1-Aryl-4,6-dihydropyrazolo[4,3-d][1]benzazepin-5(1H)-ones: A new class of antiproliferative agents with selectivity for human leukemia and breast cancer cell lines

Article abstract of DOI:10.1016/j.ejmech.2007.02.007

The synthesis of 1-aryl-4,6-dihydropyrazolo[4,3-d][1]benzazepin-5(1H)-ones by cyclization of 4-[(dimethylamino)methylidene]-3,4-dihydro-1H-[1]benzazepine-2,5-dione with arylhydrazines is reported. When tested on a panel of human cancer cell lines, the title compounds showed antiproliferative activity and a characteristic selectivity pattern of growth inhibition. Although structurally akin to established kinase inhibitors, the new compounds did not exhibit noteworthy inhibitory activity when tested on an array of cancer-related kinases.

Full text of DOI:10.1016/j.ejmech.2007.02.007

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 42 (2007) 1317e1324
http://www.elsevier.com/locate/ejmech
Original article
1-Aryl-4,6-dihydropyrazolo[4,3-d][1]benzazepin-5(1H )-ones: A new
class of antiproliferative agents with selectivity for human leukemia
and breast cancer cell lines
Simone Kohfeld a, Peter G. Jones b, Frank Totzke c, Christoph Schachtele c, Michael
¨
H.G. Kubbutat c, Conrad Kunick a,
*
a Technische Universita¨t Braunschweig, Institut fu¨r Pharmazeutische Chemie, Beethovenstraße 55, D-38106 Braunschweig, Germany
b Technische Universita¨t Braunschweig, Institut fu¨r Anorganische und Analytische Chemie, Hagenring 30, D-38106 Braunschweig, Germany
c ProQinase GmbH, Breisacher Strasse 117, 79106 Freiburg, Germany
Received 11 October 2006; received in revised form 1 February 2007; accepted 15 February 2007
Available online 1 March 2007
Abstract
The synthesis of 1-aryl-4,6-dihydropyrazolo[4,3-d][1]benzazepin-5(1H )-ones by cyclization of 4-[(dimethylamino)methylidene]-3,4-dihy-
dro-1H-[1]benzazepine-2,5-dione with arylhydrazines is reported. When tested on a panel of human cancer cell lines, the title compounds
showed antiproliferative activity and a characteristic selectivity pattern of growth inhibition. Although structurally akin to established kinase
inhibitors, the new compounds did not exhibit noteworthy inhibitory activity when tested on an array of cancer-related kinases.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Antiproliferative activity; COMPARE; Cyclization; Kinase
1. Introduction
identifying novel antiproliferative kinase inhibitors, 1-aryl-
4,6-dihydropyrazolo[4,3-d][1]benzazepin-5(1H )-ones of the
general formula 2 were designed in which the [1]benzazepinone
substructure of the paullones is combined with an 1-arylpyra-
zolo annelated ring. Apparently the only published examples
of this basic structure are four derivatives with the general for-
mula 3 bearing a 3-methyl substituent, which were synthesized
in a multi-step procedure with closure of the seven-membered
lactam ring as final step [9]. Although these compounds were de-
signed as anthramycin analogs with potential antitumor activity,
biological test results with 3 have apparently not been published.
A more general synthetic procedure for the preparation of 2 is
reported here. The new compounds 2 were tested both for anti-
proliferativeproperties in the IVCLSPand on an array ofkinases
related to cancer.
The paullones (general formula 1, Fig. 1) constitute a class of
cyclin-dependent kinase inhibitors for which antiproliferative
activity against the tumor cell lines of the in vitro cell line
screening project (IVCLSP) of the National Cancer Institute
(NCI) has been demonstrated [1]. Paullones have also been re-
ported to inhibit glycogen synthase kinase-3 (GSK-3) [2,3]
and mitochondrial malate dehydrogenase (mMDH) [4], al-
though it is currently not clear towhat extent the distinct enzyme
inhibiting properties contribute to the growth inhibitory activity
of the compound family [5e7]. A crystal structure analysis of
the complex of GSK-3 and alsterpaullone (1, R ¼ NO2) revealed
that paullones are accommodated within the ATP binding
pocket of the kinase and are held in position through a pair of hy-
drogen bonds between the lactam function of the paullone and
Val135 of the hinge region of the protein [8]. With a view to
2. Chemistry
For a generally applicable synthesis of 2 the enaminone 6 [10]
was reacted with appropriately substituted phenylhydrazines in
* Corresponding author. Tel.: þ49 531 391 2754; fax: þ49 531 391 2799.
E-mail address: c.kunick@tu-braunschweig.de (C. Kunick).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.02.007

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S. Kohfeld et al. / European Journal of Medicinal Chemistry 42 (2007) 1317e1324
Table 1
Crystal data and structure refinement of compound 2a
O
O
O
H
N
H
N
H
N
Identification code
Empirical formula
Formula weight
Temperature
kofi
C17H13N3O
275.30
133(2) K
CH3
HN
N
N
N
N
R
˚
Wavelength
0.71073 A
Monoclinic
P21/n
Crystal system
Space group
Unit cell dimensions
R
R
a ¼ 90ꢀ
˚
1
2
3
a ¼ 19.2027(15) A
b ¼ 98.540(4)ꢀ
g ¼ 90ꢀ
˚
b ¼ 7.5016(6) A
Fig. 1. Structures of paullones 1 and structurally related 1-aryl-4,6-dihydropyr-
azolo[4,3-d][1]benzazepin-5(1H )-ones 2. The 3-methyl derivatives 3 have
been described before [9].
˚
c ¼ 19.3614(15) A
3
˚
2758.1(4) A
Volume
Z
8
Density (calculated)
Absorption coefficient
F(000)
1.326 Mg/m3
0.086 mmꢁ1
1152
glacial acetic acid at 70 ꢀC to furnish the title compounds 2 in
moderate to excellent yields.
Crystal habit, size
Colorless tablet,
0.45 ꢂ 0.40 ꢂ 0.10 mm3
1.39e30.04ꢀ
Dependent on the direction of the initial nucleophilic
attack by the phenylhydrazine, both 2 and the isomeric
2-aryl-2,6-dihydropyrazolo[4,3-d][1]benzazepin-5(4H )-ones 4
are conceivable as reaction products. According to examples
from the literature, the formation of type 2 cyclization prod-
ucts was expected [11e17]. Because an unambiguous distinc-
tion between 2 and 4 based on spectral data was difficult, the
structure assignment was supported by a single crystal struc-
ture analysis of the parent compound 2a (Fig. 2, Table 1).
Compound 2a crystallizes with two independent molecules
in the asymmetric unit, which are, however, closely similar;
a least-squares fit of all non-H atoms gives an r.m.s. deviation
Theta range for data
collection
Reflections collected
Independent reflections
Data/parameters
Goodness-of-fit on F2
Final R indices [I > 2s(I )]
R Indices (all data)
Largest diff. peak
and hole
30 572
8038 [R(int) ¼ 0.0365]
8038/387
1.027
R1 ¼ 0.0468, wR2 ¼ 0.1093
R1 ¼ 0.0712, wR2 ¼ 0.1185
ꢁ3
˚
0.307 and ꢁ0.259 e A
is excluded. The conformation of the seven-membered rings is
such that atoms C5, N6, C3a and C10b are approximately co-
˚
˚
of 0.10 A, which is reduced to 0.04 A if the phenyl substituent
˚
planar, with the other three atoms lying ca. 0.6 A to the same
side of the plane thus defined (approximate mirror symmetry
about C4 and the midpoint of C6aeC10a). Each independent
molecule forms chains parallel to the y axis by hydrogen bond-
ing of the form N6eH/O1; neighbouring molecules are re-
lated by the 21 axis.
In two cases the intermediates 7h and 7m of the reaction
were isolated upon reacting 6 with the appropriate phenylhy-
drazines at room temperature. The structures 7 could be distin-
guished unequivocally from the hypothetical intermediates 5.
Cyclization of the structures 7h and 7m at 70 ꢀC led to the
1-aryl-4,6-dihydropyrazolo[4,3-d][1]benzazepin-5(1H )-ones
2h and 2m, which were identical to the products obtained from
the direct condensation of 6 with phenylhydrazines (Scheme 1).
3. Biological evaluation and discussion
For an assessment of the antiproliferative activity, the title
compounds 2ae2owereevaluatedin thevitro cell line screening
project (IVCLSP) of the American National Cancer Institute
(NCI). In this screening setting, the growth inhibition of three
cell lines, namely MCF7 cells (breast cancer), NCI-H460
(non-small cell lung cancer, NSCLC), and SF-268 (cancer of
the central nervous system; CNS cancer) by 100 mM concentra-
tion of the title compounds is determined as initial parameter.
The results given in Table 2 revealed that while the SF-268
cells were apparently not sensitive to the test compounds, both
the breast and the lung cancer cell lines were inhibited. Six of
the compounds from Table 2 (2b, 2ee2i) inhibited one of the
Fig. 2. X-ray structure of one of the two independent molecules of compound
2a. Ellipsoids correspond to 50% probability levels.

S. Kohfeld et al. / European Journal of Medicinal Chemistry 42 (2007) 1317e1324
1319
O
O
O
O
O
H
N
H
N
H
N
H
N
H
N
CH3
N
i
i
i
i
CH3
N
N
O
N
O
N
N
HN
N
NH
CH3
H3C
NH
R
R
R
R
4
5
6
7
2
Scheme 1. Cyclization of enaminone 6 with phenylhydrazines. Reagents and conditions: (i) appropriately substituted phenylhydrazine hydrochloride, sodium
acetate, glacial acetic acid, 70 ꢀC, and 1 h.
cell lines to 32% growth or less and thus fulfilled a criterion
fixed by the NCI for a further evaluation. These six derivatives
were subsequently evaluated in the full 60 cell line screening
panel of the NCI. Among other data, for a test compound the
GI50 value (concentration that inhibits 50% net cell growth) is
determined for each of the 60 cancer cell lines in the panel. As
a general parameter for in vitro antiproliferative activity, an
averaged value (Meangraph Midpoint, MG-MID) is calculated
from the results of the distinct cell lines. The selectivity pat-
terns or ‘‘fingerprints’’ found for distinct compounds over
the whole cell line panel are characteristic for individual mo-
lecular mechanisms underlying the growth inhibition. The
comparison of selectivity patterns is a suitable method for
the determination of antiproliferative agents acting by similar
molecular mechanisms. The bioinformatic tool COMPARE
can be used by the Internet for the comparison of selectivity
patterns included in the NCI database of test compound pat-
terns [18,19]. For example, the paullones 1 were identified
using a similarity search in the NCI database with the selectiv-
ity profile of the CDK inhibitor flavopiridol as a seed pattern
[20].
Testing the six title compounds 2b, 2ee2i in the 60-cell
line screening indeed revealed a noteworthy antiproliferative
activity of all compounds (Table 3). However, the 40-methoxy
derivative 2e and the 30,50-dichloro derivative 2h were clearly
superior to the other derivatives, which showed MG-MID
values in the two-digit micromolar concentration range. In
contrast, 2e and 2h exhibited averaged growth inhibition in
submicromolar concentrations, outperforming established an-
titumor drugs such as cis-platin and, with respect to distinct
cell lines, even anticancer drugs with high potency such as
adriamycin (Table 3). Furthermore, 2e and 2h show pro-
nounced selectivity patterns with preference for cell lines
from the leukemia and the breast cancer subpanel. Examples
of selectively inhibited cell lines are K-562 (leukemia) and
MDA-MB-435 (breast cancer) which are listed in Table 3.
Table 3
In vitro antitumor activity of the 1-aryl-4,6-dihydropyrazolo[4,3-d][1]benzaze-
pin-5(1H )-ones 2b, 2ee2i compared with the standard antitumor agents cis-
platin and adriamycin
Table 2
Results of the NCI prescreening using three cancer cell lines (cell growth [%]
related to controls, test compound concentration ¼ 100 mM)
R
GI50
MG-MIDa
GI50 K-562b GI50 MDA-
MB-435c
R
MCF7a
NCI-H460b
SF-268c
2a
2bd
2c
2d
2ed
2fd
2gd
2hd
2id
2j
40-H
55
51
54
64
60
57
52
40
130
66
64
66
69
61
55
33
32
41
49
26
29
28
21
11
53
39
49
44
41
62
90
98
2b
2e
40-Br
14.8
4.07
1.91
40-Br
40-OCH3
40-CH3
40-CF3
1.82/0.37d 0.35/0.14d
0.025/0.16d
2.09
11.2
40-Cl
80
98
2f
2g
12.3
8.32
1.78
12.6
20-CH3
40-OCH3
40-CH3
40-CF3
30,50-Di-Cl
40-tert.-Butyl
20-Cl
88
2h
2i
cis-Platin
30,50-Di-Cl
40-tert.-Butyl 22.4
10.3
0.51/0.52d <0.01/0.011d <0.01/<0.01d
95
31.6
12.6
18.6
12.6
n.a.e
62
(NSC 119875)e
Adriamycin
n.a.e
71
0.11
0.10
0.20
(NSC 123127)f
2k
2l
30-Cl
101
98
Given are concentrations to reduce the cancer cell growth to 50% (GI50) [mM].
a
20-F
MG-MID ¼ Meangraph Midpoint: averaged value of the concentrations
2m
2n
2o
a
20-Br
87
98
needed for 50% growth inhibition over all cell lines in the IVCLSP. The high-
est test dose was 100 mM; the lowest test dose was 0.01 mM.
20,40-Di-F
40-NO2
62
b
Leukemia cell line. Bold numbers indicate values below the threshold for
a further testing.
Breast cancer cell line.
Non-small cell lung cancer cell line.
CNS cancer cell line.
b
c
Breast cancer cell testing.
Repeated tests.
c
d
d
e
Compound was selected for testing in the full IVCLSP panel of the NCI.
n.a.: Not available.
Results from the NCI database, status of Sept. 2005, number of tests: 6.
Results from the NCI database, status of Sept. 2005, number of tests: 2.
e
f

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S. Kohfeld et al. / European Journal of Medicinal Chemistry 42 (2007) 1317e1324
Compound 2h inhibits these cancer cells in two-digit nanomo-
lar concentrations and is in this respect far more potent than
the standard agents included in Table 3.
However, the inhibition of other kinases might play a role
in the observed biological activity of 2.
´
´
As a resume, a simple synthesis for 1-aryl-4,6-dihydropyr-
azolo[4,3-d][1]benzazepin-5(1H )-ones 2 is reported, constitut-
ing a class of novel cancer cell growth inhibitors. Further
investigations are required to determine the targets pivotal
for the antiproliferative activity of 2. Once identified, these tar-
gets might direct a further rational development of 2 as anti-
cancer agents.
In order to investigate whether the antiproliferative 1-aryl-
4,6-dihydropyrazolo[4,3-d][1]benzazepin-5(1H )-ones 2b, 2ee
2i act by a similar biological mechanism, a pairwise comparison
of the selectivity patterns from the IVCLSP was performed by
a matrix COMPARE analysis [18,19]. The resulting Pearson
correlation coefficients (PCC) listed in Table 4 shows that sev-
eral pronounced correlations indeed exist within the compound
family. Although a single high correlation between two isolated
compounds might be coincidental, the concentration of highly
correlated patterns in this group of compounds is obvious and
gives reason to suppose that 2 do not act by an undefined
general toxicity but by a specific target or a specific target com-
bination. Especially noteworthy are the correlations found
between 2b and 2f (PCC ¼ 0.853) and between the compounds
showing the strongest antiproliferative activity, 2e and 2h
(PCC ¼ 0.723). In contrast, the low PCC values between 2b,
2ee2i on the one hand and alsterpaullone 1a on the other
hand lead to the hypothesis that the antiproliferative activity
of the paullones and the novel compound family 2 is not based
on identical mechanisms. However, given the close structural
relationship between the paullones 1 and the title compounds
2, a related mechanism of action appeared feasible, namely
the inhibition of protein kinases.
In order to check this possibility, all novel derivatives 2ae
2o were tested in a proprietary protein kinase assay (33Pan-
QinaseÒ Activity Assay) on an array of 23 cancer-related
protein kinases, comprising the entities EGF-R, EphB4,
ERBB2, FAK, IGF1-R, SRC, VEGFR-2, VEGFR-3, AKT1,
Aurora A, Aurora B, CDK2/cyc A, CDK4/cyc D1, CK2-
a1, PLK1, TIE2, FLT3, MET, PDGFR-b, ARK5, PAK4,
PDK1 and SAK. For the initial test run, compounds were
tested in a single concentration (10 mM). The results revealed
that, with a few exceptions, the protein kinases were not in-
hibited by more than 40%. These exceptions included (in-
hibited kinases in brackets): 2c (FLT3), 2k (CDK4) and 2i
(VEGFR-3). Further investigations showed that 2c, 2k and
2i did not inhibit any of the indicated kinases with IC50
values below 10 mM (ATP concentration ¼ 1 mM; data not
shown). Based on these results, the inhibition of one of the
kinases from the screening array is unlikely as a mechanism
underlying the antiproliferative activity of 2e and 2h.
4. Experimental protocols
4.1. General remarks
Melting points (mp) were determined on an electric variable
heater (Electrothermal 9100) and are not corrected. Infrared
spectra were recorded on a Thermo Nicolet FT-IR 200. Nuclear
magnetic resonance spectra were recorded at the NMR labora-
tories of the Chemical Institutes of the Technical University
Braunschweig on a Bruker Avance DRX-400, using tetrame-
thylsilane as internal standard. NMR signals are reported in
parts per million on a d scale. C, H, N analyses were performed
with a CE Instruments FlashEAÒ 1112 Elementar Analyzer.
Analyses indicated by the symbols of elements were within
ꢃ0.4% of the theoretical values. The purity of all compounds
used for biological assays was >95% as determined by HPLC
using a 100% method. The HPLC analyses were carried out us-
ing a Merck Hitachi LaChrom Elite system (pump: L-2130,
DAD detector: L-2450; autosampler: L-2200; column:
LiChrospher 100 RP-18 (5 mm); eluent: acetonitrile/water mix-
tures; elution rate 1.000 mL/min; detection wavelength:
254 nm and 280 nm; overall run time: 15 min).
4.2. General synthesis protocols
4.2.1. General method for the preparation of 1-aryl-4,6-
dihydropyrazolo[4,3-d][1]benzazepinones (2aeo)
A mixture of 4-[(dimethylamino)methylene]-3,4-dihydro-
1H-[1]benzazepine-2,5-dione (230 mg, 1 mmol) (6) and the
appropriately substituted phenylhydrazine (either 1.5 mmol
of the free base or 1.5 mmol of the phenylhydrazine hydro-
chloride and 1.5 mmol of sodium acetate) is stirred in glacial
acetic acid (10 mL) at 70 ꢀC for 1 h. After cooling to room
temperature the mixture is poured into 5% aqueous sodium
Table 4
PCC values resulting from a matrix COMPARE analysis comprising the selectivity patterns of alsterpaullone (1a, R ¼ NO2) and pyrazolobenzazepinones 2b, 2ee
a,b
2i
1a
0.069 (52)
2b
2e
2f
2g
2h
2i
2h
2g
2f
2e
2b
a
0.284 (52)
0.558 (55)
0.391 (52)
0.853 (55)
0.508 (55)
0.246 (52)
0.723 (57)
0.219 (52)
0.432 (55)
0.446 (52)
0.549 (55)
0.339 (52)
0.085 (49)
0.168 (52)
0.273 (52)
0.202 (58)
ꢁ0.420 (52)
0.010 (55)
ꢁ0.052 (57)
ꢁ0.131 (55)
Numbers of cell lines used for the comparison of selectivity patterns are given in brackets.
b
PCC values above 0.350 are printed bold. Due to experiences in other compound classes this value can be used as a threshold for the discrimination of com-
pound families [24].

S. Kohfeld et al. / European Journal of Medicinal Chemistry 42 (2007) 1317e1324
1321
acetate solution (20 mL). The resulting precipitate is collected,
washed with water, dried and crystallized from ethanol.
(m, 3H, ArH), 7.51e7.54 (m, 2H, ArH), 7.80 (s, 1H, ArH),
10.13 (s, 1H, NH); 13C NMR (DMSO-d6, 100.6 MHz):
d (ppm) ¼ 31.9 (CH2); 123.0, 123.5, 126.3 (2C), 128.4,
129.1, 129.3 (2C), 138.5 (tert. C); 118.2, 119.6, 132.1,
136.0, 138.6, 172.6 (quat. C, one signal missing due to peak
overlapping). Anal. C17H12ClN3O (C, H, N).
4.2.2. General method for the preparation of 4-[(2-aryl-
hydrazino)methylene]-3,4-dihydro-1H[1]benzazepine-2,5-
diones (7h and 7m)
A mixture of 4-[(dimethylamino)methylene]-3,4-dihydro-
1H-[1]benzazepine-2,5-dione (230 mg, 1 mmol) (6) and the
appropriately substituted phenylhydrazine (either 1.5 mmol
of the free base or 1.5 mmol of the phenylhydrazine hydro-
chloride and 1.5 mmol of sodium acetate) is stirred in glacial
acetic acid (15 mL) for 1 h at room temperature. Subsequently,
the mixture is poured into 5% aqueous sodium acetate solution
(30 mL). The resulting precipitate is collected, washed with
water, dried and crystallized from ethanol (70%).
4.3.4. 1-(2-Methylphenyl)-4,6-dihydropyrazolo-
[4,3-d][1]benzazepin-5(1H )-one (2d)
Preparation according to the general method described in
Section 4.2.1 yielded colorlꢁes1s crystals (212 mg; 73%); mp.:
245 ꢀC; IR (KBr): 3202 cm (NH), 3065 cmꢁ1 (CH arom.),
2980 and 2920 cmꢁ1 (CH aliph.), 1689 cmꢁ1 (C]O); 1H
NMR (DMSO-d6, 400 MHz): d (ppm) ¼ 1.81 (s, 3H, CH3),
3.35 (s, 2H, CH2), 6.71 (dd, 1H, J ¼ 7.9/1.3 Hz, ArH), 6.89
(dt, 1H, J ¼ 6.9/8.0/1.4 Hz, ArH), 7.23e7.36 (m, 5H, ArH),
7.40 (dt, 1H, J ¼ 7.5/7.1/1.5 Hz, ArH), 7.76 (s, 1H, ArH),
10.07 (s, 1H, NH); 13C NMR (DMSO-d6, 100.6 MHz):
d (ppm) ¼ 16.8 (CH3); 31.9 (CH2); 122.7, 123.3, 126.9,
127.1, 127.7, 128.8, 129.1, 131.0, 137.7 (tert. C); 115.9,
119.7, 134.6, 135.9, 137.3, 139.0, 172.7 (quat. C). Anal.
C18H15N3O (C, H, N).
4.3. Synthesis of particular compounds
4.3.1. 1-Phenyl-4,6-dihydropyrazolo[4,3-d][1]benzazepin-
5(1H )-one (2a)
Preparation according to the general method described in
Section 4.2.1. yielded light yellow crystals (152 mg, 56%);
mp.: 247e248 ꢀC; IR (KBr): 3055 cmꢁ1 (CH arom.),
2967 cmꢁ1 and 2909 cmꢁ1 (CH aliph.), 1682 cmꢁ1 (C]O);
1H NMR (CDCl3, 400 MHz): d (ppm) ¼ 3.45 (s, 2H, CH2),
6.95 (dd, 1H, J ¼ 7.9/2.0 Hz, ArH), 6.99 (dt, 1H, J ¼ 6.9/7.9/
1.0 Hz, ArH), 7.21 (dd, 1H, J ¼ 7.9/0.8 Hz, ArH), 7.30e7.42
(m, 6H, ArH), 7.67 (s, 1H, ArH), 8.59 (s, 1H, NH); 13C
NMR (CDCl3, 100.6 MHz): d (ppm) ¼ 32.5 (CH2); 123.2,
124.7, 125.3 (2C), 128.3, 129.60 (2C), 129.64, 129.66, 138.8
(tert. C); 118.0, 121.3, 135.5, 136.7, 140.3, 174.7 (quat. C).
Anal. C17H13N3O (C, H, N).
4.3.5. 1-(4-Methoxyphenyl)-4,6-dihydropyrazolo-
[4,3-d][1]benzazepin-5(1H )-one (2e)
Preparation according to the general method described in
Section 4.2.1 yielded light yellow crystals (274 mg, 89%);
mp.: 275 ꢀC; IR (KBr): 3224 cmꢁ1 (NH), 3128 cmꢁ1 (CH
1
arom.), 1669 cmꢁ1 (C]O); H NMR (DMSO-d6, 400 MHz):
d (ppm) ¼ 3.31 (s, 2H, CH2), 3.79 (s, 3H, OCH3), 6.84 (dd,
1H, J ¼ 7.9/1.3 Hz, ArH), 6.96e7.02 (m, 3H, ArH), 7.20e
7.27 (m, 3H, ArH), 7.32 (dt, 1H, J ¼ 7.5/7.7/1.5 Hz, ArH),
7.71 (s, 1H, ArH), 10.07 (s, 1H, NH); 13C NMR (DMSO-d6,
100.6 MHz): d (ppm) ¼ 31.9 (CH2); 55.4 (CH3); 114.3 (2C),
122.9, 123.3, 126.3 (2C), 128.2, 128.7, 137.6 (tert. C); 117.2,
119.9, 132.8, 136.0, 158.6, 172.6 (quat. C, one signal missing
due to peak overlapping). Anal. C18H15N3O2 (C, H, N).
4.3.2. 1-(4-Bromophenyl)-4,6-dihydropyrazolo-
[4,3-d][1]benzazepin-5(1H )-one (2b)
Preparation according to the general method described in
Section 4.2.1. yielded yellow needles (270 mg, 74%); mp.:
282 ꢀC; IR (KBr): 3226 cmꢁ1 (NH), 3123 cmꢁ1 (CH arom.),
2956 cmꢁ1 (CH aliph.), 1668 cmꢁ1 (C]O); 1H NMR
(DMSO-d6, 400 MHz): d (ppm) ¼ 3.32 (s, 2H, CH2), 6.88 (dd,
1H, J ¼ 7.9/1.4 Hz, ArH), 7.03 (dt, 1H, J ¼ 8.4/8.0/1.2 Hz,
ArH), 7.25e7.30 (m, 3H, ArH), 7.36 (dt, 1H, J ¼ 7.3/8.3/
1.5 Hz, ArH), 7.64e7.67 (m, 2H, ArH), 7.80 (s, 1H, ArH),
10.14 (s, 1H, NH); 13C NMR (DMSO-d6, 100.6 MHz):
d (ppm) ¼ 31.9 (CH2); 123.1, 123.5, 126.6 (2C), 128.4, 129.1,
132.2 (2C), 138.9 (tert. C); 118.3, 119.6, 120.5, 136.0, 136.1,
138.7, 172.6 (quat. C). Anal. C17H12BrN3O (C, H, N).
4.3.6. 1-(4-Methylphenyl)-4,6-dihydropyrazolo-
[4,3-d][1]benzazepin-5(1H )-one (2f)
Preparation according to the general method described in
Section 4.2.1 yielded a white powder (245 mg, 85%); mp.:
285e287 ꢀC; IR (KBr): 3177 cmꢁ1 (NH), 3062 cmꢁ1 (CH
arom.), 2976 cmꢁ1 and 2924 cmꢁ1 (CH aliph.), 1692 cmꢁ1
1
(C]O); H NMR (DMSO-d6, 400 MHz): d (ppm) ¼ 2.35 (s,
4.3.3. 1-(4-Chlorophenyl)-4,6-dihydropyrazolo-
[4,3-d][1]benzazepin-5(1H )-one (2c)
3H, CH3), 3.31 (s, 2H, CH2), 6.84 (dd, 1H, J ¼ 7.9/1.3 Hz,
ArH), 6.98 (dt, 1H, J ¼ 8.1/8.3/1.3 Hz, ArH), 7.17e7.19 (m,
2H, ArH), 7.25e7.28 (m, 3H, ArH), 7.33 (dt, 1H, J ¼ 8.1/
8.3/1.5 Hz, ArH), 7.74 (s, 1H, ArH), 10.10 (s, 1H, NH); 13C
NMR (DMSO-d6, 100.6 MHz): d (ppm) ¼ 20.6 (CH3); 31.9
(CH2); 123.0, 123.3, 124.6 (2C), 128.3, 128.8, 129.7 (2C),
137.3 (tert. C); 117.6, 120.0, 135.88, 135.94, 137.3, 172.7
(quat. C, one signal missing due to peak overlapping). Anal.
C18H15N3O (C, H, N).
Preparation according to the general method described in
Section 4.2.1 yielded colorless crystals (240 mg; 73%); mp.:
273e274 ꢀC; IR (KBr): 3178 cmꢁ1 (NH), 3062 cmꢁ1 (CH
arom.), 2977 cmꢁ1 (CH aliph.), 1691 cmꢁ1 (C]O); 1H
NMR (DMSO-d6, 400 MHz): d (ppm) ¼ 3.32 (s, 2H, CH2),
6.87 (dd, 1H, J ¼ 7.9/1.4 Hz, ArH), 7.03 (dt, 1H, J ¼ 7.0/8.0/
1.3 Hz, ArH), 7.28 (dd, 1H, J ¼ 8.2/1.0 Hz, ArH), 7.33e7.37

1322
S. Kohfeld et al. / European Journal of Medicinal Chemistry 42 (2007) 1317e1324
4.3.7. 1-(4-Trifluoromethylphenyl)-4,6-
dihydropyrazolo[4,3-d][1]benzazepin-5(1H )-one (2g)
135.90, 135.92, 137.3, 150.3, 172.7 (quat. C). Anal.
C21H21N3O (C, H, N).
Preparation according to the general method described in
Section 4.2.1 yielded a whꢁit1e powder (257 mg, 75%); mp.:
245 ꢀC; IR (KBr): 3172 cm (NH), 3065 cmꢁ1 (CH arom.),
2979 cmꢁ1 and 2927 cmꢁ1 (CH aliph.), 1692 cmꢁ1 (C]O);
1H NMR (DMSO-d6, 400 MHz): d (ppm) ¼ 3.34 (obscured
by the water peak), 6.89 (dd, 1H, J ¼ 7.9/1.4 Hz, ArH), 7.04
(dt, 1H, J ¼ 8.0/8.4/1.3 Hz, ArH), 7.31 (dd, 1H, J ¼ 8.2/
1.0 Hz, ArH), 7.39 (dt, 1H, J ¼ 8.3/8.5/1.5 Hz, ArH), 7.53e
7.56 (m, 2H, ArH), 7.83e7.85 (m, 2H, ArH), 7.87 (s, 1H,
ArH), 10.18 (s, 1H, NH); 13C NMR (DMSO-d6,
100.6 MHz): d (ppm) ¼ 31.9 (CH2); 123.1, 123.6, 124.9
(2C), 126.5, 128.5 (2C), 129.3, 139.3 (tert. C); 119.0, 119.5,
125.3, 136.0, 136.3, 142.7, 172.7 (quat. C, one signal missing
due to peak overlapping). Anal. C18H12F3N3O (C, H, N).
4.3.10. 1-(2-Chlorophenyl)-4,6-dihydropyrazolo-
[4,3-d][1]benzazepin-5(1H )-one (2j)
Preparation according to the general method described in
Section 4.2.1 yielded orange crystals (201 mg, 65%); mp.:
244e245 ꢀC; IR (KBr): 3286 cmꢁ1 (NH), 1687 cmꢁ1
1
(C]O); H NMR (DMSO-d6, 400 MHz): d (ppm) ¼ 3.38 (br
s, 2H, CH2), 6.74 (dd, 1H, J ¼ 7.9/1.3 Hz, ArH), 6.93 (dt,
1H, J ¼ 8.1/8.3/1.4 Hz, ArH), 7.24 (dd, 1H, J ¼ 8.2/1.1 Hz,
ArH), 7.31 (dt, 1H, J ¼ 8.3/8.4/1.5 Hz, ArH), 7.50e7.63 (m,
4H, ArH), 7.80 (s, 1H, ArH), 10.08 (s, 1H, NH); 13C NMR
(DMSO-d6, 100.6 MHz): d (ppm) ¼ 31.8 (CH2); 122.8,
123.4, 126.5, 128.4, 129.0, 130.0, 130.3, 130.9, 138.5 (tert.
C); 116.1, 119.6, 130.5, 136.0, 137.4, 138.0, 172.5 (quat. C).
Anal. C17H12ClN3O (C, H, N).
4.3.8. 1-(3,5-Dichlorophenyl)-4,6-dihydropyrazolo-
[4,3-d][1]benzazepin-5(1H )-one (2h)
Preparation according to the general method described in
4.3.11. 1-(3-Chlorophenyl)-4,6-dihydropyrazolo-
[4,3-d][1]benzazepin-5(1H )-one (2k)
Section 4.2.1 yielded yellow crysꢁta1ls (274 mg, 80%); mp.:
Preparation according to the general method described in
Section 4.2.1 yielded light yellow crystals (252 mg, 82%);
mp.: 272e273 ꢀC; IR (KBr): 3220 cmꢁ1 (NH), 3144 cmꢁ1
(CH arom.), 1684 cmꢁ1 (C]O); 1H NMR (DMSO-d6,
400 MHz): d (ppm) ¼ 3.33 (s, CH2, obscured by water peak),
6.89 (dd, 1H, J ¼ 7.9/1.4 Hz, ArH), 7.03 (dt, 1H, J ¼ 8.0/8.4/
1.3 Hz, ArH), 7.22 (dt, 1H, J ¼ 7.1/2.0/2.2 Hz, ArH), 7.29 (dd,
1H, J ¼ 8.2/1.0 Hz, ArH), 7.38 (dt, 1H, J ¼ 8.3/8.5/1.5 Hz,
ArH), 7.43e7.51 (m, 3H, ArH), 7.81 (s, 1H, ArH), 10.13 (s,
1H, NH); 13C NMR (DMSO-d6, 100.6 MHz): d (ppm) ¼ 31.9
(CH2); 123.1, 123.3, 123.4, 124.4, 127.7, 128.4, 129.2, 130.8,
138.8 (tert. C); 118.4, 119.5, 133.4, 136.0, 136.2, 140.8, 172.6
(quat. C). Anal C17H12ClN3O (C, H, N).
283e284 ꢀC; IR (KBr): 3215 cm
(NH), 3144 cmꢁ1 and
3098 cmꢁ1 (CH arom.), 1690 cmꢁ1 (C]O); 1H NMR
(DMSO-d6, 400 MHz): d (ppm) ¼ 3.34 (obscured by water sig-
nal), 6.96 (dd, 1H, J ¼ 7.9/1.4 Hz, ArH), 7.08 (dt, 1H, J ¼ 8.0/
8.4/1.2 Hz, ArH), 7.31 (dd, 1H, J ¼ 8.2/1.0 Hz, ArH), 7.38 (d,
2H, J ¼ 1.9 Hz, ArH), 7.41 (dt, 1H, J ¼ 8.3/8.5/1.5 Hz, ArH),
7.68 (t, 1H, J ¼ 1.9 Hz, ArH), 7.85 (s, 1H, ArH), 10.15 (s, 1H,
NH); 13C NMR (DMSO-d6, 100.6 MHz): d (ppm) ¼ 31.8
(CH2); 123.1, 123.2 (2C), 123.4, 127.3, 128.5, 129.4, 139.3
(tert. C); 118.8, 119.2, 134.3 (2C), 136.1, 136.4, 141.3, 172.4
(quat. C). Anal. C17H11Cl2N3O (C, H, N).
The preparation of 2h was also accomplished by stirring
a slurry of 7h (0.25 mmol, 91 mg) in glacial acetic acid
(3 mL) for 1 h at 70 ꢀC. After cooling, the mixture was poured
into 5% aqueous sodium acetate solution (6 mL). The precip-
itate was collected, washed with water, and subsequently crys-
tallized from ethanol (70%) to yield light yellow crystals
(27 mg, 32%). According to mp. and spectral data the com-
pound was identical with the material prepared by general
method described in Section 4.2.1.
4.3.12. 1-(2-Fluorophenyl)-4,6-dihydropyrazolo-
[4,3-d][1]benzazepin-5(1H )-one (2l)
Preparation according to the general method described in Sec-
tion 4.2.1 yielded yellow crystals (182 mg, 62%); mp.: 234e
236 ꢀC; IR (KBr): 3210 cmꢁ1 (NH), 3140 cmꢁ1, 3100 cmꢁ1
(CH arom.), 2972 cmꢁ1, 2916 cmꢁ1 (CH aliph.), 1685 cmꢁ1
1
(C]O); H NMR (DMSO-d6, 400 MHz): d (ppm) ¼ 3.36 (s,
2H, CH2), 6.82 (dd, 1H, J ¼ 7.8/1.3 Hz, ArH), 6.96 (dt, 1H,
J ¼ 8.0/8.3/1.3 Hz, ArH), 7.26 (dd, 1H, J ¼ 8.1/1.1 Hz, ArH),
7.31e7.41 (m, 3H, ArH), 7.52e7.58 (m, 1H, ArH), 7.62 (t, 1H,
4.3.9. 1-(4-tert-Butylphenyl)-4,6-dihydropyrazolo-
[4,3-d][1]benzazepin-5(1H )-one (2i)
Preparation according to the general method described in
J ¼ 7.3/7.6 Hz, ArH), 7.83 (s, 1H, ArH), 10.12 (s, 1H, NH); 13
C
Section 4.2.1 yielded colorless crystals (138 mg, 42%); mꢁp1.:
NMR (DMSO-d6, 100.6 MHz): d (ppm) ¼ 31.8 (CH2); 116.7
2
4
228e229 ꢀC; IR (KBr): 3204 cmꢁ1 (NH), 3134 cm
,
(d, JC,F ¼ 19.6 Hz), 122.9, 123.4, 125.4 (d, JC,F ¼ 3.6 Hz),
3099 cmꢁ1 (CH arom.), 2963 cmꢁ1, 2914 cmꢁ1, 2870 cmꢁ1
(CH aliph.), 1690 cmꢁ1 (C]O); 1H NMR (DMSO-d6,
400 MHz): d (ppm) ¼ 1.30 (s, 9H, CH3), 3.31 (s, 2H, CH2),
6.85 (dd, 1H, J ¼ 7.9/1.3 Hz, ArH), 6.96 (dt, 1H, J ¼ 8.1/8.3/
1.4 Hz, ArH), 7.21e7.24 (m, 2H, ArH), 7.45e7.48 (m, 2H,
ArH), 7.27 (dd, 1H, J ¼ 8.1/1.2 Hz, ArH), 7.33 (dt, 1H,
J ¼ 8.3/8.4/1.5 Hz, ArH), 7.75 (s, 1H, ArH), 10.10 (s, 1H,
NH); 13C NMR (DMSO-d6, 100.6 MHz): d (ppm) ¼ 31.0
(3 ꢂ CH3); 31.9 (CH2); 123.0, 123.3, 124.3 (2C), 125.9
(2C), 128.3, 128.8, 138.0 (tert. C); 34.4, 117.7, 120.0,
126.4, 128.9, 129.0, 130.9 (d, JC,F ¼ 8.1 Hz), 138.8 (tert. C);
3
1
119.6, 127.6, 127.7, 135.9, 137.9, 155.6 (d, JC,F ¼ 155.6 Hz),
172.4 (quat. C). Anal. C17H12FN3O (C, H, N).
4.3.13. 1-(2-Bromophenyl)-4,6-dihydropyrazolo-
[4,3-d][1]benzazepin-5(1H )-one (2m)
Preparation according to the general method described in
Section 4.2.1 yielded orange crystals (206 mg, 57%); mp.:
248e249 ꢀC; IR (KBr): 3281 cmꢁ1 (NH), 3158 cmꢁ1
,
3062 cmꢁ1 (CH arom.), 1686 cmꢁ1 (C]O); 1H NMR

S. Kohfeld et al. / European Journal of Medicinal Chemistry 42 (2007) 1317e1324
1323
(DMSO-d6, 400 MHz): d (ppm) ¼ 3.37 (br s, 2H, CH2), 6.74
(dd, 1H, J ¼ 7.8/1.5 Hz, ArH), 6.92 (dt, 1H, J ¼ 8.1/8.3/
1.4 Hz, ArH), 7.24 (dd, 1H, J ¼ 8.1/1.3 Hz, ArH), 7.30 (dt,
1H, J ¼ 8.3/8.6/1.5 Hz, ArH), 7.47 (ddd, 1H, J ¼ 7.8/7.8/
2.3 Hz, ArH), 7.53e7.59 (m, 2H, ArH), 7.77e7.78 (m, 2H,
mp.: 219e222 ꢀC; IR (KBr): 3188 cmꢁ1 (NH), 3070 cmꢁ1
1
(CH arom.), 2965 cmꢁ1 (CH aliph.), 1688 cmꢁ1 (C]O); H
NMR (DMSO-d6, 400 MHz): d (ppm) ¼ 2.98 (s, 2H, CH2),
6.82 (t, 1H, J ¼ 1.8 Hz, ArH), 6.91 (d, 2H, J ¼ 1.3 Hz, ArH),
7.15 (d, 1H, J ¼ 8.1 Hz, ArH), 7.20 (dt, 1H, J ¼ 7.2/8.1/
1.0 Hz, ArH), 7.40 (dt, 1H, J ¼ 6.9/8.3/1.4 Hz, ArH), 7.70
(dd, 1H, J ¼ 8.1/1.3 Hz, ArH), 8.17 (s, 1H, ArH), 9.62 (s,
1H, NH), 10.19 (s, 1H, NH), 10.57 (s, 1H, NH); 13C NMR
(DMSO-d6, 100.6 MHz): d (ppm) ¼ 31.1 (CH2); 109.7 (2C),
116.7, 121.3, 123.0, 127.8, 129.5, 138.7 (tert. C); 108.2,
127.4, 134.6 (2C), 136.6, 147.5, 151.2, 172.0 (quat. C).
Anal. C17H13Cl2N3O2 (H, N, C) calcd. 56.37; found 55.90.
1
ArH), 10.07 (s, 1H, NH); C NMR (DMSO-d6, 100.6 MHz):
d (ppm) ¼ 31.8 (CH2); 122.8, 123.3, 126.7, 128.9 (2C), 130.2,
131.2, 133.4, 138.3 (tert. C); 116.0, 119.5, 121.1, 136.0,
137.7, 139.0, 172.5 (quat. C). Anal. C17H12BrN3O (C, H, N).
The preparation of 2m was also accomplished by stirring
a slurry of 7m (0.25 mmol, 93 mg) in glacial acetic acid
(3 mL) for 1 h at 70 ꢀC. After cooling, the mixture was poured
into 5% aqueous sodium acetate solution (6 mL). The precip-
itate was collected, washed with water, and subsequently crys-
tallized from ethanol (70%) to yield orange crystals (49 mg,
54%). According to mp. and spectral data the compound
was identical with the material prepared by general method
described in Section 4.2.1.
4.3.17. 4-{[2-(2-Bromophenyl)hydrazino]methylene}-3,
4-dihydro-1H-[1]benzazepine-2,5-dione (7m)
Preparation according to the general method described in
Section 4.2.2 yielded brown crystals (223 mg; 60%); mp.:
212 ꢀC (dec.); IR (KBr): 3430 cmꢁ1, 3324 cmꢁ1, 3198 cmꢁ1
(NH), 3099 cmꢁ1, 3055 cmꢁ1 (CH arom.), 2957 cmꢁ1 (CH
1
4.3.14. 1-(2,4-Difluorophenyl)-4,6-dihydropyrazolo-
[4,3-d][1]benzazepin-5(1H )-one (2n)
aliph.), 1647 cmꢁ1 (C]O); H NMR (DMSO-d6, 400 MHz):
d (ppm) ¼ 2.99 (s, 2H, CH2), 6.72 (dt, 1H, J ¼ 7.2/7.9/
1.6 Hz, ArH), 7.15 (d, 1H, J ¼ 7.8 Hz, ArH), 7.20 (dt, 1H,
J ¼ 7.3/7.9/0.9 Hz, ArH), 7.30 (dt, 1H, J ¼ 7.2/8.2/1.0 Hz,
ArH), 7.37e7.42 (m, 2H, ArH), 7.47 (dd, 1H, J ¼ 7.8/
1.3 Hz, ArH), 7.72 (dd, 1H, J ¼ 7.9/1.4 Hz, ArH), 8.45 (s,
1H, ArH), 9.48 (s, 1H, NH), 9.81 (s, 1H, NH), 10.18 (s, 1H,
NH); 13C NMR (DMSO-d6, 100.6 MHz): d (ppm) ¼ 31.8
(CH2); 114.0, 119.9, 121.3, 123.0, 127.9, 128.5, 129.5,
132.5, 141.2 (tert. C); 106.0, 107.5, 127.7, 136.6, 142.6,
152.9, 172.1 (quat. C). Anal. C17H14BrN3O2 (C, H, N).
Preparation according to the general method described in Sec-
tion 4.2.1 yielded colorless crꢁy1stals (193 mg, 62%); mp.: 217e
218 ꢀC; IR (KBr): 3233 cm (NH), 3126 cmꢁ1, 3088 cmꢁ1
(CH arom.), 2981 cmꢁ1, 2896 cmꢁ1 (CH aliph.), 1673 cmꢁ1
1
(C]O); H NMR (DMSO-d6, 400 MHz): d (ppm) ¼ 3.33 (s,
CH2 obscured by water signal), 6.85 (dd, 1H, J ¼ 7.8/1.3 Hz,
ArH), 7.00 (dt, 1H, J ¼ 8.1/8.3/1.3 Hz, ArH), 7.26e7.37 (m,
3H, ArH), 7.47 (dt, 1H, J ¼ 10.9/10.9/2.8 Hz, ArH), 7.71 (q,
1H, J ¼ 8.6/6.3/8.3 Hz, ArH), 7.83 (s, 1H, ArH), 10.12 (s, 1H,
NH); 13C NMR (DMSO-d6, 100.6 MHz): d (ppm) ¼ 31.8
(CH2); 105.3 (t, 2JC,F ¼ 24.0/27.4 Hz), 112.6 (d,
2JC,F ¼ 26.6 Hz), 122.9, 123.5, 126.5, 129.1, 130.3 (t,
3JC,F ¼ 10.6 Hz), 139.0 (tert. C); 116.6, 119.4, 124.5, 136.0,
138.0, 172.4 (quat. C, two C not detected due to low intensity
or peak overlapping). Anal. C17H11F2N3O (C, H, N).
4.4. X-ray structure determination
Numerical details are summarised in Table 1. Data were re-
corded on a Bruker SMART 1000 CCD diffractometer. The
structure was refined on F2 using the program SHELXL-97
(G.M. Sheldrick, University of Gottingen, Germany). Hydro-
¨
4.3.15. 1-(4-Nitrophenyl)-4,6-dihydropyrazolo-
[4,3-d][1]benzazepin-5(1H )-one (2o)
gens of NH groups were refined freely, others with a riding
model.
Preparation according to the general method described in Sec-
tion 4.2.1 (reflux instead of strirring at 70 ꢀC yielded colorꢁle1ss crys-
tals (214 mg, 66%); mp.: 264 ꢀC; IR (KBr): 3196 cm (NH),
3067 cmꢁ1 (CH arom.), 2954, 2898 cmꢁ1 (CH aliph.),1681 cmꢁ1
Complete data (excluding structure factors) have been de-
posited at the Cambridge Crystallographic Data Centre under
the number CCDC-623071. These data can be obtained free of
charge from www.ccdc.cam.ac.uk/data_request/cif.
1
(C]O); H NMR (DMSO-d6, 400 MHz): d (ppm) ¼ 3.33 (s,
CH2 obscured by water peak), 6.94 (dd, 1H, J ¼ 8.0/1.4 Hz,
ArH), 7.05 (dt, 1H, J ¼ 8.1/8.3/1.3 Hz, ArH), 7.32 (dd, 1H,
J ¼ 8.1/0.9 Hz, ArH), 7.41 (dt, 1H, J ¼ 8.3/8.6/1.5 Hz, ArH),
7.57e7.61 (m, 2H, ArH), 7.91 (s, 1H, ArH), 8.30e8.33 (m, 2H,
ArH), 10.21 (s, 1H, NH); 13C NMR (DMSO-d6, 100.6 MHz):
d (ppm) ¼ 31.9 (CH2); 123.2, 123.6, 124.79 (2C), 124.82 (2C),
128.7, 129.4, 139.9 (tert. C); 119.4, 119.6, 136.1, 136.5, 144.4,
145.8, 172.6 (quat. C). Anal. C17H12N4O3 (C, H, N).
4.5. In vitro anticancer assays
4.5.1. Prescreening
At the National Cancer Institute, a 3-cell line panel primary
anticancer assay is used, consisting of the MCF7 (Breast),
NCI-H460 (Lung), and SF-268 (CNS) lines. For testing,
each cell line was inoculated and preincubated on a microtiter
plate. Test agents were then added at 10ꢁ4 M concentration
and the culture was incubated for 48 h. End-point determina-
tions were made using alamar blue as described previously
[21]. Results for each test agent are reported as the percent
of growth of the treated cells when compared to the untreated
control cells. Compounds that reduce the growth of at least
4.3.16. 4-{[2-(3,5-Dichlorophenyl)hydrazino]methylene}-3,
4-dihydro-1H-[1]benzazepine-2,5-dione (7h)
Preparation according to the general method described
in Section 4.2.2 yielded beige crystals (178 mg, 49%);

1324
S. Kohfeld et al. / European Journal of Medicinal Chemistry 42 (2007) 1317e1324
one of the cell lines to approximately 32% or less are passed
on for evaluation in the full panel of 60 cell lines over a 5-log
dose range.
European Commission (Contract No LSHB-CT-2004-503467,
to F.T., C.S., M.H. G.K., and C.K.) is gratefully acknowledged.
References
4.5.2. Screening on 60 cell lines
[1] C. Kunick, K. Lauenroth, K. Wieking, X. Xie, C. Schultz, R. Gussio,
D. Zaharevitz, M. Leost, L. Meijer, A. Weber, F.S. Jørgensen,
T. Lemcke, J. Med. Chem. 47 (2004) 22e36.
Screening compounds are tested against 60 human tumor
cell lines at five concentrations (10ꢁ8e10ꢁ4 M). After 48 h
of continuous drug exposure, a sulforhodamine B protein as-
say is used to determine cell viability or growth. The percent-
age of growth is calculated on the basis of the optical density
of the treated cultures compared to untreated control cell lines.
Details concerning the performance of this screening have
been published [22,23].
[2] M. Leost, C. Schultz, A. Link, Y.-Z. Wu, J. Biernat, E.-M. Mandelkow,
J.A. Bibb, G.L. Snyder, P. Greengard, D.W. Zaharevitz, R. Gussio,
A.M. Senderowicz, E.A. Sausville, C. Kunick, L. Meijer, Eur. J. Bio-
chem. 267 (2000) 5983e5994.
[3] C. Kunick, K. Lauenroth, M. Leost, L. Meijer, T. Lemcke, Bioorg. Med.
Chem. Lett. 14 (2004) 413e416.
[4] M. Knockaert, K. Wieking, S. Schmitt, M. Leost, K.M. Grant,
J.C. Mottram, C. Kunick, L. Meijer, J. Biol. Chem. 277 (2002)
25493e25501.
4.6. Kinase assays
[5] X. Xie, T. Lemcke, R. Gussio, D.W. Zaharevitz, M. Leost, L. Meijer,
C. Kunick, Eur. J. Med. Chem. 40 (2005) 655e661.
A proprietary protein kinase assay (33PanQinaseÒ Activity
Assay) was used for measuring the kinase activity of the 23
protein kinases. All assays were performed with a Beckman-
Coulter/Sagian robotic system and 96-well FlashPlatesÔ
from Perkin Elmer/NEN (Boston, MA, USA) in a 50 mL reac-
tion volume. The reaction cocktail was pipetted in 4 steps in
the following order: 20 mL of assay buffer; 5 mL of ATP solu-
tion (in H2O); 5 mL of test compound (in 10% DMSO); 10 mL
of substrate/10 mL of enzyme solution (premixed). The assay
for all enzymes contained 60 mM HEPESeNaOH, pH
7.5, 3 mM MgCl2, 3 mM MnCl2, 3 mM Na-orthovanadate,
1.2 mM DTT, 50 mg/mL PEG20 000, 1 mM [g-33P]-ATP (ap-
prox. 5 ꢂ 105 cpm per well). For the 23 kinases the following
substrates were used [substrate amount in square brackets
given in ng/50 mL]: poly(Glu,Tyr)4:1 [125]: EGF-R, EPHB4,
ERBB2, FAK, IGF1-R, SRC, VEGFR-2, VEGFR-3, TIE2;
GSK-3(14-27) [1000]: AKT1; tetra(LRRWSLG) [500/250, re-
spectively]: Aurora A, Aurora B; Histone H1 [125]: CDK2/
CycA; Rb-CTF [500]: CDK4/CycD1; p53-CTM [125]: CK2-
alpha1; casein [1000]: PLK1; poly(Ala,Glu,Lys,Tyr)6:2:5:1
[125]: FLT3, MET, PDGFR-beta; tetra(LRRWSLG) [500]:
PAK4, PDK1; autophosphorylation: ARK5, SAK. The reac-
tion cocktails were incubated at 30 ꢀC for 80 min. The reac-
tion was stopped with 50 mL of 2% (v/v) H3PO4, plates
were aspirated and washed two times with 200 mL of 0.9%
(w/v) NaCl. Incorporation of 33Pi was determined with a mi-
croplate scintillation counter (Microbeta Trilux, Wallac).
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We are grateful to the American National Cancer Institute for
performing the cell line screening. Funding of the project by the
2148e2153.