Indolocarbazoles: Potent, selective inhibitors of human cytomegalovirus replication

Article abstract of DOI:10.1016/S0968-0896(99)00032-2

In our search for new, safer anti-HCMV agents, we discovered that the natural product Arcyriaflavin A (1a) was a potent inhibitor of HCMV replication in cell culture. A series of analogues (symmetrical indolocarbazoles) was synthesised to investigate structure-activity relationships in this series against a range of herpes viruses (HCMV, VZV, HSV1, and 2). This identified a number of novel, selective and potent inhibitors of HCMV, 12,13-dihydro-2,10-difluoro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-(6H)-dione (1d) being the best example (IC₅₀=40nM, therapeutic index >1450). Compounds described in this series were generally poor inhibitors of protein kinase C βII, and no correlation was found between the ability to inhibit HCMV and the enzyme PKC. Copyright (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(99)00032-2

Bioorganic & Medicinal Chemistry 7 (1999) 1067±1074
Indolocarbazoles: Potent, Selective Inhibitors of Human
Cytomegalovirus Replication
Martin J. Slater,* Stuart Cockerill, Robert Baxter, Robert W. Bonser, Kam Gohil,
Clare Gowrie, J. Edward Robinson, Edward Littler, Nigel Parry, Roger Randall
and Wendy Snowden
GlaxoWellcome Medicines Research Centre, Gunnels Wood Road, Stevenage, SG1 2NY, UK
Received 7 August 1998; accepted 21 October 1998
AbstractÐIn our search for new, safer anti-HCMV agents, we discovered that the natural product Arcyria¯avin A (1a) was a
potent inhibitor of HCMV replication in cell culture. A series of analogues (symmetrical indolocarbazoles) was synthesised to
investigate structure±activity relationships in this series against a range of herpes viruses (HCMV, VZV, HSV1, and 2). This iden-
ti®ed a number of novel, selective and potent inhibitors of HCMV, 12,13-dihydro-2,10-di¯uoro-5H-indolo[2,3-a]pyrrolo[3,4-c]car-
bazole-5,7-(6H)-dione (1d) being the best example (IC50=40 nM, therapeutic index >1450). Compounds described in this series
were generally poor inhibitors of protein kinase C bII, and no correlation was found between the ability to inhibit HCMV and the
enzyme PKC. # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
and bacterial phosphotransferases, and is capable of
phosphorylating ganciclovir.12,13 We thus screened
known kinase inhibitors and discovered the simple,
unsubstituted indolocarbazole Arcyria¯avin A (1a) to
be a potent, selective inhibitor of HCMV replication in
vitro. Herein we describe the synthesis and anti-
HCMV activities of novel ring substituted Arcyria-
¯avin analogues.
Human cytomegalovirus (HCMV) disease is the most
common life-threatening opportunistic viral infection in
the immunocompromised. It is the primary cause of
death in recipients of bone marrow and renal trans-
plants,1,2 and is the most prevalent serious infection in
AIDS patients, frequently giving rise to pneumonitis
and retinitis.3,4 Current treatments are limited to ganci-
clovir (GCV), foscarnet (PFA) and cidofovir
(HPMPC).5±7 While these drugs have made a signi®cant
contribution to the treatment of HCMV disease, each
has major drawbacks, and all su€er from poor oral
bioavailability. GCV has signi®cant myelosuppressive
activity,5 whilst foscarnet and cidofovir are associated
with signi®cant renal toxicity.8,9 All three anti-HCMV
agents exert their primary antiviral e€ects by inhibition
of viral DNA polymerase. GCV and cidofovir may also
be incorporated into viral DNA.10,11 In the search for
more potent, selective, novel anti-HCMV drug candi-
dates, we therefore considered targeting alternative viral
protein functions.
Protein kinase C (PKC) isoenzymes are a family of
phospolipid-dependent serine/threonine protein kinases
that play an important role in signal transduction path-
ways. Consequently, PKC controls a large variety of
cellular processes such as proliferation, di€erentiation
and smooth muscle contraction.14 Since disruption of
these processes have severe implications for many phy-
siological functions, it was important to identify com-
pounds that are selective for HCMV and have no e€ect
on PKC. We thus chose to prepare analogues main-
taining the imide group, since the staurosporine agly-
cone (10) is known to be a better PKC inhibitor than 1a
(ꢀ20-fold).15
HCMV does not appear to code for a thymidine kinase.
The HCMV gene UL97 encodes for a protein that
shares homology with protein kinases, guanylyl kinase
Results and Discussion
Chemistry
Key words: Human cytomegalovirus; antiviral; indolocarbazoles;
protein kinase C; inhibitors.
*Corresponding author. Tel.: 44 (0) 1438 763416; fax: 44 (0) 1438
763616; e-mail: MJS40312@glaxowellcome.co.uk
In order to investigate the e€ect of substitution in the
benzene nuclei of 1a, the preparation of bis-indolylma-
leimides16 and their oxidative cyclisation17 to indolo-
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00032-2

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M. J. Slater et al. / Bioorg. Med. Chem. 7 (1999) 1067±1074
carbazoles appeared to be the most versatile meth-
od.18a±h Additionally, this method provided access to
bis-indolylmaleimides, allowing us to contrast antiviral
activities of the indolocarbazoles and bis-indolylmale-
imides in vitro. Thus, indole magnesium bromides were
readily prepared (from indoles 3) and reacted with 2,3-
dichloro-N-methylmaleimide (2) to a€ord the deep red
bis-indolyl-N-methylmaleimides (4a±f) along with the
yellow mono substitution adducts. These were readily
separated by ¯ash chromatography over silica (Scheme 1).
Conversion to the corresponding maleic anhydrides
(5a±f) with aqueous hydroxide and then to the parent
bis-indolylmaleimides (6a±f) using hot ammonium ace-
tate was performed in good yields over two steps.17
Aromatisation to the planar, yellow, ¯uorescent, indo-
locarbazole targets (1a±f) was achieved using 2,3-
dichloro-5,6-dicyanobenzoquinone (DDQ) and catalytic
p-toluene sulphonic acid (PTSA) in a mixture of toluene
and dioxan under re¯ux. The DDQ/PTSA method
failed with the 4,8-di¯uoro-bis-indolylmaleimide (6f),
resulting in considerable decomposition from which the
product could not be isolated. A possible explanation is
that aromatisation forces the ¯uorine substituents into
close proximity to the imide carbonyl atoms. We there-
fore sought an alternative procedure. Photocyclisation
in isopropanol in the presence of iodine and air19 also
gave a complex mixture of products. Fortunately, clean
conversion to the required product (1f) was accom-
plished photochemically in isopropanol using an excess
of iodine in an inert atmosphere, removing oxidative
side reactions.20 The 2,10-dihydroxy (1g) and 3,9-dihy-
droxy (1h) derivatives were prepared from the corre-
sponding dimethoxy indolocarbazoles using neat
pyridine hydrochloride in a sealed tube.18a
Figure 1. Structures of Arcyria¯avin A and Staurisporine aglycone.
Scheme 1. Synthesis of indolocarbazoles. Reagents: (i) a. EtMgBr, THF, benzene b. 2, THF, benzene; (ii) KOH, MeOH or dioxan; (iii) NH4OAc,
140 ꢁC; (iv) DDQ, PTSA, toluene, 100 ꢁC for examples 1a±1e, hn, iodine, isopropanol for example 1f. For conversions 1c into 1g and 1e into 1h,
pyridine hydrochloride, 120 ꢁC. For conversion 1a into 1i, N-chlorosuccinimide, MeCN. For conversion 1a into 1j, N-bromosuccinimide, MeCN.

M. J. Slater et al. / Bioorg. Med. Chem. 7 (1999) 1067±1074
1069
Figure 2.
It was reasoned that the 3,9-dichloro (1i) and 3,9-
dibromo (1j) analogues could be synthesised from 1a
using the appropriate N-halo succinimide in acetonitrile,
since these sites are favoured for electrophilic and radi-
cal substitution in carbazoles. This was indeed the case,
the products being less soluble than 1a and the mono-
halogenated intermediates, allowing puri®cation by
recrystallisation. To investigate the e€ect of the con-
formation of the indole groups on antiviral activity, the
cis (7) and trans (8) bis-indolylsuccinimides were pre-
pared from 6a by literature routes.21
therapeutic index of 1450 based on the CCID50 in Vero
cells. 1,11 (1b) and 4,8 (1f) disubstitution resulted in loss
of anti-HCMV activity.
As 1a was found to have a similar level of PKC inhibi-
tion against PKC from rat brain and recombinant
PKCbII, we chose to routinely use the latter as this was
experimentally more convenient. Thus, we could readily
prepare one large batch of protein with consistent
properties and providing consistent inhibition data.
Generally, the indolocarbazoles (1a±j) are poor inhibi-
tors of PKCb, and there is no clear correlation between
anti-HCMV activity and inhibition of PKCb, suggesting
that this is not the mode of action for these compounds.
Indeed, the introduction of 2,10 or 3,9 dihydroxy
groups (in 1g and 1h) appears to promote inhibition of
PKC and reduce activity against HCMV. The ability of
the 3,9-dihydroxy derivative (1h) to inhibit PKC
(IC50=0.5 mM) is particularly noteworthy. Stauro-
sporine itself was rather toxic in MDCK cells and it was
not possible to measure any antiviral activities, whilst
the bis-indolylsuccinimides (7 and 8) were inactive,
con®rming the importance of planarity for anti-HCMV
activity.
Biology
Antiviral ecacy was measured by plaque assay against
a panel of herpes viruses (HCMV, HSV-1, HSV-2,
VZV), initially at 10 and 100 mM. GCV was included for
comparison. The bis-indolylmaleimides (6a±f) were
generally toxic in the range 10±100 mM under the assay
conditions, and inactive below 10 mM. Antiviral activity
is restricted to indolocarbazoles, and these are selective
for the inhibition of HCMV (Table 1, only HCMV
antiviral data shown). The imide NH was found to be
essential for anti-HCMV activity, as replacement of the
proton by a methyl group as in 9 completely abolished
activity.18d Interestingly, the imide proton is also essen-
tial for inhibition of protein kinase enzymes. Good anti-
HCMV activity is maintained with symmetrical sub-
stituents in the 2, 10 or 3, 9 sites (see examples 1c, 1d, 1i,
1j). Indeed, the 2,10-di¯uoroindolocarbazole (1d) is the
most potent in the series with an IC50 of 40 nM and a
Arcyria¯avin A analogues represent a novel class of
antiviral compounds, selective for the inhibition of
HCMV. The mode of action is not known, but does not
appear to be related to the inhibition of PKC. To the
best of our knowledge, this is the ®rst report of indolo-
carbazoles with selective antiviral activity unrelated to
PKC inhibition. The closest literature example we are
aware of is the reported anti-HIV-1 activity of the
indolocarbazole lactam Go 6976 (Fig. 3). This antiviral
e€ect is due to the prevention of PKC-mediated induc-
tion of viral replication in latently infected cells, and is
speci®c for the U1 cell line.22 We thought it would be
interesting nevertheless to examine whether indolo-
carbazoles 1a±j were able to inhibit the replication of
Table 1. Anti-HCMV activities, PKC inhibition data and cell
cytotoxicities of indolocarbazoles and selected compounds
HCMV
(IC50/mM)
CCID50/mM
PKC: % inhibition
at 100 mM
1a
1b
1c
1d
1e
1f
1g
1h
1i
0.2
>10
0.5
0.04
10
>10
4.6
>10
0.16
0.6
T100
>10
>10
>100
3
>125
>31
<8
<50
32
43
59
58
>60
>125
84
>31
>125
>125
34
IC50=60 mM
1
IC50=50 mM
IC50=0.5 mM
18
1j
6a
7
0
IC50=5 mM
143
101
6
11
2
8
9
GCV
ꢀ100
>500
Not tested
T100=unable to measure an antiviral e€ect due to cell toxicity at
100 mM.
Figure 3. Structure of Go 6976.

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M. J. Slater et al. / Bioorg. Med. Chem. 7 (1999) 1067±1074
HIV-1 in MT4 cells.23 No activity was observed up to a
concentration of 50 mM.
were prepared by sonication in 250 mM Tris.HCl pH
7.5. Samples of diluted enzyme (8.8 mg protein/10 mL)
were added to microtubes containing 30 mL reaction
bu€er (25 mM Tris.HCl pH 7.5, 8.35 mM EGTA,
8.8 mM CaCl2, 16.7 mM MgCl2, 160 mM glycogen syn-
thase peptide substrate (PLSRTLSVAAKK), 5 mCi/mL
[g32P]-ATP), 10 mL prepared micelles and 1 mL inhibitor
or vehicle (DMSO). This mixture gives a bu€ered free
calcium level of 100 mM. After 15 min at room temp.,
40 mL samples were spotted onto squares of P81 paper
and washed three times with 75 mM orthophosphoric
acid. The amount of [32P]-labelled peptide bound to the
P81 paper was quantitated by liquid scintillation count-
ing in 10 mL Pico¯uor scintillant.
The UL97 protein was conceptually the target that lead
to the discovery of these inibitors, and has recently been
shown to be a kinase capable of autophosphorylation
on serine and threonine.24 The mode of action of indo-
locarbazoles could be inhibition of UL97, an alternative
viral kinase, or inhibition of an induced or elevated host
kinase.25,26 Further experiments to identify the mode
of action of these inhibitors are under way in our
laboratories.
In combination experiments using constant drug ratios
of 1a and GCV, the combination was found to have an
additive antiviral e€ect. Thus, it should be possible to
use indolocarbazole inhibitors in combination with
GCV to treat HCMV disease.
Antiviral assay
HCMV activity was determined by plaque reduction
assay as previously described, using strain AD169 in
MRC 5 cells.28 For IC50 determination, a dose±response
curve was obtained from which the 50% inhibitory
concentration (IC50) was calculated.
Conclusion
We have discovered Arcyria¯avin A (1a) to be a potent,
selective inhibitor of HCMV replication in cell culture.
A series of novel symmetrical analogues have been pre-
pared to investigate the SAR, a€ording a number of
very active compounds, such as the 2,10-di¯uoro deri-
vative (1d), which is approximately 100 times more
potent than GCV in vitro. Indolocarbazoles can be used
in conjunction with GCV, and warrant further investi-
gation as potential candidates for the treatment of
HCMV disease. Anti-HCMV activity does not appear
to be related to the inhibition of protein kinase C. The
synthesis and evaluation of further analogues are
underway in our laboratories and will be reported in
due course.
Cytotoxicity assay
Cytotoxicity determinations were performed in Vero
cells as previously reported.28 The concentration
required for a 50% inhibition of cell growth compared
to control cells grown in the absence of compound is
termed the CCID50. The yellow-orange color of many
of the compounds in this study interfered with the tet-
razolum dye measurement (MTT). We therefore intro-
duced a modi®cation in which the compound was
washed from the cells prior to visualisation with the dye.
In many cases, it was only possible to de®ne a lower
limit for toxicity due to the poor solubility of the com-
pounds in the assay conditions.
Experimental
PKC assay
Chemistry
General
Production of recombinant PKCbII (kindly donated by
Dr Christy King, GlaxoWellcome, RTP, North Car-
olina) was carried out by the infection of Sf9 cells with
recombinant baculovirus stocks in spinner ¯asks and
culturing for 5 days at room temperature. Cells were
harvested, washed with PBS, and suspended in 0.002
volumes of lysis bu€er (20 mM Tris.HCl pH 7.5, 2 mM
EDTA, 5 mM EGTA, 1% Triton X-100, 1 mM PMSF).
Cells were lysed by homogenisation in a Potter homo-
genizer and the soluble fraction obtained after cen-
trifugation at 100,000 g for 60 min at 4 ꢁC. This fraction
was then loaded onto a DE52 anion exchange column
equilibrated with bu€er A (25 mM Tris.HCl pH7.5,
10 mM 2-mercaptoethanol, 10% glycerol). Proteins
were eluted in a linear gradient from bu€er A to bu€er
B (bu€er A containing 500 mM NaCl). Fractions were
assayed for phorbol ester dependent PKC activity, and
the active fractions pooled and stored at 70 ꢁC.
2,3-Dichloromaleic anydride was purchased from
Aldrich. Thin-layer chromatography (TLC) was per-
formed on E. Merck 60F254 No. 5719 glass backed
plates. Flash column chromatography was conducted
using E. Merck silica gel 60 (230±400 mesh). Solvents
employed were A (hexane/ethyl acetate, 4/1), B (hexane/
ethyl acetate, 2/1), C (hexane/ethyl acetate, 1/1), D (ethyl
acetate), E (cyclohexane, ethyl acetate, 4/1). All 1H NMR
spectra were recorded on a Bruker 200 MHz spectro-
meter in DMSO-d6 unless stated otherwise, with tetra-
methylsilane as internal standard. Chemical shifts are
reported in d ppm. Elemental analyses were determined
by the Physical Sciences Department of Wellcome
Research Labs, Beckenham. Electron impact mass
spectra (EIMS) were recorded on a Kratos Concept 1S
spectrometer. Fast atomic bombardment mass spectra
(FABMS) were recorded on Kratos MS50 or MS80
spectrometers using 3-nitrobenzyl alcohol or thiogly-
cerol matrices. Melting points were obtained using a
Gallenkamp apparatus and are uncorrected. Photo-
chemical reactions were performed on a Rayonet Type
PKC activity was measured essentially as in Ref. 27.
Brie¯y, mixed micelles containing 20 mol% phosphati-
dylserine and 80 mol% Triton X-100‹200 mM PDBu

M. J. Slater et al. / Bioorg. Med. Chem. 7 (1999) 1067±1074
1071
RS Preparative Photochemical Reactor, employing 8
lamps with wavelengths of 254, 300 and 350nM
(Southern N. E. Ultraviolet Co., Middleton, Connecti-
cut, USA).
(2H, brs, NH) 7.75 (2H, s), 7.25 (2H, dd, J=10 Hz,
1.5 Hz), 6.8 (2H, dd, J=10 Hz, 5 Hz), 6.5 (2H, ddd,
J=10 Hz, 5 Hz, 1.5 Hz), 3.05 (3H, s, NMe); Anal.
C21H13F2N3O4. 1.13 CH3CO2Et requires C, 66.48%; H,
3.64%; N, 10.81%; found C, 66.35%; H, 3.53%; N,
10.78%; EIMS m/z 377 (M+).
Preparation of 3,4-dichloro-N-methylmaleimide (2). To a
suspension of 2,3-dichloromaleic anhydride (13.36 g,
80 mmol) in glacial acetic acid (25 mL) was added aqu-
eous methylamine (10.7 mL of an approx 25% solution
in water, ꢀ84 mmol) with stirring and cooling. The
solution was heated under re¯ux for 330 min, cooled,
and water (30 mL) added. The mixture was placed at
4 ꢁC overnight, a€ording the product (2) as yellow
crystals (7.75 g, 54%) after ®ltration, washing with
water and drying in vacuo. mp 82±83 ꢁC; 1H NMR 3.12
(s, N-Me); Anal. C5H3Cl2NO2 requires C, 33.36%; H,
1.68%; N, 7.78%; found C, 33.04%; H, 1.65%;
N,7.62%.
2,3-Bis-(5-methoxy-1H-indol-3-yl)-N-methylmaleimide (4e).
Chromatography solvent C; Yield 36%; Recrystallised
ꢁ
1
from EtOAc (dark-red solid); mp >320 C; H NMR
11.55 (2H, brs, NH), 7.8 (2H, d, J=2.5 Hz), 7.25 (2H, d,
J=7.5 Hz), 6.6 (2H, dd, J=7.5 Hz, 2.5 Hz), 6.2 (2H, d,
J=2.5 Hz), 3.1 (6H,s, OMe), 3.05 (3H, s, N-Me); Anal.
C23H19N3O4.0.36CH3CO2Et requires C, 67.77%; H,
5.09%; N, 9.70%; found C, 67.56%; H, 4.88%; N,
9.80%; EIMS m/z 401 (M+).
2,3-Bis-(4-¯uoro-1H-indol-3-yl)-N-methylmaleimide (4f).
Chromatography solvent C; Yield 26% (dark-red
1
General procedure for the preparation of bis-indolyl-N-
methylmaleimides (4a±4f). To a solution of ethyl mag-
nesium bromide (57 mmol) in THF (25 mL) under
nitrogen at 0 ꢁC was added a solution of the appropriate
indole 3 (57 mmol) in benzene (35 mL) over 40 min with
stirring. After heating the solution for 30 min at 50 ꢁC, a
solution of 2 (14 mmol) in benzene was added drop-
wise over 30 min at room temperature. The dark violet
solution was then heated under re¯ux for 1±4 h. After
cooling, the mixture was quenched with 20% w/v citric
acid solution or ammonium chloride solution, ethyl
acetate added, the organic layer separated, dried
(MgSO4) and evaporated. The dark-red residue was
chromatographed over ¯ash silica using the eluants
described for the individual compounds below. The
excess indole (3) was eluted ®rst, followed by the yellow
mono-indole substituted maleimides, and ®nally the red
bis-indolylmaleimides (4a±f).
solid); mp >320 ꢁC; H NMR 11.75 (2H, brs, NH), 7.5
(2H, s), 7.2 (2H, d, J=7.5 Hz), 7.0 (2H, ddd, J=10 Hz,
7.5 Hz, 5 Hz), 6.6 (2H, dd, J=10 Hz, 7.5 Hz), 3.2 (3H, s,
NMe); Anal. C21H13F2N3O2.0.2H2O requires C,
66.22%; H, 3.55%; N, 11.03%; found C, 66.13%; H,
3.47%; N, 10.80%; EIMS m/z 377 (M+).
General procedure for the preparation of 2,3-bis-(1H-
indol-3-yl)-maleic anhydrides (5a±5f). A solution of the
appropiate bis-indolyl-N-methylmaleimide 4 (1 mmol)
in 10% aqueous potassium hydroxide (20 mL) and
dioxane (10 mL) was heated under re¯ux for 4±6 h (until
starting material was absent as measured by TLC ana-
lysis on silica). The mixture was cooled, and acidi®ed to
pH 1 with 2 N HCl. If the product precipitated at this
stage, it was isolated by ®ltration. Otherwise, the pro-
duct was extracted into ethyl acetate and puri®ed by
crystallisation or column chromatography over silica.
2,3-Bis-(1H-indol-3-yl)-N-methylmaleimide (4a). Data
consistent with that reported in Ref. 16.
2,3-Bis-(1H-indol-3-yl)-maleic anhydride (5a). Data con-
sistent with that reported in Ref. 16.
2,3-Bis-(7-methyl-1H-indol-3-yl)-N-methylmaleimide (4b).
Chromatography solvent: A; Yield 30%; Recrystallised
2,3-Bis-(7-methyl-1H-indol-3-yl)-maleic anhydride (5b).
Chromatography solvent E; Yield 54%; mp 310 ꢁC;
TLC (EtOAc/Hexane, 1/1) Rf 0.37; H NMR 11.9 (2H,
from EtOAc (dark-red solid); mp >320 ꢁC; H NMR
1
1
11.6 (2H, brs, NH), 7.7 (2H, s), 6.8 (2H, d, J=7.5 Hz), 6.7
(2H, d, J=7.5 Hz), 6.6 (2H, pseudo t, J=7.5 Hz), 3.05
(3H, s, N-Me), 2.4 (6H, s); Anal. C23H19N3O2.0.2CH3
CO2Et requires C, 73.86%; H, 5.36%; N, 10.86%;
found C, 73.96%; H, 5.22% N, 11.00%; EIMS m/z 369
(M+).
brs, NH), 7.8 (2H, s), 6.85 (2H, d, J=7.5 Hz), 6.75 (2H,
d, J=7.5 Hz), 6.65 (2H, t, J=7.5 Hz), 2.5 (6H, s); Anal.
C22H16N2O3.0.2CH3CO2Et.0.3H2O requires C, 72.18%;
H, 4.84%; N, 7.38%; found C, 72.08%; H, 4.71%; N,
7.21%; EIMS m/z 356 (M+).
2,3-Bis-(6-methoxy-1H-indol-3-yl)-maleic anhydride (5c).
This product was not obtained pure. The crude material
following the ethyl acetate extraction was taken directly
into the ammonium acetate reaction below (5c into 6c).
2,3-Bis-(6-methoxy-1H-indol-3-yl)-N-methylmaleimide (4c).
Chromatography solvent C; Yield 62%; Recrystallised
from EtOAc (dark-red solid); mp >320 ꢁC; H NMR
1
11.45 (2H, brs, NH), 7.6 (2H, s), 6.85 (2H, d, J=1.5Hz),
6.7 (2H, d, J=7.5 Hz), 6.3 (2H, dd, J=7.5Hz, 1.5 Hz), 3.7
(3H, s, OMe), 3.0 (3H, s, NMe); Anal. C23H19N3O4
requires C, 68.82%.; H, 4.77%; N, 10.47%; found C,
68.71%; H, 4.66%; N, 10.37%; FABMS m/z 402 (MH+).
2,3-Bis-(6-¯uoro-1H-indol-3-yl)-maleic anhydride (5d).
Chromatography solvent B; Yield 31%; mp 291±
292 ꢁC; H NMR 11.95 (2H, brs, NH), 7.9 (2H, s), 7.2
1
(2H, dd, J=10 Hz, 1.5 Hz), 6.8 (2H, dd, J=10 Hz,
5 Hz), 6.6 (2H, ddd, J=10 Hz, 5 Hz, 1.5 Hz); Anal.
C20H10F2N2O3.0.55CH3CO2Et requires C, 64.60%; H,
3.52%; N, 6.79%; found C, 64.79%; H, 3.71%; N,
6.64%; EIMS m/z 364 (M+).
2,3-Bis-(6-¯uoro-1H-indol-3-yl)-N-methylmaleimide (4d).
Chromatography solvent C; Yield 43%; Recrystallised
from EtOAc (red solid); mp 274±276 ꢁC; 1H NMR 11.75

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M. J. Slater et al. / Bioorg. Med. Chem. 7 (1999) 1067±1074
2,3-Bis-(5-methoxy-1H-indol-3-yl)-maleic anhydride (5e).
Yield ꢀ100% following extraction from EtOAc,and
used directly in the next step. TLC (Hex/EtOAc, 1/1) Rf
0.27; 1H NMR 11.8 (2H, brs, NH), 7.9 (2H, s), 7.3 (2H,
d, J=7.5 Hz), 6.6 (2H, dd, J=7.5 Hz, 2.5 Hz), 6.25 (2H,
d, J=2.5 Hz), 3.1 (6H, s, OMe); Anal. C22H16N2
O5.0.6H2O requires C, 66.20%; H, 4.34%; N, 7.08%;
found C, 66.18%; H, 4.15%; N, 6.97%; EI-MS m/z 388
(M+).
NMR 11.55 (2H, brs, NH), 10.8 (1H, brs, NH), 7.75
(2H, s), 7.2 (2H, d, J=10 Hz), 6.55 (2H, dd, J=10 Hz,
2.5 Hz), 6.2 (2H, d, J=2.5 Hz), 3.1 (6H, s); Anal.
C22H17N3O4.0.2CH3CO2Et requires C, 67.61%; H,
4.63%; N, 10.38%; found C, 67.52%; H, 4.50%; N,
10.22%; EIMS m/z 387 (M+).
2,3-Bis-(4-¯uoro-1H-indol-3-yl)-maleimide (6f). Crude
product recrystallised from EtOAc; Yield 95%; mp
ꢁ
1
300±302 C; H NMR 11.75 (2H, brs, NH), 10.95 (1H,
brs, NH), 7.55 (2H, s), 7.25 (2H, m), 6.65 (2H, dd,
J=10 Hz, 7.5 Hz); Anal. C20H11 F2N3O2.0.26H2O
requires C, 65.28%; H, 3.16%; N, 11.42%.; found C,
65.20%; H, 3.08%; N, 11.44%; FABMS m/z 364
(MH+).
2,3-Bis-(4-¯uoro-1H-indol-3-yl)-maleic anhydride (5f).
Yield ꢀ100% (red solid) following extraction from
ꢁ
1
EtOAc. mp 161±162 C; H NMR 12.0 (2H, brs, NH),
7.65 (2H, s), 7.25 (2H, d, J=7.5 Hz), 7.1 (2H, m), 6.65
(2H, dd, J=10 Hz, 7.5 Hz); Anal. C20H10F2N2O3.H2O
requires C, 62.74%; H, 3.18%; N, 7.32%; found C,
62.95%; H, 3.24%; N, 7.08%; FABMS m/z 365 (MH+).
General procedure for the preparation of 12,13-dihydro-
5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-(6H)-diones
(1a±1e). To a solution of the 2,3-bis-(1H-indol-3-yl)-
maleimide 6a±e (0.5 mmol)) in dioxan (25 mL) and tol-
uene (75 mL) at 100 ꢁC was added DDQ (125 mg,
0.55 mmol) and PTSA (4.75 mg, 0.025 mmol), and the
mixture maintained at 100 ꢁC for 15 min. After cooling,
the solvent was evaporated in vacuo, and the products
puri®ed as described for the individual examples.
General procedure for the preparation of 2,3-bis-(1H-
indol-3-yl)-maleimides (6a±6f). A mixture of the 2,3 bis-
(1H-indol-3-yl)-maleic anhydride (5) and ammonium
acetate (100±250 equiv) were heated at 140 ꢁC (bath
temperature) until TLC (silica) indicated complete
reaction (15±200 min). The mixture was cooled, parti-
tioned between water and ethyl acetate, the organic
layer washed with 5% sodium bicarbonate solution,
separated, dried (MgSO4) and evaporated. The product
was puri®ed by ¯ash chromatography over silica unless
speci®ed otherwise.
12,13-Dihydro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-
5,7-(6H)-diones (1a). Data consistent with that reported
in Ref. 18a.
2,3-Bis-(1H-indol-3-yl)-maleimide (6a). Data consistent
with that reported in Ref. 16.
12,13-Dihydro-1,11-dimethyl-5H-indolo[2,3-a]pyrrolo[3,4-
c]carbazole-5,7-(6H)-dione (1b). The crude product was
dissolved in EtOAc, washed with 5% sodium bicarbo-
nate solution, dried (MgSO4), and evaporated. Pure
product was obtained by successive trituration with
EtOAC, acetone, and THF (25 mL each). Yield 20%
2,3-Bis-(7-methyl-1H-indol-3-yl)-maleimide (6b). Chro-
matography solvent E; Yield 86%; mp >320 ꢁC; 1H
NMR 11.69 (2H, brs, NH), 10.85 (1H, brs, NH), 7.7
(2H, s), 6.8 (2H, d, J=7.5 Hz), 6.7 (2H, d, J=7.5 Hz),
6.55 (2H, t, J=7.5 Hz), 2.45 (6H, s); Anal. C22H17N3
O2.0.1CH3CO2Et requires C, 73.87%; H, 4.93%; N,
11.54%; found C, 73.94%; H, 5.01%; N, 11.42%;
EIMS m/z 355 (M+).
(yellow solid); mp >300 ꢁC; H NMR 11.45 (2H, brs,
1
NH), 10.4 (1H, brs, NH), 8.8 (2H, d, J=7.5 Hz), 7.35
(2H, d, J=7.5 Hz), 7.25 (2H, pseudo t, J=7.5 Hz),
2.75 (6H, s); Anal. C22H15N3O2.2.33H2O requires C,
66.83%; H, 5.01%; N, 10.63%; found C, 66.97%; H,
4.75%; N, 10.68%; FABMS m/z 35 (MH+).
2,3-Bis-(6-methoxy-1H-indol-3-yl)-maleimide (6c). Chro-
matography solvent C; Recrystallised from EtOAc;
12,13-Dihydro-2,10-dimethoxy-5H-indolo[2,3-a]pyrrolo[3,4-
c]carbazole-5,7-(6H)-dione (1c). The crude product was
puri®ed directly by ¯ash chromatography over silica,
eluting with hexane/EtOAc (3/7). Yield 46% (yellow
solid); mp >330 ꢁC; 1H NMR 11.45 (2H, brs, NH), 10.8
(1H, brs, NH), 8.8 (2H, d, J=10 Hz), 7.3 (2H, d,
J=2.5 Hz), 6.95 (2H, dd, J=10 Hz, 2.5 Hz), 3.9 (6H, s);
Anal. C22H15N3O4.0.4CH3CO2Et requires C, 66.83%;
H, 4.42%; N, 9.91%; found C, 66.83%; H, 4.22%; N,
9.70%; EIMS m/z 385 (M+).
Yield 50% from 4c; mp 280 ꢁC; H NMR 11.49 (2H,
1
brs, J=2.5 Hz, NH), 10.8 (1H, brs, NH), 7.6 (2H, d,
J=2.5 Hz), 6.85 (2H, d, J=2.5 Hz), 6.7 (2H, d,
J=10 Hz), 6.3 (2H, dd, J=10 Hz, 2.5 Hz), 3.7 (6H, s);
Anal. C22H17N3O4.0.9CH3CO2Et requires C, 65.88%;
H, 5.22%; N, 9.00%; found C, 66.04%; H, 5.22%; N,
8.89%; EIMS m/z 387 (M+).
2,3-Bis-(6-¯uoro-1H-indol-3-yl)-maleimide (6d). Crude
product recrystallised from EtOAc; Yield 28% from 4d;
mp 281±282 ꢁC; 1H NMR 11.7 (2H, brs, NH), 10.9 (1H,
brs, NH), 7.75 (2H, s), 7.15 (2H, dd, J=10 Hz, 1.5 Hz),
6.7 (2H, dd, J=10 Hz, 5 Hz), 6.5 (2H, ddd, J=10 Hz,
5 Hz, 1.5 Hz); Anal. C20H11F2N3O2.1.2CH3CO2Et requires
C, 63.45%; H, 4.45%; N, 8.91%; found C, 63.18%; H,
4.19%; N, 9.90%; FABMS m/z 364 (MH+).
12,13-Dihydro-2,10-di¯uoro-5H-indolo[2,3-a]pyrrolo[3,4-
c]carbazole-5,7-(6H)-dione (1d). The crude product was
dissolved in EtOAc, washed with 5% sodium bicarbo-
nate solution, dried (MgSO4), and evaporated. Pure
product was obtained by successive trituration with EtOAC
and acetone. Yield 55% (yellow solid); mp >300 ꢁC; 1H
NMR 11.8 (2H, brs, NH), 11.1 (1H, brs, NH), 8.95 (2H,
dd, J=7.5 Hz, 5 Hz), 7.63 (2H, dd, J=7.5 Hz, 1.5 Hz),
7.15 (2H, m); Anal. C20H9F2N3O2.0.74CH3CO2Et
2,3-Bis-(5-methoxy-1H-indol-3-yl)-maleimide (6e). Chro-
matography solvent F; Yield 95%; mp ꢀ280 ꢁC; 1H

M. J. Slater et al. / Bioorg. Med. Chem. 7 (1999) 1067±1074
1073
requires C, 64.66%; H, 3.53%; N, 9.85%; found C,
64.48%; H, 3.34%; N, 9.93%; EI-MS m/z 361 (M+).
12,13-Dihydro-3,9-dichloro-5H-indolo[2,3-a]pyrrolo[3,4-
c]carbazole-5,7-(6H)-dione (1i). To a warm solution of
1a (320 mg, 0.98 mmol) in acetonitrile (120 mL) was
added N-chlorosuccinimide (262 mg, 2.2 mmol), and the
mixture heated under re¯ux for 2 h. Cooling provided
the crude product, which was isolated by ®ltration, and
puri®ed by repeated crystallisation from hot THF (3Â).
12,13-Dihydro-3,9-dimethoxy-5H-indolo[2,3-a]pyrrolo[3,4-
c]carbazole-5,7-(6H)-dione (1e). The crude product was
puri®ed directly by ¯ash chromatography over silica,
eluting with solvent C. Yield 38% (red solid); mp
>320 ꢁC; H NMR 11.6 (2H, brs, NH), 10.9 (1H, brs,
Yield 12 mg, 3.1%. mp >330 ꢁC; H NMR 11.9 (2H,
1
1
NH), 8.55 (2H, d, J=2.5 Hz), 7.7 (2H, d, J=10 Hz), 7.2
(2H, dd, J=10 Hz 2.5 Hz), 3.9 (6H, s); Anal. C22H15N3
O40.8H2O requires C, 66.09%; H, 3.78%; N,10.51%;
found C, 66.28%; H, 3.72%; N, 10.49%; EIMS m/z 385
(M+).
brs, NH), 11.1 (1H, brs, NH), 8.9 (2H, d, J=1.5 Hz),
7.85 (2H, d, J=7.5 Hz), 7.55 (2H, dd, J=7.5, 1.5 Hz);
EIMS m/z 397, 395, 393 (M+).
12,13-Dihydro-3,9-dibromo-5H-indolo[2,3-a]pyrrolo[3,4-
c]carbazole-5,7-(6H)-dione (1j). To a suspension of 1a
(300 mg, 0.92 mmol) in acetonitrile (25 mL) at room
temperature was added N-bromosuccinimide (414 mg,
2.5 mmol) and the suspension stirred for 90 min. The
dark green precipitate was ®ltered o€, and recrystallised
from hot THF (3Â), to a€ord the product (1j) as an
12,13-Dihydro-4,8-di¯uoro-5H-indolo[2,3-a]pyrrolo[3,4-
c]carbazole-5,7-(6H)-dione (1f). A solution of 6f (28 mg,
77 mmol) and iodine (7 mg) in propan-2-ol (40 mL) was
sonicated under a ¯ow of nitrogen for 10 min. The
solution was then irradiated at wavelengths of 254 nm,
300 nm, 350 nm for 48 h. The solvent was evaporated in
vacuo, and the residue triturated with acetone (2 mL) to
a€ord 1f (20 mg, 72%) as a yellow solid. mp >300 ꢁC;
1H NMR 12.1 (2H, s, NH), 10.75 (1H, s, NH), 7.65 (2H,
d, J=7.5 Hz), 7.55 (2H, m), 7.1 (2H, dd, J=10 Hz,
7.5 Hz); EIMS m/z 361 (M+).
orange solid (60 mg, 13.5%). mp >320 ꢁC; H NMR
1
11.8 (2H, brs, NH), 11.1 (1H, brs, NH), 9.1 (2H, d,
J=1.5 Hz), 7.85 (2H, d, J=7.5 Hz), 7.65 (2H, dd,
J=7.5, 1.5 Hz); EIMS m/z 485, 484, 483, 482, 481
(M+).
Acknowledgements
12,13-Dihydro-2,10-dihydroxy-5H-indolo[2,3-a]pyrrolo[3,4-
c]carbazole-5,7-(6H)-dione. A mixture of 1c (76 mg,
0.2 mmol) and pyridine hydrochloride (800 mg,
6.9 mmol) was heated at 180 ꢁC in a sealed tube for
60 min. After cooling, ethyl acetate and water were
added, the organic fraction washed successively with 2
N hydrochloric acid and brine, then dried over MgSO4.
Following evaporation in vacuo to approximately
20 mL, the mixture was applied to a ¯ash silica column,
and eluted with hexane, then hexane/ethyl acetate (4/1).
Pooling and evaporation of the appropriate fractions
a€orded the product (1g) as a yellow solid (62 mg,
approx 90% pure). A pure sample was obtained fol-
lowing crystallisation from acetone (2 mL), yield 22 mg
(31%); mp 145±147 ꢁC; TLC (EtOAc) Rf 0.35; 1H NMR
11.25 (2H, brs, NH), 10.75 (1H, brs, NH), 9.7 (2H,
brs, OH), 8.7 (2H, d, J=10 Hz), 7.1 (2H, d, J=2.5 Hz),
6.8 (2H, dd, J=10 Hz, 2.5 Hz); FABMS m/z 358
(MH+).
The authors wish to thank Alan Emerson and Chris
Roberts for excellent technical assistance with cytotoxi-
city assays, and Dr. Margaret Tisdale for HIV data.
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