Discovery of novel substituted benzo-anellated 4-benzylamino pyrrolopyrimidines as dual EGFR and VEGFR2 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2017.04.053

The quinazoline scaffold is the main part of many marketed EGFR inhibitors. Resistance developments against those inhibitors enforced the search for novel structural lead compounds. We developed novel benzo-anellated 4-benzylamine pyrrolopyrimidines with varied substitution patterns at both the molecular scaffold and the attached residue in the 4-position. The structure-dependent affinities towards EGFR are discussed and first nanomolar derivatives have been identified. Docking studies were carried out for EGFR in order to explore the potential binding mode of the novel inhibitors. As the receptor tyrosine kinase VEGFR2 recently gained an increasing interest as an upregulated signaling kinase in many solid tumors and in tumor metastasis we determined the affinity of our compounds to inhibit VEGFR2. So we identified novel dually acting EGFR and VEGFR2 inhibitors for which first anticancer screening data are reported. Those data indicate a stronger antiproliferative effect of a VEGFR2 inhibition compared to the EGFR inhibition.

Full text of DOI:10.1016/j.bmcl.2017.04.053

Accepted Manuscript
Discovery of novel substituted benzo-anellated 4-benzylamino pyrrolopyrimi-
dines as dual EGFR and VEGFR2 inhibitors
Tim Fischer, Thomas Krüger, Abdulkarim Najjar, Frank Totzke, Christoph
Schächtele, Wolfgang Sippl, Christoph Ritter, Andreas Hilgeroth
PII:
S0960-894X(17)30429-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.04.053
BMCL 24906
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
18 November 2016
13 April 2017
15 April 2017
Please cite this article as: Fischer, T., Krüger, T., Najjar, A., Totzke, F., Schächtele, C., Sippl, W., Ritter, C.,
Hilgeroth, A., Discovery of novel substituted benzo-anellated 4-benzylamino pyrrolopyrimidines as dual EGFR and
VEGFR2 inhibitors, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.
2
017.04.053
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1
Discovery of novel substituted benzo-anellated 4-benzylamino
pyrrolopyrimidines as dual EGFR and VEGFR2 inhibitors
a
a
a
b
b
Tim Fischer , Thomas Krüger , Abdulkarim Najjar , Frank Totzke , Christoph Schächtele ,
a
c
a,
Wolfgang Sippl , Christoph Ritter , Andreas Hilgeroth *
a
Institute of Pharmacy, Martin-Luther-University Halle-Wittenberg, Wolfgang-Langenbeck-
Str. 4, 06120 Halle, Germany
b
ProQinase GmbH, Breisacherstr. 117, 79106 Freiburg, Germany
c
Institute of Pharmacy, Ernst-Moritz-Arndt University Greifswald, Friedrich-Ludwig-Jahn-
Str. 17, 17489 Greifswald, Germany
Abstract – The quinazoline scaffold is the main part of many marketed EGFR inhibitors.
Resistance developments against those inhibitors enforced the search for novel structural lead
compounds. We developed novel benzo-anellated 4-benzylamine pyrrolopyrimidines with
varied substitution patterns at both the molecular scaffold and the attached residue in the 4-
position. The structure-dependent affinities towards EGFR are discussed and first nanomolar
derivatives have been identified. Docking studies were carried out for EGFR in order to
explore the potential binding mode of the novel inhibitors. As the receptor tyrosine kinase
VEGFR2 recently gained an increasing interest as an upregulated signalling kinase in many
solid tumors and in tumor metastasis we determined the affinity of our compounds to inhibit
VEGFR2. So we identified novel dually acting EGFR and VEGFR2 inhibitors for which first
anticancer screening data are reported. Those data indicate a stronger antiproliferative effect
of a VEGFR2 inhibition compared to the EGFR inhibition.
_
______________

2
Keywords: structure-dependent activity, lead structure, compound evaluation, anticancer
screening
*
Corresponding author. Tel. +49 345 55 25168; fax +49 345 55 27207; E-mail address:
andreas.hilgeroth@pharmazie.uni-halle.de (A. Hilgeroth).
The understanding of cell signaling processes in the last decades led to the identification of
1
,2
novel target structures for a more selective anticancer therapy. Early identified protein
kinases have been cyclin dependent kinases (cdks) which were found deregulated in cancer
3
,4
cells to cause enhanced proliferation rates. Then receptor tyrosine kinases (RTKs) gained
certain interest because of their central role in various cellular pathways of regulating cell
5
-7
growth and apoptosis. Such RTKs can be found either overexpressed in cancer cells or
6
,8,9
overactivated by the production of endogenous ligands.
After binding to the extracellular
region of such a RTK the membrane-located receptor half undergoes a dimerization with a
second neighboured receptor half to give a functional unit which proceeds in an intracellular
5,6
phosphorylation of amino acid residues. After such an autophosphorylation cell signaling
proteins dock at the phosphorylated RTK binding site and are phosphorylated to mediate
6
,10
further cellular processes.
One central target structure for anticancer therapies became the epidermal growth factor
1
1,12
receptor (EGFR) tyrosine kinase.
EGFR can be activated by various ligands and plays a
role in enhanced cell proliferation, escape from apoptosis that ensures a cancer cell survival
1
1,12
and, finally, in aggressive invasive tumor growth.
So EGFR became a central target
1
3
structure for the development of inhibitors as potential anticancer drugs. Early inhibitors
have been small-molecule inhibitors which bind to the ATP-binding site so that the
14,15
intracellular phosphorylation of the EGFR tyrosine residues is blocked.
Meanwhile,
resistances against such inhibitors have been described which result from amino acid

3
1
6,17
mutations within the encoding EGFR gene.
While single muations like L858R have been
tolerated by the inhibitors, certain mutations in the exon 20 gene region led to inhibitor
1
8,19
resistances, mainly the T790M mutation or a combined L858R_T790M mutation.
So
prominent marketed inhibitors are affected from such mutations like erlotinib which
reversibly binds to EGFR contrasting novel inhibitors like afatinib which bind to EGFR in an
1
9,20
irreversible manner.
Erlonitnib owns a quinazoline scaffold which can be found in several marketed EGFR
21
inhibitors. Due to the increasing resistance developments against the established EGFR
2
2
inhibitors there is a strong demand for novel lead structures. While the pyrimidine part of
the quinazoline core and the attached 4-anilino residue were maintained in all recent
alternative structures the attached phenyl residue has been exchanged with a five-ring
2
3,24
heterocycle.
Merely micromolar activites have been reported for all those alternative
structures as EGFR inhibitors without a selectivity of kinase inhibition.
We synthesized a series of novel benzo-anellated pyrrolopyrimidines as potential EGFR
inhibitors with a varied 4-benzylamino function. We evaluated the EGFR inhibiting properties
of our novel compounds and additionally investigated their potential to inhibit the vascular
endothelian growth factor (VEGFR) 2 that supports the invasive tumor growth by enforcing
2
5
tumor-angiogenesis. Moreover, recent findings of VEGFR2 in many solid tumors made it a
2
6-28
most interesting target structure for anticancer therapy.
So we identified dual inhibitors of
both EGFR and VEGFR2 which have been evaluated in first cellular anticancer studies.
Our target structures 6a-t resulted from the reaction of the 4-benzylamino varied
compounds 2a-k with the respective p-benzoquinone 3 in a solution mixture of dried ethanol
2
9
and acetic acid (4/1) under reflux conditions. The 4-substituted starting compounds 2a-k
were yielded from the reaction of the 2,6-diamino-4-chloropyrimidine 1 with the respective
3
0
varied benzylamine at a temperature of 135 °C after 2-4 h. The suggested mechanism for the

4
target compound formation started with a primary Michael addition of both starting
compounds to give the 5-substituted pyrimidine intermediate 4.
Cl
H N
2
N
NH2
1
(a)
R2
OH
OH
R2
R1
R1
R3
HN (CH2)n
HN (CH2)n
O
R
(b)
+
H N
2
N
NH2
H N
2
N
NH2
O
2a-k
3
4
O
O
R2
R3
R1
HN (CH2)n
H N
2
N
NH2
5
-
H O
2
R2
HN (CH2)n
R1
HO
R3
N
H
N
NH2
6a-t

5
Scheme 1. Reagents and conditions: (a) varied benzylamine, 135 °C, 2-4 h, yields: 48-80%;
(
b) reflux, 3-5 h, ethanol, acetic acid, yields: 2-22%.
Then the hydroquinone substructure was oxidized by the remaining excess of the original
benzoquinone 3 to give the quinone substructure in compound 5. Finally, a cycloaddition
reaction took place under elimination of water to result in the benzo-anellated derivative 6
given in an average yield of 14%.
The protein kinase inhibitory activity has been determined in an ATP competition assay for
3
1
different inhibitor concentrations. The reduced phosphorylation of a respective kinase
substrate under inhibitor application has been determined with scintillation measurements
counting the radioactive incorporated phosphorus.
Table 1. Protein kinase inhibiting data of target compounds 6a-t.
_
__________________________________________________________________________
[µM]
VEGFR2
__________________________________________________________________________
K
i
1
2
3
n
R
R
R
EGFR
_
6
6
6
6
6
6
6
6
6
a
b
c
d
e
f
0
0
0
0
0
0
0
0
0
-
H
H
H
H
H
H
H
H
H
H
0.70
0.64
0.61
0.37
0.27
0.09
5.67
0.12
5.95
2.76
4.83
13.41
-
Me
MeO
Br
-
a
-
n.a.
a
-
Cl
n.a.
4-Me
4-Me
4-OMe
4-OMe
H
3.26
a
g
h
i
Me
H
n.a.
b
n.d.
b
Me
n.d.

6
6
6
6
6
6
6
6
6
6
6
6
j
0
0
0
0
0
0
0
0
1
0
0
4-Cl
H
H
0.08
0.11
1.26
1.87
4.03
0.29
0.90
3.68
0.64
0.91
0.52
3.24
a
k
l
4-Cl
H
Me
H
n.a.
3-OMe
2-OMe
2-OMe
3-Cl
H
3.99
0.85
7.24
m
n
o
p
q
r
s
H
H
H
Me
H
b
H
n.d.
n.d.
b
3-Cl
H
Me
H
3,4-OMe
H
11.07
4.47
1.93
4.15
-
-
-
H
H
(R)Me
(S)Me
H
t
H
_
__________________________________________________________________________
a
not active.
b
not determinable.
We found a submicromolar affinity towards EGFR for the 4-benzylamine substituted
compound 6a. Then we introduced a methyl function into the 7-position of the molecular
scaffold in derivative 6b. The affinity was almost similar to that of the unsubstituted scaffold
in compound 6a. A methoxy function instead of the methyl group in derivative 6c also
resulted in an almost unchanged affinity. If halogen atoms were placed into the 7-position we
found improvements in the affinity. The bromo substitution in compound 6d led to an almost
twofold increase in affinity and the chloro substitution in derivative 6e resulted in the best
affinity of those 4-benzylamine substituted compounds with a further molecular scaffold
substitution.
We then introduced a methyl function into the 4-position of the benzylamino residue in
compound 6f. We found a main increase in affinity reaching nanomolar ranges of 90 nM

7
similar to the reversibly binding erlotinib for which 45 nM have been determined. If that 4-
methly benzylamino substitution was combined with a 7-methyl function at the molecular
scaffold in compound 6g the affinity was mainly lowered and was also almost tenfold lower
than that of the only 7-methyl substituted compound 6b. If the 4-methyl benzylamino function
was replaced with a methoxy function in derivative 6h the affinity was similar. For the
combination of the 4-methoxy benzylamino substitution with the additional 7-methyl group at
the molecular scaffold in derivative 6i we found the same tendency of a reduced affinity if
compared to the 4-methyl benzylamino substituted derivatives 6f with the nanomolar affinity
and the additionally 7-methyl substituted compound 6g with main decreases in the EGFR
affinity.
Next we investigated the influence of a 4-chloro substitution in the benzylamino residue in
derivative 6j. The compound showed the best affinity of 80 nM for the only 4-benzylamine
substituted compounds. The introduction of a methyl function into the 7-position of the
molecular scaffold in compound 6k led to an almost similar affinity with only a slight
reduction.
We then varied the substituent position within the benzylamino residue. A methoxy
function in the 3-position of the benzylamino residue in compound 6l led to a tenfold lower
affinity towards EGFR if compared to the 4-positioned residue in derivative 6h. If placed in
the 2-position of the benzylamine residue in derivative 6m the affinity was further lowered.
The combination with the additional 7-methyl function in compound 6n was not favourable
with a mainly lowered affinity.
When the 4-chloro substituent in the benzylamine residue of derivative 6j moved to the 3-
position the affinity was found decreased in derivative 6o. If combined with the 7-methyl
functions in compound 6p a further affinity reduction was observed. We also investigated one
disubstituted benzylamine compound. The 3,4-dimethoxy benzylamine derivative 6q showed

8
a mainly decreased affinity if compared to the both monomethoxy substituted derivatives 6h
and 6l, respectively.
Next we varied the benzyl residue first with a phenylethyl residue. The elongation of the 4-
amino substituent in compound 6r was not of favour with an almost similar activity than that
of the benzylamine derivative 6a. The additional (R) methyl function in compound 6s was
less favourable, whereas slight improvements in the affinity resulted for the (S) methyl
function in compound 6t.
If we summarize the results of our substituent-dependent affinities towards EGFR so far, it
can be stated that additional molecular scaffold substitutions were not favourable if combined
with substituted benzylamine residues. Best nanomolar affinities resulted for the benzylamine
derivatives substituted in the 4-position. Both 3- and 2-substituted benzylamine compounds
showed lowered EGFR affinities for the respective substituents. A benzylamine disubstitution
is not favourable and changes in the 4-aminoalkyl chain partly influenced the EGFR affinity
with slight decreases or increases, respectively.
3
2
The docking results with EGFR showed that the benzo-anellated pyrrolo[2,3-b]pyridine
scaffold is mimicking the adenine ring of ATP and represents the hinge-binding motifs. They
also showed that potent inhibitors (e.g. 6f and 6h) are able to fom two hydrogen bonds to the
hinge region residues: one to the NH of Met793 and one to the CO of Gln791. In case of 6f
and 6h the 4-methyl and 4-methoxy substituted benzyl group is interacting with the
hydrophobic pocket nearby Leu788 (Figure 1A). If the 4-methoxy or methyl substitution on
the benzylamino ring is combined with a methyl substitution on position 7 a dramatic loss of
activity is observed. The docking of 6i to EGFR showed that the 7-methyl group is facing into
the solvent region which is unfavourable (Figure 1B). In case of the compounds without a 4-
methoxy/methyl benzylamino group (e.g. 6b) the pyrrolo[2,3-b]pyridine scaffold and the 7-
methyl substitutent are more buried which might explain the similar activities of 6a and 6b.

9
Figure 1A. Interaction of the most active EGFR inhibitors 6f (cyan carbon atoms) and 6h
(
orange carbon atoms) at the active site of EGFR. The substituted benzyl ring is completely
burried in a hydrophobic pocket between Thr790 and Leu788. Hydrogen bonds are shown as
dashed lines. Only relevant amino acids are displayed.

10
Figure 1B. Docking result for the less active inhibitor 6i (orange carbon atoms) at EGFR. The
-methyl group is facing into the polar solvent region resulting in unfavourable interactions.
The molecular surface of EGFR is displayed and colored according to the hydropbhobicty
7
(
polar region = magenta, hydrophobic region = green).
With first identified nanomolar EGFR inhibitors of various substitution patterns we
decided to determine the VEGFR2 affinities of our compounds to estimate their potential for
further anticancer studies of cell proliferation. VEGFR2 not only supports the EGFR activity
in tumor growth with activity in angiogenesis, but is also found in many solid tumors like
2
8,33
bladder cancer, breast cancer, lung cancer and prostate cancer according to recent studies.
The role of VEGFR2 in such solid tumors is under discussion and indications have been
found that VEGFR2 is envolved in metastasis of such tumors. So VEGFR2 became an
interesting target structure either for combined therapies to address both EGFR and VEGFR2
or to address VEGFR2 alone.

11
Within our first compound series 6a-6e with a 7-substitution of the molecular scaffold and
an unsubstituted 4-benzylamine residue we found best affinities towards VEGFR2 for the 7-
unsubstituted derivative 6a with a lower micromolar affinity. The 7-methyl function caused a
decreased compound affinity and the 7-halogen substituted compounds 6d and 6e were not
active as VEGFR2 inhibitors. The 7-methoxy function in compound 6c was also
unfavourable. The 4-methyl function in the benzylamine compound 6f decreased the affinity
if compared to the unsubstituted derivative 6a and the additional methyl function in 7-position
of the molecular scaffold led to a loss of VEGFR2 affinity. Similar results were found for the
4
-chlorobenzylamine function in derivative 6j with decreases in the VEGFR2 affinity if
compared to the unsubstituted compound 6a and a loss of affinity for the additionally 7-
methly substituted derivative 6k.
A methoxy function in the 3-position of the benzylamine residue in compound 6l led to a
lower micromolar affinity towards VEGFR2. If moved to the 2-position of the benzylamine in
compound 6m the affinity mainly increased reaching submicromolar ranges with a K
.85 µM. An additional methyl function in the 7-position of the molecular scaffold in
derivative 6n was unfavourable with main decreases in the VEGFR2 affinity.
i
value of
0
A dimethoxy substitution of the benzylamine residue in compound 6q was also
unfavourable if compared to the monomethoxy substitution in compound 6l.
Within the series of 4-aminoalkyl side chain varied derivatives 6r-t we found best
affinities for the (R) methyl substituted compound 6s being more active than the only
benzylamine substituted compound 6a. The two other derivatives 6r and t showed similar
reduced affinities.
Next we carried out a first selectivity screening of protein kinase inhibition for compound
6
g which was not active as VEGFR2 inhibitor and showed a sufficient solubility in the used
protein kinase inhibition assays even at higher concentrations > 100 µM. We tested against
protein kinases of all the protein kinase families. We determined no activity against MARK1

12
and DAPK1 from the cAMK familiy and against MEK1 and MEKK2 of the STE family with
IC50 values > 100 µM. The compound was not active as PKCα and as DMPK inhibitor, again
with IC50 values > 100 µM. Both kinases belong to the AGC kinase family again. Similar IC50
values were determined for the inhibition of CK1α and Wee1 of the CK1 family and for
ALK1, MLK4 and, finally, for IRAK of the TKL familiy. We found no activity against c-Abl
and JAK2 of the TK family with IC50 values > 100 µM. Finally, we determined the activity
against CDK1 and CDK2 of the CMGC family and found a residual activity with values of 61
µM for CDK1 and of 48 µM for CDK2.
High expression rates of EGFR and its activating ligands as tumor-inducing agents have
1
1
been recently reported to occur mainly in breast cancer and non-small lung cancer cells.
EGFR was found as tumor-relevant growth factor in breast cancer, lung cancer and bladder
2
7-29
cancer.
So we selected relevant cancer cells lines of breast cancer and non-small lung
cancer to initially estimate the potential of our compounds to act as antiproliferative agents in
anticancer tumor growth. In these studies 10 µM of the selected inhibitors were used and the
growth inhibition of the relevant tumor cells was determined in the sulforhodamine assay at
3
4,35
the National Cancer Institute of Health (NCI).

13
Table 2. Anticancer cell screening data of NCI selected compounds.
_
__________________________________________________________________________
Growth inhibition [%]
6
e
6j
6m
_
__________________________________________________________________________
Breast cancer
MDA-MB-231/ATCC
MDA-MB-468
BT-549
34
28
21
16
65
71
24
33
73
55
37
41
HS 578T
Non-small lung cancer
HOP-92
19
20
5
57
31
25
66
63
82
HOP-62
NCI-H460
_
__________________________________________________________________________
a
not determined.
The NCI selected one submicromolar EGFR inhibitor which showed no VEGFR2 affinity
(
6e), one nanomolar EGFR inhibitor with a VEGFR2 affinity in low micromolar range (6j)
and, finally, one low micromolar active EGFR inhibitor with a submicromolar VEGFR2
affinity (6m) for first anticancer screening studies. The determined compound growth
inhibition activities in respective tumor cells are shown in table 2.
Comparing all the anticancer screening cell line results for the selected compounds we
found the lowest activity for the only EGFR-inhibiting compound 6e with best results in the
relevant breast cancer cell lines ranging from 16 to 34%. The activity was similar to that of

14
gefitinib for which a 20% growth inhibition has been reported in the breast cancer cell line HS
78T at a concentration of 4 µM. Our nanomolar active EGFR inhibitor 6j with a low
5
micromolar VEGFR2 affinity showed increased activities in the EGFR-relevant breast cancer
cell and all non-small lung cancer cell lines. Surprisingly, our submicromolar VEGFR2
inhibitor 6m with only a low micromolar EGFR affinity showed best growth inhibition rates
for three of the breast cancer and for all of the non-small lung cancer cell lines.
Novel lead compounds as EGFR inhibitors are demanded alternatively to the established
quinazoline derivatives. We discovered benzo-anellated pyrrolopyrimidines as novel class of
receptor tyrosine kinase EGFR inhibitors and identified first nanomolar active compounds
which showed a favourable 4-substitution of the 4-benzylamino residue. Furthermore, we
determined the VEGFR2 affinities of our compounds and identified first dual inhibitors of
EGFR and VEGFR2. Our dual inhibitors were initially screened at the NCI for anticancer cell
growth and proved to be effective in selected EGFR- and VEGFR2-cancer relevant cells. The
most active VEGFR2 inhibitor showed the best anticancer growth as far as investigated. The
results reveal the importance of VEGFR2 inhibition for antiproliferative cancer cell growth in
respective VEGFR2-relevant tumors. With first kinase inhibition data of selectivity we found
perspective lead compounds for further drug development studies.
Acknowledgement
Financial support of the work was given from the Deutsche Forschungsgemeinschaft
(
DFG) within the project HI 687/10-1 to Andreas Hilgeroth and to Christoph Ritter within the
DFG projects RI 1196/4-1 and, finally, to Tim Fischer as fellowship of the graduate support
program of the country saxony anhalt.

15
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[
29] General procedure for the formation of the 4-benzylamino substituted benzo-anellated
pyrrolopyrimidines 6: 2-5 mmol of the respective 4-substituted diaminopyrimidine 2
were dissolved in a mixture of dried ethanol (20 mL) and acetic acid (5 mL). Then 1.8
equiv. of the respective quinone 3 were added and the solution was refluxed for 3-5
h. The reaction procedure was followed with tlc and after finishing the solvent was
removed in vacuum. The raw product was purified by column chromatography using
silica gel and an eluent mixture of chloroform and methanol in a relation of 95 to 5
that changed to 90 to 10 during column separation. The unified product fractions were
evaporated and the remaining solid was recrystallized from dried chloroform. 2-
Amino-4-(benzylamino)-9H-pyrimido[4,5-b]indole-6-ol 6a. Yield 0.168 g (22%); grey
1
solid; mp 253-265 °C; H NMR (dmso-d
6
) δ 5.32 (s, 2H, CH
2
), 5.94 (br, 2H, 2-NH
2
),
6
7
.51 (br, 1H, NH), 6.58 (dd, J = 8.3, 2.3 Hz, 1H, 7-H), 7.01 (d, J = 8.3 Hz, 1H, 8-H),
.15-7.26 (m, 5H, phenylic H), 7.40 (d, J = 2.3 Hz, 1H, 5-H), 8.73 (br, 1H, OH), 11.92
+
(
br 1H, 9-H); MS (ESI, cation), m/z = 306 [M+H ]. IR (KBr): 3460, 3342, 3065, 3034,
-1
2
929, 1641, 1620, 1496, 1460, 1595, 1567, 1452, 1415, 1360 cm . Elemental Anal.
calcd. (%) for C17
2.87.
15 5
H N
O: C, 66.87; H, 4.95; N, 22.94. Found: C, 66.83; H, 5.25; N,
2
[
30] General procedure for the formation of the varied 4-benzylamino substituted
diaminopyrimidine 2: 10 mmol of the 2,6-Diamino-4-chloropyrimidine 1 were

18
mixed with 30 mmol of the benzylamine and heated to 135 °C for 2-4 h. The
reaction was controlled by tlc and stopped when no more of the starting compound 1
could be detected by tlc. After cooling to room temperature the raw product was
purified using column chromatography on silica gel and an eluent mixture of
ethylacetate and methanol (95/5) that switched to a relation of 90/10. The resulting
products of the collected and evaporated fractions were recrystallized from mixtures of
4
methanol and diethylether. N -benzylpyrimidine-2,4,6-triamine 2a. Yield 1.56 g
1
(
73%); white solid; mp 144-146 °C; H NMR (dmso-d
6
) δ 4.27 (s, 2H, CH
2
), 4.89 (s,
1
H, 5-H), 5.38 (br, 2H, 2-NH
2
), 5.59 (br, 2H, 6-NH
2
), 7.11 (m, 1H, 4´-H), 7.17-7.21
+
(
m, 4H, 2´-, 3´-, 5´-, 6´-H), 7.32 (br, 1H, NH); MS (ESI, cation), m/z = 216 [M+H ];
Elemental Anal. calcd. (%) for C11
H, 5.85; N, 32.33.
H
13
N
5
: C, 61.38; H, 6.09; N, 32.53. Found: C, 61.05;
TM
[
31] The measuring of protein kinase activity was performed in 96-well FlashPlates in a
5
0 µL reaction volume. The reaction mixture consisted of 20 µL of assay buffer
solution, 5 µL of an ATP solution in water, 5 µL of the used test compound in a 10%
dmso solution and finally a premixture of each 10 µL of used substrate and enzyme
solutions. The assay buffer solution contained 70 mM of HEPES-NAOH pH 7.5, each
3
mM of magnesium chloride and manganese(II) chloride, 3 µM of sodium
3
3
orthovanadate, 1.2 mM of DTT, 50 µg/mL of PEG20000 and finally 15 µM of [γ- P]-
5
ATP making approximately 7 x 10 cpm per well. The final kinase concentration had
been 20 ng/50 µL for EGFR and VEGFR2. The used substrate was Poly(Glu,Tyr)4:1 in
a concentration of 125 ng/50 µL.The reaction mixtures were incubated at 30 °C for 60
min. The reaction was stopped with 50 µL of a 2% (v/v) solution of phosphoric acid.
Then the plates were aspirated and washed twice with 200 µL of water or a 0.9%
3
3
solution of sodium chloride. The incorporation of Pi was determined with a
microplate scintillation counter. Ten different inhibitor concentrations were measured

19
in a range of 3 nM to 100 µM. The residual activity (%) and the IC50 values were
finally calculated. From the IC50 values the affinity constants K were determined using
the equation: IC50 = 1/2 [E ] + K x (1 + [S] / K ).
i
t
i
m
[
32] The 3D structure of the kinase EGFR was taken from the Protein databank. We took
from the available X-ray structures the ones which were co-crystallized with an
inhibitor structurally most similar to the benzo-anellated pyrrolo[2,3-b]pyridines under
study (PDB ID 2ITY). The protein structures were prepared as follows: water molecules
and bound small molecules were deleted, hydrogen atoms were added and the resulting
structures were minimized using the MMF94 force field and the conjugate gradient
method until a gradient of 0.01 kcal/mol was reached. We used the docking program
GOLD 5.2 (Cambridge Crystallographic Data Centre, Cambridge UK) to dock all
molecules into the ATP binding site of EGFR. For all docking runs the program default
settings were used. The binding site was defined on the gatekeeper residue with a radius
of 15 Å. Goldscore was chosen as fitness function. For each molecule 30 docking runs
were performed.
[
33] Kopparapu, P. K.; Boorjian, St. A.; Robinson, B. D.; Downes, M.; Gudas, L. J.;
Mongan, N. P.; Persson, J. L. Anticancer Res. 2013, 33, 2381.
[
[
34] Boyed, M. R. Pract. Oncol. 1989, 3, 1.
35] Monks, A.; Scudiero, D.; Skehan, P.; Boyed, M. Am. Assoc. Cancer Res. 1989, 30,
6
07.

20
R2
R1
HO
HN (CH2)n
3
R
N
H
N
NH2
6a-t
GA