Development of 5-benzylpaullones and paullone-9-carboxylic acid alkyl esters as selective inhibitors of mitochondrial malate dehydrogenase (mMDH)

Article abstract of DOI:10.1016/j.ejmech.2009.10.018

A collection of paullones was tested for inhibitory activity against mitochondrial malate dehydrogenase (mMDH) as a biological target for antiproliferative activity. Based on the results of this screening, 5-benzylpaullones and paullone-9-carboxylic acid alkyl esters were developed as selective mMDH inhibitors. The new derivatives did not show noteworthy antiproliferative activity when tested on a panel of cancer cell lines, suggesting that mMDH inhibition is of minor relevance for the growth inhibition caused by paullones.

Full text of DOI:10.1016/j.ejmech.2009.10.018

European Journal of Medicinal Chemistry 45 (2010) 335–342
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Development of 5-benzylpaullones and paullone-9-carboxylic acid alkyl esters
as selective inhibitors of mitochondrial malate dehydrogenase (mMDH)
Anja Becker a, Simone Kohfeld a, Annette Lader a, Lutz Preu a, Tanja Pies b, Karen Wieking b,
,
Yoan Ferandin c, Marie Knockaert c, Laurent Meijer c, Conrad Kunick a
*
a Technische Universita¨t Braunschweig, Institut fu¨r Pharmazeutische Chemie, Beethovenstraße 55, D-38106 Braunschweig, Germany
b Universita¨t Hamburg, Institut fu¨r Pharmazie, Bundesstraße 45, D-20146 Hamburg, Germany
c Centre National de la Recherche Scientifique, Station Biologique, BP 74, F-29682 Roscoff, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A collection of paullones was tested for inhibitory activity against mitochondrial malate dehydrogenase
(mMDH) as a biological target for antiproliferative activity. Based on the results of this screening,
5-benzylpaullones and paullone-9-carboxylic acid alkyl esters were developed as selective mMDH
inhibitors. The new derivatives did not show noteworthy antiproliferative activity when tested on a panel
of cancer cell lines, suggesting that mMDH inhibition is of minor relevance for the growth inhibition
caused by paullones.
Received 16 July 2009
Accepted 9 October 2009
Available online 14 October 2009
Keywords:
Antiproliferative activity
Enzyme inhibition
Mitochondrial malate dehydrogenase
Paullone
Ó 2009 Elsevier Masson SAS. All rights reserved.
Kinase
1. Introduction
might, at least in part, be responsible for the antiproliferative activity
of distinct paullones. In order to identify these additional targets,
The paullones are a family of ATP competitive and selective kinase
inhibitors. The parent molecular scaffold of the paullones is based on
the 7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)-one system, of
which more than 280 derivatives have been described. Paullones
substituted on distinct positions display manifold biological activi-
ties, e.g. inhibition of tumor cell growth [1], protection of insulin-
producing pancreatic beta cells [2], and antiparasitic activity against
Leishmania parasites [3]. The commercial available congener alster-
paullone is a widely used inhibitor of glycogen synthase kinase-3
(GSK-3) and cyclin-dependent kinases (CDKs) [4]. The potent anti-
proliferative acivity of alsterpaullone against human tumor cell lines
appears to be due to perturbation of the mitochondrial membrane
potential eventually leading to apoptosis [5]. Although the structure-
activity relationships are well established for both CDK1 and GSK-3
inhibition by paullones [6], the structural requirements for the
antitumor activity of paullones are still obscure. A recent publication
indicated that an obvious correlation between kinase inhibition and
tumor cell growth inhibition by paullones could not be shown [6]. Of
note, some paullones with potent kinase inhibitory properties are
devoid of noteworthy antiproliferative activity [6]. It was therefore
speculated that additional biological targets besides GSK-3 and CDKs
pull-down experiments have been carried out using paullones bound
to solid matrices. These affinity investigations revealed that paul-
lones bind mitochondrial malate dehydrogenase (mMDH) from
various tissues, and subsequent enzymatic studies showed that
alsterpaullone and kenpaullone inhibited mMDH in the low micro-
molar concentration range [7]. Although mMDH inhibition has been
suggested as mechanistic rationale for the design of antineoplastic
agents [8,9], these results were not sufficient to explain the relevance
of the mMDH inhibition for the antiproliferative activity of paullones.
To discriminate between the influence of GSK-3/CDK inhibition
and mMDH inhibition on growth inhibition by paullones, we initi-
ated a program for the development of paullones with improved
mMDH inhibitory activity but devoid of kinase inhibitory properties.
Such mMDH-selective paullones were subsequently tested for
antiproliferative activity against tumor cell lines.
In a first step, we determined the mMDH inhibition potential of
a wide array of paullones. Initial tests were carried out at a 10
mM
paullone concentration. The results of these tests are given inTable 1
together with the IC50 values for CDK1 and GSK-3. The results show
on the one hand that the majority of the paullones exhibit only low
inhibitory potency for mMDH and on the other hand that there is no
obvious correlation between mMDH inhibition and GSK-3 or CDK1/
cyclin B inhibition. 1j (paullone-9-carboxylic acid methyl ester) and
2a (5-benzylkenpaullone) showed a considerable mMDH inhibition
* Corresponding author. Fax: þ49 531 391 2799.
E-mail address: c.kunick@tu-bs.de (C. Kunick).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.10.018

336
A. Becker et al. / European Journal of Medicinal Chemistry 45 (2010) 335–342
Table 1
acid (not shown in Scheme 1). For the synthesis of 5-benzylpaul-
lones 4a–k as analogues of 2a, the corresponding paullones 1 were
selectively deprotonated by means of sodium hydride at the lactam
nitrogen and the resulting anions alkylated with appropriately
substituted benzyl bromides. To avoid additional substitution at the
indole nitrogen the reaction was stopped before completion and
the products were separated from unreacted starting materials 1 by
column chromatography. The paullone-9-carboxylic acid 1i was
used as educt for the synthesis of the corresponding alkyl esters
5a–f by a Mitsunobu reaction [13], employing the respective alco-
hols as nucleophiles in the presence of triphenylphosphane and di-
tert-butyl azodicarboxylate in dry THF.
mMDH inhibition (residual mMDH activity in the presence of 10
and kinase inhibition (IC50) by paullones
m
m test compound)
R1
O
O
H
4
N
N
3
R1
2
1
8
R2
N
HN
R2
Br
9
11
10
3. Biological evaluation and discussion
1
2
Results of the enzyme inhibition tests revealed that in compli-
ance with our working hypothesis the new 2a-related benzyl-
paullones 4a–k were devoid of kinase inhibitory activity against
GSK-3 and CDK1/cyclin B but showed improved mMDH inhibitory
.
R1
R2
residual mMDH IC50 GSK-3 IC50 CDK1/
activity @
10
M [%]a
[
m
M]
Cyclin B
m
[m
M]
activity, e.g. derivative 4k (IC50 ¼ 0.77
mM). Similarly, the paullone-
9-carboxylic esters 5a–f exhibited poor kinase inhibitory activity. In
this series, the octyl ester 5f showed a noteworthy mMDH inhibi-
1ab
1bb
1cb
1db
1eb
1fb
1gb
1hb
1i
H
H
H
H
H
H
H
H
H
H
H
H
9-Br
9-NO2
H
9-Cl
9-F
9-CF3
9-CN
9-CH3
74.5 ꢂ 0.6
54.2 ꢂ 3.1
78.7 ꢂ 4.0
73.4 ꢂ 4.3
46.2 ꢂ 5.4
88.3 ꢂ 0.9
91.3 ꢂ 5.5
87.8 ꢂ 7.4
98.7 ꢂ 0.6
60.9 ꢂ 3.1
76.6 ꢂ 1.0
96.2 ꢂ 1.3
97.7 ꢂ 0.9
83.9 ꢂ 1.4
56.3 ꢂ 2.6
58.7 ꢂ 3.0
15.0 ꢂ 8.0
82.0 ꢂ 0.1
25.9 ꢂ 3.9
89.2 ꢂ 2.7
87.6 ꢂ 3.1
65.3 ꢂ 3.1
28.7 ꢂ 3.0
8.6 ꢂ 0.6
0.023
0.004
0.62
0.024
0.080
0.030
0.010
0.13
0.40
0.035
7.0
0.60
1.6
0.40
0.024
2.0
47
4.2
0.4
0.28
0.28
0.70
0.20
0.40
0.60
tion with an IC50 value of 2.7 mM (Table 2). In both compound
families, a correlation was found between the mMDH inhibitory
activity and the lipophilicity of the molecules expressed by the
clogP values (Figs. 1 and 2, respectively). This observation suggests
that hydrophobic interactions play a major role in the paullone-
mMDH binding mode. Selected derivatives with a considerable
degree of mMDH selectivity (4b, 4k, 5d and 5f) were then tested for
antiproliferative activity in the in vitro cell line screening project
(IVCLSP) of the National Cancer Institute (NCI).
In the IVCLSP, the growth inhibitory potencies of test compounds
are determined on50–60cancercelllines derivedfrom nine different
tumor entities, representing cancers of the brain, lung, colon, ovary,
breast, prostate, kidney, as well as leukaemia and melanoma. The
screening system is a two step procedure. Initially, the test
9-COOH
9-COOCH3
9-SCH3
30
1j
1k
1l
7.2
1.2
9-SOCH3
13.0
0.075
1mb 2,3-di-OCH3 9-CF3
1n
3-OCH3
9-CF3
0.24
0.10
1ob 2,3-di-OCH3 9-Br
1pc 2-OCH3
1qc 3-OH
9-Br
9-Br
9-Br
9-Br
0.015
0.018
1rb
1sb
1tb
4-OCH3
4-OH
16
250
40
4.3
2,3-di-OCH3 9-CN
0.018
0.044
430
1.4
2.5
35
20
60
6.2
compounds are incubated in a 10 mM concentration with the cancer
1ub 4-OCH3
H
11-Cl
140
0.20
5.0
10
1vb
H
H
cells for two days. A sulforhodamine B assay is used for measurement
of the surviving cells. If a compound shows considerable growth
inhibitory activity and/or selectivity in the one-dose screening,
a continuative test procedure is performed in which five concen-
trations between 10ꢁ8 to 10ꢁ4 M are employed and GI50 values
(GI50 ¼ concentration to inhibit cell growth by 50%) are calculated for
the distinct cell lines. Furthermore, for each test compound an
averaged GI50 value for all the cell lines in the screening panel is
calculated defining the averaged antiproliferative activity. This
parameter is designated meangraph midpoint (MG-MID).
1wb
8,10-di-Cl
2ab Benzyl
2bb CH3
H
H
81.9 ꢂ 0.6
2.1
4.0
0.4
2cb
2db
H
H
–CH2CH]CH2 74.1 ꢂ 0.0
CH3
98.0 ꢂ 1.1
a
mean ꢂ SEM (¼standard error of the mean).
b
c
kinase inhibition data taken from [10].
CDK1/cyclin B inhibition data taken from [11].
at 10 mM (39% and 91%, respectively) while exhibiting comparably
Table 3 lists selected results of the IVCLSP for the mMDH-selec-
tive inhibitors 4b, 4k, 5d and 5f. For comparison, the table includes
data of the 9-cyano-2,3-dimethoxy-paullone 1t which inhibits the
kinases CDK1/cyclin B and GSK-3, but is devoid of mMDH inhibitory
activity (refer to Table 1). Since the mMDH inhibitors turned out to
be weak antiproliferative agents, only the test data of cell growth at
poor kinase inhibition. We therefore synthesized a number of new
5-benzylpaullones 4a–k and of paullone-9-carboxylic acid esters
5a–f in order to discover compounds with enhanced mMDH
inhibitory activity and selectivity.
2. Chemistry
10 mM inhibitor concentration are shown. Besides the averaged
parameter for all cell lines of the panel, the data on four cell lines are
included that are especially sensitive to characteristic kinase
inhibitory paullones like 1t, namely the colon cancer cell lines
COLO205 and HCT 116 and the ovarian cancer cell lines IGROV1 and
The synthesis of the novel 5-benzylpaullones 4 as analogues to
2a and of the paullone-9-carboxylic acid esters 5 as analogues to 1j
is depicted in Scheme 1. The paullones 1 used as synthetic inter-
mediates were prepared by a Fischer indole synthesis reacting 3,4-
dihydro-1H-1-benzazepine-2,5-dione 3 [12] with appropriately
substituted commercially available phenyl hydrazines in a mixture
of acetic and sulphuric acid at 70 ꢀC [11]. The 9-methyl-
sulfinylpaullone 1l was prepared by oxidation of the corresponding
9-methylthiopaullone 1k by oxidation with m-chloroperbenzoic
OVCAR3. As is shown in Table 3, 1t (10 mM) completely suppresses
the growth of COLO205 and of IGROV1 and provokes a net cell kill of
the COLO205 and OVCAR3 cell lines. The GI50 values for these cell
lines are in the low micromolar or submicromolar concentration
range. In contrast, the four mMDH inhibitors 4b, 4k, 5d and 5f are,
even in the high concentration of 10 mM, only modestly active or

A. Becker et al. / European Journal of Medicinal Chemistry 45 (2010) 335–342
337
R1
R2
O
O
H
N
O
H
N
N
(i)
(ii)
HN
HN
O
R3
R3
1a, c, d, f, h, i, k (for substitution refer toTable 1)
(R = OCH3),
3
4a-k
3
(R3 = tert-butyl)
1x
1y
O
O
H
H
N
N
(iii)
HN
HN
CO2H
CO2R1
1i
5a-f
Scheme 1. Synthesis of 5-benzylpaullones 4a–k and paullone-9-carboxylic acid esters 5a–f as analogues of 1j and 2a, respectively. Reagents and conditions: (i) 1. appropriately
substituted phenylhydrazine, AcOH, 70 ꢀC, 1–2 h; 2. H2SO4, 70 ꢀC, 1 h; (ii) 1.NaH, dry THF; 2. appropriately substituted benzyl bromide, reflux; (iii) R1OH, di-tert-butyl azodi-
carboxylate, triphenylphosphane, dry THF, 24 h.
completely inactive on the selected cell lines as well as on the other
cell lines of the NCI panel.
1112 Elemental Analyzer (Technische Universita¨t Braunschweig).
Analyses indicated by the symbols of elements were within ꢂ0.4%
of the theoretical values. Mass spectra were recorded on a VG
70-250S, VG Analytical, using a FAB xenon atom beam and PEG 300
or PEG 600 as matrices (Institute of Organic Chemistry, Universita¨t
Hamburg); or a Finnigan-MAT 95 instrument (department of mass
spectrometry of the Chemical Institutes of the Technische Uni-
versita¨t Braunschweig). The HPLC purity analyses were carried out
using a Merck Hitachi LaChrom Elite system (pump: L-2130, DAD
detector: L-2450; autosampler: L-2200; column: Merck LiChro-
4. Conclusion
A systematic investigation of 28 paullones failed to show
a correlation between the mMDH and the kinase inhibitory activity
within the compound family. Two series of novel paullones, namely
5-benzylpaullones 4 and paullone-9-carboxylic acid alkyl esters 5
were developed as selective inhibitors of mMDH versus kinases like
CDK1/cyclin B and GSK-3. Within the two series, the mMDH inhi-
bition was correlated to the lipophilicity of the compounds. In
contrast to kinase inhibitory paullones like 1t the mMDH inhibitors
did not exhibit noteworthy antiproliferative activity on cancer cells.
CART 125-4, LiChrospher 100 RP-18 (5 mm); eluent: acetonitrile/
water mixtures, elution rate 1.000 mL/min; detection wavelength:
254 nm and 280 nm; overall run time: 15 min.); tM ¼ hold-up time,
tN ¼ net retention time.
Hence,
a
contribution of the mMDH inhibition to the anti-
The following compounds were synthesized according to pub-
lished procedures: 1a–h, 1m, 1o, 1r–w, 2a–d, 9-methoxypaullone
(1x) [14,15],1p,1q [11],1i [16], 9-tert-butylpaullone (1y) [16], 3 [12].
proliferative activity of paullones could not be proven. In contrast,
the results suggest that mMDH inhibition is of minor relevance for
the antiproliferative activity displayed by paullones.
5.1.2. Preparation of paullones (7,12-dihydroindolo[3,2-
5. Experimental protocols
d][1]benzazepin-6(5H)-ones; (1)), general procedure
An appropriate 3,4-dihydro-1H-1-benzazepine-2,5-dione
(1 mmol) and an appropriate substituted phenylhydrazine
(1.50 mmol) [or the appropriate substituted phenylhydrazine
hydrochloride (1.05 mmol) and sodium acetate (123 mg,
1.50 mmol)] are suspended in glacial acetic acid (10 mL) and stirred
for 1 h at 70–80 ꢀC. Subsequently, concentrated sulphuric acid
(0.1 mL) is added, and stirring is continued at 70–80 ꢀC for 1 h. After
cooling to room temperature, the reaction mixture is poured into
a 5% aqueous sodium acetate solution (30 mL). The resulting
precipitate is filtered off with suction, washed with water, and
crystallized from the given solvent.
5.1. Chemistry
5.1.1. General
Melting points (mp) were determined on an electric variable
heater (Electrothermal IA 9100). Infrared spectra were recorded
using KBr pellets on a Philips PU 9712 spectrometer or on a Thermo
Nicolet FT-IR 200 spectrometer. Nuclear magnetic resonance
spectra were recorded either on a Bruker AMX 400 instrument
(Institute of Organic Chemistry, Universita¨t Hamburg); on a Bruker
Avance DRX-400 or a Bruker Avance II-600 (NMR laboratories of the
Chemical Institutes of the Technische Universita¨t Braunschweig)
using DMSO-d6 as solvent and tetramethylsilane as internal stan-
5.1.2.1. 9-(Methylthio)-7,12-dihydroindolo[3,2-d][1]benzazepin-
6(5H)-one (1k). Synthesis according to general procedure 5.1.2.
Crystallization from ethanol afforded 60% beige powder; mp:
>330 ꢀC; IR (KBr): 3210 cmꢁ1 (NH), 1640 cmꢁ1 (C]O); 1H NMR
dard. NMR signals are reported in ppm on a d scale. C, H, N analyses
were performed with a CHN-O-Rapid, Heraeus or a EA 1108, Carlo
Erba (Universita¨t Hamburg) or with a CE Instruments FlashEAÒ

338
A. Becker et al. / European Journal of Medicinal Chemistry 45 (2010) 335–342
Table 2
6,2
mMDH inhibition and kinase inhibition by 5-benzylpaullones 2a, 4a–k and paul-
lone-9-carboxylic acid esters 1j, 5a–f
6
5,8
5,6
5,4
5,2
5
R1
R2
O
O
H
N
R2 = 0,7068
N
4,8
5
6
7
8
clogP
HN
HN
Fig. 2. Correlation between lipophilicity and mMDH inhibitory activity in the series of
5-benzylpaullones 2a and 4a–k. The clogP values were calculated using ACD/Labs logP
vers. 6.0.
R3
CO2R1
2a, 4a-k
1j, 5a-f
5.1.2.2. 9-(Methylsulfinyl)-7,12-dihydroindolo[3,2-d][1]benzaze-
pin-6(5H)-one (1l). Under stirring, 9-(methylthio)-7,12-dihy-
droindolo[3,2-d][1]benzazepin-6(5H)-one (1k) (150 mg,
0.50 mmol) was dissolved in acetone (60 mL) and cooled down
to temperatures <0 ꢀC. A solution of meta-chloroperbenzoic acid
(141 mg of 70% m-CPBA, 0.57 mmol) in acetone (14 mL) was
slowly added drop by drop to the dissolved educt, maintaining
temperatures <0 ꢀC and observing the progression of the reac-
tion by TLC. After the completion of the reaction, the mixture
was poured into water (300 mL). The resulting precipitate was
filtered off with suction and washed with 7% aqueous sodium
hydrogen carbonate solution. Crystallization from ethanol
afforded 42% of a salmon colored powder, mp.: >330 ꢀC; IR
(KBr): 3200 cmꢁ1 (NH), 1650 cmꢁ1 (C]O), 1040 cmꢁ1 (S]O); 1H
.
Entry Substitution pattern
IC50 mMDH
IC50 GSK-3 IC50 CDK1-
M] cyclin B
M]
[m
M] ꢂ SEMa
[m
[
m
2a
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
1j
5a
5b
5c
5d
5e
5f
R1, R2 ¼ H; R3 ¼ Br
R1, R2 ¼ H; R3 ¼ Cl
R1, R2 ¼ H; R3 ¼ C(CH3)3
R1, R2 ¼ H; R3 ¼ CF3
R1, R2 ¼ H; R3 ¼ CH3
R1, R2 ¼ H; R3 ¼ OCH3
R1 ¼ CH3, R2 ¼ H; R3 ¼ Br
R1 ¼ OCH3, R2 ¼ H; R3 ¼ Br
R1, R2 ¼ Cl; R3 ¼ Br
R1 ¼ CH3, R2, R3 ¼ H
R1, R2 ¼ Cl; R3 ¼ H
R1, R2 ¼ Cl; R3 ¼ C(CH3)3
R1 ¼ CH3
2.4 ꢂ 0.4
2.7 ꢂ 0.05
1.6 ꢂ 0.1
4.2 ꢂ 0.1
6.2 ꢂ 1.2
10
35
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
7.2
>100
700
>10
600
>10
>1000
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
4.2
11.9 ꢂ 0.4
1.6 ꢂ 0.1
1.8 ꢂ 0.3
1.1 ꢂ 0.1
10.5 ꢂ 0.1
7.9 ꢂ 0.5
0.77 ꢂ 0.02
13.5 ꢂ 2.0
20.0 ꢂ 0.8
6.2 ꢂ 0.2
9.9 ꢂ 0.3
4.4 ꢂ 0.6
4.3 ꢂ 0.2
2.7 ꢂ 0.2
NMR (DMSO-d6, 400 MHz):
d
(ppm) ¼ 2.75 (s, 3H, SOCH3), 3.57
(d, J ¼ 2.6 Hz, 2H, CH2), 7.25–7.33 (m, 2H, ar H), 7.41 (dt, J ¼ 7.7/
7.5/1.5 Hz, 1H, ar H), 7.48 (dd, J ¼ 8.5/1.7 Hz, 1H, ar H), 7.62 (d,
J ¼ 8.4 Hz, 1H, ar H), 7.77 (dd, J ¼ 7.9/1.3 Hz, 1H, ar H), 8.04 (d,
J ¼ 1.2 Hz, 1H, ar H), 10.14 (s, 1H, lactam NH), 11.97 (s, 1H, indole
R1 ¼ C2H5
25
R1 ¼ n-C3H7
13.0
>10
25
7.0
40
R1 ¼ n-C4H9
R1 ¼ n-C5H11
NH); 13C NMR (DMSO-d6, 100.6 MHz):
d
(ppm) ¼ 31.5 (CH2), 43.6
R1 ¼ n-C6H13
R1 ¼ n-C8H17
(SOCH3), 112.3, 114.3, 117.1, 122.2, 123.6, 126.9, 128.4 (tert. C),
108.0, 126.4, 134.3, 135.6, 136.3, 138.4, 171.4 (quat. C, one signal
lacking due to peak overlapping); Anal. C17H14N2O2S (C, H, N).
a
mean of two tests. SEM ¼ standard error of the mean.
(DMSO-d6, 400 MHz):
d
(ppm) ¼ 2.51 (s, 3H, SCH3), 3.51 (s, 2H, CH2),
5.1.2.3. 3-Methoxy-9-trifluormethyl-7,12-dihydroindolo[3,2-d][1]ben-
zazepin-6(5H)-one (1n). Synthesis according to general procedure
5.1.2. Crystallization from ethꢁa1nol afforded 25% yellow powder; mp.
>330 ꢀC; IR (KBr): 3230 cm (NH), 1630 cmꢁ1 (C]O); 1H NMR
7.15 (dd, J ¼ 8.4/1.5 Hz, 1H, ar H), 7.23–7.30 (m, 2H, ar H), 7.34–7.41
(m, 2H, ar H), 7.64 (d, J ¼ 1.5 Hz,1H, ar H), 7.73 (dd, J ¼ 7.6/1.0 Hz,1H,
ar H), 10.10 (s, 1H, lactam NH), 11.63 (s, 1H, indole NH); 13C NMR
(DMSO-d6, 100.6 MHz):
117.3, 122.2, 123.1, 123.6, 126.8, 128.0 (tert. C), 107.0, 122.6, 127.0,
127.3, 133.2, 135.4, 135.9, 171.5 (quat. C); Anal. C17H14N2OS (C, H, N).
d
(ppm) ¼ 17.2 (SCH3), 31.4 (CH2), 112.0,
(DMSO-d6, 400 MHz):
d
(ppm) ¼ 3.59 (s, 2H, CH2), 3.82 (s, 3H,
Table 3
Results of the in vitro cell line screening with five selected paullones 4b, 4k, 5d, 5f,
and 1t. Values indicate percentage of growth to untreated controls at 10 M (4b, 4k,
5d, 5f, 1t) or GI50 values (1t [mM]).
5,8
5,6
5,4
5,2
5
m
Colon cancer
Ovarian Cancer
Averaged parameter
over all cell lines
COLO 205a
HCT 116a
IGROV1b
OVCAR3b
4bc
4kc
5dd
5fd
1tc
140%
98%
86%
94%
4%
91%
46%
69%
98%
ꢁ56%
107%
74%
57%
92%
2%
125%
71%
79%
100%
67%
80%
88%
31%
85%
4,8
R2 = 0,8096
ꢁ34%
1te
0.47
m
M
0.47
m
M
0.93
m
M
1.5
mM
5.6
m
Mf
4,6
a
3
4
5
clogP
6
7
Colon cancer cell line.
b
c
d
e
f
Ovarian cancer cell line.
single one-dose test run @ 10
averaged data from duplicate one-dose test runs @ 10
GI50 values, averaged data from two 5-dose test runs.
Meangraph midpoint (MG-MID).
m
M.
Fig. 1. Correlation between lipophilicity and mMDH inhibitory activity in the series of
paullone-9-carboxylic acid alkyl esters 1j and 5a–f. The clogP values were calculated
using ACD/Labs logP vers. 6.0.
m
M.

A. Becker et al. / European Journal of Medicinal Chemistry 45 (2010) 335–342
339
OCH3), 6.85 (d, 1H, 2.6 Hz, ar H), 6.95 (dd, 1H, 8.9/2.3 Hz, ar H), 7.43
(m, 1H, ar H), 7.59 (d, 1H, 8.6 Hz, ar H), 7.70 (d, 1H, 8.7 Hz, ar H), 8.09
(s, 1H, ar H), 10.01 (s, 1H, lactam NH), 11.98 (s, 1H, indole NH); 13C
2H, ar H), 7.10–7.17 (m, 3H, ar H), 7.35 (dt, 1H, J ¼ 7.5/1.1 Hz, ar H),
7.42–7.45 (m, 1H, ar H), 7.49 (dd, 1H, J ¼ 8.7/1.7 Hz, ar H), 7.61 (dd,
1H, J ¼ 8.4/1.0 Hz, ar H), 7.65 (d, 1H, J ¼ 8.5 Hz, ar H), 7.70 (dd, 1H,
J ¼ 7.8/1.6 Hz, ar H), 8.22 (s, 1H, ar H), 12.25 (s, 1H, indole NH); 13C
NMR (DMSO-d6, 100.6 MHz):
d
(ppm) ¼ 55.3 (OCH3), 31.2 (CH2),
106.9, 110.3, 111.8, 115.4 (q, JCF ¼ 4 Hz), 117.8 (q, JCF ¼ 4 Hz), 128.1
(tert. C), 106.5, 115.1, 119.9, (q, JCF ¼ 32 Hz), 125.5 (q, JCF ¼ 271 Hz),
126.0, 134.9, 137.1, 138.5, 159.4, 171.1 (quat. C); Anal. C18H13F3N2O2
(C, H, N).
NMR (DMSO-d6, 150.9 MHz):
d
(ppm) ¼ 31.3 (azepine CH2), 52.0
3
(benzyl CH2); 112.3, 116.2 (q, JC,F ¼ 3.9 Hz, C–C–CF3), 118.4 (q,
3JC,F ¼ 3.3 Hz, C–C–CF3), 124.3, 125.2, 126.2 (2C), 126.6, 127.3, 128.2
(2C), 128.6 (tert. C); 110.6, 120.0 (q, 2JC,F ¼ 31.2 Hz, C–CF3), 125.4 (q,
1JC,F ¼ 271.3 Hz, CF3), 125.5, 134.5, 137.7, 138.6, 139.2, 170.1 (1
quaternary C atom not detected under application of 512 Scans)
(quat. C); Anal. C24H17F3N2O (C,H,N); HPLC (ACN/H2O; 60:40):
99.9% at 254 nm, 100.0% at 280 nm, tN ¼ 4.62 min, tM ¼ 1.02 min.
5.1.3. Preparation of 5-benzylpaullones 4a-4k, general procedure
A solution of the appropriate 7,12-dihydroindolo[3,2-d][1]ben-
zazepin-6(5H)-one 1a, c, d, f, h, x, y (1.0 mmol) in dried THF (15 mL)
was refluxed under nitrogen atmosphere. Sodium hydride
suspension in mineral oil (60%) (0.5 mmol) was added successively.
As soon as gas formation was no longer observable, the appropriate
benzyl bromide (0.5–1 mmol) was added to the mixture. After
refluxing for the indicated reaction time, the reaction mixture was
cooled to room temperature and water (50 mL) was added. The
aqueous phase was extracted with dichloromethane (3 ꢃ 20 mL).
The organic solvent was removed under reduced pressure and the
obtained residue was purified by column chromatography and
subsequent crystallization from ethanol/water (70:30).
5.1.3.4. 5-Benzyl-9-methyl-7,12-dihydroindolo[3,2-d][1]benzazepin-
6(5H)-one (4d). Synthesis according to general procedure 5.1.3.
(benzyl bromide 173 mg, 1.00 mmol, reaction time 3 h) yielded 36%
orange crystals; mp.: 214 ꢀC; IR (KBr): 3322 cmꢁ1 (NH), 1650 cmꢁ1
(C]O); 1H NMR (DMSO-d6, 600 MHz):
d
(ppm) ¼ 2.42 (s, 3H, CH3),
3.14 (br s, 1H, azepine CH, superimposed by water peak), 3.93 (br s,
1H, azepine CH), 5.02 and 5.13 (2 br s, 2H, benzyl CH2), 6.90–6.91
(m, 2H, ar H), 7.02 (dd, 1H, J ¼ 8.3/1.7 Hz, ar H), 7.10–7.16 (m, 3H, ar
H), 7.31 (dt, 1H, J ¼ 7.5/1.2 Hz, ar H), 7.35–7.37 (m, 2H, ar H), 7.49
(d,1H, J ¼ 0.6 Hz, ar H), 7.57 (dd,1H, J ¼ 8.3/1.1 Hz, ar H), 7.66 (dd,1H,
J ¼ 7.7/1.7 Hz, ar H), 11.58 (s, 1H, indole NH); 13C NMR (DMSO-d6,
5.1.3.1. 5-Benzyl-9-chloro-7,12-dihydroindolo[3,2-d][1]benzazepin-
6(5H)-one (4a). Synthesis according to general procedure 5.1.3.
(benzyl bromide 103 mg, 0.600 mmol, reaction time 1.5 h) yielded
27% white powder, mp.: 240 ꢀC; IR (KBr): 3301 cmꢁ1 (NH),
150.9 MHz):
d
(ppm) ¼ 21.1 (CH3); 31.6 (azepine CH2), 52.0 (benzyl
CH2); 111.3, 117.7, 123.8, 124.1, 125.1, 126.2 (2C), 126.5, 127.1, 127.8,
128.1 (2C) (tert. C); 109.2,126.1, 126.3,127.7,132.4,135.7, 137.8,138.9,
170.1 (quat. C); Anal. (C24H20N2O) (C,H,N); HPLC (ACN/H2O; 60:40):
99.2% at 254 nm, 99.0% at 280 nm, tN ¼ 3.62 min, tM ¼ 1.02 min.
1646 cmꢁ1 (C]O); 1H NMR (DMSO-d6, 600 MHz):
d
(ppm) ¼ 3.16
(br s, 1H, azepine CH, superimposed by water peak), 4.03 (br s, 1H,
azepine CH), 5.02 and 5.14 (2 br s, 2H, benzyl CH2), 6.89–6.91 (m, 2H,
ar H), 7.10–7.16 (m, 3H, ar H), 7.19 (dd, 1H, J ¼ 8.7/2.1 Hz, ar H), 7.32–
7.34 (m, 1H, ar H), 7.39–7.42 (m, 1H, ar H), 7.47 (d, 1H, J ¼ 8.5 Hz, ar
H), 7.58 (d,1H, J ¼ 8.3 Hz, ar H), 7.67 (dd,1H, J ¼ 7.9/1.5 Hz, ar H), 7.83
(d, 1H, J ¼ 2.1 Hz, ar H), 11.95 (s, 1H, indole NH); 13C NMR (DMSO-d6,
5.1.3.5. 5-Benzyl-9-methoxy-7,12-dihydroindolo[3,2-d][1]benzaze-
pin-6(5H)-one (4e). Synthesis according to general procedure 5.1.3.
(benzyl bromide 86 mg, 0.50 mmol, reaction time 3 h) yielded 8%
yellow crystals; mp.: 222 ꢀC (Lit. [17] 213–215 ꢀC); IR (KBr):
3429 cmꢁ1 (NH), 1635 cmꢁ1 (C]O); 1H NMR (DMSO-d6, 600 MHz):
150.9 MHz):
d
(ppm) ¼ 30.0 (azepine CH2), 50.6 (benzyl CH2); 111.7,
116.2, 120.7, 122.9, 123.8, 124.8 (2C), 125.2, 125.9, 126.8 (2C), 127.0
(tert. C); 108.0, 122.4, 124.3, 125.6, 132.6, 134.3, 136.4, 137.8, 168.7
(quat. C); MS (EI): m/z ¼ 372 [M]þꢄ; HRMS (C23H17ClN2O) calcd.
372.1029; found 372.1035; HPLC (ACN/H2O; 60:40): 99.4% at
254 nm, 99.1% at 280 nm, tN ¼ 3.63 min, tM ¼ 1.02 min.
d
(ppm) ¼ 3.15 (br s, 1H, azepine CH, superimposed by water peak),
3.81 (s, 3H, OCH3), 3.99 (br s, 1H, azepine CH), 5.03 and 5.14 (2 br s,
2H, benzyl CH2), 6.83 (dd, 1H, J ¼ 8.7/2.5 Hz, ar H), 6.91–6.92 (m, 2H,
ar H), 7.10–7.17 (m, 3H, ar H), 7.23 (d, 1H, J ¼ 2.5 Hz, ar H), 7.31 (dt,
1H, J ¼ 7.5/1.2 Hz, ar H), 7.34–7.37 (m, 2H, ar H), 7.57 (dd, 1H, J ¼ 8.3/
1.1 Hz, ar H), 7.64 (dd, 1H, J ¼ 7.7/1.7 Hz, ar H), 11.56 (s, 1H, indole
5.1.3.2. 5-Benzyl-9-tert-butyl-7,12-dihydroindolo[3,2-d][1]benzaze-
pin-6(5H)-one (4b). Synthesis according to general procedure 5.1.3.
(benzyl bromide 173 mg, 1.00 mmol, reaction time 8 h) yielded 24%
beige powder, mp.: 211–212 ꢀC; IR (KBr): 3233 cmꢁ1 (NH),1637 cmꢁ1
NH); 13C NMR (DMSO-d6, 150.9 MHz):
d
(ppm) ¼ 30.2 (azepine CH2),
50.7 (benzyl CH2); 53.9 (OCH3); 98.2, 111.0, 111.4, 122.8, 123.7, 124.9
(2C), 125.2, 125.7, 126.4, 126.8 (2C) (tert. C); 108.3, 124.9, 125.0, 131.1,
131.5, 136.5, 137.5, 152.2, 168.9 (quat. C); HRMS (C24H20N2O2) calcd.
368.1525; found 368.1529; HPLC (ACN/H2O; 60:40): 96.3% at
254 nm, 96.6% at 280 nm, tN ¼ 2.08 min, tM ¼ 1.02 min.
(C]O); 1H NMR (DMSO-d6, 400 MHz):
d
(ppm) ¼ 1.37 (s, 9H, 3x CH3),
3.15 (br s, 1H, azepine CH), 4.00 (br s, 1H, azepine CH), 5.09 (br s, 2H,
benzyl CH2), 6.89–6.91 (m, 2H, ar H), 7.08–7.16 (m, 3H, ar H), 7.28–
7.32 (m, 2H, ar H), 7.35–7.41 (m, 2H, ar H), 7.59 (dd,1H, J ¼ 8.2/1.1 Hz,
ar H), 7.65–7.66 (m, 2H, ar H), 11.57 (s, 1H, indole NH); 13C NMR
5.1.3.6. 9-Bromo-5-(4-methylbenzyl)-7,12-dihydroindolo[3,2-d][1]ben-
zazepin-6(5H)-one (4f). Synthesis according to general procedure
5.1.3. (4-methylbenzyl bromide 93 mg, 0.50 mmol, reaction time
2.5 h) yielded 28% off-white powder; mp.: 274 ꢀC; IR (KBr):
3318 cmꢁ1 (NH), 1639 cmꢁ1 (C]O); 1H NMR (DMSO-d6, 400 MHz):
(DMSO-d6, 100.6 MHz):
(CH3); 34.4 (C(CH3)3); 51.9 (benzyl CH2); 111.2, 113.6, 120.6, 125.1,
125.8, 126.4 (2C), 126.6, 127.1, 127.7, 128.2 (2C) (tert. C); 110.0, 124.2,
d
(ppm) ¼ 30.7 (azepine CH2); 31.8 (3C)
126.5, 132.4, 135.6, 137.9, 138.8, 141.6, 170.3 (quat. C); MS (EI): m/z
d
(ppm) ¼ 2.17 (s, 3H, CH3), 3.14 (br s, 1H, azepine CH, superimposed
(%) ¼ 394 [M]þ
;
HRMS (C27H26N2O) calcd. 394.2045; found.
by water peak), 4.01 (br s, 1H, azepine CH), 4.98 and 5.09 (2 br s, 2H,
benzyl CH2), 6.76–6.78 (m, 2H, ar H), 6.92–6.94 (m, 2H, ar H), 7.29–
7.34 (m, 2H, ar H), 7.38–7.44 (m, 2H, ar H), 7.59 (dd,1H, J ¼ 8.1/1.0 Hz,
ar H), 7.66 (dd, 1H, J ¼ 7.7/1.6 Hz, ar H), 7.96 (d, 1H, J ¼ 2.0 Hz, ar H),
11.93 (s, 1H, indole NH); 13C NMR (DMSO-d6, 150.9 MHz):
ꢄ
394.2047; HPLC (ACN/H2O; 60:40): 99.8% at 254 nm, 99.4% at
280 nm, tN ¼ 6.79 min, tM ¼ 1.02 min.
5.1.3.3. 5-Benzyl-9-trifluoromethyl-7,12-dihydroindolo[3,2-d][1]ben-
zazepin-6(5H)-one (4c). Synthesis according to general procedure
5.1.3. (benzyl bromide 103 mg, 0.600 mmol, reaction time 2 h)
yielded 16% orange crystals; mp.: 224 ꢀC; IR (KBr): 3305 cmꢁ1 (NH),
d
(ppm) ¼ 20.4 (CH3); 31.3 (azepine CH2), 51.7 (benzyl CH2); 113.5,
120.6, 124.3, 124.6, 125.2, 126.3 (2C), 127.2, 128.3, 128.7 (2C) (tert. C);
109.3, 111.7, 125.7, 127.7, 133.9, 134.6, 135.6, 135.9, 139.1, 170.0 (quat.
C); HRMS (C24H19BrN2O) calcd. 430.0681; found 430.0676; HPLC
(ACN/H2O; 70:30): 97.2% at 254 nm, 97.1% at 280 nm, tN ¼ 2.48 min,
tM ¼ 1.03 min.
1648 cmꢁ1 (C]O); 1H NMR (DMSO-d6, 600 MHz):
(br s, 1H, azepine CH, superimposed by water peak), 4.18 (br s, 1H,
azepine CH), 5.02 and 5.17 (2 br s, 2H, benzyl CH2), 6.90–6.91 (m,
d
(ppm) ¼ 3.21

340
A. Becker et al. / European Journal of Medicinal Chemistry 45 (2010) 335–342
5.1.3.7. 9-Bromo-5-(4-methoxybenzyl)-7,12-dihydroindolo[3,2-d]
[1]benzazepin-6(5H)-one (4g). Synthesis according to general
procedure 5.1.3. (4-methoxybenzyl bromide 101 mg, 0.500 mmol,
reaction time 4 h) with a reaction time of 4 h yielded 28% white
powder; mp.: 236 ꢀC; IR (KBr): 3319 cmꢁ1 (NH), 1638 cmꢁ1 (C]O);
ar H), 7.57 (dd, 1H, J ¼ 8.1/1.3 Hz, ar H), 7.68–7.73 (m, 2H, ar H), 11.75
(s, 1H, indole NH); 13C NMR (DMSO-d6, 150.9 MHz):
d
(ppm) ¼ 31.4
(azepine CH2), 51.0 (benzyl CH2); 111.6, 118.2, 119.1, 122.3, 124.3,
125.4, 126.7, 127.1, 128.0, 128.2, 130.3 (tert. C); 109.6, 125.8, 126.2,
129.0, 130.8, 132.1, 137.3, 138.5, 139.1, 170.3 (quat. C); Anal.
(C23H16Cl2N2O) (C,H,N); HPLC (ACN/H2O; 60:40): 99.7% at 254 nm,
99.8% at 280 nm, tN ¼ 4.89 min, tM ¼ 1.02 min.
1H NMR (DMSO-d6, 600 MHz):
d
(ppm) ¼ 3.11 (br s, 1H, azepine CH,
superimposed by water peak), 3.64 (s, 3H, OCH3), 4.02 (br s, 1H,
azepine CH), 4.91 and 5.12 (2 br s, 2H, benzyl CH2), 6.68–6.70 (m, 2H,
ar H), 6.79–6.82 (m, 2H, ar H), 7.30 (dd,1H, J ¼ 8.7/1.9 Hz, ar H), 7.32–
7.34 (m,1H, ar H), 7.39–7.43 (m, 2H, ar H), 7.62 (dd,1H, J ¼ 8.3/0.8 Hz,
ar H), 7.65 (dd, 1H, J ¼ 7.7/1.7 Hz, ar H), 7.97 (d, 1H, J ¼ 1.5 Hz, ar H),
11.94 (s, 1H, indole NH); 13C NMR (DMSO-d6, 150.9 MHz):
5.1.3.11. 9-tert-Butyl-5-(3,4-dichlorobenzyl)-7,12-dihydroindolo[3,2-d]
[1]benzazepin-6(5H)-one (4k). Synthesis according to general
procedure 5.1.3 (3,4-dichlorobenzyl bromide 120 mg, 0.500 mmol,
reaction time 3 h) yielded 16% yellow powder; mp.: 221–222 ꢀC; IR
(KBr): 3241 cmꢁ1 (NH), 1637 cmꢁ1 (C]O); 1H NMR (DMSO-d6,
d
(ppm) ¼ 31.3 (azepine CH2), 51.2 (benzyl CH2); 54.8 (OCH3); 113.5
(2C), 113.5, 120.6, 124.4, 124.6, 125.1, 127.2, 127.6 (2C), 128.2 (tert. C);
109.3, 111.6, 125.8, 129.5, 133.8, 135.8, 139.0, 157.8, 170.0 (quat. C);
HRMS (C24H19BrN2O2) calcd. 446.0630; found 446.0633; HPLC (ACN/
H2O; 60:40): 97.3% at 254 nm, 96.6% at 280 nm, tN ¼ 4.19 min,
tM ¼ 1.02 min.
600 MHz):
d
(ppm) ¼ 1.37 (s, 9H, 3x CH3), 3.15 (br s, 1H, azepine CH),
4.01 (br s, 1H, azepine CH), 4.97 and 5.21 (2 br s, 2H, benzyl CH2),
6.89 (dd, 1H, J ¼ 8.3/2.1 Hz, ar H), 7.05 (d, 1H, J ¼ 1.9 Hz, ar H), 7.30
(dd, 1H, J ¼ 8.7/1.9 Hz, ar H), 7.34 (dt, 1H, J ¼ 7.5/1.0 Hz, ar H), 7.37–
7.42 (m, 3H, ar H), 7.57 (dd, 1H, J ¼ 8.2/0.8 Hz, ar H), 7.65–7.68
(m, 2H, ar H), 11.62 (s, 1H, indole NH); 13C NMR (DMSO-d6,
5.1.3.8. 9-Bromo-5-(3,4-dichlorobenzyl)-7,12-dihydroindolo[3,2-d]
[1]benzazepin-6(5H)-one (4h). Synthesis according to general
procedure 5.1.3. (3,4-dichlorobenzyl bromide 120 mg, 0.500 mmol,
reaction time 5.5 h) yielded 25% beige powder, mp.: 251 ꢀC; IR
(KBr): 3166 cmꢁ1 (NH), 1636 cmꢁ1 (C]O); 1H NMR (DMSO-d6,
150.9 MHz):
d
(ppm) ¼ 30.8 (azepine CH2), 31.1 (3C) (CH3); 33.7
(C(CH3)3); 50.2 (benzyl CH2); 110.6, 113.0, 120.1, 123.7, 124.8, 126.1,
126.5, 127.2, 127.7, 129.7 (tert. C); 109.3, 125.0, 125.9, 128.5, 130.2,
131.6,135.0,137.7, 138.5,141.0, 169.8 (quat. C); HRMS (C27H24Cl2N2O)
calcd. 462.1266; found 462.1255; HPLC (ACN/H2O; 70:30): 99.2% at
254 nm, 98.6% at 280 nm, tN ¼ 5.84 min, tM ¼ 1.02 min.
600 MHz):
d
(ppm) ¼ 3.18 (br s, 1H, azepine CH, superimposed by
water peak), 4.04 (br s, 1H, azepine CH), 4.97 and 5.17 (2 br s, 2H,
benzyl CH2), 6.90 (dd, 1H, J ¼ 8.3/2.1 Hz, ar H), 7.01 (d, 1H, J ¼ 1.9 Hz,
ar H), 7.31 (dd, 1H, J ¼ 8.5/2.0 Hz, ar H), 7.35–7.38 (m, 1H, ar H),
7.42–7.45 (m, 3H, ar H), 7.57 (d, 1H, J ¼ 8.3 Hz, ar H), 7.69 (dd, 1H,
J ¼ 7.7/1.5 Hz, ar H), 7.99 (d, 1H, J ¼ 1.9 Hz, ar H), 12.00 (s, 1H, indole
5.1.4. Preparation of paullone-9-carboxylic esters 1j and 5a–f,
general procedure
To
a
suspension of 6-oxo-5,6,7,12-tetrahydroindolo[3,2-
d][1]benzazepine-9-carboxylic acid (1i) (0.5 mmol) in freshly dried
THF (3.5 mL), 2.5 equiv. of triphenylphosphane (1.25 mmol) and
2.5 equiv. of an appropriate anhydrous alcohol (1.25 mmol) are
added. The reaction mixture is stirred in an ice bath for 30 min before
adding 2.5 equiv. of di-tert-butyl azodicarboxylate (1.25 mmol), and
stirring is continued for another 30 min. The ice bath is removed, and
the mixture is allowed to warm up to room temperature. The reac-
tion mixture is then stirred for a total of 24 h, adding 1 mL of the
appropriate alcohol after 20 h. The solid is filtered off with suction
and crystallized from methanol unless stated otherwise.
NH); 13C NMR (DMSO-d6,150.9 MHz):
d
(ppm) ¼ 31.2 (azepine CH2),
51.0 (benzyl CH2); 113.6, 120.7, 124.4, 124.7, 125.5, 126.7, 127.3, 128.1,
128.5, 130.3 (tert. C); 109.2, 111.7, 125.8, 127.6, 130.8, 133.7, 136.0,
138.8, 139.0, 170.2 (quat. C, 1 quaternary C atom not detected due to
peak overlapping); HRMS (C23H15BrCl2N2O) calcd. 483.9745; found
483.9747; HPLC (ACN/H2O; 70:30): 99.3% at 254 nm, 98.7% at
280 nm, tN ¼ 3.60 min, tM ¼ 1.03 min.
5.1.3.9. 5-(4-Methylbenzyl)-7,12-dihydroindolo[3,2-d][1]benzazepin-
6(5H)-one (4i). Synthesis according to general procedure 5.1.3.
(4-methylbenzyl bromide 93 mg, 0.50 mmol, reaction time 3.5 ꢁh1)
yielded 26% yellow crystals; mp.: 228–229 ꢀC; IR (KBr): 3229 cm
(NH), 1640 cmꢁ1 (C]O); 1H NMR (DMSO-d6, 600 MHz):
5.1.4.1. Methyl 6-oxo-5,6,7,12-tetrahydroindolo[3,2-d][1]benzaze-
pine-9-carboxylate (1j). Preparation according to general proce-
dure 5.1.4. yielded 23% white powder; mp.: >330 ꢀC; IR (KBr):
3370 cmꢁ1 (NH), 3200 cmꢁ1 (NH), 1690 cmꢁ1 (C]O), 1670 cmꢁ1
d
(ppm) ¼ 2.17 (s, 3H, CH3), 3.14 (br s, 1H, azepine CH), 3.97 (br s, 1H,
azepine CH), 4.96 and 5.12 (2 br s, 2H, benzyl CH2), 6.78–6.79 (m, 2H,
ar H), 6.93–6.95 (m, 2H, ar H), 7.09–7.11 (m, 1H, ar H), 7.18–7.21
(m, 1H, ar H), 7.30–7.33 (m, 1H, ar H), 7.36–7.38 (m, 1H, ar H), 7.46
(dd, 1H, J ¼ 8.1/0.8 Hz, ar H), 7.58 (dd, 1H, J ¼ 8.3/0.8 Hz, ar H), 7.67
(dd, 1H, J ¼ 7.7/1.7 Hz, ar H), 7.71 (d, 1H, J ¼ 7.7 Hz, ar H) 11.72 (s, 1H,
(C]O); 1H NMR (DMSO-d6, 400 MHz):
d
(ppm) ¼ 3.56 (s, 2H, CH2),
3.87 (s, 3H, ester–OCH3), 7.25–7.33 (m, 2H, ar H), 7.41 (dt, J ¼ 7.6/7.6/
1.5 Hz, 1H, ar H), 7.52 (d, J ¼ 8.4 Hz, 1H, ar H), 7.77 (dd, J ¼ 7.8/1.4 Hz,
1H, ar H), 7.81 (dd, J ¼ 8.7/1.5 Hz, 1H, ar H), 8.35 (d, J ¼ 1.3 Hz, 1H, ar
H), 10.14 (s, 1H, lactam NH), 12.03 (s, 1H, indole NH); 13C NMR
indole NH); 13C NMR (DMSO-d6, 150.9 MHz):
d
(ppm) ¼ 19.4 (CH3);
(DMSO-d6, 100.6 MHz):
d
(ppm) ¼ 31.4 (CH2), 51.6 (ester–OCH3),
30.5 (azepine CH2), 50.5 (benzyl CH2); 110.5, 117.1, 118.0, 121.1, 123.1,
124.0, 125.2 (2C), 126.0, 126.7, 127.6 (2C) (tert. C); 108.6, 124.8, 125.1,
131.2, 133.6, 134.5, 136.2, 137.8, 169.0 (quat. C); HRMS (C24H20N2O):
calcd. 352.1575; found 352.1565; HPLC (ACN/H2O; 65:35): 98.6% at
254 nm, 98.7% at 280 nm, tN ¼ 2.28 min, tM ¼ 1.02 min.
111.4, 120.4, 122.3, 122.9, 123.6, 126.9, 128.4 (tert. C), 108.6, 120.6,
122.1, 126.0, 134.2, 135.6, 139.8, 167.0, 171.2 (quat. C); Anal
C18H14N2O3 (H, N); C calcd. 70.58; found 70.06.
5.1.4.2. Ethyl 6-oxo-5,6,7,12-tetrahydroindolo[3,2-d][1]benzazepine-
9-carboxylate (5a). Preparation according to general procedure
5.1.4. yielded 29% white powder after crystallization from ethanol;
mp.: >330 ꢀC; IR (KBr): 3380 cmꢁ1 (NH), 3200 cmꢁ1 (NH),
1690 cmꢁ1 (C]O),1670 cmꢁ1 (C]O); 1H NMR (DMSO-d6, 400 MHz):
5.1.3.10. 5-(3,4-Dichlorobenzyl)-7,12-dihydroindolo[3,2-d][1]benza-
zepin-6(5H)-one (4j). Synthesis according to general procedure
5.1.3. (3,4-dichlorobenzyl bromide 120 mg, 0.500 mmol, reaction
time 4 h) yielded 32% yellow crystals; mp.: 232–233 ꢀC; IR (KBr):
3252 cmꢁ1 (NH), 1634 cmꢁ1 (C]O); 1H NMR (DMSO-d6, 400 MHz):
d
(ppm) ¼ 1.37 (t, J ¼ 7.1 Hz, 3H, CH3), 3.56 (s, 2H, azepine CH2), 4.34
(q, J ¼ 7.1 Hz, 2H, ester OCH2), 7.25–7.33 (m, 2H, ar H), 7.41 (dt, J ¼ 7.8/
7.6/1.4 Hz, 1H, ar H), 7.52 (d, J ¼ 8.6 Hz, 1H, ar H), 7.77 (dd, J ¼ 7.7/
1.1 Hz, 1H, ar H), 7.82 (dd, J ¼ 8.6/1.5 Hz, 1H, ar H), 8.34 (d, J ¼ 1.3 Hz,
1H, ar H), 10.15 (s, 1H, lactam NH), 12.02 (s, 1H, indole NH); 13C NMR
d
(ppm) ¼ 3.21 (br s, 1H, azepine CH, superimposed by water peak),
3.96 (br s, 1H, azepine CH), 5.07 (br s, 2H, benzyl CH2), 6.90 (dd, 1H,
J ¼ 8.3/2.3 Hz, ar H), 7.03 (d,1H, J ¼ 2.0 Hz, ar H), 7.08–7.12 (m,1H, ar
H), 7.18–7.22 (m,1H, ar H), 7.33–7.43 (m, 3H, ar H), 7.46–7.49 (m,1H,
(DMSO-d6, 100.6 MHz):
d
(ppm) ¼ 14.2 (CH3), 31.4 (azepine CH2),

A. Becker et al. / European Journal of Medicinal Chemistry 45 (2010) 335–342
341
60.1 (ester OCH2), 111.3, 120.3, 122.3, 122.9, 123.6, 126.9, 128.4 (tert.
C), 108.6,120.9, 122.2,126.0,134.2,135.6, 139.8, 166.5,171.2 (quat. C);
Anal. C19H16N2O3 (C,H,N).
128.9 (tert. C), 109.0, 121.4, 122.6, 126.5, 134.7, 136.1, 140.3, 167.0,
171.7 (quat. C); Anal (C23H24N2O3) (C, H, N).
5.1.4.7. Octyl 6-oxo-5,6,7,12-tetrahydroindolo[3,2-d][1]benzazepine-
9-carboxylate (5f). Preparation according to general procedure
5.1.4. yielded 33% white powder; mp.: 292 ꢀC (dec.); IR (KBr):
3360 cmꢁ1 (NH), 3200 cmꢁ1 (NH), 1680 cmꢁ1 (C]O), 1670 cmꢁ1
5.1.4.3. Propyl 6-oxo-5,6,7,12-tetrahydroindolo[3,2-d][1]benzaze-
pine-9-carboxylate (5b). Preparation according to general proce-
dure 5.1.4. yielded 28% white powder, mp.: 285 ꢀC (dec.); IR (KBr):
3360 cmꢁ1 (NH), 3200 cmꢁ1 (NH), 1690 cmꢁ1 (C]O), 1670 cmꢁ1
(C]O); 1H NMR (DMSO-d6, 400 MHz):
d
(ppm) ¼ 0.86 (t, J ¼ 6.7 Hz,
(C]O); 1H NMR (DMSO-d6, 400 MHz):
d
(ppm) ¼ 1.01 (t, J ¼ 7.4 Hz,
3H, CH3),1.21–1.48 (m,10H, CH2),1.75 (quint., J ¼ w7.0 Hz, 2H, CH2),
3.55 (s, 2H, azepine CH2), 4.28 (t, J ¼ 6.6 Hz, 2H, OCH2), 7.25–7.33
(m, 2H, ar H), 7.41 (dt, J ¼ 7.8/7.6/1.5 Hz, 1H, ar H), 7.52 (d, J ¼ 8.6 Hz,
1H, ar H), 7.77 (dd, J ¼ 7.6/1.0 Hz, 1H, ar H), 7.81 (dd, J ¼ 8.6/1.5 Hz,
1H, ar H), 8.32 (d, J ¼ 1.3 Hz,1H, ar H), 10.15 (s, 1H, lactam NH),12.02
3H, CH3), 1.77 (sext., J ¼ w7.1 Hz, 2H, ester CH2), 3.56 (s, 2H, azepine
CH2), 4.25 (t, J ¼ 6.7 Hz, 2H, OCH2), 7.25–7.33 (m, 2H, ar H), 7.41
(dt, J ¼ 7.8/7.6/1.4 Hz, 1H, ar H), 7.52 (d, J ¼ 8.4 Hz, 1H, ar H), 7.77
(dd, J ¼ 7.9/1.3 Hz, 1H, ar H), 7.82 (dd, J ¼ 8.5/1.7 Hz, 1H, ar H), 8.34
(d, J ¼ 1.3 Hz, 1H, ar H), 10.15 (s, 1H, lactam NH), 12.01 (s, 1H, indole
(s, 1H, indole NH); 13C NMR (DMSO-d6, 100.6 MHz):
d
(ppm) ¼ 13.8
NH); 13C NMR (DMSO-d6, 100.6 MHz):
(CH2), 31.4 (azepine CH2), 65.6 (OCH2), 111.4, 120.2, 122.3, 122.9,
123.7, 126.9, 128.4 (tert. C), 108.5, 120.9, 122.2, 126.0, 134.2, 135.6,
139.8, 166.6, 171.3 (quat. C); Anal. C20H18N2O3 (C, H, N)
d
(ppm) ¼ 10.4 (CH3), 21.7
(CH3), 22.0, 25.5, 28.2, 28.5, 28.6, 31.1 (CH2), 31.4 (azepine CH2), 64.1
(OCH2), 111.4, 120.2, 122.3, 122.9, 123.6, 126.9, 128.4 (tert. C), 108.5,
120.9, 122.2, 126.0, 134.2, 135.6, 139.8, 166.5, 171.2 (quat. C); Anal.
C25H28N2O3 (H, N); C calcd. 74.23; found 73.65; HRMS–FAB (m/z):
[M þ H]þ calcd. 405.2178, found 405.2175.
5.1.4.4. Butyl 6-oxo-5,6,7,12-tetrahydroindolo[3,2-d][1]benzazepine-
9-carboxylate (5c). Preparation according to general procedure 5.1.4.
yielded 30% white powder; mp: >330 ꢀC (dec. starting at 285 ꢀC); IR
(KBr): 3370 cmꢁ1 (NH), 3200 cmꢁ1 (NH), 1690 cmꢁ1 (C]O),
5.2. Biological assays
5.2.1. mMDH inhibition assay
1670 cmꢁ1 (C]O); 1H NMR (DMSO-d6, 400 MHz):
d
(ppm) ¼ 0.97 (t,
5.2.1.1. Reagents. Trizma buffer, pH 8.5: TrizmaÒ-base (Sigma T-
1503) (12.11 g, 100.0 mmol) is dissolved in aqua bidest. The pH is
adjusted to 8.5 with HCl (10%).
J ¼ 7.4 Hz, 3H, CH3), 1.47 (sext., J ¼ w7.4 Hz, 2H, CH2), 1.74 (quint.,
J ¼ w7.1 Hz, 2H, CH2), 3.56 (s, 2H, azepine CH2), 4.30 (t, J ¼ 6.6 Hz, 2H,
OCH2), 7.25–7.33 (m, 2H, ar H), 7.41 (dt, J ¼ 7.6/7.6/1.5 Hz, 1H, ar H),
7.52 (d, J ¼ 8.4 Hz,1H, ar H), 7.77 (dd, J ¼ 7.9/1.3 Hz,1H, ar H), 7.81 (dd,
J ¼ 8.5/1.7 Hz, 1H, ar H), 8.33 (d, J ¼ 1.3 Hz, 1H, ar H), 10.15 (s, 1H,
lactam NH), 12.01 (s, 1H, indole NH); 13C NMR (DMSO-d6,
L
-malate, 0.5 M:
(1.78 g, 10.0 mmol) is dissolved in Trizma buffer to yield 20.0 mL.
NAD, 16 mM: NAD (Fluka 43410) (10.614 mg, 16.000 mol) is
dissolved in Trizma buffer (1000 L).
mMDH solution 0.5%: mMDH (5
L-malic acid disodium salt (Sigma, M 9138)
m
m
100.6 MHz):
d
(ppm) ¼ 14.0 (CH3), 19.2, 30.8 (ester CH2), 31.9 (aze-
mL, Sigma M 2634, solution in
pine CH2), 64.3 (ester OCH2), 111.8, 120.7, 122.7, 123.4, 124.1, 127.3,
128.9 (tert. C),109.0,121.4,122.6,126.5,134.7,136.1,140.3,167.0,171.7
(quat. C); Anal. C21H20N2O3 (C, H, N).
50% glycerol) is dissolved in Trizma buffer pH 8.5 to yield 1000
The enzyme solution is prepared freshly and kept at 0 ꢀC.
mL.
Solutions of test compounds, 10 mM: test compound (1.000 mg)
is dissolved in a measured volume of DMSO to yield a 10 mM stock
solution. The stock solution is dissolved in DMSO to yield solutions
of 0.050, 0.100, 0.250, 0.500, 1000, 2500 mM. All solutions except
the enzyme solution are maintained and handled at 25 ꢀC.
5.1.4.5. Pentyl 6-oxo-5,6,7,12-tetrahydroindolo[3,2-d][1]benzaze-
pine-9-carboxylate (5d). Preparation according to general proce-
dure 5.1.4. yielded 15% white powder; mp.: 293 ꢀC (dec.); IR (KBr):
3370 cmꢁ1 (NH), 3200 cmꢁ1 (NH), 1690 cmꢁ1 (C]O), 1670 cmꢁ1
(C]O); 1H NMR (DMSO-d6, 400 MHz):
d
(ppm) ¼ 0.92 (t, J ¼ 7.1 Hz,
5.2.1.2. Assay conditions. mMDH catalyzes the conversion of L-
3H, CH3), 1.33–1.47 (m, 4H, CH2–CH2), 1.76 (quint., J ¼ w7.0 Hz, 2H,
CH2), 3.56 (s, 2H, azepine CH2), 4.29 (t, J ¼ 6.6 Hz, 2H, OCH2), 7.25–
7.33 (m, 2H, ar H), 7.41 (dt, J ¼ 7.8/7.6/1.4 Hz, 1H, ar H), 7.52
(d, J ¼ 8.4 Hz, 1H, ar H), 7.77 (dd, J ¼ 7.6/1.3 Hz, 1H, ar H), 7.81
(dd, J ¼ 8.5/1.6 Hz,1H, ar H), 8.33 (d, J ¼ 1.5 Hz,1H, ar H),10.16 (s,1H,
lactam NH), 12.03 (s, 1H, indole NH); 13C NMR (DMSO-d6,
malate to oxaloacetate, which is coupled to the formation of the
reduced cofactor NADH/Hþ. In the applied spectral photometric
assay, absorbance at 340 nm caused by NADH/Hþ was used as
a read-out. For calculation of enzyme activity, a time interval of
2.5 min, during which a linear absorbance increase was observed,
was monitored. The mMDH assays were run in a dual-trace spec-
trometer SpecordÒ 200 (Analytik Jena AG) equipped with a constant
temperature holder maintained at 25 ꢀC. Assays were performed in
disposable 1.5 mL polymethylmethacrylate cuvettes (Sarstedt AG,
Nu¨mbrecht, Germany) of 1 cm width. For the assay, Trizma buffer
100.6 MHz):
d
(ppm) ¼ 13.8 (CH3), 21.7, 27.7, 27.9 (CH2), 31.4 (aze-
pine CH2), 64.1 (OCH2), 111.4, 120.2, 122.3, 122.9, 123.7, 126.9, 128.4
(tert. C), 108.5, 120.9, 122.2, 126.0, 134.2, 135.6, 139.8, 166.6, 171.3
(quat. C); Anal C22H22N2O3 (H, N); C calcd. 72.91; found 72.04.
HRMS–FAB (m/z): [M þ H]þ calcd. 363.1709, found 363.1702.
(790
inhibitor solution (10
reaction was started by addition of NAD (50
and -malate (150 L of 0.5 M solution). The absorbance increase at
340 nm was monitored over 2.5 min against a blank solution (same
mixture of reagents without enzyme solution). The slope rate A340
t is a measure for the reaction velocity. The mMDH activity is
m
L), enzyme solution (3–10
L) were incubated in a cuvette for 3 min. The
L of 16 mM solution)
mL, depending on activity), and
m
5.1.4.6. Hexyl 6-oxo-5,6,7,12-tetrahydroindolo[3,2-d][1]benzazepine-
9-carboxylate (5e). Preparation according to general procedure
5.1.4. yielded 28% white powder; mp.: 280 ꢀC (dec.); IR (KBr):
3370 cmꢁ1 (NH), 3200 cmꢁ1 (NH), 1680 cmꢁ1 (C]O), 1670 cmꢁ1
m
L
m
D
/
(C]O); 1H NMR (DMSO-d6, 400 MHz):
d
(ppm) ¼ 0.89 (t, J ¼ 7.0 Hz,
D
3H, CH3), 1.28–1.49 (m, 6H, CH2–CH2–CH2), 1.76 (quint., J ¼ w7.1 Hz,
2H, CH2), 3.56 (s, 2H, azepine CH2), 4.29 (t, J ¼ 6.6 Hz, 2H, OCH2),
7.25–7.33 (m, 2H, ar H), 7.41 (dt, J ¼ 7.6/7.6/1.5 Hz, 1H, ar H), 7.52 (d,
J ¼ 8.7 Hz, 1H, ar H), 7.77 (dd, J ¼ 7.9/1.3 Hz, 1H, ar H), 7.81 (dd,
J ¼ 8.6/1.5 Hz, 1H, ar H), 8.33 (d, J ¼ 1.5 Hz, 1H, ar H), 10.16 (s, 1H,
lactam NH), 12.03 (s, 1H, indole NH); 13C NMR (DMSO-d6,
expressed as ratio of the velocities of the inhibited and the unin-
hibited reaction. For determination of the velocity of the uninhib-
ited reaction the assay was performed with 10 mL DMSO instead of
the inhibitor solution. Dose response-curves were constructed by
drawing the residual enzyme activities against the final inhibitor
concentrations. IC50 values were calculated from the dose response-
curves using GraphPad Prism 4.00 (Graphpad Software, San Diego,
CA, USA, 2003). Assays were performed in duplicate.
100.6 MHz):
d
(ppm) ¼ 14.3 (CH3), 22.4, 25.6, 28.6, 31.3 (CH2), 31.9
(azepine CH2), 64.6 (OCH2), 111.8, 120.7, 122.7, 123.4, 124.1, 127.3,

342
A. Becker et al. / European Journal of Medicinal Chemistry 45 (2010) 335–342
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methodology developed by the ‘‘Protein Phosphorylation & Human
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5.2.3. In vitro cell line screening
Screening compounds are tested against 60 human tumor cell
lines initially at 10ꢁ4 M test compound concentration. After 48 h of
continuous drug exposure, a sulforhodamine B protein assay is used
to determine cell viability or growth. The percentage of growth is
calculated on the basis of the optical density of the treated cultures
compared to untreated control cell lines. In case of a noteworthy
antiproliferative activity and selectivity, the tests are repeated at
five concentrations (10ꢁ8 M to 10ꢁ4 M) and dose response-curves
are calculated for each cell line. Details concerning the performance
of this screening have been published [19,20].
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Acknowledgements
We are grateful to the National Cancer Institute for performance
of the in vitro cell line screening. Funding of the project by the
European Commission (Contract No LSHB-CT-2004-503467, Protein
Kinase Research, to C.K. and L.M.) is gratefully acknowledged.
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