (1H,m, H-2eq), 2.21 (1H,m, H-2ax), 2.03
(3H, s, COCH3), 2.02 (3H, s, COCH3), 1.98 (3H, s, COCH3).
13C NMR (500 MHz, CDCl3) 21.1, 21.2, 21.6 (CH3CO), 24.7
(NCH3), 34.6 (C2′), 62.1, 68.5, 70.0, 80.7 (C1′, C3′, C4′, C5′, C6′),
111.6, 112.5, 120.6, 121.8, 122.3, 122.6, 123.1, 123.5, 129.3,
129.4 (C tert arom), 107.4, 108.4, 125.3, 125.9, 127.3, 127.5,
136.5, 136.9 (C quat arom), 170.1, 170.7, 171.0, 172.6, 172.7
(CdO).
6-Methyl-12-(2-deoxy-3,4,6-tri-O-acetyl-â-D-glucopy-
ranosyl)-6,7,12,13-tetrahydroindolo[2,3-a]pyrrolo[3,4-c]car-
bazole-5,7-dione (29). A red solution of 27 (120 mg, 0.20
mmol) in benzene (200 mL) was treated with of iodine (35 mg).
Air was continuously bubbled through the reaction which was
irradiates with a medium-pressure mercury lamp equipped
with a Vycor filter for 8 h. Benzene was added to the reaction
each 2 h to keep the solvent volume constant. The solvent was
removed, and the residue was dissolved in EtOAc and washed
with aqueous Na2S2O3 and brine. The organic phase was dried
over Na2SO4, and the residue was purified on a Prep-TLC
using EtOAc-benzene (1:4) as developing solvent to give the
product 29 (98 mg, 82% yield) as a yellow solid: HRMS (M +
Na)+ (ESI+) calcd for C33H29N3O9Na+ 634.1796, found 634.1792.
1H NMR (500 MHz, CDCl3) 10.16 (1H, s, NH), 9.32 (1H, d,
J ) 7.9), 9.24 (1H, d, J ) 7.9 Hz), 7.72 (1H, d, J ) 8.0 Hz),
7.62 (2H, m), 7.55 (1H, d, J ) 8.3 Hz), 7.46 (2H, m), 6.25 (1H,
dd, J ) 2.4 Hz, J ) 11.4 Hz, H-1′), 5.65 (1H, t, J ) 9.8 Hz,
H-4′), 5.39 (1H, m, H-3′), 4.80 (1H, dd, J ) 3.3 Hz, J ) 12.9
Hz, H-6′), 4.39 (1H, dd, J ) 2.1 Hz, J ) 12.9 Hz, H-5′), 4.29
(1H, dd, J ) 2.7 Hz, J ) 12.9 Hz, H-6′), 3.27 (3H, s, NCH3),
2.47 (1H, m, H-2eq), 2.30 (1H, m, H-2ax), 2.29 (3H, s, COCH3),
2.18 (3H, s, COCH3), 1.98 (3H, s, COCH3). 13C NMR (500 MHz,
CDCl3) 20.68, 20.69, 21.2 (CH3CO), 25.6 (NCH3), 35.5 (C2′),
61.6, 67.6, 70.7, 76.2, 82.7 (C1′, C3′, C4′, C5′, C6′), 109.5, 111.5,
121.5, 122.5, 125.8, 126.2, 127.4, 127.5 (C tert arom), 118.9,
119.5, 119.7, 122.2, 122.8, 128.1, 128.3, 129.6, 140.2, 140.6 (C
quat arom), 169.6, 169.8, 169.9, 170.0, 170.5 (CdO).
1
for C26H22N4O5Na+ 493.1482, found 493.1472. H NMR (500
MHz, DMSO+D2O) 9.10 (1H, d, J ) 8.0 Hz), 9.01 (1H, d, J )
7.9 Hz), 7.95 (1H, d, J ) 8.4 Hz), 7.76 (1H, d, J ) 8.2 Hz),
7.57 (2H, m), 7.37 (2H, m), 6.54 (1H, dd, J ) 2.1 Hz, J ) 11.0
Hz, H-1′), 3.94 (2H, m, H-5′, H-3′), 3.38 (1H, m, H-4′), 2.18
(2H, m, H-2), 1.47 (3H, d, J ) 6.1 Hz, H-6′). 13C NMR (500
MHz, DMSO+D2O) 18.7 (C6′), 38.5 (C2′), 60.5, 70.3, 76.7, 82.3
(C1′, C3′, C4′, C5′), 112.3, 112.4, 121.8, 122.2, 124.8, 125.1, 127.9,
128.1 (C tert arom), 117.1, 117.7, 117.9, 118.5, 121.3, 121.4,
128.4, 128.7, 140.2, 140.6 (C quat arom), 168.9, 169.0, (CdO).
2-Deoxy-3,4,6-trii-O-acetyl-D-glucopyranose (25). AG
50W-X2 resin (0.4 g) and water (0.6 mL) were added to a
solution 3,4,6-tri-O-acetyl-D-glucal (0.45 g) and lithium bro-
mide hydrate (0.5 g) in acetonitrile (15 mL). The mixture was
stirred at room temperature for 1 h. The product was isolated
as described for 17 and purified on a column of silica gel using
ethyl acetate-hexane (1:3) as eluant to give the compound 25
(340 mg, 71% yield) as a colorless solid.35
2,5-Dihydro-3-[1-(tert-butyloxycarbonyl)-1H-indol-3-
yl]-4-[1-(2-deoxy-3,4,6-tri-O-acetyl-D-glucopyranosyl)-1H-
indol-3-yl]-1H-pyrrole-2,5-dione (26). Triphenylphosphine
(894 mg, 3.40 mmol) and DEAD (1 g, 3.40 mmol) were added
to a solution of 13 (0.68 g, 1.80 mmol) in THF (50 mL). The
mixture was cooled to -78 °C, and then DEAD (0.54 mL., 3.40
mmol) was added dropwise. The mixture was allowed to reach
room temperature and stirred for 15 h, and then water was
added. After extraction with ethyl acetate, the combined
organic phase was dried over Na2SO4. The solvent was
removed, and the residue was purified on a column of silica
gel using ethyl acetate-hexane-trimethylamine (1:3:0.01) as
eluant to give the anomeric mixture 26 (0.38 g, 35% yield,
R:â ) 1:2.2) as an orange solid, which could not be separated
by chromatography.
2,5-Dihydro-3-(1H-indol-3-yl)-4-[1-(2-deoxy-3,4,6-tri-O-
acetyl-â-D-glucopyranosyl)-1H-indol-3-yl]-1H-pyrrole-2,5-
dione(27) and 2,5-Dihydro-3-(1H-indol-3-yl)-4-[1-(2-deoxy-
3,4,6-tri-O-acetyl-R-D-glucopyranosyl)-1H-indol-3-yl]-1H-
pyrrole-2,5-dione (28). A solution of 26 (40 mg, 0.06 mmol)
in formic acid (88%, 4.0 mL) was stirred at room temperature
and monitored by TLC every hour. After being stirred 6 h, the
reaction was completed. Trimethylamine was added dropwise
until neutralization. Saturated aqueous NaOAc was added.
After extraction with ethyl acetate, the combined organic phase
was dried over Na2SO4. The solvent was removed, and the
residue was purified on prepTLC (ethyl acetate-hexane (2:3)
to afford 27 (red solid, 20 mg) and 28 (red solid, 10 mg), the
yield was 88%.
6-Methyl-12-(2-deoxy-3,4,6-tri-O-acetyl-R-D-glucopy-
ranosyl)-6,7,12,13-tetrahydroindolo[2,3-a]pyrrolo[3,4-c]car-
bazole-5,7-dione (30). Followed the same procedure de-
scribed for 29, Compound 30 was obtained from 28 as a yellow
solid (68% yield): HRMS (M + Na)+ (ESI+) calcd for C33H29N3O9-
Na+ 634.1796, found 634.1790. 1H NMR (500 MHz, CDCl3)
10.16 (1H, s, NH), 9.28 (1H, d, J ) 7.9), 9.19 (1H, d, J ) 7.9
Hz), 7.67 (1H, d, J ) 8.4 Hz), 7.64 (1H, m), 7.51 (1H, m), 7.43
(3H, m), 6.64 (1H, dd, J ) 2.5 Hz, J ) 9.7 Hz, H-1′), 5.23 (1H,
br, H-3′), 5.05 (1H, br, H-4′), 4.78 (1H, m, H-5′), 5.38 (1H, dd,
J ) 9.8 Hz, J ) 12.3 Hz, H-5′), 4.25 (1H, dd, J ) 4.3 Hz, J )
12.3 Hz, H-6′), 3.19 (3H, s, NCH3), 2.59 (1H, m, H-2eq), 1.78
(1H, m, H-2ax), 2.27 (3H, s, COCH3), 2.25 (3H, s, COCH3), 2.24
(3H, s, COCH3). 13C NMR (500 MHz, CDCl3) 20.7, 221.0, 21.3
(CH3CO), 23.7 (NCH3), 34.2 (C2′), 59.2, 65.6, 67.8, 76.0, 798.1
(C1′, C3′, C4′, C5′, C6′), 109.8, 110.7, 121.0, 121.9, 125.8, 126.1,
127.3, 127.5 (C tert arom), 118.2, 119.2, 119.6, 121.3, 122.3,
122.9, 128.4, 129.6, 140.3, 140.5 (C quat arom), 168.7, 169.1,
169.8, 169.9, 170.9 (CdO).
27: HRMS (M + Na)+ (ESI+) calcd for C33H31N3O9Na+
636.1952, found 636.1944. 1H NMR (500 MHz, CDCl3) 8.64
(1H, s, NH), 7.69 (1H, s), 7.62 (1H, d, J ) 2.6 Hz), 7.34 (1H, d,
J ) 8.3 Hz), 7.26 (1H, d, J ) 8.1 Hz), 7.11 (1H, t, J ) 7.4 Hz),
7.05 (1H, t, J ) 7.4 Hz), 6.97 (2H, dd, J ) 2.9 Hz, J ) 8.1 Hz),
6.77 (2H, dd, J ) 8.1 Hz, J ) 15.6 Hz), 5.66 (1H, dd, J ) 1.3
Hz, J ) 11.0 Hz, H-1′), 5.20 (1H, m, H-3′), 5.10 (1H, t, J ) 9.7

2608 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Zhang et al.
6-Methyl-12-(2-deoxy-â-D-glucopyranosyl)-6,7,12,13-tet-
rahydroindolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-dione (8).
A solution of tetrabutylammonium fluoride (1.0 M in THF, 7
mL) was added to a solution of 29 (98 mg, 0.016 mmol) in THF
(20 mL). The mixture was refluxed for 15 h, diluted with
EtOAc (100 mL), washed with water, and dried over Na2SO4.
The solvent was removed, and the residue was purified on a
column of silica gel using MeOH-CH2Cl2 (1:25) as eluent to
give the product 8 (70 mg, 91% yield) as a yellow solid: HRMS
(M + Na)+ (ESI+) calcd for C27H23N3O6Na+ 508.1479, found
508.1484. 1H NMR (500 MHz, DMSO) 12.09 (1H, s, NH), 9.18
(1H, d, J ) 7.5), 9.09 (1H, d, J ) 8.0 Hz), 8.03 (1H, d, J ) 8.4
Hz), 7.73 (1H, d, J ) 8.2 Hz), 7.62 (1H, dd, J ) 7.4 Hz, J ) 8.1
Hz), 7.56 (1H, dd, J ) 7.2 Hz, J ) 8.0 Hz), 7.41 (1H, t, J )
10.8), 7.35 (1H, t, J ) 7.8), 6.63 (1H, dd, J ) 6.9 Hz, J ) 13.5
Hz, H-1′), 6.01 (1H, t, J ) 3.9 Hz, H-4′), 5.35 (1H, d, J ) 3.7,
OH-6′), 5.00 (1H, d, J ) 4.4, OH-3′), 4.11 (1H, m, H-5′), 3.86
(3H, m, OH-4′, H-3′, H-6′), 3.17 (3H, s, NCH3), 1.83 (2H, m,
H-2). 13C NMR (500 MHz, DMSO) 25.6 (NCH3), 34.5 (C2′), 58.9,
69.4, 70.7, 79.4, 81.6 (C1′, C3′, C4′, C5′, C6′), 111.4, 112.7, 120.9,
122.0, 124.8, 125.1, 127.5, 127.7 (C tert arom), 117.5, 118.6,
118.9, 121.7, 121.8, 125.4, 128.6, 129.7, 140.5, 141.3 (C quat
arom), 170.0, 170.1 (CdO).
H-6); 13C NMR (500 MHz, DMSO) 14.8, 16.6 (CH3, C6), 28.9
(C2), 61.9 (CH2), 51.9, 65.2, 70.9 (C1, C4, C5), 95.9 (C3).
4-O-Benzoxyl-3-dibenzoxy amido-2,3,6-trideoxy-R-L-
lyxo-hexose (34) and 2,5-Dihydro-3-[1-(tert-butyloxycar-
bonyl)-1H-indol-3-yl]-4-[1-(4-O-benzoxyl-3-dibenzoxy ami-
do-2,3,6-trideoxy-â-L-lyxo-hexosyl)-1H-indol-3-yl]-1H-
pyrrole-2,5-dione (35). A mixture of 33 (100 mg), 3 M
hydrochloric acid aqueous (2.2 mL), and THF (1.4 mL) was
stirred at 60 °C for 15 h. TLC showed the reaction was
completed. Solid sodium bicarbonate was added to the reaction
mixture until pH 7. After extraction with ethyl acetate, the
combined organic phase was dried over Na2SO4. The solvent
was removed, and the residue was purified on a column of
silica gel using ethyl acetate-hexane (1:4) as eluant to give
34 (75 mg, 80% yield) as a colorless solid: HRMS (M + Na)+
(ESI+) calcd for C27H25NO6Na+ 482.1580, found 482.1843.
To a solution of 13 (0.94 g, 2.14 mmol) in THF (20 mL) were
added 34 (1.19 g, 2.59 mmol) and triphenylphosphine (678 mg,
2.59 mmol). The mixture was cooled to -78 °C, and then DEAD
(0.41 mL, 2.59 mmol) was added dropwise. The mixture was
allowed to reach room temperature and stirred at room
temperature for 18 h, and then water was added. After
extraction with ethyl acetate, the combined organic phase was
dried over Na2SO4. The solvent was removed, and the residue
was purified on a column of silica gel using ethyl acetate-
benzene (1:15) as eluant to give the product 35 (0.64 g, 34%
yield) as an orange solid: HRMS (M + Na)+ (ESI+) calcd for
C53H46N4O9Na+ 905.3156, found 905.3121.
6-Methyl-12-(2-deoxy-R-L-rhamnopyranosyl)-6,7,12,13-
tetrahydroindolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-di-
one (9). Following the same procedure described for 8,
Compound 9 was obtained from 30 as a yellow solid (92%
yield): HRMS (M + Na)+ (ESI+) calcd for C27H23N3O6Na+
1
2,5-Dihydro-3-[1H-indol-3-yl]-4-[1-(4-O-benzoxyl-3-dibenz-
oxy amido-2,3,6-trideoxy-â-L-lyxo-hexosyl)-1H-indol-3-
yl]-1H-pyrrole-2,5-dione (36). A solution of 35 (630 mg, 0.72
mmol) in 88% formic acid (70 mL) and THF (3 mL) was stirred
at room temperature and monitored by TLC every hour. After
being stirred 22 h, the reaction was completed. Trimethyl-
amine was added dropwise until neutralization, and then
saturated aqueous NaOAc was added. After extraction with
ethyl acetate, the combined organic phase was dried over Na2-
SO4. The solvent was removed, and the residue was purified
on a column of silica gel (ethyl acetate-hexane (3:7) to afford
36 (454 mg, 81% yield) as a orange solid: HRMS (M + Na)+
(ESI+) calcd for C48H38N4O7Na+ 805.2632, found 805.2647. 1H
NMR (500 MHz, CDCl3) 8.57 (1H, s, NH), 7.72 (1H, s), 8.07
(1H, d, J ) 7.2 Hz), 7.84 (1H, s), 7.65 (1H, d), 7.54 (2H, m),
7.42 (2H, m), 7.27 (5H, m), 7.12 (11H, m), 6.78 (2H, m), 5.81
(1H, dd, J ) 2.1 Hz, J ) 10.8 Hz, H-1′), 5.68 (1H, br, H-4′),
5.41 (1H, m, H-3′), 4.26 (1H, m, H-5′), 3.18 (3H, s, NCH3), 3.57
(1H,m, H-2eq), 2.14 (1H,m, H-2ax), 1.30 (3H, d, J ) 6.4 Hz, H-6′).
13C NMR (500 MHz, CDCl3) 17.7 (C6′), 24.6 (NCH3), 34.6 (C2′),
68.4, 70.9, 74.2 (C1′, C4′, C5′), 84.8 (C3′), 107.4, 107.5, 111.6,
120.9, 121.3, 122.6, 122.9, 123.0, 123.1, 125.7,125.9, 127.0,
128.6, 128.8, 128.9, 129.3, 129.4, 130.2, 130.3, 132.5, 133.5,
136.0, 136.2, 136.3, 137.2 (arom), 167.0, 172.6, 172.8, 173.5
(CdO).
6-Methyl-12-(4-O-benzoxyl-3-dibenzoxyamido-2,3,6-tri-
deoxy-â-L-lyxo-hexosyl)-6,7,12,13-tetrahydroindolo[2,3-a]-
pyrrolo[3,4-c]carbazole-5,7-dione (37). A red solution of 36
(454 mg, 0.58 mmol) in benzene (400 mL) was treated with
iodine (120 mg). Air was continuously bubbled through the
reaction which was irradiates with a medium-pressure mer-
cury lamp equipped with a Vycor filter for 15 h. Benzene was
added to the reaction each 2 h to keep the solvent volume
constant. The solvent was removed, and the residue was
dissolved in EtOAc and washed with aqueous Na2S2O3 and
brine. The organic phase was dried over Na2SO4, the solvent
was removed, and the residue was purified on a column of
silica gel using EtOAc-hexane (1:4) as eluant to give the
product 37 (387 mg, 85% yield) as a yellow solid: HRMS
(M + Na)+ (ESI+) calcd for C48H36N4O7Na+ 803.2476, found
803.2457. 1H NMR (500 MHz, CDCl3) 10.73 (1H, s, NH), 9.38
(1H, d, J ) 7.3 Hz), 9.24 (1H, d, J ) 7.9 Hz), 7.79 (2H, d, J )
7.7 Hz), 7.63 (2H, br), 7.44 (2H, m), 7.30 (1H, m), 7.20 (4H,
m), 7.09 (5H, m), 6.94 (4H, m), 6.64 (1H, d, J ) 8.1 Hz), 6.41
(1H, d, J ) 10.8 Hz, H-1′), 6.04 (1H, br, H-4′), 5.59 (1H, d, J )
13.1 Hz, H-3′), 4.77 (1H, m, H-5′), 3.31 (3H, s, NCH3), 3.44
508.1479, found 508.1472. H NMR (500 MHz, DMSO) 12.06
(1H, s, NH), 9.19 (1H, d, J ) 7.8), 9.08 (1H, d, J ) 7.9 Hz),
7.77 (1H, d, J ) 8.5 Hz), 7.64 (2H, m), 7.58 (1H, m), 7.40 (1H,
m), 7.35 (1H, m), 6.79, (1H, br, OH-4′), 6.70 (1H, d, J ) 8.7
Hz, H-1′), 5.60 (1H, s, OH-3′), 4.85 (1H, s, HO-6′), 4.38 (2H,
m, H-5′, H-6′), 4.17 (1H, br, H-3′), 3.90 (1H, s, H-4′), 3.77 (1H,
m, H-6′), 3.14 (3H, s, NCH3), 2.71, 1.87 (2H,m, H-2). 13C NMR
(500 MHz, DMSO) 24.1 (NCH3), 34.8 (C2′), 58.5, 66.6, 67.4,
74.2, 83.9 (C1′, C3′, C4′, C5′, C6′), 110.3, 111.8, 120.5, 121.4, 124.9,
125.1, 127.5, 127.6 (C tert arom), 117.1, 117.7, 118.7, 120.7,
121.2, 125.4, 128.1, 129.1, 140.0, 140.8 (C quat arom), 170.0,
170.1 (CdO).
Hydrochloride of Ethyl 3-Amino-2,3,6-trideoxy-R-L-
lyxo-hexoside (32). The hydrochloride of daunorubicin (10.0
g) was heated in 0.2 M hydrochloric acid (800 mL) at 90 °C
for 1 h. On cooling, the orange precipitate was filtered off and
dried to give daunorubicinone (6.4 g, 91% yield). The filtrate
was evaporated to dryness, which was dissolved in refluxing
ethanol. The solution was cooled to room temperature and left
for overnight at room temperature. The white precipitate was
filtered off to give 32 (3.08 g, 83% yield): 1H NMR (500 MHz,
DMSO) 8.01 (3H, s, NH3), 5.40 (1H, d, J ) 6.1, HO-4), 4.82
(1H, d, J ) 2.2 Hz, H-1), 3.76 (1H, m, H-5), 3.56 (2H, m, H-4,
CH2), 3.38 (2H, m, H-3, CH2), 1.82 (1H, tt, J ) 3.4 Hz, J )
12.7 Hz, H-2eq), 1.68 (1H, dd, J ) 4.24 Hz, J ) 12.6 Hz, H-2ax),
1.12 (3H, t, J ) 7.1 Hz, CH3), 1.08, (1H, d, J ) 6.5 Hz, H-6).
13C NMR (500 MHz, DMSO) 15.8, 17.6 (CH3, C6), 28.9 (C2),
62.7, (CH2), 47.4, 66.3, 66.7 (C1, C4, C5), 96.1 (C3).
Ethyl 4-O-Benzoxyl-3-dibenzoxy amido-2,3,6-trideoxy-
R-L-lyxo-hexoside (33). Benzoxy chloride (10 mL) was added
dropwise to a cold solution of 32 (1.5 g, 7.143 mmol) in
anhydrous pyridine (30 mL). The mixture was stirred at room
temperature for overnight, then quenched with methanol (5
mL). The solvent was removed, and the residue was dissolved
in ethyl acetate, washed with 0.05 M H2SO4 solution, saturated
Na2CO3 solution, and brine, and dried over Na2SO4. Compound
33 (2.55 g, 73% yield) as a white solid was obtained by removal
of the solvent and purification on a column of silica gel using
ethyl acetate-hexane (1:9) as eluant: HRMS (M + Na)+ (ESI+)
1
calcd for C29H29NO6Na+ 510.1893, found 510.1893. H NMR
(500 MHz, DMSO) 8.01 (3H, s, NH3), 5.40 (1H, d, J ) 6.1, HO-
4), 4.82 (1H, d, J ) 2.2 Hz, H-1), 3.76 (1H, m, H-5), 3.56 (2H,
m, H-4, CH2), 3.38 (2H, m, H-3, CH2), 1.82 (1H, tt, J ) 3.4 Hz,
J ) 12.7 Hz, H-2eq), 1.68 (1H, dd, J ) 4.24 Hz, J ) 12.6 Hz,
H-2ax), 1.12 (3H, t, J ) 7.1 Hz, CH3), 1.08, (1H, d, J ) 6.5 Hz,

Rebeccamycin Analogues with Uncommon Sugars
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2609
(1H,m, H-2eq), 2.14 (1H,m, H-2ax), 1.74 (3H, d, J ) 6.4 Hz, H-6′).
13C NMR (500 MHz, CDCl3) 17.7 (C6′), 23.8 (NCH3), 32.2 (C2′),
60.4, 70.8, 76.2 (C1′, C4′, C5′), 85.1 (C3′), 109.2, 111.6, 118.4,
119.0, 119.4, 120.7, 121.3, 121.8, 122.1, 123.0, 125.3, 126.2,
126.6, 127.3, 128.1, 128.2, 128.5, 128.6, 128.9, 129.7, 130.1,
132.2, 133.3, 136.3, 139.9, 140.0 (arom), 166.8, 170.1, 170.2,
173.0 (CdO).
6-Methyl-12-(3-benzoxy amido-2,3,6-trideoxy-â-L-lyxo-
hexosyl)-6,7,12,13-tetrahydroindolo[2,3-a]pyrrolo[3,4-c]-
carbazole-5,7-dione(10). Method 1: A solution of tetrabu-
tylammonium fluoride (1.0 M in THF, 3 mL) was added to a
solution of 37 (50 mg, 0.064 mmol) in THF (8 mL). The mixture
was refluxed for 15 h, diluted with EtOAc (40 mL), washed
with water, and dried over Na2SO4. The solvent was removed,
and the residue was purified on a column of silica gel using
MeOH-CH2Cl2 (1:50) as eluent to give the product 10 (32 mg,
89% yield) as a yellow solid.
Method 2: A solution of 37 (50 mg, 0.064 mmol) and
sodium methoxide (cat.) in anhydrous methanol (10 mL) was
stirred at room-temperature overnight. The solvent was
evaporated under reduced pressure. The residue was dissolved
in dichloromethane, washed with water, and dried over Na2-
SO4. The solvent was removed, and the residue was purified
on a column of silica gel using MeOH-CH2Cl2 (1:50) as eluent
to give the product 10 (33 mg, 91% yield) as a yellow solid.
10: HRMS (M + Na)+ (ESI+) calcd for C34H28N4O5Na+
595.1952, found 595.1974. 1H NMR (500 MHz, CD3CN) 11.76
(1H, s, NH), 9.16 (1H, d, J ) 7.8 Hz), 9.03 (1H, d, J ) 7.9 Hz),
8.00 (1H, d, J ) 7.1 Hz), 7.81 (2H, d, J ) 7.3 Hz), 7.72 (1H, d,
J ) 8.4 Hz), 7.64 (1H, d, J ) 8.1 Hz), 7.48 (3H, m), 7.45-7.26
(4H, m), 6.42 (1H, dd, J ) 5.3 Hz, J ) 11.4 Hz, H-1′), 4.72
(1H, m, H-3′), 4.25 (1H, m, H-5′), 4.10 (1H, d, J ) 1.6 Hz, HO-
4′), 3.09 (3H, s, NCH3), 2.71 (1H, q, J ) 12.2 Hz, H-2eq), 2.04
(1H,m, H-2ax), 1.47 (3H, d, J ) 6.4 Hz, H-6′). 13C NMR (500
MHz, CD3CN) 17.8 (C6′), 24.1 (NCH3), 31.9 (C2′), 50.9, 69.5,
76.4 (C1′, C4′, C5′), 82.6 (C3′), 110.5, 112.4, 119.0, 121.2, 121.8,
122.0, 122.3, 122.7, 125.8, 126.1, 126.4, 128.0, 128.1, 128.3,
128.7, 129.4, 129.5, 130.0, 130.6, 132.4, 134.1, 135.5, 141.0,
141.9 (arom), 167.6, 170.9, 171.1 (CdO).
11,12-Dihydro-11-(3-benzoxy amido-2,3,6-trideoxy-â-L-
lyxo-hexosyl) indolo[2,3-a]carbazole-5,6-dicarboxylic An-
hydride (11). A mixture of 10 (10 mg, 0.017 mmol), KOH
aqueous (48%, 12 mL), and EtOH (4 mL) was stirred at reflux
for overnight. The color of the reaction solution was changed
from deep red to pale yellow. The solution was acidified with
10% citric acid to pH 6, stirred for additional 24 h at room
temperature, then extracted with EtOAc. After removal of the
solvent, the crude compound 11 was purified on a column of
silica gel using MeOH-CH2Cl2 (1:25) to give 11 (8 mg, 80%
yield): HRMS (M + Na)+ (ESI+) calcd for C33H25N3O6Na+
582.1635, found 582.1650. 1H NMR (500 MHz, pyridine-d5)
12.66 (1H, s, NH), 9.45 (2H, m), 9.28 (1H, m), 8.22 (2H, d, J )
7.6 Hz), 7.95 (2H, m), 7.69 (1H, m), 7.59 (1H, m), 7.52-7.34
(5H, m), 6.81 (1H, d, J ) 8.4, H-1′), 5.23 (1H, m, H-3′), 4.55
(1H, br, H-4′), 4.40 (1H, q, J ) 6.4 Hz, H-5′), 2.94 (1H, q, J )
12.6 Hz, H-2eq), 2.24 (1H,m, H-2ax), 1.61 (3H, d, J ) 6.4 Hz,
H-6′). 13C NMR (500 MHz, pyridine-d5) 17.8 (C6′), 31.7 (C2′),
50.9, 68.8, 76.5 (C1′, C4′, C5′), 82.4 (C3′), 110.4, 112.1, 117.9,
118.1, 118.9, 119.9, 121.3, 121.8, 121.9, 122.1, 123.1, 123.8,
123.9, 125.0, 125.3, 127.9, 128.0, 128.1, 128.6, 129.4, 130.5,
135.8, 140.5, 141.5 (arom), 165.5, 165.6, 167.8 (CdO).
11,12-Dihydro-11-(3-amono-2,3,6-trideoxy-â-L-lyxo-hex-
osyl) indolo[2,3-a]carbazole-5,6-dicarboxylic Anhydride
(12). A mixture of 37 (240 mg, 0.31 mmol), solid NaOH (1.50
g), methoxyethanol (23 mL), and water (7 mL) was stirred at
refluxing for overnight. The solution was acidified with 10%
citric acid to pH 7, stirred for additional 24 h at room
temperature, then extracted with EtOAc. After removal of the
solvent, the crude compound was purified on a column of silica
gel to give 11 (70 mg, 40% yield) using MeOH-CH2Cl2 (1:25)
as eluant, and 12 (58 mg, 42% yield) using MeOH-CH2Cl2
(1:5) as eluant. 12: HRMS (M + Na)+ (ESI+) calcd for
C26H21N3O5Na+ 478.1373, found 478.1376. 1H NMR (500 MHz,
DMSO-D2O) 8.61 (1H, d, J ) 7.9 Hz), 8.47 (1H, d, J ) 7.9
Hz), 7.90 (1H, d, J ) 8.4 Hz), 7.64 (1H, d, J ) 8.2 Hz), 7.48
(2H, m), 7.25 (1H, t, J ) 7.5 Hz), 7.16 (1H, t, J ) 7.5 Hz), 6.49
(1H, dd, J ) 2.9, J ) 10.7, H-1′), 3.94 (2H, m, H-3′, H-5′), 3.32
(1H, m, H-4′), 2.08 (2H, m, H-2′), 1.48 (3H, d, J ) 6.1 Hz, H-6′).
13C NMR (500 MHz, DMSO-D2O) 18.8 (C6′), 38.7 (C2′), 70.4,
75.9, 76.8, 82.7 (C1′, C3′, C4′, C5′), 112.5, 113.3, 121.6, 122.1,
123.9, 124.2, 128.1, 128.4 (C tert arom), 117.5, 117.7, 117.9,
118.7, 120.9, 121.8, 129.1, 129.4, 140.1, 140.5 (C quat arom),
164.58, 164.64 (CdO).
11,12-Dihydro-11-(3-amono-2,3,6-trideoxy-â-L-lyxo-hex-
osyl)indolo[2,3-a]carbazole-5,6-dicarboxylic Anhydride
(15) and 6-(3-Imidazol-1-yl-propyl)-12-(2,3,4,6-tetra-O-
benzyl-â-D-glucopyranosyl)-6,7,12,13-tetrahydroindolo-
[2,3-a]pyrrolo[3,4-c]carbazole-5,7-dione (3). Followed the
same procedure described for 23, compound 15 was obtained
from 6-(3-imidazol-1-yl-propyl)-12-(2,3,4,6-tetra-O-benzyl-â-D-
glucopyranosyl)-6,7,12,13-tetrahydroindolo[2,3-a]pyrrolo[3,4-
c]carbazole-5,7-dione (14) as a yellow solid (80% yield): HRMS
(M + Na)+ (ESI+) calcd for C54H44N2O8Na+ 871.2989, found
871.3018. THF (2 mL) and 3-(amino propanyl) imidazole (1.0
mL) was added to the crude compound 15 (50 mg, 0.06 mmol).
The mixture was stirred at 60-70 °C for 24 h, and then diluted
with EtOAc (80 mL), washed with brine, and dried over Na2-
SO4. The solvent was removed, and the residue was purified
on a column of silic gel using MeOH-CH2Cl2 (1:30) as eluant
to give the product 3 (45 mg, 80%) as a yellow solid: HRMS
(M + Na)+ (ESI+) calcd for C60H53N5O7Na+ 978.3837, found
978.3859. 1H NMR (500 MHz, CDCl3) 10.84 (1H, s, NH), 9.41
(1H, d, J ) 7.9 Hz, Ar-H), 9.28 (1H, d, J ) 7.7 Hz, Ar-H),
7.69 (3H, m, Ar-H), 7.43-7.29 (21H, m, Ar-H), 7.11 (2H, m),
7.02 (1H, t, J ) 7.5 Hz, Ar-H), 6.89 (1H, t, J ) 7.7 Hz, Ar-
H), 6.10 (1H, d, J ) 7.7 Hz, Ar-H), 6.05 (1H, d, J ) 9.0 Hz,
H-1′), 5.04 (1H, d, J ) 10.6 Hz), 4.88 (2H, br), 4.76 (2H, m),
4.66(1H, d, J ) 12.5 Hz), 4.41(1H, t, J ) 9.6 Hz), 4.15(2H, d,
J ) 7.1 Hz), 4.07-3.84 (6H, m), 2.93 (1H, d, J ) 9.6 Hz), 2.24
,
(2H, m). 13C NMR (DEPT135) (500 MHz, pyridine-d5 ) 30.8
(CH2), 35.0, 44.7 (N-CH2), 66.9 (C6′), 74.2, 75.1, 75.5, 76.1
(PhCH2), 75.9, 77.2, 77.7, 81.1, 84.8, 85.7 (C1′,C2′, C3′, C4′, C5′).
6-(3-Imidazol-1-yl-propyl)-12-â-D-glucopyranosyl-6,7,-
12,13-tetrahydroindolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-
dione (4). A mixture of 3 (40 mg, 0.067 mmol), Pd/C (40 mg,
palladium, 10 wt % on activativated carbon), and HCl (1 M, 2
mL) in MeOH-THF (60 mL) was hydrogenated at room
temperature/45 psi for 24 h, then filtered and washed with
ethanol. The filtrate was concentrated to remove the solvent.
The residue was dissolved in 80 mL EtOAc, washed with
saturated aqueous NaHCO3 and brine, and dried over Na2-
SO4. The solvent was removed, and the residue was purified
on a prepTLC using MeOH-CH2Cl2 (1:10) as developing
solvent to give the product 4 (21 mg, 80% yield) as a orange
solid: HRMS (M + Na)+ (ESI+) calcd for C26H22N4O5Na+
1
493.1482, found 493.1472. H NMR (500 MHz, DMSO+D2O)
9.13 (1H, d, J ) 7.8 Hz, Ar-H), 9.05 (1H, d, J ) 8.0 Hz, Ar-
H), 7.92 (1H, d, J ) 8.7 Hz, Ar-H), 7.71 (1H, s, imidazole-H),
7.67 (1H, m, Ar-H), 7.56 (2H, m, Ar-H), 7.38 (2H, m, Ar-
H), 7.02 (1H, t, J ) 7.5 Hz, Ar-H), 6.89 (1H, t, J ) 7.7 Hz,
Ar-H), 6.10 (1H, d, J ) 7.7 Hz, Ar-H), 7.26 (1H, s, imidazole-
H), 6.89 (1H, s, imidazole-H), 6.24 (1H, d, J ) 9.0 Hz, H-1′),
4.09 (3H, m), 3.95 (2H, m), 3.76 (1H, m), 3.72-3.48(4H, m),
2.15 (2H, m). 13C (500 MHz, DMSO+D2O) 30.5 (CH2), 35.3,
44.4 (N-CH2), 58.6, 67.9, 73.4, 76.8, 78.9, 84.8 (C1′,C2′, C3′, C4′,
C5′ C6′), 112.2, 112.6, 117.6, 118.7, 119.1, 119.9, 120.4, 121.0,
121.3, 121.4, 121.7, 124.8, 127.6, 127.8, 128.6129.9, 137.8,
141.2, 142.6 (arom), 170.1, 170.2 (CdO).
Cell Culture. Cell lines SW620 and K562 were cultured
in RPMI 1640 supplemented with 10% fetal bovine serum
(FBS), 1% nonessential amino acid, and penicillin (100 units/
mL)-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 (Reb, 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11 and 12) (MTS assay). Cells (2000-
10 000) were seeded in 96-well plates in RPMI-1640 and
incubated for 24 h. The exponentially growing cancer cells were

2610 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Zhang et al.
(13) Cavazos, C.; Keir, S. T.; Yoshinari. T.; Bigner, D. D.; Friedman,
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J-107088 [6-N-(1-hydroxymethyla-2-hydroxy) ethylamino-12, 13-
dihydro-13-(b-D-gluco pyranosyl)-5H-indolo[2,3-a]-pyrrolo[3,4-
c]-carbazole-5, 7(6H)-dione] against pediatric and adult central
nervous system tumor xenografts. Cancer Chemother. Pharma-
col. 2001, 48, 250-254.
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groups on the sugar moiety. Bioorg. Med. Chem. 2003, 11, 3709-
3722.
incubated with various concentrations of compounds for 72 h
at 37 °C (5% CO2, 95% humidity). After 72 h incubation,
tetrazolium [3-(4,5-dimethythiazol-2-yl)]-5-(3-carboxymethoxy-
phenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS, 2
mg/mL) and phenazine methosulfate (PMS, 25 µM) 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. The IC50 values of the
carbohydrate-drug conjugates for cytotoxicity were calculated
by the dose-response curves of percentage of cell growth vs
control (no compound added).
Inhibition of Topoisomerase I with ICT Bioassay. ICT
bioassay was performed according to previously described,37-39
Briefly, HeLa cells were incubated with or without 10 µM
camptothecin or tested compounds for 30 min during expo-
nential growth at 37 °C. Following detergent lysis with
sarkosyl, DNA, and denatured proteins were fractionated by
CsCl gradient centrifugation. The fraction was collected from
the bottom. The DNA plus 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 HybondECL membrane (Amersham Biosciences), and
the blot was probed with anti-topo I antibody. The immuno-
blotsignals were visualized by chemiluminescence (Amersham
Biosciences).
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Acknowledgment. This work was partially sup-
ported by the funding to D.S. from the College of
Pharmacy, The Ohio State University.
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