Novel 3-azaindolyl-4-arylmaleimides exhibiting potent antiangiogenic efficacy, protein kinase inhibition, and antiproliferative activity

Article abstract of DOI:10.1021/jm301217c

Tumor growth and metastasis are highly associated with the overexpression of protein kinases (PKs) regulating cell growth, apoptosis resistance, and prolonged cell survival. This study describes novel azaindolyl-maleimides with significant inhibition of PKs, such as VEGFR, FLT-3, and GSK-3β which are related to carcinogenesis. Furthermore, these compounds exhibit high kinase selectivity and potent inhibition of angiogenesis and cell proliferation, offering versatile options in cancer treatment strategies.

Full text of DOI:10.1021/jm301217c

Article
pubs.acs.org/jmc
Novel 3‑Azaindolyl-4-arylmaleimides Exhibiting Potent
Antiangiogenic Efficacy, Protein Kinase Inhibition, and
Antiproliferative Activity
Christopher Ganser,†,∥ Eva Lauermann,†,∥ Annett Maderer,‡ Torsten Stauder,§ Jan-Peter Kramb,†
Stanislav Plutizki,† Thomas Kindler,§ Markus Moehler,‡ and Gerd Dannhardt*,†
‡
§
†Department of Pharmacy, Department of Internal Medicine I, and Department of Internal Medicine III, Johannes Gutenberg
University of Mainz, Mainz, Germany
ABSTRACT: Tumor growth and metastasis are highly
associated with the overexpression of protein kinases (PKs)
regulating cell growth, apoptosis resistance, and prolonged cell
survival. This study describes novel azaindolyl-maleimides with
significant inhibition of PKs, such as VEGFR, FLT-3, and
GSK-3β which are related to carcinogenesis. Furthermore,
these compounds exhibit high kinase selectivity and potent
inhibition of angiogenesis and cell proliferation, offering
versatile options in cancer treatment strategies.
mamma carcinoma, prostate carcinoma, glioblastoma, karposi
sarcoma, melanoma, and leukemia for which no curative
therapies are known so far.
Beside VEGFR-2, more protein kinases such as FMS-like
tyrosine kinase 3 (FLT-3)6 or glycogen synthase kinase 3β
(GSK-3β) became attractive drug targets in cancer therapy, e.g.,
regarding the treatment of acute myeloid leukemia (AML) or
colorectal cancer.
Several small molecule inhibitors of protein kinases
(imatinib, gefitinib, erlotinib, sorafenib, and sunitinib) showed
promising efficacy and acceptable toxicity.1,7 Despite these first
achievements, many tumors are still resistant to therapy and
associated with a poor prognosis. Thus, there is an ongoing
urgent need for compounds with novel therapeutic profiles
amplifying the armamentarium against cancer.
INTRODUCTION
■
Overexpression of protein kinases (PKs), which trigger
intracellular signal transduction by phosphorylating tyrosine,
threonine, or serine residues in key proteins, is closely related
to the process of cancerogenesis. By regulating cell growth,
apoptosis resistance, survival, and angiogenesis,1 PKs such as
vascular endothelial growth factor receptor (VEGFR) are of
vital relevance in both physiological and pathophysiological
angiogenesis. VEGFRs exists in three subtypes VEGFR-1 to
VEGFR-3. VEGFR-2 (also known as KDR or FLK-1 in mice) is
pivotal for the process of vessel growth. The morphogenetic
process of angiogenesis depends on the formation of new blood
vessels from endothelial cells of already existing capillary
vessels.2 This physiological process in adolescent organisms is
also closely related to a multitude of pathological events in
humans including rheumatoid arthritis, psoriasis, ischemia, and
malignant diseases.3 Once tumors have reached a size of about
3 mm3, their growth is completely dependent on this process.4
In general, tumors with a high expression of angiogenic factors
(e.g., VEGF, basic fibroblast growth factor (bFGF), matrix
metalloproteinases, and serine proteases) and low levels of
inhibitors of angiogenesis (e.g., thrombospondin and inhibitors
of metalloproteinases) have a higher microvessel density and
aggressiveness. Also they are locally more advanced and
metastasize more frequently than tumors without these
angiogenic phenotypes. Because of the limited occurrence of
physiological angiogenesis in adults, its inhibition may lead to
tumor-specific compound capabilities with a minor toxicity
potential.5 Therefore, angiogenesis inhibitors are considered to
be potential therapeutic agents with low toxicity in the
treatment of solid tumors such as bronchial carcinoma,
In a previous article8 we described profile and molecular
modeling studies of a highly selective VEGFR-2/3 inhibitor,
which illustrate a possible binding mode of indolyl-maleimides
into the ATP pocket of VEGFR-2. By implementing electro-
negative heteroatoms at different positions in the indolyl
moiety, lowering the π-electron density, providing a new H-
bond acceptor, and increasing π−π-interaction (due to different
inhibitor alignment) of the azaindolyl moiety with Phe1045 in
the ATP-binding site of VEGFR-2, we aim to enhance the
stability of the receptor−inhibitor complex. To get additional
SAR data, the aryl moiety of the previously described 7-
azaindolyl compound8 was slightly varied, creating mono- and
dimethoxyphenyl derivatives.
Received: June 4, 2012
Published: October 22, 2012
© 2012 American Chemical Society
9531
dx.doi.org/10.1021/jm301217c | J. Med. Chem. 2012, 55, 9531−9540

Journal of Medicinal Chemistry
Article
Scheme 1. Synthesis of 4-, 5- and 6-Azaindoles9−11
a
Scheme 2. Acylation and Protection of Azaindole Moieties Using the Methods of Zhang et al.,9a Ottoni et al.,12 and Basel et
al.15
a
Reagents and conditions: (i) (a) azaindole, AlCl3, DCM, 0 °C, 0.5 h; (b) ethyl chlorooxoacetate, rt, 18 h; (c) ice, DCM; (ii) di-tert-butyl
dicarbonate, DMAP, DCM, rt, 12 h. (∗) 9d: 7-azaindole (commercially available).
These novel azaindolyl-arylmaleimides show remarkable
inhibition of angiogenesis as well as potent inhibitory efficacy
against different tyrosine kinases, related to proliferation,
survival, cell migration, and angiogenesis in malignant cells.
Inhibition of VEGFR-2 and FLT-3 was detected in nanomolar
ranges. Further investigations disclosed a likewise inhibitory
potency against GSK-3β. In addition, the compounds exhibit
potent antiproliferative and proapoptotic properties in different
carcinoma cell lines, such as HT-29, Mkn-45, and Molm-14.
Because of these results, we focus on selective VEGFR-2, FLT-
3, and GSK-3β inhibitors that may be important in the
treatment of solid tumors and acute myeloid leukemia (AML),
respectively.
RESULTS AND DISCUSSION
Synthesis of 3-(Azaindolyl)-4-arylmaleimides. 4- and 6-
■
Azaindoles (9b, 9c) were obtained by reacting 2-chloro-3-
nitropyridine (4) with dimethyl malonate, 6 M HCl, N,N-
dimethylformamide dimethyl acetal (DMFDMA), followed by
catalytic hydrogenation to yield 4-azaindole (9b) and 6-
azaindole (9c), respectively, using vinylmagnesium bromide
and catalytic hydrogenation.9 In the case of 4-azaindole,
procedures by Cash et al.10 were applied. 5-Azaindole (9a)
was prepared according to Dormoy et al.11 by nitration of 3-
picoline-1-oxide (1) followed by treatment with N,N-
dimethylformamide diethyl acetal (DMFDEA) and reduction
with palladium/C, H2 (Scheme 1). 7-Azaindole (9d) is
commercially available by Sigma Aldrich. The required
9532
dx.doi.org/10.1021/jm301217c | J. Med. Chem. 2012, 55, 9531−9540

Journal of Medicinal Chemistry
Article
a
Scheme 3. Synthesis of the Maleimide Moiety According to Faul et al.13
a
Reagents and conditions: (i) (a) potassium tert-butoxide (4 equiv), THF, rt, 14 h; (b) NH4Cl, ethyl acetate.
azaindole glyoxyl esters (10d, 11a−c) proved to be difficult to
prepare because of the decreased π-electron density of
azaindoles (9a−d) in comparison to indole and the therefore
minor reactivity toward electrophilic reagents. To acylate at the
C-3 position (Scheme 2), we used a modified procedure of
Zhang et al.9a and Ottoni et al.12 with excess of aluminum
chloride (AlCl3) as Lewis acid in dry dichloromethane (DCM)
to increase the electrophilicity. Among the several synthetic
methods for preparing maleimides, we modified a method of
Faul et al.13 This procedure results in good yields and appeared
to be efficient concerning the tolerance of functional groups.
Scheme 3 shows the maleimide formation, carried out in a one-
pot reaction with azaindole glyoxyl esters (10d, 11a−c) and
arylacetamide derivatives (12a−c) in the presence of potassium
tert-butoxide (tBuOK) and activated molecular sieves in dry
THF.14 Arylacetamides 12a−c are readily available from the
corresponding acetic acids, which are converted to the acid
chloride by reacting with phosphorus pentachloride and
hydrolyzed with ammonia.
strategy in tumor treatment have strongly influenced recent
developments of novel drugs in this field.16
In previous papers we reported the synthesis and biological
evaluation of 3,4-diarylmaleimides as angiogenesis and RTK
inhibitors.14a,8 Motivated by their significant activity, we started
to optimize these small molecule compounds with regard to
pharmacodynamic and pharmacokinetic demands as drug
candidates for the treatment of acute myeloid leukemia and
solid tumors. The prepared 3-azaindolyl-4-arylmaleimides
(Chart 1) differ from available kinase inhibitors because of
their tumor and vascular targeting properties, selective
inhibition of relevant kinases (VEGFR-2, FLT-3, GSK-3β),
and crucial effects on several cancer cell lines by promoting or
inducing apoptosis and/or decreasing cell viability.
In this section we describe the biological evaluation of the
synthesized 3-azaindolyl-4-phenylmaleimide derivatives con-
cerning their antiangiogenic activity, their inhibitory efficacy
on different protein kinases, and their effect on the viability of
vascular endothelial and various cell lines. In addition the
combination of maleimide compounds with the topoisomerase
I inhibitor irinotecan was investigated.
Biological Evaluation. Investigations concerning the
inhibition of receptor tyrosine kinases (RTKs) as an innovative
9533
dx.doi.org/10.1021/jm301217c | J. Med. Chem. 2012, 55, 9531−9540

Journal of Medicinal Chemistry
Article
To identify the molecular targets, inhibitory effects on
VEGFR-2, FLT-3, and GSK-3β were investigated using kinase
inhibition assays based on the previous results of Peifer et al.14a
(Table 2). VEGFRs are key mediators of angiogenesis and play
Chart 1. Synthesized 3-azaindolyl-4-arylmaleimide
derivatives (13-18) and reference compound 19.14a
a
Table 2. IC50 of VEGFR-2, FLT-3, and GSK-3β Inhibition
IC50 (nM)
compd
VEGFR-2
37
FLT-3
GSK-3β
≥10000
13
16
17
18
19
7
132 18
≥3000
≥1500
590 13
373 22
40
3
215 57
48
70
3
2
18
5
9
1
18
7
71
2
a
The kinase assays were performed by Millipore using Merck
Millipores KinaseProfiler. All compounds have been tested in
duplicate.
essential roles by initiating proliferation and migration of
endothelial cells, a requirement for tumor growth and
metastasis. VEGFR-2 is also strongly involved in the early
blood vessel growth in the avian embryo.18 GSK-3β is known to
modulate apoptotic cell signaling,19 whereas FLT-3 plays a
crucial role in cell proliferation and differentiation.20 The half-
maximal inhibitory concentration (IC50) values were deter-
mined (Table 2) and discussed in relation to their structural
features.
The 6- and 7-azaindole derivatives (18, 13) as well as the
indolyl derivative 19 significantly inhibited VEGFR-2 with IC50
values in a low nanomolar range. In comparison to 18 and 13,
respectively, IC50 values of 4- and 5-azaindolyl derivatives (17,
16) were about 10-fold higher. FLT-3 was most potently
inhibited by 6-azaindole derivative 18 (IC50= 18 nm), 7-
azaindole derivative 13 (IC50= 132 nm), and the reference
compound 19 (indolyl derivative, IC50 = 18 nm), whereas 16
and 17 showed only minor inhibition. Compounds 16, 17, 18,
and 19 could be identified as potent inhibitors of GSK-3β with
IC50 values in a low nanomolar range.
The antiangiogenic potency was evaluated using a
quantitative in vivo chick embryo assay17 (Table 1). The
azaindolyl compounds at 26 nM inhibit up to 61% the
microvessel formation after 24 h of incubation, thus confirming
significant antiangiogenic activity.
Table 1. Inhibition of Vessel Growth in the in Vivo Chick
Embryo Model by 3-Azaindolyl-4-arylmaleimides (c = 26
nM)
The correlation between IC50 values of VEGFR-2 inhibition
and the antiangiogenic effect in chick embryo assay is
a
compd
IVG (% inh)
13
14
15
16
17
18
61 24
25 21
30 20
40 18
10 28
32 20
82 14
Table 3. Kinase Screening of Selected Compounds 13 and
18
a
13
18
CDK1/cyclinB
CDK2/cyclinA
CDK6/cyclinD3
c-RAF
92 1.4
94 0.71
99 1.4
111 0.71
97 1.4
80 0.71
88 2.12
103 1.41
117 0.71
111 2.12
b
19
a
Inhibition of vessel growth versus control in the chick embryo model
after incubation for 24 h. Each experiment was performed with 15
samples. For further details see refs 14a and 17. Data from ref 14a.
EGFR
b
Flt-3
2
0.00
50 5.66
0.00
0
2
1
0.00
0.71
0.00
GSK-3β
VEGFR-2
Met
0
The compound bearing a 7-azaindole moiety and additional
3,4,5-trimethoxyphenyl substituent 13 is found to be the most
potent azaindole (61% inhibition of vessel growth). Related to
our previously reported 3-(indol-3-yl)-4-(3,4,5-
trimethoxyphenyl)maleimide 19 (82% inhibition of vessel
growth),14a regioisomere 6- and 5-azaindolyl derivatives as
well as monomethoxy- and dimethoxyphenyl derivatives (18,
16, 15, 14) revealed moderate inhibition (25−40% inhibition
of vessel growth). The 4-azaindole compound 17 showed no
significant inhibition in this screening.
109 8.49
84 1.41
89 0.71
93 2.1
83 2.83
106 8.49
80 3.54
83 2.12
75 2.83
99 2.12
116 5.66
PDGFR-β
PKC-α
PKC-γ
PKC-ε
77 2.8
PKC-ι
69 0.71
77 0.71
Tie2
a
Data are presented as residual activity in percent at 10 μM, related to
the 100% controls. All compounds have been tested in duplicate.
9534
dx.doi.org/10.1021/jm301217c | J. Med. Chem. 2012, 55, 9531−9540

Journal of Medicinal Chemistry
Article
a
Table 4. MTT Assay Using HT-29 Cells, Viability in %
concn (μM)
13
14
16
17
18
19
2.6
7.9
26
64.95 2.16
45.55 3.71
38.50 0.99
38.11 5.43
54.33 4.55
49.21 3.18
43.05 1.75
40.50 2.38
90.76 2.30
61.51 2.10
56.55 0.90
47.44 2.10
114.36 5.2
98.31 0.90
92.64 2.80
65.75 1.50
30.38 1.52
25.80 3.52
12.67 2.6
4.22 0.44
78.50 3.70
62.90 1.50
53.90 4.80
42.80 1.60
52
a
Samples were incubated for 3 days. Viability of untreated cells is 100%.
a
Table 5. MTT Assay Using Mkn-45 Cells, Viability in %
concn (μM)
13
14
16
18
19
0.5
113.22 6.16
110.01 4.04
86.84 5.57
58.66 5.21
57.09 4.96
44.68 0.83
117.94 4.32
118.13 5.21
60.35 3.97
57.01 0.41
62.12 5.55
59.42 2.91
99.07 5.98
100.26 6.39
99.04 5.41
98.61 6.02
89.41 6.78
82.52 1.53
99.17 2.22
94.50 0.40
98.60 5.70
93.20 1.00
81.10 6.90
75.90 2.70
65.90 0.90
1.0
106.80 5.45
104.94 4.91
107.57 7.78
100.67 5.04
68.58 1.50
5.0
10.0
20.0
50.0
a
Samples were incubated for 5 days. Viability of untreated cells is 100%.
a
remarkable for compounds 13 and 19. The expected
correlation for compound 18 could not be verified, which
might be due to its higher inhibitory potency against GSK-3β.
13 (IC50(VEGFR-2) = 37 nM) exhibited 61% inhibition of
vessel growth, whereas 18 (IC50(VEGFR-2) = 48 nM) showed
only low antiangiogenic effects (32%). In our previous study,
we reported an inhibition of 82% for 3-(indol-3-yl)-4-(3,4,5-
trimethoxyphenyl)maleimide (19) with IC50 of 70 nM
(VEGFR-2).14a
Table 6. MTT Assay Using Molm-14 Cells, Viability in %
concn (μM)
13
18
0.5
65.89 6.34
38.88 3.38
4.41 0.29
3.56 0.20
1.77 0.10
73.37 3.41
54.20 4.53
9.35 0.57
5.39 0.75
2.40 0.24
1.0
10.0
20.0
50.0
a
Samples were incubated for 3 days. Viability of untreated cells is
100%.
To determine the kinase selectivity profile of two selected
compounds, 15 kinases were chosen for screening. The kinase
panel consists of members of the CDK family (CDK1/cyclinB,
CDK2/cyclinA, CDK6/cyclinD3), based on previous molecular
modeling data,14a and several isozymes of the PKC family
(PKC-α,-γ, -ε, -ι) reported to be affected by staurosporine
related diarylmaleimides.21 Eight kinases bearing cysteine
residues in the ATP binding pocket like the addressed FLT-
3, GSK-3β, and VEGFR-2 were also included in the panel to
verify selectivity (Met, PDGFR, EGFR, c-RAF, Tie2). Residual
activity was measured at 10 μM for compounds 13 and 18, as
they both exhibit potent inhibition of VEGFR-2 but differ in
inhibiting GSK-3β and angiogenesis assay as mentioned above.
The results of the kinase screening, summarized as residual
activity at 10 μM in Table 3, confirmed the selectivity of
compound 13 toward FLT-3 and VEGFR-2 and 18 against
FLT-3, GSK-3β, and VEGFR-2, respectively. With residual
activity between 75% (PKC-ε) and 117% (c-RAF), compound
18 shows no inhibitory efficacy against other tested kinases of
the panel. With regard to a weak inhibition of GSK-3β (50%
residual activity) maleimide 13 selectively inhibits FLT-3 and
VEGFR-2, whereas the selectivity of azaindole 18 comprises
FLT-3, GSK-3β, and VEGFR-2.
Because of these results, the antiproliferative activity of new
compounds was evaluated by using a MTT assay in various cell
lines. The viability was determined 3 days (HT-29, Molm-14)
and 5 days (Mkn-45) after incubation with compounds. As
outlined in Table 4, the viability of human colon
adenocarcinoma cells (HT-29) decreases dose-dependently
for compounds 18 and 19 while applying concentrations in the
low micromolar range. Whereas 18 offers potent activity, the
previously reported compound 19 shows minor efficacy against
HT-29 cells. For maleimide 14 no dose-dependency could be
observed in the MTT assay using HT-29 cells, indicating
possible cytotoxic effects.
The human gastric carcinoma cell line Mkn-45 (Table 5)
offers marginal activity in the MTT assay and no dose-
dependency for all tested compounds. Viability was measured 5
days after treatment of compounds, as no effects could be seen
after 3 days. The herein presented data suggest a generally
higher response of synthesized maleimides against HT-29 cells
than Mkn-45 cells.
Taking the kinase inhibition and selectivity profile of the
compounds into account (see Tables 2 and 3) we subsequently
measured potential inhibitory effects of compounds 13 and 18
using the leukemic cell line Molm-14, which carries an
activating FLT3-ITD (internal tandem duplication) mutation.22
Both inhibitors were capable of remarkably reducing cell
proliferation at low micromolar concentration (Table 6).
In summary, the highest activity for compounds 13 and 18
was found in Molm-14 cells followed by the decrease of
viability using HT-29 cells.
To determine proapoptotic effects, we investigated human
umbilical vein endothelial cells (HUVECs) as well as gastric
cancer cells Mkn-45 with Nicoletti FACS analysis. As illustrated
in Table 7, a substantial, dose-dependent proapoptotic activity
in HUVECs was observed, indicated by a significant increase of
the sub-G1 cell fraction. Among the tested maleimides this
effect was most pronounced for indolyl derivative 19 (87.7%
sub-G1 fraction, 2.6 μM) and 6-azaindolyl derivative 18
(66.3%, sub-G1 fraction, 2.6 μM), whereas 7-azaindole (13)
and 5-azaindole (16) derivatives showed only moderate
induction of apoptosis. For compound 19 these results are in
agreement with the IC50 values for the VEGFR-2 inhibition
(Table 2, 19, 70 nM) and the antiangiogenic effect (chick
9535
dx.doi.org/10.1021/jm301217c | J. Med. Chem. 2012, 55, 9531−9540

Journal of Medicinal Chemistry
Article
a
Table 7. Induction of Apoptosis in HUVEC
sub-G1 fraction (%)
concn (μM)
13
16
17
18
19
0.79
2.6
26.7 1.9
49.2 2.2
31.3 1.7
46.3 3.2
14.2 2.4
16.3 1.3
48.0 2.3
66.3 1.0
25.7 0.9
87.7 2.3
a
Samples were incubated for 4 days. Sub-G1 fraction of untreated cells: 15.4 1.5%.
a
Table 8. Induction of Apoptosis in Mkn-45 Cells
sub-G1 fraction (%)
concn (μM)
13
14
16
18
19
1.0
9.3 0.8
12.7 0.9
24.8 0.6
31.8 3.2
45.8 5.6
55.7 4.2
8.8 2.4
12.8 0.3
15.8 2.5
16.5 0.6
22.9 4.1
28.3 2.7
14.2 2.4
20.3 0.7
29.8 3.5
45.9 0.9
55.3 2.6
5.0
16.3 0.6
37.8 2.4
50.7 1.6
65.9 2.7
15.7 3.6
21.5 1.2
25.7 3.3
31.6 2.1
10.0
15.0
20.0
a
Samples were incubated for 7 days. Sub-G1 fraction of untreated cells: 10.9% 3.2%.
Table 9. Enhancement of Apoptosis in HT-29 Human Colon Adenocarcinoma Cells by Combining Compounds and
a
Irinotecan
sub-G1 fraction (%)
concn
7.9 μM test compound
7.9 μM test compound plus 1.18 μM irinotecan
13
17
18
19
2.32 1.16
4.22 0.74
79.9 4.13
4.7 0.5
22.36 2.80
25.71 1.97
75.69 1.77
53.1 3.3
a
Samples were incubated for 7 days. Induction of apoptosis at 1.18 μM irinotecan single application: 18.3 1.1%. Induction of apoptosis by drug-
free control: 3.4 0.9%.
embryo assay, Table 1, 82% IVG). The expected correlation for
compounds 13 and 18 could not be verified, which might be
due to its high inhibitory potency against FLT-3 and GSK-3β.
Hence, various mechanisms may be responsible for the
induction of apoptosis.
Table 8 indicates a significant dose-dependent induction of
apoptosis in Mkn-45 cells for azaindoles 13, 14, and the
previously reported indolyl derivative 19. In comparison to
VEGF dependent HUVECs, a lower increase of sub-G1 fraction
was observed for all tested compounds in the gastric cell line
Mkn-45, e.g., 13 (2.6 μM, 49.2% sub-G1 fraction in HUVEC
versus 5.0 μM, 16.3% sub-G1 fraction in Mkn-45). The findings
of the antiproliferative and proapoptotic assays using different
cell lines support the hypothesis that the potent effects of
maleimides are mediated through different mechanisms of
action.
We further investigated the efficacy of the synthesized
maleimides and the topoisomerase I inhibitor irinotecan alone
and in combination to induce apoptosis using human HT-29
cells. As illustrated in Table 9, indolyl derivative 19
demonstrated a synergistic effect on induction of apoptotic
cell death upon combination with irinotecan. Contrary to the 4-
azaindole derivative (17) and 7-azaindole (13), both of which
slightly increase the potency of irinotecan (1.18 μM, 18.3
1%), the 6-azaindole (18) did not increase the efficacy of
irinotecan.
arylmaleimides in an in vivo chick embryo assay and kinase
assays. The 7-azaindolyl derivate 13 demonstrated a strong
inhibition of vessel growth. 18 (6-azaindole) and 13 (7-
azaindole) are highly potent inhibitors of VEGFR-2 kinase with
IC50 in the low nanomolar range, similar to the previously
reported indolyl derivative 19. Furthermore maleimide 18 was
identified as potent GSK-3β inhibitor. Kinase selectivity of
compounds 13 and 18 was confirmed representatively by
screening 15 chosen kinases of different classes.
The 6-azaindole 18 is highly effective in leukemic cells and
endothelial cells, as well as against HT-29 cells in monotherapy.
Regarding proapoptotic efficacy in combination with topo-
isomerase I inhibitor irinotecan, no synergistic increase was
detected because of its high efficacy at 7.9 μM in monotherapy.
7-Azaindole 13 shows minor potency against endothelial cells
in comparison to the above-mentioned 6-azaindole 18. In
addition, they offer no dose dependency in cell viability assays
using colon and gastric tumor cells lines. The indolyl derivative
19 in combination with irinotecan possesses a synergistic effect
on apoptosis induction using human endothelial cells.
5-Azaindole derivative 16 and 4-azaindole derivate 17
indicate moderate to weak activity in kinase and cellular assays.
On the basis of the high potency of the presented maleimides
both in vitro and in vivo, prospective studies are on the way to
improve the pharmacological profile leading to novel drug
candidates.
These results demonstrate a possible benefit of combining
synthesized maleimides and topoisomerase I inhibitors for
cancer treatment.
EXPERIMENTAL SECTION
■
Chick Embryo Assay as in Vivo Angiogenesis Model. The
assay was performed according to Peifer et al.17 Test compounds were
dissolved in DMSO and used as 26 mM stock solution. For the
preparation of agarose pellets, the stock solution was mixed with 2%
agarose solution at 80 °C and 10 μL pellets were produced (final
concentration, 26 nM). Fertilized chicken eggs (white leghorn) were
CONCLUSION
■
This study evaluated the antiangiogenic and kinase inhibition
efficacy as well as the kinase selectivity profile of 3-azaindolyl-4-
9536
dx.doi.org/10.1021/jm301217c | J. Med. Chem. 2012, 55, 9531−9540

Journal of Medicinal Chemistry
Article
incubated at 37.5 °C with 62% relative humidity in an automatically
turning egg incubator (Ehret). After 70 h of incubation time, 8 mL of
albumin was removed and a window was opened in the shell to
uncover the developing germination layer. The window was sealed
with transparent tape. The eggs were returned to the incubator and
stored at 90% relative humidity without turning. After 75 h of
incubation the angiogenic experiment was initiated (start time, 0 h)
and 15 agarose pellets were placed on the area vasculosa preparations.
After 24 h, images of the control and sample-treated areas were taken
and digitized via Leica QWin software. The angiogenic response was
measured using the automatic image analysis system programmed in a
routine. The digitized images were analyzed for total, major (capillaries
with a diameter of more than 50 μm), and minor blood vessel areas
using the customized image analysis software Leica QWin (Leica
Microsystems, Bensheim, Germany). Calculated data were sent
automatically via dynamic data exchange to an Excel table and
processed statistically. An antiangiogenic response due to agent-treated
samples was considered positive if the mean of minor blood vessel area
showed >20% inhibition compared to control samples. Differences
between treated and control samples were evaluated using Student's t
test.
RPMI 1640 supplemented with 10% FCS and L-glutamine. All cells
were maintained at 37 °C in an incubator under an atmosphere
containing 5% CO2. Each experiment was performed with
exponentially growing cells.
Chemistry. Infrared spectra were recorded on a Thermo Nicolet
Avatar 330 FT-IR spectrometer. 1H (300 MHz, digital resolution
0.3768 Hz) and 13C (75 MHz, digital resolution 1.1299 Hz) NMR
spectra were recorded on a Bruker AC 300. The data are reported as
follows: chemical shift in ppm from Me4Si as external standard,
multiplicity, and coupling constant (Hz). EI mass spectra were
recorded on a Varian MAT 44S (80 eV) and FD mass spectra on a
Finnigan MAT 7 (5 kV). For clarity only the highest measured signal
is given for FD mass spectra. Elemental analyses were performed on a
Haereus CHN rapid analyzer, Carlo Erba Strumentazione 1106.
Combustion analysis results agreed with the calculated data within
0.4% unless otherwise stated. Melting points/decomposition
temperatures were determined on a Buchi apparatus according to
̈
Dr. Tottoli and are uncorrected. Where appropriate, column
chromatography was performed for crude precursors with Merck
silica gel 60 (0.063−0.200 mm). Column chromatography for test
compounds was performed using a MPLC system B-680 (Buchi) with
̈
MTT Assay. The test compounds were determined for their
antiproliferative activity by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) assay as described by Mosmann et
al.23 Cells in the exponential growth phase were transferred to 96-well
flat-bottom plates. Then 10 000 viable cells resuspended in 200 μL of
RPMI medium were plated into each well and incubated overnight.
Cells were then exposed to various concentrations of test compounds
(100 μL/well) for 3 or 5 days at 37 °C with 5% CO2. Subsequently, 10
μL/well MTT stock solution (5 mg/mL; Biomol, Germany) was
added and the cells were incubated at 37 °C with 5% CO2 for 4 h. An
amount of 100 μL solubilization solution (10% SDS in 0.01 M HCl)
was added, and the cells were incubated at 37 °C with 5% CO2
overnight. Plates were read on an ELISA reader ELX 800 (Bio-Tek
KC4 software) at 562 nm absorbance. Each experiment was done in
triplicate.
Nicoletti Cell Cycle Analysis. HUVEC or cancer cells (HT-29)
were transferred to 12-well flat-bottomed plates. Then 1.5 × 105 viable
cells contained in 1 mL of cell suspension were plated into each well,
incubated overnight before exposure to various concentrations of
drugs. Subsequently, cells were incubated in 1 mL of medium
containing various concentrations of test compounds at 37 °C with 5%
CO2. After incubation the cells were washed with PBS, trypsinized,
pelleted, mixed with PI buffer (containing 0.1% sodium citrate, 0.1%
Triton X-100, 50 mg/mL propidium iodide (PI)), and incubated for 1
h at 4 °C. Cell cycle sub-G1 fraction analysis was performed as
described previously (Nicoletti et al.)24 using a flow cytometer (BD
FACS Calibur, BD Biosciences, Heidelberg, Germany). Each experi-
ment was done in triplicate. The same analysis was performed with
HUVEC cells. Then 1.5 × 104 cells were plated into each well and
treated with test compounds for 7 days.
Merck silica gel (0.015−0.040 mm). The progress of the reactions was
monitored by thin layer chromatography (TLC) performed with
Merck silica gel 60 F-254 plates. Where necessary, reactions were
carried out in a nitrogen atmosphere using 4 Å molecular sieves. All
reagents and solvents were obtained from commercial sources and
used as received. On the basis of NMR and combustion analysis data,
all final compounds reported are ≥95% pure.
General Procedure A for the C-3 Acylation of Azaindoles
(Modified from Zhang et al.).9a Azaindole (1 equiv) was added to a
mixture of aluminum chloride (5 equiv) in 100 mL of dry
dichloromethane at 0 °C under argon atmosphere. After 30 min at
0 °C, the mixture was warmed to room temperature and ethyl
chlorooxoacetate (5 equiv) was added dropwise. The reaction mixture
was stirred vigorously overnight, and then carefully ice was added.
After addition of dichloromethane the organic layer was separated,
washed carefully with cold NaHCO3 solution, dried over MgSO4,
filtered, and concentrated.
General Procedure B for Protection of Azaindoles (Modified
from Basel et al.).15 Di-tert-butyl dicarbonate (1.5 equiv) and DMAP
were added to a stirred suspension of acylated azaindole (1 equiv).
After being stirred for 12 h, the mixture was concentrated in vacuo and
purified by column chromatography (petroleum ether/ethyl acetate,
1/1).
General Procedure C for the Preparation of Phenyl-
acetamide Derivates (12a−c). To the phenylacetic acid derivative
(1 equiv) was added phosphorus pentachloride (5 equiv), and the
mixture was stirred for 2 h in dichloromethane at room temperature.
In a second step ammonia (25%) was added via septum and the
mixture was stirred for another 2 h, then given into ice and extracted
with dichloromethane (4 times). The combined organic layers were
dried (MgSO4) and concentrated in vacuo. The crude product was
recrystallized from ethanol.
General Procedure D for the Preparation of 3-Azaindolyl-4-
arylmaleimides (Modified from Peifer et al.).14a Potassium tert-
butoxide (4 equiv) in dry THF was added via septum to a stirred
solution of phenylacetamide derivative and acylated azaindole
derivative in dry THF, containing 2−3 g of molecular sieves (4 Å).
After the mixture was stirred overnight, the reaction was quenched
with saturated NH4Cl-solution. The residue was filtered, extracted
with ethyl acetate, and the combined organic layers were dried over
MgSO4, concentrated, and purified by column chromatography (ethyl
acetate/ethanol, 9/1).
3-Methyl-4-nitropyridine-l-oxide (2) (Modified from Dormoy
et al.).11 To 3-picoline-1-oxide 1 (10 g, 91.6 mmol) was slowly added
a solution of HNO3 (27.5 mL) and H2SO4 (35 mL) via syringe at 25
°C. The mixture was heated to 100−105 °C for 3 h, cooled to room
temperature, followed by slow addition of ice and a pH adjustment to
2−3 with sodium carbonate. The mixture was filtered and washed with
chloroform. The organic layers were dried and concentrated in vacuo
GSK-3β, VEGFR-2, FLT-3 Kinase Assays and Kinase Screen-
ing. IC50 values and residual activity determination were carried out at
Millipore UK Ltd. (Gemini Crescent, Dundee Technology Park,
Dundee DD2 1SW, U.K. (IC50 Profiler)).25 Details of each assay
protocol can be found on Millipore’s Web site at www.millipore.com/
drugdiscovery/dd3/assayprotocols. Other kinase assay data are taken
from Peifer et al.8
Cell Lines. The human colon cancer cell line HT-29 was obtained
from the German Resource Centre for Biological Material (DSMZ,
Braunschweig, Germany). The cells were routinely cultured in RPMI
1640 (Invitrogen, Karlsruhe, Germany) and supplemented with 10%
FCS (Colbe, Germany), 100 units/mL penicillin, and 100 μg/mL
̈
streptomycin (1%, Cambrex, Germany).
Human gastric cancer cell line Mkn-45 was obtained from the
DSMZ and from the European Collection of Cell Cultures (ECACC,
Salisbury, U.K.) and cultured in RPMI 1640 medium supplemented
with 20% FCS and 1% penicillin/streptomycin.
The human leukemia cell line Molm-14 was kindly provided by
Scott Armstrong (Boston Children's Hospital, U.S.) and cultured in
9537
dx.doi.org/10.1021/jm301217c | J. Med. Chem. 2012, 55, 9531−9540

Journal of Medicinal Chemistry
Article
to give a crude orange solid which was washed with acetone to give the
desired compound as yellow crystals (48.2 mmol, 52%). For further
data see ref 11.
3-Dimethylaminovinylene-4-nitropyridine-l-oxide (3) (Modi-
fied from Dormoy et al.).11 A solution of 3-methyl-4-nitropyridine-
l-oxide 2 (2.64 g, 17.1 mmol) and DMFDMA (3.82 g, 26 mmol) in
DMF was heated to 100 °C for 1.5 h. The mixture was concentrated in
vacuo, and the crude residue was washed with methanol. The title
compound was obtained as dark purple crystals (16.76 mmol, 98%).
For further data see ref 11.
Dimethyl 2-(3-Nitropyridin-2-yl)malonate (5) (Modified
from Cash et al.).10 2-Chloro-3-nitropyridine 4 (2 g, 12.5 mmol)
was added to a stirred suspension of sodium hydride (0.5 g, 12.5
mmol, mixture of 60% NaH in mineral oil) in 20 mL of dry DMF
under nitrogen. Dimethyl malonate (1.43 mL, 1.65 g, 12.5 mmol) was
added cautiously dropwise. After the mixture was stirred for 5 h at
room temperature, the solution was diluted with water. After addition
of diethyl ether, the mixture was washed four times with brine to
remove DMF. The organic phase was dried over Na2SO4, filtered,
concentrated, and purified by column chromatography (petroleum
ether/ethyl acetate, 2/1). The title compound was obtained as a pale
brown oil (5.5 mmol, 44%). For further data see ref 26.
6-Azaindole (9c) (Modified from Mahadevan et al.).9b An
amount of 0.1 g of Pd/charcoal (10% Pd) was placed in a
miniautoclave with 20 mL of alkaline ethanol. A solution of 7-
chloro-6-azaindole 8 (0.5 g, 3.3 mmol) in 80 mL of alkaline ethanol
was added. The miniautoclave was put under H2 pressure (1 bar), and
the mixture was stirred for about 8 h at room temperature. The slurry
was filtered through Celite, washed with alkaline ethanol, and
concentrated in vacuo. The crude residue was absorbed with 40 mL
of water, extracted three times with dichloromethane, dried over
Na2SO4, and concentrated in vacuo. Purification was achieved by
recrystallization (cyclohexane/dichloromethane, 1/1) to obtain light
brown crystals (2.74 mmol, 83%). Mp 131−132 °C. IR ν [cm−1] =
3060 ν(aromat CH); 1622; 1495; 1458. FD-MS: m/z (rel intens) =
118.2 (74.69%; M+•). For further data see ref 9b.
Ethyl 2-(5-Azaindol-3-yl)-2-oxoacetate (10a). General proce-
dure A was followed, using 5-azaindole 9a (0.5 g, 4.23 mmol) to obtain
the title compound as a pale yellow powder (1.9 mmol, 45%). For
further data see ref 14b.
Ethyl 2-(4-Azaindol-3-yl)-2-oxoacetate (10b). General proce-
dure A was followed, using 4-azaindole 9b (0.38 g, 4.60 mmol) to
obtain a yellow oily residue which solidified to yellow crystals. After a
washing with cold petroleum ether the title compound was obtained
(2.30 mmol, 50%) as a light yellow powder. For further data see ref 28.
Ethyl 2-(6-Azaindol-3-yl)-2-oxoacetate (10c). General proce-
dure A was followed, using 6-azaindole 9c (0.25 g, 2.12 mmol) to
obtain a yellow oily residue which solidified to a greenish-yellow
powder. After a washing with cold diethyl ether the title compound
was obtained (0.89 mmol, 42%) as light yellow powder. For further
data see ref 14b.
Ethyl 2-( 7-Azaindol-3-yl)-2-oxoacetate (10d). General proce-
dure A was followed, using 7-azaindole 9d (1.20 g, 10.16 mmol) to
obtain a yellow oily residue which solidified to a greenish-yellow
powder. After a washing with cold diethyl ether the title compound
was obtained (5.13 mmol, 50.5%) as a light yellow powder. For further
data see ref 14b.
Ethyl 2-[1-(tert-Butoxycarbonyl)-5-azaindol-3-yl]-2-oxoace-
tate (11a). General procedure B was followed, using 10a (0.3 g, 1.4
mmol) to yield the title compound as colorless crystals (1.33 mmol,
95%). For further data see ref 14b.
Ethyl 2-[1-(tert-Butoxycarbonyl)-4-azaindol-3-yl]-2-oxoace-
tate (11b). General procedure B was followed, using 10b (0.48 g,
2.20 mmol) to obtain light yellow crystals (1.9 mmol, 86%). For
further data see ref 28.
Ethyl 2-[1-(tert-Butoxycarbonyl)-6-azaindol-3-yl]-2-oxoace-
tate (11c). General procedure B was followed, using 10c (0.55 g,
2.5 mmol) to yield the title compound as colorless crystals (2.25
mmol, 90%). Mp 152 °C. For further data see ref 14b.
3-(7-Azaindol-3-yl)-4-(3,4,5-trimethoxyphenyl)maleimide
(13). General procedure D was followed using 10d (1.03 g, 4.70
mmol) and 3,4,5-trimethoxyphenylacetamide 12a (0.67 g, 2.98 mmol).
Purification was achieved by column chromatography (petroleum
ether/ethyl acetate/isopropanol, 7/2/1) to yield the title compound
(1.26 mmol, 42.6%) as yellow crystals. Anal. (C20H17N3O5·H2O),
calcd C 61.85, H 4.67, N 10.82; found C 61.76, H 4.29, N 11.12. For
further data see ref 14b.
2-Methyl-3-nitropyridine (6) (Modified from Cash et al.).10
Dimethyl 2-(3-nitropyridin-2-yl)malonate 5 (1.4 g, 5.5 mmol) was
dissolved in 70 mL of 6 M HCl and refluxed for 8 h. After
neutralization with saturated Na2CO3 solution, the aqueous portion
was extracted three times with dichloromethane. The combined
organic layers were dried over Na2SO4, filtered, concentrated, and
purified by bulb tube distillation (0.35 mbar, 70−80 °C). The title
compound was obtained as a pale yellow oil (5.1 mmol, 92%). For
further data see ref 26.
N,N-Dimethyl-2-(3-nitropyridin-2-yl)ethenamine (7) (Ac-
cording to Cash et al.).10 2-Methyl-3-nitropyridine 6 (0.7 g, 5.1
mmol) was dissolved in 15 mL of dry DMF and stirred under nitrogen.
N,N-Dimethylformamide dimethyl acetal (1.35 mL, 1.22 g, 10.2
mmol) was added dropwise. The mixture was heated to 90 °C for 4 h.
After approximately 15 min a deep reddish color appeared. After
evaporation of the solvent, the title compound was obtained as a red
oil (0.8 mmol, 73%) which can be used without further purification.
For further data see ref 26.
7-Chloro-6-azaindole (8) (According to Zhang et al.).27 2-
Chloro-3-nitropyridine 4 (5.0 g, 31 mmol) was dissolved in 200 mL of
dry THF under nitrogen atmosphere and cooled to −78 °C. After
dropwise addition of 100 mL of 1 M vinylmagnesium bromide
solution the mixture was stirred for 8 h, quenched with 20% NH4Cl
solution, extracted with ethyl acetate, dried over Na2SO4, and
concentrated in vacuo. Crude product was purified by column
chromatography (isopropyl ether/ethyl acetate, 1/1) to obtain a
brown solid (10.23 mmol, 33%). Mp 188−190 °C. IR ν [cm−1] =
3077 ν(NH); 1618; 1548; 1483. For further data see ref 27.
5-Azaindole (9a) (Modified from Dormoy et al.).11 To a
solution of 3-dimethylaminovinylene-4-nitropyridine-l-oxide 3 (1 g,
4.78 mmol) in dry ethanol was added 10% Pd/charcoal (0.93 g, 26
mmol) under argon atmosphere. Then the mixture was put in a
miniautoclave under H2 pressure (2−3 bar) and stirred for 8 h at 65
°C and overnight at room temperature. The slurry was filtered,
concentrated, and further purified by column chromatography
(ethanol) to obtain yellow crystals (3.56 mmol, 74.5%). Mp 112 °C.
IR ν [cm−1] = 3657 ν(NH); 2978; 2901; 1618; 1462. For further data
see ref 11.
3-(7-Azaindol-3-yl)-4-(3,5-dimethoxyphenyl)maleimide (14).
General procedure D was followed using 10d (1.5 g, 6.84 mmol) and
12b (1.54 g, 6.84 mmol) to yield the title compound as yellow crystals
1
(3.28 mmol, 48%). Mp >270 °C. H NMR (300 MHz, DMSO) δ
[ppm] = 12.35 (s, 1H), 11.13 (s, 1H), 8.18 (s, 1H), 8.05 (s, 1H), 6.84
(s, 1H), 6.75 (s, 1H), 6.53 (s, 3H), 3.53 (s, 6H). Anal.
(C19H15N3O4·0.5H2O), calcd C 63.68, H 4.50, N 11.73; found C
63.94, H 4.16, N 11.86.
4-Azaindole (9b) (According to Cash et al.).10 An amount of
0.2 g of 10% Pd/charcoal was flashed with nitrogen before 10 mL of a
mixture of 8.8% formic acid in methanol was added cautiously. 7 was
also dissolved in 10 mL of a mixture of 8.8% formic acid in methanol
before it was added to the reaction mixture. The mixture was stirred
for 4 h until the red color completely disappeared. The Pd catalyst was
removed by filtration through Celite, and the filtrate was concentrated.
The title compound crystallized overnight (1.8 mmol, 71%). For
further data see ref 28.
3-(7-Azaindol-3-yl)-4-(2-Methoxyphenyl)maleimide (15).
General procedure D was followed using 10d (0.5 g, 2.3 mmol) and
12c (0.51 g, 2.5 mmol). Purification with chromatography on silica gel
(petroleum ether/ethyl acetate, 1/4) gave the title compound as a
1
yellow solid (1.19 mmol, 52%). Mp >280 °C. H NMR (DMSO-d6),
300 MHz, δ [ppm] = 3.36 (s, 3 H); 6.64−6.80 (m, 2 H); 6.96−7.08
(m, 2 H); 7.27 (d, J = 8.82 Hz, 1 H); 7.38−7.48 (m, 1 H); 8.01 (s, 1
9538
dx.doi.org/10.1021/jm301217c | J. Med. Chem. 2012, 55, 9531−9540

Journal of Medicinal Chemistry
Article
H); 8.15 (d, J = 2.62 Hz, 2 H); 11.07 (s, 1 H); 12.39 (s, 1 H). IR v
[cm−1] = 3154 ν(NH); 1744 ν(CO); 1691 ν(CO). FD-MS (m/z) =
319.4 (M+•+1). Anal. (C18H13N3O3·0.5H2O), calcd C 66.76, H 4.20, N
12.98; found C 66.99, H 4.06, N 12.86
(3) Folkman, J. Angiogenesis in cancer, vascular, rheumatoid and
other disease. Nat. Med. 1995, 1 (1), 27−31.
̃
(4) Carmeliet, P.; Jain, R. K. Angiogenesis in cancer and other
diseases. Nature 2000, 407 (6801), 249−257.
3-(5-Azaindol-3-yl)-4-(3,4,5-trimethoxyphenyl)maleimide
(16). General procedure D was followed using 12a (0.32 g, 1.4 mmol)
and 11a (0.3 g, 1.4 mmol) to obtain the title compound as yellow
(5) Fan, T.-P. D.; Brem, S. Angiosuppression. In The Search for New
Anti-Cancer Drugs; Kluwer Academic Publishers: Lancaster, U.K.,
1992; Vol. 3, p 118−229.
crystals (0.5 mmol, 36%). IR v [cm−1] = 3281 ν(NH); 3060; 2993;
̃
(6) Heidel, F.; Mack, T.; Razumovskaya, E.; Blum, M.-C.; Lipka, D.;
1765, 1698. Anal. (C20H17N3O5·H2O), calcd C 60.45, H 4.82, N 10.57;
found C 60.23, H 4.45, N 10.38. For further data see ref 14b.
3-(4-Azaindol-3-yl)-4-(3,4,5-trimethoxyphenyl)maleimide
(17). General procedure D was followed, using 11b (0.54 g, 1.7 mmol)
and 12a (0.38 g, 1.7 mmol) to obtain yellow crystals (0.45 mmol,
26%). Anal. (C20H17N3O5·H2O), calcd C 60.45, H 4.82, N 10.57;
found C 60.12, H 4.53, N 10.42. For further data see ref 28.
3-(6-Azaindol-3-yl)-4-(3,4,5-trimethoxyphenyl)maleimide
(18). General procedure D was followed using 11c (0.54 g, 1.7 mmol)
and 12a (0.38 g, 1.7 mmol). Purification was achieved by column
chromatography (petroleum ether/ethyl acetate/isopropanol, 5/2/3).
The obtained oil was recrystallized from chloroform, forming yellow
crystals (0.5 mmol, 33%). Anal. (C20H17N3O5·H2O), calcd C 60.45, H
4.82, N 10.57; found C 60.13, H 4.56, N 10.24. For further data see ref
14b.
Ballaschk, A.; Kramb, J.-P.; Plutizki, S.; Ronnstrand, L.; Dannhardt, G.;
̈
Fischer, T. 3,4-Diarylmaleimidesa novel class of kinase inhibitors
effectively induce apoptosis in FLT3-ITD-dependent cells. Ann.
Hematol. 2012, 91 (3), 331−344.
(7) (a) Cunningham, D.; Humblet, Y.; Siena, S.; Khayat, D.; Bleiberg,
H.; Santoro, A.; Bets, D.; Mueser, M.; Harstrick, A.; Verslype, C.;
Chau, I.; Van Cutsem, E. Cetuximab monotherapy and cetuximab plus
irinotecan in irinotecan-refractory metastatic colorectal cancer. N. Engl.
J. Med. 2004, 351 (4), 337−345. (b) Hurwitz, H.; Fehrenbacher, L.;
Novotny, W.; Cartwright, T.; Hainsworth, J.; Heim, W.; Berlin, J.;
Baron, A.; Griffing, S.; Holmgren, E.; Ferrara, N.; Fyfe, G.; Rogers, B.;
Ross, R.; Kabbinavar, F. Bevacizumab plus irinotecan, fluorouracil, and
leucovorin for metastatic colorectal cancer. N. Engl. J. Med. 2004, 350
(23), 2335−2342. (c) Markus Moehler, O. L.; Gockel, I.; Galle, P. R.;
Lang, H. Multidisciplinary management of gastric and gastro-
esophageal cancers. World J. Gastroenterol. 2008, 14 (24), 3773−
3780. (d) Moehler, M.; Mueller, A.; Trarbach, T.; Lordick, F.;
Seufferlein, T.; Kubicka, S.; Geißler, M.; Schwarz, S.; Galle, P. R.;
Kanzler, S.; Onkologie, G. A. I. Cetuximab with irinotecan, folinic acid
and 5-fluorouracil as first-line treatment in advanced gastroesophageal
cancer: a prospective multi-center biomarker-oriented phase II study.
Ann. Oncol. 2009, 22 (6), 1358−1366. (e) Shepherd, F. A.; Rodrigues
Pereira, J.; Ciuleanu, T.; Tan, E. H.; Hirsh, V.; Thongprasert, S.;
Campos, D.; Maoleekoonpiroj, S.; Smylie, M.; Martins, R.; van
Kooten, M.; Dediu, M.; Findlay, B.; Tu, D.; Johnston, D.; Bezjak, A.;
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +49-6131-39-25726. Fax: +49-6131-3330477. E-mail:
dannhardt@uni-mainz.de.
Author Contributions
∥Authors C.G. and E.L. contributed equally.
Notes
The authors declare no competing financial interest.
Clark, G.; Santabar
non-small-cell lung cancer. N. Engl. J. Med. 2005, 353 (2), 123−132.
(8) Peifer, C.; Krasowski, A.; Hammerle, N.; Kohlbacher, O.;
́
bara, P.; Seymour, L. Erlotinib in previously treated
ACKNOWLEDGMENTS
■
̈
We gratefully acknowledge financial support by “Stiftung
Dannhardt, G.; Totzke, F.; Schachtele, C.; Laufer, S. Profile and
̈
Rheinland-Pfalz fur Innovation”.
̈
molecular modeling of 3-(indole-3-yl)-4-(3,4,5-trimethoxyphenyl)-1H-
pyrrole-2,5-dione (1) as a highly selective VEGF-R2/3 Inhibitor. J.
Med. Chem. 2006, 49 (25), 7549−7553.
ABBREVIATIONS USED
■
(9) (a) Zhang, Z.; Yang, Z.; Wong, H.; Zhu, J.; Meanwell, N. A.;
Kadow, J. F.; Wang, T. An effective procedure for the acylation of
azaindoles at C-3. J. Org. Chem. 2002, 67 (17), 6226−6227.
(b) Mahadevan, I.; Rasmussen, M. Synthesis of pyrrolopyridines
(azaindoles). J. Heterocycl. Chem. 1992, 29 (2), 359−367.
(10) Cash, M. T.; Schreiner, P. R.; Phillips, R. S. Excited state
tautomerization of azaindole. Org. Biomol. Chem. 2005, 3 (20), 3701−
3706.
bFGF, basic fibroplast growth factor; DMFDEA, N,N-
dimethylformamide diethyl acetal; DMFDMA, N,N-dimethyl-
formamide dimethyl acetal; FLK, fetal liver kinase; FLT-3,
FMS-like tyrosine kinase 3; GSK, glycogen synthase kinase;
HT-29, human colon adenocarcinome grade II cell line;
HUVEC, human umbilical vein endothel cell; ITD, internal
tandem duplication; KDR, kinase insert domain receptor; Mkn-
45, human gastric cancer cell line; Molm-14, human leukemia
cell line; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide; NaH, sodium hydride; RTK, receptor tyrosine
(11) Dormoy, J.-R.; Heymes, A. Synthese industrielle en serie
ellipticine. I: Elaboration d’une nouvelle voie d’acces
pyrido[4,3:b]carbazoles et analogues: A. Synthese et et
precurseurs. Tetrahedron 1993, 49 (14), 2885−2914.
̀
aux 6H-
ude des
̀
́
́
t
kinase; BuOK, potassium tert-butoxide
(12) Ottoni, O.; Neder, A. d. V. F.; Dias, A. K. B.; Cruz, R. P. A.;
Aquino, L. B. Acylation of indole under Friedel−Crafts conditions an
improved method to obtain 3-acylindoles regioselectively. Org. Lett.
2001, 3 (7), 1005−1007.
REFERENCES
■
(1) (a) Moore, M. J.; Goldstein, D.; Hamm, J.; Figer, A.; Hecht, J. R.;
Gallinger, S.; Au, H. J.; Murawa, P.; Walde, D.; Wolff, R. A.; Campos,
D.; Lim, R.; Ding, K.; Clark, G.; Voskoglou-Nomikos, T.; Ptasynski,
M.; Parulekar, W. Erlotinib plus gemcitabine compared with
gemcitabine alone in patients with advanced pancreatic cancer: a
phase III trial of the National Cancer Institute of Canada Clinical
Trials Group. J. Clin. Oncol. 2007, 25 (15), 1960−1966. (b) Ma, W.
W.; Adjei, A. A. Novel agents on the horizon for cancer therapy. CA
Cancer J. Clin. 2009, 59 (2), 111−137.
(13) Faul, M. M.; Winneroski, L. L.; Krumrich, C. A. A new one step
synthesis of maleimides by condensation of glyoxylate esters with
acetamides. Tetrahedron Lett. 1999, 40 (6), 1109−1112.
(14) (a) Peifer, C.; Stoiber, T.; Unger, E.; Totzke, F.; Schachtele, C.;
̈
́
Marme, D.; Brenk, R.; Klebe, G.; Schollmeyer, D.; Dannhardt, G.
Design, synthesis, and biological evaluation of 3,4-diarylmaleimides as
angiogenesis inhibitors. J. Med. Chem. 2006, 49 (4), 1271−1281.
(b) Dannhardt, G.; Peifer, C.; Plutizki, S.; Kramb, J., 3-(Indolyl)-4-
Arymaleimide Derivatives and Their Use as Angiogenesis Inhibitors.
EP Patent 1819697, 2007. (c) Dannhardt, G.; Fischer, T.; Heidel, F.
H.; Kramb, J. P.; Plutizki, S. Use of 3-(Indolyl)- or 3-(Azaindolyl)-4-
arylmaleimide Derivatives in Leukemia Management. EP Patent
08858282.1, 2011.
(2) (a) Risau, W. Differentiation of endothelium. FASEB J. 1995, 9
(10), 926−933. (b) Conway, E. M.; Collen, D.; Carmeliet, P.
Molecular mechanisms of blood vessel growth. Cardiovasc. Res. 2001,
49 (3), 507−521. (c) Carmeliet, P. Mechanisms of angiogenesis and
arteriogenesis. Nat. Med. 2000, 6 (4), 389−395.
9539
dx.doi.org/10.1021/jm301217c | J. Med. Chem. 2012, 55, 9531−9540

Journal of Medicinal Chemistry
Article
(15) Basel, Y.; Hassner, A. Di-tert-butyl dicarbonate and 4-
(dimethylamino)pyridine revisited. Their reactions with amines and
alcohols. J. Org. Chem. 2000, 65 (20), 6368−6380.
(16) (a) Lemmon, M. A.; Schlessinger, J. Cell signaling by receptor
tyrosine kinases. Cell 2010, 141 (7), 1117−1134. (b) Bennasroune, A.;
́
Gardin, A.; Aunis, D.; Cremel, G.; Hubert, P. Tyrosine kinase
receptors as attractive targets of cancer therapy. Crit. Rev. Oncol./
Hematol. 2004, 50 (1), 23−38. (c) Zhang, H.-C.; Ye, H.; Conway, B.
R.; Derian, C. K.; Addo, M. F.; Kuo, G.-H.; Hecker, L. R.; Croll, D. R.;
Li, J.; Westover, L.; Xu, J. Z.; Look, R.; Demarest, K. T.; Andrade-
Gordon, P.; Damiano, B. P.; Maryanoff, B. E. 3-(7-Azaindolyl)-4-
arylmaleimides as potent, selective inhibitors of glycogen synthase
kinase-3. Bioorg. Med. Chem. Lett. 2004, 14 (12), 3245−3250.
(17) Peifer, C.; Dannhardt, G. A novel quantitative chick embryo
assay as an angiogenesis model using digital image analysis. Anticancer
Res. 2004, 24 (3A), 1545−1552.
(18) Aitkenhead, M.; Christ, B.; Eichmann, A.; Feucht, M.; Wilson,
D. J.; Wilting, J. Paracrine and autocrine regulation of vascular
endothelial growth factor during tissue differentiation in the quail. Dev.
Dyn. 1998, 212 (1), 1−13.
(19) Gotschel, F.; Kern, C.; Lang, S.; Sparna, T.; Markmann, C.;
̈
Schwager, J.; McNelly, S.; von Weizsacker, F.; Laufer, S.; Hecht, A.;
̈
Merfort, I. Inhibition of GSK3 differentially modulates NF-κB, CREB,
AP-1 and β-catenin signaling in hepatocytes, but fails to promote
TNF-α-induced apoptosis. Exp. Cell Res. 2008, 314 (6), 1351−1366.
(20) Zhang, W.; Konopleva, M.; Shi, Y.-x.; McQueen, T.; Harris, D.;
́
Ling, X.; Estrov, Z.; Quintas-Cardama, A.; Small, D.; Cortes, J.;
Andreeff, M. Mutant FLT3: a direct target of sorafenib in acute
myelogenous leukemia. J. Natl. Cancer Inst. 2008, 100 (3), 184−198.
(21) Davis, P. D.; Hill, C. H.; Lawton, G.; Nixon, J. S.; Wilkinson, S.
E.; Hurst, S. A.; Keech, E.; Turner, S. E. Inhibitors of protein kinase C.
1. 2,3-Bisarylmaleimides. J. Med. Chem. 1992, 35 (1), 177−184.
(22) Matsuo, Y.; MacLeod, R.; Uphoff, C.; Drexler, H.; Nishizaki, C.;
Katayama, Y.; Kimura, G.; Fujii, N.; Omoto, E.; Harada, M.; Orita, K.
Two acute monocytic leukemia (AML-M5a) cell lines (MOLM-13
and MOLM-14) with interclonal phenotypic heterogeneity showing
MLL-AF9 fusion resulting from an occult chromosome insertion,
ins(11;9)(q23;p22p23). Leukemia 1997, 11, 1469−1477.
(23) Mosmann, T. Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays. J. Immunol.
Methods 1983, 65 (1−2), 55−63.
(24) Nicoletti, I.; Migliorati, G.; Pagliacci, M. C.; Grignani, F.;
Riccardi, C. A rapid and simple method for measuring thymocyte
apoptosis by propidium iodide staining and flow cytometry. J.
Immunol. Methods 1991, 139 (2), 271−279.
(25) Millipore. Lead DiscoveryKinases and Phosphatases. http://
www.millipore.com/life_sciences/flx4/ld_kinases.
(26) Sloan, M. J.; Phillips, R. S. Enzymatic synthesis of aza-l-
tryptophans: the preparation of 5- and 6-aza-l-tryptophan. Bioorg. Med.
Chem. Lett. 1992, 2 (9), 1053−1056.
(27) Zhang, Z.; Yang, Z.; Meanwell, N. A.; Kadow, J. F.; Wang, T. A
general method for the preparation of 4- and 6-azaindoles. J. Org.
Chem. 2002, 67 (7), 2345−2347.
(28) Dannhardt, G.; Kramb, J.; Plutizki, S. 3-(Indolyl)- or 3-
(Azaindolyl)-4-arylmaleimide Compounds and Their Use in Tumor
Treatment. WO 2011/73092 A1, 2011.
9540
dx.doi.org/10.1021/jm301217c | J. Med. Chem. 2012, 55, 9531−9540