Structure-activity relationships in a series of substituted indolocarbazoles: Topoisomerase I and protein kinase C inhibition and antitumoral and antimicrobial properties

Article abstract of DOI:10.1021/jm9603779

A series of compounds structurally related to staurosporine, rebeccamycin, and corresponding aglycones was synthesized, and their activities toward protein kinase C and topoisomerases I and II were tested together with their in vitro antitumor efficiency against murine B16 melanoma and P388 leukemia cells. Their antimicrobial activities were also examined against a Gram-negative bacterium (Escherichia coli), a yeast (Candida albicans), and three Gram-positive bacteria (Bacillus cereus, Streptomyces chartreusis, and Streptomyces griseus). To avoid side effects expected with protein kinase C inhibitors, we introduced substitution on the maleimide nitrogen and/or a sugar moiety linked to one of the indole nitrogens to obtain specific inhibitors of topoisomerase I with minimal activities on protein kinase C. As expected, these structures were inefficient on topoisomerase II, and some of them exhibited a strong activity against topoisomerase I. Generally, dechlorinated compounds were found to be more active than chlorinated analogues against both purified topoisomerase I and protein kinase C. On the other hand, opposite results were obtained in the cell antiproliferative assays. These results suggest lack of cell membrane permeability in the absence of the chlorine residue or cleavage of carbon- chlorine bonds inside the cell.

Full text of DOI:10.1021/jm9603779

J . Med. Chem. 1996, 39, 4471-4477
4471
Str u ctu r e-Activity Rela tion sh ip s in a Ser ies of Su bstitu ted In d oloca r ba zoles:
Top oisom er a se I a n d P r otein Kin a se C In h ibition a n d An titu m or a l a n d
An tim icr obia l P r op er ties
Elisabe`te Rodrigues Pereira,† Laure Belin,† Martine Sancelme,† Michelle Prudhomme,*,† Monique Ollier,‡
Maryse Rapp,‡ Danie`le Seve`re,§ J ean-Franc¸ois Riou,§ Doriano Fabbro,∇ and Thomas Meyer∇
Synthe`se et Etude de Syste`mes a` Inte´reˆt Biologique, Universite´ Blaise Pascal, URA 485 du CNRS, 63177 Aubie`re, France,
Unite´ INSERM U71, Rue Montalembert, 63005 Clermont-Ferrand, France, Rhoˆne-Poulenc Rorer, 13 Quai J ules Guesde, 93403
Vitry sur Seine, France, and De´partement d’Oncologie, CIBA-GEIGY Limited, K-125-409, CH-4002 Baˆle, Switzerland
Received May 24, 1996X
A series of compounds structurally related to staurosporine, rebeccamycin, and corresponding
aglycones was synthesized, and their activities toward protein kinase C and topoisomerases I
and II were tested together with their in vitro antitumor efficiency against murine B16
melanoma and P388 leukemia cells. Their antimicrobial activities were also examined against
a Gram-negative bacterium (Escherichia coli), a yeast (Candida albicans), and three Gram-
positive bacteria (Bacillus cereus, Streptomyces chartreusis, and Streptomyces griseus). To avoid
side effects expected with protein kinase C inhibitors, we introduced substitution on the
maleimide nitrogen and/or a sugar moiety linked to one of the indole nitrogens to obtain specific
inhibitors of topoisomerase I with minimal activities on protein kinase C. As expected, these
structures were inefficient on topoisomerase II, and some of them exhibited a strong activity
against topoisomerase I. Generally, dechlorinated compounds were found to be more active
than chlorinated analogues against both purified topoisomerase I and protein kinase C. On
the other hand, opposite results were obtained in the cell antiproliferative assays. These results
suggest lack of cell membrane permeability in the absence of the chlorine residue or cleavage
of carbon-chlorine bonds inside the cell.
Ch a r t 1
In tr od u ction
Protein kinase C (PKC) plays a key role in cell signal
transduction, controlling a large variety of cell responses
including gene expression and cell proliferation.1 PKC
consists of at least 12 isoenzymes requiring different
cofactors and showing differential tissue distribution
and substrate specificities. Altered expression of PKC
isoenzymes has been reported in a wide range of
neoplastic and preneoplastic tissues. In these tissues,
some of the PKC isoforms concerned are overexpressed
and some others underexpressed.2
Among the known PKC inhibitors possessing an
indolocarbazole moiety and interacting with the ATP
binding site of the enzyme are microbial metabolites
such as staurosporine, K-252a, and UCN-01 together
with the K-252a derivative KT6006 and related
aglycones3-9 (Chart 1). However, these nonselective
PKC inhibitors, compared with other kinases and dif-
ferent PKC isoforms, may be responsible for serious side
effects. In this series, the K-252a derivatives KT6124
and KT65284,10 (Chart 1), substituted on the nitrogen
of the upper heterocycle with a hydroxy or an amino
group, are much less potent PKC inhibitors. They exert
a broad spectrum of antiproliferative activity against
human tumor cell lines in vitro and are also inhibitors
of topoisomerase I.
inhibitor. Its antitumor activity seems rather to be
correlated to its inhibitory potency against topoi-
somerase I.12 ED-110 and NB-506, semisynthetic de-
rivatives of antibiotic BE-13793C, and a rebeccamycin
derivative 2 (Chart 2) were reported to exhibit antitu-
moral properties.13-15 ED-110, NB-506, and 2, like
rebeccamycin, bear a sugar unit attached to one indole
nitrogen. In addition, NB-506 and 2 bear a substituent
on the maleimide nitrogen. The cumulative toxicity of
NB-506 was found to be much lower than those of some
anticancer drugs on the market, and a phase I trial has
shown reduction of tumors resistant to taxol.16
Rebeccamycin (1) (Chart 2), a structurally related
antitumor antibiotic,11 in which the sugar moiety is
linked to only one indole nitrogen, is not a PKC
† Universite´ Blaise Pascal.
‡ Unite´ INSERM U71.
§ Rhoˆne-Poulenc Rorer.
∇ CIBA-GEIGY Ltd.
X Abstract published in Advance ACS Abstracts, October 1, 1996.
S0022-2623(96)00377-9 CCC: $12.00 © 1996 American Chemical Society

4472 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 22
Rodrigues Pereira et al.
Ch a r t 2
13, and 15 were obtained from rebeccamycin by struc-
tural modifications.9,17 11, 14, and 16 were synthesized
from 2,3-dibromo-N-methylmaleimide and indolyl-Mg-
Br.7,8,18 2 and 3 were prepared from rebeccamycin and
6 and 7 from dechlorinated rebeccamycin by reaction
with hydrazine hydrate and hydroxylamine hydrochlo-
ride, respectively. 4 and 8 were prepared from 2 and
6, respectively, by reaction with dimethylformamide and
hydrochloric acid (Scheme 1).
22 was obtained from 13 by reaction with hydrazine
hydrate and 23 from 22 by reaction with dimethylfor-
mamide and hydrochloric acid. 17-21 were prepared
from the corresponding anhydride by reaction with
hydrazine hydrate,19 hydroxylamine hydrochloride,19
formic hydrazide, ethanolamine, and carbohydrazide,
respectively (Scheme 2). For the preparation of the
chlorinated analogue of 18, our classical method by
reaction of hydroxylamine hydrochloride on imide 13
failed even though it was applied successfully to rebec-
camycin (1). To have a more reactive starting material,
we tried, unsuccessfully, to prepare the corresponding
anhydride from 13 in a basic medium followed by ring
closure by acidic treatment.20
Ch a r t 3
Resu lts a n d Discu ssion
The inhibitory activities of compounds 1-23 toward
PKC, topoisomerase I, and topoisomerase II were de-
termined. The inhibition of protein kinase A (PKA) was
examined for some of them to evaluate their selectivity.
The antiproliferative activities against two different
murine cell lines, B16 melanoma cells and P388 leuke-
mia cells, were also examined in vitro. The results are
reported in Table 1.
In h ibitor y P oten cies tow a r d P KC. Maleimide
indolocarbazoles bearing a sugar linked to one of the
indole nitrogens were inactive against PKC except for
compounds 3 and 7 substituted with a hydroxy group.
9 and 10 bearing an amide function in the upper
heterocycle such as staurosporine were found to be PKC
inhibitors. In the aglycone series, substitution on the
upper heterocycle produced an activity in the same
range as or lower than that of the unsubstituted
analogue; in series with the sugar moiety, substitution
with a hydroxy group enhanced activity toward PKC.
Except for 17 and 18, the compounds tested against both
PKC and PKA exhibited a stronger activity against
PKC. Dechlorinated compounds, bearing a sugar moi-
ety or in the aglycone series, were always more active
against PKC than their chlorinated analogues (except
when both were inactive).
From these literature data, it appears that in male-
imide indolocarbazole compounds the presence of a
sugar moiety linked to one of the two indole nitrogens,
with or without a substitution on the maleimide nitro-
gen with a functional group bearing a labile hydrogen,
may induce a weaker or absence of PKC inhibitory effect
and a marked topoisomerase I inhibitory potency. The
lower toxicity of these compounds may be due to their
more specific action against topoisomerase I.
To extend knowledge in this field, we prepared a
range of maleimide and maleamide indolocarbazoles
with and without sugar moieties and with and without
substitution on the nitrogen of the upper heterocycle by
substituents bearing a labile hydrogen (Chart 3).
Top oisom er a se In h ibition . As expected from lit-
erature data,4,10 compounds 1-23 were inactive toward
topoisomerase II. Concerning topoisomerase I inhibi-
tion, the same results were obtained as for PKC inhi-
bition:Dechlorinated compounds had an identical or
greater activity than the dichloro analogues. Compared
with the corresponding aglycones, the presence of the
sugar moiety enhanced the activity toward topoi-
somerase I. Compared with 5, analogues 6-8 substi-
tuted on the maleimide nitrogen were 10 times more
active. Amide 10 was the most active toward topoi-
somerase I but was also a strong PKC inhibitor.
Ch em istr y
Rebeccamycin (1) was isolated from cultures of Sac-
charotrix aerocolonigenes (ATCC 39243).11 5, 9, 10, 12,
In Vitr o An tip r olifer a tive Activity. Compared
with reference compounds 1 and 2, no further improve-

SAR of Substituted Indolocarbazoles
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 22 4473
Sch em e 1
Sch em e 2
Ta ble 1. Inhibitory Activities of Compounds 1-23 toward PKC, PKA, Topoisomerase I, and Topoisomerase II and Antiproliferative
Activities in Vitro against Murine B16 melanoma and P388 leukemia Cells
IC50 (µM)
PKA
MIC (µg/mL)
compd
PKC
B16
0.48
0.3
0.7
nd
17.5
4.1
5.8
nd
>125
nd
4.8
n.d.
24
>100
8.4
nd
P388
topoisomerase I
topoisomerase II
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
> 100a
>100b
68b
> 100
nd
nd
nd
>100
nd
nd
nd
>100
nd
25.7
>100
>100
60
>100
34
0.3
0.1
0.5
0.3
3.5
0.3
3
0.3
3
3
1
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.1
0.3
1
>100b
>100a
>100b
40b
1
0.1
0.1
0.1
1
0.01
10
>10
>10
10
>100
28.8a
3.7a
2.45a
4
>100a
>100a
44.7a
>100a
3.5
>10
4
9
>10
22.1a
0.3
>10
>10
3.5
7
>10
>10
>10
1
43.5a
79.2a
44.6
41.5
nd
nd
nd
>250
4.6
>100
>150
75-15*
17
10
>10
>100b
1
>100b
78b
>10
>10
>10
>10
>100b
>100
nd
nd
nd
a
b
PKC and PKA inhibition measured according to procedure a described in the Experimental Section. PKC inhibition measured
according to procedure b. *21 partially precipitated in DMSO. The IC50 value for 11 determined by procedure b was 0.64 µM.
ment of the cytotoxic properties against B16 melanoma
or P388 leukemia was found for the new derivatives
synthesized. Cytotoxic activity was found to be weaker
for dechlorinated compounds than for the dichloro
analogues in the series bearing the sugar, which cor-
responds to an opposite structure-activity relationship
(SAR) compared to in vitro results against topoi-
somerase I and PKC.

4474 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 22
Rodrigues Pereira et al.
Ta ble 3. MIC Values (µg/mL) Determined on B. cereus
Ta ble 2. In Vivo Efficacy Study on B16 Melanoma and P388
Leukemia Cellsa
compd
B. cereus
compd
B. cereus
compd
B. cereus
dose
mean survival
in days
1
2
3
4
5
6
7
8
6.25
0.8
3.1
9
10
11
12
13
14
15
16
3.1
12.5
>50
6.25
>50
>50
>50
6.25
17
18
19
20
21
22
23
>50
>50
50
(mg/kg/day, 1-5-9)
T/C × 100
1. In Vivo Efficacy Study on B16 Melanoma Cells
control
24.5
30.5
27.5
26
23
30
100
124
112
106
94
122
106
112
128
112
3.1
>50
1,
2
16
64
>50
50
25
25
50
nda
>50
11, 2
16
64
18, 2
a
The insolubility of 22 prevented the determination of its MIC
value.
26
27.5
31.5
27.5
16
64
Gram-positive bacteria tested. 11 was only active on
B. cereus, and 23 was only active on S. griseus. The
other compounds exhibited moderate to strong antibac-
terial activities on the Gram-positive bacteria tested.
MIC values were determined on B. cereus (Table 3).
The activity of 22 could not be determined because of
its insolubility. As observed on melanoma B16 and
leukemia P388 cells, chlorinated compounds were more
active than their dechlorinated analogues. In the
maleimide series, MIC values for the aglycones were
>50 mM, while a strong antimicrobial effect was
observed for the chlorinated analogues of rebeccamycin.
These results raise the question of the role of the
chlorine. It may induce the lipophilicity necessary to
facilitate the membrane crossing. Enzymatic carbon-
chlorine bond cleavage may subsequently occur inside
the cells.
2. In Vivo Efficacy Study on P388 Leukemia Cells
control
11
15
16.5
18
11
100
136
150
164
100
100
104
104
100
100
1,
2
16
64
11, 2
16
64
18, 2
11
11.5
11.5
11
16
64
11
a
Drugs were administred intraperitoneally on days 1, 5, and
9. Antitumor activity was determined by comparing the median
survival time of treated animals (T) with that of controls (C) and
is expressed as an oncostatic index: T/C × 100.
In all cases, for maleimide indolocarbazoles, com-
pounds bearing the sugar moiety (1, 2, 4-10) were more
active than their corresponding aglycones (13, 22, 23,
14, 17-19, 12, 11). For the dechlorinated analogues of
rebeccamycin, substitution on the maleimide nitrogen
afforded a stronger activity (6-8 compared to 5). For
10 and 11, which were the strongest PKC inhibitors,
and 10, which was the strongest topoisomerase I inhibi-
tor, there was no evidence for any additive effect that
might enhance antiproliferative activity in cells.
In Vivo An titu m or Assa y. The antitumor activities
of 1, 11, and 18 were examined in vivo on mice
inoculated with B16 melanoma cells (Table 2.1) and
P388 leukemia cells (Table 2.2). DL50 values also
determined for 1 and 11 were found to be >120 mg/kg.
The antiproliferative activity obtained in vitro on B16
melanoma cells was not confirmed by the in vivo tests.
Only 1 exhibited a significant activity on mice injected
with P388 leukemia cells. None of the three compounds
was significantly active on mice bearing B16 cells. Some
marginal activity was observed with 18 at 16 mg/kg
against B16 melanoma (T/C × 100 ) 128) but was not
confirmed against P388 leukemia. This discrepancy
between in vivo and in vitro tests may be due to poor
membrane crossing or a metabolic process inactivating
these drugs. The nonsignificant results obtained in vivo
could also be due to the poor solubility of the compounds
(soluble in DMSO), which could prevent the transport
of the drug. We are investigating the synthesis of
analogues of 18 bearing lipophilic substituents to en-
hance the membrane-crossing ability.
Exp er im en ta l Section
Ch em istr y. IR spectra were recorded on a Perkin-Elmer
881 spectrometer (ν in cm-1) and NMR spectra on a Bruker
AC 400 spectrometer (1H, 400 MHz; 13C, 100 MHz) (chemical
shifts δ in ppm, abbreviations: singlet (s), doublet (d), triplet
(t), multiplet (m), tertiary carbons (C tert), quaternary carbons
(C quat)). Mass spectra (EI and FAB+) were determined at
CESAMO (Talence, France) on a high-resolution FISONS
Autospec-Q spectrometer. Chromatographic purifications were
performed with flash Geduran SI 60 (Merck) 0.040-0.063 mm.
For purity tests (compounds 19-23), TLC was performed on
fluorescent silica gel plates (60 F254 from Merck) and visualized
by UV light.
6-Am in o-1,11-d ich lor o-12-(4-O-m eth yl-â-D-glu cop yr a n -
n osyl)-6,7,12,13-tetr a h yd r oin d olo[2,3-a ]pyr r olo[3,4-c]ca r -
ba zole-5,7-d ion e (2). To rebeccamycin (352 mg, 0.618 mmol)
in THF (2 mL) was added H2N-NH2, H2O (2.1 mL, 43.2 mmol).
The mixture was stirred at room temperature for 3 h. Water
(140 mL) was then added, and the mixture was stirred
overnight. The precipitate was collected and dried in a
desiccator to give 2 as a dark yellow powder (211 mg, 0.361
mmol, 58% yield). IR (KBr): νNH,OH 3300-3600 cm-1, νCdO
1720 cm-1
. Mp: 254-257 °C. HRMS (FAB+): calcd for
C27H23Cl2N4O7, 585.0944; found, 585.0896. 1H NMR (400 MHz,
DMSO-d6): 3.55-4.20 (6H, m, C2′-H, C3′-H, C4′-H, C5′-H, C6′-
H2), 3.65 (3H, s, OCH3), 5.08 (3H, s, NH2, OH), 5.40 (1H, s,
OH), 5.50 (1H, s, OH), 6.99 (1H, d, J ) 9.2 Hz, C1′-H), 7.51
(2H, t, J ) 7.8 Hz), 7.75 (1H, d, J ) 7.8 Hz), 7.78 (1H, d, J )
7.8 Hz), 9.14 (1H, d, J ) 7.8 Hz), 9.34 (1H, d, J ) 7.8 Hz),
10.67 (1H, s, Nindole-H). 13C NMR (100 MHz, DMSO-d6): 59.5
(C6′), 60.1 (OCH3), 72.1, 77.3, 79.2, 80.2, 84.4 (C2′, C3′, C4′, C5′,
C1′), 116.2, 116.3, 117.6 (2C), 119.4, 119.7, 123.1, 125.0, 129.6
(2C), 137.2, 137.7 (C quat arom), 122.1, 122.6, 123.4, 124.0,
127.1, 130.1 (C tert arom), 167.9, 168.1 (CdO).
An tim icr obia l Activity. Antibiogram tests on a
Gram-negative bacterium (Escherichia coli), a yeast
(Candida albicans), and three Gram-positive bacteria
(Bacillus cereus, Streptomyces chartreusis, and Strep-
tomyces griseus) (data not shown) showed that 1-23
were all inactive on E. coli. All were inactive against
yeast C. albicans, except aglycones 12, 13, and 16.
Aglycones 13-15, 17, 18, and 20 were inactive on the
1,11-Dich lor o-6-h yd r oxy-12-(4-O-m eth yl-â-D-glu cop yr -
a n n osyl)-6,7,12,13-tetr a h yd r oin d olo[2,3-a ]p yr r olo[3,4-c]-
ca r ba zole-5,7-d ion e (3). To rebeccamycin (200 mg, 0.35
mmol) in DMF (2 mL) were added NH2OH, HCl (1.70 g, 24.5
mmol), and NEt3 (3.4 mL, 24.5 mmol). The mixture was
heated at 80 °C for 4 h. It was then poured into water and
extracted with AcOEt. The organic phase was washed with

SAR of Substituted Indolocarbazoles
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 22 4475
brine and dried over MgSO4. After removal of the solvent and
purification by flash chromatography (eluent, cyclohexane-
AcOEt, 30:70), compound 3 was isolated as a yellow solid (120
J ) 7.3 Hz), 7.64 (2H, t, J ) 7.6 Hz), 7.83 (1H, d, J ) 8.1 Hz),
8.03 (1H, d, J ) 8.5 Hz), 9.11 (1H, d, J ) 8.0 Hz), 9.18 (1H, d,
J ) 7.9 Hz), 10.82 (1H, s, N-OH), 11.65 (1H, s, Nindole-H). 13C
NMR (100 MHz, DMSO-d6): 58.6 (C6′), 60.2 (OCH3), 73.4, 76.5,
77.3 (2C), 84.2 (C2′, C3′, C4′, C5′, C1′), 111.9, 112.4, 120.5, 120.7,
124.5 (2C), 127.1, 127.3 (C tert arom), 115.3, 117.1, 117.3,
118.6, 121.0, 121.4, 128.2, 129.7, 141.0, 142.3 (C quat arom),
166.9, 167.0 (CdO).
6-F or m a m id o-12-(4-O-m et h yl-â-D-glu cop yr a n n osyl)-
6,7,12,13-tetr a h yd r oin d olo[2,3-a ]p yr r olo[3,4-c]ca r ba zole-
5,7-d ion e (8). A mixture of 6 (124 mg, 0.240 mmol), DMF
(1.5 mL), and concentrated HCl (0.04 mL) was stirred at 60
°C for 4 h. A further 0.02 mL of concentrated HCl was added
and the mixture warmed at 37 °C for 16 h. AcOEt was added,
and the same workup as for 4 yielded 8 as a yellow powder
mg, 0.21 mmol, 60% yield). IR (KBr): νNH,OH 3200-3600 cm-1
,
ν CdO 1670 cm-1. Mp: >300 °C. 1H NMR (400 MHz, DMSO-
d6): 3.35-4.12 (6H, m, C2′-H, C3′-H, C4′-H, C5′-H, C6′-H2), 3.62
(3H, s, OCH3), 5.15 (1H, s, OH), 5.40 (2H, s, OH), 6.97 (1H, d,
J ) 9.1 Hz, C1′-H), 7.50 (2H, t, J ) 7.8 Hz), 7.75 (1H, d, J )
7.8 Hz), 7.79 (1H, d, J ) 7.7 Hz), 9.08 (1H, d, J ) 7.9 Hz),
9.27 (1H, d, J ) 7.9 Hz), 10.65 (1H, s, Nindole-H), 10.88 (1H br
s, N-OH). 13C NMR (100 MHz, DMSO-d6): 60.0 (C6′), 60.2
(OCH3), 72.4, 77.5, 79.2, 80.2, 84.3 (C2′, C3′, C4′, C5′, C1′), 116.0,
116.1, 116.6, 117.8, 118.8, 119.3, 123.0, 124.9, 129.3, 129.5,
137.2, 137.6 (C quat arom), 121.8, 122.3, 123.1, 123.8, 127.0,
130.0 (C tert arom), 166.1, 166.5 (CdO).
(53 mg, 0.098 mmol, 41% yield). IR (KBr): νNH,OH
cm-1, νCdO 1710, 1720 cm-1
Mp: 265-267 °C. HRMS
H24N4O8, 544.1594; found, 544.1591. H
NMR (400 MHz, acetone-d6): 3.75 (3H, s), 3.87-4.25 (6H, m),
4.53 (1H, d, J ) 4.8 Hz), 4.59 (1H, d, J ) 4.0 Hz), 5.35 (1H, s),
6.34 (1H, d, J ) 8.5 Hz), 7.28 (1H, t, J ) 7.5Hz), 7.32 (1H, t,
J ) 7.5 Hz), 7.50 (1H, t, J ) 7.5 Hz), 7.53 (1H, t, J ) 7.5 Hz),
7.72 (1H, d, J ) 8.2 Hz), 7.88 (1H, d, J ) 8.4 Hz), 8.53 (1H, s,
CHO), 9.07 (1H, d, J ) 8.1 Hz), 9.18 (1H, d, J ) 8.1 Hz), 9.77
(1H, s, NHCHO), 11.55 (1H, s, Nindole-H). 13C NMR (100 MHz,
3300-3550
1,11-Dich lor o-6-for m am ido-12-(4-O-m eth yl-â-D-glu copy-
r a n n osyl)-6,7,12,13-t et r a h yd r oin d olo[2,3-a ]p yr r olo[3,4-
c]ca r ba zole-5,7-d ion e (4). A mixture of 2 (183 mg, 0.312
mmol), DMF (2 mL), and concentrated HCl (0.06 mL) was
stirred at 60 °C for 4 h. A further 0.03 mL of concentrated
HCl was added and the mixture warmed at 37 °C for 16 h.
AcOEt was added and the mixture washed successively with
2% aqueous bicarbonate solution and brine. The organic phase
was dried over MgSO4, the solvent was removed, and the
residue was purified by flash chromatography (eluent, AcOEt)
to yield 4 as a yellow powder (146 mg, 0.238 mmol, 76% yield).
IR (KBr): νNH,OH 3200-3550 cm-1, νCdO 1710, 1720 cm-1. Mp:
.210-212 °C. HRMS (FAB+): calcd for C28H23N4O8Cl2,
613.0892; found, 613.0985. 1H NMR (400 MHz, DMSO-d6):
3.57-4.11 (6H, m, C2′-H, C3′-H, C4′-H, C5′-H, C6′-H2), 3.65 (3H,
s, OCH3), 5.05 (1H, m, OH), 5.55 (2H, m, 2 OH), 6.95 (1H, d,
J ) 9.2 Hz, C1′-H), 7.46 (1H, t, J ) 7.0 Hz), 7.48 (1H, t, J )
6.5 Hz), 7.72 (1H, d, J ) 7.6 Hz), 7.78 (1H, d, J ) 7.7 Hz),
8.56 (1H, s, CHO), 9.03 (1H, d, J ) 8.0 Hz), 9.21 (1H, d, J )
7.9 Hz), 10.77 (1H, s, NHCHO), 10.97 (1H, s, Nindole-H). 13C
NMR (100 MHz, DMSO-d6): 59.9 (OCH3), 60.2 (C6′), 72.1, 77.4,
79.1, 80.3, 84.5 (C2′, C3′, C4′, C5′, C1′), 116.4, 116.5, 117.1, 119.1,
119.8, 122.9, 124.8 (2C), 130.1, 130.2, 137.3, 138.0 (C quat
arom), 122.4, 122.9, 123.2, 123.8, 127.5, 130.3 (C tert arom),
160.7 (CHO), 165.8, 166.0 (CdO).
.
1
(FAB+): calcd for C28
acetone-d6
): 59.3 (OCH3), 60.1 (C6′), 73.8, 77.3, 77.4, 78.0, 84.9
(C2′, C3′, C4′, C5′, C1′), 111.3, 112.1, 120.3, 120.5, 121.7, 121.8,
124.7, 124.8 (C tert arom), 116.4, 118.0, 118.2, 119.4, 120.9,
121.0, 128.9, 130.5, 141.4, 142.5 (C quat arom), 159.9 (CHO),
166.1, 166.6 (CdO).
6-For m a m id o-6,7,12,13-tetr a h yd r o-5,7-d ioxoin d olo[2,3-
a ]p yr r olo[3,4-c]ca r ba zole (19). To 6,7,12,13-tetrahydro-5,7-
dioxo-5H-indolo[2,3-a]furo[3,4-c]carbazole18 (100 mg, 0.306
mmol) in DMF (10 mL) was added formic hydrazide (184 mg,
3.06 mmol). The mixture was stirred at 140 °C for 1 h. After
cooling, addition of water (12 mL) caused the precipitation of
an orange solid that was collected and washed successively
with water and ethyl ether to give 19 (106 mg, 0.280 mmol,
94% yield). 19 was isolated as a single compound. Only one
spot was obtained by TLC (Rf ) 0.28; eluent, cyclohexane-
AcOEt, 30:70). IR (KBr): νNH,OH 3200-3500 cm-1 νCdO 1690,
6-Am in o-12-(4-O-m eth yl-â-D-glu cop yr a n n osyl)-6,7,12,-
13-tetr a h yd r oin d olo[2,3-a ]p yr r olo[3,4-c]ca r ba zole-5,7-d i-
on e (6). To compound 5 (100 mg, 0.200 mmol) was added
H2N-NH2, H2O (0.7 mL, 14 mmol). The mixture was stirred
at 50 °C for 1.5 h and then poured into water and extracted
with AcOEt. The organic phase was dried over MgSO4, the
solvent was removed, and the residue was purified by flash
chromatography (eluent, cyclohexane-AcOEt, 10:90) to give
6 as a red solid (94 mg, 0.183 mmol, 91% yield). IR (KBr):
,
1760 cm-1. Mp: >300 °C. HRMS (EI): calcd for C21H12N4O3,
368.0909; found, 368.0913. 1H NMR (400 MHz, DMSO-d6):
7.44 (2H, t, J ) 7.3 Hz), 7.65 (2H, t, J ) 7.3 Hz), 7.88 (2H, d,
J ) 8.0 Hz), 8.51 (1H, s), 9.01 (2H, d, J ) 7.8 Hz), 10.82 (1H,
s), 11.96 (2H, s, Nindole-H). 13C NMR (100 MHz, DMSO-d6):
112.3, 120.5, 124.0, 127.2 (C tert arom), 115.8, 116.4, 121.3,
129.4, 140.5 (C quat arom), 160.7, 166.9 (CdO).
6-(2-Hydr oxyeth yl)-6,7,12,13-tetr ah ydr o-5,7-dioxoin dolo-
[2,3-a ]p yr r olo[3,4-c]ca r ba zole (20). A mixture of 6,7,12,-
13-tetrahydro-5,7-dioxo-5H-indolo[2,3-a ]furo[3,4-c]carba-
νNH,OH 3330, 3420 cm-1, νCdO 1750 cm-1
. Mp: 260-262 °C.
HRMS (FAB+): calcd for C27H25N4O7, 517.1723; found,
517.1680. 1H NMR (400 MHz, DMSO-d6): 3.55-4.10 (6H, m,
C2′-H, C3′-H, C4′-H, C5′-H, C6′-H2), 3.71 (3H, s, OCH3), 5.05 (2H,
s, NH2), 5.10 (1H, d, J ) 7.7 Hz, OH), 5.36 (1H, d, J ) 5.5 Hz,
OH), 6.22 (1H, s, OH), 6.35 (1H, d, J ) 8.9 Hz, C1′-H), 7.43
(2H, t, J ) 7.5 Hz), 7.64 (2H, t, J ) 7.5 Hz), 7.77 (1H, d, J )
8.1 Hz), 8.01 (1H, d, J ) 8.4 Hz), 9.17 (1H, d, J ) 7.9 Hz),
9.23 (1H, d, J ) 7.9 Hz), 11.65 (1H, s, Nindole-H). 13C NMR
(100 MHz, DMSO-d6): 58.5 (C6′), 60.1 (OCH3), 73.2, 76.3, 77.1,
77.3, 84.1 (C2′, C3′, C4′, C5′, C1′), 111.8, 112.3, 120.5, 120.7, 124.4
(2C), 126.9, 127.1 (C tert arom), 116.6, 117.1, 118.3, 118.5,
121.0, 121.4, 128.2, 129.6, 140.9, 142.2 (C quat arom), 168.7,
168.8 (CdO).
zole18
(100 mg, 0.306 mmol) in ethanolamine (1.3 mL, 1.29 g,
21.2 mmol) was stirred at room temperature for 1 h and then
poured into water and extracted with AcOEt. The organic
phase was washed with brine and dried over MgSO4
. Removal
of the solvent yielded 20 as a yellow solid (85 mg, 0.230 mmol,
75% yield). TLC (Rf ) 0.66; eluent, cyclohexane-AcOEt, 30:
70) revealed there was only a single compound. IR (KBr):
νNH,OH 3300-3500 cm-1, νCdO 1750 cm-1
. Mp: >300 °C.
HRMS (EI): calcd for C22H15N3O3, 369.1113; found, 369.1110.
1H NMR (400 MHz, DMSO-d6): 3.70 (2H, t, J ) 5.4 Hz), 3.78
(2H, t, J ) 5.4 Hz), 4.90 (1H br s, OH), 7.35 (2H, t, J ) 7.6
Hz), 7.54 (2H, t, J ) 7.4 Hz), 7.79 (2H, d, J ) 8.2 Hz), 9.02
(2H, d, J ) 8.0 Hz), 11.75 (2H, br s, Nindole-H). 13C NMR (100
MHz, DMSO-d6): 40.1 (CH2), 58.4 (CH2OH), 112.1, 120.2,
124.2, 126.8 (C tert arom), 115.6, 118.8, 121.5, 128.9, 140.4 (C
quat arom), 169.9 (CdO).
6-H yd r oxy-12-(4-O-m et h yl-â-D-glu cop yr a n n osyl)-6,7,-
12,13-tetr a h yd r oin d olo[2,3-a ]p yr r olo[3,4-c]ca r ba zole-5,7-
d ion e (7). To 5 (100 mg, 0.200 mmol) in DMF (2 mL) were
added NH2OH, HCl (970 mg, 13.9 mmol), and NEt3 (1.40 g,
13.9 mmol). The mixture was stirred at 70 °C for 2 h and then
treated as for 3. Purification by flash chromatography (eluent,
AcOEt-MeOH, 90:10) gave 7 as an orange solid (103 mg, 0.194
mmol, 99% yield). IR (KBr): νNH,OH 3200-3550 cm-1, νCdO
6-(4-Sem ica r ba zid o)-6,7,12,13-tetr a h yd r o-5,7-d ioxoin -
d olo[2,3-a ]p yr r olo[3,4-c]ca r ba zole (21). A mixture of 6,7,-
12,13-tetrahydro-5,7-dioxo-5H-indolo[2,3-a]furo[3,4-c]carba-
zole18 (100 mg, 0.306 mmol), DMF (5 mL), and carbohydrazide
(160 mg, 1.77 mmol) was stirred at 80 °C for 3 h. After cooling,
addition of water (12 mL) caused the precipitation of an orange
solid. After collection and washing with water and then
petroleum ether, 21 was isolated (57 mg, 0.143 mmol, 47%
yield). TLC (Rf ) 0.13; eluent, cyclohexane-AcOEt, 30:70)
1710 cm-1
. Mp: 298-300 °C. HRMS (FAB+): calcd for
C27H24N3O8, 518.1563; found, 518.1551. 1H NMR (400 MHz,
DMSO-d6): 3.50-4.15 (6H, m, C2′-H, C3′-H, C4′-H, C5′-H, C6′-
H2), 3.71 (3H, s, OCH3), 5.10 (1H, s, OH), 5.38 (1H, s, OH),
6.25 (1H, s, OH), 6.35 (1H, d, J ) 8.9 Hz, C1′-H), 7.37 (2H, t,

4476 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 22
Rodrigues Pereira et al.
revealed there was only a single compound. IR (KBr): νNH
3200-3400 cm-1, νCdO 1720, 1760 cm-1. Mp: >300 °C. HRMS
(EI): calcd for C20H12N4O2 (M - (CO-NH-NH2) + H), 340.0960;
found, 340.0951. 1H NMR (400 MHz, DMSO-d6): 4.45 (2H,
br s, NH2), 7.40 (2H, t, J ) 7.4 Hz), 7.64 (2H, t, J ) 7.2 Hz),
7.85 (2H, d, J ) 8.1 Hz), 8.04 (1H, br s), 9.02 (3H, d, J ) 7.8
Hz, br s), 12.45 (2H, s, Nindole-H). 13C NMR (100 MHz, DMSO-
d6): 111.9, 120.3, 124.1, 127.0 (C tert arom), 115.4, 116.7,
121.3, 129.4, 140.2 (C quat arom), 158.4, 168.0 (CdO).
6-Am in o-1,11-d ich lor o-6,7,12,13-t et r a h yd r o-5,7-d iox-
oin d olo[2,3-a ]p yr r olo[3,4-c]ca r ba zole (22). To 13 (200 mg,
0.5 mmol) in DMF (20 mL) was added H2N-NH2, H2O (1.72
mL, 35.5 mmol). The mixture was stirred at 70 °C for 1 h.
After cooling, addition of water (25 mL) caused the precipita-
tion of a yellow solid which was collected and washed with
water and ethyl ether to give 22 (186 mg, 0.454 mmol, 90%
yield). 22 was isolated as a single compound. Only one spot
was obtained by TLC (Rf ) 0.23; eluent, cyclohexane-AcOEt,
50:50). IR (KBr): νNH 3340 cm-1, νCdO 1710 cm-1. Mp: >300
°C. HRMS (EI): calcd for C20H10Cl2N4O2, 408.0181; found,
408.0179. 1H NMR (400 MHz, DMSO-d6): 4.96 (2H, s, NH2),
7.37 (2H, t, J ) 7.9 Hz), 7.65 (2H, d, J ) 7.6 Hz), 8.89 (2H, d,
J ) 7.9 Hz), 11.85 (2H, br s, Nindole-H). 13C NMR (100 MHz,
DMSO-d6): 121.3, 123.1, 126.1 (C tert arom), 115.6, 115.8,
117.8, 123.0, 128.7, 137.0 (C quat arom), 168.5 (CdO).
were washed with PBS. Fresh medium was added, and
incubation was performed at 37 °C in a CO2 incubator for 12
days. Dishes were then rinsed with PBS, fixed with methanol,
and stained with 0.2% crystal violet solution, and colonies (>50
cells) were counted.
The antiproliferative activity is expressed as IC50 (inhibiting
concentration 50%), the drug concentration giving a 50%
cloning efficiency compared to untreated cells.
P r otein Kin a se In h ibition . (a ) Mea su r em en ts of P KC
a n d P KA In h ibition (P r oced u r e a ). Histones IIIs and IIa,
phosphatidylserine, and diacylglycerol were purchased from
Sigma; [γ32P]ATP was from Amersham. PKA was purchased
from Sigma and PKC from Calbiochem.
PKC phosphorylation assays were performed in a reaction
mixture (80 µL) containing histone IIIs (2.4 mg/mL), MgCl2
(10 mM), CaCl2 (0.1 mM), phosphatidylserine (10 mg/mL),
diacylglycerol (10 mg/mL), ATP (10 µM), [γ32P] ATP (106 cpm/
80 µL), Tris buffer (50 mM, pH 7.5), PKC (0.5 µg/mL), and
inhibitors at different concentrations. PKA phosphorylation
assays were performed in a reaction mixture (80 µL) containing
histone IIa (1 mg/mL), MgCl2 (5 mM), ATP (10 µM), [γ32P]-
ATP (106 cpm/80 µL), Tris buffer (50 mM, pH 7.0), PKA (1
µg/mL), and inhibitors at different concentrations. For each
kinase, reactions were run at 30 °C for 12 min and stopped
with trichloroacetic acid (12%, w/v) in the presence of bovine
serum albumin (0.9 mg) as a carrier protein. After centrifuga-
tion (10 min at 3000 rpm), the pellet was dissolved in 1 M
NaOH and precipitated a second time with trichloroacetic acid.
Radioactivity incorporated into histones was counted by
scintillation spectrometry (Tri-Carb 4530, Packard). All ex-
periments were carried out in triplicate.
(b) Mea su r em en ts of P KC-r In h ibition (P r oced u r e b).
Protamine sulfate was from Merck (Darmstadt, Germany).
Unless specified, chemicals were from Sigma (St. Louis, MO).
[γ-33P]ATP (1000-3000 Ci/mmol) was obtained from Amer-
sham. Recombinant baculoviruses from PKC subtypes were
supplied by Dr. Silvia Stabel, Ko¨ln, Germany.
Expression and partial purification of PKCs together with
measurements of activities were carried out as previously
described.25 Stock solutions of compounds (in DMSO) were
diluted in serial 10-fold dilutions using DMSO/water (v/v 50:
50) as the solvent. PKC isoenzyme activity was assayed using
protamine sulfate as a substrate in the absence of phosphati-
dylserine and diacylglycerol.26 Incorporation of γ-33P onto
protamine sulfate was determined by spotting 50 µL aliquots
on P81 chromatography paper (Whatman).27 Compounds were
tested on PKC-R in two independent experiments. Data show
IC50 values (half-maximum inhibitory concentrations) ex-
pressed in micromolar.
In Vivo An titu m or Assa y. Male DBA/2J co mice and male
B6D2F1/J co mice were purchased from IFFA CREDO
(L’Arbresle, France). Murine P388 leukemia cells, obtained
from ICIG, Villejuif, were maintained by weekly transplanta-
tion of the tumor cells into the peritoneal cavity of male DBA/2
mice. For antitumor testing, 1 × 106 cells were intraperito-
neally injected into male B6D2F1 mice on day 0. The B16
melanoma cell line was maintained in culture in MEM
medium;28 0.5 × 106 cells were subcutaneously implanted on
the dorsum of B6D2F1 mice on day 0. Each treated group
comprised six and the control group 12 mice. Drugs were
administered intraperitoneally on days 1, 5, and 9. Control
animals were treated with solvent (DMSO-olive oil). Median
survival times (MST) were determined for the respective
groups. Antitumor activity is expressed as an oncostatic
index: T/C × 100.29,30
6-F or m a m id o-1,11-d ich lor o-6,7,12,13-t et r a h yd r o-5,7-
d ioxoin d olo[2,3-a ]p yr r olo[3,4-c]ca r b a zole (23). To 22
(100 mg, 0.244 mmol) in DMF (10 mL) was added concentrated
HCl (0.05 mL), and the mixture was stirred at 60 °C for 4 h.
Concentrated HCl (0.02 mL) was then added and the mixture
stirred at 37 °C for 16 h. After cooling, water was added, and
the solid was collected, washed with water and then Et2O, and
dried in a desiccator to yield 23 (100 mg, 0.228 mmol, 93%
yield) as a yellow powder. 23 was isolated as a single
compound. Only one spot was obtained by TLC (Rf ) 0.26;
eluent, cyclohexane-AcOEt, 50:50). IR (KBr): νNH 3300 cm-1
,
νCdO 1660, 1720 cm-1. Mp: >300 °C. HRMS (EI): calcd for
C21H10N4O3Cl2, 436.0123; found, 436.0123. 1H NMR (400 MHz,
DMSO-d6): 7.40 (2H, t, J ) 7.8 Hz), 7.68 (2H, d, J ) 7.6 Hz),
8.51 (1H, s, CHO), 8.84 (2H, d, J ) 7.9 Hz), 10.85 (1H, s, NH),
11.90 (2H, s, Nindole-H). 23 was too insoluble in DMSO to get
the 13C NMR spectrum.
Biologica l Tests. Rebeccamycin was from our laboratory
stock sample.
Top oisom er a se In h ibition . Topoisomerases I and II were
prepared from calf thymus as already described.21,22 Topoi-
somerase I or II inhibitions were evaluated using the DNA
cleavage assay carried out according to the procedure previ-
ously described.23 Each compound was evaluated for its
minimal inhibitory concentration (MIC), corresponding to the
lowest concentration (µg/mL) that would produce a detectable
stimulation of the DNA cleavage reaction.
Gr ow t h In h ib it ion Assa ys: P 388 Mu r in e Leu k em ia
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 according to a
published procedure.24 The concentrations of drugs giving 50%
of growth inhibition (IC50) were determined.
B16 Cell Cu ltu r e. B16, a mouse melanoma cell line
derived from spontaneous skin tumor in C57BI/6 mice, was
supplied by the Institut de Cance´rologie et Immunoge´ne´tique,
Villejuif, France. Stock cell cultures were maintained as
monolayers in 25 cm3 culture flasks in Eagle’s minimum
essential medium (Gibco, Paisly, Scotland) supplemented with
10% fetal calf serum (Sigma Chemical Co.), vitamin solution
(100×; Gibco), 100 mM sodium pyruvate (Gibco), nonessential
amino acids (100×; Gibco), 200 µM L-glutamine, and genta-
mycin (Schering-Plough). The cells were grown at 37 °C in a
humidified atmosphere containing 5% CO2. In these condi-
tions, the doubling time was 15 h.
B16 Cell Cytotoxic Assa y. B16 cells were plated into 60
mm Petri dishes (200 cells/dish) and allowed to adhere for 20
h before treatment. Culture medium containing increasing
concentrations of drugs was added, and incubation was
conducted for 24 h at 37 °C in a CO2 incubator. After this
time, the drug-containing medium was discarded, and the cells
An tibiogr a m Tests a n d MIC Deter m in a tion s. Five
strains were tested, three Gram-positive bacteria (B. cereus
ATCC 14579, S. chartreusis NRRL 11407, S. griseus ATCC
23345), 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) diffusion method using
the following nutrient media: Mueller-Hilton broth (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

SAR of Substituted Indolocarbazoles
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 22 4477
droxy-13-(â-D-glucopyranosyl)-5H-indolo[2,3-a]pyrrolo[3,4-c]car-
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I-mediated DNA cleavage and mechanisms of cell line-selective
cytotoxicity. Cancer Res. 1995, 55, 1310-1315.
strains. Compounds 1-23 were dissolved in DMSO, and a
paper disk containing each one (300 µg) was placed on Petri
dishes. Growth inhibition was examined after 24 h incubation
at 27 °C.
MIC values of 1-23 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
to 0.05 µg/mL.
(15) Kojiri, K.; Kondo, H.; Arakawa, H.; Mitsuru, O.; Suda, H.
European Patent 0 545 195 A1, 1993.
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