Synthesis and biological evaluation of rebeccamycin analogues modified at the imide moiety

Article abstract of DOI:10.1016/j.bmcl.2012.06.016

Glycosylated indolocarbazoles related to the antibiotic rebeccamycin represent an important class of antitumour drugs. In the course of our structure-activity relationship studies, new rebeccamycin analogues modified at the imide moiety were synthesised. The antiproliferative activity of the compounds was evaluated on three human cancer cell lines, A2780 (ovarian cancer), H460 (lung cancer), and GLC4 (small-cell lung cancer). The in vitro cytotoxicity of compounds 2 and 4, characterised respectively by a 1,3-dioxolan and (1,3-dioxolan-4-yl)methylene groups linked to the imide moiety, was higher than the reference compound, edotecarin. The effect of compound 2 in inducing tumour regression in the A2780 xenograft model was also investigated.

Full text of DOI:10.1016/j.bmcl.2012.06.016

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5013–5017
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of rebeccamycin analogues modified
at the imide moiety
⇑
Fabio Animati, Marco Berettoni, Mario Bigioni, Monica Binaschi, Amalia Cipollone , Clelia Irrissuto,
Federica Nardelli, Lauso Olivieri
Menarini Ricerche Pomezia, via Tito Speri 10, 00040 Pomezia (Rome), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 January 2012
Revised 6 June 2012
Accepted 7 June 2012
Available online 15 June 2012
Glycosylated indolocarbazoles related to the antibiotic rebeccamycin represent an important class of
antitumour drugs. In the course of our structure–activity relationship studies, new rebeccamycin ana-
logues modified at the imide moiety were synthesised. The antiproliferative activity of the compounds
was evaluated on three human cancer cell lines, A2780 (ovarian cancer), H460 (lung cancer), and GLC4
(small-cell lung cancer). The in vitro cytotoxicity of compounds 2 and 4, characterised respectively by
a 1,3-dioxolan and (1,3-dioxolan-4-yl)methylene groups linked to the imide moiety, was higher than
the reference compound, edotecarin. The effect of compound 2 in inducing tumour regression in the
A2780 xenograft model was also investigated.
Keywords:
Indolocarbazole
Rebeccamycin
Imide substituents
Disaccharides
Cytotoxicity
Ó 2012 Elsevier Ltd. All rights reserved.
Glycosylated [2,3-a] indolocarbazoles have attracted attention
for their antitumour properties.1 The microbial metabolite rebec-
camycin has shown remarkable activity in the poisoning of topoi-
somerase I, a nuclear enzyme that resolves topological problems
arising from numerous vital nuclear processes including DNA rep-
lication, transcription, recombination, and chromatin remodel-
ling.2–4 The crystal structure of reconstituted human
topoisomerase I in covalent and noncovalent complexes with 22-
base pair DNA duplexes was published5,6 and a model for the inter-
action of this complex with the known topoisomerase I inhibitor,
camptothecin,7 and with other classes of inhibitors, indenoisoquin-
olines, indolocarbazoles and phenantridines,8 proposed. Since the
isolation of rebeccamycin, a large number of analogues has been
designed to elucidate structure–activity relationships and to obtain
compounds with better pharmacological properties.9,10 In addition,
the DNA-binding properties and the DNA sequence selectivity of
this class of compounds were investigated.11,12 The sugar moiety
has been revealed to be fundamental for the biological activity,
the indolocarbazole aglycone being completely devoid of activity;
a b configuration at the glycosidic linkage is mandatory for topoi-
able DNA intercalation and enzyme inhibition than those with
chlorine atoms in the position 1 and 11; substitution with a methyl
group on the indole nitrogen abolished both topoisomerase I inhi-
bition and DNA intercalation. A few synthetic compounds structur-
ally related to rebeccamycin, like NCS655649,13 edotecarin14 and
BMS-25074915 (Fig. 1) have entered clinical trials.
On the basis of these studies, a model for the drug-topoisomer-
ase I-DNA ternary complex has been proposed in which the planar
indolocarbazole residue is inserted between two consecutive base
pairs disposing the sugar moiety into the major groove of the dou-
ble helix. The substituent on the imide nitrogen is supposed to pro-
trude toward the opposite groove where it can interact with
topoisomerase I.16 Later, an X-ray crystal structure of the indoloc-
arbazole monosaccharide SA315F bound to a topoisomerase I-DNA
complex, confirmed the intercalative binding mode of this class of
compounds.17 The solved structure revealed that the pyranosyl
substiutent lay on the major groove side of the binding site while
the maleimmide ring protruded into the minor groove side of the
intercalation binding pocket with the substituted nitrogen, pre-
sumably protonated, positioned approximately 4 Å from Arg364,
a residue that if mutated, confers resistance to different classes
of topoisomerase I inhibitors.
somerase I inhibition, as the
a-N-glycosidic analogues do not be-
have as intercalating agents and have much less effect on
enzyme inhibition; the nature and position of the substituents on
the indolo rings influenced target interaction, as derivatives with
hydroxy groups in the 2 and 10 positions showed a more favour-
Our interest in this class of compounds prompted us to synthe-
sise indolocarbazole analogues modified both at the carbohydrate
moiety—results on this work have been already reported18—and
on the residue linked to the imide nitrogen, (the subject of this
study), in order to evaluate the influence of these structural mod-
ifications on the biological activity of the new compounds.
⇑
Corresponding author. Tel.: +39 06 91184414; fax: +39 06 9106137.
E-mail address: acipollone@menarini-ricerche.it (A. Cipollone).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.06.016

5014
F. Animati et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5013–5017
H
N
OH
tartrate
OH
HN
N
H
N
H
H
N
N6
N
O
O
O
O
O
O
O
O
O
O
7
5
4
1
8
F
F
9
3
2
10
HO
OH
HO
OH
N
N
H
N
N
H
N
N
H
N
N
H
N
N
H
11
Cl
Cl
Cl
Cl
OH
OH
O
OH
OH
OH
O
O
O
O
HO OH
F
OH
OCH3
HO OH
OH
HO OH
OH
HO
HO OH
OCH3
BMS-250749
Rebeccamycin
Edotecarin
SA315F
NSC 655649
Figure 1. Chemical structures of some natural (rebeccamycin) and synthetic glycosylated indolocarbazole compounds.
R1
R1
N
R2
O
N
R2
O
O
N
O
O
N
O
O
i.
HO
OH
HO
OH
HO
OH
N
N
H
N
N
N
N
H
H
OH
OH
O
OH
O
O
OH
OR
HO
HO OH
OR
OH
HO
OR
R1
R2
H
9
R = H
Compounds
R
H
1-8
β
10
R = -D-glucosyl
α
11 R = -D-glucosyl
1
H3CO
OCH3
12a-e
, satd. aq. NaHCO3, DMF, 70°C.
i.
O
O
2
3
H
H
H
-
R2
R1
R1R2NNH2
12a
H
H3CO
OCH3
O
N
O
O
H
-
12b
12c
O
4
5
H
H
H
H
O
O
N
HO
OH
HO
OH
12d
12e
H
H
O
HO
HO
HO
O
O
O
O
OH
6
H
Scheme 1. General synthetic scheme for compounds 1–8.
HO
HO
HO
O
O
7
8
H
H
O
a methyl group on the imide nitrogen inhibited topoisomerase I
inhibition while the anhydride group left the enzyme activity
unaffected.
The new derivatives are listed in Figure 2. They all share the
2,10-dihydroxy indolocarbazole framework found in edotecarin.
Compounds 1–5 bear the same b-linked glucosyl residue also pres-
ent in edotecarin, while compounds 6–8 have either a b-glucosyl-
OH
HO
HO
HO
O
O
O
OH
Figure 2. Structures of the compounds 1–8.
Substitutions on the original unsubstituted imide group of rebec-
camycin, and their influence on the biological activity of indolocar-
bazole derivatives, have been reviewed.9,10,19 Analogues with
amino, hydroxy, formylamino, methyl and imidazolylpropyl20
groups introduced in the rebeccamycin or in the dechlorinated
series, in most cases maintained the enzyme inhibition and the cyto-
toxic activity. In compounds like edotecarin and the rebeccamycin
analogue NCS655649, in which respectively, a 2-hydroxy-1-(hydro-
xy-methyl)ethyl and a diethylaminoethyl group, are linked to the
imide function, an increased potency with respect to the parent
compounds, was observed. On the other hand, as previously noted,
b-glucose (6, 7) or an a-glucosyl-b-glucose (8) disaccharide moiety
linked to the indolocarbazole aglycone. The main concern in the
selection of the residues linked to the imide function, was the
retention of the hydrophilic character of these groups. Polar groups
were introduced in more constrained structures with respect to
edotecarin, with the aim of preserving the solubility of the new
derivatives, and to evaluate how these modifications would affect
the interactions in the binding pocket and hence the biological
activity of these derivatives.
The synthesis of compounds 1–8 is outlined in Scheme 1. The
key precursors in this synthetic sequence were the anhydrides

F. Animati et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5013–5017
5015
O
N
N
O
O
O
O
O
O
ii., iii.
i.
HO
OH
BnO
OBn
BnO
OBn
N
N
H
N
H
N
H
N
N
H
OBn
13
O
OH
O
+
OH
OR
HO
OBn
OR
BnO
BnO
RO
O
BnO
Cl
OBn
14 R = Bn
17 R = Bn
9
R = H
15 R = 2,3,4,6-tetra-O-benzyl- β-D-glucosyl
16 R = 2,3,4,6-tetra-O-benzyl-α-D-glucosyl
18 R = 2,3,4,6-tetra-O-benzyl- β-D-glucosyl
19 R = 2,3,4,6-tetra-O-benzyl-α-D-glucosyl
10
R = β-D-glucosyl
R = α-D-glucosyl
11
i. KOH, Na2SO4, CH3CN, r.t.; ii. 10% Pd/C, H2, CH2Cl2/CH3OH 2/1, r.t.; iii. 2M aq. KOH, r.t.
Scheme 2. General synthetic scheme for anhydrides 9–11.
9–11 bearing either the mono or disaccharide moiety. These were
reacted with hydrazines 12a–e in DMF at 60–70 °C. After standard
work-up (dilution with water, extraction of the product in ethyl
acetate/methylethylketone and silica gel chromatography) the
final compounds 1–8 were obtained in yields ranging from 70%
to 80%.
tetra-O-benzyl-
D-glucose by treatment with oxalyl chloride under
standard conditions. 1-Chloro disaccharides 15 and 16 were ob-
tained by elaboration of D-glucose and tetra-O-benzyl-D-glucose
as already reported.18 Deprotection of the benzyl groups, followed
by standard elaboration of the N-methyl imide function, gave the
corresponding glycosylated anhydrides.
Anhydrides 9–11 were obtained following a convergent syn-
thetic strategy in which the indolopyrrolocarbazole moiety 13,
easily obtained using the method of Ohkubo and co-workers21
was coupled with different mono- and disaccharides (Scheme 2).
N-Glycosylation of aglycone 13 was achieved using 1-chloro glyco-
sides as glycosyl donors in heterogeneous basic media.22 This gly-
cosylation protocol is reported to promote almost exclusively the
The hydrazines 12a–e were either purchased (12c) or synthes-
ised from commercially available precursors following synthetic
procedures reported in the literature (Scheme 3). 2-Methylene-
1,3-propandiol was elaborated to (1,3-dimethoxypropan-2-
yl)hydrazine 12a, by methylation of the hydroxyl groups, double
bond oxidation and treatment of the resulting ketone with tert-
butylhydrazine carboxylate. A mixture of 1,3-dioxan-5-ol and
(1,3-dioxolan-4-yl)methanol was oxidised with oxalyl chloride,
DMSO and triethylamine to the corresponding carbonyl com-
formation of the b-glycoside (ratio
a/b anomer = 1/10). Glycosyl
donor 14 was quantitatively obtained from commercially available
NH2
O
HN
i., ii.
iii., iv., v.
HO
OH
H3CO
OCH3
H3CO
OCH3
i. KOH, MeI, DMSO, r.t., 3h; ii. KMnO4, Al2O3, H2O, CH2Cl2, r.t.; iii. BocNHNH2, EtOH, r.t., 12h; iv. NaCNBH3, 6M HCl, MeOH, 5h; v. 6M HCl, H2O, 4h r.t.
O
O
O
O
O
O
O
O
O
O
O
O
v.
v.
2 HCl
NH2
OH
N
+
HN
O
+
HN
iii., iv
i.
ii.
NHBoc
O
NHBoc
+
75/25
OH
O
O
O
O
H
N
H
N
N
O
O
O
NHBoc
NH2
CHO
NHBoc
2 HCl
i. (COCl)2, DMSO, Et3N, r.t.; ii. BocNHNH2, EtOH, r.t., 4h; iii. BH3.THF,THF, r.t; iv. Flash chromatography (SiO2, toluene/AcOEt 6/4); v. AcCl, MeOH, r.t., 1.5h
NHBoc
OH
NHBoc
OH
NH2
OH
OH
HN
N
HN
O
HO
i.
iii.
ii.
O
HO
HO
HO
OH
2HCl
HO
i.BocNHNH2, EtOH, r.t., 4h; ii. NaCNBH3, 6M HCl, MeOH, 5h; iii. 2M HCl
Scheme 3. General synthetic scheme for hydrazides 12a–e.

5016
F. Animati et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5013–5017
Table 2
pounds. Their reaction with tert-butoxycarbonylhydrazine gave
the corresponding hydrazones, which were reduced and separated
by flash chromatography. Deprotection of the tert-butoxycarbonyl
group afforded hydrazides 12b and 12e. In an analogous fashion,
the reaction of 2-hydroxycyclohexanone dimer with tert-but-
oxycarbonylhydrazine and the reduction of the resulting hydra-
zone with sodium cyanoborohydride gave, after deprotection of
the Boc group, hydrazine 12d.
Antitumour efficacy of compound 2 on A 2780 xenograft model
Compound
Dose (mgKgꢁ1
)
Schedule, Route TVIa (%) Deaths/ Totalb
2
90
q4dx4, i.v.
q4dx4, i.v
99.7
99.8
0/5
0/5
Edotecarin 90
a
Tumour volume inhibition in treated versus control mice determined at the
nadir of tumour volume in the treated group.
b
Number of deaths out of the total number of mice.
In order to verify drug target inhibition, a preliminary evalua-
tion of the new compounds was performed in agarose gels using
a DNA cleavage assay based on human recombinant topoisomerase
I (data not shown). Next, the in vitro antiproliferative activity of
compounds 1–8 was determined against a panel of three human
cancer cell lines—A2780 (ovarian cancer), H460 (lung cancer) and
GLC-4 (small-cell lung cancer)- using edotecarin and camptothecin
as reference compounds. The Sulforhodamine B (SRB) assay23 was
used to determine the activity of the new indolocarbazole deriva-
tives and the reference compounds. All compounds were dissolved
in sterile DMSO and diluted with saline (0.9% NaCl) immediately
before use. A2780 (2000 cells/well), H460 (1500 cells/well) and
GLC-4 (1500 cells/well) cell lines were seeded in 96-well microtiter
plates and incubated for 24 h at 37 °C in a 5% CO2 incubator. Drugs
were then added to the wells to achieve final drug concentrations
a considerable loss of activity both in the A2780 (IC50 127 nM)
and in the GLC4 cell line (IC50 70 nM). Derivatives 7 and 8, bearing
the (1,3-dioxolan-4-yl)methylene group and a b-
glucose or an -glucosyl-b- -glucose respectively, showed de-
creased activity in both the cell lines used. It is noteworthy that,
the -1,3 configuration between the two glucosyl moieties (8)
D-glucosyl-b-D-
a
-D
D
a
seemed slightly more favourable than the b-1,3 one (7) in both
the cell lines (7: IC50 55 nM in A2780 cell line; IC50 74 nM in
GLC4 cell line; 8: IC50 11.7 nM in A2780 cell line, IC50 5.3 nM in
GLC4 cell line).
Compound 2 was evaluated in a preliminary experiment for its
effect in inducing tumour regression in the A2780 human ovarian
carcinoma xenograft model. Human tumour cell lines A2780
(ECACC) were originated from subcutaneous (s.c.) in vivo injection
of tumour cells (10 ꢀ 106 cells/flank/0.2 ml). Tumour cells were
suspended in a 0.9% NaCl sterile solution and 0.2 ml of tumour
cells suspension was injected into the right flank of female nude
mice. Female athymic nude mice, 6–8 weeks old, were purchased
from Harlan Italy, maintained in microisolator cages and supplied
with sterile materials under standard conditions, according to
UKCCCR guideline.24 For anti-tumour activity experiments, each
experimental group included five mice bearing a subcutaneous tu-
mour in the right flank. Tumour growth was followed by calibre
measurement of length and width at predetermined (weekly or
twice weekly) times. Tumour volume (TV) was calculated using
the formula: volume in mm3 = width2 ꢀ length/2 (from Ref. 25).
The compound was dissolved in sterile water for injection and di-
luted with saline (0.9% NaCl) immediately before use. It was
administrated i.v. with a twice weekly schedule (q4dx4) at the
same dose as edotecarin and at a dose volume of 10 ml/kg
(Table 2). Drug treatment started when the tumours were approx-
imately 50 mm3 in volume.
ranging from 0.001 to 10 lM. After 24 h of drug exposure, cells
were washed twice with phosphate buffer saline (PBS) and incu-
bated in drug-free medium for about three doubling times (72 h),
at which point the cellular viability was measured using the SRB
assay. The IC50 (the concentration achieving 50% cellular mortality
compared to untreated control) was evaluated by a curve in which
the surviving percentage of cells was reported as a function of the
drug concentration. The H460 cell line responded weakly to the
tested compounds, while in the A2780 and GLC4 cell lines the
new compounds showed similar cytotoxicity profiles (Table 1).
Among the monosaccharides derivatives, compounds 2 and 4,
displayed a higher cytotoxicity with respect to edotecarin. Com-
pound 2, characterised by a 1,3-dioxolan moiety linked to the
imide nitrogen, was 10-fold more potent than edotecarin (IC50
0.7 nM and IC50 6.5 respectively) in the A2780 cell line and equipo-
tent in the GLC4 cell line (IC50 0.7 nM and IC50 0.8 nM respectively),
while compound 4, bearing a (1,3-dioxolan-4-yl)methylene group,
was 3-fold more potent (IC50 1.7 nM) in the A2780 cell line and
equipotent in the GLC4 cell line (IC50 0.8 nM). The other monosac-
charide analogues 1, 3, and 5, exhibited reduced cytotoxicity in
comparison to edotecarin (respectively 2-fold, 10-fold and 20-fold
less than the reference compound in the A2780 cell line).
Compound 2 showed an efficacy comparable to that of edotec-
arin in terms of tumour volume inhibition, and no significant tox-
icity was observed at the schedule and dosage used.
In summary, a series of indolocarbazole glycosides (1–8)26
modified in the upper heterocycle and bearing both mono- and
disaccharide residues were synthesised and their biological activ-
ity was investigated on a panel of tumour cell lines. The preserva-
tion of the hydrophilic character was the main concern in the
choice of the groups linked to the imide nitrogen. Two compounds
(2 and 4), bearing respectively the 1,3-dioxolan and the (1,3-dioxo-
lan-4-yl)methylene groups linked at the imide nitrogen and the
same glucose residue as edotecarin, exhibited a higher in vitro
activity with respect to this reference compound. One of them
(2) was also evaluated on an A2780 xenograft model. These results
showed that introduction of hydrophilic substituents at position 6
on the nitrogen imide may represent a profitable route for the de-
sign of a new class of anticancer compounds. The planned studies
on the binding mode of these new indolocarbazole glycosides
could aid in making further improvements.
When the promising 1,3-dioxolan and the (1,3-dioxolan-4-
yl)methylene groups were introduced in anhydrides 10–11 bearing
disaccharide residues, the resulting compounds 6–8 did not main-
tain the activity of the corresponding monosaccharide analogues.
Compound 6, characterised by a 1,3-dioxolan moiety and a b-D-
glucosyl-b-D-glucose linked to the indolocarbazole core, exhibited
Table 1
Cytotoxic activities of compounds 1–8 (IC50, in nM)
Compound
A2780
H460
GLC4
1
2
3
4
5
6
7
12.7 7.3
0.7 0.1
54 9.5
1.7 1.1
>125
>250
>1000
>1000
>250
3.8 0.8
0.7 0.3
90 9.5
0.8 0.4
101 19
70 16
90
9
127 28
55 22
700 50
113
8
74
3
Acknowledgments
8
11.7 6.3
6.5 0.4
>250
193 145
5.3 1.3
0.8 0.1
Edotecarin
Cpta
7
6
5
4
10
8
The authors wish to thank A. Ettorre for analytical assistance
and C. Fincham for helpful preview of the manuscript.
a
Cpt = Camptothecin.

F. Animati et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5013–5017
5017
indolo[2,3-a]-pyrrolo-[3,4-c]carbazole-5,7(6H)-dione (2): 1H NMR (300 MHz,
DMSO-d6) 11.21 (1H, s), 9.79 (2H, br s), 8.88 (1H, d, J = 6 Hz), 8.81(1H, d,
J = 6 Hz), 7.19 (1H, s), 7.01 (1H, s), 6.83 (2H, dt), 5.98 (1H, d, J = 6 Hz), 5.90 (1H,
br s), 5.85 (1H, d, J = 3 Hz), 5.37 (1H, br s), 5.14 (1H, br s), 4.92 (1H, s), 4.83(1H,
d, J = 6 Hz), 4.66 (1H, d, J = 6 Hz), 4.11 (1H, dd), 4.06–4.01 (2H, m), 3.98–3.90
(2H, m), 3.79 (1H, d, J = 6 Hz), 3.72–3.64 (1H, dd), 3.57–3.48 (2H, m); ESI-MS
(m/z): ESꢁ 619.2 [M-H]ꢁ, (Calcd 620.18). 2,10-Dihydroxy-6-morpholino-12-b-
References and notes
1. Prudhomme, M. Curr. Pharm. Des. 1997, 3, 265.
2. Wang, J. C. Annu. Rev. Biochem. 1996, 65, 635.
3. Pommier, Y.; Pourquier, P.; Fan, Y.; Strumberg, D. Biochim. Biophys. Acta 1998,
1400, 83.
4. Capranico, G.; Marinello, J.; Baranello, L. Biochim. Biophys. Acta 2010, 1806, 240.
5. Redimbo, M. R.; Stewart, L.; Kuhn, P.; Champoux, J. J.; Hol, W. G. J. Science 1998,
279, 1504.
6. Redimbo, M. R.; Champoux, J. J.; Hol, W. G. J. Biochemistry 2000, 39, 6832.
7. Kerrigan, J. E.; Pilch, D. S. Biochemistry 2001, 40, 9792.
8. Pommier, Y. Chem. Rev. 2009, 109, 2894.
9. Prudhomme, M. Eur. J. Med. Chem. 2003, 38, 123.
10. Prudhomme, M. Curr. Med. Chem. Anticancer Agents 2004, 4, 509.
11. Bailly, C.; Colson, P.; Houssier, C.; Rodrigues-Pereira, E.; Prudhomme, M.;
Waring, M. J. Mol. Pharmacol. 1998, 53, 77.
12. Bailly, C.; Riou, J.-F.; Colson, P.; Houssier, C.; Rodrigues-Pereira, E.; Prudhomme,
M. Biochemistry 1997, 36, 3917.
13. Dowlati, A. et al J. Clin. Oncol. 2001, 19, 2309.
14. Danny, W. IDrugs 2004, 7, 137.
15. Saulnier, M. G.; Balasubramanian, B. N.; Long, B. H., et al J. Med. Chem. 2005, 48,
2258.
16. Bailly, C.; Carrasco, C.; Hamy, F.; Vezin, H.; Prudhomme, M.; Saleem, A.; Rubin,
E. Biochemistry 1999, 38, 8605.
17. Staker, B. L.; Feese, M. D.; Cushman, M.; Pommier, Y.; Zembo-wer, D.; Stewart,
L.; Burgin, A. B. J. Med. Chem. 2005, 48, 2336.
18. Animati, F.; Berettoni, M.; Bigioni, M.; Binaschi, M.; Felicetti, P.; Gontrani, L.;
Incani, O.; Madami, A.; Monteagudo, E.; Olivieri, L.; Resta, S.; Rossi, C.;
Cipollone, A. Chem. Med. Chem. 2008, 3, 266.
19. Prudhomme, M. Curr. Med. Chem. 2000, 7, 1189.
20. Zhang, G.; Shen, J.; Cheng, H.; Zhu, L.; Fang, L.; Luo, S.; Muller, M.; Lee, G. E.; Du,
Y.; Sun, D.; Wang, P. G. J. Med. Chem. 2005, 48, 2600.
21. Okhubo, M.; Nishimura, T.; Jona, H.; Honma, T.; Morishima, H. Tetrahedron
1996, 52, 8099.
22. Okhubo, M.; Kawamoto, H.; Ohno, T.; Nakano, M.; Morishima, H. Tetrahedron
1997, 53, 585.
23. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.;
Warren, J. T.; Bokesh, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82,
1107.
24. United Kingdom Co-ordinating Committee on Cancer Research (UKCCCR)
Guidelines for the Welfare of Animals in Experimental Neoplasia, 2nd Ed., Br. J.
Cancer 1998, 77, 1.
25. Geran, R. I.; Greenberg, N. H.; Macdonald, M. M.; Schumacher, A. M.; Abbott, B.
J. Cancer Chemother. Rep. 1972, 3, 1.
D
-glucopyranosyl-12,13-dihydro-5H-indolo-[2,3-a]pyrrolo[3,4-c]carbazole-
5,7(6H)-dione (3): 1H NMR (300 MHz, DMSO-d6) 11.24 (1H, s), 9.84–9.72 (2H,
br s), 8.88 (1H, d, J = 6 Hz), 8.78 (1H, d, J = 6 Hz), 7.20 (1H, s), 7.00 (1H, s), 6.82
(2H, t like), 5.98 (1H, d, J = 6 Hz), 5.89 (1H, br s), 5.34(1H, br s), 5.12 (1H, br s),
4.90 (1H, br s), 4.08–3.99 (1H, m), 3.97–3.89 (2H, br s), 3.81–3.73 (4H, br s),
3.54–3.27 (7H, m); ESI-MS (m/z): ES+ 605.2 [M+H]+, ESꢁ 603.1 [MꢁH]ꢁ, (Calcd
604.18).
6-((1,3-dioxolan-4-yl)methylamino)-2,10-di-hydroxy-12-b-D-
glucopyra-nosyl-12,13-dihydro-5H-indolo[2,3-a]-pyrrolo[3,4-c]carbazole-
5,7(6H)-dione (4): 1H NMR (300 MHz, DMSO-d6) 11.21 (1H, s), 9.84 (2H, br s),
8.88 (1H, d, J = 6 Hz), 8.79 (1H, d, J = 6 Hz), 7.18 (1H, s), 7.00 (1H, s), 6.82 (2H,
dt), 5.97 (1H, d, J = 6 Hz), 5.91 (1H, t), 5.47 (1H, br s), 5.23 (1H, br s), 4.99 (1H, br
s), 4.89 (2H, s), 4.75 (2H, s), 4.18 (1H, p), 4.03–3.99 (2H, m), 3.97–3.87 (2H, m),
3.77 (1H, d like), 3.74–3.71 (4H, dd, J = 6 Hz), 3.56–3.46 (2H, m), 3.10–3.03 (1H,
m); ESI-MS (m/z): ESꢁ 619.2 [MꢁH]ꢁ, (Calcd 620.18). 6-(2,6-Dihydroxycyclo-
hexylamino)-2,10-dihydroxy-12-b-D-glucopyranosyl-12,13-dihy-dro-5H-
indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione (5): 1H NMR (300 MHz,
DMSO-d6) 11.23 (1H, s), 9.88–9.69 (4H, m), 8.93–8.74 (4H, m), 7.20 (2H, s),
7.00 (2H, s), 6.88–6.77 (4H, m), 6.02–5.84 (4H, m), 5.67 (1H, m), 5.40–5.29 (2H,
m), 5.12 (1H, br s), 4.96–4.88 (1H, m), 4.87–4.82 (1H, m), 4.73–4.65 (1H, m),
4.60–4.52(1H, m), 4.07–3.98 (1H, m), 3.88–3.76 (2H, m), 3.75–3.57 (1H, m),
3.01–2.92 (2H, m), 1.99–1.51 (4H, m); ESI-MS (m/z): ESꢁ 647.2 [MꢁH]ꢁ, (Calcd
648.62).
6-(1,3-Dioxan-5-ylami-no)-12-[30-O-(b-
D-glucopyranosyl)-b-D-
glucopyranosyl]-2,10-di-hydroxy-12,13-dihydro-5H-indolo[2,3-a]pyrrolo[3,4-
c]carbazole-5,7(6H)-dione (6): 1H NMR (300 MHz, DMSO-d6) 11.18 (1H, s), 9.83
(2H, br s), 8.87 (1H, d, J = 6 Hz), 8.79 (1H, d, J = 6 Hz), 7.21 (1H, s), 7.00 (1H, s),
6.84 (2H, t), 6.16 (1H, d, J = 6.0 Hz), 6.05 (1H, br s), 5.87 (1H, d, J = 3.0 Hz), 5.76
(2H, s), 5.05–4.97 (4H, m), 4.88–4.82 (2H, m), 4.67–4.62 (2H, m), 4.32 (1H, d,
J = 6 Hz), 4.12–3.98 (6H, m), 3.88–3.81 (1H, m), 3.80–3.63 (6H, m), 3.45–3.36
(1H, m), 3.24–3.17 (1H, t), 3.15–2.93 (3H, m); ESI-MS (m/z): ESꢁ 781.3 [MꢁH]ꢁ,
(Calcd
782.23).
6-((1,3-Dioxolan-4-yl)-methyl-amino)-12-[30-O-(b-
D-
glucopyrano-syl)-b-
D
-glucopyranosyl]-2,10-dihydroxy-12,13-dihydro-5H-
indolo[2,3-a]pyrrolo-[3,4-c]-carbazole-5,7(6H)-dione (7): 1H NMR (300 MHz,
DMSO-d6) 11.18 (1H, s), 9.83 (2H, br s), 8.89 (1H, d, J = 6 Hz), 8.80 (1H, d,
J = 6 Hz), 7.21 (1H, s), 7.00 (1H, s), 6.84 (2H, t), 6.15 (1H, d, J = 6.9 Hz), 6.05 (1H,
br s), 5.89 (1H, t), 5.06–4.92 (4H, m), 4.89 (1H, s), 4.83 (1H, br s), 4.76 (1H, s),
4.63 (1H, br s), 4.33 (1H, d, J = 6 Hz), 4.19 (1H, p), 4.04–3.98 (3H, m), 3.85–3.65
(4H, m), 3.46–3.32 (1H, m), 3.24–3.17 (1H, m), 3.14–2.93 (4H, m); ESI-MS (m/
z): ESꢁ 781.2 [MꢁH]ꢁ, (Calcd 782.23). 6-((1,3-Dioxolan-4-yl)-methylamino)-
26. Data for compounds 1–8: 6-(1,3-Dimethoxypropan-2-ylamino)-2,10-
12-[30-O-(
a-D-glucopyranosyl)-b-D-glucopyrano-syl]-2,10-dihydroxy-12,13-
dihydroxy-12-b-D-glucopyranosyl-12,13-dihydro-5H-indolo-[2,3-
dihydro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione (8): 1H NMR
(300 MHz, DMSO-d6) 11.19 (1H, s), 9.88 (2H, br s), 8.88 (1H, d, J = 6 Hz), 8.80
(1H, d, J = 6 Hz), 7.21 (1H, s), 7.02 (1H, s), 6.83 (2H, t), 6.11 (4H, d, J = 6 Hz), 5.91
(1H, t), 5.71 (1H, br s), 5.33 (1H, br s), 4.98 (1H, d, J = 3 Hz), 4.88 (1H, s), 4.75
(1H, s), 4.62 (1H, m), 4.25–4.12 (2H, m), 4.06–3.98 (3H, m), 3.84–3.69 (2H, m),
3.62–3.54 (1H, m), 3.11–2.97 (2H, m); ESI-MS (m/z): ESꢁ 781.3 [MꢁH]ꢁ, (Calcd
782.23).
a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione (1): 1H NMR (300 MHz, DMSO-d6)
11.20 (1H, s), 9.77 (2H, br s), 8.89 (1H, d, J = 6 Hz), 8.81(1H, d, J = 6 Hz), 7.18
(1H, s), 7.00 (1H, s), 6.82 (2H, dt), 5.98 (1H, d, J = 6 Hz), 5.89 (1H, br s), 5.54(1H,
s), 5.37 (1H, br s), 5.14 (1H, br s), 4.03 (1H, d, J = 6 Hz), 3.98–3.88 (2H, m), 3.79
(1H, d, J = 6 Hz), 3.67–3.60 (2H, m), 3.56–3.50 (2H, br s), 3.44 (4H, d, J = 6 Hz),
3.24 (6H, s); ESI-MS (m/z): ESꢁ 635.2 [M-H]ꢁ, (Calcd 636.21). 6-(1,3-Dioxan-
5-ylamino)-2,10-di-hydroxy-12-b-D-glucopyranosyl-12,13-dihydro-5H-