Synthesis and cytotoxic activity of different open indolocarbazole alkaloid analogues

Article abstract of DOI:10.1016/S0968-0896(03)00308-0

An array of 4-(aryl or indolyl)pyrrolo[3,4-c]carbazole-1,3-diones (open analogues of indolocarbazole alkaloids), 10-(aryl or indolyl)pyrrolo[3,4-b]carbazole-1,3-diones, and different derivatives have been prepared using a Diels-Alder plus Fischer indolization approach and tested as cytotoxic agents. Some representative compounds display interesting cytotoxic profiles.

Full text of DOI:10.1016/S0968-0896(03)00308-0

Bioorganic & Medicinal Chemistry 11 (2003) 3413–3421
Synthesis and Cytotoxic Activity of Different Open
Indolocarbazole Alkaloid Analogues
Esther Caballero,a,* Marta Adeva,a Suzanne Calderon,a Heidi Sahagun,a
Fernando Tome,a Manuel Medarde,a Jose Luis Fernandez,b Miguel Lopez-Lazaroc
and Maria Jesus Ayusoc
a
´
Laboratorio de Quımica Organica y Farmaceutica, Facultad de Farmacia, 37007 Salamanca, Spain
´
Instituto Biomar SA.24231 Onzonilla, Leo n, Spain
´
b
´
Departamento de Farmacologıa, Facultad de Farmacia, Sevilla, Spain
c
´
Received 4 March 2003; accepted 8 May 2003
Abstract—An array of 4-(aryl or indolyl)pyrrolo[3,4-c]carbazole-1,3-diones (open analogues of indolocarbazole alkaloids), 10-(aryl
or indolyl)pyrrolo[3,4-b]carbazole-1,3-diones, and different derivatives have been prepared using a Diels–Alder plus Fischer indo-
lization approach and tested as cytotoxic agents. Some representative compounds display interesting cytotoxic profiles.
# 2003 Elsevier Ltd. All rights reserved.
Indolocarbazole alkaloids are a class of natural pro-
ducts isolated from microorganisms, slime molds, and
marine organisms that show an array of interesting
biological activities.1
staurosporine, are mainly protein kinase C (PKC)
inhibitors.6 The mechanism by which Arcyriaflavin-A
and simple related compounds inhibit human cytome-
galovirus (HCMV) replication seems to be through
blockade of the activity of protein kinase pUL97.7 Cur-
rently, several indolocarbazole alkaloids or analogues,
Three representative members of this family are Arcyri-
aflavin-A,2 an inhibitor of human cytomegalovirus
replication,3 which was originally isolated from the
slime mold Arcyria denudata (myxomycetes), rebecca-
mycin4 and staurosporine5 (Fig. 1), both displaying
antitumour activity, which were respectively isolated
from Nocardia aerocoligenes and Streptomyces
staurosporeus (both actinomycetes).
The considerable research interest in these compounds
lies in the potent antitumour properties of many of
them. This has given rise to a huge amount of research
aimed at their synthesis, the preparation of analogues,
and an understanding of their mechanism of action.
Compounds whose basic structure is the pyrroloindolo-
carbazole system with one N-glycosidic bond, such as
rebeccamycin, act by DNA topoisomerase I inhibition,
whereas those with two N-glycosidic bonds, such as
*Corresponding author. Tel.: +34-923-214528; fax: +34-923-294515;
e-mail: escab@usal.es
Figure 1.
0968-0896/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00308-0

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E.Caballero et al./ Bioorg.Med.Chem.11 (2003) 3413–3421
such as the topoisomerase I poison NB-506, are in
clinical trials for their potential use in cancer therapy.8
order to use the corresponding analogues as inter-
mediates in the synthesis of the natural product Arcyri-
aflavin A and its unsymmetrical analogues.
Attracted by the novel structural features and biological
relevance of this family of alkaloids, together with our
interest in applying novel aryl-trialkylsiloxy-dienes to
the synthesis of natural products and analogues, some
time ago we set up a research line aimed at the pre-
paration of analogues of indolocarbazole alkaloids and
other natural products, following a Diels–Alder method-
ology in all cases. The results of this research can be
broken down into four different parts: (a) Synthesis and
reactivity of novel aryl/aryloxy/heteroaryl dienes,9 (b)
Synthesis of anthracycline analogues,10 (c) New synthesis
of Arcyriaflavin-A11 and its analogues,12 (d) Cytotoxic
evaluation.10a,12a
Finally, we considered the preparation of Family III,
due to the importance of the indolyl ring in many
bioactive compounds. Moreover, in this case we took
some natural marine products such as wakayin16 and
eudistomin U17 as models (Fig. 3), all of them display-
ing cytotoxic activity (topoisomerase I/II inhibitors).
We expected that the combination of a planar system
and a unit (in this case indolyl) able to interact with
DNA through hydrophobic or H-bond forces, as pos-
tulated for topoisomerase inhibitors,18 would lead to
compounds having some interesting bioactivity.
We now wish to report the cytotoxic activity of the
broad number of open analogues that we have synthe-
sized to date, together with some hitherto unpublished
synthetic procedures for obtaining those compounds.
All compounds prepared are 4-(aryl or indolyl)-
pyrrolo[3,4-c] or 10-(aryl or indolyl)pyrrolo[3,4-b] car-
bazole-1,3-diones and derivatives, and can be divided
into three families (Fig. 2).
Figure 3.
Family I was designed taking into account the presence
of the trimethoxyphenyl unit in a variety of cytotoxic
agents such as colchicine and podophyllotoxin13 (tubu-
lin polymerisation inhibitors), etoposide (a topoisomer-
ase II inhibitor, with 4-hydroxy-3, 5-dimethoxyphenyl
unit) and duocarmycin SA14 (a DNA alkylating and
minor groove binder). We also focused on the tubulin
polymerisation inhibitor 1069C,15 in which the best
antitumour profile is provided by the 3,4,5-trimethoxy-
and 2,5-dimethoxyphenyl groupings. Thus, we attemp-
ted to modulate the possible cytotoxic activity by
introducing these characteristic structural features.
Many approaches to the synthesis of indolocarbazole
alkaloids and analogues have been described and are
summarised in different reviews and publications19
(Scheme 1). By contrast, there are few references to the
synthesis of analogues with structures similar to those
described in this paper, and most of them use a Diels–
Alder methodology.
Moody’s group reported20 an intramolecular cycload-
dition between an alkyne and a pyranoindolone, yield-
ing an open 4-(2-nitrophenyl)pyrrolo[3,4-c]carbazole
analogue, which was transformed into the staurosporine
aglycone via nitrene insertion. Starting from a C-4 ester
of a pyridazino[4,5-b]indole, a similar approach to open
analogues has also been reported.21 The Hoffman-La
Roche and Goedecke groups developed a strategy based
on the use of maleimides as dienophiles and 2-aryl/het-
eroarylvinyl-substituted indoles as dienes, leading to
Family II was chosen because the nitro group also con-
fers cytotoxic activity in many antitumour agents and in
4-aryl/heteroarylpyrrolo[3,4-c]carbazoles22,23
(which
show cytotoxicity through PKC inhibition). McCort et
al. have recently synthesised a series of other 4-aryl/
heteroarylpyrrolo[3,4-c]carbazoles, introducing the aryl/
heteroaryl moiety by means of a palladium catalysed
cross-coupling, after a Diels–Alder reaction similar to
that of the two latter references.24
Further examples of the synthesis of this kind of analo-
gues are a series of 4-[2-(N,N-dimethylaminoethoxy)-
phenyl]pyrrolo[3,4-c]carbazoles (some of them with
thrombopoietic activity)25 and the formation of 4-(1-
aminophenyl)pyrrolo[3,4-c]carbazoles from bisindolyl-
maleimides.26
We followed an original strategy that was also based on
the Diels–Alder reaction, in this case between novel
aryl/heteroaryl-dienes and maleimides, followed by a
Figure 2.

E.Caballero et al./ Bioorg.Med.Chem.11 (2003) 3413–3421
3415
Scheme 1. Diels–Alder synthetic approaches to 4-aryl/heteroarylpyrrolo[3,4-c]carbazoles.
Fischer indolization with para-substituted phenylhy-
drazines. In addition to its utility for obtaining Arcyr-
iaflavin-A and unsymmetrical derivatives,11 this
methodology is useful for the synthesis of new families
of open analogues,12 as we describe below.
In our case, the separation of both regioisomers (A, B)
by CC or crystallisation followed by aromatization with
DDQ gave representative planar compounds in the C and
D series. Since we were more interested in type C analo-
gues, we prepared different N-substituted derivatives in
order to test their cytotoxicities against representative
cancer cell lines. According to structure–activity
relationship studies described in the literature for staur-
osporine and rebeccamycin analogues, a planar system
is required, whereas substitution on the carbazole
nitrogen is variable, and sugar, aminoalkyl or cya-
noalkyl residues have been reported—among others—as
substituents that confer good activity.27 On the basis of
this, we proposed the modifications indicated for the
families I, II and III.
Chemistry
The methodology used for the synthesis of these com-
pounds is similar to that established by us for the
synthesis of Arcyriaflavin-A and other grooved analo-
gues. The first step in the synthesis (Scheme 2) is the
Diels–Alder reaction of dienes IVa and IVb with N-
substituted maleimides, which is quantitative after 2–4
days, using benzene (toluene) as a solvent for IVa and
CH2Cl2/acetone for IVb. As reported in our previous
studies endo products were always obtained and differ-
ent conformations were observed, depending on the
type of substitution of the aromatic moiety.12
The synthesis of these compounds is shown in Scheme 2
and the compounds synthesized are summarised in
Table 1 (for experimental details, see this and previous
papers). We decided to synthesize epoxides E (33–35),
aminoalcohols F (36–38), the cyano derivative 39, and
the glycoside 43 to obtain a representation of most
common substituents on the carbazole nitrogen
encountered among the indolocarbazole derivatives.
Since compound 1 proved to be the most potent of the
non-planar compounds of type A or B, we also trans-
formed it into the 40-hydroxy derivative 7 in order to
know the influence of this moiety on the activity of non-
planar systems.
Fischer indolization is usually carried out in acidic
medium on ketones VI, obtained from the enolcyclo-
adduct V by HCl hydrolysis. Since the indolization
reaction is carried out under acidic conditions, which
could also produce the hydrolysis of the enolether to the
ketone, we have now checked the direct indolization
reaction on the silylenolether V by modifying the
Fischer conditions. To the glacial acetic acid/EtOH 1:1
solution some drops of HCl 37% followed by hydrazine
were added. For example, from the corresponding
enolcycloadduct Vb and p-methoxyphenylhydrazine,
regiosomers 13 and 21 were obtained in good yields.
The regioisomeric ratio in this reaction changed,
depending on whether compound V or VI was used, but
in all the cases pyrrolo[3,4-c]carbazoles A were the
major products and pyrrolo[3,4-b]carbazoles B the
minor ones.
Biological Assays
All the products synthesized were subjected to cyto-
toxicity assays against neoplastic cell lines representa-
tive of solid tumours and leukaemia in order to know
their potential as antineoplastic agents. The results
obtained for representative compounds are presented in

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E.Caballero et al./ Bioorg.Med.Chem.11 (2003) 3413–3421
Scheme 2. Synthesis of 1–43: (a) R2–MeO–C6H4–NH–NH2, HOAc/EtOH 1:1, drops HCl (37%), reflux; (b) DDQ, C6H6, rt or reflux; (c) NaOH,
Bu4NSO4H, RX, C6H6, rt; (d) NaH, DMF, RX; rt; (e) Me2NH, EtOH, rt; (f) tetraacetyl-a-d-glucopyranosyl bromide, Ag2O, C6H6, reflux; (g)
NH4OH/MeOH, rt
Table 2, from which several conclusions can be
deduced. The compounds not included in Table 2 were
inactive.
although no important increase in their activity was
observed. The most cytotoxic derivatives (IC50=0.3–5.0
mM in at least two of the cell lines tested) are those with
a non-aromatic C ring (1 , 41) or aromatic derivatives
with different substitutions: imido-N-phenyl (33, 36) or-
N-methyl (27, 37, 38), and carbazole- N–H (25, 27, 28)
or carbazole-N-substituted (36), although in general the
C-aromatised derivatives (series C–F) are more cyto-
toxic than their non-aromatic analogues (series A–B).
Among the C-aromatised carbazole with free N–H
(series C), the most active are those presenting ortho-
substituents on the phenyl group (25 R6=OMe; 28
R6=NO2).
The initially synthesized compound, 1, is among the
most active compounds in the series, with several new
derivatives with the same order of cytotoxicity,
Table 1. Structure of compounds 1–38. See Scheme 2 for general
structures A–F
Compounds
Substitution
A
B C D E F R1 R2 R3 R4 R5 R6
R7 Ar/HetAr
1 14 22
33 36 Ph OMe OMe OMe OMe
Me OMe OMe OMe OMe
H OMe OMe OMe OMe
H
H
H
OMe
OMe
H
H
H
H
NO2
Ar
Ar
Ar
Ar
Ar
Ar
Ar
Ar
Ar
Ar
Ar
This variability in structure and cytotoxicity allowed us
to propose two general observations: the presence of the
3,4,5-trimethoxyphenyl group as the aryl (Ar) moiety is
associated with the active (1, 33, 36, 41, 42) or moder-
ately active (14, 15, 22, 31) compounds, and the intro-
duction of a 3-(N,N-dimethylamino)-2-hydroxypropyl
moiety on the carbazole nitrogen (series F) is accom-
panied by a noticeable increase in cytotoxicity in com-
parison with the corresponding carbazole free N–H
analogues (series C). In the light of the lower cytotoxi-
city of these derivatives (12, 13, 21, 29, 30), the replace-
ment of the phenyl-substituted moiety (Ar) by the
2
3
4
5
6
7
8
9
1
1
5 23 31
6 24
25
Ph OMe OMe
H
H
H
H
H
1 7 26 32 34 37Me OMe OMe
8 27
1
35 38Me OMe OMe OMe
Ph OMe OMe OH OMe
Ph Me OMe OMe OMe
Me Br OMe OMe OMe
1 0 1 9 28
11 20
Ph OMe
H
H
H
H
H
Ph OMe NO2
Me OMe
Me OMe
1
1 3 21
2
29
Me
SO2Ph HetAr
HetAr
HetAr
30
Me OMe
H

E.Caballero et al./ Bioorg.Med.Chem.11 (2003) 3413–3421
3417
Table 2. Cytotoxicity of representative compounds against cancer cell lines. IC50 (mM)
Compd
Structural type
P-388
A-549
HT-29
MEL-28
UACC-62
TK-10
MCF-7
1
7
A
A
A
B
B
B
C
C
C
C
C
D
D
E
F
0.5
10
7.4
9.8
11.1
12
1.9
10
7.4
9.8
22.2
12
1.9
10
7.4
9.8
11.1
1.2
9.8
10.5
1.9
10
7.4
9.8
22.2
12
12
14
15
20
22
25
27
28
29
31
33
35
36
37
38
41
42
43
9.8
2.1
9.8
2.1
9.8
2.1
>10
>10
>10
2.3
6.6
2.7
>10
8.9
11.5
2.8
>5
11.2
2.2
8.2
0.8
4.8
1.0
3.0
5.9
0.3
>5
11.2
2.2
8.2
4.1
4.8
2.4
2.2
>5
11.2
17.7
8.2
4.1
4.8
2.4
2.2
>5
11.2
2.2
8.2
4.1
4.8
2.4
3.0
5.9
>10
>10
>10
F
F
11.3
2.3
10.9
>10
5.9
>5
>10
5.9
>5
>5
>5
5.2
9.9
6.4
P-388: mouse lymphoma. A-549 human lung carcinoma. HT-29: human colon adenocarcinoma.
MEL-28 and UACC-62: human melanoma. TK-10: human renal adenocarcinoma. MCF-7: human breast adenocarcinoma.
3-indolyl residue (HetAr) is not a worthwhile modifi-
cation.
are expressed in cmꢀ1. NMR spectra were recorded on
Bruker WP 200 SY spectrometer in CDCl3 or the
reported solvent. Mass spectra were obtained by EI or
FAB methods on a VGTS-250 mass spectrometer.
Microanalyses were carried out on Perkin–Elmer 2400
CHN. Flash chromatography was performed with
Merck 60 silica gel (0.063–0.2 or 0.040–0.063 mm). Sol-
vents of analytical grade were used as purchased and,
when necessary, dried using standard procedures.
Thus, there is a certain degree of cytotoxicity associated
with some of the compounds with the pyrrolo[3,4-c]-
carbazole skeleton, and this cytotoxicity is mainly dis-
played by compounds having a highly hydrophobic
residue (3,4,5-trimethoxyphenyl) and/or a solubilising
moiety (3-(N,N-dimethylamino)-2-hydroxypropyl) on
the carbazole nitrogen. The planarity of the indolo-car-
bazole system is not directly associated with cytotoxic
compounds, but the non-planar non-aromatic C-ring
derivatives are in general less active.
Fischer indolization. Preparation of 13 and 21. To a
solution of 2.18 g (3.7 mmol) of cycloadduct V (R=iPr,
R1=Me, R7=SO2Ph) in 300 mL of AcOH/EtOH
(1:1 vol/vol) and 3.3 mL of HCl (37%), 7.4 mmol of
p-methoxyphenylhydrazine were added. The reaction
mixture was refluxed for 6 h and then rendered basic
with solid Na2CO3 and extracted with EtOAc, washed
with brine, and dried. Evaporation of the solvent, fol-
lowed by flash chromatography (silica gel, hexane/
AcOEt 1:1) and crystallization gave 13 (1.18 g, 59%)
and 21 (0.55g, 27%).
The activity of our first model compound 1 was not
improved but did persist in some of the derivatives
described in this paper. This suggests that compound 1
is a particular case of a folded molecule whose cyto-
toxicity may be due to a mechanism of action other than
those of planar molecules: that already described for
indolocarbazoles (DNA topoisomerase I inhibition or
PKC inhibition) or that one to be investigated for our
open analogues of the natural alkaloids.
(ꢁ)-(3aS,4S,10cS)-4-(N-phenylsulphonylindol-3-yl)-2-
methyl-9-methoxy-3a,4,5,10c-tetrahydro-6H-pyrrolo[3,4-
c]carbazole-1,3-dione (13). Yellow solid; Rf (hexane/
AcOEt 1:1) 0.53; mp 280 ꢂC (ether/MeOH). IR (KBr):
3426, 1758, 1706, 1175 cmꢀ1; 1H NMR (DMSO) d 10.94
(s, 1H), 7.94 (d, J=7.8 Hz, 2H), 7.90 (d, J=8.0 Hz,
1H), 7.75 (d, J=7.6 Hz, 1H), 7.67 (d, J=7.8 Hz, 1H),
7.65 (s, 1H), 7.57 (dd, J=7.8, 7.8 Hz, 2H), 7.34 (dd,
J=8.0, 7.6 Hz, 1H), 7.31 (d, J=2.4 Hz, 1H), 7.27 (dd,
J=7.6, 7.6 Hz, 1H), 7.22 (d, J=8.8 Hz, 1H), 6.73 (dd,
J=8.8, 2.4 Hz, 1H), 4.43 (d, J=7.6 Hz 1H), 3.95 (dd,
J=7.6, 4.3 Hz, 1H), 3.77 (s, 3H), 3.68 (m,1H), 3.11 (m,
2H), 2.58 (s, 3H), 13C NMR (DMSO) d 177.1, 176.7,
153.3, 137.2, 135.6, 134.5, 134.2, 131.1, 130.2, 129.8
(2C), 126.9, 126.7 (2C), 124.9, 124.4, 123.3 (2C), 120.2,
113.2, 111.6, 110.5, 102.9, 101.9, 55.4, 43.2, 40.8, 30.7,
25.4, 24.1; HRMS (FAB) calcd for C30H25N3O5S
In conclusion, the methodology applied in this work is
highly suitable for the synthesis of open indolocarbazole
analogues. Although not highly potent, many of these
derivatives displayed cytotoxicity against the cell lines
assayed, thereby stimulating the preparation of new
related compounds.
Experimental
General methods
Melting points were determined on a Buchi 510 appa-
ratus and are uncorrected. IR spectra were recorded on
Nicolet Impact 410 spectrophotometer and the values

3418
E.Caballero et al./ Bioorg.Med.Chem.11 (2003) 3413–3421
539.1515, found m/z 540.1593. Anal. calcd for
C30H25N3O5S: C, 66.78, H, 4.67, N, 7.79, S, 5.94, found:
C, 66.83, H, 4.80, N, 7.96, S, 5.77.
allowed to react for 14 h at room temperature, diluted
in CHCl3, washed with brine, and dried over Na2SO4.
Solvent evaporation and purification by diethyl ether
precipitation or column chromatography yielded the
corresponding N-alkylated product.
(ꢁ)-(3aR,10R,10aS)-10-(N-phenylsulphonylindol-3-yl)-2-
methyl-8-methoxy-3a,4,10,10a-tetrahydro-5H-pyrrolo[3,4-
b]carbazole-1,3-dione (21). Rf (hexane/AcOEt 1:1) 0.24;
mp 265 ꢂC (ether/MeOH). IR (KBr): 3421, 1745, 1697,
6-(2,3-epoxypropyl)-9-methoxy-2-phenyl-4-(3,4,5-trime-
thoxyphenyl)-6H-pyrrolo[3,4-c]carbazole-1,3-dione (33).
Using procedure A and epichlorohydrin (2 mmol),
after CC (hexane/EtOAc 4:6) product 33 was obtained
in 23% yield. Using procedure B, 33% of 33 was
obtained; yellow solid; IR (KBr): 1761, 1710, 1587
1
1173 cmꢀ1; H NMR (DMSO) d 8.32 (brs, 1H), 6.8–7.9
(m, 11H), 6.76 (dd, J=8.6, 2.2 Hz, 1H), 6.56 (d, J=2.2
Hz, 1H), 5.07 (d, J=6.8 Hz, 1H), 3.65 (dd, J=17.8, 3.0
Hz, 1H), 3.60 (s, 3H), 3.53 (dd, J=9.8, 6.8 Hz, 1H), 3.38
(td, J=9.8, 3.0 Hz, 1H), 3.12 (dd, J=17.8, 9.8 Hz, 1H),
1.93 (s, 3H). 13C NMR (DMSO) d 178.9, 177.5, 154.1,
137.6, 135.1, 133.8, 132.6, 131.4, 130.6, 129.1 (2C), 127.1
(2C), 126.1, 125.7, 125.1, 123.1, 122.2, 121.1, 114.1,
113.2, 111.7, 110.6, 100.1, 55.8, 46.1, 38.9, 29.8, 23.7,
18.8. HRMS (FAB) calcd for C30H25N3O5S 539.1515,
found m/z 540.1291. Anal. calcd for C30H25N3O5S: C,
66.78; H, 4.67, N: 7.79; S, 5.94, found: C, 66.36; H, 4.33;
N, 7.36; S, 5.41.
1
cmꢀ1; H NMR d 8.73 (d, J=2.6 Hz, 1H), 7.66 (s, 1H),
7.40–7.60 (m, 5H), 7.45 (d, J=8.8 Hz, 1H), 7.27 (dd,
J=8.8, 2.6 Hz, 1H), 6,89 (s, 2H), 4.81 (dd J=15.9, 2.6
Hz, 1H), 4.36 (dd J=15.9, 5.5 Hz, 1H), 3.98 (s, 3H),
3.93 (s, 9H), 3.36 (m, 1H), 2.85 (t, J=4.6 Hz, 1H), 2.55
(dd, J=4.6, 2.6 Hz, 1H); 13C NMR d 168.1, 167.9,
155.3, 152.8 (2C), 145.2, 138.4 (2C), 137.0, 133.2 (3C),
129.2 (2C), 128.0, 127.2 (2C), 121.2 (2C), 119.3, 119.0,
115.3, 110.0, 107.7, 107.4 (2C), 61.1, 56.4 (2C), 56.1,
50.6, 45.0, 44.9; MS (FAB) m/z: 564 (M+, 32).
Aromatisation and deprotection of the N-sulphonyl
group. 250 mg (0.46 mmol) of tetrahydro-
pyrrolocarbazole 13 dissolved in 20 mL of toluene was
reacted with 1 mmol of DDQ for 3 h at reflux. The
crude reaction mixture was diluted in EtOAc, washed
with NaHCO3 and brine, dried, and evaporated. By
precipitation in CHCl3, 176 mg (70%) of dehydro-
genated product were obtained. This product was trea-
ted, in THF, with 4 equiv of TBAF and maintained
under reflux for 24 h. By evaporation of the solvent and
chromatography (silica gel, hexane/AcOEt 1:1), 30 (68
mg, 61%) was isolated. Mp 210 ꢂC (ether/MeOH). IR
(KBr): 3311, 1750, 1691, 1026 cmꢀ1; 1H NMR (DMSO)
d 8.48 (d, J=2.0, 1H), 7.85 (s, 1H), 7.84 (s, 1H), 7.66
(m, 2H), 7.66 (d, J=7.6 Hz, 1H), 7.49 (d, J=8.1 Hz,
1H), 7.48 (d, J=7.6 Hz, 1H), 7.17 (dd, J=8.1, 2.0 Hz,
1H), 7.16 (t, J=7.1 Hz, 1H), 7.07 (t, J=6.8 Hz, 1H),
3.87 (s, 3H), 3.06 (s, 3H). 13C NMR (DMSO) d 168.9,
168.8, 153.6, 144.8, 136.8, 136.1, 130.8, 127.5, 127.2,
126.1, 121.4, 120.6, 119.6, 118.8, 118.2, 117.3, 116.9,
115.6, 112.2, 111.9, 110.9, 107.1, 55.4, 23.1; HRMS
(FAB) calcd for C24H17N3O3 395.1270, found m/z
396.1348.
4-(2,5-dimethoxyphenyl)-6-(2,3-epoxypropyl)-2-methyl-9-
methoxy-6H-pyrrolo[3,4-c]carbazole-1,3-dione (34). Using
procedure A and epichlorohydrin (2 mmol), by CC
(hexane/EtOAc 4:6) 42% of 34 was obtained; yellow
solid; IR (KBr): 1703, 1593, 1503, 1126 cmꢀ1; 1H NMR
d 8.67 (d, J=2.6 Hz, 1H), 7.43 (s, 1H), 7.41 (d, J=8.8
Hz, 1H); 7.24 (dd, J=8.8, 2.6 Hz, 1H), 6.95 (m, 3H),
4.68 (dd, J=15.7, 4.6 Hz, 1H), 4.32 (dd, J=15.7, 5.1
Hz, 1H), 4.01 (s, 3H), 3.81 (s, 3H), 3.71 (s, 3H), 3.34 (m,
1H), 3.18 (s, 3H), 2.81 (t, J=4.6 Hz, 1H), 2.54 (dd,
J=4.6, 2.6 Hz, 1H) ; 13C NMR d 170.9, 170.1, 155.0,
153.4, 151.2, 144.9, 138.6, 135.2, 133.6 (2C), 121.8 (3C),
118.6, 117.2, 114.9 (2C), 111.9, 110.0, 107.5, 56.3, 56.0
(2C), 50.4, 45.1 (2C), 23.8.
4-(3,4-dimethoxyphenyl)-6-(2,3-epoxypropyl)-2-methyl-9-
methoxy-6H-pyrrolo[3,4-c]carbazole-1,3-dione (35). Using
procedure A and epichlorohydrin (2 mmol), by CC
(hexane/EtOAc 4:6), 41% of 35 was obtained); yellow
solid; IR (KBr): 1751, 1603, 1519, 1120 cmꢀ1; 1H NMR
d 8.71 (d, J=2.2 Hz, 1H), 7.54 (s, 1H), 7.42 (d, J=8.8
Hz, 1H), 7.16–7.28 (m, 3H), 7.00 (d, J=8.4 Hz, 1H),
4.75 (dd, J=16.1, 2.6 Hz, 1H), 4.32 (dd, J=16.1, 5.1
Hz, 1H), 4.03 (s, 3H), 3.97 (s, 6H), 3.35 (m, 1H), 3.22 (s,
3H), 2.84 (t, J=4.7 Hz, 1H), 2.54 (dd, J=4.7, 2.6 Hz,
1H); 13C NMR d 169.2, 169.1, 155.1, 149.3, 149.0,
145.5, 137.9, 137.7, 130.4, 128.4, 122.3, 121.3, 120.0,
119.0, 118.7 114.5, 113.5, 110.8, 109.9, 107.7, 56.2 (3C),
50.6, 45.0 (2C), 23.9.
General procedures for N-alkylation of pyrrolo[3,4-c]car-
bazole: (A) Phase transfer conditions. To a suspension of
the corresponding pyrrolo[3,4-c]carbazole (1 mmol) in
benzene (1 mL) were added a solution of NaOH (50%)
(0.5 mL), tetra-N-butyl ammonium hydrogen sulphate
(0.5 mmol), and the alkylating agent. The reaction mix-
ture was allowed to react for 14 h at room temperature,
after which it was diluted in CHCl3, washed with brine,
and dried over Na2SO4. Solvent evaporation followed
by column chromatography afforded the N-alkylated
product.
6-(2-cyanoethyl)-2-phenyl-9-methoxy-4-(3,4,5-trimethoxy-
phenyl)-6H-pyrrolo[3,4-c]
carbazole-1,3-dione
(39).
Using procedure A and 1,1 mmol of 3-bromopropio-
nitrile, by CC (hexane/EtOAc 3:7) 23% of 39 was
obtained; yellow solid; IR (KBr): 2260, 1709, 1586
1
(B) NaH in DMF. To a solution of the corresponding
pyrrolo[3,4-c]carbazole (1 mmol) in dry DMF (3.3 mL),
under Ar, was added NaH (80%) (1.5 mmol). The
reaction mixture was stirred for 50 min and then the
alkylating agent (2 mmol) was added. The mixture was
cmꢀ1; H NMR d 8.76 (d, J=2.5 Hz, 1H), 7.68 (s, 1H),
7.40–7.55 (m, 5H), 7.37 (d, J=8.9 Hz, 1H), 7.29 (dd,
J=8.9, 2.5 Hz, 1H), 6.90 (s, 2H), 4.73 (t, J=6.5 Hz,
2H), 3.98 (s, 3H), 3.92 (s, 3H), 3.91 (s, 6H), 2.93 (t,
J=6.5 Hz, 2H); 13C NMR d 167.8, 167.4, 155.5, 152.8

E.Caballero et al./ Bioorg.Med.Chem.11 (2003) 3413–3421
3419
(2C), 144.1, 138.6, 138.2, 136.2, 133.7, 132.7, 131.9,
129.1 (2C), 128.0, 127.0 (2C), 121.7, 119.5, 119.4, 119.0,
117.1 (2C), 114.7, 109.1, 108.1, 107.3 (2C), 60.9, 56.3
(2C), 56.0, 39.3, 17.7; HRMS (FAB) calcd for
C33H27N3O6 561.1900, found m/z 562.1978.
(m, 1H), 4.01 (s, 3H), 3.97 (s, 6H), 3.20 (s, 3H), 2.45 (dd,
J=11.1, 9.9 Hz, 1H); 2.32 (dd, J=11.1, 4.0 Hz, 1H),
2.25 (s, 6H); 13C NMR d 169.4, 169.0, 155.0, 149.3,
148.4, 145.5, 137.9, 137.7, 131.0, 128.9, 122.3, 121.3
(2C), 118.7, 118.5, 114.9, 113.7, 110.8, 110.2, 107.6,
66.9, 62.8, 56.2, 56.0 (2C), 47.8, 45.4 (2C), 23.8; HRMS
(FAB) calcd for C29H31N3O6 517.2213, found m/z
518.2284.
6-methyl-9-methoxy-4-(2-nitrophenyl)-2-phenyl-6H-pyr-
rolo[3,4-c]carbazole-1,3-dione (40). Using procedure B,
and purification by precipitation in diethyl ether 70% of
40 was obtained, yellow powder; IR (KBr): 1708, 1608,
1520 cmꢀ1; 1H NMR (DMSO) d 8.46 (d, J=2.7, 1.5 Hz,
1H), 8.23 (dd, J=8.0, 1.5 Hz, 1H), 8.00 (s, 1H), 7.87 (td,
J=8.0, 1.5 Hz, 1H), 7.75 (td, J=8.0, 1.5 Hz, 1H), 7.30–
7.70 (m, 8H), 3.98 (s, 3H), 3.86 (s, 3H); 13C NMR
(DMSO) d 167.5, 167.3, 154.3, 148.2, 145.0, 137.9,
133.8, 133.2, 132.8 (4C), 129.7, 128.8 (2C), 127.9, 127.3
(2C), 125.6, 124.2, 119.9 (2C), 118.0, 115.0, 111.2, 106.8,
55.5, 29.9; MS (FAB) m/z: 478 (M++H, 4).
General procedure for the preparation of ꢀ-tetra-O-acet-
ylglucosides. A mitxture of the corresponding pyr-
rolo[3,4-c]carbazole (1 mmol) in benzene (375 mL),
2,3,4,6-tetraacetyl-a-d-glucopyranosyl
bromide
(5
mmol) and Ag2O (10 mmol) was refluxed for 3 h and
then filtered off. The filtrate was evaporated and pur-
ified by flash chromatography to give the N-a-tetra
acetylglucoside.
(ꢁ)-(3aS,4S,10cS)-9-methoxy-2-phenyl-6-(2,3,4,6-tetra-
O-acetyl-ꢀ-D-glucopyranosyl)-4-(3,4,5-trimethoxyphenyl)-
3a,4,5,10c- tetrahydro- 6H -pyrrolo[3,4- c]carbazole- 1,3-
dione (41). CC (hexane/EtOAc 3:7) 27%; yellow solid;
IR (KBr): 1748, 1592, 1586, 1502 cmꢀ1; 1H NMR d 7.81
(d, J=9.1 Hz, 1H), 7.46 (d, J=2.2 Hz, 1H), 7.40 (m,
3H), 6.88 (dd, J=9.1, 2.2 Hz, 1H), 6.71 (m, 2H), 6.48 (s,
2H), 5.75 (d, J=5.9 Hz, 1H), 5.27 (m, 1H), 4.96 (bd,
J=9.9 Hz, 1H), 4.42 (d, J=7.3 Hz, 1H), 4.40–4.15 (m,
3H), 4.00–3.40 (m, 5H), 3.92 (s, 3H), 3.79 (s, 3H), 3.61
(s, 6H), 2.00–2.15 (s, 12H); 13C NMR d 176.4, 175.5,
171.5, 170.8, 170.2, 169.7, 154.9, 153.1 (2C), 136.9,
136.0, 134.6, 131.9, 131.0, 129.1 (2C), 128.6, 127.3, 126.4
(2C), 112.5, 111.4, 105.7 (2C), 104.0, 102.1, 96.9, 73.2,
69.9, 68.3, 67.4, 66.7, 60.9, 56.0 (2C), 55.7, 46.2, 40.7,
40.3, 27.2, 24.6, 20.8 (3C); MS (EI) m/z: 842 (M+, 12).
General procedure for the preparation of N-(3-dimethyl-
amino-2-hydroxypropyl)-pyrrolo[3,4-c]carbazoles. A solu-
tion of the corresponding epoxide (1 mmol) in EtOH
and another solution of dimethylamine in EtOH (30%)
(3.8 mL) was stirred at room temperature for 3 h. Sol-
vent evaporation and—sometimes—purification by
flash chromatography afforded the amino-alcohol.
6-(3-dimethylamino-2-hydroxypropyl)-9-methoxy-2-phenyl-
4-(3,4,5-trimethoxyphenyl)-6H-pyrrolo[3,4-c]carbazole-
1,3-dione (36). By CC (EtOAc/MeOH 1:1) 76% of 36
was obtained; yellow solid; IR (KBr): 3446, 1709, 1586
1
cmꢀ1; H NMR d 8.79 (d, J=2.2 Hz, 1H), 7.72 (s, 1H),
7.20–7.65 (m, 7H), 6.61 (s, 2H), 4.45 (m, 2H), 4.23 (m,
1H), 4.01 (s, 3H), 3.95 (s, 3H), 3.94 (s, 6H), 2.52 (t,
J=11.1, 1H), 2.41 (dd, J=11.1, 2.2 Hz, 1H), 2.31 (s,
6H); 13C NMR d 168.1, 167.6, 155.0, 152.7 (2C), 145.5,
138.1 (2C), 138.0, 133.2 (2C), 132.1, 129.0 (2C), 127.8,
127.0 (2C), 121.1, 119.1, 118.7, 118.6, 115.7, 110.2,
107.6, 107.4 (2C), 66.9, 62.7, 60.9, 56.1 (2C), 55.9, 47.7,
45.7 (2C); HRMS (FAB) calcd for C35H35N3O7
609.2475, found m/z 610.2552.
9-methoxy-2-phenyl-6-(2,3,4,6-tetra-O-acetyl-ꢀ-D-gluco-
pyranosyl)-4-(3,4,5-trimethoxyphenyl)-6H-pyrrolo[3,4-c]-
carbazole-1,3-dione (42). CC (hexane/EtOAc 4:6) 50%;
;
yellow solid; IR (KBr): 1750, 1712, 1586 cmꢀ1 1H
NMR d 8.80 (d, J=2.8 Hz, 1H), 8.31 (s, 1H), 8.03 (d,
J=9.3 Hz, 1H), 7.35–7.60 (m, 5H), 7.21 (dd, J=9.3, 2.8
Hz, 1H), 6.91 (s, 2H), 5.81 (d, J=5.5 Hz, 1H), 5.34 (m,
1H), 4.99 (bd, J=9.5 Hz, 1H), 4.25–4.30 (m, 1H), 4.15–
4.25 (m, 3H), 3.94 (s, 3H), 3.93 (s, 3H), 3.92 (s, 6H),
2.05–2.20 (s, 12H); 13C NMR d 170.7, 169.7 (2C), 169.0,
167.9, 167.4, 155.4, 152.8 (2C), 143.5, 138.7, 138.3,
136.0, 133.2 (2C), 132.0, 129.1 (2C), 128.0, 127.1 (2C),
122.7, 121.7, 120.0, 119.1, 118.6, 113.8, 107.5 (3C), 97.1,
73.1, 69.7, 68.4, 67.1, 63.0, 61.0, 56.4 (2C), 55.9, 22.6,
20.9, 20.7 (2C); MS (FAB) m/z: 839 (M++H, 12); [a]D25
1.50 (c 0.07 in CH2Cl2).
6-(3-dimethylamino-2-hydroxypropyl)-4-(2,5-dimethoxy-
phenyl)-9-methoxy-2-methyl-6H-pyrrolo[3,4-c]carbazole-
1,3-dione (37). (95%); yellow solid; IR (KBr): 3377,
1
1699, 1605, 1518 cmꢀ1; H NMR d 8.68 (d, J=2.6 Hz,
1H), 7.59 (s, 1H), 7.44 (d, J=8.8 Hz), 7.23 (dd, J=8.8
Hz, J=2.6 Hz, 1H), 6.95 (m, 3H), 4.35 (m, 2H), 4.15 (m,
1H), 4.02 (s, 3H), 3.81 (s, 3H), 3.71 (s, 3H), 3.18 (s, 3H),
2.42 (t, J=10.9 Hz, 1H), 2.32 (dd, J=10.9, 6.9 Hz, 1H),
2.23 (s, 6H); 13C NMR d 170.0, 169.0, 154.8, 153.3,
151.5, 145.3, 138.1 (2C), 133.3, 133.2, 121.3 (2C), 119.0,
118.4, 117.2, 115.4, 113.9, 111.9, 110.4, 107.3, 66.9, 62.8,
56.3, 55.9 (2C), 47.6, 45.6 (2C), 23.7; HRMS (FAB)
calcd for C29H31N3O6 517.2213, found m/z 518.2263.
Preparation of 9-methoxy-6-ꢀ-glucopyranosyl-4-(3,4,5-
trimethoxyphenyl)-6H-pyrrolo[3,4-c]carbazole-1,3-dione
(43). To a solution of 42 (1 mmol) in MeOH (220 mL)
was added NH4OH (33% in water) (100 mL). The
reaction mixture was stirred for 4 h at room tempera-
ture. The solvent was removed and the residue dissolved
in EtOAc and washed with brine. The organic phase
was dried over Na2SO4 and the solvent was evaporated.
Purification by column chromatography (EtOAc/
MeOH 97:3, 39%) led to 43 as a yellow powder; IR
6-(3-dimethylamino-2-hydroxypropyl)-4-(3,4-dimethoxy-
phenyl)-9-methoxy-2-methyl-6H-pyrrolo[3,4-c]carbazole-
1,3-dione (38). (95%); yellow solid; IR (KBr): 3338,
1
1702, 1633 cmꢀ1; H NMR d 8.70 (d, J=2.7 Hz, 1H),
7.61 (s, 1H), 7.42 (d, J=9.1 Hz, 1H), 7.24 (dd, J=9.1,
2.7 Hz, 1H), 7.21 (d, J=2.6 Hz, 1H), 7.20 (dd, J=8.5,
2.6 Hz, 1H), 7.00 (d, J=8.5 Hz, 1H), 4.37 (m, 2H), 4.17
1
(KBr): 3350, 1713, 1588 cmꢀ1; H NMR (CD3OD) d

3420
E.Caballero et al./ Bioorg.Med.Chem.11 (2003) 3413–3421
8.39 (d, J=2.7 Hz, 1H), 8.23 (s, 1H), 7.97 (d, J=9.1 Hz,
1H), 7.10 (dd, J=9.1, 2.7 Hz, 1H), 6.91 (s, 2H), 5.81 (d,
J=5.2 Hz, 1H), 3.20–4.20 (m, 5H), 3.76 (s, 9H), 3.73 (s,
3H); 13C NMR (CD3OD) d 169.8, 169.6, 156.1, 153.9
(2C), 140.9, 139.2, 139.0 (2C), 137.4, 135.2, 123.7, 121.8,
119.5, 119.0, 118.9, 114.9, 108.4 (3C), 99.5, 77.8, 75.0,
73.9, 70.5, 61.2 (2C), 56.8 (2C), 56.0; MS (FAB) m/z:
594 (M+, 10); [a]D25 1.20 (c 0.06 in CH2Cl2).
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Financial support came from the Spanish MCYT and
the European FEDER Funds for the Project PPQ2000-
1179, and from Junta de Castilla y Leon (Consejerıa de
Educacion y Cultura) for the Project SA048/03. S. Cal-
deron thanks the Spanish MECyD for a predoctoral
grant.
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