Pyrrolo[3,4-c]-β-carboline-diones as a novel class of inhibitors of the platelet- derived growth factor receptor kinase

Article abstract of DOI:10.1016/S0223-5234(00)00140-9

Members of the structurally diverse family of β-carbolines have previously been shown to exhibit a wide range of biological activities. A novel synthetic strategy for generation of β-carbolines was developed, allowing imido-β-carbolines to be created in three steps from known compounds. The compounds were screened for inhibition of platelet-derived growth factor (PDGF)-stimulated tyrosine phosphorylation in Swiss 3T3 fibroblasts. A number of the newly synthesized β-carbolines with moderate to potent inhibitory activity were revealed. The most active derivative, 2,3- dihydro-8,9-dimethoxy-5-(2-methylphenyl)-1H,6H-pyrrolo[3,4-c]pyrido[3,4- b]indole-1,3-dione 2ee, inhibited purified PDGF receptor kinase and PDGF- receptor autophosphorylation in intact cells with IC₅₀ values of 0.4 and 2.6 μM, respectively. Dione 2ee also inhibited PDGF-stimulated DNA synthesis in Swiss 3T3 fibroblasts with an IC₅₀ of 3.2 μM. The compound had no effect on Src or epidermal growth factor (EGF) receptor kinase activity and a six-seven-fold higher IC₅₀ for inhibition of basic fibroblast growth factor (bFGF)-stimulated tyrosine phosphorylation or Kit/stem cell factor (SCF) receptor autophosphorylation, indicating a reasonable extent of kinase specificity. Thus, β-carbolines present a new lead of tyrosine kinase inhibitors with the capacity to selectively interfere with PDGF receptor signal transduction and PDGF-dependent cell growth. (C) 2000 Editions scientifiques et medicales Elsevier SAS.

Full text of DOI:10.1016/S0223-5234(00)00140-9

Eur. J. Med. Chem. 35 (2000) 413−427
© 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
S0223523400001409/FLA
413
Original article
Pyrrolo[3,4-c]-ꢀ-carboline-diones as a novel class of inhibitors of the platelet-
derived growth factor receptor kinase
Steffen Tellera, Stella Eluwab, Markus Kollerb, Andrea Ueckera, Thomas Beckersc, Silke Baasnerc,
Frank-D. Böhmera*, Siavosh Mahboobib**
aResearch Unit Molecular Cell Biology, Medical Faculty, Friedrich Schiller University, D-07747 Jena, Germany
bFaculty of Chemistry and Pharmacy, Institute of Pharmacy, University Regensburg, D-93040 Regensburg, Germany
cASTA Medica AG, D-60314 Frankfurt/Main, Germany
Received 3 August 1999; revised 15 November 1999; accepted 19 November 1999
Abstract – Members of the structurally diverse family of ꢀ-carbolines have previously been shown to exhibit a wide range of biological
activities. A novel synthetic strategy for generation of ꢀ-carbolines was developed, allowing imido-ꢀ-carbolines to be created in three steps
from known compounds. The compounds were screened for inhibition of platelet-derived growth factor (PDGF)-stimulated tyrosine
phosphorylation in Swiss 3T3 fibroblasts. A number of the newly synthesized ꢀ-carbolines with moderate to potent inhibitory activity were
revealed. The most active derivative, 2,3-dihydro-8,9-dimethoxy-5-(2-methylphenyl)-1H,6H-pyrrolo[3,4-c]pyrido[3,4-b]indole-1,3-dione 2ee,
inhibited purified PDGF receptor kinase and PDGF-receptor autophosphorylation in intact cells with IC50 values of 0.4 and 2.6 µM,
respectively. Dione 2ee also inhibited PDGF-stimulated DNA synthesis in Swiss 3T3 fibroblasts with an IC50 of 3.2 µM. The compound had
no effect on Src or epidermal growth factor (EGF) receptor kinase activity and a six–seven-fold higher IC50 for inhibition of basic fibroblast
growth factor (bFGF)-stimulated tyrosine phosphorylation or Kit/stem cell factor (SCF) receptor autophosphorylation, indicating a reasonable
extent of kinase specificity. Thus, ꢀ-carbolines present a new lead of tyrosine kinase inhibitors with the capacity to selectively interfere with
PDGF receptor signal transduction and PDGF-dependent cell growth. © 2000 Éditions scientifiques et médicales Elsevier SAS
ꢀ-carbolines / PDGF receptor / tyrosine kinase / signal transduction
1. Introduction
tors (for reviews see [1–3]). Aberrant activation of PDGF
receptors has been implicated in various disease states. A
well-investigated example is the autocrine activation of
resident PDGF receptors by aberrantly expressed PDGF
in human glioma cells [4–6]. Also, evidence for activa-
tion of PDGF receptors in situ in human meningioma was
recently obtained [7]. An example of ligand-independent
activation of PDGFꢀ-receptor in a human tumour is the
TEL–PDGFR fusion protein, detected in human chronic
myelomonocytic leukaemia [8, 9]. In other forms of
human cancer, tumour-derived PDGF may be involved in
stroma cell stimulation and thereby indirectly drive tu-
mour growth [10]. A role of PDGF for the development of
atherosclerotic lesions and restenosis following coronary
balloon angioplasty and also for the development of
several forms of fibrosis has been well documented
[11–14]. Thus, inhibitors of PDGF receptor signalling
activity are likely to be valuable for the treatment of these
Platelet-derived growth factors (PDGFs) comprise a
family of dimeric proteins with potent effects on prolif-
eration and migration of mesenchymal cells such as
fibroblasts and vascular smooth muscle cells. They exert
their cellular actions by activating their cognate class III
receptor tyrosine kinases, the PDGFα or PDGFꢀ recep-
* Correspondence regarding functional aspects
** Correspondence regarding chemical aspects and reprints:
siavosh.mahboobi@chemie.uni-regensburg.de
Dedicated to Professor W. Schunack on the occasion of his 65th
birthday.
Abbreviations: bFGF: basic fibroblast growth factor; DMSO:
dimethylsulfoxide; EGF: epidermal growth factor; FCS: foetal calf
serum; FRS2: FGF receptor substrate 2; c-KIT: stem cell factor;
PDGF: platelet-derived growth factor; PMSF: phenylmethylsulfo-
nyl fluoride; SCF: stem cell factor; TFA: trifluoroacetic acid; TMS:
tetramethylsilane; THF: tetrahydrofurane; XTT: 2,3-bis(2-methoxy-
4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide.

414
Figure 1. Imidocarbazoles 1 and imido-ꢀ-carbolines 2.
pathological conditions. Several classes of direct inhibi-
tors of the PDGF receptor tyrosine kinase activity have
been described, including quinoxalines [15–17], pheny-
laminopyrimidines [18], quinolines [19–22], pyridopyri-
midines [23], phenylbenzimidazoles [24] and lefluno-
mide [25]. They have been shown to suppress disease-
relevant PDGFR signalling in experimental models. Given
the potential medical importance of blockade of this
signalling system, the development of novel leads for
PDGF receptor blockers is highly warranted.
Figure 2. Synthesis of 3-bromo-4-(3-indolyl)pyrroline-2,5-
diones 3. Key: (a) toluene/THF/room temperature to reflux.
spite of several attempts, we were unable to apply these
methods even to synthesis of the originally described
compounds [40].
Starting materials for our ꢀ-carboline syntheses are the
well-known 3-bromo-4-(3-indolyl)pyrroline-2,5-diones 3
[41, 42], which can be obtained easily by quenching
Alkaloids with a ꢀ-carboline nucleus possess wide-
spread and potent biological activities [26–30]. The
reported effects of this class of compounds comprise
antineoplastic (tubulin binding) [28, 29, 31], anticonvul-
sive, hypnotic and anxiolytic (benzodiazepine receptor
ligands) [26–28, 32, 33], antiviral [30], antimicrobial [29]
as well as topoisomerase-II inhibition [34] and inhibition
of cGMP-dependent processes [35]. On the other hand,
the synthesis and evaluation of imidocarbazole deriva-
tives related to structure 1 [36] have recently received
much attention (figure 1). Compounds of this type act
inter alia as inhibitors of protein kinase C and different
protein tyrosine kinases [37–39].
Therefore, imido-ꢀ-carbolines 2 (figure 1) which com-
bine structural features of both classes of compounds
were synthesized and screened as effectors of PDGF
receptor tyrosine kinase activity in intact cells. Several
compounds of this class were found to exert inhibitory
activity. Direct inhibition was demonstrated at the level of
purified kinase and extended to inhibition of PDGF-
stimulated cell growth. Further characterization revealed
structure–activity relationships and defined selectivity in
this novel group of PDGF receptor blockers.
excess
indolylmagnesium
bromides
4
with
N-unsubstituted or N-substituted 3,4-dibromopyrroline-
2,5-dione 5 in toluene/THF (figure 2).
In order to introduce the pyridine nitrogen of the target
ꢀ-carbolines, we chose the azido group as a precursor of
the activated imino group. After several unsuccessful
attempts with several dipolar aprotic solvents, sodium
azide in DMSO at ambient temperature yielded 6 as a
precipitate on addition of H2O. Due to their apparent
instability (every attempt to obtain an analytically pure
sample led to decomposition), the moist 3-azido-4-(3-
indolyl)-pyrroline-2,5-diones 6 were dissolved in THF
immediately without any purification and, after drying
over Na2SO4, tri-n-butylphosphine was added at 0 °C.
When evolution of nitrogen ceased, removal of the
solvent afforded the 3-tributylphosphanimino-4-(3-
indolyl)-pyrroline-2,5-diones 7, which in principle should
be more reactive than the triphenylphosphanimines which
can only be cleaved by strong acids [28, 29]. However,
compounds 7 are reactive enough to undergo a Staudinger
reaction [30, 43] with benzaldehydes or phenylacetalde-
hyde R2CHO in o-xylene under catalysis of Pd/C to
furnish the desired pyrrolo[3,4-c]-ꢀ-carbolin-1,3-diones
2a–gg (figure 3). The yields are low (relative to 3,
because compounds 7 as well as 6 did not provide
satisfactory analytical data due to instability) and strongly
depend on the nature and position of the substituents on
the R2 phenyl ring. A further reason is the formation of
the 3-amino-4-(3-indolyl)pyrroline-2,5-diones 8 with
yields of 10–15%, which are easily separable from 2 by
column chromatography. Compound 2ee could only be
2. Synthesis of pyrrolo[3,4-c]-ꢀ-carboline-diones (2)
Though there are several synthetic approaches to
ꢀ-carbolines [27, 40], none of them are applicable to the
preparation of the desired products of type 2. One
reported synthesis of compounds of structure 2 would
lead to non-removable groups R1 or R3 [33]. However, in

415
Figure 3. Synthesis of pyrrolo[3,4-c]-ꢀ-carboline-1,3-diones 2 and pyrrolo[3’,4’:2,3]-azepino[4,5,6-cd]indole-8,10-diones 10. Keys:
(a) NaN3/DMSO/room temperature; (b) (nBu)3P/THF/room temperature; (c) R2CHO/Pd(C)/o-xylene/reflux; (d) R2CHO/
dichloromethane/methanol/room temperature; (e) TFA/dichloromethane/–78 °C.
obtained in satisfactory quantities by a modified proce-
dure using higher temperature and shorter reaction time,
but this method was not able to raise the yields of other
ꢀ-carbolines (e.g. 2v and 2z). Condensation of 8 with
various aldehydes gave the corresponding imines 9.
Under Pictet-Spengler conditions the latter did not cy-
clize to pyrrolo-ꢀ-carbolines 11, which we intended to
fibroblasts as the primary assay. Among the compounds
listed in table I, compounds 2k, 2t, 2w and 2bb displayed
inhibitory activity in this test (table I), whereas the other
compounds had little effect (IC50 > 30 µM). When the
inhibition was analysed qualitatively by immunoblotting,
compounds 2w and 2t revealed inhibition of PDGF
receptor autophosphorylation (table II, figure 5A and B)
and inhibition of other PDGF-dependent tyrosine phos-
phorylations in these cells, whereas 2bb and 2k had
comparatively weak effects on PDGF receptor autophos-
phorylation (not shown).
dehydrogenate
to
compounds
2.
Instead,
pyrrolo[3’,4’:2,3]azepino[4,5,6-cd]indole-8,10-diones 10
are obtained [44].
The 9-benzyloxy derivatives 2ff and 2gg could be
transformed to the 9-hydroxy compounds 2hh and 2ii by
heating under reflux with ammonium formate and Pd/C in
ethanol/THF [45] (figure 4). Compounds 2 are fluorescent
yellow solids crystallizing from ethyl acetate. The various
compounds prepared are shown in table I.
These results revealed that certain ꢀ-carbolines display
inhibitory activity for the PDGF receptor. Although the
3. Characterization of pyrrolo[3,4-c]-ꢀ-carboline-
diones as inhibitors of the PDGF receptor tyrosine
kinase
The newly synthesized compounds were screened for
PDGF receptor antagonist activity by measuring PDGF-
stimulated tyrosine phosphorylation in intact Swiss 3T3
Figure 4. Synthesis of 9-hydroxy-pyrrolo[3,4-c]-ꢀ-carboline-
1,3-diones 2hh–ii. Key: (a) HCO2NH4/Pd(C)/ethanol/THF/
reflux.

416
Table I. Structure of the ꢀ-carboline derivatives 2a–2ii and effects on PDGF-stimulated phosphorylation in Swiss 3T3 cells (reference
compound: AG 1295 [16]).
R2a
R3
R4
IC50 (µM)
IC50 (µM)
b
c
Number
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
2r
2s
2t
2u
2v
2w
2x
2y
2z
2aa
2bb
2cc
2dd
2ee
2ff
2gg
2hh
2ii
Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
Me
H
H
H
H
H
H
–
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
> 30
> 30
> 30
> 30
> 30
> 30
> 30
> 30
> 30
> 30
3–10
> 30
> 30
> 30
> 30
> 30
> 30
> 30
> 30
0.1–1
> 30
> 30
3–10
> 30
> 30
> 30
> 30
0.1–1
> 30
–
> 30
> 30
> 30
> 30
> 30
> 30
> 30
> 30
> 30
> 30
3–10
> 30
> 30
> 30
> 30
> 30
> 30
> 30
> 30
3–10
> 30
> 30
3–10
> 30
> 30
> 30
> 30
1–10d
> 30
1–10d
1–3
4-CN-Ph
4-tert-BuO-Ph
4-EtO-Ph
4-MeO-Ph
4-Me-Ph
4-F-Ph
CH2-Ph
2-OH-3-MeO-Ph
2-MeO-Ph
2-Me-Ph
3-Me-Ph
3-OH-4-MeO-Ph
2,4-di-MeO-Ph
3,4-di-MeO-Ph
4-OH-3-MeO-Ph
4-NMe2-Ph
3-NO2-Ph
3-MeO-Ph
Ph
4-MeO-Ph
3-MeO-Ph
2-MeO-Ph
2,4-di-MeO-Ph
3,4-di-MeO-Ph
4-CN-Ph
Ph
Ph
Ph
2-Me-Ph
2-Me-Ph
2-MeO-Ph
2-Me-Ph
2-MeO-Ph
2-Me-Ph
H
H
H
9-MeO
9-MeO
9-MeO
9-MeO
9-MeO
9-MeO
9-MeO
9-MeO
8,9-di-MeO
H
9-MeO
8,9-di-MeO
9-PhCH2O
9-PhCH2O
9-OH
9-OH
–
–
–
–
–
–
–
> 30
> 30
10–30
> 30
0.5–1e
AG 1295
–
a R1 = H; bPDGF-stimulated tyrosine phosphorylation in total cell lysates; cautophosphorylation of PDGF receptor evaluated by
immunoblotting, ranges for the IC50 were defined based on at least three independent experiments; dvery shallow dose–response curve; etaken
from [16].
number of active compounds is limited, preliminary
conclusions for SAR can be made: methoxy-substitution
in R4 at C-9 or at C-8 and C-9, respectively, seems
favourable (compounds 2t and 2bb versus 2a). Substitu-
tion of the imido NH affects activity negatively (com-
pound 2aa versus compound 2t). Generally, substitution
of the R2 phenyl ring had negative effects, except in the
R2 o-position, where Me substitution is beneficial (com-
pound 2k versus 2a). On the basis of these consider-
ations, further compounds were synthesized. To explore
the effects of alternative substitutions at C-9 in R4, OH-
or benzyloxy- groups were introduced. This generally
gave inactive or only weakly active compounds, regard-
less of the substitutions in R2 (table I, compounds,
2ff–2ii). In particular, the OH derivatives 2hh and 2ii or
the benzyloxy- derivatives 2ff and 2gg had little or no
detectable activity compared to their 9-methoxy-
substituted counterparts 2w or 2dd. However, the com-
bination of the favourable structural features identified in
the first series of compounds, namely Me-substitution in
o-position of ring R2 and MeO-substitution in C-9 (2dd)
or C-8 and C-9 (2ee) yielded active compounds, with 2ee

417
Table II. Effect of the ꢀ-carbolines 2t, 2w and 2ee on the phosphorylation of the PDGF receptor in intact cells and in vitro and on other
tyrosine kinases.
Test
IC50 (µM)
2t
2w
2ee
PDGF receptor phosphorylation (Swiss 3T3 cells)a
7.4
1.7
0.4
> 30
> 30
> 30
15.4
> 30
4.8
1.9
0.2
> 30
> 30
11.8
> 30
> 30
2.6
n.d.
0.4
> 30
> 30
> 30
17.3
16.4
PDGF receptor phosphorylation (Swiss 3T3 membranes)a
PDGF receptor phosphorylation (purified human PDGFꢀ-receptor)
EGF receptor phosphorylation (A 431 cells)
Src-dependent tyrosine phosphorylation in src-transformed NIH3T3 cells
p60c-src kinase activity in vitro
FGF receptor-dependent tyrosine phosphorylation (FRS-2; Swiss 3T3 cells)
c-Kit/SCF receptor phosphorylation (CHRF cells)
a Quantification of IC50 values was based on the intensity of the signal for autophosphorylated PDGF receptor. Titration was done using
four–eight inhibitor concentrations within a range of two orders of magnitude which were selected on the basis of preliminary experiments.
Results of triplicate determinations were subjected to curve fitting.
Figure 5. Inhibition of PDGF receptor (PDGFR) autophosphorylation in intact Swiss 3T3 cells and in vitro by ꢀ-carbolines 2t and
2w. Swiss cells were cultured in 24-well plates to confluence and made quiescent. Cells were then incubated with 2t (A) or 2w (B)
for 2 h in serum free medium in the indicated concentrations, stimulated with PDGF (100 ng/mL) for 10 min and extracted. The
extracts were subjected to SDS-PAGE and immunoblotting with anti-phosphotyrosine antibodies. Purified PDGFꢀ-receptor samples
were pre-incubated with compound 2t (C) or 2w (D) for 15 min on ice. Then, PDGF was added for 20 min and receptor
autophosphorylation was allowed to proceed for 3 min in the presence of 2 µM [γ-32P]ATP. The reaction was quenched with
SDS-PAGE sample buffer and the samples were subjected to electrophoresis and autoradiography.

418
Table III. Effect of the ꢀ-carbolines 2t and 2w and 2ee on DNA synthesis in Swiss 3T3 cells stimulated by different agents.
DNA synthesis (Swiss 3T3 cells)a
IC50 (µM)
2t
2w
2ee
PDGF-stimulated
FGF-stimulated
EGF/insulin-stimulated
10 % FCS-stimulated
5.4
2.7
8.9
5.7
3.2
3.2
21.4
> 30
20
11.4
> 30
4.5
a Quiescent Swiss 3T3 cells were stimulated with the different growth-promoting agents in the absence or presence of inhibitors. Four–five
inhibitor concentrations were used in the range of activity defined by preliminary experiments. Results are means of three independent
experiments run with duplicate determinations. IC50 values were determined by curve-fitting.
being the most active compound identified among
ꢀ-carboline structures so far (tables II and III, figure 6A).
Compound 2dd displayed very shallow dose–response
curves (not shown), possibly because of low solubility
and was therefore precluded from further analysis.
Compounds 2t, 2w and 2ee were then subjected to a
more detailed characterization. All three compounds
inhibited PDGF receptor autophosphorylation in isolated
Swiss 3T3 cell membranes (not shown) and PDGF
receptor autokinase reaction with a purified PDGF recep-
tor preparation (figure 5C and D and figure 6B). Thus, the
inhibition of PDGF receptor phosphorylation observed in
intact cells is based on a direct kinase inhibition. Inhibi-
tion of PDGF receptor autophosphorylation by 2ee oc-
curred with similar efficacy in the murine Swiss 3T3 cells
(figure 6A) and in porcine aortic endothelial (PAE) cells,
stably transfected with the human PDGFꢀ receptor
(figure 6C).
We further evaluated the specificity of kinase inhibi-
tion by the active ꢀ-carboline derivatives. For compari-
son we chose, on the one hand, the epidermal growth
factor (EGF) receptor tyrosine kinase and the cytoplasmic
tyrosine kinase pp60src (Src kinase). These two enzymes
are only very distantly related to PDGF receptors at the
level of kinase domain sequence homology. They are
widely expressed and represent tyrosine kinase families
implicated in various types of cancer. For a more rigorous
selectivity judgement we employed the fibroblast growth
factor (FGF) receptor tyrosine kinase and the stem cell
factor (SCF) receptor tyrosine kinase, also known as
c-Kit. FGF receptors and PDGF receptors are moderately
related and frequently expressed in the same type of cells,
while the c-Kit/SCF receptor is a very close relative of
the PDGF receptor. EGF receptor kinase activity was
measured as receptor autophosphorylation in intact A431
cells, pp60src kinase activity was assayed with recombi-
nant kinase in vitro or as pp60src-dependent tyrosine
phosphorylation in src-transformed NIH3T3 cells, FGF
receptor activity was measured as bFGF-stimulated phos-
phorylation of the 92 kDa FGF receptor substrate 2
(FRS2) in Swiss 3T3 cells and c-Kit/SCF receptor kinase
activity was measured as SCF-stimulated autophospho-
rylation of c-Kit in CHRF cells (table II, figure 7), a
haematopoietic cell line with high c-Kit/SCF receptor
levels.
Compound 2t did not show activity in any of these
assays, whereas 2w displayed some activity on pp60src
kinase activity in vitro and moderately inhibited Src-
dependent phosphorylation in intact cells (table II, fig-
ure 6).
Compound 2ee partially inhibited bFGF-stimulated
phosphorylation of FRS2 in Swiss 3T3 cells and Kit/SCF
receptor phosphorylation at 30 µM but had no effect on
these kinases at lower concentrations or on EGF receptor
kinase or Src kinase (table II, figure7).
We further tested whether the compounds would affect
PDGF-dependent cell growth. We observed inhibition of
PDGF-dependent DNA synthesis (table III) by 2t, 2w and
2ee in Swiss 3T3 cells, with 2ee having the highest
potency. Interestingly, 2t exhibited little selectivity with
respect to DNA synthesis stimulated by different growth
promoting agents (table III), whereas 2w had a quite
selective effect on PDGF-stimulated growth. Compound
2ee displayed intermediate selectivity, with similar inhi-
bition of DNA synthesis stimulated by PDGF or FCS,
moderate inhibition of FGF-stimulated DNA synthesis
and no effect on DNA synthesis stimulated by the
combination of EGF and insulin. Compounds 2t and 2w
were also tested for their capacity to inhibit proliferation
of sis-transformed NIH3T3 fibroblasts. Both compounds
inhibited colony formation of sis-transformed NIH3T3
fibroblasts but not of src- or BxB raf-transformed NIH3T3
fibroblasts in semi-solid agar (table IV).
4. Discussion
A synthetic approach for the generation of imido-ꢀ-
carbolines 2 via phosphanimines 7 is presented. Members

419
compared (table II). Under these conditions, cellular
uptake behaviour and the capacity of the compounds to
compete with high intracellular ATP concentrations con-
tribute to activity, which may possibly explain the differ-
ential result. Even more obvious are the effects of the
structural differences of the three compounds on selec-
tivity in DNA synthesis assays (table III). While 2t
inhibits DNA synthesis stimulated with all agents tested,
2w preferentially inhibits PDGF-stimulated DNA synthe-
sis and 2ee exhibits an intermediate selectivity. The low
selectivity of 2t in these assays does not match the results
obtained when testing susceptibility of other tyrosine
kinases. Thus, 2t may have other cellular targets which
are common to the pathways of mitogenic stimulation for
the investigated mitogenic agents. Compound 2w is
rather selective in all assays including a lack of inhibition
of other tested tyrosine kinases, in particular c-Kit/SCF
receptor. Thus, ꢀ-carbolines may be superior to previ-
ously described compounds in that they provide selectiv-
ity of inhibition distinguishing even between these two
closely related receptor tyrosine kinases.
5. Experimental protocols
Uncorrected melting points were measured on a Re-
ichert 300419 hot-stage microscope. Rf values were
determined by TLC on Merck Art. 5554 silica 60 F254
coated aluminium sheets with 254 nm UV detection.
Elemental analyses were performed by Analytical Lab.
Univ. Regensburg, and all were within ± 0.4% of the
theoretical values. FT-IR spectra were recorded in KBr
1
pellets on a Nicolet 510 spectrometer. H-NMR were
Figure 6. Inhibition of PDGF receptor autophosphorylation in
intact cells and in vitro by ꢀ-carboline 2ee. Inhibition of PDGF
receptor (PDGFR) autophosphorylation in intact Swiss 3T3
cells (A) and with purified PDGFꢀ-receptor (B) by 2ee was
measured as described in the legend to figure 5. PAE cells
stably expressing human PDGFꢀ-receptor were cultured in
6-well plates to confluence and made quiescent. Cells were then
incubated with 2ee in the indicated concentrations for 2 h in
serum free medium, stimulated with PDGF (50 ng/mL) for
10 min and extracted. The extracts were subjected to an affinity
precipitation with wheat germ agglutinin beads, SDS-PAGE
and immunoblotting with anti-phosphotyrosine antibodies (C).
recorded in DMSO-d6 at 250 MHz on a Bruker 250
spectrometer. Chemical shifts were expressed in parts per
million downfield relative to TMS as an internal standard.
Electron impact mass spectra were recorded at 70 eV on
a Varian MAT 112S spectrometer. All reactions were
carried out under nitrogen that had been dried over
self-indicating silica gel, conc. H2SO4 and KOH. Anhy-
drous ethanol was prepared using sodium and diethyl
phthalate. Other solvents were used in commercially
available quality without further purification.
5.1. General procedure
for compounds 2a–2dd, 2ff and 2gg
of this structural family exhibit inhibition of PDGF-
receptor autophosphorylation in vitro and in intact cells
and block PDGF-dependent cell growth. The most active
compounds 2t, 2w and 2ee, which are structurally rather
similar, exhibit little differences in their potency towards
the purified PDGFꢀ-receptor kinase (table II). In con-
trast, 2ee is more potent than 2w and even more than 2t
when cellular PDGF receptor phosphorylation assays are
A solution of the corresponding 3-bromo-4-(3-
indolyl)pyrroline-2,5-dione 3 [35, 36] (17.18 mmol) and
NaN3 (1.23 g, 18.89 mmol) in anhydrous DMSO (50 mL)
is stirred for 1 h at ambient temperature. After addition of
ice/H2O (500 mL), the resulting red precipitate is filtered
and rinsed with H2O. The solid 6 is dissolved in THF

420
Figure 7. Effects of ꢀ-carbolines 2t, 2w and 2ee on other tyrosine kinases. Effects on epidermal growth factor receptor (EGFR) (A),
fibroblast growth factor (FGF) receptor (B) and c-Kit/stem cell factor (SCF) receptor (C) were measured in A431 cells, Swiss 3T3 cells
and CHRF cells, respectively. Cells were treated with the inhibitors in the indicated concentrations as described in the legend to figure 1
and were left unstimulated (–) or were stimulated (+) with 100 ng/mL EGF for 5 min, 100 ng/mL bFGF for 10 min or 40 ng/mL SCF
for 5 min, respectively. Ligand-dependent autophosphorylation of EGFR was directly detectable in cell lysates. Autophosphorylation
of c-Kit/SCF receptor was analysed in specific immunoprecipitates. Activity of FGF receptor was judged from phosphorylation of the
92kDa FGF receptor substrate 2 (FRS2), which was detectable in cell lysates. NIH3T3 cells transformed with a constitutively active
variant of the src oncogene exhibit strongly elevated tyrosine phosphorylation, detectable in lysates of unstimulated cells. This was
taken as a measure for effects on Src-kinase activity (D).

421
Table IV. Effect of the ꢀ-carbolines 2t and 2w on the soft agar
colony formation and proliferation of cell lines transformed with
different oncogenes.
1 735, 1 715 (C=O); 1 610, 1 580, 1 565, 1 450 (C=C);
1H-NMR: δ 0.88 (s; 9 H, C(CH3)3), 7.22; 8.03 (AA’BB’,
JAB = 8.7 Hz; 4 H phenyl), 7.38–8.80 (m; 4 H aromat.),
11.35 (br s; 1 H (exch.), NH imid), 12.12 (br s; 1 H
(exch.), NH indole); MS: m/z (%): 385 (0.11) [M+·], 357
(100), 329 (37). Anal. (C23H19N3O3) C, H, N.
Assay
Cell line
IC50 (µM)
2t
2w
Colony
Formation
sis-transformed NIH3T3
src-transformed NIH3T3
BxB raf-transformed NIH3T3
8.3
9
> 30
> 30
3.7
> 30
> 30
6.6
5.1.4. 5-(4-Ethoxyphenyl)-2,3-dihydro-
1H,6H-pyrrolo[3,4-c]pyrido[3,4-b]indole-1,3-dione 2d
Proliferation sis-transformed NIH3T3
Yield 1.76 g (29%): m.p. 244 °C; Rf 0.43 (ethyl acetate/
hexane 1:1); IR: ν (cm–1) 3 370 (NH); 3 070–2 975 (CH);
1 735, 1 715 (C=O); 1 625, 1 605, 1 580, 1 450 (C=C);
1H-NMR: δ 0.88 (t, J = 7.0 Hz; 3 H, CH3CH2), 4.19 (q,
(100 mL) and dried over Na2SO4 for 1 h. The latter is
filtered off, and tri-n-butylphosphine (4.30 mL,
17.18 mmol) is added to the filtrate, while cooling in an
ice bath. After 1 h of stirring at ambient temperature the
solvent is evaporated in vacuo. The resulting crude
product 7 is dissolved in o-xylene (250 mL), the corre-
sponding aldehyde (17.18 mmol) and 10% Pd/C (1 g) are
added and the reaction mixture is refluxed for 48 h. The
catalyst is removed by filtration through Celite, the
solvent is removed in vacuo, and the oily residue is
purified by flash column chromatography (column 5 cm ×
10 cm, silica gel, ethyl acetate/hexane 1:1) to give yellow
crystals (yields stated relative to 3). Analytical samples
were obtained from ethyl acetate.
J = 7.0 Hz; 2 H, CH3CH2), 7.22; 8.02 (AA’BB’, JAB
=
8.8 Hz; 4 H phenyl), 7.38–8.79 (m; 4 H aromat.), 11.38
(br s; 1 H (exch.), NH imid), 12.20 (br s; 1 H (exch.), NH
indole); MS: m/z (%): 357 (80) [M+·], 329 (100). Anal.
(C21H15N3O3) C, H, N.
5.1.5. 2,3-Dihydro-5-(4-methoxyphenyl)-
1H,6H-pyrrolo[3,4-c]pyrido[3,4-b]indole-1,3-dione 2e
Yield 0.64 g (11%): m.p. 289 °C; Rf 0.40 (ethyl acetate/
hexane 1:1); IR: ν (cm–1) 3 200 (NH); 3 050–2 960 (CH);
1 765, 1 725 (C=O); 1 630, 1 610, 1 580, 1 455 (C=C);
1H-NMR: δ 3.91 (s; 3 H, OCH3), 7.24; 8.08 (AA’BB’,
JAB = 8.8 Hz; 4 H phenyl), 7.38–8.79 (m; 4 H aromat.),
11.40 (br s; 1 H (exch.), NH imid), 12.11 (br s; 1 H
(exch.), NH indole); MS: m/z (%): 343 (100) [M+·]. Anal.
(C20H13N3O3) C, H, N.
5.1.1. 2,3-Dihydro-5-phenyl-1H,6H-
pyrrolo[3,4-c]pyrido[3,4-b]indole-1,3-dione 2a
Yield 1.1 g (20%): m.p. > 350 °C; Rf 0.48 (ethyl
acetate/hexane 1:1); IR: ν (cm–1) 3 470, 3 380 (NH);
3 180–3 050 (CH); 1 765, 1 720 (C=O); 1 625, 1 560,
1
1 495 (C=C); H-NMR: δ 7.42–7.46 (m; 1 H aromat.,
5.1.6. 2,3-Dihydro-5-(4-methylphenyl)-
1H,6H-pyrrolo[3,4-c]pyrido[3,4-b]indole-1,3-dione 2f
7–H), 7.64–7.78 (m; 5 H phenyl), 8.01–8.07 (m; 2 H
aromat., 8-H and 9-H), 8.79 (m; 1 H aromat., 10-H),
11.44 (br s; 1 H (exch.), NH imid), 12.27 (br s; 1 H
(exch.), NH indole); MS: m/z (%): 313 (100) [M+·], 77
(3). Anal. (C19H11N3O2) C, H, N.
Yield 0.38 g (7%): m.p. > 350 °C; Rf 0.51 (ethyl
acetate/hexane 1:1); IR: ν (cm–1) 3 330 (NH); 3 035–2 990
(CH); 1 775, 1 725 (C=O); 1 630, 1 585, 1 505, 1 465
(C=C); 1H-NMR: δ 2.51 (s; 3 H, CH3), 7.50; 7.95
(AA’BB’, JAB = 8.1 Hz; 4 H phenyl), 7.38–8.79 (m; 4 H
aromat.), 11.40 (br s; 1 H (exch.), NH imid), 12.19 (br s;
1 H (exch.), NH indole); MS: m/z (%): 327 (100) [M+·].
Anal. (C20H13N3O2) C, H, N.
5.1.2. 5-(4-Cyanophenyl)-2,3-dihydro-
1H,6H-pyrrolo[3,4-c]pyrido[3,4-b]indole-1,3-dione 2b
Yield 0.40 g (7%): m.p. > 350 °C; Rf 0.47 (ethyl
acetate/hexane 1:1); IR: ν (cm–1) 3 385, 3 230 (NH);
3 090–3 025 (CH); 2 245 (C≡N); 1 775, 1 740 (C=O);
1 625, 1 585, 1 495, 1 450 (C=C); 1H-NMR: δ 7.41–8.80
(m; 4 H aromat.), 8.15; 8.23 (AA’BB’, JAB = 8.4 Hz; 4 H
phenyl), 11.54 (br s; 1 H (exch.), NH imid), 12.33 (br s;
1 H (exch.), NH indole); MS: m/z (%): 338 (100) [M+·].
Anal. (C20H10N4O2) C, H, N.
5.1.7. 5-(4-Fluorophenyl)-2,3-dihydro-
1H,6H-pyrrolo[3,4-c]pyrido[3,4-b]indole-1,3-dione 2g
Yield 10 mg (0.2%): m.p. 271 °C; Rf 0.45 (ethyl
acetate/hexane 1:1); IR: ν (cm–1) 3 365, 3 210 (NH);
3 170–3 020 (CH); 1 765, 1 720 (C=O); 1 640, 1 610,
1
5.1.3. 5-(4-tert-Butoxyphenyl)-2,3-dihydro-
1 535, 1 450 (C=C); H-NMR: δ 7.43–8.89 (m; 8 H
1H,6H-pyrrolo[3,4-c]pyrido[3,4-b]indole-1,3-dione 2c
Yield 1.79 g (27%): m.p. 247 °C; Rf 0.50 (ethyl acetate/
hexane 1:1); IR: ν (cm–1) 3 450 (NH); 3 050–2 960 (CH);
aromat.), 11.43 (br s; 1 H (exch.), NH imid), 12.25 (br s;
1 H (exch.), NH indole); MS: m/z (%): 331 (100) [M+·].
Anal. (C19H10N3O2F) C, H, N.

422
5.1.8. 5-Benzyl-2,3-dihydro-1H,6H-
pyrrolo[3,4-c]pyrido[3,4-b]indole-1,3-dione 2h
5.1.13. 2,3-Dihydro-5-(3-hydroxy-4-methoxyphenyl)-
1H,6H-pyrrolo[3,4-c]pyrido[3,4-b]-indole-1,3-dione 2m
Yield 0.10 g (2%): m.p. 329 °C; Rf 0.33 (ethyl acetate/
hexane 1:1); IR: ν (cm–1) 3 285 (NH, OH); 3 050–3 960
(CH); 1 765, 1 740 (C=O); 1 630, 1 580, 1 495, 1 460
Yield 0.10 g (2%): m.p. 306 °C; Rf 0.51 (ethyl acetate/
hexane 1:1); IR: ν (cm–1) 3 450 (NH); 3 190–2 960 (CH);
1 760, 1 720 (C=O); 1 630, 1 580, 1 495, 1 460 (C=C);
1H-NMR: δ 4.63 (s; 2 H, CH2), 7.16–8.73 (m; 9 H
aromat.), 11.31 (br s; 1 H (exch.), NH imid), 12.55 (br s;
1 H (exch.), NH indole); MS: m/z (%): 327 (100) [M+·],
1
(C=C); H-NMR: δ 3.90 (s; 3 H, OCH3), 7.18–8.79 (m;
7 H aromat.), 9.47 (br s; 1 H (exch.), OH), 11.31 (br s; 1
H (exch.), NH imid), 12.13 (br s; 1 H (exch.), NH indole);
MS: m/z (%): 359 (100) [M+·]. Anal. (C20H13N3O4) C, H,
N.
·
326 (81) [M – H]+. Anal. (C20H13N3O2) C, H, N.
5.1.9. 2,3-Dihydro-5-(2-hydroxy-3-methoxyphenyl)-
1H,6H-pyrrolo[3,4-c]pyrido[3,4-b]-indole-1,3-dione 2i
5.1.14. 5-(2,4-Dimethoxyphenyl)-2,3-dihydro-
1H,6H-pyrrolo[3,4-c]pyrido[3,4-b]indole-1,3-dione 2n
Yield 0.43 g (7%): m.p. 308 °C; Rf 0.37 (ethyl acetate/
hexane 1:1); IR: ν (cm–1) 3 370, 3 205 (NH); 3 060–2 980
(CH); 1 770, 1 735 (C=O); 1 615, 1 580, 1 495, 1 450
Yield 1.20 g (19%): m.p. 335 °C; Rf 0.30 (ethyl acetate/
hexane 1:1); IR: ν (cm–1) 3 340 (NH, OH); 3 050–2 960
(CH); 1 765, 1 720 (C=O); 1 625, 1 580, 1 490, 1 475
1
1
(C=C); H-NMR: δ 3.92 (s; 3 H, OCH3), 6.98–8.78 (m;
(C=C); H-NMR: δ 3.78 (s; 3 H, OCH3), 3.91 (s; 3 H,
7 H aromat.), 9.53 (br s; 1 H (exch.), OH), 11.39 (br s; 1
H (exch.), NH imid), 11.82 (br s; 1 H (exch.), NH indole);
MS: m/z (%): 359 (100) [M+·]. Anal. (C20H13N3O4) C, H,
N.
OCH3), 6.76–8.77 (m; 7 H aromat.), 11.34 (s; 1 H (exch.),
NH imid), 12.13 (s; 1 H (exch.), NH indole); MS: m/z
(%): 373 (100) [M+·]. Anal. (C21H15N3O4) C, H, N.
5.1.15. 5-(3,4-Dimethoxyphenyl)-2,3-dihydro-
1H,6H-pyrrolo[3,4-c]pyrido[3,4-b]indole-1,3-dione 2o
Yield 0.55 g (9%): m.p. 288 °C; Rf 0.36 (ethyl acetate/
hexane 1:1); IR: ν (cm–1) 3 300, (NH); 3 070–2 960
(CH); 1 765, 1 735 (C=O); 1 630, 1 605, 1 520, 1 460
5.1.10. 2,3-Dihydro-5-(2-methoxyphenyl)-1H,6H-
pyrrolo[3,4-c]pyrido[3,4-b]indole-1,3-dione 2j
Yield 0.51 g (9%): m.p. 264 °C; Rf 0.46 (ethyl acetate/
hexane 1:1); IR: ν (cm–1) 3 335, 3 210 (NH); 3 050–2 970
(CH); 1 765, 1 720 (C=O); 1 630, 1 605, 1 580, 1 500
1
(C=C); H-NMR: δ 3.91 (s; 3 H, OCH3), 3.92 (s; 3 H,
OCH3), 7.24–8.80 (m; 7 H aromat.), 11.41 (br s; 1 H
(exch.), NH imid), 12.21 (br s; 1 H (exch.), NH indole);
MS: m/z (%): 373 (100) [M+·]. Anal. (C21H15N3O4) C, H,
N.
1
(C=C); H-NMR: δ 3.78 (s; 3 H, OCH3), 7.20–8.78 (m;
8 H aromat.), 11.38 (br s; 1 H (exch.), NH imid), 11.86
(br s; 1 H (exch.), NH indole); MS: m/z (%): 343 (100)
[M+·]. Anal. (C20H13N3O3) C, H, N.
5.1.16. 2,3-Dihydro-5-(4-hydroxy-3-methoxyphenyl)-
1H,6H-pyrrolo[3,4-c]pyrido[3,4-b]-indole-1,3-dione 2p
Yield 0.10 g (2%): m.p. 345 °C; Rf 0.29 (ethyl acetate/
hexane 1:1); IR: ν (cm–1) 3 415, 3 310 (NH, OH);
3 050–2 980 (CH); 1 765, 1 715, (C=O); 1 625, 1 610,
5.1.11. 2,3-Dihydro-5-(2-methylphenyl)-1H,6H-
pyrrolo[3,4-c]pyrido[3,4-b]indole-1,3-dione 2k
Yield 0.41 g (7%): m.p. 314 °C; Rf 0.50 (ethyl acetate/
hexane 1:1); IR: ν (cm–1) 3 275 (NH); 3 070–2 950 (CH);
1 765, 1 715 (C=O); 1 625, 1 580, 1 500, 1 455 (C=C);
1H-NMR: δ 2.18 (s; 3 H, CH3), 7.39–8.79 (m; 8 H
aromat.), 11.43 (br s; 1 H (exch.), NH imid), 11.98 (br s;
1 H (exch.), NH indole); MS: m/z (%): 327 (100) [M+·].
Anal. (C20H13N3O2) C, H, N.
1
1 575, 1 520 (C=C); H-NMR: δ 3.93 (s; 3 H, OCH3),
7.03–7.78 (m; 7 H aromat.), 9.63 (br s; 1 H (exch.), OH),
11.32 (br s; 1 H (exch.), NH imid), 12.17 (br s; 1 H
(exch.), NH indole); MS: m/z (%): 359 (100) [M+·]. Anal.
(C20H13N3O4) C, H, N.
5.1.17. 5-(4-N,N-Dimethylaminophenyl)-2,3-dihydro-
1H,6H-pyrrolo[3,4-c]pyrido[3,4-b]-indole-1,3-dione 2q
Yield 1.08 g (18%): m.p. 184 °C; Rf 0.30 (ethyl acetate/
hexane 1:1); IR: ν (cm–1) 3 450 (NH); 3 050–2 960 (CH);
1 765, 1 735 (C=O); 1 610, 1 565, 1 535, 1 495 (C=C);
1H-NMR: δ 3.07 (s; 6 H, N(CH3)2), 6.98; 7.99 (AA’BB’,
JAB = 8.9 Hz; 4 H phenyl), 7.37–8.78 (m; 4 H aromat.),
11.30 (br s; 1 H (exch.), NH imid), 12.11 (br s; 1 H
(exch.), NH indole); MS: m/z (%): 356 (100) [M+·]. Anal.
(C21H16N4O2) C, H, N.
5.1.12. 2,3-Dihydro-5-(3-methylphenyl)-1H,6H-
pyrrolo[3,4-c]pyrido[3,4-b]indole-1,3-dione 2l
Yield 0.27 g (5%): m.p. 321 °C; Rf 0.52 (ethyl acetate/
hexane 1:1); IR: ν (cm–1) 3 400, 3 230 (NH); 3 060–2 970
(CH); 1 770, 1 715 (C=O); 1 605, 1 580, 1 570, 1 455
1
(C=C); H-NMR: δ 2.52 (s; 3 H, CH3), 7.39–8.80 (m; 8
H aromat.), 11.51 (br s; 1 H (exch.), NH imid), 12.18 (br
s; 1 H (exch.), NH indole); MS: m/z (%): 327 (100)
[M+·], 312 (8) [M – ·CH3]+. Anal. (C20H13N3O2) C, H, N.

423
5.1.18. 2,3-Dihydro-5-(3-nitrophenyl)-1H,6H-
pyrrolo[3,4-c]pyrido[3,4-b]indole-1,3-dione 2r
1H-NMR: δ 3.90 (s; 3 H, OCH3), 3.91 (s; 3 H, OCH3),
7.15–7.23 (m; 1 H, phenyl), 7.36 (dd, Jo = 9.0 Hz, Jm =
2.6 Hz; 1 H, 8–H), 7.53–7.63 (m; 3 H, phenyl), 7.67 (d,
Jo = 9.0 Hz; 1 H, 7-H), 8.27 (d, Jm = 2.5 Hz; 1 H, 10-H),
11.42 (br s; 1 H (exch.), NH imid), 12.08 (br s; 1 H
(exch.), NH indole); MS: m/z (%): 373 (100) [M+·], 358
Yield 0.1 g (2%): m.p. 322 °C; Rf 0.46 (ethyl acetate/
hexane 1:1); IR: ν (cm–1) 3 370 (NH); 3 070–2 980 (CH);
1 770, 1 720 (C=O); 1 630, 1 585 (C=C); 1 535, 1 350
(NO2); 1H-NMR: δ 7.42–8.81 (m; 8 H aromat.), 11.53 (br
s; 1 H (exch.), NH imid), 12.41 (br s; 1 H (exch.), NH
indole); MS: m/z (%): 358 (100) [M+·], 312 (70) [M –
·NO2]+, 241 (44) [312–(HNCO + CO)]+, 214 (25) [241 –
HCN]+. Anal. (C19H10N4O4) C, H, N.
·
(39) [M – CH3]+. Anal. (C21H15N3O4) C, H, N.
5.1.23. 2,3-Dihydro-9-methoxy-5-(2-methoxyphenyl)-
1H,6H-pyrrolo[3,4-c]pyrido[3,4-b]-indole-1,3-dione 2w
Yield 0.96 g (15%): m.p. 307 °C; Rf 0.27 (ethyl acetate/
hexane 1:1); IR: ν (cm–1) 3 330, 3 200 (NH); 3 070–2 835
(CH); 1 775, 1 730 (C=O); 1 635, 1 605, 1 575 (C=C);
1H-NMR: δ 3.77 (s; 3 H, 2’-OCH3), 3.90 (s; 3 H,
9-OCH3), 7.19 (m; 1 H, phenyl), 7.30 (d, J = 8.1 Hz; 1 H,
phenyl), 7.34 (dd, Jo = 8.9 Hz, Jm = 2.5 Hz; 1 H, 8-H),
7.52–7.64 (m; 2 H, phenyl), 7.60 (d; Jo = 8.6 Hz; 1 H,
7–H), 8.26 (d, Jm = 2.5 Hz; 1 H, 10-H), 11.36 (br s; 1 H
(exch.), NH imid), 11.71 (br s; 1 H (exch.), NH indole);
MS: m/z (%): 373 (100) [M+·], 358 (9) [M – ·CH3]+, 355
5.1.19. 2,3-Dihydro-5-(3-methoxyphenyl)-
1H,6H-pyrrolo[3,4-c]pyrido[3,4-b]indole-1,3-dione 2s
Yield 0.63 g (11%): m.p. 256 °C; Rf 0.34 (ethyl acetate/
hexane 1:1); IR: ν (cm–1) 3 350, 3 240 (NH); 3 040–2 980
(CH); 1 765, 1 740 (C=O); 1 650, 1 625, 1 600, 1 580
1
(C=C); H-NMR: δ 3.91 (s; 3 H, OCH3), 7.18–8.80 (m;
8 H aromat.), 11.41 (br s; 1 H (exch.), NH imid), 12.20
(br s; 1 H (exch.), NH indole); MS: m/z (%): 343 (100)
[M+·], 342 (78) [M – ·H]+. Anal. (C20H13N3O3) C, H, N.
·
(19) [M – H2O]+·, 340 (21) [355 – CH3]+. Anal.
5.1.20. 2,3-Dihydro-9-methoxy-5-phenyl-
(C21H15N3O4) C, H, N.
1H,6H-pyrrolo[3,4-c]pyrido[3,4-b]indole-1,3-dione 2t
Yield 0.94 g (16%): m.p. 288 °C; Rf 0.40 (ethyl acetate/
hexane 1:1); IR: ν (cm–1) 3 450, 3 360 (NH); 3 060–2 835
(CH); 1 770, 1 710 (C=O); 1 630, 1 570, 1 490 (C=C);
1H-NMR: δ 3.90 (s; 3 H, OCH3), 7.35 (dd, Jo = 8.9 Hz,
Jm = 2.0 Hz; 1 H, 8–H), 7.58–7.72 (m; 3 H, 3’,4’,5’–H),
7.66 (d, Jo = 8.9 Hz; 1 H, 7–H), 8.03 (d, Jo = 6.9 Hz; 2
H, 2’,6’-H), 8.25 (δ, Jm = 1.9 Hz; 1 H, 10–H), 11.39 (br
s; 1 H (exch.), NH imid), 12.07 (br s; 1 H (exch.), NH
indole); MS: m/z (%): 343 (100) [M+·], 328 (67) [M –
·CH3]+, 300 (43) [328 – CO]+, 171.5 (3) M2+. Anal.
(C20H13N3O3) C, H, N.
5.1.24. 5-(2,4-Dimethoxyphenyl)-2,3-dihydro-9-
methoxy-1H,6H-pyrrolo[3,4-c]pyrido-
[3,4-b]indole-1,3-dione 2x
Yield 0.83 g (12%): m.p. 308 °C; Rf 0.20 (ethyl acetate/
hexane 1:1); IR: ν (cm–1) 3 395, 3 280 (NH); 3 090–2 840
(CH); 1 770, 1 745 (C=O); 1 615, 1 575, 1 515 (C=C);
1H-NMR: δ 3.77 (s; 3 H, 2’-OCH3), 3.90 (s; 3 H, OCH3),
3.91 (s; 3 H, OCH3), 6.77 (dd, Jo = 8.4 Hz, Jm = 2.3 Hz;
1 H, 5’-H), 6.82 (d, Jm = 2.3 Hz; 1 H, 3’-H), 7.33 (dd,
Jo = 9.0 Hz, Jm = 2.6 Hz; 1 H, 8-H), 7.48 (d, Jo = 8.4 Hz;
1 H, 6’-H), 7.60 (dd, Jo = 9.0 Hz, Jp = 0.6 Hz; 1 H, 7-H),
8.25 (dd, Jm = 2.6 Hz, Jp = 0.6 Hz; 1 H, 10-H), 11.32 (br
s; 1 H (exch.), NH imid), 11.65 (br s; 1 H (exch.), NH
indole); MS: m/z (%): 403 (100) [M+·], 388 (7) [M –
·CH3]+, 385 (16) [M – H2O]+·, 373 (8) [M – H2C=O]+·,
370 (17) [385 – ·CH3]+, 201.5 (6) M2+. Anal.
(C22H17N3O5) C, H, N.
5.1.21. 2,3-Dihydro-9-methoxy-5-(4-methoxyphenyl)-
1H,6H-pyrrolo[3,4-c]pyrido[3,4-b]-indole-1,3-dione 2u
Yield 0.90 g (14%): m.p. 341 °C; Rf 0.30 (ethyl acetate/
hexane 1:1); IR: ν (cm–1) 3 450, 3 365 (NH); 3 120–2 835
(CH); 1 770, 1 735 (C=O); 1 610, 1 580, 1 570 (C=C);
1H-NMR: δ 3.89 (s; 3 H, OCH3), 3.91 (s; 3 H, OCH3),
7.22; 8.01 (AA’BB’, JAB = 8.9 Hz; 4 H, 2’,3’,5’,6’-H),
7.33 (dd, Jo = 9.0 Hz, Jm = 2.6 Hz; 1 H, 8–H), 7.65 (d,
Jo = 8.9 Hz; 1 H, 7–H), 8.24 (d, Jm = 2.5 Hz; 1 H, 10–H),
11.42 (br s; 1 H (exch.), NH imid), 11.98 (br s; 1 H
(exch.), NH indole); MS: m/z (%): 373 (100) [M+·], 358
5.1.25. 5-(3,4-Dimethoxyphenyl)-2,3-dihydro-9-
methoxy-1H,6H-pyrrolo[3,4-c]pyrido-
[3,4-b]indole-1,3-dione 2y
Yield 0.55 g (8%): m.p. 228 °C; Rf 0.36 (ethyl acetate/
hexane 1:1); IR: ν (cm–1) 3 435, 3 275 (NH); 3 060–2 870
(CH); 1 750, 1 700 (C=O); 1 630, 1 610, 1 585 (C=C);
1H-NMR: δ 3.90 (s; 3 H, OCH3), 3.90 (s; 3 H, OCH3),
3.91 (s; 3 H, OCH3), 7.24 (d, Jo = 8.3 Hz; 1 H, 5’-H), 7.35
(dd, Jo = 9.0 Hz, Jm = 2.7 Hz; 1 H, 8–H), 7.57 (d, Jm =
2.1 Hz; 1 H, 2’-H), 7.61 (dd, Jo = 8.3 Hz, Jm = 2.1 Hz; 1
H, 6’-H), 7.67 (dd, Jo = 9.0 Hz, Jp = 0.5 Hz; 1 H, 7–H),
8.26 (dd, Jm = 2.7 Hz, Jp = 0.5 Hz; 1 H, 10–H), 11.34 (br
·
(51) [M – CH3]+. Anal. (C21H15N3O4) C, H, N.
5.1.22. 2,3-Dihydro-9-methoxy-5-(3-methoxyphenyl)-
1H,6H-pyrrolo[3,4-c]pyrido[3,4-b]-indole-1,3-dione 2v
Yield 0.45 g (7%): m.p. 309 °C; Rf 0.38 (ethyl acetate/
hexane 1:1); IR: ν (cm–1) 3 360, 3 285 (NH); 3 075–2 835
(CH); 1 765, 1 740 (C=O); 1 630, 1 610, 1 570 (C=C);

424
s; 1 H (exch.), NH imid), 12.06 (br s; 1 H (exch.), NH
indole); MS: m/z (%): 403 (100) [M+·], 388 (40) [M –
·CH3]+. Anal. (C22H17N3O5) C, H, N.
5.1.30. 2,3-Dihydro-9-methoxy-5-(2-methylphenyl)-1H,
6H-pyrrolo[3,4-c]pyrido[3,4-b]-indole-1,3-dione 2dd
Yield 0.74 g (12%): m.p. 243 °C; Rf 0.45 (ethyl acetate/
hexane 1:1); IR: ν (cm–1) 3 375, 3 225 (NH); 3 085–2 835
(CH); 1 770, 1 735 (C=O); 1 570, 1 495, 1 465 (C=C);
1H-NMR: δ 2.17 (s; 3 H, CH3), 3.90 (s; 3 H, OCH3), 7.35
(dd, Jo = 9.0 Hz, Jm = 2.7 Hz; 1 H, 8-H), 7.40–7.56 (m;
4 H phenyl), 7.59 (dd, Jo = 9.0 Hz, Jp = 0.6 Hz; 1 H,
7–H), 8.26 (dd, Jm = 2.7 Hz, Jp = 0.6 Hz; 1 H, 10-H),
11.40 (br s; 1 H (exch.), NH imid), 11.84 (br s; 1 H
(exch.), NH indole); MS: m/z (%): 357 (100) [M+·], 342
5.1.26. 5-(4-Cyanophenyl)-2,3-dihydro-9-methoxy-
1H,6H-pyrrolo[3,4-c]pyrido[3,4-b]-indole-1,3-dione 2z
Yield 0.13 g (2%): m.p. 335 °C (dec.); Rf 0.39 (ethyl
acetate/hexane 1:1); IR: ν (cm–1) 3 455, 3 375 (NH);
3 100–2 835 (CH); 2 230 (C· N); 1 770, 1 720 (C=O);
1 655, 1 630, 1 575, 1 510 (C=C); 1H-NMR: δ 3.90 (s; 3
H, OCH3), 6.88 (dd, Jo = 8.8 Hz, Jm = 2.4 Hz; 1 H, 8–H),
7.41 (d, Jo = 8.8 Hz; 1 H, 7–H), 8.14; 8.21 (AA’BB’,
JAB = 8.0 Hz; 4 H, 2’,3’,5’,6’-H), 8.30 (d, Jm = 2.5 Hz;
1 H, 10–H), 11.49 (br s; 1 H (exch.), NH imid), 12.21 (br
s; 1 H (exch.), NH indole); MS: m/z (%): 368 (100)
[M+·], 353 (79) [M – ·CH3]+, 325 (36) [353 – CO]+. Anal.
(C21H12N4O3) C, H, N.
·
(29) [M – CH3]+. Anal. (C21H15N3O3) C, H, N.
5.1.31. 2,3-Dihydro-8,9-dimethoxy-5-(2-methylphenyl)-
1H,6H-pyrrolo[3,4-c]pyrido-[3,4-b]indole-1,3-dione 2ee
Prepared analogous to the previous general method,
but heating under reflux for 1 h in tetralin instead of
o-xylene as solvent gives yellow crystals. Yield 0.67 g
(10%): m.p. > 350 °C (ethyl acetate); Rf 0.21 (ethyl
acetate/hexane 1:1); IR: ν (cm–1) 3 300 (NH); 3 085–2 840
(CH); 1 770, 1 730 (C=O); 1 630, 1 575, 1 490 (C=C);
1H-NMR: δ 2.17 (s; 3 H, CH3), 3.89 (s; 3 H, OCH3), 3.90
(s; 3 H, OCH3), 7.11 (s; 1 H, 8-H), 7.39–7.55 (m; 4 H
phenyl), 8.17 (s; 1 H, 10-H), 11.32 (br s; 1 H (exch.), NH
imid), 11.75 (br s; 1 H (exch.), NH indole); MS: m/z (%):
387 (100) [M+·], 372 (24) [M – ·CH3]+. Anal.
(C22H17N3O4) C, H, N.
5.1.27. 2,3-Dihydro-9-methoxy-2-methyl-5-phenyl-1H,
6H-pyrrolo[3,4-c]pyrido[3,4-b]-indole-1,3-dione 2aa
Yield 0.74 g (12%): m.p. 305 °C; Rf 0.62 (ethyl acetate/
hexane 1:1); IR: ν (cm–1) 3 400 (NH); 3 100–2 835 (CH);
1 760, 1 705 (C=O); 1 635, 1 570, 1 495 (C=C); 1H-
NMR: δ 3.13 (s; 3 H, NCH3), 3.88 (s; 3 H, OCH3), 7.32
(dd, Jo = 9.0 Hz, Jm = 2.6 Hz; 1 H, 8-H), 7.59–7.70 (m;
3 H, 3’,4’,5’-H), 7.62 (d, Jo = 8.9 Hz; 1 H, 7–H), 8.01 (dd,
Jo = 7.6 Hz, Jm = 7.7 Hz; 2 H, 2’,6’-H), 8.19 (d, Jm = 2.6
Hz; 1 H, 10-H), 12.05 (br s; 1 H (exch.), NH indole); MS:
m/z (%): 357 (100) [M+·], 342 (48) [M – ·CH3]+, 314 (20)
[342 – CO]+. Anal. (C21H15N3O3) C, H, N.
5.1.32. 9-Benzyloxy-2,3-dihydro-5-(2-methoxyphenyl)-
1H,6H-pyrrolo[3,4-c]pyrido-[3,4-b]indole-1,3-dione 2ff
Yield 1.39 g (18%): m.p. 279 °C; Rf 0.68
(dichloromethane/methanol 10:1); IR: ν (cm–1) 3 415
(NH); 3 040–2 890 (CH); 1 775, 1 730 (C=O); 1 600,
5.1.28. 2,3-Dihydro-8,9-dimethoxy-5-phenyl-
1H,6H-pyrrolo[3,4-c]pyrido[3,4-b]indole-1,3-dione 2bb
Yield 0.11 g (2%): m.p. > 350 °C; Rf 0.14 (ethyl
acetate/hexane 1:1); IR: ν (cm–1) 3 465, 3 310 (NH);
3 090–2 835 (CH); 1 770, 1 715 (C=O); 1 660, 1 590,
1
1 570, 1 490, 1 460 (C=C); H-NMR: δ 3.77 (s; 3 H,
OCH3), 5.24 (s; 2 H, CH2), 7.16–7.22 (m; 1 H aromat.),
7.28–7.64 (m; 10 H aromat.), 8.35 (d, J = 2.5 Hz; 1 H,
10-H), 11.36 (s; 1 H (exch.), NH imid), 11.72 (s; 1 H
(exch.), NH indole); MS: m/z (%): 449 (49) [M+·], 358
(100) [M – ·C7H7]+, 91 (51) [C7H7]+. Anal. (C27H19N3O4)
C, H, N.
1
1 490 (C=C); H-NMR: δ 3.90 (s; 3 H, OCH3), 3.92 (s;
3 H, OCH3), 7.18 (s; 1 H, 7–H), 7.58–7.70 (m; 3 H,
3’,4’,5’-H), 8.02 (dd, Jo = 8.0 Hz, Jm = 1.6 Hz; 2 H,
2’,6’-H), 8.17 (s; 1 H, 10-H), 11.32 (br s; 1 H (exch.), NH
imid), 11.99 (br s; 1 H (exch.), NH indole); MS: m/z (%):
373 (100) [M+·], 358 (50) [M – ·CH3]+. Anal.
(C21H15N3O4) C, H, N.
5.1.33. 9-Benzyloxy-2,3-dihydro-5-(2-methylphenyl)-1H,
6H-pyrrolo[3,4-c]pyrido[3,4-b]-indole-1,3-dione 2gg
Yield 0.52 g (7%): m.p. 264 °C; Rf 0.64
(dichloromethane/methanol 10:1); IR: ν (cm–1) 3 450
(NH); 3 095–2 965 (CH); 1 770, 1 700 (C=O); 1 620,
5.1.29. 2,3-Dihydro-2-methyl-5-phenyl-
1H,6H-pyrrolo[3,4-c]pyrido[3,4-b]indole-1,3-dione 2cc
Yield 0.05 g (1%): m.p. 317 °C; Rf 0.66 (ethyl acetate/
hexane 1:1); IR: ν (cm–1) 3 385 (NH); 3 060–2 940 (CH);
1 765, 1 705 (C=O); 1 630, 1 605, 1 585 (C=C); 1H-
NMR: δ 3.15 (s; 3 H, CH3), 7.39–8.78 (m; 9 H aromat.),
12.23 (s; 1 H (exch.), NH indole); MS: m/z (%) 327 (100)
[M+·]. Anal. (C20H13N3O2) C, H, N.
1
1 570, 1 485, 1 465 (C=C); H-NMR: δ 2.17 (s; 3 H,
CH3), 5.24 (s; 2 H, CH2), 7.34–7.61 (m; 11 H aromat.),
8.35 (d, J = 2.4 Hz; 1 H, 10-H), 11.37 (br s; 1 H (exch.),
NH imid), 11.84 (br s; 1 H (exch.), NH indole); MS: m/z
·
(%): 433 (33) [M+·], 342 (80) [M – C7H7]+, 91 (100)
[C7H7]+. Anal. (C27H19N3O3) C, H, N.

425
5.2. General procedure for compounds 2hh and 2ii
To solution of the 9-benzyloxy compound
protease inhibitors as described [16]. Equal amounts of
lysate were diluted 1:10 in 10 mM Tris/HCl, pH 7.6, and
were applied to a nitrocellulose membrane in a dot-blot
apparatus (BioRad). The membrane was blocked with 1%
BSA and developed with anti-phosphotyrosine antibodies
(RC20–HRP conjugate, Dianova, Transduction Laborato-
ries) and the ECL reagent (Amersham). The films were
scanned, and the phosphorylation signals were quantified
using the program NIH Image 1.54. IC50 values were
calculated referring to the difference of non-PDGF stimu-
lated samples (0%) and DMSO treated PDGF stimulated
samples (100%). Ranges for the IC50 values were defined
based on at least three independent experiments. For a
qualitative analysis of the effects on tyrosine phosphory-
lation, 10 µg protein of identically treated cell extracts
were subjected to SDS-PAGE with 7.5% gels and immu-
noblotting with anti-phosphotyrosine antibodies. To evalu-
ate PDGF receptor levels, total cell lysates of one well
were subjected to incubation with wheat germ agarose
beads (Pharmacia, 30 µL 1:1 suspension per extract) for
1 h at 4 °C with end-over-end rotation. The beads were
washed once with lysis buffer and extracted with SDS-
PAGE sample buffer. SDS-PAGE and immunoblotting
were performed as above except that the blots were
developed with anti-PDGF receptor antibodies DIG 1
[16]. Quantification of IC50 values was based on the
intensity of the signal for autophosphorylated PDGF
receptor. Titration was done using four–eight inhibitor
concentrations within a range of two orders of magnitude
which was selected on the basis of preliminary experi-
ments. Results of triplicate determinations were subjected
to curve fitting using the program Sigma Plot 2.0 (Jandel
Corporation). Porcine aortic endothelial (PAE) cells ex-
pressing human PDGFb receptors were kindly provided
by Dr L. Claesson-Welsh (Uppsala). Testing inhibition of
human PDGFb receptor kinase in these cells was done as
with Swiss 3T3 cells except that the cells were starved in
0.5% FCS-containing medium for 24 h before the experi-
ment. In vitro kinase reactions with isolated Swiss 3T3
cell membranes or purified PDGF receptor were per-
formed as described earlier [16, 17]. To measure effects
on EGF receptor phosphorylation, either in vitro kinase
reactions with Swiss 3T3 membranes were performed
[16], or A431 cells (ATCC CRL 1555) were starved
overnight in serum-free medium, treated for 2 h with the
compounds, thereafter stimulated with 100 ng/mL EGF
(TEBU/Peprotech) for 10 min and subjected to extraction
and immunoblotting as described above. FGF-dependent
tyrosine phosphorylation was measured in Swiss 3T3
cells, cultivated and treated with compounds as described
above and stimulated for 10 min with 100 ng/mL basic
FGF (TEBU/Peprotech). Cell extracts were analysed by
a
(0.12 mmol) in anhydrous ethanol (5 mL) and THF
(5 mL) are added ammonium formate (15.8 mg,
0.22 mmol) and 10% Pd/C (10 mg). This mixture is
heated under reflux for 3 h. After cooling to room
temperature the catalyst is removed by filtration through
Celite, the solvent is removed in vacuo, and the oily
residue is purified by column chromatography (column
1 cm × 2 cm, silica gel, dichloromethane/methanol 10:1)
to yield yellow crystals. Analytical samples were ob-
tained as yellow crystals from ethyl acetate.
5.2.1. 2,3-Dihydro-9-hydroxy-5-(2-methoxyphenyl)-1H,
6H-pyrrolo[3,4-c]pyrido[3,4-b]-indole-1,3-dione 2hh
Yield 30 mg (70%): m.p. 262 °C; Rf 0.60
(dichloromethane/methanol 10:1); IR: ν (cm–1) 3 425
(NH, OH); 3 070–2 960 (CH); 1 765, 1 705 (C=O);
1 640, 1 605, 1 570, 1 500 (C=C); 1H-NMR: δ 3.77 (s; 3
H, OCH3), 7.15–7.20 (m; 2 H aromat.), 7.29 (d, J =
8.2 Hz; 1 H aromat.), 7.48–7.63 (m; 3 H aromat.), 8.16 (d,
J = 2.4 Hz; 1 H, 10-H), 9.45 (s; 1 H (exch.), OH), 11.31
(s; 1 H (exch.), NH imid), 11.57 (s; 1 H (exch.), NH
indole); MS: m/z (%): 359 (100) [M+·]. Anal.
(C20H13N3O4) C, H, N.
5.2.2. 2,3-Dihydro-9-hydroxy-5-(2-methylphenyl)-
1H,6H-pyrrolo[3,4-c]pyrido[3,4-b]-indole-1,3-dione 2ii
Yield 25 mg (61%): m.p. > 350 °C; Rf 0.41
(dichloromethane/methanol 10:1); IR: ν (cm–1) 3 320,
3 265 (NH, OH); 3 080–2 975 (CH); 1 780, 1 705 (C=O);
1 640, 1 570, 1 510, 1 460 (C=C); 1H-NMR: δ 2.17 (s; 3
H, CH3), 7.19 (dd, J1 = 8.8 Hz, J2 = 2.4 Hz; 1 H, 8–H),
7.39–7.55 (m; 5 H aromat.), 8.17 (d, J = 2.4 Hz; 1 H,
10-H), 9.49 (s; 1 H (exch.), OH), 11.35 (s; 1 H (exch.),
NH imid), 11.70 (s; 1 H (exch.), NH indole); MS: m/z
(%): 343 (100) [M+·]. Anal. (C20H13N3O3) C, H, N.
5.3. Assays for tyrosine kinase inhibition
Swiss 3T3 fibroblasts (ATCC CCL 92) were cultivated
in DMEM/10% FCS (Life Technologies). The effect on
PDGF-stimulated tyrosine phosphorylation was measured
by subjecting quiescent cultures in 24-well plates (NUNC)
to a medium change into serum-free DMEM (0.4 mL per
well) and treating them with the test compounds in
DMSO (final DMSO concentration 1%) or solvent for
2 h. The cells were subsequently stimulated with 100 ng/
mL human recombinant PDGF-BB (TEBU/Peprotech) or
mock-treated for 10 min at room temperature, washed
twice with PBS and extracted with lysis buffer containing
Hepes, pH 7.5, 1% Triton X-100, phosphatase and

426
immunoblotting with anti-phosphotyrosine antibodies. A
prominent phosphoprotein band of 90–92 kDa, probably
representing FRS2 [45-46], appeared only in FGF-
stimulated cells and was taken as a measure of FGF
receptor activity. pp60src Kinase activity and Src-
dependent tyrosine phosphorylation in src-transformed
NIH-fibroblasts were determined as described elsewhere
[47]. SCF-dependent c-Kit phosphorylation was mea-
sured in CHRF cells [48] (generously provided by Dr M.
Lieberman, Cincinnati), cultivated in DMEM/10% HS
(Life Technologies). For the assay, CHRF cells were
treated for 2 h in serum-free medium with inhibitors, then
stimulated with 40 ng/mL human SCF (TEBU/Peprotech)
for 5 min, washed twice with buffer containing 50 mM
Tris, pH 8.0, 250 mM NaCl, 100 mM NaF and 10%(v/v)
glycerol, and followed by centrifugation at 1 500 rpm for
5 min. The cells were extracted by lysis buffer containing
50 mM Tris, pH 8.0, 250 mM NaCl, 100 mM NaF, 10%
(v/v) glycerol, 1% (v/v) NP-40, 1 mM PMSF, 1 µM
leupeptin, 1 µM aprotinin and 1 mM Na-orthovanadate.
The lysates were subjected to an immunoprecipitation
with anti-c-Kit antibodies (Santa Cruz). Thereafter, the
receptor was immunoblotted with antiphosphotyrosine
antibodies and re-blotted with anti-c-Kit antibodies to
check the unchanged receptor content.
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