Synthesis and biological activities of 7-aza rebeccamycin analogues bearing the sugar moiety on the nitrogen of the pyridine ring

Article abstract of DOI:10.1016/j.bmc.2006.07.013

The synthesis of a new family of 7-aza-rebeccamycin analogues in which the sugar moiety is attached to the nitrogen of the pyridine ring is described. The capacity of the newly synthesized compounds to bind to DNA and to inhibit topoisomerase I has been e

Full text of DOI:10.1016/j.bmc.2006.07.013

Bioorganic & Medicinal Chemistry 14 (2006) 7551–7562
Synthesis and biological activities of 7-aza rebeccamycin analogues
bearing the sugar moiety on the nitrogen of the pyridine ring
a
a
b
b
´ `
Samir Messaoudi, Fabrice Anizon, Paul Peixoto, Marie-Helene David-Cordonnier,
c
c
c
a,
*
´
´
Roy M. Golsteyn, Stephane Leonce, Bruno Pfeiffer and Michelle Prudhomme
a
`
`
`
´ ˆ
`
Universite´ Blaise Pascal, Synthese et Etude de Systemes a Interet Biologique, UMR 6504 du CNRS, 63177 Aubiere, France
bINSERM U814, Institut de Recherches sur le Cancer de Lille, Place de Verdun, 59045 Lille, France
cInstitut de Recherches Servier, 125 Chemin de Ronde, 78290 Croissy-sur-Seine, France
Received 24 March 2006; revised 28 June 2006; accepted 3 July 2006
Available online 4 August 2006
Abstract—The synthesis of a new family of 7-aza-rebeccamycin analogues in which the sugar moiety is attached to the nitrogen of
the pyridine ring is described. The capacity of the newly synthesized compounds to bind to DNA and to inhibit topoisomerase I has
been evaluated. Their cytotoxicities toward four tumor cell lines, one murine leukemia L1210 and three human tumor cell lines, one
prostate carcinoma DU145, one colon carcinoma HT29, and one non-small cell lung carcinoma A549, have been determined. Their
abilities to inhibit the checkpoint kinase Chk1 have been evaluated.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
NCI-H69 small cell lung carcinoma, and A431 epi-
dermo¨ıd carcinoma cells with IC50 values in the nano-
molar range. Interestingly, in spite of major differences
in DNA binding and topoisomerase I inhibitory capac-
ities between the compounds bearing the sugar moiety
either on the indole or on the azaindole, in both series
strong cytotoxicities were observed, suggesting other
targets than DNA and topoisomerase I for compounds
in which the sugar is attached to the azaindole. More
recently, 7-aza-rebeccamycins bearing substituents on
the aromatic framework, in various positions of the su-
gar part and on the imide nitrogen, have been
synthesized.8
Rebeccamycin and related compounds have triggered
considerable interest in the past 15 years because of their
ability to inhibit topoisomerase I, a nuclear enzyme
essential for DNA replication and transcription.1–5 It
has been shown that topoisomerase I is the major target
for many rebeccamycin derivatives, however, various
rebeccamycin analogues may have several other targets
such as DNA or kinases.5 Recently, with the aim of
improving the binding properties to their possible tar-
gets, new rebeccamycin analogues, in which one or both
indole moieties have been replaced by a 7-azaindole
unit, have been synthesized.6,7 In mono 7-azaindole
compounds, the carbohydrate moiety has been linked
either to the indole or to the azaindole (Fig. 1). The in vi-
tro antiproliferative activities against a panel of nine tu-
mor cell lines of these 7-aza-rebeccamycin have been
determined. Compared to the parent compounds with-
out 7-azaindole unit, the aza-rebeccamycins were much
more selective toward the tumor cell lines tested. The
most sensitive cells were SK-N-MC neuroblastoma,
In the course of our syntheses of 7-aza-rebeccamycins,
in the Mitsunobu coupling reaction carried out with
aglycone A, we observed the formation of a by-product
which could not be purified by chromatography. It was
isolated in mixture with triphenylphosphine oxide
(Scheme 1). But because of the known tautomerization
of 7-azaindole,9,10 the formation of compound 10 with
the carbohydrate attached to the nitrogen of the pyri-
dine heterocycle was suspected. This compound could
be of interest as an intermediate for the synthesis of a
new family of 7-aza-rebeccamycin analogues. The posi-
tion of the sugar moiety may have major effects on
DNA affinity and on topoisomerase I poisoning.
Keywords: Rebeccamycin; 7-Azaindole; Antitumor compounds; Chk1
inhibitors; DNA binding agents; Topoisomerase I inhibitors.
*
Corresponding author. Tel.: +33 4 73 40 71 24; fax: +33 4 73 40 77
17; e-mail: Michelle.PRUDHOMME@univ-bpclermont.fr
0968-0896/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.07.013

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S. Messaoudi et al. / Bioorg. Med. Chem. 14 (2006) 7551–7562
R
2
N
R
N
R
N
O
O
O
O
O
O
A
B
N
N
H
N
N
N
H
N
N
H
N
R
R
1
1
OH
OH
OH
OH
O
O
O
6 R = H
7 R = CH3
4 R = H
5 R = CH3
HO
HO
HO
OH
OH
OH
OCH
3
OH
1 R1=Cl, R2=H rebeccamycin
2 R1= R2=H dechlorinated rebeccamycin
3 R1=H, R2=CH3
R
N
O
O
H
N
R
O
N
N
N
H
N
OH
8 R = H
O
9 R = CH3
N
N
O
HO
H C
3
H
OH
OH
H CO
3
NHCH
3
Staurosporine R = H
UCN-01 R = OH
Figure 1. Structures of rebeccamycin and aza analogues previously synthesized.
In this paper, the syntheses of 7-aza-rebeccamycins
bearing the sugar moiety on the pyridine ring are
described. Their cytotoxicities against four tumor cell
lines, their DNA binding properties, and their anti-topo-
isomerase I activities are reported. Moreover, since
structurally related natural products staurosporine and
UCN-01 (Fig. 1) are potent Checkpoint 1 kinase
(Chk1) inhibitors,11 the capacities of the newly synthe-
sized compounds to inhibit Chk1 have been investigated.
7-azaindole. An axial–axial coupling constant of 9 Hz
0
0
between H1 and H2 is consistent with a b-coupling.
Deprotection of the hydroxyl group of the sugar unit
by aminolysis in methanol afforded compound 12. Oxi-
dative photocyclization of 11, followed by the removal
of the acetyl groups, led to 14, the first aza-rebeccamycin
analogue of this series.
With the aim of introducing various substituents on the
imide nitrogen,13 N-methylated compound 12 was treat-
ed with aqueous NaOH followed by a work-up in an
acidic medium affording 15 with concomitant hydrolysis
of the acetates of the sugar moiety (Scheme 3). Like in
N-methylated series, the Mitsunobu reaction carried
out in N-benzyloxymethyl (N-BOM) series with agly-
cone C6 to obtain analogues without substituents on
the imide nitrogen, either with 3,4,6-tri-O-benzyl-2-tos-
yl-a-D-glucopyranose D14 or with 2,3,4,6-tetra-O-ben-
zyl-a-D-glucopyranose E, led to compounds F14 and G,
respectively, as the major products. The minor products
of the reactions were 16 and 17 isolated in 17% and 25%
yields, respectively (Scheme 4). Deprotection of the in-
dole nitrogen with tetrabutylammonium fluoride15 gave
compounds 18 and 19 which were further photocyclized
leading to compounds 20 and 21. Removal of the BOM
and benzyl protective groups, using boron tribromide
then aqueous NH4OH, yielded compounds 22 and 23.
2. Results and discussion
2.1. Chemistry
The Mitsunobu reaction performed with aglycone A12
protected on the indole nitrogen with a Boc group and
a-D-2,3,4,6-tetra-O-acetyl-glucopyranose led to com-
pound B as the major product (Scheme 1), whereas com-
pound 10, the minor product, could only be isolated in
mixture with triphenylphosphine oxide. Compound 10
was partly purified by flash chromatography. Removal
of the Boc protective group was performed by reaction
of compound 10 (in mixture with triphenylphosphine
oxide) with formic acid giving compound 11. The
position of the sugar moiety was assigned from NMR
experiments (1H–1H COSY and 1H–13C correlations,
0
INEPT-DN) (Scheme 2). The anomeric proton H1 at
7.01 ppm is coupled with the two carbons at
125.7 ppm (CH) and at 150.0 ppm (C quat) of the
Via a Mitsunobu coupling reaction, compound 11 was
isolated with only 8% yield. To enhance the coupling

S. Messaoudi et al. / Bioorg. Med. Chem. 14 (2006) 7551–7562
7553
CH3
N
CH3
N
CH3
N
O
O
O
O
O
O
DIAD
THF
PPh3
+
N
N
N
Boc
N
N
N
OH
OAc
N
N
H
N
Boc
O
AcO
Boc
O
O
A
10
in mixture
with PPh O
OAc
OAc
AcO
AcO
B
OAc
AcO
3
OAc
OAc
AcO
OAc
HCO2H
CH3
N
CH3
N
O
O
NH4OH
MeOH
93%
O
O
N
N
N
H
N
N
N
H
O
OH
O
HO
OAc
AcO
hν, Ι
benzene
70%
12
2
11
OH
OH
OAc
OAc
CH3
N
CH3
N
O
O
O
O
NH4OH
MeOH
69%
N
N
N
H
N
N
N
H
O
O
OAc
OH
AcO
HO
14
13
OAc
OH
AcO
OH
Scheme 1.
d, 6.78 ppm
CH3
N
d, 7.67 ppm
deprotection of the indole nitrogen using formic acid,
compound 24 was obtained as the major product in
63% yield, whereas compound F was the minor product
in only 20% yield. Photocyclization of 24 led to 25 in
69% yield.
O
O
H
H
107.2
107.5
7.83 ppm
125.7 ppm
122.7 ppm
N
H
N
H
N
2.2. DNA binding
H
1O' Ac
135.9 ppm
O
150.0 ppm
As part of the determination of structure–activity rela-
tionships, compounds 14 and 22 were first tested for
their ability to interact with DNA. Either calf thymus
(CT-DNA) or poly(dAdT)2 was used in melting temper-
ature studies in the presence of increasing drug/base ra-
tios of the derivatives 14 and 22. From comparison of
the melting temperature (Tm) obtained in the presence
or absence (control) of the tested drugs, Figure 2A clear-
ly evidences compound 14 as a potent DNA helix stabi-
lizing molecule, whereas compound 22 clearly does not.
The deduced DTm values obtained using increasing
drug/DNA ratios of compound 14 in the presence of
poly(dAdT)2 are much higher (up to a DTm value
of 10 °C for R = 1) than those obtained in the presence
AcO
7.01 ppm
83.8 ppm
2'
OAc
AcO
11
Scheme 2.
on the pyridine nitrogen, the reaction was carried out
with aglycone A and a 1,2-anhydro sugar according to
the method reported by Danishefsky et al.16 in a total
synthesis of rebeccamycin (Scheme 5). The 3,4,6-tri-O-
benzyl-1,2-anhydro sugar was prepared from 3,4,6-tri-
O-acetyl-glucal in three steps according to the literature
procedure.17,18 After coupling of the carbohydrate then

7554
S. Messaoudi et al. / Bioorg. Med. Chem. 14 (2006) 7551–7562
CH3
N
O
O
O
O
O
1. NaOH, H2O
THF
N
N
N
2. HCl
N
N
N
H
H
O
O
OH
OAc
HO
AcO
12
15
OH
OAc
AcO
HO
Scheme 3.
BOM
N
BOM
N
BOM
N
O
O
O
O
O
O
DIAD
THF
PPh3
+
N
N
N
N
N
N
OH
OR
N
N
H
N
SO2Ph
O
SO2Ph
BnO
SO2Ph
O
O
OR
OR
BnO
BnO
C
16 R = Ts
17 R = Bn
OBn
F R = Ts
BnO
OBn
OBn G R = Bn
BnO
BnO
D R = Ts
E R = Bn
TBAF
THF
H
N
BOM
N
BOM
N
O
O
O
O
O
O
1) BBr3, CH2Cl2
2) NH4OH, THF
hν, I
2
benzene
N
N
N
H
N
N
N
H
N
N
N
H
O
O
O
18 R = Ts
19 R = Bn
OR
OR
20 R = Ts
21 R = Bn
OR
HO
BnO
BnO
22 R = Ts
23 R = H
OH
OBn
OBn
HO
BnO
BnO
Scheme 4.
of CT-DNA (DTm value of 4 °C for R = 1). By contrast,
the presence of a tosyl group on the sugar ring totally
abolished this DNA binding propensity.
weakly changes the DNA relaxation profile induced by
topoisomerase I in correlation with its weak DNA bind-
ing efficiency.
Interestingly, the presence of the sugar ring on the first
ring (A-ring) of the rebeccamycin structure enhances
the DNA binding propensity since the same structure
but bearing the sugar ring on the classical B-ring (com-
pound 7) failed to interact with DNA as previously
shown.6
2.3. Topoisomerase I poisoning
These selected rebeccamycin derivatives were further
studied for their ability to poison topoisomerase I en-
zyme in order to address their mechanism of action.
By comparison with the reference molecule camptothe-
cin, increasing concentrations of compounds 14 or 22
were incubated with supercoiled DNA plasmid as de-
scribed previously for Figure 2B but the samples were
separated on an ethidium bromide-containing agarose
gel (Fig. 3A). An efficient DNA cleavage (Nck form)
is only observed using camptothecin (CPT lane) suggest-
ing that either 14 and 22 are not efficient topoisomerase
poisons. An efficient gel shift of the supercoiled DNA
form appears with increasing concentrations of 14 in
In order to get an insight into the nature of the DNA
binding, we performed topoisomerase I induced gel
relaxation experiments (Fig. 2B). Incubation of increas-
ing concentrations of compound 14 correlates with an
increase in the relaxation of the circular plasmid to a ful-
ly relaxed form and using a larger amount of compound
to a positively supercoiled form typical of the effect of an
intercalating compound. By contrast, compound 22 only

S. Messaoudi et al. / Bioorg. Med. Chem. 14 (2006) 7551–7562
7555
CH
N
CH
3
N
3
CH
N
3
O
O
O
O
O
O
1- NaH / THF
+
OBn
2-
N
N
N
N
N
N
H
H
N
N
N
BnO
BnO
O
H
Boc
O
O
BnO
OH
BnO
OH
O
H
63%
24
20%
A
3- HCO H
2
OBn
OBn
I
BnO
BnO
hν, Ι
2
69%
benzene
CH
N
3
O
O
N
N
N
H
O
BnO
OH
25
OBn
BnO
Scheme 5.
correlation with its efficient DNA binding effect. Migra-
tion on a denaturing polyacrylamide gel of a radiola-
beled DNA fragment incubated in the presence of
increasing concentrations of 14 or 22 reveals that either
compounds failed to poison topoisomerase I, only a
weak cleavage site being observed using 50 lM of com-
pounds. By contrast, the control compound camptothe-
cin and the topoisomerase I poisoning rebeccamycin
derivative NB-506 used as controls efficiently induced
cleavage sites (Fig. 3B).
2.5. Chk1 inhibitory properties
The transitions in the cell division cycle are regulated
predominantly by the activities of cyclin-dependent
kinases (CDKs). In the case of DNA damage, however,
checkpoints are engaged and will block the cell cycle by
eventually inhibiting CDKs to allow time for DNA re-
pair. The activity of the G1/S phase checkpoint requires
the p53 protein, which is either absent or mutated in
more than 50% of human tumors. In the absence of a
G1/S phase checkpoint, tumors become more depen-
dent upon the G2/M phase checkpoint, which is regu-
lated, in part, by Checkpoint 1 kinase (Chk1). An
inhibitor of Chk1 should, therefore, selectively force
cancer cells to bypass the G2 checkpoint and enter a
premature and lethal mitosis. Staurosporine and
UCN-01, isolated from cultures of Streptomyces, inhib-
it Chk1 with IC50 values of 8 and 7 nM, respectively.11
Clinical trials are currently undergoing combining
UCN-01 with a DNA damaging agent such as cisplatin
and topotecan.19 Compounds 14, 22, and 23 are car-
bazoles structurally related to the potent Chk1 inhibi-
tors staurosporine and UCN-01. Therefore, the
inhibitory properties of compounds 14, 22, and 23 were
determined and compared to that of rebeccamycin 1.
The percentages of Chk1 inhibition at a drug concen-
tration of 10 lM were evaluated, and for the most effi-
cient inhibitor 23, the IC50 value was determined (Table
2). Rebeccamycin 1 and compounds 14 and 22 are
weak Chk1 inhibitors, whereas compound 23 is a
strong Chk1 inhibitor with an IC50 value toward this
enzyme of 61 nM. Interestingly, Compound 23 with
2.4. In vitro antiproliferative activities
The in vitro antiproliferative activities toward four
tumor cell lines: one murine leukemia L1210, and three
human tumors: prostate carcinoma DU145, colon car-
cinoma HT29, and non-small cell lung carcinoma A549
were evaluated. The IC50 values in lM are reported in
Table 1. Compared with the parent compounds 6 and
7 in which the sugar part is attached to the nitrogen of
the B-ring, compounds 23 and 14 with the glucosyl
moiety attached to the nitrogen of the A-ring were less
cytotoxic against L1210 cells. Compound 14 is also less
cytotoxic than compound 7 toward all the other tumor
cell lines tested. At first sight, it seems that the shifting
of the sugar unit from the B-ring to the A-ring decreas-
es the cytotoxicity. Bis-indolyl compound 12 is less
cytotoxic than the fully aromatized analogue 7. The
presence of a tosyl group in the 20 position of the sugar
increases the cytotoxicity (compare 22 and 23), this
effect could be due to a better penetration into the
cells.

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S. Messaoudi et al. / Bioorg. Med. Chem. 14 (2006) 7551–7562
A
Topo I
A
12
10
R=0.1
R=0.25
R=0.5
R=1
14
22
14
8
6
4
2
Nck
22
Sc
Rel
0
Topo I
NB506 14
B
22
Topo I
B
14
22
Rel+Nck
topo
Sc
Figure 2. DNA binding efficiencies and mode of binding to the DNA
helix of compounds 14 and 22. (A) Melting temperature studies were
performed using either calf thymus DNA or a synthetic poly(dAdT)2
double stranded oligonucleotide and increasing concentrations of 14 or
22 to a drug/DNA ratio R of 0.1 (white boxes), 0.25 (dashed boxes),
0.5 (black boxes) or 1 (gray boxes). The various Tm values were
deduced from the first derivative plots and were subtracted from the
value obtained using either DNA alone to obtain the various DTm
values presented in the diagrams. (B) Topoisomerase I DNA relaxa-
tion studies were obtained by incubating supercoiled plasmid DNA
with increasing concentrations of compounds 14 or 22 prior to the
addition of topoisomerase I enzyme as described in the experimental
section. The DNA samples were then separated on an agarose gel and
stained post-electrophoresis using ethidium bromide. Sc, Rel, Nck, and
topo refer to supercoiled, relaxed, nicked, and topoisomer DNA
plasmid forms, respectively. Lanes DNA refer to the native plasmid
DNA alone.
the glucosyl moiety attached to the nitrogen of the pyr-
idine ring is a considerably stronger Chk1 inhibitor
than the parent compound 6 in which the sugar is
linked to the nitrogen of the pyrrole. Concerning com-
pound 22, it may be possible that the bulky tosyl group
of compound 22 prevents the binding to the ATP bind-
ing site of the enzyme. The weak inhibitory effect of
compound 14 is not surprising since the imide nitrogen
is not free. In the crystal structures of Chk1 in complex
with staurosporine, UCN-01, and isogranulatimide, a
natural product with a carbazole structure containing
an imide upper heterocycle, two hydrogen bonds are
conserved: one between a carbonyl of the upper hetero-
cycle of the drugs and the Cys87 residue, the other
between the lactam or imide NH and the Glu85 resi-
due.11,20 The N-methylated imide prevents the
stabilization of compound 14 inside the ATP binding
site of Chk1.
Figure 3. Topoisomerase I DNA poisoning. Increasing concentrations
of 14, 22, NB506 or 20 lM of CPT were incubated with supercoiled
pUC19 plasmid DNA (A) or the 117 bp radiolabeled DNA fragment
(B) prior to further addition of 4 U of human recombinant topoiso-
merase I enzyme. The DNA samples were then separated on a 1%
agarose ethidium bromide-containing gel (A) or a 8% denaturing
polyacrylamide gel (B). Sc, Rel, and Nck refer to supercoiled, relaxed,
and nicked DNA plasmid forms, respectively. G-track lanes reveal the
guanines position on the radiolabeled 117 bp DNA fragment. Lanes
DNA correspond to the relative DNA alone.
3. Conclusion
In conclusion, we have prepared a novel series of aza
rebeccamycin derivatives in which the glucosyl moiety
is attached to the nitrogen of the pyridine ring. It ap-
peared that the shifting of the sugar from the nitrogen

S. Messaoudi et al. / Bioorg. Med. Chem. 14 (2006) 7551–7562
7557
Table 1. In vitro antiproliferative activities (IC50 lM) toward several
tumor cell lines: one murine leukemia L1210, and four human tumors
prostate carcinoma DU145, colon carcinoma HT29, non-small cell
lung carcinoma A549, and colon carcinoma HCT116
4.1.1. 3-(1H-Indol-3-yl)-1-methyl-4-[7-(2,3,4,6-tetra-O-
acetyl-b-D-glucopyranos-1-yl)-7H-pyrrolo[2,3-b]pyridin-3-
yl]-pyrrole-2,5-dione (11). To a solution of aglycone A
(384 mg, 0.863 mmol) in THF (27 mL) were added
2,3,4,6-O-acetyl-a-D-glucopyranose (672 mg, 1.93 mmol)
and triphenylphosphine (505 mg, 1.93 mmol). The mix-
ture was cooled to ꢀ78 °C then diethyl azodicarboxylate
(DEAD) (304 lL, 1.93 mmol) was added dropwise. The
mixture was allowed to reach room temperature, then
was stirred at room temperature for 18 h. Water
(50 mL) was added. After extraction with EtOAc, the
organic phase was dried over MgSO4, and the solvent
was removed. The residue was purified by flash chroma-
tography (eluent: cyclohexane/AcOEt 7:3 containing
NEt3 20%) to give a mixture of N-Boc 10 and triphenyl-
phosphine oxide (600 mg).
Compound
L1210
DU145
HT29
A549
HCT116
1
2
0.14
0.11
0.58
0.06
1.3
ne
ne
0.3
2.5
0.3
2
nd
nd
nd
nd
nd
nd
nd
nd
nd
13.4
16
3
0.42
0.59
6
0.43
4.8
0.46
5.3
4
5
17.8
>100
67.2
76.2
>100
57.9
100
47.2
>100
59.9
>100
>100
nd
6
0.13
0.34
85
0.36
0.20
82.6
83
7
12
14
22
23
17.6
15.4
48
nd
nd
nd
This mixture was dissolved in formic acid (250 mL) and
stirred at room temperature for 24 h. After evaporation,
the residue was purified by flash chromatography
(eluent: cyclohexane/AcOEt from 7:3 to 5:5) to give 11
(48 mg, 0.071 mmol, 8% yield) as a red solid.
Table 2. Percentages of Chk1 inhibition at a drug concentration of
10 lM
Compound
% of Chk1 inhibition at 10 lM
IC50 in lM
1
6
62
15
nd
nd
14
22
23
31.8
45.5
85
nd
nd
Mp: 155 °C. IR (KBr) mC@O 1700, 1752 cmꢀ1, mNH 3300–
3500 cmꢀ1
.
HRMS (ESI+) [M + H]+ calcd for
0.061
C34H33N4O11 673.2146, found 673.2118. 1H NMR
(400 MHz, CDCl3): 1.62 (3H, s, CH3CO), 2.01 (3H, s,
CH3CO), 2.08 (6H, s, CH3CO), 3.19 (3H, s, N–CH3),
0
4.11–4.20 (2H, m, H5 , H6 ), 4.34 (1H, dd, J1 = 13.0
0
of the B-ring to the nitrogen of the A-ring enhances
binding to the DNA. However, as observed with other
rebeccamycin derivatives, the DNA binding affinity is
not correlated with topoisomerase I inhibitory proper-
ties. The shifting of the sugar from the nitrogen of the
B-ring to the nitrogen of the A-ring considerably
increases the Chk1 inhibitory effect. Indeed, com-
pound 23 is a potent Chk1 inhibitor. The work
reported here provides a new avenue to the design
of Chk1 inhibitors.
0
Hz, J2 = 5.0 Hz, H6 ), 5.27 (1H, t, J = 10.0 Hz, H4 ),
0
0
5.37 (1H, t, J = 10.0 Hz, H2 ), 5.58 (1H, t, J = 9.5 Hz,
0
H3 ), 6.64–6.72 (2H, m), 6.78 (1H, d, J = 8.0 Hz), 7.01
0
(1H, d, J = 9.0 Hz, H1 ), 7.05 (1H, t, J = 7.5 Hz), 7.34
(1H, d, J = 8.0 Hz), 7.67 (1H, d, J = 8.0 Hz), 7.79–7.84
(2H, m), 8.38 (1H, s), 8.82 (1H, s, NHind). 13C NMR
(100 MHz, CDCl3): 20.0, 20.5, 20.6, 20.7 (CH3CO),
0
0
24.2 (N–CH3), 61.6 (C6 ), 67.9, 71.6, 72.5, 75.5 (C2 ,
0
0
0
0
C3 , C4 , C5 ), 83.8 (C1 ), 111.0, 111.4, 120.4, 121.7,
122.7, 125.7, 127.9, 134.7, 149.9 (C tert arom), 107.2,
107.5, 124.9, 125.1, 127.9, 128.5, 136.0, 150.0 (C quat
arom), 169.0, 169.6 (2C), 170.5, 172.6 (2C) (C@O).
4. Experimental
4.1. Chemistry
4.1.2. 3-(1H-Indol-3-yl)-1-methyl-4-[(7-b-D-glucopyranos-
1-yl)-7H-pyrrolo[2,3-b]pyridin-3-yl]-pyrrole-2,5-dione (12).
To a solution of 11 (20 mg, 0.0297 mmol) in methanol
(10 mL) was added 28% aqueous NH4OH (16 mL). The
mixture was stirred at 65 °C for 22 h. The solvent was re-
moved, then water and EtOAc were added to the residue.
After extraction with EtOAc, the organic phase was dried
over MgSO4, and the solvent was removed to give 12
(14 mg, 0.0277 mmol, 93% yield) as a red solid.
IR spectra were recorded on a Perkin-Elmer 881
spectrometer. NMR spectra were recorded on
a
Bruker AVANCE 400 (1H: 400 MHz, 13C:
100 MHz) chemical shifts d in ppm, the following
abbreviations are used: singlet (s), doublet (d), triplet
(t), pseudo-triplet (pt), doubled triplet (dt), multiplet
(m), br s (broad signal), tertiary carbons (C tert),
and quaternary carbons (C quat). The signals were
1
assigned from H–1H COSY and 13C–1H correlations.
Mp: 182–185 °C. IR (KBr) mC@O 1695, 1750 cmꢀ1
,
NH,OH 3100–3600 cmꢀ1. HRMS (FAB+) [M+H]+ calcd
Low resolution mass spectra (ESI+ and APCI+) were
determined on a MS Hewlett Packard engine. HRMS
spectra (FAB+) were determined on a high resolution
Fisons Autospec-Q spectrometer at CESAMO
(Talence, France). Chromatographic purifications
were performed by flash silicagel Geduran SI 60
(Merck) 0.040–0.063 mm or Kieselgel 60 (Merck)
0.063–0.200 mm column chromatography. For purity
tests, TLC were performed on fluorescent silica gel
plates (60 F254 from Merck).
m
1
for C26H24N4O7 505.1723, found 505.1719. H NMR
(400 MHz, DMSO-d6): 3.07 (3H, s, CH3), 3.48–3.56
(4H, m), 3.72–3.85 (2H, m), 4.68 (1H, t, J = 6.0 Hz,
OH), 5.25 (1H, d, J = 4.5 Hz, OH), 5.39 (2H, d,
J = 5.5 Hz, OH), 6.53 (1H, d, J = 8.5 Hz), 6.71 (1H, d,
J = 7.0 Hz), 6.77 (1H, d, J = 8.0 Hz), 6.87 (1H, t,
J = 7.0 Hz), 7.05 (1H, t, J = 7.0 Hz), 7.45 (1H, d,
J = 8.5 Hz), 7.66 (1H, d, J = 8.0 Hz), 7.79 (1H, d,
J = 1.5 Hz), 8.08 (1H, s), 8.26 (1H, d, J = 6.5 Hz),

7558
S. Messaoudi et al. / Bioorg. Med. Chem. 14 (2006) 7551–7562
11.72 (1H, s, NHind). 13C NMR (100 MHz, DMSO-d6):
72.6, 77.1, 81.1, 86.5 (C1 , C2 , C3 , C4 , C5 ), 109.8,
111.9, 119.8, 124.0, 126.5, 128.3, 135.6 (C tert arom),
114.9, 116.1, 118.1, 119.6, 121.6, 124.1, 132.6, 140.8,
142.7, 154.1 (C quat arom), 170.3 (2 C@O).
0
0
0
0
0
0
23.9 (N–CH3), 60.8 (C6 ), 69.5, 72.5, 76.9, 80.7, 86.6 (C1 ,
0
0
0
0
0
C2 , C3 , C4 , C5 ), 110.5, 111.8, 119.4, 120.9, 121.6, 128.3,
128.6, 133.5, 148.7 (C tert arom), 105.5, 106.2, 123.8,
124.8, 126.7, 128.2, 136.0, 150.2 (C quat arom), 171.9,
172.0 (C@O).
4.1.5. 3-[7-(b-D-Glucopyranos-1-yl)-7H-pyrrolo[2,3-
b]pyridin-3-yl]-4-(1H-indol-3-yl)-furane-2,5-dione (15).
4.1.3. 6-Methyl-1-(2,3,4,6-tetra-O-acetyl-b-D-glucopyr-
anos-1-yl)-1H-pyrido[30,20:4,5]pyrrolo[2,3-a]pyrrolo[3,4-
c]carbazole-5,7(12H)-dione (13). A mixture of 11 (70 mg,
0.104 mmol) and iodine (290 mg, 1.14 mmol) in benzene
(150 mL) was irradiated for 4 h with a medium-pressure
mercury lamp (400 W). The solvent was removed, and
the residue was dissolved in EtOAc and washed with sat-
urated aqueous sodium thiosulfate (100 mL), then with
brine (100 mL). The organic phase was dried over
MgSO4. After removal of the solvent, the residue was
purified by flash chromatography (eluent: EtOAc/cyclo-
hexane 5:5) to give 13 (49 mg, 0.073 mmol, 70% yield) as
an orange solid.
To a suspension of 12 (50 mg, 0.074 mmol) in water
(40 mL) were added NaOH (44.5 mg, 1.1 mmol) and
THF (5 mL). The mixture was stirred at room tempera-
ture for 2 h, then was acidified to pH 1 with 2 N HCl.
The mixture was stirred overnight at room temperature.
After evaporation, the residue was purified by flash
chromatography (eluent: THF/methanol 95:5) to yield
15 (21.8 mg, 0.044 mmol, 60% yield) as a red solid.
Mp: 168–170 °C. IR (KBr) mC@O 1750, 1820 cmꢀ1
,
m
NH,OH 3000–3600 cmꢀ1. HRMS (ESI+) [M + H]+ calcd
1
for C25H22N3O8 492.1407, found 492.1403. H NMR
(400 MHz, DMSO-d6): 3.45–3.55 (3H, m), 3.72–3.85
(2H, m), 4.10 (1H, m), 4.64 (1H, t, J = 5.5 Hz, OH),
5.22 (1H, d, J = 5.5 Hz, OH), 5.30–5.40 (2H, m, 2OH),
Mp: 128–130 °C. IR (KBr) mC@O 1700, 1750 cmꢀ1, mNH
3200–3600 cmꢀ1. HRMS (ESI+) [M + H]+ calcd for
C34H31N4O11 671.1989, found 671.1982. 1H NMR
(400 MHz, CDCl3): 1.33 (3H, s, CH3CO), 1.97 (3H, s,
CH3CO), 2.10 (3H, s, CH3CO), 2.11 (3H, s, CH3CO),
3.15 (3H, s, N–CH3), 4.34–4.43 (3H, m), 5.29 (1H, t,
J = 9.5 Hz), 5.38 (1H, t, J = 9.5 Hz), 5.65 (1H, t,
J = 9.5 Hz), 6.72 (1H, t, J = 7.0 Hz), 6.79 (1H, d,
0
6.51 (1H, d, J = 9.5 Hz, H1 ), 6.75 (1H, d, J = 7.5 Hz),
6.80 (1H, d, J = 8.0 Hz), 6.96 (1H, t, J = 7.0 Hz), 7.07
(1H, t, J = 8.0 Hz), 7.46 (1H, d, J = 8.0 Hz), 7.73 (1H,
d, J = 7.5 Hz), 7.88 (1H, d, J = 3.0 Hz), 8.10 (1H, s),
8.33 (1H, d, J = 6.5 Hz), 12.10 (1H, s, NHind). 13C
0
NMR (100 MHz, DMSO-d6): 60.7 (C6 ), 69.5, 72.5,
0
0
0
0
0
76.9, 80.7, 86.7 (C1 , C2 , C3 , C4 , C5 ), 111.4, 112.2,
119.8, 121.1, 122.0, 129.0, 129.7, 134.1, 149.8 (C tert
arom), 104.7, 105.4, 124.3, 124.4, 126.3, 129.1, 136.1,
150.7 (C quat arom), 166.6, 166.7 (2C, C@O).
0
J = 9.0 Hz, H1 ), 7.27 (1H, dt, J1= 8.0 Hz, J2= 1.5 Hz),
7.42 (2H, t, J = 6.5 Hz), 7.79 (1H, d, J = 7.0 Hz), 8.89
(1H, d, J = 8.0 Hz), 8.93 (1H, d, J = 7.0 Hz), 9.66 (1H,
s, NHind). 13C NMR (100 MHz, CDCl3): 19.8, 20.5,
0
20.7, 20.9 (CH3CO), 23.6 (N–CH3), 62.2 (C6 ), 67.8,
4.1.6. 1-Benzyloxymethyl-3-[1-phenylsulfonyl-indol-3-yl]-
4-[7-(2-O-tosyl-3,4,6-tri-O-benzyl-b-D-glucopyranos-1-
yl)-7H-pyrrolo[2,3-b]pyridin-3-yl]-pyrrole-2,5-dione (16).
To a solution of aglycone C (200 mg, 0.341 mmol) in
THF (18 mL) were added D (459 mg, 0.76 mmol) and
triphenylphosphine (199 mg, 0.76 mmol). The mixture
was cooled to ꢀ78 °C, then diisopropyl azodicarboxy-
late (DIAD) (147 lL, 0.76 mmol) was added dropwise.
The mixture was allowed to reach room temperature,
then was stirred at room temperature for 18 h. Water
(50 mL) was added. After extraction with EtOAc, the
organic phase was dried over MgSO4, and the solvent
was removed. The residue was purified by flash chroma-
tography (eluent: CH2Cl2/EtOAc 9:1) to give 16 (69 mg,
0.058 mmol, 17% yield) as a red solid.
0
0
0
0
0
72.4, 73.6, 75.5, 83.7 (C1 , C2 , C3 , C4 , C5 ), 109.9,
111.0, 120.6, 125.3, 127.0, 128.5, 136.3 (C tert arom),
115.0, 117.7, 119.3, 120.1, 122.3, 124.9, 132.6, 140.1,
141.1, 152.9 (C quat arom), 169.0, 169.6, 169.9, 170.1,
170.3, 170.8 (C@O).
4.1.4.
1-(b-D-Glucopyranos-1-yl)-6-methyl-1H-pyri-
do[30,20:4,5]pyrrolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(12H)-
dione (14). To a solution of 13 (45 mg, 0.067 mmol) in
methanol (20 mL) was added 28 % aqueous NH4OH
(31 mL). The mixture was stirred at 65 °C for 22 h. After
removal of the solvent, water and EtOAc were added
and the mixture was filtered off. The solid residue was
washed with EtOAc to give 14 (23 mg, 0.046 mmol, 69
% yield) as a red solid.
Mp: 63–65 °C. IR (KBr) mC@O 1706 cmꢀ1
.
1H NMR
Mp: 290–295 °C (decomposition). IR (KBr) mC@O 1690,
1740 cmꢀ1, mNH,OH 3200–3600 cmꢀ1. HRMS (FAB+)
[M+H]+ calcd for C26H23N4O7 503.1566, found:
(400 MHz, CDCl3): 2.11 (3H, s, CH3), 3.51–3.60 (1H,
m), 3.63 (1H, dd, J1 = 11.0 Hz, J2= 3.5 Hz), 3.70 (1H,
d, J = 10.0 Hz), 3.82 (1H, t, J = 9.0 Hz), 3.90 (1H, t,
J = 9.0 Hz), 4.34 (1H, d, J = 12.0 Hz), 4.42 (1H, d,
J = 12.0 Hz), 4.43 (1H, d, J = 10.5 Hz), 4.56 (1H, d,
J = 10.5 Hz), 4.62 (2H, s), 4.68 (1H, d, J = 11.0 Hz),
1
503.1569. H NMR (400 MHz, DMSO-d6): 3.18 (3H,
s, CH3), 3.48–3.60 (4H, m), 3.81 (1H, d, J = 4.0 Hz),
3.96 (1H, m), 4.71 (1H, br s, OH), 5.33 (1H, br s,
OH), 5.48 (1H, br s, OH), 5.57 (1H, d, J = 4.5 Hz,
0
4.69 (1H, d, J = 10.5 Hz), 4.84 (1H, t, J = 9.0 Hz, H2 ),
0
OH), 6.78 (1H, d, J = 9.0 Hz, H1 ), 7.33 (1H, t,
5.14 (2H, AB system, J = 10.5 Hz, Dt = 7.5 Hz), 6.37
(1H, t, J = 7.0 Hz), 6.64–6.73 (2H, m), 6.76 (1H, d,
0
J = 9.0 Hz, H1 ), 6.82 (2H, d, J = 8.5 Hz), 6.92 (2H, d,
J = 8.0 Hz), 6.96–7.02 (3H, m), 7.05 (1H, dt,
J1 = 8.5 Hz, J2 = 1.5 Hz), 7.09–7.23 (14Harom), 7.24
(1H, d, J = 7.5 Hz), 7.28 (2H, d, J = 7.5 Hz), 7.38 (2H,
J = 7.5 Hz), 7.39 (1H, t, J = 7.0 Hz), 7.52 (1H, t,
J = 7.5 Hz), 7.68 (1H, d, J = 8.0 Hz), 8.67 (1H, d,
J = 6.5 Hz), 8.97 (1H, d, J = 7.5 Hz), 9.40 (1H, d,
J = 7.0 Hz), 12.55 (1H, s, NHind). 13C NMR
0
(100 MHz, DMSO-d6): 23.5 (N–CH3), 60.9 (C6 ), 69.5,

S. Messaoudi et al. / Bioorg. Med. Chem. 14 (2006) 7551–7562
7559
t, J = 7.5 Hz), 7.48 (1H, t, J = 7.0 Hz), 7.64 (1H, m),
7.68 (1H, d, J = 6.5 Hz), 7.86 (3H, d, J = 7.5 Hz), 7.95
(1H, s), 8.27 (1H, s). 13C NMR (100 MHz, CDCl3):
5.25 (2H, AB system, J = 11.0 Hz, Dt = 10.0 Hz), 6.07
(2H, d, J = 8.5 Hz), 6.70 (2H, d, J = 8.0 Hz), 6.74 (1H,
t, J = 8.0 Hz), 6.80 (1H, m), 7.05–7.50 (23Harom), 7.77
(1H, d, J = 6.5 Hz), 8.94–8.99 (2H, m), 10.27 (1H, s,
NH). 13C NMR (100 MHz, CDCl3): 21.9 (CH3), 66.9,
0
16.3 (CH3), 62.2, 62.6, 64.8, 66.5, 70.1, 70.6 (C6
+
0
0
0
0
5CH2), 71.8, 73.1, 74.6, 77.4, 78.6 (C1 , C2 , C3 , C4 ,
0
C5 ), 106.3, 108.2, 117.0, 118.5, 119.8, 121.3-127.5,
0
68.0, 71.6, 73.5, 75.1, 75.9 (C6 +5CH2), 70.1, 77.1,
0
0
0
0
0
128.9, 146.5 (C tert arom), 101.5, 107.6, 114.8, 127.3,
127.5, 128.9, 129.3, 132.1, 132.2, 132.3, 132.5, 132.6,
139.6, 145.7 (C quat arom), 165.6, 165.9 (C@O).
78.6, 80.7, 81.9 (C1 , C2 , C3 , C4 , C5 ), 112.1, 121.2,
125.1, 126.0, 127.8–129.0, 130.0, 130.9 (C tert arom),
118.3, 118.8, 121.3, 130.6, 132.4, 137.6 (several C),
137.7, 141.0, 144.4, 156.7 (C quat arom), 168.9 (2C,
C@O).
4.1.7.
1-Benzyloxymethyl-3-(1H-indol-3-yl)-4-[7-(2-O-
tosyl-3,4,6-tri-O-benzyl-b-D-glucopyranos-1-yl)-7H-pyr-
rolo[2,3-b]pyridin-3-yl]-pyrrole-2,5-dione (18). To a solu-
tion of 16 (69 mg, 0.059 mmol) in THF (3 mL) was
added 1.1 M n-Bu4NF in THF (177 mL). The mixture
was stirred at room temperature for 2.5 h. Water
(45 mL) was added. After extraction with EtOAc, the
organic phase was dried over MgSO4. The solvent was
removed and the residue was purified by flash chroma-
tography (eluent: CH2Cl2 100% to CH2Cl2/EtOAc 8:2)
to give 18 (36 mg, 0.035 mmol, 59% yield) as a red solid.
4.1.9.
1-(2-O-Tosyl-b-D-glucopyranos-1-yl)-1H-pyri-
do[30,20:4,5]pyrrolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-
(6H,12H)-dione (22). To a suspension of 20 (120 mg,
0.115 mmol) in CH2Cl2 (12 mL) at ꢀ78 °C was added
1M BBr3 in CH2Cl2 (1.87 mL). The mixture was stirred
at ꢀ78 °C for 30 min, then water was added. After
extraction with EtOAc, the organic phase was dried over
MgSO4, and the solvent was removed. To a solution of
the residue (41 mg) in THF (6 mL) was added 28%
aqueous NH4OH (12 mL). The mixture was stirred at
room temperature overnight. After evaporation, the res-
idue was purified by flash chromatography (eluent:
EtOAc 100% to EtOAc/methanol 95:5) to give 22
(30 mg, 0.047 mmol, 41% yield) as an orange solid.
Mp: 93–95 °C. IR (KBr) mC@O 1706, 1755 cmꢀ1, mNH
3500 cmꢀ1. Mass (ESI+) [M+H]+ 1035. 1H NMR
(400 MHz, CDCl3): 2.14 (3H, s, CH3), 3.61 (1H, d,
J = 10.5 Hz), 3.67 (1H, dd, J1 = 11.0 Hz, J2 = 3.0 Hz),
3.75 (1H, m), 3.82–3.92 (2H, m), 4.38 (1H, d,
J = 12.0 Hz), 4.45 (1H, d, J = 12.0 Hz), 4.47 (1H, d,
J = 11.0 Hz), 4.57 (1H, d, J = 10.5 Hz), 4.66 (2H, s),
4.71 (1H, d, J = 10.5 Hz), 4.73 (1H, d, J = 9.5 Hz),
Mp: 200 °C (degradation). IR (KBr) mC@O 1705,
1741 cmꢀ1
, . HRMS (FAB+)
mNH 3018–3642 cmꢀ1
[M+H]+ calcd for C32H26N4O9S 669.0846, found
669.0856. H NMR (400 MHz, DMSO-d6): 1.83 (3H,
1
0
4.91 (1H, t, J = 8.5 Hz, H2 ), 5.16 (2H, AB system,
J = 11.0 Hz, Dt = 8.0 Hz), 6.55 (1H, t, J = 7.0 Hz),
0
6.66 (1H, t, J = 7.5 Hz), 6.80–6.90 (3H + H1 , m), 6.90–
s, CH3), 3.50–3.61 (2H, m), 3.64 (1H, m), 3.74 (1H, m,
0 0
H3 ), 3.81 (1H, dd, J1 = 11.5 Hz, J2 = 4.0 Hz, H6 ), 4.74
0 0
(1H, t, J = 5.5 Hz, OH6 ), 5.15 (1H, t, J = 9.0 Hz, H2 ),
0
5.63 (1H, br s, OH), 5.92 (1H, d, J = 4.0 Hz, OH3 ),
5.60 (2H, d, J = 8.0 Hz), 6.92 (2H, d, J = 8.5 Hz), 6.93
7.00 (3H, m), 7.00–7.08 (3H, m), 7.10–7.30 (17Harom),
7.33 (2H, d, J = 7.5 Hz), 7.37 (1H, d, J = 7.5 Hz), 7.65
(1H, d, J = 2.5 Hz), 7.70 (1H, d, J = 6.5 Hz), 8.32 (1H,
s), 8.90 (1H, s). 13C NMR (100 MHz, CDCl3): 21.5
0
(1H, d, J = 9.0 Hz, H1 ), 7.28 (1H, t, J = 7.0 Hz), 7.32
0
(CH3), 67.2, 67.9, 71.6, 73.4, 75.4, 75.9 (C6 + 5CH2),
(1H, t, J = 7.0 Hz), 7.53 (1H, t, J = 7.5 Hz), 7.68 (1H,
d, J = 8.0 Hz), 8.70 (1H, d, J = 6.5 Hz), 8.98 (1H, d,
J = 8.0 Hz), 8.19 (1H, d, J = 7.5 Hz), 10.94 (1H, br s,
NH), 12.45 (1H, s, NH). 13C NMR (100 MHz, CDCl3):
0
0
0
0
0
77.2, 78.4, 80.0, 82.8, 83.8 (C1 , C2 , C3 , C4 , C5 ),
111.0, 111.4, 120.7, 122.1, 122.7, 126.1, 126.8, 126.9-
128.6, 129.5 (2C), 134.3, 150.5 (C tert arom), 107.0,
107.2, 124.5, 125.3, 128.9, 132.6, 135.9, 137.5, 137.6,
137.7, 137.8, 144.8, 150.5 (C quat arom), 171.9, 172.0
(C@O).
0
0
20.3 (CH3), 60.7 (C6 ), 69.4, 74.6, 81.0, 81.9, 83.1 (C1 ,
0
0
0
0
C2 , C3 , C4 , C5 ), 109.9, 111.8, 119.7, 124.1, 125.6
(2C), 126.5, 128.6 (2C), 131.8, 135.7 (C tert arom),
114.9, 116.1, 119.1, 120.4, 121.6, 124.2, 133.0, 133.5,
140.8, 142.7, 143.5, 153.2 (C quat arom), 171.5, 171.7
(C@O).
4.1.8. 6-Benzyloxymethyl-1-(2-O-tosyl-3,4,6-tri-O-ben-
zyl-b-D-glucopyranos-1-yl)-1H-pyrido[30,20:4,5]pyrrolo[2,3-
a]pyrrolo[3,4-c]carbazole-5,7(12H)-dione (20). A mixture
of 18 (180 mg, 0.174 mmol) and iodine (66 mg,
0.259 mmol) in benzene (300 mL) was irradiated accord-
ing to the procedure described for the synthesis of com-
pound 13. After identical work-up, the residue was
purified by flash chromatography (eluent: cyclohexane/
EtOAc 7:3) to give 20 (135 mg, 0.130 mmol, 75% yield)
as a yellow solid.
4.1.10. 1-Benzyloxymethyl-4-(1-phenylsulfonyl-indol-3-
yl)-3-[7-(2,3,4,6-tetra-O-benzyl-b-D-glucopyranos-1-yl)-
7H-pyrrolo[2,3-b]pyridin-3-yl]-pyrrole-2,5-dione (17). To
a solution of aglycone C (500 mg, 0.852 mmol) in
THF (5 mL) were added 2,3,4,6-tetra-O-benzyl-a-D-
glucopyranose E (1.364 g, 2.25 mmol) and triphenyl-
phosphine (786 mg, 2.25 mmol). The mixture was
cooled to ꢀ78 °C, then DIAD (500 lL, 2.25 mmol)
was added dropwise. After identical work-up as de-
scribed for the synthesis of compound 16, the residue
was purified by flash chromatography (eluent:
CH2Cl2/EtOAc 95:5) to give G (in mixture with start-
ing products) and 17 (234 mg, 0.215 mmol, 25% yield)
as red solids. Compound G cannot be purified by
chromatography.
Mp: 53–55 °C. IR (KBr) mC@O 1707, 1753 cmꢀ1, mNH
2926 cmꢀ1. Mass (APCI+) [M-OTs]+ 861. 1H NMR
(400 MHz, CDCl3): 1.54 (3H, s, CH3), 3.70–3.81 (2H,
m), 3.88–4.10 (3H, m), 4.48 (1H, d, J = 12.5 Hz), 4.53
(1H, d, J = 11.0 Hz), 4.54 (1H, d, J = 11.5 Hz), 4.57
(1H, d, J = 9.5 Hz), 4.71 (2H, s), 4.79 (1H, d,
J = 11.0 Hz), 4.82 (1H, d, J = 10.0 Hz), 4.99 (1H, br s),

7560
S. Messaoudi et al. / Bioorg. Med. Chem. 14 (2006) 7551–7562
Mp: 83–85 °C. IR (KBr) mC@O 1707, 1762 cmꢀ1. Mass
(ESI+) [M + Na]+ 1133, [M + K]+ 1149. 1H NMR
(400 MHz, CDCl3): 3.57–3.61 (2H, m), 3.70 (1H, dd,
J1 = 11.0 Hz, J2 = 3.0 Hz), 3.73–3.81 (3H, m), 3.94
(1H, t, J = 8.5 Hz), 4.19 (1H, d, J = 11.0 Hz), 4.38
(1H, d, J = 12.0 Hz), 4.45 (1H, d, J = 12.0 Hz), 4.53
(1H, d, J = 11.0 Hz), 4.65 (2H, s), 4.78 (1H, d,
J = 11,0 Hz), 4.81 (2H, s), 5.15 (2H, s), 6.29 (1H, t,
J = 7.0 Hz), 6.39–6.49 (3H, m), 6.64 (1H, d, J = 8.0
Hz), 6.90 (2H, t, J = 7.5 Hz), 6.97 (1H, t,
J = 8.0 Hz), 7.02 (1H, t, J = 8.0 Hz), 7.09–7.13 (2H,
m), 7.16–7.28 (17Harom), 7.31 (2H, d, J = 7.5 Hz),
7.37-7.43 (3H, m), 7.48 (1H, d, J = 7.5 Hz), 7.52
(1H, d, J = 7.0 Hz), 7.86 (1H, s), 7.88 (2H, d,
J = 8.0 Hz), 8.00 (1H, s), 8.39 (1H, s). 13C NMR
(100 MHz, CDCl3): 67.4, 68.2, 71.7, 73.5, 74.3, 75.2,
identical work-up, the residue was purified by flash
chromatography (eluent: cyclohexane/EtOAc 7:3) to
give 21 (53 mg, 0.055 mmol, 76% yield) as a yellow solid.
Mp 68-73 °C. IR (KBr) mC@O 1702, 1751 cmꢀ1, mNH
3396 cmꢀ1 1H NMR (400 MHz, CDCl3): 3.63–3.89
.
(6H, m), 4.28 (1H, d, J = 11.5 Hz), 4.41 (1H, d,
J = 12.0 Hz), 4.50 (1H, d, J = 12.0 Hz), 4.54 (1H, d,
J = 11.0 Hz), 4.61–4.68 (2H, m), 4.70 (2H, s), 4.77
(1H, d, J = 10.0 Hz), 5.25 (2H, s), 6.34 (2H, d,
J = 7.5 Hz), 6.63 (2H, t, J = 7.0 Hz), 7.04–7.32 (23Harom),
7.36 (2H, d, J = 7.5 Hz), 7.38–7.42 (2H, m), 7.68 (1H, d,
J = 6.5 Hz), 9.01 (1H, d, J = 8.0 Hz), 9.22 (1H, br s),
10.47 (1H, br s, NH). 13C NMR (100 MHz, CDCl3):
0
66.9, 68.3, 71.5, 73.6, 74.5, 75.1, 75.7 (C6 +CH2), 77.2,
0
0
0
0
0
78.1, 82.0, 85.7 (C1 , C2 , C3 , C4 , C5 ), 111.5, 116.2,
120.4, 120.7, 125.3, 127.2, 127.5–128.5, 129.7, 132.4,
136.7 (C tert arom), 115.2, 118.1, 119.4, 119.7, 122.4,
136.3, 137.8, 137.9, 138.1, 140.5 (C quat arom), 169.9,
170.2 (C@O).
0
75.9 (CH2 + C6 ), 70.0, 77.2, 78.0, 81.1, 85.4 (C1 ,
0
0
0
0
0
C2 , C3 , C4 , C5 ), 111.5, 113.5, 122.1, 123.3, 125.1,
126.9, 127.2-129.0, 129.5, 133.7, 134.1, 152.1 (C tert
arom), 106.5, 112.9, 120.0, 133.0, 134.6, 136.7, 137.7,
137.8, 138.0, 151.1, 156.4 (C quat arom), 170.9,
171.2 (C@O).
4.1.13. 1-(b-D-Glucopyranos-1-yl)-1H-pyrido[30,20:4,5]pyr-
rolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H,12H)-dione
(23). To a suspension of 21 (53 mg, 0.055 mmol) in
CH2Cl2 (5.5 mL) at ꢀ78 °C was added 1 M BBr3 in
CH2Cl2 (1.1 mL). The reaction was carried out as de-
scribed for the synthesis of compound 22.
4.1.11. 1-Benzyloxymethyl-3-(1H-indol-3-yl)-4-[7-(2,3,4,6-
tetra-O-benzyl-b-D-glucopyranos-1-yl)-7H-pyrrolo[2,3-
b]pyridin-3-yl]-pyrrole-2,5-dione (19). To a solution of 17
(223 mg, 0.201 mmol) in THF (18 mL) was added 1.1 M
n-Bu4NF in THF (0.9 mL). The mixture was stirred
overnight at room temperature. Water (45 mL) was add-
ed. After extraction with EtOAc, the organic phase was
dried over MgSO4, then the solvent was removed and
the residue was purified by flash chromatography (elu-
ent: cyclohexane/EtOAc 8:2) to give 19 (109 mg,
0.112 mmol, 56% yield) as a red solid.
After removal of the solvent, the solid residue was
washed with CH2Cl2 then with water to give 23
(9.7 mg, 0.020 mmol, 36% yield) as a red solid.
Mp > 200 °C (decomposition). IR (KBr) mC@O 1703,
1743 cmꢀ1
, . HRMS (FAB+)
mNH 3004–3654 cmꢀ1
[M+H]+ calcd for C25H20N4O7 489.1410, found
489.1419. H NMR (400 MHz, DMSO-d6): 3.46 (1H,
Mp: 93–95 °C. IR (KBr) mC@O 1704, 1760 cmꢀ1, mNH
3406 cmꢀ1. Mass (ESI+) [M+H]+ 971. 1H NMR
(400 MHz, CDCl3): 3.65 (1H, d, J = 10.0 Hz), 3.72–
3.80 (2H, m), 3.80–3.88 (2H, m), 3.94 (1H, t, J = 8.5
Hz), 4.24 (1H, d, J = 11.0 Hz), 4.44 (1H, d,
J = 12.0 Hz), 4.52 (1H, d, J = 12.0 Hz), 4.58 (1H, d,
J = 11.0 Hz), 4.71 (2H, s), 4.80–4.88 (3H, m), 5.21
(2H, s), 6.41 (1H, t, J = 7.5 Hz), 6.48 (1H, t,
J = 7.0 Hz), 6.53 (2H, d, J = 8.0 Hz), 6.78 (1H, d,
J = 8.0 Hz), 6.90 (1H, t, J = 8.0 Hz), 6.99 (2H, t,
J = 7.5 Hz), 7.09 (1H, t, J = 7.0 Hz), 7.13–7.18 (2H,
m), 7.19–7.33 (20Harom), 7.38 (2H, d, J = 7.5 Hz),
7.57 (1H, d, J = 6.5 Hz), 7.60 (1H, d, J = 7.5 Hz),
7.77 (1H, d, J = 2.5 Hz), 9.34 (1H, s, NH). 13C
NMR (100 MHz, CDCl3): 67.2, 68.3, 71.6, 73.5,
1
0
0
0
0
m, H6 ), 3.52–3.64 (3H, m, H3 , H5 , H4 ), 3.84 (1H, dd,
J1 = 10.5 Hz, J2 = 5.5 Hz), 3.94 (1H, dt, J1 = 9.0 Hz,
0
J2 = 5.0 Hz, H2 ), 4.74 (1H, t, J = 6.0 Hz, OH6 ), 5.33
0
0
(1H, d, J = 6.0 Hz, OH4 ), 5.46 (1H, d, J = 4.5 Hz,
0
OH3 ), 5.57 (1H, d, J = 5.0 Hz, OH2 ), 6.82 (1H, d,
0
0
J = 9.5 Hz, H1 ), 7.35 (1H, t, J = 7.0 Hz), 7.39 (1H, t,
J = 7.0 Hz), 7.55 (1H, t, J = 7.5 Hz), 7.71 (1H, d,
J = 8.0 Hz), 8.68 (1H, d, J = 6.5 Hz), 9.00 (1H, d,
J = 8.0 Hz), 9.43 (1H, d, J = 7.0 Hz), 10.94 (1H, br s,
NH), 12.56 (1H, s, NH). 13C NMR (100 MHz, CDCl3):
0
0
0
0
0
60.9 (C6 ), 69.5, 72.6, 77.2, 81.1, 86.5 (C1 , C2 , C3 , C4 ,
0
C5 ), 109.8, 111.8, 119.7, 124.2, 126.4, 132.5, 135.7 (C
tert arom), 114.8, 116.0, 119.1, 120.6, 121.7, 124.1,
133.6, 140.7, 142.9, 154.0 (C quat arom), 171.5, 171.7
(C@O).
0
74.3, 75.1, 75.8 (C6 + CH2), 77.2, 78.1, 81.2, 85.4
0
0
0
0
0
(C1 , C2 , C3 , C4 , C5 ), 110.9, 111.5, 120.3, 121.8,
122.6, 127.5–128.7, 133.9, 150.7 (C tert arom), 106.9,
107.1, 124.6, 125.1, 128.8, 136.0, 136.5, 137.8, 137.9,
138.0, 138.2, 150.6 (C quat arom), 172.0, 172.1
(C@O).
4.1.14. 3-(1H-Indol-3-yl)-1-methyl-4-[1-(3,4,6-tri-O-ben-
zyl-b-D-glucopyranos-1-yl)-1H-pyrrolo[2,3-b]pyridin-3-
yl]-pyrrole-2,5-dione (I) and 3-(1H-indol-3-yl)-4-[7-(3,4,
6-tri-O-benzyl-b-D-glucopyranos-1-yl)-7H-pyrrolo[2,3-b]pyri-
din-3-yl]-1-methyl-pyrrole-2,5-dione (24). To a solution of
3,4,6-tri-O-benzyl-glucal (280 mg, 0.680 mmol) in
CH2Cl2 (4 mL) was added at 0 °C a solution of 0.07–
0.09 M dimethyldioxirane in acetone (15 mL).17,18 The
mixture was stirred at 0 °C for 40 min. After evapora-
tion under reduced pressure at 0 °C, the residue was
dried under vacuum for 2 h. To a solution of aglycone
4.1.12. 6-Benzyloxymethyl-1-(2,3,4,6-tetra-O-benzyl-b-D-
glucopyranos-1-yl)-1H-pyrido[30,20:4,5]pyrrolo[2,3-a]pyr-
rolo[3,4-c]carbazole-5,7(12H)-dione (21). A mixture of 19
(70 mg, 0.072 mmol) and iodine (29 mg, 0.106 mmol) in
benzene (150 mL) was irradiated according to the proce-
dure described for the synthesis of compound 13. After

S. Messaoudi et al. / Bioorg. Med. Chem. 14 (2006) 7551–7562
7561
A (100 mg, 0.225 mmol) in THF (5 mL) was added NaH
(11.8 mg, 60% dispersion in mineral oil). The mixture
was stirred for 30 min at room temperature before drop-
wise addition of a solution of H in THF (4 mL). After
refluxing for 48 h, the solvent was removed and the res-
idue was dried under vacuum for 2 h. The residue was
dissolved in formic acid and stirred at room temperature
for 24 h. After evaporation, the residue was purified by
flash chromatography (eluent: cyclohexane/EtOAc from
7:3 to 5:5) to give I (34.8 mg, 0.045 mmol, 20% yield)
and 24 (111 mg, 0.143 mmol, 63% yield) as red solids.
69% yield) as a yellow solid. Mp: 237–240 °C (decompo-
sition). IR (KBr) mC@O 1690, 1745 cmꢀ1, mNH,OH 3200–
3600 cmꢀ1
.
HRMS (FAB+) [M+H]+ calcd for
C47H41N4O7 773.2975, found 773.2956. 1H NMR
(400 MHz, CDCl3): 3.18 (3H, s, N–CH3), 3.72–3.86
(5H, m), 3.90 (1H, m), 4.47 (1H, d, J = 12.5 Hz), 4.54
(1H, d, J = 12.0 Hz), 4.63 (1H, d, J = 10.5 Hz), 4.85
(1H, d, J = 11.0 Hz), 4.87 (1H, d, J = 11.0 Hz), 5.02
(1H, d, J = 11.5 Hz), 5.93 (1H, d, J = 6.0 Hz, OH),
0
6.81 (1H, d, J = 9.5 Hz, H1 ), 7.20–7.38 (15Harom), 7.41
(2H, t, J = 7.0 Hz), 7.52 (1H, dt, J1 = 8.0 Hz, J2=
1.0 Hz), 7.69 (1H, d, J = 8.0 Hz), 8.74 (1H, d,
J = 6.0 Hz), 8.98 (1H, d, J = 8.0 Hz), 9.43 (1H, dd,
J1 = 7.5 Hz, J2 = 1.0 Hz), 12.50 (1H, s, NHind). 13C
Compound I
Mp: 72–75 °C. IR (KBr), mC@O 1700, 1751 cmꢀ1, mNH,OH
3300–3500 cmꢀ1. Mass (FAB+) [M + H]+ 775. 1H NMR
(400 MHz, CDCl3): 3.15 (3H, s, N–CH3), 3.65–3.84 (5H,
NMR (100 MHz, CDCl3): 23.5 (N–CH3), 68.5 (C6 ),
72.3, 74.3, 74.4 (CH2 of OBn), 72.7, 76.7, 77.7, 85.2,
0
0
0
0
0
0
86.8 (C1 , C2 , C3 , C4 , C5 ), 109.9, 111.9, 119.8, 124.0,
126.5, 127.3-128.3, 132.3, 135.8 (C tert arom), 115.0,
116.2, 118.2, 119.6, 121.6, 124.3, 133.5, 137.9, 138.0,
138.8, 140.9, 142.7, 154.1 (C quat arom), 170.2, 170.3
(C@O).
0
m), 3.94 (1H, t, J = 9.0 Hz, H2 ), 4.44 (1H, d,
J = 12.0 Hz), 4.53 (1H, d, J = 12.0 Hz), 4.60 (1H, d,
J = 11.0 Hz), 4.87 (1H, d, J = 11.0 Hz), 4.89 (1H, d,
J = 11.5 Hz), 4.94 (1H, d, J = 11.5 Hz), 5.96 (1H, d,
0
J = 9.0 Hz, H1 ), 6.77 (2H, m), 6.95 (1H, d,
J = 8.5 Hz), 7.03 (1H, t, J = 7.5 Hz), 7.17–7.43 (17Harom),
7.47 (1H, d, J = 3.0 Hz), 7.80 (1H, s), 8.21 (1H, dd,
J1 = 4.5 Hz, J2= 1.5 Hz), 9.15 (1H, d, J = 2.5 Hz,
NHind). 13C NMR (100 MHz, CDCl3): 24.2 (N–CH3),
4.2. Melting temperature studies
The concentrations of CT-DNA and double stranded
poly(dAdT) oligonucleotide (Sigma–Aldrich) were
determined from their molar extinction coefficients of
6600 Mꢀ1cmꢀ1. Compounds 14 and 22, NB-506, and
camptothecin (Sigma) were prepared in DMSO
(10 mM) and more diluted solutions were freshly pre-
pared in the appropriate experimental buffers.
0
68.6 (C6 ), 73.5, 75.0, 75.5 (CH2 of OBn), 73.6, 77.4,
0
0
0
0
0
77.6, 82.9, 85.5 (C1 , C2 , C3 , C4 , C5 ), 111.8, 117.2,
120.4, 121.9, 122.7, 127.5-128.7, 129.2, 130.9, 142.0,
143.7 (C tert arom), 106.3, 118.9, 124.7, 126.0, 129.1,
136.1, 137.9, 138.1, 138.6, 148.0 (C quat arom), 172.0
(2C, C@O).
CT-DNA or poly(dAdT)2 (20 lM) was incubated with
increasing concentrations of compounds 14 or 22 in
1 mL of BPE buffer for a final drug/base pair ratio of
0.1, 0.25, 0.5 or 1. The absorbency at 260 nm was mea-
sured using an Uvikon 943 spectrophotometer thermo-
stated with a Neslab RTE111 cryostat with a point
measured every min over a range of 20–100 °C with an
increment of 1 °C per min. The Tm values were deduced
from the midpoint of the hyperchromic transition and
the variation of melting temperature (DTm) calculated
Compound 24
Mp: 92–95 °C. IR (KBr) mC@O 1690, 1750 cmꢀ1
,
mNH,OH 3200–3600 cmꢀ1
.
HRMS (ESI+) [M + H]+
calcd for C47H43N4O7 775.3132, found 775.3115. 1H
NMR (400 MHz, CDCl3): 3.08 (3H, s, N–CH3),
3.64–3.88 (6H, m), 4.42 (1H, d, J = 12.0 Hz), 4.48
(1H, d, J = 12.0 Hz), 4.51 (1H, d, J = 11.0 Hz), 4.79
(1H, d, J = 11.0 Hz), 4.80 (1H, d, J = 11.0 Hz), 4.91
0
(1H, d, J = 11.0 Hz), 6.53 (1H, d, J = 9.0 Hz, H1 ),
from the formula : DTm = Tm[drug+DNA] ꢀ Tm[DNA alone]
.
6.62–6.74 (3H, m), 6.97 (1H, dt, J1 = 8.0 Hz,
J2 = 1.5 Hz), 7.10–7.14 (2H, m), 7.19–7.30 (14Harom),
7.63 (1H, d, J = 2.5 Hz), 7.67 (1H, d, J = 7.5 Hz),
7.83 (1H, d, J = 6.0 Hz), 8.11 (1H, s), 9.10 (1H, s,
NHind). 13C NMR (100 MHz, CDCl3): 24.1 (N–CH3),
4.3. Topoisomerase I-mediated DNA cleavage
Graded concentrations of compounds 14, 22, NB506
or 20 lM of CPT were incubated with supercoiled
pUC19 plasmid DNA (120 ng) or 117 base pairs 30-
end-labeled DNA fragments in 20 lL of relaxation
buffer for 15 min at 37 °C to ensure binding equilib-
rium prior to adding human recombinant topoisomer-
ase I enzyme (4 U) for a further 30 min incubation at
37 °C. The cleavage reactions were then stopped
through digestion of the topoisomerase I protein by
adding SDS (0.25%) and proteinase K (250 lg/mL)
for 30 min at 50 °C. The DNA samples were then
differently separated depending on the size of the var-
ious DNA substrates. On the one hand, the plasmid
DNA samples were loaded onto a 1% agarose ethi-
dium bromide-containing gel after addition of 2 lL
of the loading buffer. After a 2 h electrophoresis
in TBE buffer at 120 V at room temperature, the
0
68.4 (C6 ), 73.4, 74.9, 75.5 (CH2 of OBn), 76.3, 76.6,
0
0
0
0
0
78.2, 85.7, 86.8 (C1 , C2 , C3 , C4 , C5 ), 111.1, 111.5,
120.3, 121.8, 122.4, 126.0, 127.7-128.4, 134.4, 148.9
(C tert arom), 106.7, 107.2, 124.9, 125.2, 127.5, 128.1,
136.0, 137.7, 137.9, 138.4, 150.2 (C quat arom),
172.6, 172.7 (C@O).
4.1.15. 1-(3,4,6-Tri-O-benzyl-b-D-glucopyranos-1-yl)-6-
methyl-1H-pyrido[30,20:4,5]pyrrolo[2,3-a]pyrrolo[3,4-c]car-
bazole-5,7(12H)-dione (25). A mixture of 24 (100 mg,
0.129 mmol) and iodine (388 mg, 1.52 mmol) in benzene
(150 mL) was irradiated as described for the synthesis of
compound 13. After identical work-up, the residue was
purified by flash chromatography (eluent: EtOAc/cyclo-
hexane from 5:5 to 8:2) to give 25 (68 mg, 0.088 mmol,

7562
S. Messaoudi et al. / Bioorg. Med. Chem. 14 (2006) 7551–7562
´
by grants from the Ligue contre le Cancer, Comite du
Nord (N° 1FI3558FEAR).
agarose gel was photographed under UV light. On
the other hand, the radiolabeled DNA samples were
precipitated to be then diluted in 5 lL of denaturing
loading buffer. The samples were heated at 90 °C for
3 min and subsequently cooled on ice to then be sep-
arated on a 8% denaturing polyacrylamide gel for
90 min at 65 W in TBE buffer. The cleaved bands
were identified using the Molecular Dynamics 445SI
PhosphorImager. The precise localization of the bases
on the DNA fragment was established from compar-
ison with the position of guanines in the G-track lane
obtained from Maxam and Gilbert classical
procedure.
References and notes
1. Prudhomme, M. Curr. Med. Chem. 2000, 7, 1189–1212.
2. Long, B. H.; Rose, W. C.; Vyas, D. M.; Matson, J. A.;
Forenza, S. Curr. Med. Chem. AntiCancer Agents 2002, 2,
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4.4. Growth inhibition assays
Tumor cells were provided by American Type Culture
Collection (Frederik, MD, USA). They were cultivated
in RPMI 1640 medium (Life Science technologies, Cer-
gy-Pontoise, France) supplemented with 10 % fetal calf
serum, 2 mM L-glutamine, 100 U/mL penicillin,
100 lg/mL streptomycin, and 10 mM Hepes buffer (pH
7.4). Cytotoxicity was measured by the microculture tet-
razolium assay as described.21 Cells were continuously
exposed to graded concentrations of the compounds
for four doubling times, then 15 lL of 5 mg/mL 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
was added to each well and the plates were incubated
for 4 h at 37 °C. The medium was then aspirated and
the formazan solubilized by 100 lL DMSO. Results
are expressed as IC50, concentration which reduced by
50% the optical density of treated cells with respect to
untreated controls.
´
´
´
6. Marminon, C.; Pierre, A.; Pfeiffer, B.; Perez, V.; Leonce,
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´
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7. Marminon, C.; Pierre, A.; Pfeiffer, B.; Perez, V.; Leonce,
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8. Messaoudi, S.; Anizon, F.; Leonce, S.; Pierre, A.; Pfeiffer,
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12. Routier, S.; Coudert, G.; Merour, J. Y.; Caignard, D. H.
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4.5. Chk1 inhibitory assays
13. Rodrigues Pereira, E.; Belin, L.; Sancelme, M.; Prud-
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Fabbro, D.; Meyer, T. J. Med. Chem. 1996, 39, 4471–4477.
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Human Chk1 full-length enzyme with an N-termi-
nal GST sequence was purchased from Upstate
Biochemicals (No. 14-346). Assays for compound
testing were based upon the method described by
Davies et al.22 except that the final ATP concen-
tration was 15 lM. Compounds (10 lM final con-
centration) were tested in duplicate and the
average inhibition was calculated as a % relative
to samples without compound. The reproducibility
of assays was monitored by testing a control com-
pound in every experiment.
20. Jiang, X.; Zhao, B.; Britton, R.; Lim, L. Y.; Leong, D.;
Sanghera, J. S.; Zhou, B. B. S.; Piers, E.; Andersen, R. J.;
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´
´
`
21. Leonce, S.; Perez, V.; Casabianca-Pignede, M. R.; Anstett,
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We thank the Institut de Recherche sur la Cancer de
´
Lille and the Conseil Regional Nord-Pas-de-Calais for
a PhD fellowship to Paul Peixoto and Brigitte Baldey-
rou for technical expertise. This work was supported
22. Davies, S. P.; Reddy, H.; Caivano, M.; Cohen, P.
Biochem. J. 2000, 351, 95–105.