Syntheses, biochemical and biological evaluation of staurosporine analogues from the microbial metabolite rebeccamycin

Article abstract of DOI:10.1016/S0968-0896(98)00096-0

The indolocarbazole antibiotics staurosporine and rebeccamycin (1) are potent antitumor drugs targeting protein kinase C and topoisomerase I, respectively. To obtain staurosporine analogues from rebeccamycin, different structural modifications were perfor

Full text of DOI:10.1016/S0968-0896(98)00096-0

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 1597±1604
Syntheses, Biochemical and Biological Evaluation of
Staurosporine Analogues from the Microbial Metabolite
Rebeccamycin
Fabrice Anizon,a Pascale Moreau,a Martine Sancelme,a Aline Voldoire,a
Michelle Prudhomme,a,* Monique Ollier,b Daniele Severe,c
Jean-Francois Riou,c Christian Bailly,d Doriano Fabbro,e Thomas Meyere
,
and A. M. Aubertinf
a
 Á Á
Universite Blaise Pascal, Synthese, Electrosynthese et Etude de Systemes a Interet Biologique, UMR 6504, 63177 Aubiere, France
Á
Á
Á
 Ã
bINSERM U71, Rue Montalembert, 63005 Clermont-Ferrand, France
cRhoÃne-Poulenc Rorer, 13, Quai Jules Guesde, 93403 Vitry sur Seine, France
dCentre Oscar Lambret et INSERM U124, Place de Verdun, 59045 Lille, France
eNovartis, Department of Oncology, K-125-409, CH-4002, Basle, Switzerland
f
Â
INSERM U74, Universite Louis Pasteur, Strasbourg, France
Received 5 February 1998; accepted 8 April 1998
AbstractÐThe indolocarbazole antibiotics staurosporine and rebeccamycin (1) are potent antitumor drugs targeting
protein kinase C and topoisomerase I, respectively. To obtain staurosporine analogues from rebeccamycin, di€erent
structural modi®cations were performed: coupling of the sugar moiety to the second indole nitrogen, dechlorination
and then reduction of the imide function to amide. The newly synthesized compounds (3±6) were tested for their abil-
ities to bind to DNA and to inhibit topoisomerase I and protein kinase C. Their antiproliferative e€ects in vitro against
B16 melanoma and P388 leukemia (including the related P388CPT cell line resistant to camptothecin) as well as their
anti-HIV-1 and antimicrobial activities against various strains of microorganisms were determined. The cytotoxicity of
the dechlorinated imide analogue 5 correlates well with its DNA binding and anti-topoisomerase I activities. These
®ndings provide guidance for the development of new topoisomerase I-targeted antitumor indolocarbazoles equipped
with a carbohydrate attached to the two indole nitrogens. # 1998 Elsevier Science Ltd. All rights reserved.
Introduction
rebeccamycin does not inhibit this enzyme.6,7 The two
main di€erences between the structures of rebeccamycin
and staurosporine are the functionality of the upper
heterocycle (imide function in rebeccamycin, amide
function in staurosporine) and, most importantly, the
sugar moiety which is attached to both indole nitrogens
in staurosporine but to only one indole nitrogen in
rebeccamycin, ED-110 and NB-506 (Fig. 1). These
factors could explain the discrepancy in the enzyme
inhibition.
Rebeccamycin is an antitumor antibiotic isolated from
cultures of Saccharotrix aerocolonigenes.1,2 Its structure
contains an indolocarbazole framework onto which a
sugar unit is attached via a b-N-glycosyl bond with one
of the indole nitrogens. Its antitumor activity, as well
as those of related compounds ED-110, NB-506, and
derivatives of the antibiotic BE-13793C, is attributed
to topoisomerase
I
inhibition.3±5 Unlike the well
known protein kinase C (PKC) inhibitor staurosporine,
In the course of structure±activity relationship studies
on rebeccamycin analogues, we investigated the possi-
bilities to obtain, from rebeccamycin, compounds for
which the sugar moiety would be linked to both
indole nitrogens. In this paper, we report the synthesis,
Key words: Topoisomerase I; protein kinase C; indolocarb-
azoles; rebeccamycin; staurosporine.
*Corresponding author. Tel.: 33 4 73 40 71 24; Fax: 33 4 73 40
77 17.
0968-0896/98/$Ðsee front matter # 1998 Elsevier Science Ltd. All rights reserved
PII: S0968-0896(98)00096-0

1598
F. Anizon et al./Bioorg. Med. Chem. 6 (1998) 1597±1604
Figure 1.
biochemical (DNA binding and PKC and topoisome-
rase I inhibition) and biological (antimicrobial, anti-
tumor and anti-HIV-1 activities) evaluation of
compounds 3±6 prepared by semi-synthesis from
rebeccamycin (Fig. 2).
Results and Discussion
Chemistry
Compound 2 was prepared from rebeccamycin by O-
tosylation at the 20 position using p-toluenesulfonyl-
chloride and potassium carbonate as a base. The 20
position of the ester was assigned from 1H-1H COSY
and exchange with D2O. Reaction of tosylate 2 with
sodium azide led to a mixture of azide 3 and compound
4. Azide 3 is probably formed via 20,30-epoxide resulting
from the elimination of p-toluenesulfonic acid before the
nucleophilic attack by the azide anion. A nucleophilic
attack by the indole nitrogen leads to 4. Compound 4
could be formed either by a direct nucleophilic sub-
stitution on tosylate 2 leading to inversion of the con-
®guration at C20 or by nucleophilic attack at the 20
position of the transient epoxide leading to retention of
con®guration at C20. In compound 4, C2 and C3 , at
0
0
56.6 and 68.7 ppm, respectively, are shielded compared
0
0
to C2 and C3 of rebeccamycin (72.0 and 77.5 ppm); H3
0
0
is coupled with OH3 . The weak coupling constant
(6 Hz) between protons at the 10 and 20 positions
observed in cyclized compound 4 is in the favor of the
nucleophilic attack of the epoxide. However the weak
coupling constants between protons at the 20 and 30
positions in compounds 4 and 5 (3.2 and about 0 Hz,
respectively) are in the favor of an axial-equatorial cou-
pling resulting from a direct nucleophilic substitution.
Molecular modeling experiments (Program MOPAC
version 6.0, QCPE no 455) were carried out on com-
pounds 4 and 5 with both con®gurations R and S at the
C20. The coupling constants J1 ,2 and J2 ,3 were 2.0 and
3.2 Hz for 4S, 9.0 and 10.2 Hz for 4R, 2.4 and 3.6 Hz for
5S, 8.7.and 10.8 for 5R, respectively. These values ®t
0
0
0
0
Figure 2.

F. Anizon et al./Bioorg. Med. Chem. 6 (1998) 1597±1604
1599
0
0
better with a S con®guration except for J1 ,2 . The cor-
rect assignment of the structures could be obtained from
cristallographic data (under investigations). Compound
4 was the major product (53% yield) whereas azide 3
was obtained in 36% yield. The position of the azide
group in compound 3 and the stereochemistry on the
sugar moiety were determined from 1H-1H COSY,
exchange with D2O and 13C-1H correlations. The posi-
tion of N3 group was assigned from the chemical shift of
addition of puri®ed DNA has practically no e€ect on
the absorption band of compounds 3 or 4 centered
326 nm. Similarly, the main absorption band of com-
pound 6 at 288 nm remains unmodi®ed in the presence
of DNA, even in the presence of a large excess of DNA.
In sharp contrast, compound 5 has signi®cant interac-
tion with DNA as judged from the 5 nm bathochromic
shift and the 39% hypochromism observed upon addi-
tion of DNA. Rebeccamycin can also interact with
DNA but its absorption spectrum shows no red shift
and the hypochromism is weak (8%). We have shown
previously that the presence of bulky chlorine atoms on
the indolocarbazole chromophore (as with rebeccamycin)
markedly hinders the capacity of the drug to intercalate
into DNA and to recognise speci®c sequences.11,12 The
¯uorescence data in Figure 4 provide complementary
information. The ¯uorescence spectrum of compound 3
or 4 shows very little variation in the presence of DNA.
Only a slight decrease of the ¯uorescence peak at
550 nm is detected. Conversely, under identical condi-
tions the spectrum of compound 5 is drastically chan-
ged. Addition of DNA causes a signi®cant increase in
the ¯uorescence peak at 540 nm. The absorption and
¯uorescence spectral changes with compound 5 re¯ect
the perturbation of the complexed indolocarbazole
chromophore system upon binding to DNA. This
dechlorinated imide analogue 5 is apparently the only
drug in the series capable of detectable complex forma-
tion with DNA.
0
C3 (59.9 ppm) compared to the shielded C3 in rebecca-
0
0
mycin (77.5 ppm) whereas the chemical shift of C2
(69.3 ppm) remains almost unchanged (72.0 in rebecca-
0
mycin). The coupling constants H2 -H3 (4.0 Hz) and
0
0
0
H3 -H4 (3.5 Hz) are consistent with axial-equatorial and
equatorial-equatorial coupling. Dechlorination of 4 was
achieved using ammonium formate and palladium on
activated carbon in methanol. Reduction of 5 was per-
formed with zinc-amalgam in ethanol/HCl,8,9 a mixture
of regioisomers 6±60 which could not be separated by
chromatography was obtained. The isomeric ratio cal-
culated from 1H NMR spectrum was 1.2:1.
Protein kinase C inhibition
The inhibitory properties toward PKC-a were tested
using protamine sulfate as a substrate. The IC50 values
are reported in Table 1. Unexpectedly, all the com-
pounds in this series are inactive toward PKC. A very
weak e€ect was noted with compound 4 at a high con-
centration but the e€ect is extremely weak compared to
what can be achieved with staurosporine (IC50 PKC-
a:28 nM).10 This suggests that the rigid tetra-
hydropyrazine ring of compounds 4±6 in which are
located both indole nitrogens is not consistent with
PKC inhibitory e€ect. In staurosporine, this heterocycle
contains seven atoms, instead of six, including an
oxygen atom and therefore is more ¯exible.
Topoisomerase I inhibition
The topoisomerase I inhibitory properties were tested
on puri®ed calf thymus topoisomerase I using the 32P-
labelled EcoRI-HindIII restriction fragment of pBR322
as a substrate. The labelled DNA fragment was incu-
bated with topoisomerase I in the presence and absence
of the drugs at concentrations ranging from 0.01 mg/mL
to 10 mg/mL and the resulting cleavage products were
analysed by agarose gel electrophoresis. The relative
ecacy of the drugs to stimulate DNA cleavage varies
considerably from one congener to one another. For
each drug, we determined the MIC which corresponds
DNA binding
The capacity of the test compounds to interact with
DNA was investigated by means of absorption and
¯uorescence spectroscopy. As shown in Figure 3,
Table 1. Inhibitory activities toward PKC, topoisomerase I, in vitro antiproliferative activities against murine B16 melanoma and
P388 leukemia cells, antimicrobial activities against B. cereus, and anti HIV-1 activities in HIV-1 Lai infected CEM-SS cells (Selectivity
index SI=CC50/IC50
)
Compd
PKC
IC50 mM
B16
IC50 mM
P388
IC50 mM
B. cereus
MIC mM
Topoisomerase I
MIC mM
HIV-1 Lai
CEM-SS IC50-CC50 (mM)-SI
1
>175
nd
118
0.48
nd
2.9
2
1.22
3.95
5.44
0.70
6.82
10.9
>84
>90
1.75
17
0.52±1.05±2.0
nd
3
4
18.1
2.07
21.3
0.58±4.2±7.2
3.7±6.6±1.8
5.7±6.3-1.1
5
6±60
>207
>213
>93
>106
8.9

1600
F. Anizon et al./Bioorg. Med. Chem. 6 (1998) 1597±1604
to the minimum drug concentration at which topoisome-
rase I-mediated DNA cleavage was detected (Table 1). As
expected from previously results,7,13 dechlorinated imide
5 exhibited the strongest topoisomerase I inhibitory
e€ect. This compound is 10 times more active than the
corresponding reduced analogues 6±60. There is no
doubt that the carbonyl group that distinguishes these
compounds must be important for both DNA binding
and topoisomerase I inhibition.
In vitro antiproliferative activity
The antiproliferative activities were tested in vitro
against two murine cell lines, B16 melanoma and P388
leukemia. IC50 values are reported in Table 1. The most
ecient compound was dechlorinated imide 5. In order
to have an insight into the involvement of topoisome-
rase I inhibition in the cytotoxicity, the e€ects of com-
pounds 1±6 on the growth of P388 CPT cells resistant to
the topoisomerase I inhibitor camptothecin were eval-
uated (Table 2). From the resistance index measured
Figure 3. Absorption spectra of compounds 4, 5, and 6±60 in
the absence and presence of calf thymus DNA. To 3 mL of
drug solution (10 mM in 10 mM Tris, 10 mM NaCl bu€er, pH
7.1) were added successive amounts of DNA. The DNA/drug
ratio increased as follows (bottom to top curves at 260 nm): 0,
1, 2, 3, and 4. With compound 5, at higher P/D (phosphate±
DNA/drug) ratios no further qualitative changes occurred and
the hypochromism at 320 nm plateaued at about 40%.
Figure 4. Fluorescence emission spectra of compounds 4 and 5
in the absence and presence of increasing amounts of calf thy-
mus DNA. To 3 mL of a drug solution (10 mM drug, 80 mM
CT-DNA, in 10 mM Tris, 10 mM NaCl bu€er, pH 7), succes-
sive amounts of DNA were added from a stock solution.
Spectra were recorded after 10 min of equilibration with an
excitation wavelength of 320 nm. The DNA/drug ratio
increased from 1 to 4 (the ®rst curve corresponds to free DNA).

F. Anizon et al./Bioorg. Med. Chem. 6 (1998) 1597±1604
1601
Table 2. Toxicities of compounds 1±6±60 towards P388 cells
and P388 CPT cells, resistant to the topoisomerase I inhibitor
camptothecin. Resistance index, R=IC50 P388 CPT/IC50 P388
Table 3. Antimicrobial activities of compounds 1±6±60 against
two Gram-positive bacteria, B. cereus and S. chartreusis, a
Gram-negative bacterium E. coli and a yeast C. albicans
Compd
IC50 P388 mM
IC50 P388 CPT mM
R
Compd
B. cereus
ATCC
14579
S. chartreusis E. coli C. albicans
NRRL
11407
ATCC
11303
IP 444
1
1.22
3.95
5.44
0.70
6.82
10.5
17
8.5
4.2
3
4
5.08
>20
>21
0.93
1
++
Ð
++
Ð
Ð
Ð
Ð
‹
Ð
Ð
Ð
Ð
‹
Ð
5
6±60
>29
>3.1
3
4
Ð
‹
++
‹
5
6±60
Ð
Ð
with compound 4, it seems obvious that topoisomerase I
plays little or no role in the cytotoxicity of this com-
pound. Its cytotoxic e€ect may be attributed to its
action at a protein kinase level because it is the only
drug in the series exhibiting a weak inhibitory e€ect
toward PKC. The high resistance index determined
for compound 5 suggests a major contribution of
topoisomerase I inhibition to the cytotoxicity of this
compound.
The size of zones of growth inhibition was 8±9 mm (++), 7±8mm
(+),6±7 mm (‹).
which produces detectable complexes with DNA and
stabilizes DNA-topoisomerase I cleavable complexes
and, as a result, exhibits marked cytotoxic activities. As
a corollary, the other compounds which fail to form
complexes with DNA, do not inhibit topoisomerase I,
or very weakly, and are not cytotoxic. It is therefore
tempting to conclude that DNA binding is a pre-
Anti-HIV-1 activity
requisite for topoisomerase
I inhibition and anti-
Since topoisomerase I is known to activate HIV-1
reverse transcriptase activity,14,15 the anti-HIV-1 activ-
ity of compounds 2±6 was tested. For that purpose, we
quantitated the reverse transcriptase activity associated
with virus particles released from HIV-1 infected CEM-
SS cells in the culture medium. The cytotoxicity of the
drugs (CC50) was evaluated in parallel to the IC50 values
and the selectivity index (CC50/IC50) was calculated
(Table 1). The best selectivity index was obtained for the
chlorinated imide 4. Compound 5 presents no particular
e€ect and therefore there seems to be no straightforward
correlation between topoisomerase I inhibition and anti-
HIV-1 activity in this series.
proliferative activity. In contrast, no obvious correlation
can be established between topoisomerase I poisoning
and the anti-HIV-1 or antimicrobial e€ects.
Compound 5 represents a useful starting point for the
future design of potent antitumor indolocarbazoles act-
ing speci®cally on topoisomerase I without unwanted
e€ects on PKC. We are now introducing polar groups
to increase its solubility.
Experimental
Chemistry
Antimicrobial properties
IR spectra were recorded on a Perkin-Elmer 881 spec-
trometer (n in cm 1). NMR spectra were performed on
a Bruker AC 400 (1H: 400 MHz, 13C: 100 MHz) (che-
mical shifts d in ppm, the following abbreviations are
used: singlet (s), doublet (d), doubled doublet (dd), tri-
plet (t), multiplet (m), tertiary carbons (C tert.), qua-
ternary carbons (C quat.)). The signals were assigned
The antimicrobial activities against two Gram-positive
bacteria (Bacillus cereus and Streptomyces chartreusis), a
Gram-negative bacterium (Escherichia coli) and a yeast
(Candida albicans) were tested (Table 3). All the com-
pounds were inactive against C. albicans. Compound 5
was weakly active against the two Gram-positive strains
tested and weakly active against E. coli. The activity of
chlorinated imide 4 against S. chartreusis was similar to
that observed with rebeccamycin.
1
from H-1H COSY, 13C-1H correlations, exchange with
D2O and inverse gate decoupling. Mass spectra (FAB+)
were determined at CESAMO (Talence, France) on a
high resolution Fisons Autospec-Q spectrometer. Chro-
matographic puri®cations were performed by ¯ash sili-
cagel Geduran SI 60 (Merck) 0.040±0.063 mm or
Kieselgel 60 (Merck) 0.063±0.200 mm column chroma-
tography. For purity tests, TLC were performed on
¯uorescent silica gel plates (60 F254 from Merck).
Rebeccamycin was from our laboratory stock sample.
Conclusion
A direct correlation can be established between DNA
binding, topoisomerase I inhibition and the cytotoxic
e€ects. Indeed, compound 5 is the only drug in the series

1602
F. Anizon et al./Bioorg. Med. Chem. 6 (1998) 1597±1604
1,11-Dichloro-12(4-O-methyl-ꢀ-D-(20-O-tosyl)-glucopyr-
anosyl)-6,7,12,13-tetrahydro(5H)-indolo[2,3-a]-pyrrolo-
[3,4-c]-carbazole-5,7-dione (2). Potassium carbonate
0
H3 , J=3.5 Hz), 7.11 (1H, s, H1 ), 7.41 (1H, t,
0
J=7.9 Hz), 7.48 (1H, t, J=7.9 Hz), 7.50 (1H, broad s,
OH), 7.72 (1H, d, J=7.4 Hz), 7.74 (1H, dd, J1=7.9 Hz,
J2=1.0 Hz), 9.08 (1H, d, J=7.9 Hz), 9.40 (1H, dd,
J1=7.9 Hz, J2=1.0 Hz), 11.30 (1H, s, Nimide-H), 12.00
(1H, s, Nindole-H); 13C NMR (100 MHz, DMSO-d6) d
(235 mg,
1
equiv) and p-toluenesulfonylchloride
(323 mg, 1.7 mmol) were added to a solution of rebec-
camycin (1 g, 1.7 mmol) in THF (200 mL). The mixture
was re¯uxed for 48 h. After removal of the solvent, the
residue was puri®ed by chromatography (eluent, cyclo-
hexane:EtOAc, 70:30) to give 2 (579 mg, 0.80 mmol,
47% yield) as a yellow powder. Melting point 168±
170 ꢀC; IR(KBr): nC=O 1720, 1770 cm 1, nNH,OH 3200±
3600 cm 1; HRMS (FAB+) M+ calcd for C34H27
N3O3SCl2 723.0845 found 723.0787; 1H NMR
(400 MHz, acetone-d6) d 2.20 (3H, s), 3.78 (3H, s,
0
0
0
0
57.3 (OCH3), 59.9 (C3 ); 60.5 (C6 ); 69.3 (C2 ); 73.6 (C4 );
0
0
78.1 (C5 ); 84.0 (C1 ); 115.5, 115.8, 117.0, 118.4, 119.4,
122.2, 122.5, 125.4, 130.5, 130.6, 136.4, 137.0 (C quat.
arom.); 121.0, 122.7, 123.5, 124.0, 126.3, 129.7 (C tert.
arom.); 170.4, 170.7 (C=O).
Compound 4: mp 296±298 ꢀC; IR(KBr): nC=O 1700,
1760 cm 1, nNH,OH 3200±3600 cm 1; HRMS (FAB+)
M+: calcd for C27H19Cl2N3O6 551.0650 found
551.0647; 1H NMR (400 MHz, DMSO-d6) d 3.46 (3H, s,
0
0
0
OCH3), 4.08 (1H, H4 , t, J=9.0 Hz), 4.17 (2H, H3 , H5 ,
0
m), 4.27 (2H, H6 , m), 4.86 (1H, t, J=5.4 Hz, OH), 5.10
0
(1H, H2 , dd, J1=9.3 Hz, J2=8.4 Hz), 5.12 (1H, d,
0 0 0
OCH3), 3.47 (3H, m, 2H6 +H4 ), 3.75 (1H, m, H5 ), 4.53
0
(1H, d, J=7.1 Hz, OH3 ), 4.61 (1H, m, H3 ), 4.92 (1H, t,
0
J=4.5 Hz, OH6 ), 5.48 (1H, dd, J1=6.3 Hz, J2=3.2 Hz,
0
J=5.9 Hz, OH), 6.58 (2H, d, J=8.5 Hz), 6.68 (2H, d,
J=7.9 Hz), 7.17 (1H, t, J=7.9 Hz), 7.28 (1H, t,
J=7.9 Hz), 7.37 (1H, dd, J1=7.9 Hz, J2=1.0 Hz), 7.56
0
0
H2 ), 6.39 (1H, d, J=6.3 Hz, H1 ), 7.29 (1H, t, J
=8.0 Hz), 7.32 (1H, t, J=8.0 Hz), 7.52 (1H, d, J
=7.9 Hz), 7.57 (1H, d, J=7.9 Hz), 8.45 (1H, d, J
0
(1H, d, H1 , J=9.3 Hz), 7.58 (1H, dd, J1=7.9 Hz,
J2=1.0 Hz), 8.86 (1H, d, J=7.9 Hz), 8.89 (1H, dd,
J1=7.8 Hz, J2=1.4 Hz), 10.06 (1H, s, NH), 10.40 (1H, s,
NH); 13C NMR (100 MHz, acetone-d6) d 20.5 (CH3 of
the tosyl group); 60.2 (OCH3); 60.3 (CH2); 75.5, 79.4,
80.4, 81.1, 81.4 (CH); 115.2, 116.6, 118.4, 119.2, 120.1;
122.2, 123.6, 124.9, 128.2, 129.1, 132.8, 135.4, 137.7,
144.1 (C quat. arom.); 121.7, 121.8, 123.5, 124.6, 125.6
(2C), 126.9, 128.9 (2C), 129.8 (C tert. arom.); 170.0,
170.1 (C=O).
=7.9 Hz), 8.51 (1H, d, J=7.9 Hz), 10.98 (1H, s, Nindole-H);
13
0
0
C NMR (100 MHz, DMSO-d6) d 62.1 (C6 ); 56.6 (C2 ),
0
0
0
0
57.1 (OCH3), 68.7 (C3 ), 77.4 (C5 ), 78.3 (C4 ), 80.7 (C1 );
111.0, 111.1, 116.5, 117.5, 120.1, 120.9, 126.1, 126.5,
128.0, 129.7, 136.2, 136.8 (C quat. arom.); 122.2, 122.6,
122.9, 123.5, 127.7, 127.9 (C tert. arom.); 170.3 (C=O).
12,13-[1,2-(4-O-Methyl-D-mannopyranosyl)]-6,7,12,13-
tetrahydro(5H)-indolo[2,3-a]-pyrrolo[3,4-c]-carbazole-5,7-
dione (5). A mixture of compound 4 (472 mg,
1,11-Dichloro-12(4-O-methyl-ꢀ-D-(30-azido)-altropyrano-
syl)-6,7,12,13-tetrahydro(5H)-indolo[2,3-a]-pyrrolo[3,4-c]-
carbazole-5,7-dione (3) and 1,11-dichloro-12,13-[1,2-(4-
O-methyl-D-mannopyranosyl)]-6,7,12,13-tetrahydro(5H)-
indolo[2,3-a]-pyrrolo[3,4-c]-carbazole-5,7-dione (4). To a
solution of compound 2 (449 mg, 0.620 mmol) in DMF
(16 mL) was added sodium azide (403 mg, 6.2 mmol, 10
equiv). The mixture was stirred at 70 ꢀC for 6 days, then
cooled, poured into water and extracted with EtOAc.
The organic phase was washed with saturated aqueous
NaHCO3 and brine, then dried over MgSO4. The sol-
vent was removed and the residue puri®ed by chroma-
tography (eluent, EtOAc:CH2Cl2, 10:90) a€ording 3
(69.4 mg, 0.117 mmol, 19% yield) as a yellow±orange
solid and 4 (226.1 mg, 0.401 mmol, 65% yield) as a yel-
low solid.
0.855 mmol), palladium 5% on activated carbon
(570 mg) and ammonium formate (570 mg) in methanol
(200 mL) was stirred at room temperature for 48 h. The
mixture was ®ltered o€ over celite and the solid washed
with THF. The solvent was removed and the residue
puri®ed by ¯ash chromatography (eluent, cyclohex-
ane:EtOAc, 40:60) to give 5 (342 mg, 0.708 mmol, 83%
yield). Melting point 284±286 ꢀC; IR(KBr): nC=O 1710,
1750 cm 1, nNH,OH 3200±3600 cm 1; HRMS (FAB+)
(M+H)+: calcd for C27H22N3O6 484.1508, found
1
484.1516. H NMR (400 MHz, DMSO-d6) d 3.40±3.56
0
(3H, m, 2H6 , H4 ), 3.63 (3H, s, OCH3), 3.83 (1H, m,
0
0
H5 ), 4.60 (2H, m, H3 , OH6 ), 5.23 (1H, s, H2 ), 6.76
0
0
0
0
0
(1H, d, J=5.0 Hz, OH3 ), 6.85 (1H, s, H1 ), 7.46 (1H, t,
J=7.4 Hz), 7.49 (1H, t, J=7.4 Hz), 7.64 (1H, t,
J=8.3 Hz), 7.67 (1H, t, J=7.4 Hz), 7.98 (1H, d,
J=8.3 Hz), 8.71 (1H, d, J=7.9 Hz), 8.84 (1H, d,
J=7.9 Hz), 8.91 (1H, d, J=8.3 Hz), 11.10 (1H, s, NH);
13C NMR (100 MHz, DMSO-d6) d 59.9 (OCH3); 60.0
Compound 3: mp >300 ꢀC; IR(KBr): nC=O 1710,
1
1750 cm 1, nN=N 2110 cm 1, nNH,OH 3200±3600 cm
;
HRMS (FAB+) (M+H)+ calcd for C27H21N6O6Cl2
595.0899 found 595.0937; H NMR (400 MHz, DMSO-
d6) d 3.58 (1H, m), 3.59 (3H, s, OCH3), 3.68 (1H, dd,
1
0
0
0
0
0
0
(C6 ); 63.2, 71.8, 76.1, 78.7, 80.2 (C1 , C2 , C3 , C4 , C5 );
111.8, 115.2, 120.8, 121.7, 124.4, 124.5, 126.8, 126.9 (C
tert. arom.); 112.3, 112.7, 120.5, 120.9, 123.6, 123.8,
130.2, 131.0, 140.6, 142.5 (C quat. arom.) 171.0, 171.2
(C=O).
0
J1=10.8 Hz, J2=4.0 Hz), 4.07 (1H, m, H5 ), 4.19 (1H,
0
0
dd, H4 , J1=9.9 Hz, J2=3.0 Hz), 4.64 (1H, t, OH6 ,
0
J=5.0 Hz), 4.77 (1H, d, H2 , J=4.0 Hz), 4.94 (1H, pt,

F. Anizon et al./Bioorg. Med. Chem. 6 (1998) 1597±1604
1603
12,13-[1,2-(4-O-Methyl-D-mannopyranosyl)]-6,7,12,13-
to the lowest concentration (mg/mL) producing
detectable DNA cleavage.
a
tetrahydro-5-oxo(5H)-indolo[2,3-a]-pyrrolo[3,4-c]-carb-
azole (6) and 12,13-[1,2-(4-O-methyl-D-mannopyrano-
syl)]-6,7,12,13-tetrahydro-7-oxo(5H)-indolo[2,3-a]-pyrrolo-
[3,4-c]-carbazole (60). Zinc-amalgam (1 g) was added to 5
(100 mg, 0.207 mmol) in a solution of ethanol (14 mL)
and 6 N HCl (2.3 mL). The mixture was re¯uxed for 4 h
then ®ltered o€ and the solid washed with EtOAc. After
identical work up as for 4, puri®cation by ¯ash chro-
matography (eluent, EtOAc) gave the isomeric mixture
of 6 and 60 (62 mg, 0.132 mmol, 64% yield) as an o€-
Growth inhibition assay. P388 murine leukemia cells:
P388 murine leukemia cells were incubated at 37 ꢀC for
96 h in the presence of various concentrations of drug
and evaluated for viability by neutral red staining as
previously described.13 The concentrations of drugs
giving 50% of growth inhibition (IC50) were determined.
B16 cells cytotoxic assay: The antiproliferative activity
was expressed as IC50 and determined as previously
described.13
white powder. IR(KBr): nC=O 1670 cm 1, nNH,OH 3200±
1
3600 cm
HRMS (FAB+) (M+H)+: calcd for
C27H24N3O5 470.1716, found 470.1730. 1H NMR
(400 MHz, DMSO-d6) d 3.44±3.56 (6H, m), 3.62 (3H, s,
OCH3), 3.63 (3H, s, OCH3), 3.79 (2H, m), 4.53 (1H, t,
Protein kinase C inhibition. Protamine sulphate was
from Merck (Darmstadt, Germany). Unless speci®ed,
chemicals were from Sigma (St. Louis, MO). [g-33P]
ATP (1000±3000 Ci/mmol) was obtained from Amer-
sham. Recombinant baculoviruses from protein kinase
C subtypes were supplied by Dr. Silvia Stabel, Koln,
Germany. Expression and partial puri®cation of PKCs
together with measurements of activities were carried
out as previously described.13 Data show IC50 values
expressed in mM.
0
0
J=6.3 Hz, OH6 ), 4.57 (1H, t, J=6.3 Hz, OH6 ), 4.63
(2H, m), 4.98 (4H, m, 2 CH2), 5.12 (1H, m), 5.17 (1H,
m), 6.76 (1H, d, J=4.8 Hz, OH), 6.82 (3H, m, 1
0
OH+2H1 ), 7.34 (1H, t, J=7.1 Hz), 7.39 (1H, t,
J=7.1 Hz), 7.41 (1H, t, J=7.1 Hz), 7.43 (1H, t,
J=7.1 Hz), 7.51 (1H, t, J=7.9 Hz), 7.53 (1H, t,
J=8.0 Hz), 7.55 (1H, t, J=7.1 Hz), 7.58 (1H, t,
J=7.2 Hz), 7.90 (1H, d, J=8.7 Hz), 7.94 (1H, d,
J=8.7 Hz), 8.14 (2H, d, J=7.9 Hz), 8.58 (2H, s, 2NH),
8.86 (1H, d, J=8.0 Hz), 8.90 (1H, d, J=8.0 Hz), 8.96
(1H, d, J=8.7 Hz), 9.04 (1H, d, J=8.0 Hz); 13C NMR
Antibiogram tests and MIC determination. Four strains
were tested, two Gram-positive bacteria (B. cereus
ATCC 14579, S. chartreusis NRRL 11407), a Gram-
negative bacterium (E. coli ATCC 11303) and a yeast
(C. albicans 444 from Pasteur Institute). Antimicrobial
activity was determined by the conventional paper disk
(Durieux No 268; 6 mm in diameter) di€usion method
using the following nutrient media: Mueller±Hinton
(Difco) for B. cereus and E. coli, Sabouraud agar
(Difco) for C. albicans and Emerson agar (0.4% beef
extract, 0.4% peptone, 1% dextrose, 0.25% NaCl, 2%
agar, pH 7.0 ) for the Streptomyces strains. Paper disks
impregnated with solutions of 1±6 in DMSO (300 mg of
drug per disk) were placed on Petri dishes. Growth
inhibition was examined after 24 h incubation at 27 ꢀC.
0
(100 MHz, DMSO-d6) d 45.5, 45.6 (CH2); 60.0 (C6 );
60.1 (OCH3); 63.5, 63.8, 72.5, 76.1, 76.2, 78.8, 78.9, 80.4
0
0
0
0
0
(C1 , C2 , C3 , C4 , C5 ); 111.3, 111.4, 114.9, 115.4, 119.7,
120.4, 120.7, 121.3, 121.7, 122.1, 125.1, 125.2 (C tert.
arom.); 111.5, 111.6, 112.6, 112.9, 119.7, 120.1, 124.6,
124.8, 125.0, 125.3, 127.1, 127.6, 129.4, 130.0, 133.4,
133.7, 139.7, 141.4, 141.5 (C quat. arom.), 172.0, 172.2
(C=O).
Absorption and ¯uorescence measurements. Absorption
spectra were recorded on a Uvikon 943 spectro-
photometer. The ¯uorescence emission spectra were
recorded on a Perkin‹Elmer LS50B ¯uorometer and
are uncorrected. In both cases, the cell holder (10 mm
pathlength) was thermostated with a Neslab RTE 111
cryostat. The DNA from calf thymus (highly poly-
merized sodium salt from Sigma Chemical Co.) was
deproteinized twice with sodium dodecyl sulphate.
DNA concentrations were determined applying a molar
extinction coecient of 6600 M 1 cm 1. Titrations of
the drugs with DNA were performed by adding aliquots
of a concentrated DNA solution to a dilute drug solu-
tion (10 mM).
MIC of 1±6 were determined classically on B. cereus
ATCC 14579 in Mueller±Hilton broth, pH 7.4 (Difco),
after 24 h incubation at 27 ꢀC. The compounds diluted
in DMSO were added to 12 tubes; the concentration
range was from 100 mg/mL to 0.05 mg/mL.
Antiviral HIV-1 activity. The cultures of CEM-SS cells
were maintained at 37 ꢀC in 5% CO2 atmosphere in
RPMI 1 640 medium supplemented with 10% decom-
plemented fetal bovine serum (FBS). The antiviral HIV-
1 activity of a given compound in CEM-SS cells was
measured by quanti®cation of the reverse transcriptase
activity (RT) associated with virus particles released
from HIV-1 Lai infected cells in the culture medium.
CEM-SS cells were infected with 100 TCID50 (the
virus stock was titrated under the same experimental
Topoisomerase I inhibition. Topoisomerase I was pre-
pared from calf thymus as already described.1617
Topoisomerase I inhibition was evaluated using the
DNA cleavage assay carried out according to the pre-
viously described method.18 The MIC values correspond

1604
F. Anizon et al./Bioorg. Med. Chem. 6 (1998) 1597±1604
conditions); after 30 min adsorption, free virus particles
were washed out and cells were resuspended in RPMI
10% SVF at the ®nal concentration of 105 cells/mL in
the presence of di€erent concentrations of test com-
pounds. After 5 days, virus production was measured by
RT assay as already described.19 The 50% inhibitory
concentration (IC50) was derived from the computer-
generated median e€ect plot of the dose±e€ect data.20
The cytotoxicity of the drugs was evaluated in parallel
by incubating uninfected cells in the presence of di€er-
ent concentrations of antiviral products. The cell viabi-
lity was determined by a measure of mitochondrial
deshydrogenase activity, enzymes reducing 3-(4,5-di-
5. Kojiri, K.; Kondo, H.; Yoshinari, T.; Arakawa, H.;
Nakajima, S.; Satoh, F.; Kawamura, K.; Okura, A.; Suda, H.;
Okanishi, M. J. Antibiotics 1991, 44, 723.
6. Tamaoki, T.; Nomoto, H.; Takahashi, I.; Kato, Y.;
Morimoto, M.; Tomita, F. Biochem. Biophys. Res. Commun.
1986, 135, 397.
7. Rodrigues-Pereira, E.; Belin, L.; Sancelme, M.; Prudhomme,
M.; Ollier, M.; Rapp, M.; Severe, D.; Riou, J. F.; Fabbro, D.;
Meyer, T. J. Med. Chem. 1996, 39, 4471.
8. Toullec, D.; Pianetti, P.; Coste, H.; Bellevergue, P.; Grand-
Perret, T.; Ajakane, M.; Baudet, V.; Boissin, P.; Boursier, E.;
Loriolle, F.; Duhamel, L.; Charon, D.; Kirilovsky, J. J. Biol.
Chem. 1991, 266, 15771.
9. Fabre, S.; Prudhomme, M.; Sancelme, M.; Rapp, M.
Bioorg. Med. Chem. 1994, 2, 73.
methylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
(MTT) into formazan (which quantity was given by the
optical density at 540 nm).21 The 50% cytotoxic con-
centration (CC50) is the concentration of drug which
reduces cell viability by 50% and was calculated with
the program used in the determination of IC50. The
CEM-SS cells were obtained from P. Nara through the
AIDS Research and Reference Reagent Program, Divi-
sion of AIDS, NIAID, NIH.
10. Wilkinson, S. E.; Parker, P. J.; Nixon, J. S. Biochem. J.
1993, 294, 335.
11. Bailly, C.; Riou, J. F.; Colson, P.; Houssier, C.; Rodrigues
Pereira, E.; Prudhomme, M. Biochemistry 1997, 36, 3917.
12. Bailly, C.; Colson, P.; Houssier, C.; Rodrigues Pereira, E.;
Prudhomme, M.; Waring, M. C. Mol. Pharmacol. 1998, 53, 77.
13. Anizon, F.; Belin, L.; Moreau, P.; Sancelme, M.; Voldoire,
A.; Prudhomme, M.; Ollier, M.; Severe, D.; Riou, J. F.; Bailly,
C.; Fabbro, D. ; Meyer, T. J. Med. Chem. 1997, 40, 3456.
14. Takahashi, H.; Matsuda, M.; Kojima, A.; Sata, T.; Andoh,
T.; Kuruta, T.; Nagashima, K.; Hall, W. W. Proc. Natl. Acad.
Sci. U.S.A 1995, 92, 5694.
Acknowledgements
The authors are grateful to Nicole Grangemare for
technical assistance in microbiology and to Jacques
Guyot for molecular modeling experiments.
15. Pommier, Y.; Poddevin, B.; Gupta, M.; Jenkins, J. Bio-
chem. Biophys. Res. Commun. 1994, 205, 1601.
16. Halligan, B. D.; Edwards, K. A.; Liu, L. F. J. Biol. Chem.
1985, 260, 2475.
17. Riou, J. F.; Helissey, P.; Grondard, L.; Giorgi-Renaud, S.
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