Syntheses and antiproliferative activities of 7-azarebeccamycin analogues bearing one 7-azaindole moiety

Article abstract of DOI:10.1021/jm0210055

Rebeccamycin analogues containing one azaindole unit, with and without a methyl group on the imide nitrogen and with the sugar moiety coupled either to the indole nitrogen or to the azaindole nitrogen were synthesized. To increase the solubility and induc

Full text of DOI:10.1021/jm0210055

J . Med. Chem. 2003, 46, 609-622
609
Syn th eses a n d An tip r olifer a tive Activities of 7-Aza r ebecca m ycin An a logu es
Bea r in g On e 7-Aza in d ole Moiety
Christelle Marminon,† Alain Pierre´,‡ Bruno Pfeiffer,§ Vale´rie Pe´rez,‡ Ste´phane Le´once,‡ Alexandra J oubert,|
Christian Bailly,| Pierre Renard,§ J ohn Hickman,‡ and Michelle Prudhomme†,*
Universite´ Blaise Pascal, Synthe`se et Etude de Syste`mes a` Inte´reˆt Biologique, UMR 6504, 63177 Aubie`re, France, Institut de
Recherches SERVIER, Division de Cance´rologie, 11 Rue des Moulineaux, 92150 Suresnes, France, Les Laboratoires SERVIER,
1 Rue Carle He´bert, 92415 Courbevoie, France, and INSERM U-524 et Laboratoire de Pharmacologie Antitumorale du Centre
Oscar Lambret, IRCL, Place de Verdun, 59045 Lille, France
Received J uly 30, 2002
Rebeccamycin analogues containing one azaindole unit, with and without a methyl group on
the imide nitrogen and with the sugar moiety coupled either to the indole nitrogen or to the
azaindole nitrogen were synthesized. To increase the solubility and induce stronger interactions
with the target macromolecules, a bromo or nitro substitutent was introduced on the indole
unit. The DNA binding and topoisomerase I inhibition properties were investigated together
with the antiproliferative activities toward nine tumor cell lines. In addition, the effect of the
compounds on the cell cycle of L1210 leukemia cells was examined. The nonaza analogues
were found to be cytotoxic against all cell lines of the panel whereas the aza-analogues showed
a selective action toward certain cell lines. They strongly inhibited the proliferation of SK-N-
MC neuroblastoma, A431 epidermoid carcinoma and NCI-H69 small cell lung carcinoma cells,
but showed little or no cytotoxic effect against IGROV ovary carcinoma, HT29 colon carcinoma,
and A549 non small cell lung carcinoma cells. Whatever their cytotoxicity profile, all compounds
induce similar cell cycle effects, with a marked G2+M block observed with L1210 leukemia
cells. The data suggest that the molecular mechanism of action of the aza-analogue derivatives
is different from that of rebeccamycin.
In tr od u ction
available. The bromo compounds 11 and 23 and nitro
compound 24 have been prepared with the aim of
enhancing the water solubility. Rebeccamycin is known
as a nonselective cytotoxic agent, and it is too toxic to
be used in the clinic.11 To investigate whether the
compounds could present some degree of selectivity
against certain cell lines, the newly designed compounds
were tested on a panel of nine tumor cell lines including
seven human solid tumors. In an attempt to gain
information on their mechanism of action, the effect of
the compounds on the cell cycle was studied. The
activities of the new compounds were compared with
their parent compounds, rebeccamycin A, dechlorinated
rebeccamycin B, and a synthetic compound C,16 methyl-
ated at the imide nitrogen.
Indolocarbazole compounds bearing a carbohydrate
moiety attached to one of the indole nitrogens have been
largely studied for their antiproliferative activities and
their ability to inhibit topoisomerase I.1 These com-
pounds are related to the antibiotics BE-13793C and
rebeccamycin. Extensive structure-activity relation-
ships studies have led to the development of analogues
which are currently undergoing clinical trials, in par-
ticular NB-506, a glycosylated derivative of BE-13793C,
and the rebeccamycin derivative NCS655649.2-11 Our
previous studies on rebeccamycin analogues have re-
vealed that topoisomerase is a privileged but not unique
target for these drugs. The structural analogy with
staurosporine, a nonselective kinases inhibitor which
interacts with the ATP binding site of the enzymes,12
raises the possibility that some of the rebeccamycin
analogues could also function as protein kinase inhibi-
tors.
Resu lts a n d Discu ssion
Ch em istr y. The synthesis of compound 10 bearing
a methyl group at the imide nitrogen is outlined in
Scheme 1. Aglycone 5 was prepared according to the
classical method13 modified by Routier et al.14 to intro-
duce a 7-azazindole. Indolylmagnesium bromide was
coupled to N-methyldibromomaleimide, and then the
indole nitrogen was protected with a tert-butyloxycar-
bonyl group (Boc) before a second nucleophilic attack
by lithiated azaindole. Oxidative cyclization of com-
pound 3 in the presence of iodine led to carbazole 4
which was further deprotected using tetrabutylammo-
nium fluoride (TBAF) to yield aglycone 5. Various
methods of N-glycosylations of indole or indolocarbazole
moieties are described in the literature:15-22 (i) coupling
of indolocarbazoles to an R-D-glucopyranosyl bromide via
In this paper, we report the syntheses of rebeccamycin
analogues bearing a azaindole unit and a carbohydrate
residue attached either to the azaindole nitrogen (com-
pounds 10, 11, 22-24, Figure 1) or the indole nitrogen
(compounds 32 and 14, Figure 1). The compounds were
synthesized from 7-azaindole which is commercially
* To whom correspondence should be addressed. Phone: (33) 4 73
40 71 24. Fax: (33) 4 73 40 77 17. E-mail: mprud@chimtp.univ-
bpclermont.fr.
† Universite´ Blaise Pascal.
‡ Institut de recherches SERVIER.
§ Les Laboratoires SERVIER.
| INSERM U-524.
10.1021/jm0210055 CCC: $25.00 © 2003 American Chemical Society
Published on Web 01/18/2003

610 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
Marminon et al.
F igu r e 1.
the Koenigs-Knorr method but it provides essentially
R-N-glycosylated compounds; (ii) coupling of an anhy-
drosugar to an indole or bis-indole moiety and in this
case, the â-N-glycosylated compound is favored; (iii)
reaction in heterogeneous basic medium with an in-
dolocarbazole and R-tetra-O-benzyl-glucopyranosyl chlo-
ride. This reaction is highly stereoselective, with the
â-N-glycosylated compound as the major product; (iv) a
Mitsunobu reaction for the coupling of a bis-indole or
an indole with R-2,3,4,6-tetra-O-benzyl-glucose. In this
case, the â-N-glycosylated compound is favored but an
electron-withdrawing group is required on the substrate.
leading to 8. Oxidative cyclization of 8 by irradiation in
the presence of iodine yielded 9, and the aminolysis of
this compound gave the required rebeccamycin analogue
10. To improve the solubility and/or to increase the
stability of the complex drug-target macromolecule,
bromination of 9 was performed with N-bromosuccin-
imide followed by regeneration of the hydroxyl functions
of the sugar. In rebeccamycin analogues of previous
series,7 bromination was found to enhance both topo-
isomerase I inhibitory potency and in vitro antiprolif-
erative activity. Bromination occurred at position 9 as
already observed in the previous series and only on the
indole moiety yielding 11.
Compound 5 been particularly insoluble, a Mitsunobu
reaction was first performed with compound 3 using
R-2,3,4,6-tetra-O-acetyl glucose prepared according to
the method described by Bayle et al.23 in the presence
of diethylazodicarboxylate (DEAD) and triphenylphos-
phine. â-N-Glycosylated 6 was obtained as the major
product of the reaction in 61% yield. Its stereochemistry
was assigned from the coupling constant J 1′-2′. The
value (9 Hz) is in agreement with an axial-axial
coupling. Treatment of 6 with TBAF led to compound 7
with simultaneous removal of the Boc group and the
acetyl groups of the sugar moiety. Due to the insolubility
of 7, the presence of the acetyl groups on the carbohy-
drate part were required for further steps. Conse-
quently, an alternative method of deprotection of the
indole nitrogen was investigated. Removal of the Boc
protecting group only was achieved using formic acid
N-Glycosylation was performed from aglycone 5 in
heterogeneous medium according to the method de-
scribed by Ohkubo et al.19,20 with tetra-O-benzyl-glu-
copyrasosyl chloride. N-Glycosylations occurred both on
the indole (â-N-glycosylated compound 13, 42% yield)
and the azaindole (â-N-glycosylated compound 12, 10%)
nitrogens. Removal of the benzyl groups of the sugar
moiety of the major product by hydrogenolysis in a
EtOAc/MeOH mixture in the presence of catalytic
amounts of Pd/C provided compound 14 in 85% yield
(Scheme 2).
For the synthesis of 22, the analogue of 10 with a free
imide nitrogen, a synthetic scheme similar to that
presented in Scheme 1 was investigated. The removable
imide protecting group, benzyloxymethyl (BOM), was
used. The synthetic procedure is outlined in Scheme 3.

7-Azarebeccamycin Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4 611
Sch em e 1
The phenylsulfonyl protecting group on the indole
nitrogen was introduced using sodium hydride as a base.
The phenylsulfonyl group was removed after the Mit-
sunobu reaction. Final deprotection of the imide nitro-
gen was performed in two steps: first, hydrogenolysis
gave compound 21 bearing a hydroxymethyl group at

612 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
Marminon et al.
Sch em e 2
the imide nitrogen and second, aminolysis served to
remove both the imide and sugar protecting groups,
thus providing compound 22. Bromination and nitration
were achieved from the more soluble acetylated com-
pound 21 yielding compounds 23 and 24, respectively.
Aromatic substitution was observed exclusively on the
indole moiety. Nitration of the indole part of these
rebeccamycin analogues has been found to promote
biological activity.11
melting temperature measurements were performed
with calf thymus DNA and the polynucleotide poly-
(dAT)2. The results of these Tm measurements, carried
out at a drug/DNA-phosphate (D/P) ratio of 0.5, are
presented in Figure 2. The position of the newly
incorporated nitrogen atom in the indolocarbazole frame-
work has a major effect on the DNA binding capacity
of the drug. Added to the unsubstituted indole, it
promotes DNA binding. Indeed, both compounds 14 and
32 give high ∆Tm values, even superior to those of
dechlorinated rebeccamycin B. In contrast, when added
to the sugar-substituted indole, it abolishes the DNA
binding capacity. Compounds 10 and 22 as well as their
bromo (11, 23) and nitro (24) derivatives fail to elevate
the melting temperature of DNA, suggesting that they
have little, if any, interaction with DNA.
To complete this study, the analogue 32, in which the
imide nitrogen is free and the sugar moiety attached to
the indole nitrogen, was synthesized (Scheme 4). The
nitrogen of the azaindole was protected with a phenyl-
sulfonyl group to allow N-glycosylation at the indole
part. Compound 26 was prepared by substitution of 25,
the aza-analogue of 13, with indolylmagesium bromide
in toluene. N-glycosylation via a Mitsunobu reaction
allowed the formation of â-N-glycosylated compound 27
with a 37% yield. A Koenigs-Knorr reaction with
2,3,4,6-tetra-O-acetyl-R-D-glucopyranosyl bromide, fol-
lowed by deprotection of the azaindole nitrogen using
TBAF to separate the required coupling compound from
sugar residues, provided 28 in only 6% yield. The R-N-
glycosylated compound 29, the major product of the
reaction was isolated in 51% yield. Compound 27 was
treated with TBAF, then photocyclized in the presence
of iodine to yield 30. The final deprotections were
performed as already shown in Scheme 3, hydrogenoly-
sis and then aminolysis.
The effect of the drug on human topoisomerase I was
investigated using a DNA sequencing assay to identify
the site of DNA cleavage in a 32P-labeled 117-bp DNA
restriction fragment. The DNA substrate was incubated
with each drug at 10 or 50 µM prior to initiating DNA
cleavage by topoisomerase I. The cleavage patterns
observed with the indolocarbazole drugs were compa-
rable, but however slightly distinct, to those seen in the
presence of the reference topoisomerase I poison camp-
tothecin. The gel in Figure 3b compares the effects of
the azaindole 22 with those of its bromo (23) and nitro
(24) derivatives. The cleavage intensities at the promi-
nent TG26 site vary in the order 24>23>22. The
substitution of the indole ring with a nitro group
markedly reinforces the anti-topoisomerase I activity.
The azaindole 32 is significantly more potent than the
azaindole 22 at stimulating DNA cleavage by topo-
isomerase I (Figure 3a). At first sight, this may be
correlated with the higher DNA affinity of 32 vs 22, but
we know from previous studies that in general there is
DNA Bin d in g a n d Top oisom er a se I In h ibition .
DNA and topoisomerase I represent potential (but not
unique) targets for the aza-rebeccamycins. We evaluated
the DNA binding properties of the new compounds and
their capacity to interfere with the DNA breakage-
reunion reaction catalyzed by human topoisomerase I.
To compare the relative affinities of the drugs for DNA,

7-Azarebeccamycin Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4 613
Sch em e 3
no direct relationships between DNA binding affinities
and topoisomerase I inhibition.
MC), colon carcinoma (HT29), non small cell lung
carcinoma (A549), small-cell lung carcinoma (H69), and
two epidermo¨ıd carcinomas (A431 and KB-3-1). The IC50
values are collated in Table 1. Non cyclized compound
7 is inactive. Comparison of 10 and 22, both bearing
the sugar moiety attached to the azaindole nitrogen,
showed that the NH compound is more active against
In Vitr o An tip r olifer a tive Activities. The in vitro
antiproliferative activities were tested against nine
tumor cell lines: murine leukemia (L1210), human
leukemia (K-562), and seven human solid tumors:
ovarian carcinoma (IGROV1), neuroblastoma (SK-N-

614 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
Marminon et al.
Sch em e 4
compounds must be considered as selective cytotoxic
agent because some cell lines are highly sensitive to
these two compounds (A431, SK-N-MC, NCI-H69) and
other are resistant (IGROV1, HT29, A549). In contrast,
the three compounds possessing two indole units, A, B,
and C are highly cytotoxic against all cell lines. Bro-
minated derivatives 11 and 23 are about 2 times more
cytotoxic against L1210, SK-N-MC, and NCI-H69 cells
than the parent compounds 10 and 22. The high
cytotoxicity of these bromo derivatives may be at-
tributed to their increased solubility. Interestingly,
almost identical profiles of cytotoxicity are observed with
all the aza derivatives, suggesting a common mecha-
nism of action.
DNA alone
F igu r e 2. Variation of the ∆Tm (Tmdrug-DNA complex - Tm
)
of the complexes between DNA and the test compounds.
Melting temperature measurements were performed in BPE
(6 mM Na2HPO4, 2 mM NaH2PO4, 1 mM EDTA) buffer at pH
7.0. with a drug/DNA ratio of 0.5, using calf thymus DNA
(hatched bars) or poly(dAT)2 (black bars).
The comparison between 22 and 32 with the free
imide nitrogen and 10 and 14 with a methyl group at
the imide nitrogen suggests that compounds for which
L1210, SK-N-MC and NCI-H69 cells than the N-
methylated analogue. However, compound 10 is more
cytotoxic toward KB-3-1 and A431 than 22. Both

7-Azarebeccamycin Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4 615
F igu r e 3. DNA cleavage by human topoisomerase I in the presence of the aza-indolocarbazoles. Gel (a) compares the two azaindoles
22 and 32; gel (b) refers to the bromo and nitro derivatives. The 117-bp 3′-end labeled fragment (DNA) was incubated in the
absence (lane TopoI) or presence of the test drug at the indicated µM concentration. Camptothecin (CPT) was used at 40 µM.
Topoisomerase I cleavage reactions were analyzed on 8% denaturing polyacrylamide gels. Numbers at the left side of the gels
show the nucleotide positions, determined with reference to the guanine tracks labeled G. The nucleotide positions and sequences
to the cleavage sites are indicated.
Ta ble 1. In Vitro Antiproliferative Activities against Nine Tumor Cell Lines: Murine Leukemia (L1210), Human Leukemia (K-562),
and Seven Human Solid Tumors: Ovarian Carcinoma (IGROV1), Neuroblastoma (SK-N-MC), Colon Carcinoma (HT29), Non Small
Cell Lung Carcinoma (A549), Small Cell Lung Carcinoma (H69), and Two Epidermo¨ıd Carcinomas (A431 and KB-3-1) (IC50 µM)
compd
L1210
IgROV
SK-N-MC
HT29
A549
A431
NCI-H69
K-562
KB-3-1
A
B
C
14
7
10
11
22
23
24
32
0.14
0.11
0.67
1.3
51.3
0.45
0.27
0.13
0.061
0.07
0.066
0.25
2.6
<0.1
<0.1
0.25
ne
0.3
2.5
0.3
2
0.25
3.8
0.84
ne
29.1
0.012
0.053
0.238
0.229
10
<0.1
<0.1
0.33
ne
11.8
0.11
0.043
0.01
0.007
0.017
ne
0.2
<0.1
ne
0.3
0.3
0.6
ne
32.6
0.11
1.2
2.56
0.93
0.552
ne
0.88
0.86
17.8
58.3
67.2
27.5
>100
32.3
>100
4.8
0.94
47.2
98.7
59.9
32
>100
85.2
>100
5.3
nea
ne
78.8
8.5
28
70.5
0.7
ne
ne
ne
0.18
0.041
0.059
0.018
ne
19.5
68.8
24.7
33.4
ne
ne
ne
ne
ne
a
ne: not evaluated.
the sugar part is linked to the indole nitrogen are less
selective than those having the carbohydrate attached
to the azaindole nitrogen. Either different targets or
different expression of these targets in the various
tumor cell lines may account for the difference of
selectivity observed between compounds in which the
sugar moiety is linked to the indole nitrogen and
compounds in which the sugar moiety is linked to the
aza-indole nitrogen.
Effect on L1210 Cell Cycle. The compounds highly
toxic toward the L1210 leukemia cell line were used to
investigate the cell cycle effects. Bromo 23 and nitro 24
derivatives as well as compound 32 induce a marked
accumulation of the cells in the G2+M phase of the cell

616 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
Ta ble 2. L1210 Cytotoxicity and Cell Cycle Effects
Marminon et al.
the sugar was attached to the azaindole (10, 11, 22, 23,
24), DNA binding properties were abolished. Concerning
topoisomerase I inhibition, 32 was more efficient than
22. This effect seems to be correlated with its higher
DNA affinity and its stronger cytotoxicity against the
tumor cell lines tested. Bromo 23 and nitro 24 analogues
were more potent topoisomerase I inhibitors than the
unsubstituted analogue 22; however, there is no clear
correlation between the anti-topoisomerase I activities
and the cytotoxicities toward the various tumor cell lines
tested.
Do these compounds inhibit kinases involved in the
transformed phenotype and/or the proliferative capabil-
ity? If they do, is this inhibition specific toward some
kinases which could explain at least partially their
specificity against the different tumor cell lines tested.
To answer these questions, the inhibitory activities of
these compounds against various cyclin-dependent ki-
nases are being investigated.
antiproliferative
activities IC50, µM
cells in the G2+M phase (%)
compd
(drug concentration)
control
A
B
-
24
0.14
0.11
0.67
1.3
69 (1 µM)
71 (1 µM)
77 (2.5 µM)
nea
26 (5-20 µM)
78 (5 µM)
79 (0.5 µM)
85 (0.25 µM)
87 (0.25 µM)
78 (0.25 µM)
C
14
10
11
22
23
24
32
0.45
0.27
0.13
0.061
0.07
0.066
a
ne: not evaluated
cycle (about 80% at 0.25 µM). Although four times less
potent, compounds A, B, and C similarly modify the cell
cycle. Globally, the effect on the cell cycle appears to be
linked to the antiproliferative properties, with the
exception of compound 10 which is relatively potent but
does not significantly perturb the cell cycle.
Exp er im en ta l Section
In conclusion, in contrast to the compounds possessing
two indole units, some of the new compounds described
here exhibit a very interesting profile of cytotoxicity,
with a high selectivity for some tumor lines. A wide IC50
range was observed, from 10 to 20 nM to >100 µM. Such
important differences are not commonly observed with
the majority of cytotoxic agents. What are the molecular
events underlying these effects? Although the tumor cell
lines used in this panel could present different perme-
ability properties and/or cellular metabolism, it seems
unlikely that they can account for the dramatic selectiv-
ity observed for some cell lines versus other ones.
Rather, it is possible that the molecular targets of the
compounds (for example kinases) were differently ex-
pressed in the cell lines, and differently affected by
analogues, even though they are structurally related.
Interestingly, the replacement of indole unit by its
bioisostere azaindole led to quite different profiles of
cytotoxicity. The aza-analogues are much more selective.
From our previous works, it could be concluded that
topoisomerase I is not the only target for rebeccamycin
analogues. The differences in the selectivity seem to
indicate more specific targets for the aza-analogues.
Compounds 10 and 14 unsubstituted on the indole ring
and possessing a methyl group at the imide nitrogen
are less selective than their parent compounds with a
free imide nitrogen 22 and 32. However, 10 was strongly
cytotoxic against epidermoid carcinoma A431. At this
stage, it is necessary to investigate the biological targets
of these molecules. Unfortunately, the cell cycle effect
of these compounds gave no clues to answer these
questions, the G2+M arrest being observed for all the
cytotoxic compounds, whatever the degree of selectivity
they present. It should be kept in mind that a G2+M
arrest could be induced by many compounds having
unrelated mechanisms of action. Experiments are in
progress to study the cell cycle effect on the sensitive
human tumor cell lines identified in this work.
Ch em istr y. IR spectra were recorded on a Perkin-Elmer
881 spectrometer (ν in cm-1). NMR spectra were performed
on a Bruker AC 400 (1H: 400 MHz, 13C: 100 MHz) (chemical
shifts δ in ppm, the following abbreviations are used: singlet
(s), broad singlet (br s), doublet (d), doubled doublet (dd), triplet
(t), doubled triplet (dt), pseudo-triplet (pt), multiplet (m),
tertiary carbons (C tert), quaternary carbons (C quat). Mass
spectra (FAB+) were determined at CESAMO (Talence, France)
on a high-resolution Fisons Autospec-Q spectrometer. Chro-
matographic purifications were performed by flash silica gel
Geduran SI 60 (Merck) 0.040-0.063 mm or Kieselgel 60
(Merck) 0.063-0.200 mm column chromatography. TLC was
performed on fluorescent silica gel plates (60 F254 from Merck).
HPLC analyses were carried out on Perkin-Elmer (series 200)
equipment using a Nucleosil 5 µm C18 column (250 × 4.6 mm)
and a flow rate of 1.00 mL/min at 25 °C (UV detection at 280
nm with a diode array detector) eluting with (1) MeCN/
H2O+H3PO4 at pH 2 (60:40), (2) MeCN/H2O+H3PO4 at pH 2
(80:20). HPLC results are presented as retention times (min),
purity (%).
ter t-Bu tyl 3-{4-[1-(2,3,4,6-Tetr a -O-a cetyl-â-D-glu cop y-
r a n osyl)-1H-p yr r olo[2,3-b]p yr id in -3-yl]-1-m eth yl-2,5-d i-
oxo-2,5-d ih yd r o-1H -p yr r ol-3-yl}-1H -in d ole-1-ca r b oxyl-
a te (6). To a solution of 3 (217 mg, 0.491 mmol) in THF (15
mL) were added 2,3,4,6-O-acetylglucopyranose (380 mg, 1.09
mmol) and triphenylphosphine (286 mg, 1.09 mmol). The
mixture was cooled to -78 °C, and then DEAD (172 µL, 1.09
mmol) was added dropwise. The mixture was stirred at room
temperature for 15 h, and then water was added. After
extraction with EtOAc, the organic phase was dried over
MgSO4, the solvent was removed, and the residue was purified
by flash chromatography (eluent cyclohexane:EtOAc:triethyl-
amine, 4:1:1%) to give â-glycosylated compound 6 (231 mg,
0.299 mmol, 61% yield) as a yellow solid.
6: mp 89-91 °C. IR (KBr) νCO 1700, 1750 cm-1 1H NMR
.
(400 MHz, CDCl3) 1.66 (3H, s), 1.70 (9H, s, t-Bu), 2.03 (3H, s),
2.06 (3H, s), 2.09 (3H, s), 3.20 (3H, s, NCH3), 4.04 (1H, m),
4.12 (1H, m), 4.24 (1H, dd, 1J ) 12.6 Hz, 2J ) 7.9 Hz), 5.25
(1H, pt, J ) 9.7 Hz), 5.48 (1H, pt, J ) 9.9 Hz), 5.53 (1H, pt, J
) 9.7 Hz), 6.26 (1H, d, J ) 8.8 Hz, H1′), 6.67 (4H, m), 7.13
(1H, m), 7.27 (1H, m), 8.06 (1H, s), 8.14 (1H, s), 8.18 (1H, dd,
2
1J ) 4.7 Hz, J ) 1.3 Hz). 13C NMR (100 MHz, CDCl3) 20.1,
20.6, 20.8, 21.1 (CH3CO), 24.4 (NCH3), 28.2 (3C) (CH3 of t-Bu),
62.2 (C6′), 68.1, 70.7, 73.2, 74.8, 80.2 (C1′, C2′, C3′, C4′, C5′), 84.8
(C-CH3 of t-Bu), 115.2, 117.7, 121.4, 122.8, 124.7, 128.9, 129.5,
130.4, 143.9 (C tert arom), 106.6, 110.3, 119.0, 126.5, 127.7,
128.8, 135.1, 147.6 (C quat arom), 149.2, 168.9 (2C), 169.5,
170.0, 170.8, 171.3 (CdO).
Surprisingly, DNA binding experiments showed major
differences between the compounds bearing the sugar
part either on the azaindole moiety or on the indole
moiety. Compounds with the sugar linked to the indole
(14 and 32) exhibited higher affinity for DNA than the
parent dechlorinated rebeccamycin B. In contrast, when
3-(1H-In dol-3-yl)-4-[1-(2,3,4,6-tetr a-O-acetyl-â-D-glu copy-
r a n osyl)-1H-p yr r olo[2,3-b]p yr id in -3-yl]-1-m eth yl-1H-p yr

7-Azarebeccamycin Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4 617
r ole-2,5-d ion e (8). A mixture of â-glycosylated compound 6
(88 mg, 0.114 mmol) in HCOOH (10 mL) was stirred at room
temperature for 24 h. Triethylamine was added dropwise until
neutralization. Saturated aqueous HCO3Na (20 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 chromatography (eluent cyclohexane:
EtOAc, 1:1) to give 8 (55 mg, 0.082 mmol, 72% yield) as an
a solution of 6 (40 mg, 0.051 mmol) in THF (6 mL) was added
a solution of tetrabutylammonium fluoride (1.1 M in THF) (2.1
mL, 2.3 mmol). The mixture was refluxed for 15 h. After
removal of the solvent, the residue was purified by flash
chromatography (eluent EtOAc:methanol, 93:7) to give 7 (21
mg, 0.042 mmol, 82% yield) as a red solid: mp 176-178 °C;
IR (KBr) νCO 1690, 1700 cm-1, νNH,OH 3200-3600 cm-1. HRMS
(FAB+) (M + H)+ calcd for C26H25N4O7 505.1723, found
1
orange solid: mp 119-121 °C; IR (KBr) νCO 1700, 1752 cm-1
,
505.1725. H NMR (400 MHz, DMSO-d6) 3.10 (3H, s, NCH3),
νNH 3300-3500 cm-1. 1H NMR (400 MHz, CDCl3) 1.69 (3H, s),
2.02 (3H, s), 2.06 (3H, s), 2.08 (3H, s), 3.17 (3H, s, NCH3), 4.03
(1H, m), 4.11 (1H, m), 4.25 (1H, dd, 1J ) 4.6 Hz, 2J ) 12.5
Hz), 5.26 (1H, pt, J ) 9.7 Hz), 5.48 (1H, pt, J ) 9.4 Hz), 5.58
(1H, pt, J ) 9.4 Hz), 6.27 (1H, d, J ) 9.3 Hz, H1′), 6.68 (1H, t,
J ) 7.1 Hz), 6.70 (1H, m), 6.83 (1H, d, J ) 8.1 Hz), 7.01 (1H,
t, J ) 7.5 Hz), 7.19 (1H, d, J ) 7.9 Hz), 7.29 (1H, d, J ) 8.1
Hz), 7.79 (1H, d, J ) 1.0 Hz), 8.00 (1H, s), 8.16 (1H, d, J ) 4.3
Hz), 9.24 (1H, s, NH). 13C NMR (100 MHz, CDCl3) 20.2, 20.6,
20.8, 21.1 (CH3CO), 24.3 (NCH3), 61.9 (C6′), 68.2, 70.6, 73.3,
74.7, 80.2 (C1′, C2′, C3′, C4′, C5′), 111.4, 117.3, 120.5, 121.6, 122.7,
127.9, 128.9, 130.6, 143.6 (C tert arom), 106.6, 107.0, 119.2,
125.3, 125.6, 129.3, 136.0, 147.6 (C quat arom), 168.9, 169.5,
170.0, 170.8, 171.8, 172.1 (CdO).
3.29-3.54 (3H, m), 3.71 (1H, t, J ) 3.3 Hz), 3.75 (1H, dd, 1J )
2
10.4 Hz, J ) 4.5 Hz), 3.86 (1H, m), 4.64 (1H, t, J ) 5.5 Hz,
OH), 5.18 (1H, d, J ) 5.5 Hz, OH), 5.27 (1H, d, J ) 5.0 Hz,
OH), 5.30 (1H, d, J ) 5.9 Hz, OH), 5.87 (1H, d, J ) 9.4 Hz,
H1′), 6.70-6.77 (2H, m), 6.95 (1H, d, J ) 8.0 Hz), 6.99 (1H,
dd, 1J ) 8.0 Hz, 2J ) 1.5 Hz), 7.00 (1H, t, J ) 7.3 Hz), 7.43
(1H, d, J ) 8.2 Hz), 7.82 (1H, d, J ) 2.8 Hz), 8.15 (1H, s), 8.16
(1H, dd, 1J ) 4.6 Hz, 2J ) 1.5 Hz), 11.88 (1H, s, NH). 13C NMR
(100 MHz, DMSO-d6) 24,0 (NCH3), 52.0 (C6′), 70.0, 72.0, 77.5,
80.0, 82.3 (C1′, C2′, C3′, C4′, C5′), 111.8, 116.5, 119.8, 121.0, 121.8,
128.7, 129.2, 131.5, 143.0 (C tert arom), 104.9, 105.3, 118.3,
125.2, 125.3, 128.8, 135.9, 147.7 (C quat arom), 171.4 (2C) (Cd
O). HPLC >96% pure, 1) tR ) 3.48 min, 2) tR ) 2.76 min.
13-(2,3,4,6-Tetr a-O-acetyl-â-D-glu copyr an osyl)-9-br om o-
6-m eth yl-12,13-d ih yd r o-5H-p yr id o[3′,2′:4,5]p yr r olo[2,3-a ]-
p yr r olo[3,4-c]ca r ba zole-5,7(6H)-d ion e (11A). To a solution
of 9 (41 mg, 0.061 mmol) in THF (2 mL) at 0 °C was added
dropwise a solution of N-bromosuccinimide (109 mg, 0.61
mmol) in THF (2 mL). The mixture was stirred at room
temperature for 4 days with light protection. Then a solution
of N-bromosuccinimide (109 mg, 0.61 mmol) in THF (2 mL)
was added dropwise. Water was added. After hydrolysis for
15 min, saturated aqueous sodium thiosulfate (20 mL) was
poured into the mixture. After extraction with EtOAc, the
organic phase was dried over MgSO4, the solvent was removed,
and the residue was purified by flash chromatography (eluent
cyclohexane:EtOAc, 3:2) to give 11A (20 mg, 0.027 mmol, 44%
yield) as a yellow solid and a mixture of brominated compounds
partially deacetylated.
13-(2,3,4,6-Tetr a -O-a cetyl-â-D-glu cop yr a n osyl)-6-m eth -
yl-12,13-dih ydr o-5H-pyr ido[3′,2′:4,5]pyr r olo[2,3-a ]pyr r olo-
[3,4-c]ca r ba zole-5,7(6H)-d ion e (9). A mixture of 8 (55 mg,
0.083 mmol) and iodine (252 mg, 0.99 mmol) in benzene (150
mL) was irradiated for 1.5 h with a medium-pressure mercury
lamp (400 W). The solvent was removed, and the residue
dissolved in EtOAc (50 mL) and washed with aqueous sodium
thiosulfate and then with brine. The organic phase was dried
over MgSO4, the solvent was removed, and the residue was
purified by flash chromatography (eluent cyclohexane:EtOAc,
1:1) to give 9 (47 mg, 0.070 mmol, 84% yield) as a yellow
solid: mp 302-304 °C; IR (KBr) νCO 1700, 1750 cm-1, νNH
3200-3600 cm-1. 1H NMR (400 MHz, CDCl3) 1.11 (3H, s), 1.93
(3H, s), 2.15 (3H, s), 2.30 (3H, s), 3.23 (3H, s, NCH3), 4.44 (2H,
1
2
d, J ) 10.9 Hz), 4.82 (1H, dd, J ) 13.4 Hz, J ) 3.6 Hz), 5.48
11A: mp 280-282 °C; IR (KBr) νCO 1700, 1760 cm-1, νNH
3360-3400 cm-1. 1H NMR (400 MHz, CDCl3) 1.11 (3H, s), 1.93
(3H, s), 2.15 (3H, s), 2.28 (3H, s), 3.25 (3H, s, NCH3), 4.41-
4.47 (2H, m), 4.85 (1H, dd, 1J ) 13.0 Hz, 2J ) 3.5 Hz), 5.44
(1H, pt, J ) 9.3 Hz), 5.58-5.66 (2H, m), 6.92 (1H, d, J ) 9.4
(1H, pt, J ) 9.2 Hz), 5.61 (1H, pt, J ) 9.4 Hz), 5.68 (1H, pt, J
1
) 9.5 Hz), 6.90 (1H, d, J ) 9.4 Hz, H1′), 7.37 (1H, dd, J ) 7.8
2
Hz, J ) 4.8 Hz), 7.44 (1H, t, J ) 7.5 Hz), 7.59 (1H, t, J ) 7.8
Hz), 7.64 (1H, d, J ) 8.0 Hz), 8.56 (1H, d, J ) 4.3 Hz), 9.19
1
2
(1H, d, J ) 8.0 Hz), 9.34 (1H, dd, J ) 7.0 Hz, J ) 0.9 Hz),
9.88 (1H, s, NH). 13C NMR (100 MHz, CDCl3) 19.2, 20.5, 21.0,
21.4 (CH3CO), 23.8 (NCH3), 61.3 (C6′), 67.4, 70.6, 72.8, 75.9,
82.1 (C1′, C2′, C3′, C4′, C5′), 111.6, 118.3, 121.7, 125.9, 127.8,
134.1, 146.9 (C tert arom), 115.4, 116.2, 119.8, 119.9, 122.0,
122.6, 127.2, 129.8, 140.9, 152.0 (C quat arom), 168.0, 169.5,
169.6, 169.7, 169.9, 170.6 (CdO).
1
2
Hz, H1′), 7.40 (1H, dd, J ) 7.9 Hz, J ) 4.9 Hz), 7.54 (1H, t,
1
2
J ) 8.5 Hz), 7.67 (1H, dd, J ) 8.7 Hz, J ) 2.0 Hz), 8.60 (1H,
1
2
1
dd, J ) 4.6 Hz, J ) 1.6 Hz), 9.32 (1H, s), 9.33 (1H, dd, J )
7.9 Hz, 2J ) 1.6 Hz), 9.95 (1H, s, NH). 13C NMR (100 MHz,
CDCl3) 19.2, 20.5, 20.7, 20.8 (CH3CO), 23.9 (NCH3), 61.4 (C6′),
67.4, 70.6, 73.0, 76.3, 82.2 (C1′, C2′, C3′, C4′, C5′), 113.1, 118.5,
128.2, 130.7, 134.2, 147.2 (C tert arom), 114.5, 115.3, 116.6,
118.6, 120.2, 122.0, 124.1, 127.2, 130.0, 139.5, 152.1 (C quat
arom), 168.1, 169.4, 169.5, 169.6, 169.9, 170.5 (CdO).
13-(â-D-Glu cop yr a n osyl)-6-m et h yl-12,13-d ih yd r o-5H -
p yr id o[3′,2′:4,5]p yr r olo[2,3-a ]p yr r olo[3,4-c]ca r ba zole-5,7-
(6H)-d ion e (10). A mixture of 9 (45 mg, 0.067 mmol) and 28%
aqueous NH4OH (31 mL) in methanol (20 mL) was stirred for
22 h at 65 °C. After removal of the solvents, water and EtOAc
were added to the residue. After filtration, the solid was
washed with EtOAc. Compound 10 was obtained (23 mg, 0.046
mmol, 69% yield) as a yellow solid: mp > 300 °C. IR (KBr)
13-(â-D-Glu cop yr a n osyl)-9-br om o-6-m eth yl-12,13-d ih y-
d r o-5H-p yr id o[3′,2′:4,5]p yr r olo[2,3-a ]p yr r olo[3,4-c]ca r ba -
zole-5,7(6H)-d ion e (11). A solution of 11A (20 mg, 0.026
mmol) and 28% aqueous NH4OH (8 mL) in methanol (8 mL)
was stirred for 22 h at room temperature with light protection.
The solvents were removed, and then water and EtOAc were
added to the residue. After fitration, the solid was washed with
EtOAc to give 11 (8 mg, 0.014 mmol, 53% yield) as a yellow
solid: mp > 300 °C, IR (KBr) νCO 1680, 1760 cm-1, νNH,OH
3300-3600 cm-1. HRMS (FAB+) (M + H)+ calcd for C26H22N4O7-
ν
CO 1690, 1750 cm-1, νNH,OH 3300-3600 cm-1. HRMS (M + H)+
(FAB+) calcd for C26H23N4O7 503.1566, found 503.1560. 1H
NMR (400 MHz, DMSO-d6) 3.25 (3H, s, NCH3), 3.58 (2H, m),
3.87 (1H, d, J ) 10.3 Hz), 3.95 (1H, d, J ) 10.8 Hz), 4.02 (1H,
m), 4.12 (1H, d, J ) 9.4 Hz), 4.99 (1H, br s, OH), 5.22 (1H, br
s, OH), 5.42 (1H, d, J ) 5.4 Hz, OH), 6.12 (1H, br s, OH), 6.64
(1H, d, J ) 8.4 Hz, H1′), 7.44 (1H, t, J ) 7.4 Hz), 7.53 (1H, dd,
1
Br 581.0672, found 581.0668. H NMR (400 MHz, DMSO-d6)
3.25 (3H, s, NCH3), 3.52-3.62 (2H, m), 3.88 (1H, d, J ) 10.5
Hz), 3.95-4.04 (2H, m), 4.12 (1H, d, J ) 10.3 Hz), 4.99 (1H, d,
J ) 4.9 Hz, OH), 5.21 (1H, d, J ) 5.0 Hz, OH), 5.42 (1H, d, J
) 5.0 Hz, OH), 6.18 (1H, m, OH), 6.65 (1H, d, J ) 8.6 Hz, H1′),
1J ) 7.9 Hz, J ) 4.9 Hz), 7.64 (1H, t, J ) 7.9 Hz), 7.75 (1H,
2
d, J ) 8.4 Hz), 8.67 (1H, d, J ) 4.0 Hz), 9.12 (1H, d, J ) 7.9
Hz), 9.36 (1H, d, J ) 7.4 Hz), 11.71 (1H, s, NH). 13C NMR
(100 MHz, DMSO-d6) 23.8 (NCH3), 58.4 (C6′), 67.6, 72.7, 76.6,
78.7, 83.1 (C1′, C2′, C3′, C4′, C5′), 112.3, 117.4, 120.7, 124.4, 127.5,
132.7, 147.1 (C tert arom), 114.2, 115.4, 117.8, 119.0, 121.2
(2C), 127.2, 129.5, 141.1, 152.5 (C quat arom), 169.6 (2C) (Cd
O). HPLC >97% pure, 1) tR ) 5.41 min, 2) tR ) 3.61 min.
1
2
7.55 (1H, dd, J ) 7.7 Hz, J ) 4.8 Hz), 7.71 (1H, d, J ) 8.7
1
2
1
Hz), 7.81 (1H, dd, J ) 8.7 Hz, J ) 1.6 Hz), 8.69 (1H, dd, J
2
) 4.6 Hz, J ) 1.3 Hz), 9.28 (1H, d, J ) 1.3 Hz), 9.36 (1H, dd,
1J ) 6.6 Hz, 2J ) 1.3 Hz), 11.83 (1H, s, NH). 13C NMR (100
MHz, DMSO-d6) 23.8 (NCH3), 58.3 (C6′), 67.6, 72.7, 76.5, 78.7,
83.1 (C1′, C2′, C3′, C4′, C5′), 114.3, 117.6, 126.4, 129.9, 132.7,
147.4 (C tert arom), 112.5, 114.1, 115.8, 116.5, 119.5, 120.8,
3-(1H-In d ol-3-yl)-4-[1-(â-D-glu cop yr a n osyl)-1H-p yr r olo-
[2,3-b]p yr id in -3-yl]-1-m eth yl-1H-p yr r ole-2,5-d ion e (7). To

618 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
Marminon et al.
.
(KBr), νCdO 1700, 1770 cm-1, νNH 3200-3500 cm-1 1H NMR
123.0, 127.2, 129.8, 139.7, 152.4 (C quat arom), 169.4, 169.5
(CdO). HPLC >95% pure, 1) tR ) 13.55 min, 2) tR ) 9.28 min.
(400 MHz, CDCl3): 4.70 (2H, s, CH2), 5.19 (2H, s, CH2), 7.27-
1
7.44 (8H, m), 7.94 (1H, d, J ) 2.5 Hz), 8.04 (1H, dd, J ) 6.3
12-(2,3,4,6-Tetr a -O-ben zyl-â-D-glu cop yr a n osyl)-6-m eth -
yl-12,13-dih ydr o-5H-pyr ido[3′,2′:4,5]pyr r olo[2,3-a ]pyr r olo-
[3,4-c]ca r ba zole-5,7(6H)-d ion e (13). To a suspension of KOH
(55 mg) and Na2SO4 (338 mg) in acetonitrile (10 mL) was
added aglycone 5 (45 mg, 0.132 mmol). The mixture was
stirred for 30 min at room temperature, before addition of
2,3,4,6-tetra-O-benzyl-R-D-glucopyranosyl chloride (221 mg,
0.396 mmol) in acetonitrile (3 mL). After stirring for 15 h at
room temperature, water (20 mL) was added and the mixture
was acidified with 1 N HCl. After extraction with EtOAc, the
organic phase was washed with brine and then dried over
MgSO4, and the solvent was removed. The residue was purified
by flash chromatography (eluent cyclohexane:EtOAc, 1:9) to
give compound 13 (48 mg, 0.056 mmol, 42% yield) and 12 (12
mg, 0.014 mmol, 11% yield) as yellow solids.
Hz, 2J ) 2.0 Hz), 9.25 (1H, s, NH). 13C NMR (100 MHz, CDCl3)
67.6, 71.9 (CH2), 112.0, 121.5, 122.9, 123.5, 127.8 (2C), 128.0,
128.5 (2C), 130.5 (C tert arom), 105.1, 114.9, 124.6, 136.3,
137.2, 137.9 (C quat arom), 166.5, 169.1 (CdO).
1-[(Ben zyloxy)m eth yl]-3-br om o-4-[1-(p h en ylsu lfon yl)-
1H-in d ol-3-yl]-1H-p yr r ole-2,5-d ion e (16). A suspension of
NaH (60% in mineral oil) (218 mg, 5.45 mmol) in THF (10 mL)
was cooled to 0 °C, and then a solution of 15 (1.038 g, 2.53
mmol) in THF (20 mL) was added dropwise. The mixture was
stirred at 0 °C for 1 h, and then benzenesulfonyl chloride (516
mL, 4.04 mmol) was added dropwise. The mixture was stirred
at room temperature for 4 h before addition of saturated
aqueous NH4Cl. After extraction with EtOAc, the organic
phase was dried over MgSO4, the solvent was removed, and
the residue was purified by flash chromatography (eluant
cyclohexane:EtOAc, 85:15) to give 16 (702 mg, 1.27 mmol, 50%
yield) as a yellow solid. Mp 49-51 °C. IR (KBr), νCdO 1740,
1790 cm-1. HRMS (FAB+) (M + H)+ calcd C26H20N2O5BrS 550,-
13: mp > 80-82 °C, IR (KBr) νCO 1700, 1750 cm-1, νNH
3200-3600 cm-1 1H NMR (400 MHz,CDCl3) 3.33 (3H, s),
.
3.88-3.91 (2H, m), 3.95-4.08 (4H, m), 4.38 (1H, d, J ) 10.4
Hz), 4.49 (1H, d, J ) 9.2 Hz), 4.73 (1H, d, J ) 12.6 Hz), 4.86-
4.92 (3H, m), 5.40 (1H, d, J ) 12.4 Hz), 6.05 (1H, d, J ) 8.2
Hz, H1′), 6.10 (1H, d, J ) 7.6 Hz), 6.80 (2H, t, J ) 7.6 Hz),
7.00 (1H, t, J ) 7.4 Hz), 7.09-7.11 (2H, m), 7.27-7.40 (15H,
m), 7.51 (1H, t, J ) 7.7 Hz), 7.60-7.68 (3H, m), 8.68 (1H, dd,
1
0198, found 550.0203. H NMR (400 MHz, CDCl3): 4.68 (2H,
s, CH2), 5.18 (2H, s, CH2), 7.24-7.38 (6H, m), 7.42 (1H, t, J )
7.5 Hz), 7.52 (2H, t, J ) 7.7 Hz), 7.61 (1H, t, J ) 7.5 Hz), 7.80
(1H, d, J ) 8.0 Hz), 7.99 (2H, d, J ) 7.8 Hz), 8.05 (1H, d, J )
8.3 Hz), 8.22 (1H, s). 13C NMR (100 MHz, CDCl3): 67.6, 71.6
(CH2), 113.2, 122.4, 122.8, 123.7, 125.4, 126.7, 126.8, 127.2,
127.5, 127.7, 127.9, 128.1, 129.0, 131.6, 134.4 (C tert arom),
109.7, 121.7, 125.5, 135.6, 137.0, 137.2, 146.4 (C quat arom),
165.0, 167.6 (CdO).
2
1J ) 4.7 Hz, J ) 1.5 Hz), 9.40 (1H, d, J ) 7.9 Hz), 9.48 (1H,
d, J ) 7.8 Hz), 11.47 (1H, s, NH). 13C NMR (100 MHz, CDCl3)
23.9 (CH3), 65.5, 73.3, 75.0, 75.4, 76.0 (CH2), 76.3, 77.8, 81.0,
84.6, 85.7 (C1′, C2′, C3′, C4′, C5′), 110.6, 117.2, 121.7, 125.9, 127.7
(2C), 127.75 (2C), 127.8 (2C), 127.9 (2C), 128.0 (2C), 128.2 (2C),
128.3 (2C), 128.5 (2C), 128.6 (2C), 129.4 (3C), 133.5, 148.2 (C
tert arom), 115.2, 116.6, 119.7, 120.2, 121.6, 122.2, 128.9,
135.9, 137.7 (2C), 137.9, 138.1, 141.8, 153.5 (C quat arom),
170.0 (2C) (CdO).
1-[(Ben zyloxy)m eth yl]-3-[1-(p h en ylsu lfon yl)-1H-in d ol-
3-yl]-4-(1H-p yr r olo[2,3-b]p yr id in -3-yl)-1H-p yr r ole-2,5-d i-
on e (17). To a solution of 7-azaindole (182 mg, 1.542 mmol)
in toluene (10 mL) at -15 °C was added dropwise a solution
of LiHMDS (1 M in hexane) (1.78 mL). The mixture was stirred
at -15 °C for 1 h, and then a solution of 16 (351 mg, 0.637
mmol) in toluene (10 mL) was added dropwise at -20 °C. The
mixture was stirred for 24 h at room temperature before
addition of saturated aqueous NH4Cl (20 mL), and then the
pH was adjusted to pH 7. After extraction with EtOAc, the
organic phase was dried over MgSO4, the solvent was removed,
and the residue was purified by flash chromatography (eluent
cyclohexane:EtOAc, 3:2) to give 17 (173 mg, 0.294 mmol, 46%
yield) as an orange solid. Mp 94-96 °C. IR (KBr), νCdO 1710,
12-(â-D-Glu cop yr a n osyl)-6-m et h yl-12,13-d ih yd r o-5H -
p yr id o[3′,2′:4,5]p yr r olo[2,3-a ]p yr r olo[3,4-c]ca r ba zole-5,7-
(6H)-d ion e (14). A mixture of compound 13 (47 mg, 0.054
mmol), catalytic amounts of 10% Pd/C in EtOAc (2 mL), and
methanol (5 mL) was hydrogenated (1 bar) for 3 days. The
solvents were removed, and the residue was purified by flash
chromatography (eluent EtOAc:MeOH, 98:2 then 9:1) to give
compound 14 (23 mg, 0.046 mmol, 85% yield) as a yellow solid.
mp > 275-277 °C, IR (KBr) νCO 1690, 1750 cm-1, νNH,OH 3100-
3600 cm-1. HRMS (FAB+) (M + H)+ calcd for C26H23N4O7
503.1567, found 503.1568. 1H NMR (400 MHz, DMSO-d6) 3.06
(3H, s, CH3), 3.38 (1H, m), 3.51 (1H, m), 3.61-3.71 (3H, m),
1
1770 cm-1, νNH 3300-3600 cm-1. H NMR (400 MHz, CDCl3):
4.73 (2H, s, CH2), 5.24 (2H, s, CH2), 6.48 (1H, dd, 1J ) 7.9 Hz,
2J ) 4.4 Hz), 6.81 (1H, d, J ) 1.0 Hz), 6.82 (1H, d, J ) 3.0
1
2
4.40 (1H, d, J ) 3.2 Hz), 4.58 (2H, dt, J ) 17.3 Hz, J ) 3.7
Hz), 4.73 (1H, d, J ) 3.8 Hz), 4.91 (1H, t, J ) 2.9 Hz), 5.40
(1H, d, J ) 6.2 Hz), 6.21-6.29 (2H, m), 6.41 (1H, t, J ) 5.6
1
2
Hz), 6.95 (1H, dd, J ) 8.4 Hz, J ) 1.5 Hz), 7.17-7.25 (2H,
m), 7.29 (2H, t, J ) 6.9 Hz), 7.39 (2H, d, J ) 6.9 Hz), 7.53 (2H,
1
2
t, J ) 6.9 Hz), 7.64 (1H, dt, J ) 6.9 Hz, J ) 1.5 Hz), 7.97-
1
2
Hz), 6.70 (1H, d, J ) 6.4 Hz), 7.15 (1H, dd, J ) 3.5 Hz, J )
1
8.01 (3H, m), 8.10 (1H, s), 8.14 (1H, s), 8.18 (1H, dd, J ) 4.4
1.0 Hz), 7.57 (1H, d, J ) 6.0 Hz), 7.69 (1H, dd, J ) 5.9 Hz, 2J
1
Hz, 2J ) 1.5 Hz), 12.05 (1H, s, NH). 13C NMR (100 MHz,
CDCl3): 67.5, 71.8 (CH2), 113.6, 116.6, 121.9, 123.5, 125.3,
127.1 (2C), 127.7 (3C), 127.8, 128.5 (3C), 129.5 (2C), 130.2,
134.3, 143.2 (C tert arom), 104.8, 112.4, 118.5, 124.2, 127.8,
131.5, 134.6, 137.7, 137.8, 148.7 (C quat arom), 170.5, 170.9
(CdO).
) 0.8 Hz), 9.25 (1H, d, J ) 5.2 Hz), 9.30 (1H, d, J ) 5.3 Hz),
11.60 (1H, s, NH). 13C NMR (100 MHz, DMSO-d6) 23.8 (CH3),
58.2 (C6′), 67.9, 73.2, 76.7, 79.0, 84.4 (C1′, C2′, C3′, C4′, C5′), 111.8,
117.0, 120.8, 124.3, 127.2, 132.3, 147.9 (C tert arom), 114.1,
114.9, 117.3, 119.1, 120.4, 120.6, 128.0, 128.3, 142.2, 152.5 (C
quat arom), 169.5 (2C) (CdO). HPLC >98% pure, 1) tR ) 4.70
min, 2) tR ) 3.51 min.
1-[(Ben zyloxy)m eth yl]-3-[1-(p h en ylsu lfon yl)-1H-in d ol-
3-yl]-4-[1-(2,3,4,6-tetr a -O-a cetyl-â-D-glu cop yr a n osyl)-1H-
p yr r olo[2,3-b]p yr id in -3-yl]-1H-p yr r ole-2,5-d ion e (18). To
a solution of 17 (545 mg, 0.927 mmol) in THF (40 mL) were
added 2,3,4,6-O-acetylglucopyranose (677 mg, 1.95 mmol) and
triphenylphosphine (510 mg, 1.95 mmol). The mixture was
cooled to -78 °C, and then DEAD (306 µL, 1.95 mmol) was
added dropwise. The mixture was allowed to reach room
temperature and then stirred for 15 h. Water (100 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 chromatography (eluent cyclohexane:
EtOAc, 65:35, then toluene:EtOAc, 3:2) to give â-glycosylated
compound 18 (519 mg, 0.565 mmol, 61% yield) as a yellow
solid. Mp 105-107 °C. IR (KBr), νCdO 1720, 1760 cm-1. HRMS
(FAB+) (M + H)+ calcd C47H43N4O14S 919.4296, found 919.2501.
1H NMR (400 MHz, CDCl3): 1.66 (3H, s, CH3CO), 2.05 (3H, s,
CH3CO), 2.10 (3H, s, CH3CO), 2.13 (3H, s, CH3CO), 4.05 (1H,
1-[(Ben zyloxy)m et h yl]-3-b r om o-4-(1H -in d ol-3-yl)-1H -
p yr r ole-2,5-d ion e (15). A solution of ethylmagnesium bro-
mide was prepared from magnesium (164 mg, 6.75 mmol) and
bromoethane (509 µL, 6.75 mmol) in THF (5 mL). The mixture
was stirred for 15 min at room temperature and then at 40 °C
for 20 min. A solution of indole (790 mg, 6.75 mmol) in THF
(40 mL) was then added dropwise. The mixture was stirred
at 40 °C for 1 h and then cooled to room temperature before
dropwise addition of a solution of N-benzyloxymethyl-2,3-
dibromomaleimide (1.27 g, 3.38 mmol) in THF (40 mL). The
mixture was stirred for 15 h at room temperature, before
addition of saturated aqueous NH4Cl. After extraction with
EtOAc, the organic phase was dried over MgSO4, the solvent
was removed, and the residue was purified by flash chroma-
tography (eluant cyclohexane:EtOAc, 4:1) to give 15 (1.043 g,
2.54 mmol, 75% yield) as a yellow solid. Mp 115-117 °C. IR

7-Azarebeccamycin Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4 619
1
2
1
m), 4.18 (1H, dd, J ) 12.5 Hz, J ) 1.5 Hz), 4.30 (1H, dd, J
mL), and 10% Pd/C (18 mg) was hydrogenated (1 bar) at room
temperature for 24 h. 10% Pd/C (21 mg) was added and the
mixture hydrogenated (1 bar) for 48 h. After filtration over
Celite, the solid was washed with methanol and EtOAc. After
removal of the solvent, the residue was purified by flash
chromatography (eluent cyclohexane:EtOAc, 65:35) to give
unreacted compound 20 (10.5 mg, 0.0135 mmol), 21 (21 mg,
0.031 mmol, 46% yield) (eluent cyclohexane:EtOAc, 3:2) as a
pale yellow solid, and a mixture of partially deacetylated
compounds.
2
) 12.6 Hz, J ) 4.7 Hz), 4.72 (2H, s, CH2), 5.23 (2H, s, CH2),
5.30 (1H, m), 5.51 (2H, m), 6.25 (1H, m), 6.48 (1H, dd, 1J )
8.0 Hz, 2J ) 4.7 Hz), 6.83 (1H, s), 6.84 (1H, s), 6.85 (1H, dd, 1J
) 8.1 Hz, 2J ) 1.0 Hz), 7.16-7.32 (4H, m), 7.39 (2H, d, J )
7.4 Hz), 7.52 (2H, t, J ) 7.9 Hz), 7.63 (1H, t, J ) 7.6 Hz), 7.95-
7.99 (3H, m), 8.06 (1H, br s), 8.15 (2H, m), 12.05 (1H, s, NH).
13C NMR (100 MHz, CDCl3): 20.0, 20.5 (2C), 20.7 (CH3CO),
61.7 (C6′), 67.4, 71.7 (CH2), 68.0, 70.9, 72.9, 74.8, 80.3 (C1′, C2′,
C3′, C4′, C5′), 113.4, 117.5, 121.9, 123.4, 125.1, 126.8 (2C), 127.5
(3C), 127.6, 128.3 (3C), 129.4 (2C), 129.8, 134.2, 143.9 (C tert
arom), 105.8, 112.1, 118.2, 125.3, 128.2, 130.4, 134.4, 137.6
(2C), 147.4 (C quat arom), 168.7, 169.4, 169.8, 170.1, 170.3,
170.6 (CdO).
21: Mp 154-156 °C. IR (KBr), νCdO 1705, 1760 cm-1, νNH
3200-3600 cm-1. HRMS (FAB+) (M + H)+ calcd C34H31N4O12
1
687.1938, found 687.1939. H NMR (400 MHz, CDCl3): 0.81
(3H, s, CH3CO), 1.80 (3H, s, CH3CO), 2.11 (3H, s, CH3CO),
2.43 (3H, s, CH3CO), 4.53 (1H, m), 4.88 (1H, m), 5.08 (1H, dd,
1-[(Ben zyloxy)m et h yl]-3-(1H -in d ol-3-yl)-4-[1-(2,3,4,6-
tetr a -O-a cetyl-â-D-glu cop yr a n osyl)-1H-p yr r olo[2,3-b]p y-
r id in -3-yl]-1H-p yr r ole-2,5-d ion e (19). To a solution of com-
pound 18 (519 mg, 0.565 mmol) in THF (20 mL) was added a
1.1 M solution of nBu4NF in THF (1.70 mL, 1.86 mmol). The
mixture was stirred for 2.5 h at room temperature. 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 chromatography (eluent
cyclohexane:EtOAc, 2:3) to give 19 (318 mg, 0.409 mmol, 72%
yield) as an orange solid. Mp 117-119 °C. IR (KBr), νCdO 1710,
2
1J ) 13.1 Hz, J ) 2.4 Hz), 5.17 (1H, d, J ) 12.3 Hz), 5.26-
5.32 (3H, m, 2H + OH), 5.42-5.48 (2H, m), 6.65 (1H, d, J )
1
9.6 Hz, H1′), 6.93 (1H, m), 7.26-7.31 (2H, m), 7.47 (1H, dd, J
) 7.8 Hz, 2J ) 4.9 Hz), 8.70 (1H, dd, 1J ) 4.8 Hz, 2J ) 1.5
Hz), 8.75 (1H, dd, 1J ) 7.8 Hz, 2J ) 1.6 Hz), 9.03 (1H, m), 9.36
(1H, s, NH). 13C NMR (100 MHz, CDCl3): 18.8, 20.4, 20.5, 21.7
(CH3CO), 60.7, 61.1 (CH2OH, C6′), 66.9, 70.2, 73.1, 76.8, 83.0
(C1′, C2′, C3′, C4′, C5′), 111.2, 118.4, 121.0, 124.8, 127.9, 135.1,
147.5 (C tert arom), 115.7, 115.9, 117.7, 118.8, 121.4, 121.6,
126.2, 129.0, 140.1, 151.7 (C quat arom), 167.5 (2C), 168.3,
169.5, 169.8, 170.6 (CdO).
1760 cm-1, νNH 3300-3500 cm-1. H NMR (400 MHz, CDCl3):
1
1.71 (3H, s, CH3CO), 2.03 (3H, s, CH3CO), 2.07 (3H, s, CH3-
CO), 2.09 (3H, s, CH3CO), 4.05 (1H, m), 4.15 (1H, J ) 11.7
Hz), 4.27 (1H, dd, 1J ) 12.6 Hz, 2J ) 4.7 Hz), 4.72 (2H, s, CH2),
5.22 (2H, d, J ) 2.0 Hz, CH2), 5.28 (1H, t, J ) 9.9 Hz), 5.50
(1H, t, J ) 9.4 Hz), 5.59 (1H, t, J ) 9.4 Hz), 6.29 (1H, d, J )
9.3 Hz, H1′), 6.69 (1H, t, J ) 7.8 Hz), 6.70 (1H, t, J ) 7.7 Hz),
6.80 (1H, d, J ) 8.1 Hz), 7.02 (1H, t, J ) 7.4 Hz), 7.16-7.33
(5H, m), 7.39 (2H, d, J ) 7.4 Hz), 7.82 (1H, d, J ) 2.1 Hz),
8.00 (1H, s), 8.17 (1H, dd, J ) 3.7 Hz, J ) 1.0 Hz), 9.35 (1H,
s, NH). 13C NMR (100 MHz, CDCl3): 20.1, 20.5, 20.6, 20.8 (CH3-
CO), 61.9 (C6′), 67.3, 71.7 (CH2), 68.1, 70.6, 73.3, 74.7, 80.3
(C1′, C2′, C3′, C4′, C5′), 111.5, 117.3, 120.6, 121.6, 122.7, 127.7
(2C), 127.8, 128.1, 128.4 (2C), 129.2, 130.6, 143.7 (C tert arom),
106.3, 106.8, 119.1, 125.2, 125.6, 129.4, 136.0, 137.7, 147.6 (C
quat arom), 168.9, 169.5, 170.0, 170.8, 171.2, 171.5 (CdO).
13-(â-D-Glu copyr an osyl)-12,13-dih ydr o-5H-pyr ido[3′,2′f:
4,5]p yr r olo[2,3-a ]p yr r olo[3,4-c]ca r b a zole-5,7(6H )-d ion e
(22). A mixture of 21 (21 mg, 0.030 mmol), methanol (9.4 mL),
and 28% aqueous NH4OH (8 mL) was stirred for 19 h at room
temperature. After removal of the solvents, a mixture of water/
EtOAc was added to the residue. After filtration, the solid was
washed successively with EtOAc and then methanol. Com-
pound 22 (8,4 mg, 0.017 mmol, 57% yield) was obtained as a
1
2
yellow solid. Mp > 300 °C. IR (KBr), νCdO 1710, 1740 cm-1
νNH,OH 3100-3600 cm-1. HRMS (FAB+) (M + H)+ calcd
25H21N4O7 489.1410, found 489.1404. 1H NMR (400 MHz,
,
C
DMSO-d6): 3.58 (2H, m), 3.87 (1H, d, J ) 10.1 Hz), 3.95 (1H,
d, J ) 9.5 Hz), 4.04 (1H, m), 4.12 (1H, d, J ) 10.3 Hz), 4.97
(1H, br s, OH), 5.22 (1H, br s, OH), 5.44 (1H, d, J ) 4.2 Hz,
OH), 6.14 (1H, br s, OH), 6.64 (1H, d, J ) 6.6 Hz, H1′), 7.43
(1H, t, J ) 7.4 Hz), 7.53 (1H, t, J ) 6,5 Hz), 7.64 (1H, t, J )
7.4 Hz), 7.76 (1H, d, J ) 8.2 Hz), 8.67 (1H, d, J ) 3.6 Hz),
9.11 (1H, d, J ) 7.9 Hz), 9.36 (1H, d, J ) 7.6 Hz), 11.25 (1H,
s, NH), 11.72 (1H, s, NH). 13C NMR (100 MHz, DMSO-d6): 58.3
(C6′), 67.6, 72.7, 76.6, 78.7, 83.1 (C1′, C2′, C3′, C4′, C5′), 112.2,
117.4, 120.6, 124.5, 127.5, 132.8, 147.0 (C tert arom), 114.3,
115.3, 117.7, 120.0, 121.3, 121.6, 127.4, 129.6, 141.0, 152.4 (C
quat arom), 170.9 (2C) (CdO). HPLC >97% pure, 1) tR ) 3.77
min, 2) tR ) 3.05 min.
6-[(Ben zyloxy)m et h yl]-13-(2,3,4,6-t et r a -O-a cet yl-â-D-
glu copyr an osyl)-12,13-dih ydr o-5H-pyr ido[3′,2′:4,5]pyr r olo-
[2,3-a ]p yr r olo[3,4-c]ca r ba zole-5,7(6H)-d ion e (20). A mix-
ture of 19 (318 mg, 0.409 mmol), benzene (500 mL), and iodine
(1.245 g, 4.90 mmol) was irradiated for 1.5 h with a medium-
pressure mercury lamp (400 W). The solvent was removed, and
the residue dissolved in EtOAc (150 mL) was washed with
saturated aqueous sodium thiosulfate and then brine. The
organic phase was dried over MgSO4. After removal of the
solvent, the residue was purified by flash chromatography
(eluent cyclohexane:EtOAc, 3:2) to give 20 (247 mg, 0.318
mmol, 78% yield) as a pale yellow solid. Mp 109-111 °C. IR
9-Br om o-13-(â-D-glu cop yr a n osyl)-12,13-d ih yd r o-5H-p y-
r id o[3′,2′:4,5]p yr r olo[2,3-a ]p yr r olo[3,4-c]ca r b a zole-5,7-
(6H)-d ion e (23). To a solution of 21 (30 mg, 0.043 mmol) in
THF (2 mL) cooled to 0 °C was added dropwise a solution of
N-bromosuccinimide (154 mg, 0.866 mmol) in THF (1.5 mL).
The light-protected mixture was stirred for 5 days at room
temperature. Water (30 mL) was added, and the mixture was
stirred for 15 min before addition of saturated aqueous sodium
thiosulfate (20 mL). After extraction with EtOAc, the organic
phase was dried over MgSO4. After removal of the solvent,
brominated derivative was obtained as a yellow solid which
was further dissolved in methanol (8 mL), and then 28%
aqueous NH4OH (9 mL) was added. The light-protected
mixture was stirred for 22 h at room temperature. After
removal of the solvents, a mixture of water/EtOAc was added
to the residue. After filtration, the solid was washed with
EtOAc. Compound 23 (13 mg, 0.023 mmol, 53% yield) was
obtained as a yellow solid. Mp > 300 °C. IR (KBr), νCdO 1710,
1750 cm-1, νNH,OH 3200-3600 cm-1. HRMS (FAB+) (M + H)+
(KBr), νCdO 1710, 1760 cm-1, νNH 3300-3500 cm-1 1H NMR
.
(400 MHz, CDCl3): 1,87 (3H, s, CH3CO), 2.01 (3H, s, CH3CO),
1
2.11 (3H, s, CH3CO), 2.32 (3H, s, CH3CO), 4.34 (1H, dd, J )
2
9.9 Hz, J ) 2.0 Hz), 4.55 (1H, d, J ) 11.6 Hz), 4.75 (2H, AB
1
system, J ) 11.9 Hz, ∆ν ) 20.8 Hz), 4.88 (1H, dd, J ) 13.1
2
Hz, J ) 3.1 Hz), 5.14 (2H, AB system, J ) 11.0 Hz, ∆ν ) 9.2
Hz), 5.30 (1H, pt, J ) 9.6 Hz), 5.50 (1H, pt, J ) 9.2 Hz), 5.57
(1H, pt, J ) 9.7 Hz), 6.84 (1H, d, J ) 9.5 Hz, H1′), 7.23 (1H, t,
J ) 7.3 Hz), 7.29-7.37 (4H, m), 7.43-7.49 (4H, m), 8.54 (1H,
1
2
dd, J ) 4.6 Hz, J ) 1.5 Hz), 9.11 (1H, d, J ) 8.0 Hz), 9.15
(1H, dd, J ) 7.8 Hz, J ) 1.6 Hz), 9.80 (1H, s, NH). 13C NMR
(100 MHz, CDCl3): 18.9, 20.3, 20.4, 21.2 (CH3CO), 61.1 (C6′),
66.6, 71.4 (CH2), 67.1, 70.4, 72.8, 76.1, 81.9 (C1′, C2′, C3′, C4′,
C5′), 111.4, 118.1, 121.4, 125.5, 127.7, 127.9, 128.3 (2C), 128.7
(2C), 133.9, 146.9 (C tert arom), 115.0, 116.0, 118.9, 119.3,
121.2, 122.1, 127.1, 129.5, 137.6, 140.5, 151.8 (C quat arom),
167.8, 168.6, 168.8, 169.3, 169.6, 170.4 (CdO).
1
2
1
calcd C25H20N4O7Br 567.0515, found 567.0519. H NMR (400
6-(H yd r oxym et h yl)-13-(2,3,4,6-t et r a -O-a cet yl-â-D-glu -
cop yr a n osyl)-12,13-d ih yd r o-5H-p yr id o[3′,2′:4,5]p yr r olo-
[2,3-a ]p yr r olo[3,4-c]ca r ba zole-5,7(6H)-d ion e (21). A mix-
ture of 20 (52 mg, 0.067 mmol), methanol (3 mL), EtOAc (1
MHz, DMSO-d6): 3.48-3.61 (2H, m), 3.87 (1H, d, J ) 10.4
Hz), 3.93-4.01 (2H, m), 4.11 (1H, d, J ) 12.2 Hz), 4.98 (1H, d,
J ) 4.8 Hz, OH), 5.21 (1H, d, J ) 5.1 Hz, OH), 5.42 (1H, d, J
) 5.0 Hz, OH), 6.17 (1H, br s, OH), 6.65 (1H, d, J ) 8.5 Hz,

620 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
Marminon et al.
1
2
H1′), 7.54 (1H, dd, J ) 7.3 Hz, J ) 4.7 Hz), 7.71 (1H, d, J )
arom), 107.0, 119.8, 121.5, 135.4, 137.3, 137.5, 146.9 (C quat
arom), 165.2, 167.9 (CdO).
1
2
8.7 Hz), 7.80 (1H, dd, J ) 8.7 Hz, J ) 1.3 Hz), 8.68 (1H, d,
1
J ) 4.7 Hz), 9.26 (1H, d, J ) 1.5 Hz), 9.35 (1H, dd, J ) 7.7
1-[(Ben zyloxy)m et h yl]-3-(1H -in d ol-3-yl)-4-[1-(p h en yl-
su lfon yl)-1H-p yr r olo[2,3-b]p yr id in -3-yl]-1H-p yr r ole-2,5-
d ion e (26). A solution of ethylmagnesium bromide was
prepared from Mg (15 mg, 0.62 mmol) and bromoethane (47
µL, 0.62 mmol) in THF (0.4 mL). The solution was stirred at
room temperature for 15 min and then at 40 °C for 20 min. A
solution of indole (76 mg, 0.65 mmol) in toluene (3 mL) was
then added dropwise. After stirring for 1 h at 40 °C, the
mixture was cooled, then a solution of X (140 mg, 0.254 mmol)
in toluene (5 mL) was added dropwise. The mixture was stirred
for 15 h and then hydrolyzed with saturated aqueous NaCl
(15 mL). After extraction with EtOAc, the organic phase was
dried over MgSO4, the solvent was removed, and the residue
was purified by flash chromatography (eluent cyclohexane:
EtOAc, 3:2) to give 26 (122 mg, 0.208 mmol, 82% yield) as a
red solid: mp 86-88 °C; IR (KBr) νCO 1710, 1730 cm-1, νNH
3300-3440 cm-1. 1H NMR (400 MHz, CDCl3) 4.75 (2H, s, CH2),
Hz, 2J ) 1.4 Hz), 11.84 (1H, s, NH). 13C NMR (100 MHz,
DMSO-d6): 58.3 (C6′), 67.6, 72.7, 76.5, 78.7, 83.1 (C1′, C2′, C3′,
C4′, C5′), 114.2, 117.6, 126.5, 129.9, 132.9, 147;3 (C tert arom),
112.5, 114.1, 115.7, 116.4, 120.5, 121.7, 123.1, 127.4, 129.8,
139.6, 152.4 (C quat arom), 170.8, 170.9 (CdO). HPLC >95%
pure, 1) tR ) 4.64 min, 2) tR ) 3.28 min.
9-Nitr o-13-(â-D-glu cop yr a n osyl)-12,13-d ih yd r o-5H-p y-
r id o[3′,2′:4,5]p yr r olo[2,3-a ]p yr r olo[3,4-c]ca r b a zole-5,7-
(6H)-d ion e (24). To Mallinckrodt ether (a solution of fuming
nitric acid (2,1 mL) in THF (13 mL)) at 0 °C was added
dropwise 21 (42 mg, 0.061 mmol) cooled to 0 °C. After stirring
for 10 min, the mixture was allowed to reach room temperature
and then stirred for 21 h. Water (40 mL) was added. After
extraction with EtOAc, the organic phase was dried over
MgSO4, the solvent was removed to give the nitrated com-
pound which was further dissolved in methanol (10 mL), and
then 28% aqueous NH4OH (17 mL) was added. The light-
protected mixture was stirred for 16 h at room temperature.
After removal of the solvents, a mixture of water/EtOAc was
added to the residue. After filtration, the solid was washed
with EtOAc. Compound 24 (12.5 mg, 0.023 mmol, 38% yield)
was obtained as a brown solid. Mp > 300 °C. IR (KBr), νCdO
1710, 1760 cm-1, νNH,OH 3300-3600 cm-1. HRMS (FAB+) (M
+ H)+ calcd C25H20N5O9 534.1261, found 534.1268. 1H NMR
(400 MHz, DMSO-d6): 3.56-3.59 (2H, m), 3.91 (1H, d, J )
7.9 Hz), 4.00 (2H, m), 4.13 (1H, m), 4.99 (1H, br s, OH), 5.20
(1H, br s, OH), 5.44 (1H, br s, OH), 6.29 (1H, br s, OH), 6.66
(1H, d, J ) 7.9 Hz, H1′), 7,55 (1H, m), 7.87 (1H, d, J ) 8.7 Hz),
8.54 (1H, m), 8.69 (1H, m), 9.35 (1H, d, J ) 7.1 Hz), 10.00
(1H, m), 11.47 (1H, s, NH), 12.19 (1H, s, NH). 13C NMR (100
MHz, DMSO-d6): 58.3 (C6′), 67.6, 72.8, 76.4, 78.7, 83.1 (C1′,
C2′, C3′, C4′, C5′), 112.6, 117.7, 121.1, 122.6, 133.0, 147.6 (C tert
arom), 114.0, 116.5, 117.6, 120.9, 121.5, 121.7, 127.2, 130.7,
141.2, 144.0, 152.4 (C quat arom), 170.5 (2C) (CdO). HPLC
>96% pure, (1) tR ) 3.75 min, (2) tR ) 3.47 min.
1
2
5.24 (2H, s, CH2), 6.53 (1H, dt, J ) 7.1 Hz, J ) 0.6 Hz), 6.60
1
2
(1H, d, J ) 7.9 Hz), 6.89 (1H, dd, J ) 8.0 Hz, J ) 4.7 Hz),
7.07 (1H, td, 1J ) 7.2 Hz, 2J ) 0.9 Hz), 7.21-7.40 (7H, m),
7.51 (2H, t, J ) 8.0 Hz), 7.63 (1H, td, 1J ) 7.4 Hz, 2J ) 0.9
Hz), 7.91 (1H, d, J ) 2.9 Hz), 8.04 (1H, s), 8.12 (1H, d, J ) 1.3
Hz), 8.16 (1H, br s), 8.34 (1H, dd, J ) 4.8 Hz, J ) 1.5 Hz),
9.11 (1H, s, NH). 13C NMR (100 MHz, CDCl3) 67.3, 71.7 (CH2),
112.0, 118.9, 120.9, 121.2, 122.8, 127.6 (2C), 127.7, 127.8,
128.1, 128.2, 128.3 (2C), 129.1, 129.2, 130.4, 131.0, 134.3, 145.3
(C tert arom), 105.6, 109.4, 121.1, 122.8, 124.2, 132.2, 136.2,
137.5, 137.7, 146.6 (C quat arom), 170.7, 170.9 (CdO).
1
2
1-[(Ben zyloxy)m eth yl]-3-[1-(2,3,4,6-tetr a -O-a cetyl-â-D-
glu cop yr a n osyl)-1H-in d ol-3-yl]-4-[1-(p h en ylsu lfon yl)-1H-
p yr r olo[2,3-b]p yr id in -3-yl]-1H-p yr r ole-2,5-d ion e (27). To
a solution of 26 (650 mg, 1.105 mmol) in THF (10 mL) were
added 2,3,4,6-O-acetylglucopyranose (616 mg, 1.769 mmol) and
PPh3 (464 mg, 1.769 mmol). The mixture was cooled to -78
°C, and then DEAD (282 µL, 1.769 mmol) was added dropwise.
The mixture was allowed to reach room temperature and then
stirred at room temperature for 15 h. Water (80 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 chromatography (cyclohexane:EtOAc, 1:1,
then toluene:EtOAc, 3:2) to give â-glycosylated compound 27
(373 mg, 0.40 mmol, 37% yield), a red solid as the major
product of the reaction. Mp 80-82 °C. IR (KBr), νCdO 1710,
1-[(Ben zyloxy)m eth yl]-3-br om o-4-[1-(p h en ylsu lfon yl)-
1H-p yr r olo[2,3-b]p yr id in -3-yl]-1H-p yr r ole-2,5-d ion e (25).
A solution of ethylmagnesium bromide was prepared from Mg
(146 mg, 6.00 mmol) and C2H5Br (452 µL, 6.00 mmol) in THF
(2.5 mL). The mixture was stirred for 1 h at room temperature,
and then commercially available 7-azaindole (708 mg, 6.00
mmol) in toluene (20 mL) was added dropwise. The mixture
was stirred at room temperature for 1.5 h, and then a solution
of N-benzyloxymethyl-2,3-dibromomaleimide (756 mg, 2.01
mmol) in toluene (20 mL) was added dropwise. After stirring
for 20 min, dichloromethane (30 mL) was added then the
mixture was stirred for 65 h at 40 °C. Saturated aqueous NH4-
Cl was added. After extraction with EtOAc, the organic phase
was dried over MgSO4, the solvent was removed, and the
residue was purified by flash (eluent cyclohexane:EtOAc, 3:2,
then toluene:EtOAc, 7:3) to give 1-[(benzyloxy)methyl]-3-
bromo-4-[1H-pyrrolo[2,3-b]pyridin-3-yl]-1H-pyrrole-2,5-dione
(471 mg, 1.14 mmol, 57% yield). To a suspension of HNa (60%
in mineral oil) (51 mg, 1.27 mmol) in THF (5 mmol) at 0 °C
was added dropwise a solution of the previous isolated
compound (330 mg, 0.800 mmol) in THF (10 mL). The mixture
was stirred at 0 °C for 1 h, and then benzenesulfonyl chloride
(125 mL, 0.98 mmol) was added dropwise. The mixture was
stirred at room temperature for 4 h, and then saturated
aqueous NH4Cl was added. After extraction with EtOAc, the
organic phase was washed with brine and dried over MgSO4,
the solvent was removed, and the residue was purified by flash
chromatography (eluent cyclohexane:EtOAc, 4:1) to give 25
(333 mg, 0.60 mmol, 75% yield) as a yellow solid: mp123-
1760 cm-1. HRMS (FAB+) (M + H)+ calcd C47H43N4O14
S
1
919.2496, found 919.2501. H NMR (400 MHz, CDCl3): 1.72
(3H, s, CH3CO), 2.00 (3H, s, CH3CO), 2.09 (3H, s, CH3CO),
2.12 (3H, s, CH3CO), 4.11-4.27 (2H, m), 4.35 (1H, dd, 1J )
12.6 Hz, 2J ) 4.7 Hz), 4.69 (2H, s, CH2), 5.20 (2H, s, CH2),
5.48-5.50 (2H, m), 5.66 (1H, m), 6.43-6.51 (2H, m), 6.88 (1H,
dd, 1J ) 8.0 Hz, 2J ) 4.8 Hz), 7.09 (1H, t, J ) 7.4 Hz), 7.21
(1H, d, J ) 7.4 Hz), 7.25 (3H, t, J ) 7.2 Hz), 7.36 (3H, d, J )
7.5 Hz), 7.44-7.54 (3H, m), 7.61 (1H, t, J ) 4.6 Hz), 7.98 (1H,
s), 8.01 (1H, s), 8.10 (2H, d, J ) 7.6 Hz), 8.30 (1H, d, J ) 4.6
Hz). 13C NMR (100 MHz, CDCl3): 20.0, 20.4, 20.6, 20.7 (CH3-
CO), 61.7 (C6′), 67.0, 71.8 (CH2), 68.0, 70.9, 72.8, 75.0, 84.3
(C1′, C2′, C3′, C4′, C5′), 110.9, 119.0, 121.6, 121.7, 123.3, 127.6
(2C), 127.8, 128.2, 128.3 (2C), 128.4 (2C), 129.1 (2C), 130.0,
130.9, 134.3, 145.4 (C tert arom), 106.7, 109.2, 121.1, 124.5,
125.5, 130.8, 135.7, 137.6, 137.8, 146.7 (C quat arom), 168.5,
169.4, 170.1, 170.4, 170.6, 171.2 (CdO).
1-[(Ben zyloxy)m eth yl]-3-[1-(2,3,4,6-tetr a -O-a cetyl-â-D-
glu cop yr a n osyl)-1H-in d ol-3-yl]-4-(1H-p yr r olo[2,3-b]p yr i-
d in -3-yl)-1H-p yr r ole-2,5-d ion e (28) a n d 1-[(Ben zyloxy)-
m eth yl]-3-[1-(2,3,4,6-tetr a -O-a cetyl-r-D-glu cop yr a n osyl)-
1H -in d o l-3-y l]-4-(1H -p y r r o lo [2,3-b ]p y r id in -3-y l)-1H -
p yr r ole-2,5-d ion e (29). To a solution of 26 (50 mg, 0.085
mmol) in toluene (7 mL) were added Ag2O (197 mg, 0.85 mmol)
and 2,3,4,6-tetra-O-acetyl-R-D-glucopyranosyl bromide (175
mg, 0.425 mmol). The mixture was refluxed for 3 h and then
cooled and filtered over Celite. After removal of the solvent,
the residue was purified by flash chromatography (eluent
cyclohexane:EtOAc, 65:35) to give a mixture of R-glycosylated
125 °C. IR (KBr) νCO 1730, 1780 cm-1 1H NMR (400 MHz,
.
CDCl3) 4.68 (2H, s, CH2), 5.18 (2H, s, CH2), 7.24-7.37 (6H,
m), 7.55 (2H, t, J ) 7.5 Hz), 7.65 (1H, t, J ) 7.5 Hz), 8.24 (1H,
1
2
dd, J ) 8.0 Hz, J ) 1.3 Hz), 8.29 (1H, br s), 8.30 (1H, br s),
8.48 (1H, s), 8.51 (1H, dd, J ) 4.7 Hz, J ) 1.3 Hz). 13C NMR
(100 MHz, CDCl3) 67.9, 72.1 (CH2), 119.6, 127.6 (2C), 128.0,
128.5 (3C), 128.6 (2C), 129.3 (2C), 132.0, 134.8, 146.0 (C tert
1
2

7-Azarebeccamycin Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4 621
compound 29A and unreacted sugar (73 mg), and then (eluent
cyclohexane:EtOAc, 1:1) a mixture of â-glycosylated compound
28A and unreacted sugar (6 mg).
CO), 4.31 (1H, d, J ) 10.1 Hz), 4.51 (1H, d, J ) 12.4 Hz), 4.80
(2H, AB system, J ) 12.0 Hz, ∆ν ) 7.8 Hz), 4.88 (1H, d, J )
11.8 Hz), 5.23-5.31 (3H, m), 5.49 (1H, t, J ) 9.5 Hz), 5.66 (1H,
t, J ) 9.7 Hz), 6.03 (1H, d, J ) 8.9 Hz, H1′), 7.27-7.42 (5H,
m), 7.48-7.51 (3H, m), 7.61 (1H, t, J ) 7.7 Hz), 8.48 (1H, d, J
) 3.7 Hz), 9.12 (1H, d, J ) 7.9 Hz), 9.33 (1H, d, J ) 7.6 Hz),
10.23 (1H, s, NH). 13C NMR (100 MHz, CDCl3): 18.5, 20.5,
20.6, 21.1 (CH3CO), 60.5 (C6′), 66.9, 71.7 (CH2), 67.0, 70.8, 72.8,
76.7, 84.3 (C1′, C2′, C3′, C4′, C5′), 110.1, 117.6, 122.3, 126.1, 127.7,
128.0, 128.1 (2C), 128.5 (2C), 133.6, 147.8 (C tert arom), 114.8
(2C), 116.5, 119.1, 120.6 (2C), 121.6, 128.4, 137.7, 141.2, 152.3
(C quat arom), 167.8, 168.8, 169.0, 169.1, 170.0, 171.9 (CdO).
To a solution of a mixture of R-glycosylated compound 29A
and R-D-acetobromoglucose (73 mg) in THF (5 mL) was added
a 1.1 M solution of nBu4NF in THF (217 mL, 0.238 mmol).
The mixture was stirred at room temperature for 3 h. Water
(20 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 chromatography (eluent
cyclohexane:EtOAc, 1:1) to give 29 (34 mg, 0.043 mmol, 51%
yield over 2 steps) as an orange solid.
To a solution of a mixture of â-glycosylated compound 28A
and R-D-acetobromoglucose (6 mg) in THF (2 mL) was added
a 1.1 M solution of nBu4NF in THF (18 mL, 0.016 mmol). The
mixture was stirred at room temperature for 3 h. Water (5
mL) was added, and after identical workup as described above,
a purification by flash chromatography (eluent cyclohexane:
EtOAc, 1:1) gave 28 (4 mg, 0.005 mmol, 6% yield for two steps)
as a red solid.
6-(H yd r oxym et h yl)-12-(2,3,4,6-t et r a -O-a cet yl-â-D-glu -
cop yr a n osyl)-12,13-d ih yd r o-5H-p yr id o[3′,2′:4,5]p yr r olo-
[2,3-a ]p yr r olo[3,4-c]ca r ba zole-5,7(6H)-d ion e (31). A mix-
ture of 30 (70 mg, 0.090 mmol), methanol (40 mL), EtOAc (20
mL), and 10% Pd/C (60 mg) was hydrogenated (1 bar) at room
temperature for 17 h. Pd/C (10%) (31 mg) was added and the
mixture hydrogenated for 21 h. After filtration over Celite, the
residue was washed with methanol and chloroform. The
solvents were removed, and the residue was purified by flash
chromatography (eluent cyclohexane:EtOAc, 1:1) to give 31 (44
mg, 0.063 mmol, 70% yield) as a pale yellow solid. Mp 264-
28 was also obtained from 27 in 51% yield by treatment
with nBu4NF in THF according to identical procedure as
above-described.
28: Mp 115-117 °C. IR (KBr), νCdO 1700, 1760 cm-1, νNH
3100-3600 cm-1. 1H NMR (400 MHz, CDCl3): 1.79 (3H, s, CH3-
CO), 2.05 (3H, s, CH3CO), 2.11 (3H, s, CH3CO), 2.14 (3H, s,
CH3CO), 4.06 (1H, m), 4.26 (1H, dd, 1J ) 12.5 Hz, 2J ) 2.0
Hz), 4.36 (1H, dd, 1J ) 12.6 Hz, 2J ) 4.7 Hz), 4.73 (2H, s, CH2),
5.23 (2H, s, CH2), 5.35 (1H, t, J ) 9.8 Hz), 5.50 (1H, t, J ) 9.4
Hz), 5.57 (1H, t, J ) 9.3 Hz), 5.67 (1H, d, J ) 8.8 Hz), 6.69-
6.78 (3H, m), 7.11 (1H, t, J ) 7.1 Hz), 7.24 (2H, d, J ) 7.3 Hz),
7.32 (2H, t, J ) 7.3 Hz), 7.40 (2H, d, J ) 7.3 Hz), 7.46 (1H, d,
J ) 8.4 Hz), 7.90 (1H, s), 8.05 (1H, s), 8.21 (1H, d, J ) 4.5 Hz),
11.97 (1H, s, NH). 13C NMR (100 MHz, CDCl3): 20.0, 20.5 (2C),
20.7 (CH3CO), 61.7 (C6′), 67.1, 71.5 (CH2), 67.9, 70.6, 72.9, 74.9,
84.3 (C1′, C2′, C3′, C4′, C5′), 110.5, 116.3, 121.3, 122.0, 123.0,
127.5 (2C), 127.6 (2C), 128.3, 128.7, 129.6, 130.6, 143.0 (C tert
arom), 105.2, 107.4, 118.8, 126.5, 126.6, 128.0, 135.4, 137.6,
148.4 (C quat arom), 168.5, 169.3, 170.0, 170.5, 171.2, 171.4
(CdO).
266 °C. IR (KBr), νCdO 1710, 1760 cm-1, νNH 3300-3600 cm-1
.
HRMS (FAB+) (M + H)+ calcd C34H31N4O12 687.1938, found
1
687.1946. H NMR (400 MHz, CDCl3): 0.89 (3H, s, CH3CO),
1.87 (3H, s, CH3CO), 2.12 (3H, s, CH3CO), 2.62 (3H, s, CH3-
CO), 4.44 (1H, d, J ) 9.9 Hz), 4.64 (1H, d, J ) 12.5 Hz), 4.99
(1H, d, J ) 11.4 Hz), 5.12 (1H, t, J ) 9.3 Hz), 5.30 (2H, AB
system, J ) 11.2 Hz, ∆ν ) 7.0 Hz), 5.51 (1H, t, J ) 9.5 Hz),
5.62 (1H, t, J ) 9.7 Hz), 5.97 (1H, d, J ) 9.3 Hz, H1′), 7.25
(1H, dd, 1J ) 7.7 Hz, 2J ) 4.8 Hz), 7.32-7.35 (2H, m), 7.49
(1H, t, J ) 7.5 Hz), 8.49 (1H, d, J ) 4.1 Hz), 9.06 (1H, d, J )
6.6 Hz), 9.07 (1H, d, J ) 7.3 Hz), 10.23 (1H, s, NH). 13C NMR
(100 MHz, CDCl3): 18.8, 20.3, 20.5, 21.2 (CH3CO), 60.5, 61.3
(CH2OH, C6′), 67.2, 70.5, 72.8, 77.0, 84.4 (C1′, C2′, C3′, C4′, C5′),
110.0, 117.5, 122.0, 125.8, 127.7, 133.8, 147.8 (C tert arom),
114.9, 116.1, 119.0, 120.6, 121.3, 122.2, 128.2, 128.3, 141.2,
152.0 (C quat arom), 167.8, 168.3, 168.8, 169.2, 170.1, 171.8
(CdO).
29: Mp 107-109 °C. IR (KBr), νCdO 1710, 1750 cm-1, νNH
3300-3600 cm-1. 1H NMR (400 MHz, CDCl3): 1.98 (3H, s, CH3-
CO), 2.05 (3H, s, CH3CO), 2.13 (3H, s, CH3CO), 2.16 (3H, s,
CH3CO), 4.02 (1H, dd, 1J ) 5.1 Hz, 2J ) 2.4 Hz), 4.08 (1H, m),
4.22 (1H, dd, 1J ) 12.2 Hz, 2J ) 5.7 Hz), 4.28 (1H, dd, 1J )
12-(â-D-Glu copyr an osyl)-12,13-dih ydr o-5H-pyr ido[3′,2′f:
4,5]p yr r olo[2,3-a ]p yr r olo[3,4-c]ca r b a zole-5,7(6H )-d ion e
(32). A mixture of 31 (28 mg, 0.040 mmol), methanol (13 mL),
and 28% aqueous NH4OH (9 mL) was stirred for 15 h at room
temperature. After removal of the solvents, a mixture EtOAc/
water was added to the residue. After fitration, the solid was
washed successively with EtOAc and methanol to give 32 (8
mg, 0.016 mmol, 39% yield) as a yellow solid. Mp > 250 °C
(degradation). IR (KBr), νCdO 1720, 1760 cm-1, νNH,OH 3100-
3600 cm-1. HRMS (FAB+) (M + H)+ calcd C25H21N4O7 489.1410,
2
12.2 Hz, J ) 2.7 Hz), 4.73 (2H, s, CH2), 4.94 (1H, d, J ) 9.4
Hz), 5.24 (2H, s, CH2), 5.27 (1H, pt, J ) 1.8 Hz), 5.52 (1H, d,
1
2
J ) 5.1 Hz, H1′), 6.73 (1H, dd, J ) 8.1 Hz, J ) 4.8 Hz), 7.00
(1H, t, J ) 7.7 Hz), 7.04 (1H, dd, 1J ) 7.8 Hz, 2J ) 1.0 Hz),
7.20-7.34 (5H, m), 7.41 (2H, d, J ) 7.4 Hz), 7.58 (1H, s), 7.76
1
2
(1H, d, J ) 8.3 Hz), 8.09 (1H, s), 8.25 (1H, dd, J ) 4.7 Hz, J
) 1.0 Hz), 11.69 (1H, s, NH). 13C NMR (100 MHz, CDCl3):
20.7, 20.8, 20.9, 24.1 (CH3CO), 63;2 (C6′), 67.3, 71.7 (CH2), 67.2,
68.3, 69.4, 73.0, 97.3 (C1′, C2′, C3′, C4′, C5′), 112.7, 116.0, 121.5,
122.1, 123.5, 127.7 (2C), 127.8, 128.2, 128.4 (2C), 130.2, 130.8,
143.2 (C tert arom), 105.1, 107.0, 113.1, 117.8, 127.4, 128.6,
134.4, 137.8, 148.9 (C quat arom), 169.0, 169.6, 170.8, 171.2,
171.3, 171.4 (CdO).
1
found 489.1416. H NMR (400 MHz, DMSO-d6): 3.65 (1H, d,
J ) 5.5 Hz), 3.81 (1H, d, J ) 10.2 Hz), 3.87-4.60 (8H, m),
6.32 (1H, d, J ) 6.0 Hz, H1′), 7.41-7.55 (2H, m), 7.66 (1H, t,
J ) 7.0 Hz), 8.05 (1H, d, J ) 8.3 Hz), 8.69 (1H, d, J ) 3.8 Hz),
9.21 (1H, d, J ) 7.8 Hz), 9.36 (1H, d, J ) 7.3 Hz), 11.28 (1H,
s, NH), 11.66 (1H, s, NH). 13C NMR (100 MHz, DMSO-d6): 58.2
(C6′), 67.8, 73.2, 76.7, 78.9, 84.4 (C1′, C2′, C3′, C4′, C5′), 111.8,
117.0, 120.8, 124.5, 127.2, 132.7, 147.6 (C tert arom), 114.3,
114.7, 119.1, 120.3 (2C), 121.3, 128.2, 128.4, 142.1, 152.2 (C
quat arom), 170.9 (2C) (CdO). HPLC >96% pure, 1) tR ) 3.28
min, 2) tR ) 3.31 min.
6-[(Ben zyloxym et h yl)]-12-(2,3,4,6-t et r a -O-a cet yl-â-D-
glu copyr an osyl)-12,13-dih ydr o-5H-pyr ido[3′,2′:4,5]pyr r olo-
[2,3-a ]p yr r olo[3,4-c]ca r ba zole-5,7(6H)-d ion e (30). To a
solution of 28 (194 mg, 0.249 mmol) in benzene (150 mL) was
added iodine (758 mg, 2.99 mmol). The mixture was irradiated
for 1.5 h with a medium-pressure mercury lamp (400 W). The
solvent was removed, and the residue dissolved in EtOAc (80
mL) was washed with aqueous sodium thiosulfate then with
brine. The organic phase was dried over MgSO4, the solvent
was removed, and the residue was purified by flash chroma-
tography (eluent cyclohexane:EtOAc, 65:35) to give 30 (131
mg, 0.168 mmol, 68% yield) as a pale yellow solid. Mp 166-
Meltin g Tem p er a tu r e Stu d ies. Melting curves were
measured using an Uvikon 943 spectrophotometer coupled to
a Neslab RTE111 cryostat. For each series of measurements,
12 samples were placed in a thermostatically controlled cell-
holder, and the quartz cuvettes (10 mm path length) were
heated by circulating water. Measurements were performed
in BPE buffer pH 7.1 (6 mM Na2HPO4, 2 mM NaH2PO4, 1 mM
EDTA). The temperature inside the cuvette was measured
with a platinum probe; it was increased over the range 20-
168 °C. IR (KBr), νCdO 1710, 1760 cm-1, νNH 3360-3420 cm-1
.
HRMS (FAB+) (M + H)+ calcd C41H37N4O12 777.2408, found
100 °C with
a heating rate of 1 °C/min. The “melting”
1
temperature Tm was taken as the midpoint of the hyperchro-
777.2409. H NMR (400 MHz, CDCl3): 0.99 (3H, s, CH3CO),
1.91 (3H, s, CH3CO), 2.14 (3H, s, CH3CO), 2.67 (3H, s, CH3-
mic transition.

622 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
Marminon et al.
(5) Long, B. H.; Balasubramanian, B. N. Non camptothecin topo-
isomerase I active compounds as potential anticancer agents.
Expert Opin. Ther. Pat. 2000, 10, 635-666.
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Prudhomme, M.; Ollier, M.; Seve`re, D.; Riou, J .-F.; Bailly, C.;
Fabbro, D.; Meyer, T. Syntheses and biological activities (topo-
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Sequencing of topoisomerase I-mediated DNA cleavage sites.
The 117 base pairs DNA fragment was prepared by 3′-[32P]-
end labeling of the EcoRI-PvuII double digest of the plasmid
pBS using R-[32P]-dATP (3000 Ci/mMol) and AMV reverse
transcriptase. For the topoisomerase I experiments, each
reaction mixture contained 2 µL of 3′-end [32P] labeled DNA
(∼1 µM), 5 µL of water, 2 µL of 10X topoisomerase I buffer,
and 10 µL of drug solution at the desired concentration (10-
50 µM). After 10 min incubation to ensure equilibration, the
reaction was initiated by addition of 2 µL (20 units) calf thymus
topoisomerase I (Life Technologies). Samples were incubated
for 45 min at 37 °C prior to adding SDS to 0.25% and
proteinase K to 250 µg/mL to dissociate the drug-DNA-
topoisomerase I cleavable complexes. The DNA was precipi-
tated with ethanol and then resuspended in 5 µL of formamide-
TBE loading buffer, denatured at 90°C for 4 min, and then
chilled in ice for 4 min prior to loading on to the sequencing
gel. DNA cleavage products were resolved by polyacrylamide
gel electrophoresis under denaturing conditions.
Gr ow th In h ibition Assa ys. Tumor cells were provided by
American Type Culture Collection (Frederik, MD). Nine cell
lines were used: one murine leukemia (L1210), one human
leukemia (K-562), and seven human solid tumors: one ovarian
carcinoma (IGROV1), one neuroblastoma (SK-N-MC), one
colon carcinoma (HT29), one non small cell lung carcinoma
(A549), one small cell lung carcinoma (H69), and two epider-
moid carcinomas (A431 and KB-3-1). They were cultivated in
RPMI 1640 medium (Life Science Technologies, Cergy-Pon-
toise, France) supplemented with 10% fetal calf serum, 2 mM
L-glutamine, 100 units/mL penicillin, 100 µg/mL streptomycin,
and 10 mM HEPES buffer (pH ) 7.4). Cytotoxicity was
measured by the microculture tetrazolium assay as described.24
Cells were continuously exposed to graded concentrations of
the compounds for four doubling times, 15 µL 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
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inhibition by an analogue of the antitumor antibiotic rebecca-
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Pfeiffer, B.; Renard, P.; Prudhomme, M. Syntheses and anti-
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sugar unit linked to both indole nitrogens. J . Med. Chem. 2002,
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of arcyriarubin D and related bisindolylmaleimides. Tetrahedron
1988, 44, 2887-2892.
(14) Routier, S.; Coudert, G.; Merour, J . Y.; Caignard, D. H.; First
synthesis of symmetrical and nonsymmetrical aza indolocarba-
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(15) Kaneko, T.; Wong, H.; Okamato, K. T.; Clardy, J . Two synthetic
approaches to rebeccamycin. Tetrahedron Lett. 1985, 26, 4015-
4018.
(16) Anizon, F.; Belin, L.; Moreau, P.; Sancelme, M.; Voldoire, A.;
Prudhomme, M.; Ollier, M.; Seve`re, D.; Riou, J .-F.; Bailly, C.;
Fabbro, D.; Meyer, T. Syntheses and biological activities (topo-
isomerase inhibition and antitumor and antimicrobial proper-
ties) of rebeccamycin analogues bearing modified sugar moieties
and substituted on the imide nitrogen with a methyl group. J .
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solubilized by 100 µL of DMSO. Results are expressed as IC50
,
the concentration which reduced by 50% the optical density
of treated cells with respect to untreated controls.
Cell Cycle An a lysis. For the cell cycle analysis, L1210 cells
(2.5 × 105 cells/mL) were incubated for 21 h with various
concentrations of the compounds and then fixed by 70%
ethanol (v/v), washed, and incubated in PBS containing 100
µg/mL RNAse and 25 µg/mL propidium iodide for 30 min at
20 °C. For each sample, 104 cells were analyzed on a XL/MCL
flow cytometer (Beckman Coulter). The fluorescence of pro-
pidium iodide was collected through a 615 nm long-pass filter.
Ack n ow led gm en t. This work was supported by
grants (to C.B.) from the Ligue Nationale Contre le
Cancer (Equipe labellise´e LA LIGUE) and (to M.P.) by
a SERVIER research grant.
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