Novel inhibitors of epidermal growth factor receptor: (4-(Arylamino)-7H- pyrrolo[2,3-d]pyrimidin-6-yl)(1H-indol-2-yl)methanones and (1H-indol-2-yl)(4- (phenylamino)thieno[2,3-d]pyrimidin-6-yl)methanones

Article abstract of DOI:10.1016/j.bmc.2011.11.023

Several members of the quinazoline class of known tyrosine kinase inhibitors are approved anticancer agents, often showing selectivity for receptors of the HER/ErbB-family. Combining structural elements of this class with the bisindolylmethanone-structure

Full text of DOI:10.1016/j.bmc.2011.11.023

Bioorganic & Medicinal Chemistry 20 (2012) 125–136
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel inhibitors of epidermal growth factor receptor: (4-(Arylamino)-7H-pyrr-
olo[2,3-d]pyrimidin-6-yl)(1H-indol-2-yl)methanones and (1H-indol-2-yl)
(4-(phenylamino)thieno[2,3-d]pyrimidin-6-yl)methanones
Thomas Beckers ,z, Andreas Sellmer a, Emerich Eichhorn a, Herwig Pongratz a, Christoph Schächtele b,
,
⇑
Frank Totzke b, , Gerhard Kelter c,à, Rebekka Krumbach c,à, Heinz-Herbert Fiebig c,à
,
Frank-D. Böhmer d,§, Siavosh Mahboobi a,
⇑
a Department of Pharmaceutical Chemistry I, University of Regensburg, D-93040 Regensburg, Germany
b ProQinase GmbH, Freiburg Breisacher Str. 117, D-79106 Freiburg, Germany
c Oncotest GmbH, Institute for Experimental Oncology, Am Flughafen 12-14, D-79108 Freiburg, Germany
d Institute of Molecular Cell Biology, Jena University Hospital, D-07747 Jena, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 June 2011
Revised 11 November 2011
Accepted 12 November 2011
Available online 20 November 2011
Several members of the quinazoline class of known tyrosine kinase inhibitors are approved anticancer
agents, often showing selectivity for receptors of the HER/ErbB-family. Combining structural elements
of this class with the bisindolylmethanone-structure led to a series of novel compounds. These com-
pounds inhibited EGFR in the nanomolar range. Moreover, inhibition of EGFR autophosphorylation in
intact A431 cells was shown, with IC50 values ranging form 0.3–1
lM for compound 42, and
0.1–0.3 M for 45. In a panel of 42 human tumor cell lines the sensitivity profile of the novel com-
l
Keywords:
Tyrosine kinase inhibitor
Anticancer agents
Bisindolylmethanone
Quinazoline
pounds was shown to be similar to that of the quinazoline class of tyrosine kinase inhibitors lapatinib
and erlotinib (TarcevaÒ).
Ó 2011 Elsevier Ltd. All rights reserved.
Epidermal growth factor receptor (EGFR)
1. Introduction
Roche, EGFR inhibitor), ZD6474 4 (Astra Zeneca, VEGFR and EGFR
inhibitor), lapatinib 5 (TykerbÒ, Glaxo Smith Kline, EGFR and
Due to the diversity in structure, synthetic and naturally occur-
ring indoles exhibit antibacterial, fungicide, cytotoxic and antipro-
liferative activity. We recently reported on 2-indolylmethanones
(e.g., 1a and 1b) as potent inhibitors of FMS-like tyrosine kinase
3 (FLT3) and platelet-derived growth factor receptor (PDGF-R)
tyrosine kinase (Fig. 1).1 Many anticancer agents which act as tyro-
sine kinase inhibitors selectively inhibit receptors of the HER/ErbB-
family. They comprise the pyrimidine group as a core moiety,
either as part of a quinazoline increment [for example see gefitinib
2 (IressaÒ, Astra Zeneca, EGFR inhibitor), erlotinib 3 (TarcevaÒ,
ErbB2) and tandutinib 6 (Millenium Pharmaceuticals, FLT3 inhibi-
tor)] (Fig. 1), or, incorporated into an indole related system as
shown by PKI-166 7 (Novartis, EGFR inhibitor).
The human epidermal growth factor receptor (EGFR, HER1) and
another member of this family, HER2 (ErbB2), have been linked to
various human malignancies, for example breast,2 head and neck,3
gastric,4 and non-small cell lung cancer.5–8 Chronic medication of-
ten induces selection of inhibitor-resistant mutants of tyrosine ki-
nases, thus resulting in relapse of the tumor.9 Consequently, we are
in need of compounds with novel therapeutic profiles based on
alternative binding modes, well-defined kinase selectivity, and
activity against clinically relevant kinase mutants.
Therefore, we prepared a series of compounds, incorporating
structural elements of the quinazoline class of known tyrosine ki-
nase inhibitors into the bisindolylmethanone-structure and
investigated their biological properties. It turned out that the
arylamino-increment, taken from the quinazoline based com-
pounds described above, was essential for biological activity. The
novel compounds (Fig. 1), are potent EGFR inhibitors in vitro and
in intact cells.
⇑
Corresponding authors. Tel.: +49 0 941 9434824; fax: +49 0 941 9431737.
E-mail addresses: f.totzke@proqinase.com (F. Totzke), gerhard.kelter@onco
test.de (G. Kelter), Boehmer@med.uni-jena.de (Frank-D. Böhmer), siavosh.mah
boobi@chemie.uni-regensburg.de (S. Mahboobi).
z
Due to untimely death, our highly respected colleague Dr. Thomas Beckers cannot
any longer be corresponding author of the pharmacological part of this publication.
R.i.p.
Tel.: +49 (0) 761 206 1723; fax: +49 (0) 761 206 1781.
Tel.: +49 (0) 761 51559 20.
Tel.: +49 (0) 3641 9395631.
à
§
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.11.023

126
T. Beckers et al. / Bioorg. Med. Chem. 20 (2012) 125–136
OH
R
F
Cl
A
D
B
C
NH
N
O
N
N
H
O
O
N
H
N
O
1a
: R = 4-CH3
1b: R = 5-OH
1c: R = H
2
Gefitinib ( )
Br
NH
NH
O
O
N
N
O
O
F
O
O
N
N
N
3
Erlotinib ( )
4
ZD6474 ( )
F
H
N
O
O
S
O
O
N
HN
Cl
NH
O
O
N
N
N
N
O
O
N
Tandutinib (6)
5
Lapatinib ( )
N
R1
NH
N
NH
N
R2
D
A
N
C
Y
B
N
OH
X
N
H
O
X = NH, S; Y = NH, O, S
7
PKI-166 ( )
General structure of novel designed compounds
Figure 1. Structures of 2-indolylmethanones (e.g., 1a and 1b), well known tyrosine kinase inhibitors containing the pyrimidine core structure as part of a quinazoline system,
and general structure of newly designed compounds.
the 2-arylboronic acids 52 and 5311 led to 54 and 55, respectively.
2. Results and discussion
Chlorine substitution by 3-chloro-4-fluoroaniline (56) led to the
final compounds 57 and 58.
2.1. Chemistry
2.2. Biology
Novel compounds were synthesized as outlined in Scheme 1:
Commercially available 4-chloro-7H-pyrrolo[2,3-d]pyrimidine (8)
was phenylsulfonated in THF solution using NaH as a base.10
Lithiation of the resulting 4-chloro-7-(phenylsulfonyl)-7H-pyrrol-
o[2,3-d]pyrimidine (9), or the sulfur analog 4-chlorothieno[2,3-
d]pyrimidine (10) and carboxylation according to procedures
described,1 yielded the carboxylic acids 11 and 12, respectively.
Heating in an excess of thionyl chloride led to the corresponding
In a first screening, the new compounds were checked for their
activities against a panel of kinases, namely EGFR, ErbB2, VEGFR2,
ABL1 wt, MET wt, and FLT3 at a concentration of 1
set the IC50 of the most potent compounds, exhibiting at least 90%
inhibition of EGFR protein kinase at 1 M was determined. The IC50
lM. In a second
l
values reach from 5.54 nM of 44 to 43.7 nM of 43.
1
acid chlorides and coupling with the lithiated species 15a0–15e0
The data compiled in Tables 1 and 2 show potent inhibiton of
EGFR, ErbB2, and some activity at VEGFR2. No inhibitons of ABL 1
wt and MET wt were observed (not shown). Interestingly, by switch-
ing from the bisindolylemethanone system of compounds 1a–1c to
the (1H-indol-2-yl)(7H-pyrrolo[2,3-d]pyrimidin-6-yl)methanone
48, by introducing two nitrogen atoms into the benzene ring of
one indole system, the inhibitory activity on FLT3 was completely
deleted. Moreover, as shown by comparison of the data of 48 with
those bearing an arylamino- or benzylamino-system connected to
C-4 of ring A this substitution pattern proved to be essential for
activity. By the way this modification did not restore any activity
at FLT3.
generated the central intermediates 16–21. After modification by
an aromatic SN-reaction the desired compounds 22–46 and 51
were obtained by cleavage of the phenylsulfonyl-protecting
groups. Cleavage of the phenylsulfonyl-protecting group in 18 by
tetrabutylammonium fluoride in THF, followed by hydrogenolytic
cleavage of the benzyloxy-group and simultaneous removing of
chlorine led to 48.
The reaction of 14 with benzofuran-2-yllithium and benzo
[b]thiophen-2-yllithium according to Scheme 1 failed. This step
was performed as shown in Scheme 2. Pd catalyzed reaction of
4-chlorothieno[2,3-d]pyrimidine-6-carbonyl chloride (14) with

T. Beckers et al. / Bioorg. Med. Chem. 20 (2012) 125–136
127
Cl
N
Cl
Cl
N
OH
O
1) n-BuLi, THF, -78°C
N
N
1) NaH, THF, 0°C
2) PhSO2Cl, 20°C
N
2) CO2
3) H2O, HCl
X
X
X
N
8: X = NH
9: X = NSO2Ph
11: X = NSO2Ph
12: X = S
10: X = S
R
Li
SO2Ph
N
Y
R
Cl
N
Cl
N
Cl
O
SOCl2
THF, -78°C
Y
N
SO2Ph
N
X
X
N
15a´-15d´: Y = CH
15e´:
Y = N
O
13: X = NSO2Ph:
14: X = S
X = NSO2Ph; Y = CH
X = NSO2Ph; Y = N
16b: R = H
16a
: R = H
17
:
R = OCH3
;
18: R = OCH2Ph
b
r
o
a
1
,
H
,
2
N
X = S: Y = CH
1 R
%)
H
0
T
19
: R = H
R
(
F
)
1
H
O
O
,
20: R = OCH3
a
H
N
TBAF,
THF
)
21: R = OtBDMS
2
Me
F
120 °C,
R1
N
48 h
Cl
R2
R
N
N
NH2
F
Cl
Y
N
H
X
O
NH
S
22-46: X = NH, S
O
N
N
Cl
Y = CH, N
N
N
N
N
H
c)
SO2Ph
N
H
O
47
49
O
F
Cl
NH4HCOO,
PdC,
MeOH,
THF,
OH
N
S
CH3
N
N
N
N
N
N
H
N
48
51
H
H
O
O
Scheme 1. Synthetic path for preparation of (4-(arylamino)-7H-pyrrolo[2,3-d]pyrimidin-6-yl)(1H-indol-2-yl)methanones and (1H-indol-2-yl)(4-(phenylamino)thieno
[2,3-d]pyrimidin-6-yl)methanones. For definitions of X, R, R1 and R2 see Tables 1 and 2. Conditions: (a) 2-butanone, 2 d; (b) n-butanol, 12 h; (c) (1) NaH, THF, CH3I, (2) NaOH
(10%), MeOH, THF. Formulas for 15a0–15e0show the lithiated species corresponding to indole-derivatives 15a–15e.
Modifications of the arylamine system itself are of minor influ-
ence. Replacement by a lipophilic substituent in position 3 of the
arylamino-system, however, seems to enhance activity, as is dem-
onstrated by compounds 23, 24, 25, and 26 in comparison with 22,
28, 29 or 30 for X = N. Substituents at C-5 of ring D have no effect
on the potency towards EGFR in this series, but reduce the activity
at ErbB2 as shown by comparison of data of 25 with those of 26.
Modifications of the ring D slightly reduce the inhibition at EGFR
(27 vs 26). In the (substituted) benzylamino compounds 31 and
series with X = N, the most active compounds 42 and 44 com-
pletely inhibit EGFR at 1.0 M. The additional 4-fluorine substitu-
l
ent of the arylamino-system of 44 in comparison with 42,
enhanced the activity, as shown by the IC50 values (16.6 nM of
42 and 5.54 nM of 44). To draw a comparison: the IC50 values of
the reference compounds erlotinib and lapatinib are 3.10 and
8.90 nM, respectively.
Thus, further modifications of 44 were performed, regar-
ding the heterocycle C, the substituent R2 in the benzene ring D,
and the alkylation of the arylamino-substituent R1. Modification
of the substituents of the ring D (H replaced by OH or OCH3)
was well tolerated for OCH3 (compound 46; IC50 EGFR = 5.93 nM)
or caused enhanced activity at VEGFR in case of 45. However, as
can be seen for 46, N-alkylation in the arylamino substituent re-
duced the activity significantly (51). Exchange of N for S in the ring
32, an additional a-methyl-group seems to be of benefit.
By introduction of sulfur instead of nitrogen in the ring B, in
nearly all cases the activities at EGFR were increased (e.g. com-
pound 34 vs 24, 36 vs 28, 37 vs 29, or 38 vs 31). Taking into account
the additional influence of substitution patterns of the arylamino
substituent, which in general follows the trend discussed for the

128
T. Beckers et al. / Bioorg. Med. Chem. 20 (2012) 125–136
Cl
N
PdCl2(PPh3)2
Cl
O
N
(HO)2B
K2HPO4 x 3H2O
Toluene, 110°C
X
S
14
52: X = O
53
: X = S
F
Cl
F
Cl
56
NH2
Cl
S
N
N
NH
S
N
N
X
O
X
O
54: X = O
55
57: X = O
58
: X = S
: X = S
Scheme 2. Synthesis of 57 and 58, respectively.
C was well tolerated in 56, introduction of oxygen in the case of 55,
however, was not.
Of compounds 42 and 45, showing IC50 values of 16.6 0.4 and
24.0 0.2 nM, respectively, as well as most potent inhibition of
ErbB2 protein kinase, EGFR autophosphorylation in intact A431
cells was determined. This phosphorylation was inhibited with
structure led to a series of novel compounds, which inhibit EGFR
in biochemical assays in the nanomolar range. These compounds
also potently inhibit EGFR autophosphorylation in intact A431 cells
with IC50 values in the submicromolar range. As shown by the sen-
sitivity profile in a panel of 42 human tumor cell lines, the novel
compounds are similar to lapatinib as expressed by the Spearman
rank coefficient (rho 0.6–0.67). This suggests the biological activi-
ties of the novel compounds to be close to the dual ErbB2/EGFR
inhibitor lapatinib.
IC50 values in a range of 0.3–1
respectively (see Fig. 2).
lM (42) and of 0.1–0.3 lM (45),
To study whether the kinase inhibition by the novel compounds
provokes growth inhibition in cancer cell lines, we compared a
selection of the compounds exhibiting the most potent dual mode
of action, namely 42, 44, 45 and 46 with lapatinib and erlotinib in
42 sensitive and resistant lines. These new compounds exhibitited
4. Experimental section
4.1. General
at least 95% inhibition at EGFR at a concentration of 1 lM and in
addition an inhibition of at least 60% at ErbB2. The cell lines repre-
sent 15 different tumor entities: bladder, colorectal, head and neck,
liver, non-small cell lung, mammary, melanoma, ovarian, pancre-
atic, pleuramesothelioma, renal, sarcoma and urethral carcinoma.
The data of Table 3 show the sensitivity profile of 42, 44, 45 and
46 to be similar to those of lapatinib and erlotinib being represen-
tatives of the quinazoline class of tyrosine kinase inhibitors. The co-
lon cancer cell line DiFi and the head and neck cancer cell line Cal27
were the most sensitive to lapatinib and to 42, 44, 45, and 46. The
cell lines most sensitive for erlotinib were DiFi, Cal27, the gastric
cancer cell line GXF 251L and the non-small cell lung adenocarci-
noma line LXFA 629L. Sensitivity towards EGFR inhibition has been
published for all four tumor models.12,13 This may be due to ampli-
fied EGFR (copy numbers measured by SNP6.0 array or FISH).14
By comparing the activity profiles of the new compounds in all
42 cell lines tested with those of erlotinb and lapatinib by a Spear-
man rank correlation, a higher score of similarity to lapatinib com-
pared erlotinib was found. For lapatinib a Spearman rank coefficient
of rho = 0.67 (42), rho = 0.67 (45), and rho = 0.60 (44) was found.
For erlotinib, rho = 0.51 was found for 45, and rho 60.4 for 42, 44,
and 46. This suggests the biological activities of the novel com-
pounds to be closer to those of the dual ErbB2/EGFR inhibitor lapat-
inib than to erlotinib, a very potent EGFR specific inhibitor.
Comparing their biological activities, 42, 44, and 45 revealed
higher similarity between each other (rho 0.76–0.85) than with
46 (rho 0.46–0.63).
NMR spectra were recorded with a Bruker Avance 300 MHz
spectrometer at 300 K, using TMS as an internal standard. IR spec-
tra (KBr) were measured with a Bruker Tensor 27 spectrometer;
melting points were determined with a Büchi B545 device. MS
spectra were measured with a Finnigan MAT 95 (EI, 70 eV) or with
a Finnigan Thermo Quest TSQ 7000 (ESI). All reactions were carried
out under nitrogen. Elemental analyses were performed by the
Analytical Laboratory of the University of Regensburg and are in
a range of 0.4% of the calculated values if not stated otherwise.
Chemical names were created using ChemDraw Ultra 10.0
software.
4.2. Procedures
4.2.1. 4-Chloro-7-(phenylsulfonyl)-7H-pyrrolo[2,3-d]pyrimidine
(9)10
89% Yield; colourless crystals, mp 169–170 °C; 1H NMR
(300 MHz, CDCl3): d 6.73 (d, J = 4.1 Hz, 1H), 7.55 (t, J = 7.5 Hz,
2H), 7.65 (t, J = 7.6 Hz, 1H), 7.80 (d, J = 4.1 Hz, 1H), 8.22 (d,
J = 7.4 Hz, 2H), 8.78 (s, 1H), ppm. MS (EI+): 293.1 ([M]+ꢀ, 13; 35Cl),
229.2 ([MꢁSO2]+, 24; 35Cl), 141.1.([MꢁC6H5SO2]+, 27; 35Cl), 77.1
([C6H5]+, 100). Anal. (C12H8ClN3O2S; 293.73): C, H, N.
4.2.2. 4-Chloro-7-(phenylsulfonyl)-7H-pyrrolo[2,3-d]pyrimidine
-6-carboxylic acid (11)
Preparation analogous to Lit.15,16 as follows: 4-Chloro-7-(phen-
ylsulfonyl)-7H-pyrrolo[2,3-d]pyrimidine (9) (10 g, 34.0 mmol) was
dissolved in dry THF (300 mL) under nitrogen, cooled to ꢁ78 °C,
n-butyllithium (1.1 equiv, 23.4 mL 1.6 M in hexane) was added
and the mixture stirred at ꢁ78 °C for 4 h. Dry CO2 gas was intro-
duced into the solution for 2 h, then the mixture was allowed to
3. Conclusions
Combining structural elements of the quinazoline class of
known tyrosine kinase inhibitors with the bisindolylmethanone

T. Beckers et al. / Bioorg. Med. Chem. 20 (2012) 125–136
129
Table 1
Influence of modifications in the ring B on inhibition of EGFR, ErbB2, and VEGFR2 by test compounds at a concentration of 1.0
l
M. Data were determined at least as duplicates and
are given as mean values standard deviation of the mean value. For compounds exhibiting inhibition >90%, IC50 values were determined in addition
R2
R1
N
D
A
C
B
X
N
N
H
O
No. R1
R2
X
EGFR inhibition in % at c = 1.0
lM
ErbB2 inhibition in % at c = 1.0
lM
VEGFR2 inhibition in % at c = 1.0 lM
48
H
OH
N
0
0
0
22
33
Cl
H
H
N
S
68
80
5
0
24
46
3
2
47
19
2
6
HN
23
44
F
H
H
N
S
88
3
22
80
5
0
47
15
2
7
100 0 IC50 = 5.54 nM
HN
HN
HN
Cl
24
34
H
H
N
S
83
1
70
60
3
2
5
9
3
5
93 1 IC50 = 18.7 nM
25
35
H
H
N
S
88
73
1
2
52
75
1
1
10
0
7
10
I
I
26
27
OCH3
N
88
77
0
1
25
20
6
3
17
1
HN
O
N
H
N
N
NH
—
—
20
22
3
6
N
O
I
NH
28
36
Br
H
H
N
S
50
82
1
1
14
51
6
3
46 8
HN
F
29
37
H
H
N
S
77
2
13
57
2
2
53
29
3
5
91 1 IC50 = 18.5 nM
O
F
HN
HN
Cl
OH
30
H
N
57
4
7
3
5
1
31
5
31
38
H
H
N
S
55
77
2
2
13
17
5
2
H
N
38 20
32
39
H
H
N
S
82
88
2
3
37
61
1
3
28
5
3
3
H3C
HN
NH
CF3
40
H
S
43
3
34
6
24
6
(continued on next page)

130
T. Beckers et al. / Bioorg. Med. Chem. 20 (2012) 125–136
Table 1 (continued)
No. R1
R2
H
X
S
EGFR inhibition in % at c = 1.0
lM
ErbB2 inhibition in % at c = 1.0
lM
VEGFR2 inhibition in % at c = 1.0 lM
41
79
1
42
9
29
20
1
4
HN
CF3
42
H
H
S
S
99 1 IC50 = 16.6 nM
96 2 IC50 = 43.7 nM
73 4.
HN
Cl
F
43
50
1
45
1
HN
Table 2
Inhibition of EGFR, ErbB2 and VEGFR2 by compounds 44–56 at a concentration of 1.0
l
M. Data were determined at least as duplicates and are given as mean values standard
deviation of the mean value.
Cl
F
R2
R1
N
N
N
D
A
C
X
B
S
O
No.
R1
R2
X
EGFR inhibition in % at c = 1.0
lM
ErbB2 inhibition in % at c = 1.0
l
M
VEGFR2 inhibition in % at c = 1.0 lM
44
45
46
51
55
56
H
H
H
CH3
H
H
NH
NH
NH
NH
O
100 0 IC50 = 5.54 nM
98 0 IC50 = 24 nM
96 1 IC50 = 5.93 nM)
80
87
60
83
88
76
0
0
2
1
3
2
15
10
55
23
51
22
7
1
8
5
5
3
OH
OCH3
H
H
H
22
8
85 14
H
S
99 1 IC50 = 10.8 nM
IC50 = 3.10 nM
IC50 = 8.90 nM
Erlotinib 3
Lapatinib 5
IC50 = 300 nM
IC50 = 60.0 nM
IC50 = 820 nM
IC50 >10
lM
(ESI+): 379.0 ([MH++CH3CN]+,53; 35Cl), 337.9 ([M+H; 35Cl]+, 100).
Anal. (C12H8ClN3O3S; 337.74): C, H, N.
EGF
+
0
-
+
+
+
1
+
+
+
pEGFR
EGFR
anti-PY
4.2.3. 4-Chlorothieno[2,3-d]pyrimidine-6-carboxylic acid (12)
4-Chlorothieno[2,3-d]pyrimidine (10) (5.80 g, 34.0 mmol) was
dissolved in dry THF (300 mL) under nitrogen, cooled to ꢁ78 °C,
n-butyllithium (1.1 equiv, 23.4 ml, 1.6 M in hexane) was added
and the mixture stirred at ꢁ78 °C for 4 h. An excess of solid carbon
dioxide was added and the mixture allowed to warm up to room
temperature overnight. The solvents were removed under reduced
pressure, and the remaining solid was suspended in water
(300 mL). The suspension was filtered over a pad of celite and
the clear solution was acidified with conc. HCl under stirring. The
precipitating colourless crystals were collected by filtration and
dried. 65% yield; colourless crystals; mp 195 °C (decomp.); 1H
NMR (300 MHz, DMSO-d6): d 9.01 (s, 1H), 8.07 (s, 1H) ppm. MS
(ESI+): 256 ([MH++CH3CN]+, 100; 35Cl), 215 ([M+H]+, 12; 35Cl). IR
anti-EGFR
0
10 3
0.3 0.1 0.03
Concentration (µM)
Figure 2. Inhibition of EGFR autophosphorylation in intact A431 cells by compound
45. Cells were serum-starved over night (0.5% FCS), treated with the inhibitor at the
indicated concentrations for 1 h, and then stimulated by 100 ng/ml EGF for 10 min
(or left unstimulated, as indicated). Total cell lysates were analyzed by immuno-
blotting to detect tyrosine phosphorylation (upper panel, anti-PY), then stripped
and reprobed for EGFR (lower panel). Note dose-dependent stabilization of EGFR
levels by inhibitor treatment, as revealed in the anti-EGFR blot.
(KBr):
H, N.
m
3442, 3091, 1724 cmꢁ1. Anal. (C7H3ClN2O2S; 214.63): C,
warm up to room temperature overnight. The solvents were re-
moved in vacuo, and the remaining solid was suspended in water
(300 mL). The suspension was filtered over a pad of Celite, the clear
solution acidified with HCl by stirring; the precipitating colorless
crystals were removed by filtration and dried in vacuo. If neces-
sary, crystallization from EtOH/H2O was performed in addition.
68% yield; colorless crystals, mp 176 °C; 1H NMR (300 MHz,
4.2.4. 4-Chloro-7-(phenylsulfonyl)-7H-pyrrolo[2,3-d]pyrimidine
-6-carbonyl chloride (13)
4-Chloro-7-(phenylsulfonyl)-7H-pyrrolo[2,3-d]pyrimidine-6-
carboxylic acid (11) (6.75 g; 20 mmol) was suspended in SOCl2
(50 mL) and refluxed for 3 h. Excess of SOCl2 was removed under
reduced pressure and the product dried in vacuo. Quantitative
yield; colorless crystals, mp 124 °C; 1H NMR (300 MHz, CDCl3): d
DMSO-d6):
d 7.73 (s, 1H), 7.73 (t, J = 7.5 Hz, 2H), 7.82 (t,
J = 6.7 Hz, 1H), 8.31 (d, J = 7.1 Hz, 2H), 8.31 (s, 1H) ppm. MS

T. Beckers et al. / Bioorg. Med. Chem. 20 (2012) 125–136
131
Table 3
4.2.6. 1-(Phenylsulfonyl)-1H-indoles
Sensitivity profile of selected {(1H-indol-2-yl)(4-(phenylamino)thieno[2,3-d]pyrimi-
din-6-yl}methanones (44, 42, 46 and 45) towards a panel of different tumor cell lines
by comparison with lapatinib and erlotinib (TarcevaÒ).
65.1 mmol starting material were dissolved in dry THF (300 mL)
under nitrogen, cooled to 0 °C and 1.1 equiv NaH (71.6 mmol)
2.86 g, 60% oil dispersion), were added in portions. Then the mix-
ture was stirred for an additional h. Benzene sulfonylchloride
(71.6 mmol; 9.14 mL) was added drop wise, the mixture allowed
to warm to room temperature and stirred for 4 h. The suspension
was poured into water (1.2 L) by stirring. After 1 h, the precipitated
product was removed by filtration, dried and dissolved in the nec-
essary amount of CH2Cl2. An aliquot of heptane was added, half of
the solvent removed under reduced pressure and the product, pre-
cipitating as colorless crystals, removed by filtration.
IC50 values [μM] in a panel of 42 human tumor cell lines (mean of 3
independent experiments)
Cell line
BXF
BXF
BXF
1218L
1352L
T24
11.77
11.53
9.65
8.36
0.235
13.14
4.62
5.35
1.48
11.48
0.530
7.18
13.46
5.79
4.21
2.87
7.73
3.51
5.80
4.83
7.70
4.85
11.37
10.13
3.35
13.44
11.11
6.12
8.12
6.06
2.99
3.68
4.55
6.86
9.46
9.49
7.67
7.77
9.10
9.38
10.95
4.40
96.0
87.8
13.9
16.0
0.140
65.8
100
9.13
11.30
11.43
10.77
1.39
21.5
15.9
23.7
28.9
1.8
15.4
24.8
17.8
11.7
1.0
6.34
5.97
9.56
7.58
0.94
7.69
13.73
6.01
4.09
7.26
1.28
7.04
6.00
3.25
4.36
2.65
6.02
4.87
4.09
4.22
7.65
5.89
6.28
6.02
4.07
9.08
9.08
8.85
7.95
5.61
2.96
3.01
5.45
9.59
8.19
9.23
4.87
5.87
3.37
8.20
10.05
3.45
CXF 269L
CXF DIFI
CXF HCT116
CXF HT29
CXF RKO
GXF 251L
GXA MKN45
HNXF CAL27
LIXF 575L
4.2.6.1. 1-(Phenylsulfonyl)-1H-indole (15a).
from 1H-indole as described above. 76% yield.16
Preparation
8.39
18.9
17.7
18.1
4.3
12.1
9.8
7.83
100
7.29
15.8
3.9
4.2.6.2.
5-Methoxy-1-(phenylsulfonyl)-1H-indole
(15b)15
.
.
0.369
76.7
0.239
47.2
74.3
1.78
100
2.76
12.54
2.61
20.2
3.0
38.2
1.6
Compound 15b was prepared as described above.
8.15
18.1
17.7
4.7
9.3
4.2.6.3. 5-(Benzyloxy)-1-(phenylsulfonyl)-1H-indole (15c)15
Compound 15c was prepared as described above.
LXF
H460
5.82
15.0
7.1
LXFA 289L
6.68
LXFA 526L
10.72
4.88
13.6
6.0
28.5
5.0
LXFA 629L
0.660
34.6
17.4
25.0
100
4.2.6.4. 5-(tert-Butyldimethylsilyloxy)-1-(phenylsulfonyl)-1H-
LXFL 1121L
LXFL 529L
9.77
16.0
7.6
9.0
indole (15d)1.
Compound 15d was prepared as described
8.61
14.8
4.2
above.
MAXF 401NL
MAXF MCF7
MAXF MDA231
MEXF 1341L
MEXF 276L
MEXF 462NL
OVXF 899L
OVXF OVCAR3
PAXF 1657L
PAXF 546L
3.89
7.3
6.96
12.5
23.4
20.1
27.1
24.0
13.9
15.3
47.6
25.1
14.8
14.0
9.2
11.3
17.1
14.4
31.0
16.7
19.0
24.6
24.6
14.1
28.4
11.1
19.4
32.5
46.3
62.9
36.0
26.7
30.4
21.1
10.2
30.1
55.0
4.9
34.5
30.1
18.6
100
8.04
4.2.6.5. 1-(Phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (15e).
Compound 15e was prepared from 1H-pyrrolo[2,3-b]pyridine
as described above. 90% yield; colorless crystals, mp 129 °C; 1H
NMR (300 MHz, CDCl3): d 6.59 (d, J = 3.8 Hz, 1H), 7.16 (dd,
J1 = 4.8 Hz, J2 = 7.8 Hz, 1H) 7.43–7.48 (m, 2H), 7.55 (t, J = 7.4 Hz,
1H), 7.72 (d, J = 4.1 Hz, 1H), 7.83 (dd, J1 = 1.6 Hz, J2 = 7.7 Hz, 1H),
8.17–8.21 (m, 2H), 8.42 (dd, J1 = 1.5 Hz, J2 = 4.8 Hz, 1H) ppm. MS
(EI+): 258.0 ([M]+ꢀ, 41), 194.1 ([M-SO2]+ꢀ, 94.3), 117.1.([M–
C6H5SO2]+, 24.9), 77.1 ([C6H5]+, 100) Remark: The observed loss
of SO2 in the mass spectra is in accordance with Wiegrebe, W.;
Schlunegger, U. P.; Herrmann, E. G., Umlagerungen von Prolinder-
ivaten. Pharm. Acta. Helv. 1974, 49, (7/8), 253–258. Anal.
(C13H10N2O2S; 258.30): C, H, N.
10.85
10.21
8.11
8.77
100
5.56
8.45
64.3
10.0
75.8
100
17.62
11.63
7.01
PAXF PANC1
PRXF 22RV1
PRXF DU145
PRXF LNCAP
PRXF PC3M
5.48
0.966
6.63
17.2
76.7
31.6
36.4
76.7
45.6
10.0
21.0
100
4.36
6.11
11.3
14.1
49.7
30.7
31.0
20.0
26.4
13.1
19.3
24.8
8.6
5.77
PXF
PXF
PXF
1118L
1752L
698L
17.95
13.45
14.28
9.05
RXF 1781L
RXF 393NL
RXF 486L
11.76
5.50
4.2.7. Preparation of (4-chloro-7-(phenylsulfonyl)-7H-pyrrolo
[2,3-d]pyrimidin-6-yl)(1-(phenylsulfonyl)-1H-indol-2-yl)
methanones 16–18
SXF
SXF
SAOS2
TE671
11.43
14.05
4.42
UXF 1138L
geomean IC50
24.5
The respective N-phenylsulfonated indole derivative 15
(10 mmol) was dissolved in dry THF (50 mL) under nitrogen,
cooled to ꢁ78 °C, n-butyllithium (1.1 equiv, 6.88 mL 1.6 M in
hexane) was added and the mixture stirred at ꢁ78 °C for 1 h. A
solution of 4-chloro-7-(phenylsulfonyl)-7H-pyrrolo[2,3-d]pyrimi-
dine-6-carboxylic acid chloride (13) (10 mmol, 3.56 g) in THF
(50 mL) was cooled to ꢁ78 °C, was added rapidly in one portion
and the mixture allowed to stir for half an hour at ꢁ78 °C. The solu-
tion was poured into satd. NH4Cl solution under stirring, the organ-
ic layer separated, dried (Na2SO4), the solvent removed under
reduced pressure and the remaining solid dissolved in a small
amount of CH2Cl2. The solution was filtered and added to diethyl
ether by stirring. The precipitated product was removed by filtra-
tion and crystallized twice form CH2Cl2/diethyl ether. Alternatively
purification by CC (SiO2; CH2Cl2/ethyl acetate 10:1) is possible.
5.87
21.6
7.70
15.0
14.9
5.48
-fold mean IC50
1/32 1/16 1/8 1/1 1/2
sensitive cell lines
1
2
4
8
16 32
resistant cell lines
IC50 values (lM) in a panel of 42 human tumor cell lines (mean of three indepen-
dent experiments).
7.26 (s, 1H), 7.62 (t, J = 7.4 Hz, 2H), 7.65 (d, J = 8.0 Hz, 2H), 7.74 (t,
J = 6.9 Hz, 2H), 9.00 (s, 1H) ppm. EI-MS (70 eV) m/z (%) = 355 [M]+ꢀ
(0.3), 320 [M-Cl.]+ (2.7), 291 [M-SO2]+. (1.9), 180 (100). The product
was used without further purification.
4.2.5. 4-Chlorothieno[2,3-d]pyrimidine-6-carbonyl chloride (14)
4-Chlorothieno[2,3-d]pyrimidine-6-carboxylic acid (12) (2.15 g,
20.0 mmol) was suspended in SOCl2 (50.0 mL) and heated to reflux
for 3 h. Excess of SOCl2 was removed under reduced pressure, the
product dried in vacuo and used without further purification.
Quantitative yield; beige powder, mp 88.2 °C; 1H NMR (300 MHz,
CDCl3): d 9.01 (s, 1H), 8.34 (s, 1H) ppm. EI-MS (70 eV) m/z (%) =
235.9 [M]+ꢀ (1.58), 233.9 [M]+ꢀ (9.9), 232 [M]+ꢀ (13.7), 197 [MꢁCl.]+
(100).
4.2.7.1. (4-Chloro-7-(phenylsulfonyl)-7H-pyrrolo[2,3-d]pyrimi-
din-6-yl)(1-(phenylsulfonyl)-1H-indol-2-yl)methanone
(16a).
17% yield; colorless crystals; mp 261–263 °C; 1H NMR (300 MHz,
CDCl3): d 7.07 (s, 1H), 7.36–7.41 (m, 2H), 7.53–7.75 (m, 8H), 8.13
(d, J = 7.8 Hz, 2H), 8.27 (d, J = 8.8 Hz, 1H), 8.48 (d, J = 7.8 Hz, 2H),
8.96 (s, 1H) ppm. MS (ESI+): 576.9 ([M+H]+, 100; 35Cl). Anal.
(C27H17ClN4O5S2; 577.03): C, H, N.

132
T. Beckers et al. / Bioorg. Med. Chem. 20 (2012) 125–136
4.2.7.2. (4-Chloro-7-(phenylsulfonyl)-7H-pyrrolo[2,3-d]pyrimi-
din-6-yl)(1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine-2-yl)-
8.01–7.94 (m, 3H), 7.78–7.71 (m, 1H), 7.67–7.58 (m, 3H), 7.20 (d,
J = 2.5 Hz, 1H), 7.11 (dd, J = 2.5, 8.9 Hz, 1H), 0.96 (s, 9H), 0.20 (s,
6H) ppm. MS (ESI+): 625 ([MH+CH3CN]+, 100; 35Cl), 584 ([MH]+,
23). IR (KBr):
(C27H26ClN3O4S2Si; 584.18): C, H, N.
methanone (16b)
.
24% yield; beige crystals; mp 227 °C
decomp.; 1H NMR1H NMR (300 MHz CDCl3): d 7.24 (s, 1H), 7.29
(s, 1H), 7.32–7.36 (m, 1H), 7.59–7.70 (m, 5H), 7.72–7.78 (m, 1H),
8.01 (dd, 1H, J1 = 7.9 Hz, J2 = 1.6 Hz), 8.49 -8.56 (m, 4H), 8.70 (dd,
1H, J1 = 4.6 Hz, J2 = 1.6 Hz), 8.98 (s, 1H) ppm. MS (ESI+): 578.0
([M+H]+, 100; 35Cl). Anal. (C26H16ClN5O5S2: 578.02): C, H, N.
m . Anal. Calcd for
3069, 2940, 1648 cmꢁ1
4.2.9. Preparation of (1H-indol-2-yl)(4-(phenylamino)-1H-
indol-2-yl)methanone-derivatives 22–32, general procedure
A mixture of (4-chloro-7-phenylsulfonyl)-7H-pyrrolo[2,3-d]-
pyrimidin-6-yl)(1-(phenylsulfonyl)-1H-indol-2-yl)methanone (16)
(0.7 mmol) and the suitably substituted aniline (1.0 mmol) in
16 g n-butanol was heated to 120 °C under nitrogen atmosphere
for 10–12 h, cooled to rt and poured into light petroleum
(100 mL). The precipitated product was filtered and the solid added
to a mixture of THF: MeOH (50 mL: 50 mL) and 50 mL 10% NaOH
(in water). The mixture was refluxed for 6 h. The organic solvent
was removed under reduced pressure, the precipitating product
was removed by filtration, dried and stirred in an organic solvent
given for the respective product.
4.2.7.3. (4-Chloro-7-(phenylsulfonyl)-7H-pyrrolo[2,3-d]pyrimi-
din-6-yl)(5-methoxy-1-(phenylsulfonyl)-1H-indol-2-yl)meth-
anone (17).
51% yield; colorless crystals; mp 245–249 °C; 1H
NMR (300 MHz CDCl3): d 3.85 (s, 3H), 7.01 (s, 1H), 7.02 (d,
J = 2.5 Hz, 1H), 7.20 (dd, J1 = 2.6 Hz, J2 = 9.2 Hz, 1H), 7.34 (s, 1H),
7.52 (t, J = 7.5 Hz, 2H), 7.95–7.65 (m, 3H), 7.72 (t, J = 7.4 Hz, 1H),
8.06 (d, J = 7.1 Hz, 2H), 8.17 (d, J = 9.3 Hz, 1H), 8.49 (d, J = 7.5 Hz,
2H), 8.94 (s, 1H) ppm. MS (ESI+): 607.0 ([M+H]+, 100; 35Cl). Anal.
(C28H19ClN4O6S2; 607.06): C, H, N.
4.2.7.4. (5-(Benzyloxy)-1-(phenylsulfonyl)-1H-indol-2-yl)(4-chl
oro-7-(phenylsulfonyl)-7H-pyrrolo[2,3-d]pyrimidin-6-yl) meth-
4.2.9.1. (4-(4-Chlorophenylamino)-7H-pyrrolo[2,3-d]pyrimidin-
anone (18).
26% yield; colorless crystals; mp 217.3–222.4 °C;
6-yl)(1H-indol-2-yl)methanone (22).
92% yield from ethyl
1H NMR (300 MHz DMSO-d6): d 5.17 (s,2H), 7.34–7.49 (m, 7H),
7.52 (s, 1H), 7.66–7.90 (m, 7H), 8.05 (d, J = 8.1 Hz, 2H), 8.14 (d,
J = 9.9 Hz, 1H), 8.29 (d, J = 7.4 Hz,2H), 9.04 (s, 1H) ppm. MS (ESI+):
683 ([M+H]+, 100; 35Cl). Anal.(C34H23ClN4O6S2; 683.15): C, H, N.
acetate; yellow crystals; mp 340 °C decomp.; 1H NMR
(300 MHz,DMSO-d6): d 7.14 (t, J = 7.4 Hz, 1H), 7.33 (t, J = 7.7 Hz,
1H), 7.45 (d, J = 8.7 Hz, 2H), 7.53 (d, J = 7.4 Hz, 2H), 7.78 (d,
J2 = 8.0 Hz, 1H), 7.99 (d, J = 9 Hz, 2H), 8.04 (s, 1H), 8.44 (s, 1H),
9.91 (s, 1H) 12.03 (s, 1H), 12.48 (s, 1H), ppm. MS (ESI+): 428.9
([MH++CH3CN]+, 56; 35Cl), 387.9 ([M+H]+, 100; 35Cl). Anal. (C21H14
ClN5O ꢂ 3/2 H2O; 387.82): C, H, N.
4.2.8. Preparation of (4-chlorobenzo[b]thiophen-2-yl)
(1-(phenylsulfonyl)-1H-indol-2-yl)methanones 19–21
The N-phenylsulfonated indole derivate 15a–15d (10.0 mmol)
was dissolved in dry THF (50 mL) under nitrogen and cooled to
ꢁ78 °C. n-Butyllithium (1.1 equiv., 6.88 mL, 1.6 M in hexane) was
added and the mixture stirred at ꢁ78 °C for 1 h. A solution of 4-
chlorothieno[2,3-d]pyrimidine-6-carbonyl chloride (14) (2.33 g,
10.0 mmol) in THF (50.0 mL), cooled to ꢁ78 °C, was added in one
portion and the mixture was stirred for half an hour at ꢁ78 °C.
The solution was poured into satd. NH4Cl solution under stirring.
The organic layer was separated, dried (Na2SO4) and the solvent
was removed under reduced pressure. The remaining solid was
dissolved in a small amount of CH2Cl2. The solution was filtered
and added to diethyl ether by stirring. The precipitated product
was collected by filtration and precipitated twice from CH2Cl2/
diethyl ether as described above.
4.2.9.2.
]pyrimidin-6-yl)(1H-indol-2-yl)methanone (23).
from ethyl acetate; yellow crystals; mp 377 °C decomp.; 1H NMR
(300 MHz, DMSO-d6): 6.84 (t, J = 6.85 Hz, 1H), 7.01 (t,
J = 6.87 Hz, 1H), 7.31 (t, J = 9.18 Hz, 1H), 7.39 (s, 1H), 7.51 (d,
J = 8.2 Hz, 1H), 7.56–7.61 (m, 2H), 7.85 (s, 1H), 8.19 (s, 1H), 8.43
(d, J = 5.49 Hz, 1H) ppm. MS (ESI+): 446.9 ([M+CH3CN]+, 64),
405.9 ([M+H]+, 100). Anal. (C21H13ClFN5Ox2/3 H2O; 405.81): C, H,
N.
(4-(3-Chloro-4-fluorophenylamino)-7H-pyrrolo[2,3-d
99% yield
d
4.2.9.3.
(4-(3-Ethynylphenylamino)-7H-pyrrolo[2,3-d]pyrimi-
61% from ethyl-
din-6-yl)(1H -indol-2-yl)methanone: (24).
acetate; yellow crystals; mp 390 °C decomp.; 1H NMR (300 MHz,
DMSO-d6): d 4.23 (s, 1H), 7.12–7.20 (m, 2H), 7.34 (t, J = 7.4 Hz,
1H), 7.41 (t, J = 7.9 Hz, 1H), 7.54 (d, J = 7.1 Hz, 2H), 7.79 (d,
J = 8.0 Hz, 1H), 7.95 (d, J = 8.0 Hz, 1H), 8.05 (s, 1H), 8.19 (s, 1H),
8.47 (s, 1H), 9.87 (s, 1H), 12,04 (s, 1H), 12,71 (s, 1H), ppm. MS
(ESI+): 418.9 ([M+CH3CN]+, 26), 377.9 ([M+H]+, 100). Anal.
(C23H15 N5O; 377.40): C, H, N.
4.2.8.1. (4-Chlorothieno[2,3-d]pyrimidin-6-yl)(1-(phenylsulfo-
nyl)-1H-indol-2-yl)methanone (19).
53% yield; beige crys-
tals; mp 224–225 °C; 1H NMR (300 MHz, DMSO-d6): d 9.15 (s,
1H), 8.26 (s, 1H), 8.11 (d, J = 8.2 Hz, 1H), 8.07–8.01 (m, 2H), 7.83–
7.71 (m, 3H), 7.69–7.56 (m, 3H), 7.42 (t, 7.6 Hz, 1H) ppm. MS
(ESI+): 495 ([M+CH3CN]+, 100; 35Cl), 454 ([M+H]+, 32; 35Cl). IR
(KBr):
m
3090, 1643 cmꢁ1. Anal. (C21H12ClN3O3S2; 453.92): C, H, N.
4.2.9.4. (1H-Indol-2-yl)(4-(3-iodophenylamino)-7H-pyrrolo[2,3-
d]pyrimidin-6-yl)methanone (25).
83% yield from ethylace-
4.2.8.2.
4-Chlorothieno[2,3-d]pyrimidin-6-yl)(5-methoxy-1-
tate; yellow crystals; mp 365 °C decomp.; 1H NMR (300 MHz,
DMSO-d6): d 7.12–7.22 (m, 2H), 7.33 (t, J = 7.5 Hz, 1H), 7.43 (d,
J = 7.9 Hz, 1H), 7.53 (d, J = 7.9 Hz, 2H), 7.79 (d, J = 7.9 Hz, 1H),
8.00–8.04 (m, 2H), 8.42 (s, 1H), 8.47 (s, 1H), 9.84 (s, 1H) 12.03 (s,
1H), 12.71 (s, 1H), ppm. MS (ESI+): 521.0 ([MH++CH3CN]+, 63),
479.9 ([M+H]+, 100). Anal. (C21H14 IN5O; 479.27): C, H, N..
(phenylsulfonyl)-1H-indol-2-yl)methanone (20).
43% yield;
light yellow powder; mp 125–130 °C; 1H NMR (300 MHz, DMSO-
d6): d 9.14 (s, 1H), 8.17 (s, 1H), 8.03–7.91 (m, 3H), 7.77–7.70
(m, 1H), 7.66–7,58 (m, 3H), 7.26 (d, J1 = 2.5 Hz, 1H), 7.18 (dd,
J1 = 2.5 Hz, J2 = 9.2 Hz, 1H), 3.79 (s, 3H) ppm. MS (ESI+): 525
([MH+CH3CN]+, 100; 35Cl), 484 ([MH]+, 20). IR (KBr):
m
2923,
1647, 1525 cmꢁ1
483.95): C, H, N.
.
Anal. Calcd for (C22H14ClN3O4S2 ꢂ CH3OH;
4.2.9.5. (4-(3-Iodophenylamino)-7H-pyrrolo[2,3-d]pyrimidin-6-
yl)(5-methoxy-1H-indol-2-yl)methanone (26).
65% yield
from CH2Cl2/MeOH; yellow crystals; mp 355.2–356.1 °C decomp.;
1H NMR (300 MHz, DMSO-d6): d 3.80 (s, 3H), 6.99–7.01 (m, 1H),
7.13–7.21 (m, 2H), 7.40–7.50 (m, 3H), 8.06–8.10 (m, 1H), 8.19 (s,
1H), 8.45–8.50 (m, 2H), 10.08 (s, 1H, exchangeable), 11.9 (s, 1H,
exchangeable), 12.65 (s, 1H, exchangeable) ppm. MS (ESI+):
4.2.8.3. (5-(tert-Butyldimethylsilyloxy)-1-(phenylsulfonyl)-1H-
indol-2-yl)(4-chlorothieno[2,3-d]pyrimidin-6-yl)methanone
(21).
NMR1H NMR (300 MHz, DMSO-d6): d 9.16 (s, 1H), 8.23 (s, 1H),
54% yield; light yellow powder; mp 204–205 °C; 1H

T. Beckers et al. / Bioorg. Med. Chem. 20 (2012) 125–136
133
509.9 ([M+H]+, 100). Anal. (C22H16 IN5O2 x.4/5 CH2Cl2; 509.30):
C, H, N.
4.2.10.1. (4-(4-Chlorophenylamino)thieno[2,3-d]pyrimidine-6-
yl)(1H-indol-2yl)methanone (33). (4-Chlorothieno[2,3-d]-
pyrimidine-6-yl)(1-(phenylsulfonyl)-1H-indol-2yl)methanone (19)
(0.70 g, 1.54 mmol) and 4-Chloroaniline (0.35 g, 2.74 mmol) were
suspended in 2-butanone (25 mL). The mixture was heated to re-
flux for 2 days. After cooling to room temperature the solvent
was removed under reduced pressure. The residue was dissolved
in a mixture of methanole (10 mL), THF (10 mL) and aqueous NaOH
(10%, 10 mL) and heated to reflux for 1.5 h. THF and methanole
were removed under reduced pressure and the residue was ex-
tracted with ethyl acetate. The organic layer was separated, dried
(Na2SO4) and the solvent removed under reduced pressure. The
precipitated product was collected by filtration and dried in vacuo.
62% Yield from CH2Cl2/MeOH; yellow crystals; mp 342–345 °C;
1H NMR (300 MHz, DMSO-d6): d 12.15 (s, 1H), 10.20 (s, 1H), 9.03 (s,
1H), 8.65 (s, 1H), 7.96–7.89 (m, 2H), 7.83 (d, J = 8.2 Hz, 1H), 7.70 (s,
1H), 7.58–7.47 (m, 3H), 7.38 (t, J = 7.4 Hz, 1H), 7.15 (t, J = 7.4 Hz,
1H) ppm. MS (ESI+): 446 ([MH++ CH3CN]+, 72; 35Cl), 405 ([M+H]+,
4.2.9.6. (4-(3-Iodophenylamino)-7H-pyrrolo[2,3-d]pyrimidin-6-
yl)(1H-pyrrolo[2,3-b]pyridin-2-yl)methanone
yield from ethyl acetate; yellow crystals; mp 365 °C decomp.; 1H
NMR (300 MHz, DMSO-d6): 7.16–7.25 (m, 2H), 7.42 (d,
(27).
60%
d
J = 7.7 Hz, 1H), 7.48 (s, 1H), 7.99–8.01 (m, 2H), 8.24 (d,
J = 8.01 Hz, 1H), 8.42 (s, 1H), 8.48 (s, 2H), 9.85 (s, 1H), 12.59 (s,
1H), 12.76 (s, 1H) ppm. MS (ESI+): 522.0 (MH++CH3CN]+, 64),
480.9 ([M+H]+, 80), 282.0 ([M+2H +2CH3CN]2+
(C20H13 IN6Ox.3/2 H2O; 480.26): C, H, N.
, 100). Anal.
4.2.9.7. (4-(4-Bromo-2-fluorophenylamino)-7H-pyrrolo[2,3-d]
pyrimidin-6-yl)(1H-indol-2-yl)methanone (28). 86% yield
from ethyl acetate; yellow crystals; mp 323 °C decomp.; 1H NMR
(300 MHz, DMSO-d6): d 7.14 (t, J = 7.5 Hz, 1H), 7.33 (t, J = 7.3 Hz,
1H), 7.45–7.54 (m, 3H), 7.69 (dd, J1 = 2.2 Hz, J2 = 10.1 Hz, 1H),
7.71 (d, J = 8.0 Hz, 1H), 7.85 (t, J = 8.6 Hz, 1H), 8.00 (s, 1H), 8.33
(s, 1H), 9.79 (s, 1H), 12.02 (s, 1H), 12.68 (s, 1H), ppm. MS (ESI+):
492.9 ([MH++CH3CN]+, 79), 451.8 ([M+H]+, 100). Anal (C21H13
BrFN5Ox ½ H2O; 450.26): C, H, N.
100; 35Cl). IR (KBr):
m
3428, 3324, 1617 cmꢁ1
.
Anal.
(C21H13ClN4OS ꢂ 0.33 CH2Cl2, 404.87): C, H, N.
4.2.10.2. (4-(3-Ethynylphenylamino)thieno[2,3-d]pyrimidin-6-
yl)(1H-indol-2-yl)methanone (34).
60% yield from metha-
4.2.9.8.
7H-pyrrolo[2,3-d]pyrimidin-6-yl)(1H-indol-2-yl)methanone
(29). 2-Chloro-4-(3-fluorobenzyloxy)aniline used was pre-
(4-(2-Chloro-4-(3-fluorobenzyloxy)phenylamino)-
nole; yellow crystals; mp 215–219 °C; 1H NMR (300 MHz, DMSO-
d6): d 12.15 (s, 1H), 10.17 (s, 1H), 9.02 (s, 1H), 8.68 (s, 1H), 8.07
(br s, 1H), 7.94–7.88 (m, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.72 (s, 1H),
7.54 (d, J = 8.2 Hz, 1H), 7.45, (t, J = 8.0 Hz, 1H), 7.40–7.33 (m, 1H),
7.29–7.24 (m, 1H), 7.16 (t, J = 7.5 Hz, 1H), 4.25 (s, 1H) ppm. MS
pared according to Lit..17 67% Yield from chloroform; yellow
crystals; mp 315 °C decomp.; 1H NMR (300 MHz, DMSO-
d6DMSO-d6): d 5.24 (s, 2H), 7.11–7.21 (m, 2H), 7.26–7.34 (m,
4H), 7.43–7.54 (m, 3H), 7.73–7.79 (m, 2H), 7.98 (s, 1H), 8.17 (d,
J = 2.5 Hz, 1H), 8.42 (s, 1H), 9.80 (s, 1H), 12.03 (s, 1H), 12.67 (s,
1H) ppm. MS (ESI+): 553 ([MH++CH3CN]+, 37), 512.1 ([M+H]+,
100). Anal.(C28H19ClFN5O2 11/10 CHCl3; 511.93): C, H, N.
(ESI+): 436 ([M+CH3CN]+, 26), 395 ([M+H]+, 100). IR (KBr):
m
3473, 3294 1593 cmꢁ1. Anal. Calcd for (C23H14N4OS ꢂ 1.33 CH3OH;
394,45): C, H, N.
4.2.10.3.
(1H-Indol-2-yl)(4-(3-iodophenylamino)thieno[2,3-
d]pyrimidin-6-yl)methanone (35).
47% yield; beige powder;
mp 249–254 °C; 1H NMR (300 MHz, DMSO-d6): d 12.14 (s, 1H),
10.13 (s, 1H), 9.01 (s, 1H), 8.67 (s, 1H), 8.31 (s, 1H), 7.97 (d,
J = 8.2 Hz, 1H), 7.82 (d, J = 8.1 Hz, 1H), 7.70 (br s, 1H), 7.53 (t,
J = 7.9 Hz, 2H), 7.37 (t, J = 7.7 Hz, 1H), 7.28–7.13 (m, 2H) ppm. MS
4.2.9.9. (4-(4-Hydroxyphenylamino)-7H-pyrrolo[2,3-d ]pyrimi-
din-6-yl)(1H-indol-2-yl)methanone: (30).
50% yield from
ethyl acetate; yellow crystals; mp 312 °C decomp.; 1H NMR
(300 MHz, DMSO-d6): d 6.92 (d, J = 8.2 Hz, 2H), 7.14 (t, J = 7.4 Hz,
1H), 7,33 (t, J = 7.3 Hz, 1H), 7.45–7.53 (m, 4H), 7.76 (d, J = 7.9 Hz,
1H), 8.34 (s, 2H), 9.81 (s, 1H), 11.19 (s, 1H), 12.05 (s, 1H), 13.22
(ESI+): 538 ([MH++CH3CN]+, 42), 497 ([M+H]+, 100). IR (KBr):
m
3447, 1622, 1591 cmꢁ1. Anal. Calcd for (C21H13IN4OS ꢂ 1.5 H2O;
(s, 1H) ppm. MS (ESI+): 410.9 (MH+
+
CH3CN]+, 34), 369.9
496.32): C, H, N.
([M+H]+, 100). Anal.(C21H15N5O2;369.38): C, H, N.
4.2.10.4. (4-(4-Bromo-2-fluorophenylamino)thieno[2,3-d]pyr-
imidin-6-yl)(1H-indol-2-yl)methanone (36).
68% yield; yel-
4.2.9.10.
(1H-indol-2-yl)methanone (31).
(4-(Benzylamino)-7H-pyrrolo[2,3-d]pyrimidin-6-yl)
low crystals from ethyl acetate; mp 277–278 °C; 1H NMR
(300 MHz, DMSO-d6): d 12.13 (s, 1H), 10.23 (s, 1H), 8.98 (s, 1H),
8.53 (s, 1H), 7.83–7.60 (m, 4H), 7.56–7.48 (m, 2H), 7.36 (t,
J = 7.2 Hz, 1H), 7.15 (t, J = 7.2 Hz, 1H) ppm. MS (ESI+): 508/510
70% yield from ethyl ace-
tate; yellow crystals; mp 320 °C decomp.; 1H NMR (300 MHz,
DMSO-d6): d 4.90 (d, J = 5,7 Hz, 2H), 7.12 (t, J = 7.5 Hz, 1H), 7.30–
7.36 (m, 2H), 7.38–7.43 (m, 2H), 7.48–7.53 (m, 4H), 7.73 (d,
J = 8.2 Hz, 1H), 8.22 (s, 1H), 8.41 (s, 1H), 10.19 (s, 1H), 12.06 (s,
1H), 13.34 (s, 1H) ppm. MS (ESI+): 409.0 ([MH++CH3CN]+, 20),
368.0 ([M+H]+, 100). Anal.(C22H17 N5O; 367.40): C, H, N.
([MH++CH3CN]+, 53), 469/467 ([M+H]+, 100). IR (KBr):
m 3440,
3323, 1617 cmꢁ1. Anal. Calcd for (C21H12BrFN4OS ꢂ 0.5 ethyl ace-
tate; 467.31): C, H, N.
4.2.10.5. (4-(3-Chloro-4-(3-fluorobenzyloxy)phenylamino)thie-
4.2.9.11.
(R)-(1H-Indol-2-yl)(4-(1-phenylethylamino)-7H-pyr-
30% yield from
no[2,3-d]pyrimidin-6-yl)(1H-indol-2-yl)methanone
(37).
rolo[2,3-d]pyrimidin-6-yl)methanone (32).
2-Chloro-4-(3-fluorobenzyloxy)aniline used was prepared accord-
ing to Lit.17 37 was obtained in 69% yield; yellow crystals from
methanole; mp 267–269 °C; 1H NMR (300 MHz, DMSO-d6): d
12.13 (s, 1H), 10.11 (s, 1H), 8.97 (s, 1H), 8.61 (s, 1H), 8.03 (d,
J = 2.5 Hz, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.74–7.66 (m, 2H), 7.56–
7.43 (m, 2H), 7.40–7.28 (m, 4H), 7.23–7.12 (m, 2H), 5.26 (s, 2H)
ethyl acetate; yellow crystals; mp 288 °C decomp.; 1H
NMR(300 MHz, DMSO-d6): d 1.16 (d, J = 7.1 Hz, 3H), 5.52–5.57
(m, 1H), 7.13 (t, J = 7.4 Hz, 1H), 7.23 (t, J = 7.1 Hz, 1H), 7.28–7.36
(m, 3H), 7.43–7.53 (m, 4H), 7.78 (d, J2 = 8.0 Hz, 1H), 7.99 (s, 1H),
8.19 (s, 1H), 8.36 (d, J = 8.0 Hz, 1H), 11.96 (s, 1H), 12.39 (s, 1H)
ppm. MS (ESI+): 423.0 ([MH++CH3CN]+, 13), 382.0 ([M+H]+, 100).
Anal.(C23H19 N5O; 381.43): C, H, N.
35
ppm. MS (ESI+): 570 ([MH++CH3CN]+, 33; Cl), 529 ([M+H]+, 100;
35Cl). IR (KBr):
m . Anal. Calcd for
3443, 3319, 1612 cmꢁ1
(C28H18ClFN4O2S ꢂ 1.25 CH3OH; 528,98): C, H, N.
4.2.10. Preparation of (1H-indol-2-yl)(4-(phenylamino)benzo[b]
thiophen-2-yl)methanones 33–46
Example:
4.2.10.6. (4-(Benzylamino)thieno[2,3-d]pyrimidin-6-yl)(1H-
indol-2-yl)methanone (38). 58% yield; yellow crystals; mp

134
T. Beckers et al. / Bioorg. Med. Chem. 20 (2012) 125–136
235–236 °C; 1H NMR (300 MHz, DMSO-d6): d 12.07 (s, 1H), 9.07 (t,
J = 5.7 Hz, 1H), 8.88 (s, 1H), 8.47 (s, 1H), 7.78 (d, J = 8.0 Hz, 1H), 7.63
(d, J = 1.4 Hz, 1H), 7.52 (d, J = 8.3 Hz, 1H), 7.44–7.24 (m, 6H), 7.14 (t,
J = 7.5 Hz, 1H), 4.82 (d, J = 5.7 Hz, 2H) ppm. MS (ESI+): 426
(KBr):
m
3456,
3216,
1576 cmꢁ1
.
Anal.
Calcd.
for
(C21H12ClFN4OS ꢂ 1.5 CH3OH; 422.86): C, H, N.
4.2.10.13. (4-(3-Chloro-4-fluorophenylamino)thieno[2,3-d]pyr-
([MH++CH3CN]+, 24), 385 ([M+H]+, 100). IR (KBr):
m
3426, 3383,
imidin-6-yl)(5-hydroxy-1H-indol-2-yl)methanone
(45).
1590 cmꢁ1. Anal. Calcd for (C22H16N4OS ꢂ 0.2 CH2Cl2; 384,45): C,
57% yield; yellow crystals; mp 293–294 °C; 1H NMR (300 MHz,
DMSO-d6): d 11.87 (s, 1H), 10.20 (s, 1H), 9.10 (s, 1H), 8.94 (s, 1H),
8.65 (s, 1H), 8.20 (dd, J1 = 2.5; J2 = 6.9 Hz, 1H), 7.83–7.77 (m, 1H),
7.55–7.46 (m, 2H), 7.35 (d, J = 8.8 Hz, 1H), 7.05 (br s, 1H), 6.92
(dd, J = 2.2, 8.8 Hz, 1H) ppm. MS (ESI+): 480 ([M+H + CH3CN]+,
H, N.
4.2.10.7. (R)-(1H-Indol-2-yl)(4-(1-phenylethylamino)thieno[2,3-
d]pyrimidin-6-yl)methanone (39).
Preparation from R-(+)
alpha-methylbenzylamin (ee P99%) (Aldrich). 65% yield; yellow
powder from ethyl acetate; mp 204–208 °C; 1H NMR (300 MHz,
DMSO-d6): d 12.09 (s, 1H), 8.95 (s, 1H), 8.81 (d, J = 8.0 Hz, 1H),
8.42 (s, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.65 (br s, 1H), 7.56–7.42 (m,
3H), 7.39–7.31 (m, 3H), 7.24 (t, J = 7.2 Hz, 1H), 7.15 (t, J = 7.4 Hz,
1H), 5.58 (quint, J = 7.2 Hz, 1H), 1.61 (d, J = 7.2 Hz, 3H) ppm. MS
100; 35Cl), 439 ([M+H]+, 86; 35Cl). IR (KBr):
m 3423, 3314,
1574 cmꢁ1. Anal. Calcd for (C21H12ClFN4O2S; 438.86): C, H, N.
4.2.10.14. (4-(3-Chloro-4-fluorophenylamino)thieno[2,3-d]pyr-
imidin-6-yl)(5-methoxy-1H-indol-2-yl)methanone
(46).
63% yield; yellow crystals; mp 265–266 °C; 1H NMR (300 MHz,
DMSO-d6): d 12.03 (s, 1H), 10.21 (s, 1H), 8.95 (s, 1H), 8.66 (s,
1H), 8.21 (dd, J = 2.5, 6.9 Hz, 1H), 7.85–7.78 (m, 1H), 7.59–7.41
(m, 3H), 7.20 (d, J = 2.3 Hz, 1H), 7.03 (dd, J = 2.3, 9.1 Hz, 1H), 3.80
(s, 3H) ppm. MS (ESI+): 494 ([MH++CH3CN]+, 100; 35Cl), 453
(ESI+): 440 ([M+CH3CN]+, 20), 399 ([M+H]+, 100). IR (KBr):
m
3418, 1580 cmꢁ1. Anal. Calcd for (C23H18N4OS ꢂ 0.25 ethyl acetate;
398,48): C, H, N.
([M+H]+, 88; 35Cl). IR (KBr):
for (C22H14ClFN4O2S ꢂ 0.75 H2O; 452.89): C, H, N.
m
3445, 3312, 1581 cmꢁ1. Anal. Calcd.
4.2.10.8.
(1H-Indol-2-yl)(4-(4-(trifluoromethyl)phenylami-
26% yield;
no)thieno[2,3-d]pyrimidin-6-yl)methanone (40).
brown crystals from THF; mp 138 °C decomp.; 1H NMR (300 MHz,
DMSO-d6): d 7.17 (t, J = 7.4 Hz, 1H), 7.37 (t, J = 8.1 Hz, 1H), 7.54 (d,
J = 8.5 Hz, 1H), 7.72 (s, 1H), 7.79–7.84 (m, 3H), 8.16 (d, J = 8.5 Hz,
2H), 8.72 (s, 1H), 9.07 (s, 1H), 10.36 (s, 1H), 12.15 (s, 1H) ppm.
MS (ESI+): 480.0 ([MH++CH3CN]+, 100), 438.9 ([M+H]+,86). Anal
Calcd. for (C22H13F3N4OSx1/3THF; 438,43): C, H, N.
4.2.10.15.
(5-(Benzyloxy)-1H-indol-2-yl)(4-chloro-7H-pyrrol-
o[2,3-d]pyrimidin-6-yl)methanone (47).
(5-(Benzyloxy)-1-
(phenylsulfonyl)-1H-indol-2-yl)(4-chloro-7-(phenylsulfonyl)-7H-
pyrrolo[2,3-d]pyrimidin-6-yl)methanone (18) (1.37 g; 2.0 mmol)
was dissolved in THF (75 mL), tetrabutylammoniumfluoride trihy-
drate (8.0 mmol; 2.52 g) was added and the mixture heated to
120 °C for 2 d in a steel vessel. The mixture was cooled to room
temperature, poured into water, the precipitated product removed
by filtration, dried and purified by cc (SiO2; CH2Cl2, ethyl acetate
1:1). 86% yield; yellow crystals; mp 282.6–283.3 °C; 1H NMR
(300 MHz, DMSO-d6): d 13.42 (s, 1H, exchangeable), 12.0 (s, 1H,
exchangeable), 8.77 (s, 1H), 7.60–7.30 (m, 9H), 7.10 (dd,
J1 = 8.9 Hz, J2 = 2.1 Hz, 1H), 5.14 (s, 2H) ppm. MS (ESI+): 444.0
([MH++CH3CN]+, 100; 35Cl), 402.9 ([M+H+]+, 44; 35Cl). Anal. Calcd
for (C22H15ClN4O2; 402,83): C, H, N
4.2.10.9.
(1H-Indol-2-yl)(4-(3-(trifluoromethyl)phenylami-
23% yield
no)thieno[2,3-d]pyrimidin-6-yl)methanone (41).
from ethyl acetate; brown crystals; mp 180 °C decomp..; 1H NMR
(300 MHz, DMSO-d6): d 7.16 (t, J = 7.5 Hz, 1H), 7.37 (t, J = 7.7 Hz,
1H), 7.53 (t, J = 9.6 Hz, 2H), 7.65–7.71 (m, 2H), 7.82 (d, J = 7.9 Hz,
1H), 8.25 (d, J = 8.2 Hz, 1H),8.29 (s, 1H), 8.71 (s, 1H), 9.04 (s, 1H),
10.33 (s, 1H), 12.15 (s, 1H) ppm. MS (ESI+): 480.0 ([MH++CH3CN]+,
100), 438.9 ([M+H]+, 81). Anal. (C22H13F3N4O ꢂ ½ ethyl acetate;
438,43): C, H, N.
4.2.10.16. (5-Hydroxy-1H-indol-2-yl)(7H-pyrrolo[2,3-d]pyrimi-
4.2.10.10. (4-(3-Chlorophenylamino)thieno[2,3-d]pyrimidin-6-
din-6-yl)methanone 48: 47
.
(0.50 g; 1.24 mmol) was dis-
yl)(1H-indol-2-yl)methanone (42).
46% yield; brown crys-
solved in MeOH/THF (1:1; 300 mL), NH4HCOO (12.4 mmol;
0.78 g) and Pd on charcoal (0.5 g; 10%) was added. The mixture
was heated to reflux for 1 h, cooled to room temperature and the
catalyst was removed by filtration over a pad of sodium sulfate.
To the clear solution thus obtainedwater (100 mL) was added,
the organic solvent removed under reduced pressure and the pre-
cipitated product removed by filtration. Purification by cc on silica
gel (THF, ethyl acetate 1:4) gave the product: 0.20 g (58%) yellow
crystals. mp 240 °C (decomp.); 1H NMR (300 MHz, DMSO-d6): d
12.95 (s, 1H, exchangeable), 9.24 (s, 1H, exchangeable), 9.05 (s,
1H, exchangeable), 8.93 (s, 1H), 7.64 (s, 1H), 7.44 (d, J = 1.4 Hz,
1H), 7.34 (d, J = 9.0 Hz, 1H), 7.02 (d, J = 2.2 Hz, 1H), 6.89 (dd,
J1 = 9.0 Hz, J2 = 2.2 Hz, 1H) ppm. MS (ESI+): 320.1 ([MH++CH3CN]+,
58), 279.0 ([M+H+]+, 100). Anal. Calcd for (C15H10N4O2 ꢂ 1/3 H2O;
278,27): C, H, N.
tals; mp 302.7 °C decomp.; 1H NMR (300 MHz, DMSO-d6): d 7.05
(d, J = 8.0 Hz, 1H), 7.13 (t, J = 7.3 Hz, 1H), 7.30–7.37 (m, 2H) 7,52
(d, J = 8.2 Hz, 1H), 7.56–7.58 (m, 2H), 7.69 (d, J = 7.9 Hz, 1H), 7.87
(s, 1H), 8.42 (s, 1H), 8.75 (s, 1H) ppm. MS (ESI+): 445.9
([MH++CH3CN]+, 78; 35Cl), 404.9 ([M+H]+, 100; 35Cl). Anal.
(C21H13Cl N4OS; 494.87): C, H, N.
4.2.10.11. (4-(4-Fluorophenylamino)thieno[2,3-d]pyrimidin-6-
yl)(1H-indol-2-yl)methanone (43).
26% yield from ethyl ace-
tate; brown crystals; mp 332.4 °C decomp..; 1H NMR (300 MHz,
DMSO-d6): d 7.16 (t, J = 7.4 Hz, 1H), 7.25–7.31 (m, 2H), 7.36 (t,
J = 7.7 Hz, 1H), 7.54 (d, J = 8.5 Hz, 1H), 7.69 (s, 1H), 7.80–7.87 (m,
3H), 8.59 (s, 1H), 8.99 (s, 1H), 10.18 (s, 1H), 12.19 (s, 1H) ppm.
MS (ESI+): 429.9 ([MH++CH3CN]+,78), 388.9 ([M+H]+,100). Anal.
1
(C21H13F N4O ꢂ ethyl acetate; 388,42): C, H, N.
4
4.2.10.17.
pyrimidin-6-yl)(1-(phenylsulfonyl)-1H-indol-2-yl)methanone
(49). (4-Chlorothieno[2,3-d]pyrimidine-6-yl)(1-(phenylsulfo-
(4-(3-Chloro-4-fluorophenylamino)thieno[2,3-d]
4.2.10.12. (4-(3-Chloro-4-fluorophenylamino)thieno[2,3-d]pyr-
imidin-6-yl)(1H-indol-2-yl)methanone (44).
66% yield; yel-
nyl)-1H-indol-2yl)methanone (19) (1.80 g, 3.97 mmol) and
3-chloro-4-fluoroaniline (1.16 g, 8.00 mmol) were suspended in 2-
butanone (75 mL). The mixture was heated to reflux for 2 days. After
cooling to room temperature the solvent was removed under re-
duced pressure. Purification by cc on silica gel (CH2Cl2) gave the
product: 1.45 g, 65% yield; light yellow powder; mp 273–274 °C;
low crystals; mp 313–316 °C; 1H NMR (300 MHz, DMSO-d6): d
12.14 (s, 1H), 12.21 (s, 1H), 9.98 (s, 1H), 8.66 (s, 1H), 8.20 (dd
J = 2.5, 6.9 Hz, 1H), 7.86–7.77 (m, 2H), 7.68 (br s, 1H), 7.57–7.45
(m, 2H), 7.37 (t, J = 7.4 Hz, 1H), 7.16 (t, J = 7.4 Hz, 1H) ppm. MS
(ESI+): 464 ([MH++CH3CN]+, 82; 35Cl), 423 ([M+H]+, 100; 35Cl). IR

T. Beckers et al. / Bioorg. Med. Chem. 20 (2012) 125–136
135
1H NMR (300 MHz, DMSO-d6): d 10.17 (s, 1H), 8.69 D, J = 5.8 Hz, 2H),
(ESI+): 372 ([MH++CH3CN; 35Cl]+, 100; 35Cl). IR (KBr):
m
3092,
8.15–8.05 (m, 4H), 7.83–7.38 (m, 9H) ppm. MS (ESI+): 604 ([MH +
1615, 1551 cmꢁ1. Anal. Calcd for (C15H7ClN2OS2; 330.81): C, H, N.
CH3CN]+, 100; 35Cl), 563 ([MH]+, 65; 35Cl). IR (KBr):
m 3356, 1641,
1618 cmꢁ1. Anal. Calcd for (C27H16ClFN4O3S2; 563.02): C, H, N.
4.2.10.23. Benzofuran-2-yl(4-(3-chloro-4-fluorophenylamino)
thieno[2,3-d]pyrimidin-6-yl)methanone (56).
According to
4.2.10.18. (4-((3-Chloro-4-fluorophenyl)(methyl)amino)thieno
[2,3-d ]pyrimidin-6-yl)(1-(phenylsulfonyl)-1H-indol-2-yl)meth-
(4-(3-Chloro-4-fluorophenylamino)thieno[2,3-d]pyrimidin-6-yl)(1
-(phenylsulfonyl)-1H-indol-2-yl)methanone (49) from Benz ofur
an-2-yl(4-chlorothieno[2,3-d]pyrimidin-6-yl)methanone (54), (0.
65 g, 2.07 mmol) and 3-Chloro-4-fluoroaniline (0.61 g, 4.20 m
mol): 0.56 g, 64% yield; yellow crystals; mp 217–218 °C; 1H NMR
(300 MHz, DMSO-d6): d 10.28 (s, 1H), 9.12 (s, 1H), 8.66 (s, 1H),
8.20 (dd, J1 = 2.6, J2 = 6.7 Hz, 1H), 8.12 (s, 1H), 7.95 (d, J = 8.10 Hz,
1H), 7.87 (d, J = 8.2 Hz, 1H), 7.83–7.77 (m, 1H), 7.69–7.61 (m,
1H), 7.55–7.42 (m, 2H) ppm. MS (EI): 423 ([M]+ꢀ, 100, 35Cl), 388
anone (50).
(4-(3-Chloro-4-fluorophenylamino)thieno[2,3-d]
pyrimidin-6-yl)(1-(phenylsulfonyl)-1H-indol-2-yl)methanone HP-
1149 (49) (0.2 g, 0.36 mmol) were dissolved in THF (15 ml) and
NaH 60% (0.02 g, 0.5 mmol) werde added. After 5 minutes CH3I
(0.5 ml, 0.8 mmol) werde added and the reaction monitored by
tlc on silica gel (ethyl acetate, light petroleum 1:2). After the disa-
pearance of the starting material 0.5 ml MeOH were added. The
solvent was removed under reduced pressure and the residue
was purified by cc on silica gel (ethyl acetate:light petroleum
1:2): 0.15 g, 70% yield; yellow powder; mp 224–226 °C; 1H NMR
(300 MHz, DMSO-d6): d 8.70 (s, 1H), 8.05 (d, J = 8.2 Hz, 1H),
7.88–7.81 (m, 3H), 7.75–7.68 (m, 2H), 7.63–7.48 (m, 4H), 7.41
(16), 194 (25). IR (KBr):
(C21H11ClFN3O2S ꢂ 0.33 CH3OH; 423.85): C, H, N.
m
3430, 1578 cmꢁ1. Anal. Calcd for
4.2.10.24. Benzo[b]thiophen-2-yl(4-(3-chloro-4-fluorophenyla-
mino)thieno[2,3-d]pyrimidin-6-yl)methanone (57).
(m, 1H), 7.30 (m, 1H), 7.05 (s, 1H), 6.23 (s, 1H), 3.54 (s, 3H) ppm.
According to (4-(3-Chloro-4-fluorophenylamino)thieno[2,3-d]
pyrimidin-6-yl)(1-(phenylsulfonyl)-1H-indol-2-yl)methanone (49)
35
MS (ESI+): 618 ([MH+CH3CN;
Cl]+, 25), 577 ([MH]+, 100; 35Cl).
IR (KBr):
m
2963, 1635, 1543 cmꢁ1
.
Anal. Calcd for
from
Benzo[b]thiophen-2-yl(4-chlorothieno[2,3-d]pyrimidin-6-
(C28H18ClFN4O3S2; 577.05): C, H, N.
yl)methanone (55), (0.55 g, 1.66 mmol) and 3-Chloro-4-fluoroani-
line (0.49 g, 3.40 mmol): 0.28 g, 38% yield; yellow crystals; mp
234–235 °C; 1H NMR (300 MHz, DMSO-d6): d 10.19 (s, 1H), 8.95 (s,
1H), 8.68 (s, 1H), 8.55 (s, 1H), 8.21–8.13 (m, 3H), 7.80–7.74 (m,
1H), 7.65–7.44 (m, 3H) ppm. MS (EI): 439 ([M]+ꢀ, 100, 35Cl), 404
4.2.10.19.
(4-((3-Chloro-4-fluorophenyl)(methyl)amino)thie-
(51).
no[2,3-d]pyrimidin-6-yl)(1H-indol-2-yl)methanone
(4-((3-Chloro-4-fluorophenyl)(methyl)amino)thieno[2,3-d]pyrimi-
din-6-yl)(1-(phenylsulfonyl)-1H-indol-2-yl)methanone (50) (0.5 g,
0.87 mmol) were dissolved in a mixture of methanole (10 mL), THF
(10 mL) and aqueous NaOH (10%, 10 mL) and heated to reflux for
1.5 h. THF and methanole were removed under reduced pressure
and the residue was extracted with ethyl acetate. The organic layer
was separated, dried (Na2SO4) and the solvent removed under re-
duced pressure. The precipitated product was collected by filtra-
tion and dried in vacuo. 0.28 g, 74% yield; yellow powder; mp
293–294 °C; 1H NMR (300 MHz, DMSO-d6): d 12.02 (s, 1H), 8.69
(s, 1H), 8.06 (dd, J1 = 2.5, J2 = 6.5 Hz, 1H), 7.82–7.62 (m, 3H), 7.47
(d, J = 8.2 Hz, 1H), 7.34 (t, J = 7.10 Hz, 1H), 7.17 (t, J = 7.4 Hz, 1H),
6.60 (d, J = 8.8 Hz, 2H), 3.59 (s, 3H) ppm. MS (EI): 436 ([M]+, 100;
(17). IR (KBr):
m . Anal. Calcd for
3444, 3091, 1621 cmꢁ1
(C21H11ClFN3OS2; 439.91): C, H, N.
4.3. Biochemical and biological assays
4.3.1. Kinase activity assays
All biochemical protein kinase activity assays were performed
in 96-well FlashPlatesTM (Perkin Elmer, Boston, USA) in a 50
reaction volume. The reaction cocktail contained 20 L of assay
buffer, 5 L of ATP solution (in H2O), 5 L of test compound (in
10% DMSO), 10 L of substrate and 10 L of purified recombinant
protein kinase. Final concentration of ATP was 1 M. The assay
for all enzymes contained 70 mM HEPES-NaOH, pH 7.5,
l
L
l
l
l
l
l
l
35Cl), 279 (22). IR (KBr):
m
3446, 3331, 1541 cmꢁ1. Anal. Calcd for
(C22H14ClFN4OS; 436.89): C, H, N.
3 mM MgCl2, 3 mM MnCl2, 3
50 g/mL PEG2 0 0 0 0, 1 M [
well). Amount of protein kinases used per 50 lL assay were:
lM Na-orthovanadate,1.2 mM DTT,
l
l
c
-33P]-ATP (approx. 5 ꢂ 105 cpm per
4.2.10.20. Benzo[b]furan-2-yl(4-chlorothieno[2,3-d]pyrimidine-
6-yl)methanone (54) and Benzo[b]thiophen-2-yl(4-chlorothie-
ABL1 wt: 10 ng; EGF-R wt: 10 ng; Erb2: 100 ng; MET wt: 12 ng;
no[2,3-d]pyrimidine-6-yl)methanone (55).
To a mixture of
VEGF-R2: 15 ng. The following substrates were used at the speci-
benzo[b]thiophen-2-ylboronic acid (0.64 g, 3.6 mmol), PdCl2(PPh3)2
(42 mg, 0.06 mmol) and K2HPO4 ꢂ 3H2O (1.0 g, 4.5 mmol) in dry
toluene (15 mL), 4-chlorothieno[2,3-d]pyrimidine-6-carbonyl chlo-
ride (0.7 g, 3.0 mmol) was added. The mixture was heated to 110 °C
for 3 h, cooled to room temperature and diluted with ethyl acetate
(30 mL). The organic layer was washed with satd. NaHCO3 solution
and dried (Na2SO4). After removing of the solvent the product was
purified by cc (SiO2, light petroleum/ethyl acetate 4:1.
fied concentrations: poly(Glu, Tyr)4:1, 0.125
ErbB2, VEGF-R2; poly(Ala, Glu, Lys, Tyr)6:2:5:1, 0.125
ABL1 wt, MET wt.
l
g/50
l
L: EGF-R wt,
l
g/50 L:
l
The reaction cocktails were incubated at 30 °C for 60 min. The
reaction was stopped with 50 l of 2%(v/v) H3PO4, plates were
aspirated and washed two times with 200 l 0.9% (w/v) NaCl.
l
l
Incorporation of 33Pi was determined with a MicrobetaTM micro-
plate scintillation counter (Perkin Elmer, Boston, MA, USA).
The inhibition for each concentration and the compound IC50
values were calculated with Quattro Workflow V3.0.3 (Quattro Re-
search GmbH, Munich, Germany). The model used for IC50 determi-
nation was ‘Sigmoidal response (variable slope)’ with parameters
‘top’ fixed at 100% and ‘bottom’ at 0%.
4.2.10.21. Benzofuran-2-yl(4-chlorothieno[2,3-d]pyrimidin-6-
yl)methanone (54).
50% yield; yellow foam; mp 166 °C; 1H
NMR (300 MHz, DMSO-d6): d 9.13 (s, 1H), 8.62 (s, 1H), 8.22 (s,
1H), 7.96–7.84 (m, 2H), 7.64 (m, 1H), 7.45 (m, 1H) ppm. MS (EI):
314 ([M]+, 100; 35Cl), 286 (25), 197 (28), 145 (77). IR (KBr):
3127, 1625. Anal. Calcd for (C15H7ClN2O2S; 314.75): C, H, N.
m
4.3.2. Biological assays
A431 cells were cultivated in DMEM (4.5 g glucose/l), supple-
mented with glutamine, and 10% fetal bovine serum (FBS). For
measuring autophosphorylation of EGFR, cells were seeded at
about 50% confluence into 24-well plates. After adherence, the
medium was replaced with DMEM with 0.5% FBS and starvation
was allowed over night. Inhibitors were then added to the plates
4.2.10.22. Benzo[b]thiophen-2-yl(4-chlorothieno[2,3-d]pyrimi-
din-6-yl)methanone (55).
53% yield; yellow powder; mp
155–158 °C; 1H NMR (300 MHz, DMSO-d6): d 9.13 (s, 1H), 8.71 (s,
1H), 8.50 (s, 1H), 8.19–8.12 (m, 2H), 7.75–7.51 (m, 2H) ppm. MS

136
T. Beckers et al. / Bioorg. Med. Chem. 20 (2012) 125–136
(final DMSO concentration 0.1%) for 1 h. EGF was added at a final
concentration of 100 ng/ml for 10 min, then cells were washed
the Cytofluor 4000 microplate reader (excitation k = 530 nm, emis-
sion k = 620 nm) to quantify the amount of attached viable cells.
Each compound was tested in three independent experiments
using all 42 cancer cell lines. Relative IC50 values were determined
by non-linear regression (log [conc. of inhibitor] versus response (%
T/C)) using the GraphPad PrismÒ analysis software (Prism 5 for
Windows, version 5.01, GraphPad Software Inc., CA). For calcula-
tion of mean IC50 values over the 42 cell lines tested, the geometric
mean (‘geomean’) was used.
with phosphate-buffered saline, and lyzed with 100
(20 mM HEPES, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM
EDTA, 10 mN sodium pyrophosphate, 2 mM EGTA, 20 M Zn-Ace-
tat, 50 mM NaF, freshly added: 1 mM PMSF, 5 g/ml leupeptin,
ll lysis buffer
l
l
2 mM sodium orthovanadate) per well. The extracts were sub-
jected to SDS-polyacrylamide gel electrophoresis, and blotting to
PVDF membranes. Blots were developed with anti-phosphotyro-
sine antibodies (4G10, Upstate, Lake Placid, USA), then stripped
and probed with anti-EGFR antibodies (Transduction Laboratories,
E12020; BD Biosciences, Heidelberg, Germany) using the enhanced
chemiluminescence method. Films were scanned and quantified
using Fuji Multi Gauge software. Ranges of IC50 values indicate
the two tested concentrations, in between which half-maximal
inhibition was observed in all experiments.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.11.023.
References and notes
1. Mahboobi, S.; Uecker, A.; Sellmer, A.; Cénac, C.; Höcher, H.; Pongratz, H.;
Eichhorn, E.; Hufsky, H.; Trümpler, A.; Sicker, M.; Heidel, F.; Fischer, T.;
Stocking, C.; Elz, S.; Böhmer, F.-D.; Dove, S. J. Med. Chem. 2006, 49, 3101.
2. Rimawi, M. F.; Shetty, P. B.; Weiss, H. L.; Schiff, R.; Osborne, C. K.; Chamness, G.
C.; Elledge, R. M. Cancer 2010, 116, 1234.
3. Sundvall, M.; Karrila, A.; Nordberg, J.; Grenman, R.; Elenius, K. Expert. Opin.
Emerg. Drugs 2010, 15, 185.
4. Kanwar, S. S.; Nautiyal, J.; Majumdar, A. P. N. Curr. Drug Targets 2010, 11, 682.
5. Yoshida, T.; Zhang, G.; Haura, E. B. Biochem. Pharmacol. 2010, 80, 613.
6. Okamoto, I.; Mitsudomi, T.; Nakagawa, K.; Fukuoka, M. Ther. Adv. Med. Oncol.
2010, 2, 301.
4.3.3. Sensitivity profile of selected ((1H-indol-2-yl)(4-(phenyl
amino)thieno[2,3-d]pyrimidin-6-yl)methanones
Cell lines of the panel comprised 15 different tumor histotypes,
each represented by one to six cell lines. They were established
from cancer of the bladder (3), colon (5), head and neck (1), liver
(1), lung (6), breast (3), pancreas (3), prostate (4), ovary (2), kidney
(3), stomach (2) and the uterine body (1), as well as from malignant
melanoma (3), sarcoma (2) and pleuramesothelioma (3). The 24 cell
lines BXF 1218L, BXF 1352L, CXF 269L, GXF 251L, LIXF 575L, LXFL
1121L, LXFA 289L, LXFA 526L, LXFL 529L, LXFA 629L, MAXF
401NL, MEXF 1341L, MEXF 276L, MEXF 462NL, OVXF 899L, PAXF
1657L, PAXF 546L, PXF 1118L, PXF 1752L, PXF 698L, RXF 1781L,
RXF 393NL, RXF 486L and UXF 1138L were established at Oncotest
from patient-derived tumor xenografts.18 The origin of the donor
xenografts was described by Fiebig et al. 1992 and 1999.19,20 The
other 18 cell lines were either kindly provided by the NCI (Bethes-
da; MD), or were purchased from ATCC (Rockville, MD), DSMZ
(Braunschweig, Germany) or JCRB (Osaka, Japan). Authenticity of
all cell lines was proven at the by STR (short tandem repeat) analy-
sis, a PCR based DNA-fingerprinting methodology.21,22 All cells were
grown at 37 °C in a humidified atmosphere with 5% CO2 in RPMI
1640 medium, supplemented with 10% (v/v) fetal calf serum and
0.1 mg/mL gentamicin (medium and all components from PAA,
Cölbe, Germany).
7. Mountzios, G.; Owonikoko, T.; Karamouzis, M.; Argiris, A. Open Lung Cancer J.
2010, 3, 57.
8. Kotsakis, A.; Georgoulias, V. Expert opin. Pharmacother. 2010, 11, 2363.
9. Daub, H.; Specht, K.; Ullrich, A. Nat. Rev. Drug Discov. 2004, 3, 1001.
10. Cossrow, J.; Guan, B.; Ishchenko, A.; Jones, J. H.; Kumaravel, G.; Lugovskoy, A.;
Peng, H.; Powell, N.; Raimundo, B. C.; Tanaka, H.; Vessels, J.; Wynn, T.; Xin, Z.
US 20090005359 A1, 2009.
11. Urawa, Y.; Ogura, K. Tetrahedron Lett. 2003, 44, 271.
12. Kalish, L. H.; Kwong, R. A.; Cole, I. E.; Gallagher, R. M.; Sutherland, R. L.;
Musgrove, E. A. Clin. Cancer Res. 2004, 10, 7764.
13. Krumbach, R.; Schüler, J.; Hofmann, M.; Giesemann, T.; Fiebig, H. H.; Beckers, T.
Eur. J. Cancer 2011, 47, 1231.
14. Licitra, L.; Perrone, F.; Tamborini, E.; Bertola, L.; Ghirelli, C.; Negri, T.; Orsenigo,
M.; Filipazzi, P.; Pastore, E.; Pompilio, M.; Bossi, P.; Locati, L. D.; Cantu’, G.;
Scaramellini, G.; Pilotti, S.; Tagliabue, E. Ann. Oncol. 2011, Epub ahead of print.
15. Mahboobi, S.; Teller, S.; Pongratz, H.; Hufsky, H.; Sellmer, A.; Botzki, A.; Uecker,
A.; Beckers, T.; Baasner, S.; Schachtele, C.; Ueberall, F.; Kassack, M. U.; Dove, S.;
Böhmer, F.-D. J. Med. Chem. 2002, 45, 1002.
16. Sundberg, R. J.; Russel, H. F. J. Org. Chem. 1973, 38, 3324.
17. Mahboobi, S.; Sellmer, A.; Winkler, M.; Eichhorn, E.; Pongratz, H.; Ciossek, T.;
Baer, T.; Maier, T.; Beckers, T. J. Med. Chem. 2010, 53, 8546.
A propidium iodide based cytoxicitiy assay was used to assess
the anti-cancer activity of the compounds. Cells were harvested
from exponential phase cultures, counted and plated in 96 well
flat-bottom microtiter plates at a cell density of 4000–40,000
cells/well. After a 24 h recovery period to allow the cells to resume
exponential growth, test compounds were added in duplicates at
ten concentrations and treatment continued for four days. Cells
18. Roth, T.; Burger, A. M.; Dengler, W.; Willmann, H. Fiebig, H. H. In: Fiebig HH,
Burger AM (eds). Relevance of Tumor Models for Anticancer Drug Development.
Contributions of Oncology; S. Karger AG: Basel, 1999; Vol. 54, p. 145.
19. Fiebig, H. H.; Dengler, W. A. Roth, T. In: Fiebig HH, Burger AM (eds). Relevance of
Tumor Models for Anticancer Drug Development. Contributions of Oncology; S.
Karger AG: Basel, 1999; Vol. 54, p. 29.
20. Fiebig, H. H.; Berger, D. P.; Dengler, W. A.; Wallbrecher, E.; Winterhalter, B. R.
In: Fiebig HH, Berger DP (eds). Immunodeficient Mice in Oncology. Contributions
of Oncology; S. Karger AG: Basel, 1992; Vol. 42, p. 321.
21. Masters, J. R.; Thomson, J. A.; Daly-Burns, B.; Reid, Y. A.; Dirks, W. G.; Packer, P.;
Toji, L. H.; Ohno, T.; Tanabe, H.; Arlett, C. F.; Kellandm, L. R.; Harrison, M.;
Virmani, A.; Ward, T. H.; Ayres, K. L.; Debenham, P. G. Proc. Natl. Acad. Sci. U.S.A.
2001, 98, 8012.
were washed with 200
ll PBS to remove dead cells, then 200
ll
of a solution containing 7
l
g/ml propidium iodide (PI) and 0.1%
(v/v) Triton X-100 was added. After an incubation period of 1–2
22. Dirks, W. G.; Faehnrich, S.; Estella, I. A.; Drexler, H. G. ALTEX 2005, 22, 103.
hours at room temperature, fluorescence (FU) was measured using