Discovery of novel dual inhibitors of receptor tyrosine kinases EGFR and PDGFR-β related to anticancer drug resistance

Article abstract of DOI:10.1080/14756366.2017.1370583

With ongoing resistance problems against the marketed EGFR inhibitors having a quinazoline core scaffold there is a need for the development of novel inhibitors having a modified scaffold and, thus, expected lower EGFR resistance problems. An additional problem concerning EGFR inhibitor resistance is an observed heterodimerization of EGFR with PDGFR-β that neutralises the sole inhibitor activity towards EGFR. We developed novel pyrimido[4,5-b]indoles with varied substitution patterns at the 4-anilino residue to evaluate their EGFR and PDGFR-β inhibiting properties. We identified dual inhibitors of both EGFR and PDGFR-β in the nanomolar range which have been initially screened in cancer cell lines to prove a benefit of both EGFR and PDGFR-β inhibition.

Full text of DOI:10.1080/14756366.2017.1370583

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Discovery of novel dual inhibitors of receptor
tyrosine kinases EGFR and PDGFR-β related to
anticancer drug resistance
Tim Fischer, Karim Najjar, Frank Totzke, Christoph Schächtele, Wolfgang
Sippl, Christoph Ritter & Andreas Hilgeroth
To cite this article: Tim Fischer, Karim Najjar, Frank Totzke, Christoph Schächtele, Wolfgang
Sippl, Christoph Ritter & Andreas Hilgeroth (2018) Discovery of novel dual inhibitors of receptor
tyrosine kinases EGFR and PDGFR-β related to anticancer drug resistance, Journal of Enzyme
Inhibition and Medicinal Chemistry, 33:1, 1-8, DOI: 10.1080/14756366.2017.1370583
To link to this article: http://dx.doi.org/10.1080/14756366.2017.1370583
© 2017 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
Published online: 03 Nov 2017.
Submit your article to this journal
Article views: 39
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=ienz20
Download by: [University of Florida]
Date: 05 November 2017, At: 22:33

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2017
VOL. 33, NO. 1, 1–8
https://doi.org/10.1080/14756366.2017.1370583
SHORT COMMUNICATION
Discovery of novel dual inhibitors of receptor tyrosine kinases EGFR and PDGFR-b
related to anticancer drug resistance
a
a
b
b
a
c
€
Tim Fischer , Karim Najjar , Frank Totzke , Christoph Schachtele , Wolfgang Sippl , Christoph Ritter and
Andreas Hilgerotha
b
aDepartment of Pharmaceutical Chemistry, Institute of Pharmacy, Martin Luther University, Halle, Germany; ProQinase GmbH Freiburg, Freiburg,
c
Germany; Department of Clinical Pharmacy, Institute of Pharmacy, University of Greifswald, Greifswald, Germany
ABSTRACT
ARTICLE HISTORY
Received 22 May 2017
Revised 8 August 2017
Accepted 14 August 2017
With ongoing resistance problems against the marketed EGFR inhibitors having a quinazoline core scaffold
there is a need for the development of novel inhibitors having a modified scaffold and, thus, expected
lower EGFR resistance problems. An additional problem concerning EGFR inhibitor resistance is an
observed heterodimerization of EGFR with PDGFR-b that neutralises the sole inhibitor activity towards
EGFR. We developed novel pyrimido[4,5-b]indoles with varied substitution patterns at the 4-anilino residue
to evaluate their EGFR and PDGFR-b inhibiting properties. We identified dual inhibitors of both EGFR and
PDGFR-b in the nanomolar range which have been initially screened in cancer cell lines to prove a benefit
of both EGFR and PDGFR-b inhibition.
KEYWORDS
Benzo-anellated com-
pounds; protein kinase
inhibitory activity; biological
activity
Introduction
Beside so far unknown mutations which may affect the inhibitor
binding to EGFR, another mechanism of drug resistance may lower
the inhibitor activity: a receptor heterodimerization12. Normally,
the ligand-activated EGFR undergoes a receptor dimerisation with
a neighboured EGFR receptor13. That dimerisation causes conform-
ational changes that allow an autophosphorylation of tyrosine resi-
dues of the receptor dimer itself. Those phosphate groups are
transferred to the intracellular EGFR substrates which mediate the
further cellular signalling14. However, it is known that EGFR may
Insight in deregulated processes of cancer cells allowed the identi-
fication of novel target structures for a perspective anticancer
therapy with less suspected side effects due to a more specific tar-
geted therapy1. So various protein kinases have been identified as
promising target structures which were found overactive in such
deregulated cancer cells2. Being mostly overexpressed they con-
tribute to uncontrolled cell growth or to the suppression of apop-
tosis3. Early small molecule inhibitors have been identified to
inhibit the protein kinase activity in a competitive manner by
binding to the ATP binding site of the respective protein kinase4.
In order to minimise prospected side effects the first inhibitors
were expected to selectively act with a single protein kinase5.
With a specific binding mode to the protein backbone the inhibi-
tors were found sensitive to mutations of the gene that encodes
also undergo
a heterodimerization with the related platelet
derived growth factor receptor (PDGFR)-b15,16. That receptor is
found in many types of cancer similar to EGFR1. A dual inhibition
of both EGFR and PDGFR-b would be of favour to prevent a kinase
inhibitor resistance caused by
a receptor heterodimerization.
Recent research development concentrated on the inhibition of
the protein to cause amino acid changes, so that certain inhibitors only PDGFR-b without affecting EGFR17
.
lost their effectiveness6. Alternatively, monoclonal antibodies have
been developed which block the extracellular ligand binding site
of the protein kinase7. With enormous costs of such antibody
therapies there is still a need to develop novel small molecule
inhibitors with alternative binding modes to the protein backbone
We developed novel pyrimido[4,5-b]indoles which promise an
alternative EGFR binding mode due to the additional indole nitro-
gen next to the N1 of the pyrimidine. The indole phenyl ring with
its substitution and the 2-amino function of the molecular scaffold
may support a target structure bonding. We varied the substitu-
tion patterns of the 4-anilino residue and determined the affinity
to EGFR and PDGFR-b in an ATP competition assay using radio-
actively marked ATP which is incorporated into the kinase sub-
strate and measured using scintillation counting technique.
and, thus, a lowered resistance development7,8
.
One of the most prominent protein kinases is the endothelial
growth factor receptor (EGFR) because that kinase is known to be
involved in the various cancer-associated processes of uncon-
trolled cell growth, suppressed apoptosis, and, finally, metastasis9.
Some mutations and resulting amino acid changes of EGFR are
known to be tolerated by the inhibitors, so that the inhibitors
maintain their activities, whereas, other mutations cause a loss of
the inhibitory activity10. So recent research developments concen-
trated on developing alternative EGFR inhibitors11.
Experimental
General
Commercial reagents were used without further purification. The
4-chloro pyrimidine 1b was prepared according to literature start-
However, novel inhibitors of EGFR are suspected to suffer from
similar anticancer drug resistances than the established inhibitors. ing from the prepared 6-amino-2-mercatopyrimidine-4-ol given by
CONTACT Andreas Hilgeroth andreas.hilgeroth@pharmazie.uni-halle.de Department of Pharmaceutical Chemistry, Institute of Pharmacy, Martin Luther
University, Wolfgang-Langenbeck-Strasse 4, Halle 06120, Germany
ß 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distri-
bution, and reproduction in any medium, provided the original work is properly cited.

2
T. FISCHER ET AL.
the reaction of cyano acetic acid methyl ester and thiourea in compound 2f and of 2h (Scheme 1). For those reactions 1.5 equiv-
alents (2f) and 4 equivalents of the aniline (2h) were used. In case
of the formation of products 2c, 2f, and 2h 1 ml of N-methyl pyr-
rolidone was additionally added. The reaction mixture was heated
to 135 ꢀC and maintained at that temperature for 2–4 h depending
on the reaction proceeding followed by tlc until no more of the
pyrimidine starting compound was detectable. After cooling to
room temperature the raw product was purified by column chro-
matography using silica gel and a mixture of ethyl acetate and
methanol 95/5 to 90/10 in a gradient elution. The isolated yellow,
oily products were partly crystallised from methanol/diethyl ether
mixtures.
sodium methanolate, a following thioether hydrolysis using raney
nickel and a final 4-chlorination reaction using phosphorus oxy-
chloride in DMF18. The 1H-NMR spectra (400 MHz) were measured
using tetramethylsilane as internal standard. TLC was performed
on E. Merck 5554 silica gel plates. The ESI spectra were recorded
on a Finnigan LCQ Classic mass spectrometer. Elemental analysis
indicated by the symbols of the elements was within 0.4% of the
theoretical values and was performed using a Leco CHNS-932
apparatus.
N4–(3-ethoxyphenyl)pyrimidine-2,4,6-triamine 2a Yield 1.89 g
(77%); yellow oil; 1H NMR (DMSO-d6) d 1.30 (t, J ¼ 7.03 Hz, 3H,
OCH2CH3), 3.98 (q, J ¼ 7.0 Hz, 2H, OCH2CH3), 5.27 (s, 1H, 5-H), 6.33
Formation of the N4 -anilino substituted pyrimidines 2
One Equivalent of the 4-chloropyrimidine 1 was mixed with three
equivalents of the aniline compound except of the formation of (br, 2H, C2-NH2), 6.40 (br, 2H, C6-NH2), 6.50 (ddd, J ¼ 7.99 Hz, 2.00,
Cl
N
1a, R = NH2
1b, R = H
H2N
N
R
1a, b
(a)
O
R1
R1
OH
HN
N
HN
N
N
N
(b)
OH
H2N
+
R2
R2
H2N
O
3
2a-m
2a, 5a, R1 = 3-EtO, R2 = NH2
2b, 5b, R1 = 3-(CH3)2CHO, R2 = NH2
2c, 5c, R1 = 3-HC
R1
O
O
CH, R2 = NH2
2d, 5d, R1 = 4-EtO, R2 = NH2
HN
N
2e, 5e, R1 = 4-(CH3)2CHO, R2 = NH2
CH, R2 = NH2
2g, 5g, R1 = 4-Cl, R2 = NH2
2h, 5h, R1 = 4-Br, R2 = NH2
2f, 5f, R1 = 4-HC
N
R2
H2N
2i, 5i, R1 = 4-(4-MeO-BnO), R2 = NH2
2j, 5j, R1 = 4-(3-MeO-BnO), R2 = NH2
2k, 5k, R1= 4-(3-F-BnO), R2 = NH2
2l, 5l, R1 = 3-Cl, 4-(4-MeO-BnO), R2 = NH2
2m, 5m, R1 = 4-(3-F-BnO), R2 = H
4
- H2O
R1
HO
HN
N
N
R2
N
H
5a-m
Scheme 1. Formation of the 4-anilino substituted pyrimidines 2 and the 4-anilino substituted target compounds 5: (a) 2–4 h, 135 ꢀC; (b) 3–6 h, reflux, EtOH, and AcOH.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
3
1.0 Hz, 1H, 40-H), 7.04 (ddd, J ¼ 7.99, 2.00, 1.0 Hz, 1H, 60-H), 7.11 (t, C2-NH2, C6-NH2), 6.94 (AA0BB0, 2H, 300-, 500-H), 7.15 (d, J ¼ 8.98 Hz,
J ¼ 7.99 Hz, 1H, 50-H), 7.16 (t, J ¼ 2.00 Hz, 1H, 20-H), 8.90 (br, 1H,
NH-Ph); MS (ESI), m/z ¼ 246 [M þ Hþ].
1H, 50-H), 7.36 (m, 3H, 60-, 200-, 600-H), 7.64 (d, J ¼ 2.33 Hz, 1H, 20-H),
8.48 (br, 1H, NH-Ph); MS (ESI), m/z ¼ 372 [M þ Hþ].
N4–(3-isopropoxyphenyl)pyrimidine-2,4,6-triamine 2b Yield 2.01 g
(76%); yellow oil; 1H NMR (DMSO-d6) d 1.24 (d, J ¼ 5.96 Hz, 6H,
OCH(CH3)2), 4.54 (hep, J ¼ 5.96 Hz, 1H, OCH(CH3)2), 5.29 (s, 1H, 5-
H), 6.49–6.51 (m, 3H, 40-H, C2-NH2), 6.56 (br, 2H, C6-NH2), 7.04–7.11
(m, 3H, 20-, 50-, 60-H), 8.90 (br, 1H, NH-Ph); MS (ESI), m/z ¼ 260
[M þ Hþ].
N4–(4-((3-fluorobenzyl)oxy)phenyl)pyrimidine-4,6-triamine
2m
Yield 1.05 g (86%); mp 184–185 ꢀC; 1H NMR (DMSO-d6) d 5.08 (s,
2H, OCH2), 5.65 (s, 1H, 5-H), 6.21 (br, 2H, NH2), 6.94 (AA0BB0, 2H, 30-,
50-H), 7.15 (m, 1H, 400-H), 7.25–7.29 (m, 2H, 200-, 600-H), 7.35 (AA0BB0,
2H, 20-, 60-H), 7.43 (dt, J ¼ 7.58, 6.58 Hz, 1H, 500-H), 7.96 (br, 1H, NH-
Ph), 8.61 (s, 1H, 2-H); MS (ESI), m/z ¼ 311 [M þ Hþ].
N4–(3-ethinylphenyl)pyrimidine-2,4,6-triamine 2c Yield 0.63 g
1
(56%); yellow oil; H NMR (DMSO-d6) d 4.10 (s, 1H, CHꢁC), 5.21 (s,
Formation of the 4-anilino substituted 9H-pyrimido[4,5-b]indol-
6-ols 5
1H, 5-H), 5.88 (br, 2H, C2-NH2), 6.03 (br, 2H, C6-NH2), 6.97 (dt,
J ¼ 7.60 Hz, 1.25 Hz, 1H, 40-H), 7.21 (t, J ¼ 7.60 Hz, 1H, 50-H), 7.62 (t,
J ¼ 1.25 Hz, 1H, 20-H), 7.04 (dt, J ¼ 7.60, 1.25 Hz, 1H, 60-H), 8.72 (br,
1H, NH-Ph); MS (ESI), m/z ¼ 226 [M þ Hþ].
One Equivalent of the N4-anilino substituted pyrimidine 2 reacted
with 1.2 to 1.8 equivalents of p-benzoquinone in volumes of 20 ml
of dried ethanol and 5 ml of dried acetic acid per 1 mmol of the
used pyrimidine 2 (Scheme 1). The solution was heated under
reflux for 3–6 h depending on the reaction proceeding that was
followed by tlc. After reaction completion the solvent was evapo-
rated in vacuum. The remaining oil was purified using column
chromatography with silica gel and an eluent mixture of chloro-
form and methanol in a relation of 95 to 5 that changed gradually
to a relation of 90 to 10.
2-Amino-4-((3-ethoxyphenyl)amino)-9H-pyrimido[4,5-b]indol-6-ol
5a Yield 0.065 g (5%); greyish powder; mp 154–157 ꢀC; 1H NMR
(DMSO-d6) d 1.33 (t, J ¼ 7.0 Hz, 3H, OCH2CH3), 4.06 (q, J ¼ 7.0 Hz,
2H, OCH2CH3), 5.93 (br, 2H, NH2), 6.57 (br, 1H, NH-Ph), 6.63 (dd,
J ¼ 8.57, 2.21 Hz, 1H, 7-H), 6.50 (dd, J ¼ 8.18, 1.90 Hz, 1H, 40-H), 6.96
(d, J ¼ 8.57, 1H, 8-H), 7.01 (d, J 1.90 Hz, 1H, 20-H), 7.05–7.04 (m, 1H,
50-H), 7.42 (d, J ¼ 8.09, 1H, 60-H), 7.45 (d, J ¼ 2.21 Hz, 1H, 5-H), 8.85
(br, 1H, OH), 11.92 (br,1H, NH); MS (ESI), m/z ¼ 336 [M þ Hþ]; Anal.
(C18H17N5O2) Calc. C 64.47, H 5.11, N 20.88; Found C 64.07, H 5.20,
N 20.55.
2-Amino-4-((3-isopropoxyphenyl)amino)-9H-pyrimido[4,5-b]indol-6-
ol 5b Yield 0.088 g (6%); greyish powder; mp 147–149 ꢀC; 1H NMR
(DMSO-d6) d 1.29 (d, J ¼ 6.0 Hz, 6H, OCH(CH3)2), 4.64 (hept,
J ¼ 6.0 Hz, 1H, OCH(CH3)2), 5.92 (br, 2H, NH2), 6.55 (br, 1H, NH-Ph),
6.61 (dd, J ¼ 8.63, 2.20 Hz, 1H, 7-H), 6.93 (dd, J ¼ 8.1, 1.98 Hz, 1H,
40-H), 6.97 (d, J ¼ 8.63, 1H, 8-H), 7.02 (d, J ¼ 1.98 Hz, 1H, 20-H), 7.06
(t, J ¼ 8.1 Hz, 1H, 50-H), 7.42 (d, J ¼ 8.1, 1H, 60-H), 7.46 (d, J ¼ 2.2 Hz,
1H, 5-H), 8.87 (br, 1H, OH), 11.93 (br, 1H, NH); MS (ESI), m/z ¼ 350
[M þ Hþ]; Anal. (C19H19N5O2) Calc. C 65.32, H 5.48, N 20.04; Found
C 64.95, H 5.10, N 20.32.
2-Amino-4-((3-ethinylphenyl)amino)-9H-pyrimido[4,5-b]indol-6-ol
5c Yield 0.020 g (2%); greyish powder; mp 176–178 ꢀC; 1H NMR
(DMSO-d6) d 4.27 (s, 1H, CHꢁC), 5.97 (br, 2H, NH2), 6.60 (br, 1H,
NH-Ph), 6.65 (dd, J ¼ 8.66, 2.30 Hz, 1H, 7-H), 6.94 (d, J ¼ 8.66, 1H, 8-
H), 7.46 (d, J ¼ 2.30 Hz, 1H, 5-H), 7.51 (ddd, J ¼ 6.14, 3.01, 1.30 Hz,
1H, 60-H), 7.55–7.58 (m, 3H, 20-, 40-, 50-H), 8.89 (br, 1H, OH), 11.93
(br,1H, NH); MS (ESI), m/z ¼ 316 [M þ Hþ]; Anal. (C18H13N5O) Calc. C
68.56, H 4.16, N 22.21; Found C 68.15, H 3.85, N 22.30.
N4–(4-ethoxyphenyl)pyrimidine-2,4,6-triamine 2d Yield 1.62 g
(66%); yellow oil; 1H NMR (DMSO-d6) d 1.29 (t, J ¼ 6.92 Hz, 3H,
OCH2CH3), 3.95 (q, J ¼ 6.92 Hz, 2H, OCH2CH3), 5.11 (s, 1H, 5-H),
5.85 (br, 2H, C2-NH2), 5.98 (br, 2H, C6-NH2), 6.80 (AA0BB0, 2H, 30-,
50-H), 7.35 (AA0BB0, 2H, 20, 60-H), 8.39 (br, 1H, NH-Ph); MS (ESI), m/
z ¼ 246 [M þ Hþ].
N4–(4-isopropoxyphenyl)pyrimidine-2,4,6-triamine 2e Yield 1.88 g
(73%); yellow oil; 1H NMR (DMSO-d6) d 1.25 (d, J ¼ 6.02 Hz, 6H,
OCH(CH3)2), 4.56 (hep, J ¼ 6.02 Hz, 2H, OCH(CH3)2), 5.13 (s, 1H, 5-
H), 5.92 (br, 2H, C2-NH2), 6.03 (br, 2H, C6-NH2), 6.83 (AA0BB0, 2H, 30-
, 50-H), 7.36 (AA0BB0, 2H, 20, 60-H), 8.50 (br, 1H, NH-Ph); MS (ESI), m/
z ¼ 260 [M þ Hþ].
N4–(4-ethinylphenyl)pyrimidine-2,4,6-triamine 2f Yield 0.59 g
1
(52%); yellow oil; H NMR (DMSO-d6) d 2.82 (s, 1H, CHꢁC), 5.17 (s,
1H, 5-H), 5.64 (br, 2H, C2-NH2), 5.82 (br, 2H, C6-NH2), 7.33 (AA0BB0,
2H, 30-, 50-H), 7.60 (AA0BB0, 2H, 20, 60-H), 8.63 (br, 1H, NH-Ph); MS
(ESI), m/z ¼ 226 [M þ Hþ].
N4–(4-chlorophenyl)pyrimidine-2,4,6-triamine 2g Yield 1.43 g
1
(61%); mp 145–147 ꢀC; H NMR (DMSO-d6) d 5.38 (s, 1H, 5-H), 4.82
(br, 4H, C2-NH2, C6-NH2), 7.32 (AA0BB0, 4H, 20-, 60-, 30-, 50-H), 7.50
(br, 1H, NH-Ph); MS (ESI), m/z ¼ 236 [M þ Hþ].
N4–(4-bromophenyl)pyrimidine-2,4,6-triamine 2h Yield 1.13 g
(81%); yellow oil; 1H NMR (DMSO-d6) d 5.20 (s, 1H, 5-H), 5.95 (br,
2H, C2-NH2), 6.07 (br, 2H, C6-NH2), 7.36 (AA0BB0, 2H, 30-, 50-H), 7.59
(AA0BB0, 2H, 20, 60-H), 8.82 (br, 1H, NH-Ph); MS (ESI), m/z ¼ 280
[M þ Hþ].
N4–(4-((4-methoxybenzyl)oxy)phenyl)pyrimidine-2,4,6-triamine 2i
Yield 2.66 g (79%); mp 179–181 ꢀC; 1H NMR (DMSO-d6) d 3.74 (s,
3H, OCH3), 4.95 (s, 2H, OCH2), 5.10 (s, 1H, 5-H), 5.88 (br, 2H, C2-
NH2), 6.01 (br, 2H, C6-NH2), 6.88 (AA0BB0, 2H, 30-, 50-H), 6.93
(AA0BB0, 2H, 20-, 60-H), 7.35 (m, 4H, 200-, 300-, 500-,600-H), 7.35 (br, 1H,
NH-Ph); MS (ESI), m/z ¼ 338 [M þ Hþ].
N4–(4-((3-methoxybenzyl)oxy)phenyl)pyrimidine-2,4,6-triamine 2j
Yield 2.96 g (88%); mp 172–174 ꢀC; 1H NMR (DMSO-d6) d 3.74 (s,
3H, OCH3), 5.02 (s, 2H, OCH2), 5.11 (s, 1H, 5-H), 5.93 (br, 2H, C2-
NH2), 6.05 (br, 2H, C6-NH2), 6.85–6.88 (m, 1H, 400-H), 6.89 (AA0BB0,
2H, 30-, 50-H), 6.97–7.00 (m, 2H, 200-, 600-H), 7.28 (t, J ¼ 8.15 Hz, 1H,
50-H), 7.37 (AA0BB0, 2H, 20-, 60-H), 8.48 (br, 1H, NH-Ph); MS (ESI), m/
z ¼ 338 [M þ Hþ].
2-Amino-4-((4-ethoxyphenyl)amino)-9H-pyrimido[4,5-b]indol-6-ol
5d Yield 0.096 g (7%); greyish powder; mp 255–257 ꢀC; 1H NMR
(DMSO-d6) d 1.36 (t, J ¼ 6.95 Hz, 3H, OCH2CH3), 4.09 (q, J ¼ 6.95 Hz,
2H, OCH2CH3), 5.89 (br, 2H, NH2), 6.54 (br, 1H, NH-Ph), 6.61 (dd,
J ¼ 8.54, 2.23 Hz, 1H, 7-H), 6.83 (d, J ¼ 8.54, 1H, 8-H), 7.07 (AA0BB0,
2H, 30, 50-H), 7.34 (AA0BB0, 2H, 20-, 60-H), 7.44 (d, J ¼ 2.23 Hz, 1H, 5-
H), 8.82 (br, 1H, OH), 11.93 (br,1H, NH); MS (ESI), m/z ¼ 336
[M þ Hþ]; Anal. (C18H17N5O2) Calc. C 64.47, H 5.11, N 20.88; Found
C 64.12, H 5.05, N 20.48.
N4–(4-((3-fluorobenzyl)oxy)phenyl)pyrimidine-2,4,6-triamine
2k
Yield 2.58 g (79%); mp 96–98 ꢀC; 1H NMR (DMSO-d6) d 5.08 (s, 2H,
OCH2), 5.12 (s, 1H, 5-H), 6.07 (br, 2H, C2-NH2), 6.16 (br, 2H, C6-NH2),
6.91 (AA0BB0, 2H, 30-, 50-H), 7.14 (dt, J ¼ 8.65, 2.52 Hz, 1H, 400-H),
7.23–7.28 (m, 2H, 200-, 600-H), 7.37 (AA0BB0, 2H, 20-, 60-H), 7.42 (dt,
J ¼ 8.65, 6.10 Hz, 1H, 500-H), 8.48 (br, 1H, NH-Ph); MS (ESI), m/
z ¼ 326 [M þ Hþ].
N4–(3-chloro-4-((4-methoxybenzyl)oxy)phenyl)pyrimidine-2,4,6-tria-
2-Amino-4-((4-isopropoxyphenyl)amino)-9H-pyrimido[4,5-b]indol-6-
mine 2l Yield 2.76 g (74%); mp 140–143 ꢀC; 1H NMR (DMSO-d6) d ol 5e Yield 0.100 g (7%); greyish powder; mp 215–217 ꢀC; 1H NMR
3.74 (s, 3H, OCH3), 5.06 (s, 2H, OCH2), 5.17 (s, 1H, 5-H), 6.66 (br, 4H, (DMSO-d6) d 1.31 (d, J ¼ 5.97 Hz, 6H, OCH(CH3)2), 4.66 (hept,

4
T. FISCHER ET AL.
J ¼ 5.97 Hz, 1H, OCH(CH3)2), 5.89 (br, 2H, NH2), 6.54 (br, 1H, NH- 2.09 Hz, 1H, 7-H), 6.86 (d, J ¼ 8.51, 1H, 8-H), 6.97 (AA0BB0, 2H, 300-,
Ph), 6.61 (dd, J ¼ 8.59, 2.25 Hz, 1H, 7-H), 6.84 (d, J ¼ 8.59, 1H, 8-H), 500-H), 7.39 (m, 2H, 20-, 60-H), 7.43 (AA0BB0, 2H, 200-, 600-H), 7.44 (s,
7.06 (AA0BB0, 2H, 30, 50-H), 7.33 (AA0BB0, 2H, 20, 60-H), 7.44 (d,
J ¼ 2.25 Hz, 1H, 5-H), 8.82 (br, 1H, OH), 11.94 (br, 1H, NH); MS (ESI),
m/z ¼ 350 [M þ Hþ]; Anal. (C19H19N5O2) Calc. C 65.32, H 5.48, N
20.04; Found C 65.15, H 5.08, N 19.85.
2-Amino-4-((4-ethinylphenyl)amino)-9H-pyrimido[4,5-b]indol-6-ol
5f Yield 0.008 g (1%); greyish powder; mp >320 ꢀC; 1H NMR
(DMSO-d6) d 2.63 (s, 1H, CHꢁC), 5.97 (br, 2H, NH2), 6.63 (br, 1H,
NH-Ph), 6.65 (dd, J ¼ 8.70, 2.45 Hz, 1H, 7-H), 7.08 (d, J ¼ 8.70, 1H, 8-
H), 7.48 (d, J ¼ 2.45 Hz, 1H, 5-H), 7.71 (AA0BB0, 2H, 30, 50-H), 8.12
(AA0BB0, 2H, 20-, 60-H), 8.94 (br, 1H, OH), 11.97 (br, 1H, NH); MS
(ESI), m/z ¼ 316 [M þ Hþ]; Anal. (C18H13N5O) Calc. C 68.56, H 4.16,
N 22.21; Found C 68.35, H 3.97, N 22.04.
1H, 5-H), 7.45–7.65 (m, 1H, 50-H), 8.85 (br, 1H, OH), 11.92 (br, 1H,
NH); MS (ESI), m/z ¼ 462 [M þ Hþ]; Anal. (C24H20ClN5O3) Calc. C
62.41, H 4.36, N 7.67; Found C 62.25, H 4.34, N 7.45.
4-((4–(3-Fluorobenzyl)oxy)phenyl)amino)-9H-pyrimido[4,5-b]indol-
6-ol 5m Yield 0.041 g (2%); greyish powder; mp 190–192 ꢀC; 1H
NMR (DMSO-d6) d 5.11 (s, 2H, OCH2), 6.32 (br, 1H, NH-Ph), 6.65
(dd, J ¼ 8.76, 2.14 Hz, 1H, 7-H), 6.84 (d, J ¼ 2.14, 1H, 5-H), 7.02
(AA0BB0, 2H, 30-, 50-H), 7.14 (t, J ¼ 8.24 Hz, 1H, 500-H), 7.25–7.31 (m,
2H, 200-, 600-H), 7.32 (AA0BB0, 2H, 20-, 60-H), 7.44 (m, 1H, 400-H), 7.91
(d, J ¼ 8.76 Hz, 1H, 8-H), 9.04 (br, 1H, OH), 9.37 (br, 1H, NH), 9.45 (s,
1H, 2-H); MS (ESI), m/z ¼ 401 [M þ Hþ]; Anal. (C23H17FN4O2) Calc. C
68.99, H 4.28, N 3.50; Found C 68.75, H 4.15, N 3.26.
2-Amino-4-((4-chlorophenyl)amino)-9H-pyrimido[4,5-b]indol-6-ol
5g Yield 0.105 g (9%); greyish powder; mp 295–297 ꢀC; 1H NMR
(DMSO-d6) d 5.99 (br, 2H, NH2), 6.63 (dd, J ¼ 8.87, 2.24 Hz, 1H, 7-H),
6.66 (br, 1H, NH-Ph), 6.96 (d, J ¼ 8.87, 1H, 8-H), 7.46 (d, J ¼ 2.24 Hz,
1H, 5-H), 7.54 (AA0BB0, 2H, 30, 50-H), 7.61 (AA0BB0, 2H, 20-, 60-H), 8.91
(br, 1H, OH), 11.95 (br, 1H, NH); MS (ESI), m/z ¼ 326 [M þ Hþ]; Anal.
(C18H12ClN5O) Calc. C 58.99, H 3.71, N 21.50; Found C 58.65, H
3.46, N 21.35.
Receptor tyrosine kinase inhibition
The protein kinases were expressed by means of the baculovirus
expression system in Sf9 insect cells as human recombinant GST
fusion proteins and purified by affinity chromatography using
GSH-agarose. The kinase identity was confirmed by mass spec-
trometry using LC-ESI-MS/MS technique.
2-Amino-4-((4-bromophenyl)amino)-9H-pyrimido[4,5-b]indol-6-ol
5h Yield 0.090 g (6%); greyish powder; mp 303–305 ꢀC; 1H NMR
(DMSO-d6) d 5.94 (br, 2H, NH2), 6.60 (br, 1H, NH-Ph), 6.64 (dd,
J ¼ 8.64, 2.30 Hz, 1H, 7-H), 6.97 (d, J ¼ 8.64, 1H, 8-H), 7.45 (d,
J ¼ 2.30 Hz, 1H, 5-H), 7.48 (AA0BB0, 2H, 30, 50-H), 7.73 (AA0BB0, 2H,
20-, 60-H), 8.89 (br, 1H, OH), 11.89 (br, 1H, NH); MS (ESI), m/z ¼ 370
[M þ Hþ]; Anal. (C18H12BrN5O) Calc. C 51.91, H 3.27, N 18.92; Found
C 51.75, H 3.14, N 18.55.
2-Amino-4-((4–(4-methoxybenzyl)oxy)phenyl)amino)-9H-pyri-
mido[4,5-b]indol-6-ol 5i Yield 0.083 g (5%); greyish powder; mp
252–254 ꢀC; 1H NMR (DMSO-d6) d 3.75 (s, 3H, OCH3), 5.09 (s, 2H,
OCH2), 5.90 (br, 2H, NH2), 6.54 (br, 1H, NH-Ph), 6.62 (dd, J ¼ 8.54,
2.15 Hz, 1H, 7-H), 6.82 (d, J ¼ 8.54, 1H, 8-H), 6.96 (AA0BB0, 2H, 300-,
500-H), 7.14 (AA0BB0, 2H, 30-, 50-H), 7.34 (AA0BB0, 2H, 20-, 60-H), 7.41
(AA0BB0, 2H, 200-, 600-H), 7.44 (d, J ¼ 2.15 Hz, 1H, 5-H), 8.81 (br, 1H,
OH), 11.92 (br, 1H, NH); MS (ESI), m/z ¼ 428 [M þ Hþ]; Anal.
(C24H21N5O3) Calc. C 67.44, H 4.95, N 16.38; Found C 67.13, H 4.75,
N 15.98.
The measuring of protein kinase activity was performed in 96-
well FlashPlatesTM from Perkin Elmer in a 50 mL reaction volume.
The reaction mixture consisted of 20 mL of assay buffer solution,
5 mL of ATP solution in water, 5 mL of used test compound in a
10% dmso solution and, finally, a premixture of each 10 mL of
used substrate and enzyme solutions. The assay buffer solution
contained 70 mM of HEPES-NAOH pH 7.5, each 3 mM of magne-
sium chloride and manganese(II) chloride, 3 mM of sodium ortho-
vanadate, 1.2 mM of DTT, 50 mg/mL of PEG20000 and finally 15 mM
of [c-33 P]-ATP making approximately 9 ꢂ 105 cpm per well. The
final kinase concentration has been 10 ng/50 mL for EGFR and
20 ng/50 mL for PDGFR-b. The substrate was Poly(Glu,Tyr)4:1 for
EGFR and Poly(Ala, Glu, Lys, Tyr)6:2:5.1 for PDFGR-b, both used in a
concentration of 125 ng/50 mL. The reaction mixtures were incu-
bated at 30 ꢀC for 60 min. The reaction was stopped with 50 mL of
a 2% (v/v) solution of phosphoric acid. Then the plates were aspi-
rated and washed twice with 200 mL of water and a 0.9% solution
33
of sodium chloride. The incorporation of P was determined with
2-Amino-4-((4–(3-methoxybenzyl)oxy)phenyl)amino)-9H-pyrimido-
[4,5-b]indol-6-ol 5j Yield 0.047g (2%); greyish powder; mp
173–175 ꢀC; 1H NMR (DMSO-d6) d 3.76 (s, 3H, OCH3), 5.16 (s, 2H,
OCH2), 5.91 (br, 2H, NH2), 6.54 (br, 1H, NH-Ph), 6.62 (dd, J ¼ 8.60,
2.03 Hz, 1H, 7-H), 6.83 (d, J ¼ 8.60, 1H, 8-H), 6.91 (dd, J ¼ 7.75,
2.14 Hz, 1H, 400-H), 7.04 (s, 1H, 200-H), 7.05 (d, J ¼ 7.25, 1H, 600-H), 7.16
(AA0BB0, 2H, 30-, 50-H), 7.32 (t, J ¼ 7.75 Hz, 1H, 500-H), 7.35 (AA0BB0,
2H, 20-, 60-H), 7.44 (d, J ¼ 2.03Hz, 1H, 5-H), 8.81 (br, 1H, OH), 11.95
(br, 1H, NH); MS (ESI), m/z ¼ 428 [M þ Hþ]; Anal. (C24H21N5O3) Calc.
C 67.44, H 4.95, N 16.38; Found C 67.14, H 5.17, N 16.24.
a microplate scintillation counter. Ten different inhibitor concen-
trations were measured in a range of 3 nM to 100 mM. The residual
activity (%) and the IC50 values were finally calculated. From the
IC50 values the affinity constants Ki were determined using the
equation: IC50 ¼1/2 [Etotal]þKi ꢂ (1 þ [S]/Km) following a competi-
tive inhibitor binding mode19 (Table 1). The used Km values for
Table 1. Protein kinase inhibitory activity as determined Ki values of our target
compounds 5a-m for the tyrosine receptor kinases EGFR and PDGFR-b.
Ki values [nM]
2-Amino-4-((4–(3-fluorobenzyl)oxy)phenyl)amino)-9H-pyrimido[4,5-
b]indol-6-ol 5k Yield 0.087 g (4%); greyish powder; mp 199–201 ꢀC;
1H NMR (DMSO-d6) d 5.21 (s, 2H, OCH2), 5.90 (br, 2H, NH2), 6.54
(br, 1H, NH-Ph), 6.62 (dd, J ¼ 8.58, 2.29 Hz, 1H, 7-H), 6.83 (d,
J ¼ 8.58, 1H, 8-H), 7.15 7.19 (m, 1H, 200-H), 7.17 (AA0BB0, 2H, 30-, 50-
H), 7.31–7.35 (m, 2H, 400-, 600-H), 7.37 (AA0BB0, 2H, 20-, 60-H), 7.44 (d,
J ¼ 2.29 Hz, 1H, 5-H), 7.47 (t, J ¼ 7.89 Hz, 1H, 500-H), 8.82 (br, 1H,
OH), 11.93 (br, 1H, NH); MS (ESI), m/z ¼ 416 [M þ Hþ]; Anal.
(C23H18FN5O2) Calc. C 66.50, H 4.57, N 16.86; Found C 66.15, H
4.25, N 16.74.
Compound
EGFR
PDGFR-b
n.a.a
5a
5b
5c
5d
5e
5f
5g
5h
5i
181 14
n.a.a
2750 184
3530 200
1910 150
1944 218
n.a.a
149 66
935 84
574 110
421 66
686 82
475 143
413 111
132 99
170 85
103 37
189 27
n.a.a
3230 195
72 15
209 15
81 10
213 35
3660 210
5j
5k
5l
2-Amino-4-((3-chloro-4-((4-methoxybenzyl)oxy)phenyl)amino)-9H-
pyrimido[4,5-b]indol-6-ol 5l Yield 0.111 g (5%); greyish powder; mp
141–143 ꢀC; 1H NMR (DMSO-d6) d 3.76 (s, 3H, OCH3), 5.21 (s, 2H,
5m
OCH2), 5.95 (br, 2H, NH2), 6.59 (br, 1H, NH-Ph), 6.63 (dd, J ¼ 8.51, anot active.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
5
ATP have been measured with 1.3 mM for EGFR and with 0.989 mM
for PDGFR-b.
Results and discussion
The proceeding compound synthesis started with the formation of
the 4-anilino substituted pyrimidines 2, which were obtained by
the reaction of the 4-chloro pyrimidine 1 with a set of various ani-
lines. The alkoxy substituted anilines were prepared by alkylation
of the corresponding aminophenol, using alkyl chloride and potas-
sium tert. butoxide in DMF22. The ethinyl anilines were prepared
using a Sonogashira reaction, from the corresponding bromo
anilines dissolved in diethyl amine in the presence of methyl-3-
butyn-2-ol, triphenylphosphine, copper(I) iodide, palladium(II)
acetate, and, finally, with sodium hydroxide in toluene under
reflux conditions23.
The starting compound 2 then reacted with p-benzoquinone in
an ethanol/acetic acid mixture to give a Michael adduct 3 in the
first reaction step that was further oxidised by an excess of the
p-benzoquinone to give intermediate 4 as shown in Scheme 1.
The nucleophilic amino function attack and consequent water
elimination then led to our target compounds 5a–m.
Docking studies
The 3D structures of EGFR were taken from the Protein databank.
We took from the available X-ray structure the ones co-crystallised
with an inhibitor structurally most similar to the pyrimido[4,5-
b]indoles under study (PDB ID 2ITY). In addition the X-ray structure
of erlotinib bound to EGFR (PDB 1M17) was taken for comparison.
The protein structures were prepared as follows: water molecules
and bound small molecules were deleted, hydrogen atoms were
added and the resulting structures were minimised using the
MMF94 force field and the conjugate gradient method until a gra-
dient of 0.01 kcal/mol was reached. We used the docking pro-
gramme GOLD 5.2 (Cambridge Crystallographic Data Centre,
Cambridge, United Kingdom) to dock all compounds into the ATP
binding site of EGFR. For all docking runs the programme default
settings were used. The binding site was defined on the gate-
keeper residue with a radius of 15 Å. Goldscore was chosen as fit-
ness function. For each molecule, 30 docking runs were
performed.
From our measured ATP competition assay with the varied tar-
get compound concentrations the EGFR affinities were calculated
and presented in Table 1.
First we evaluated the various 3-anilino substituted compounds
5a–c. The inhibitory activity of compound 5a with the 3-ethoxy
substituent was determined with a Ki value of 181 nM. If the
ethoxy substituent was replaced with a more bulky isopropoxy
substituent the affinity for compound 5b was lost. An ethinyl func-
tion placed in the 3-position of compound 5c resulted in the best
affinity with a Ki value of 149 nM. So it can be stated that sterically
demanding substituents in the 3-position of the aniline residue
are unfavourable.
Cellular growth inhibition assay20,21
Cells were inoculated into 96 well microtiter plates in 100 ml at
plate densities ranging from 5000 to 40.000 cells per well depend-
ing from the individual cell doubling time. Then the cell- contain-
ing plates were incubated at 37 ꢀC under an air atmosphere
containing 5% of carbon dioxide and with a relative humidity for
24 h. After that time two plates of each cell line were fixed in situ
with TCA as control before drug addition. Then the drug contain-
ing dmso stock solutions were used and mixed with the cell cul-
ture medium containing 50 mg/mL of gentamicin. Aliquots of
100 ml of the respective dilution were added to the preincubated
plates reaching a final drug concentration of 10 mM. The plates
were incubated again under the preincubation conditions as
described for 48 h. Then cells were fixed under addition of 50 ml
of a cold 50% solution of TCA reaching a final concentration of
10% TCA. Incubation continued for 60 min at 4 ꢀC. The supernatant
was discarded and the plates were washed with water for five
times and dried at air. Then a sulforhodamine (SRB) solution
(0.4%) in acetic acid (1%) was added to each well and plates were
incubated again for 10 min. The unbound dye was removed by
washing for five times with an acetic acid solution (1%) and the
plates were dried at air. The bound stain was solubilised with a
10 mM solution of trizma base and the absorbance was measured
with a plate reader at a wavelength of 515 nm. The growth inhib-
ition measured as lowered net protein increase after SRB staining
was determined in relation to the untreated control cells (Table 2).
If the 3-ethoxy substituent moved to the 4-position of the anil-
ine residue in compound 5d the affinity towards EGFR was found
decreased. If the isopropoxy substituent moved to the 4-position
in derivative 5e the affinity increased with a Ki value of 574 nM.
The ethinyl substituent in the 4-position also lowered the com-
pound affinity for derivative 5f. Halogen atoms placed in the 4-
position of the aniline resulted in compound affinities of 686 nM
for the 4-chloro substitution of compound 5g and of 475 nM for
the 4-bromo substitution of derivative 5h. As indicated that more
sterically demanding substituents in the 4-position of the aniline
residue seem to be of little favour for the EGFR affinity we intro-
duced benzyloxy substituents in compounds 5i–k. The 4-methoxy
benzyloxy substituent of derivative 5i resulted in an affinity of
413 nM. If the 4-methoxy substituent moved to the 3-position of
the benzyloxy residue the affinity increased to a value of 132 nM
for compound 5j. If replaced with a 3-fluoro substituent the affin-
ity was almost similar with a value of 170 nM of compound 5k. So
the 3-benzyloxy substituted compounds were found as active as
the 3-aniline substituted compounds. In compound 5l we com-
bined a 4-methoxy benzyloxy substitution with a 3-chloro aniline
substitution and found the best affinity of all derivatives so far
with a Ki value of 103 nM. That activity almost reached the activity
of erlotinib with a value of 45 nM. Finally, derivative 5m with a 3-
fluoro benzyloxy substitution and without the 2-amino function of
the molecular scaffold reached a lowered affinity towards EGFR
with a Ki value of 189 nM.
Table 2. Tumour cell growth inhibition at a concentration of 10 mM of the
respective inhibitors 5a, 5k and 5i in screened non-small cell lung and prostate
cancer cell lines.
Compound growth inhibition [%]
Cancer cell line
5a
5k
5i
The docking results showed that the pyrimido[4,5-b]indole scaf-
fold is mimicking the adenine ring of ATP and represents the
hinge-binding motifs. Potent inhibitors (e.g. 5l) are able to form
two hydrogen bonds to the hinge region residues: one to the NH
of Met793 and one to the CO of Gln791. In case of compound 5m
only one hydrogen bond to the NH of Met793 is possible. The
4–(4-MeO-BnO) substituent of derivative 5l is interacting with
Non-small cell lung cancer
EKV X
NCI-H23
NCI-H322M
Prostate cancer
PC-3
71
32
35
90
93
96
29
9
1
25
51
95
90
25
15
DU-145

6
T. FISCHER ET AL.
Figure 1. (A) Docking solution for 5l at EGFR. The inhibitor structure is coloured orange, hydrogen bonds are shown as green dashed lines. Only amino acid residues
of the ATP-binding pocket are shown. (B) Visualization of the molecular surface of the ATP-binding pocket coloured according to the hydrophobicity (polar regions are
coloured magenta, hydrophobic regions are coloured green). (C) X-ray structure of EGFR in complex with erlotinib (coloured green). Hydrogen bonds are shown as
green dashed lines.
Lys745 as well as with hydrophobic residues at the P-loop the 4-position of the aniline we found improvements in the PDFG-
(Phe723, Leu747, Figure 1(A)). The 3-chloro substituent is facing b affinities with 1910 nM for derivative 5d and 1944 nM for com-
towards the back-hydrophobic pocket nearby Met766 that is pound 5e. The 4-ethinyl compound 5f was not active. The smaller
also addressed by the cyano phenyl substituent of erlonitinb chloro halogen atom in the 4-aniline position was less favourable
than the more bulky bromo atom which caused an inhibitory
affinity of 323 nM for compound 5h, whereas compound 5g was
not active. These first results indicated that more sterically
(Figure 1(B,C)).
Next we evaluated our compounds to inhibit PDGFR-b in the
ATP competition assay. The 3-ethoxy aniline compound 5a was
not active as PDGFR-b inhibitor. The more bulky 3-isopropoxy sub- demanding substituents in the 4-position of the aniline residue
stituent in compound 5b resulted in a poor affinity of 2750 nM. If may be of favour for the PDGFR-b affinity. Indeed, we reached
replaced with the 3-ethinyl group in derivative 5c the affinity was nanomolar PDFG-b affinities for our 4-benzyloxy substituted com-
lowered to 3530 nM. If both alkoxy substituents were moved to pounds 5i–k. The 4-methoxy benzyloxy aniline substituent in

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
7
Figure 1 Continued.
compound 5i resulted in a PDGFR-b affinity of 72 nM similar to growth inhibition with rates ranging from just 1 to 29% in the
recently reported PDFG-b inhibitors with an indolin-2-one based NSCLC cell lines and of 15 to 25% in the prostate cancer cell lines.
structure17. If moved to the 3-position of the benzyloxy substitu-
These results reveal the importance of an EGFR inhibition in anti-
ent the affinity of derivative 5j was found decreased with 209 nM.
The 3-fluoro substituent, however, was more favourable in that
position with an affinity constant of 81 nM for compound 5k. If
the most favourable 4-methoxybenzyloxy substitution was com-
bined with a 3-aniline substituent we found a decrease in affinity
with a value of 213 nM for derivative 5l. Compound 5m without
the 2-amino function at the molecular scaffold showed a PDGFR-b
affinity of 3660 nM which meant a decrease in the affinity by a fac-
tor of almost fifty of compared to the 2-amino compound 5k.
It can be stated that such a 2-amino function is essential for a
PDFGR-b kinase affinity.
cancer cell line growth and prove a certain potency of that inhib-
ition with a combined inhibition of PDGFR-b.
It can be shortly summarised for our novel compound class
that large substituents in the 4-position of the aniline residue
favour both the inhibition of EGFR and PDGFR-b. The dual inhibi-
tors with favourable EGFR-affinities show an effective anticancer
growth as far as investigated. So they may be of benefit to com-
bat an anticancer drug resistance resulting from a receptor hetero-
dimerization as suggested by our first results.
Disclosure statement
So we identified dual inhibitors of EGFR and PDFG-b and, thus,
concentrated nanomolar affinities similar to sole reported EGFR
and PDGFR-b inhibitors on one compound.
No potential conflict of interest was reported by the authors.
Next the most active compounds 5a, 5k, and, finally, 5i have
been investigated to inhibit the growth of cancer cell types
related to EGFR overexpression according to literature4,24. In first
screening studies carried out with an initial concentration of
10 mM by the National Cancer Institute of Health (NCI) the growth
inhibition of respective non-small cell lung cancer (NSCLC) cell
lines and of prostate cancer cell lines was determined. The results
are shown in Table 2.
Funding
The authors acknowledge the financial support of their work by
the country Saxony-Anhalt for a scholarship to Tim Fischer and by
the German Research Foundation (DFG) within the project HI687/
10–1 to Andreas Hilgeroth.
The only EGFR inhibiting compound 5a resulted in growth
inhibition rates of 32 to 71% in the NSCLC cell lines and of 25 and
51% in the prostate cancer cell lines. For erlotinib for comparison
growth inhibition rates of 20% in the NSCLC cell line NCI-H23 and
of 40 and 45% in the prostate cancer cell lines PC-3 and DU-145
have been reported25,26. If the EGFR inhibition is combined with
the PDGFR-b drug affinity in derivative 5k we found main
increases in the growth inhibition of the respective cell lines up to
96% in the NSCLC cell line representing NCI-H322M and of 95% in
the prostate cancer cell line PC-3. Compound 5i with a similar
PDGFR-b affinity than derivative 5k, but a reduced EGFR affinity,
also lower than that of compound 5a, resulted in a reduced
References
1. Giancotti FG. Deregulation of cell signaling in cancer. FEBS
Lett 2014;588:2558.
2. Giamas G, Stebbing J, Vorgias CE, Knippschild U. Protein kin-
ases as targets for cancer treatment. Pharmacogenomics
2007;8:1005.
3. Tsai Ch J, Nussinov R. The molecular basis of targeting pro-
tein kinases in cancer therapeutics. Semin Cancer Biol
2013;23:235.
4. Zhang J, Yang PL, Gray NS. Targeting cancer with small mol-
ecule kinase inhibitors. Nat. Rev Cancer 2009;9:28.

8
T. FISCHER ET AL.
5. Knight ZA, Shokat KM. Features of selective kinase inhibitors. 17. Zhang L, Zheng Q, Yang Y, et al. Synthesis and in vivo SAR
Chem Biol 2005;12:621.
study of indolin-2-one-based multi-targeted inhibitors as
potential anticancer agents. Eur J Med Chem 2014;82:139.
ꢀ
6. Balzano D, Santaguida S, Musacchio A, Villa F. A general
framework for inhibitor resistance in protein kinases. Chem 18. Ouwerkerk N, Boom J, Lugtenburg J, Raap J. Synthesis of
Biol 2011;18:966.
7. Imai K, Takaoka A. Comparing antibody and small-molecule
therapies for cancer. Nat Rev Cancer 2006;6:714.
[1ꢀ,20,50,2-13C4]-20- deoxy-D-adenosine by a chemoenzymatic
strategy to enable labelling of any of the 215 carbon-13 and
nitrogen-15 isotopomers. Eur J Org Chem 2002;14:2356.
€
8. Shaughnessy A. Monoclonal antibodies: magic bullets with a 19. Voigt B, Krug M, Schachtele C, et al. Probing novel 1-aza-9-
hefty price tag. BMJ 2012;345:e8346.
9. Tiash S, Chowdhury EH. Growth factor receptors: promising
drug targets in cancer. J Cancer Metastasis Treat 2015;1:190.
oxafluorenes as selective GSK-3beta inhibitors. Chem Med
Chem 2008;3:120.
20. Boyed MR. Status of the NCI preclinical antitumor drug dis-
covery screen. Princ Pract Oncol 1989;3:1.
10. Gazdar AF. Activating and resistance mutations of EGFR in
non-small lung cancer: role in clinical respons to EGFR tyro- 21. Monks A, Scudiero D, Skehan P, Boyed M. Implementation of
sine kinase inhibitors. Oncogene 2009;28:S24–S31.
11. Landi L, Cappuzzo F. Irreversible EGFR-TKIs: dreaming perfec-
tion. Transl Lung Cancer Res 2013;2:40.
a pilot-scale, high flux anticancer drug screen utilizing dis-
ease-oriented panels of human tumor cell lines in culture.
Proc Am Assoc Cancer Res 1989;30:607.
12. Wang DD, Ma L, Wong MP, et al. Contribution of EGFR and 22. Kuramochi T, Kakefuda A, Hiroyoshi Y, et al. Synthesis and
ErB-3 heterodimerization to the EGFR muatation-induced
gefitinib- and erlotinib-resistance in non-small-cell lung car-
cinoma treatment. PLoS One 2015;10:e0128360.
structure-activity relationships of benzyloxyphenyl deriva-
tives as a novel class of NCX inhibitors: effects on heart fail-
ure. Bioorg Med Chem 2005;13:725.
13. Dawson JP, Berger MB, Lin CH-C, et al. Epidermal growth 23. Jeong K-S, Cho YL, Chang S-Y, et al. Assembly and binding
factor receptor dimerization and activation require ligand-
induced conformational changes in the dimer interface. Mol
Cell Biol 2005;25:7734.
properties of osmate ester-bridged binuclear macrocycles.
J Org Chem 1999;64:9459.
ꢀ
24. Guerin O, Fischel JL, Ferrero J-M, et al. EGFR targeting in
14. Normanno N, De Luca A, Bianco C, et al. Epidermal growth
factor receptor (EGFR) signaling in cancer. Gene 2006;366:2.
hormone-refractory prostate cancer: current appraisal and
prospects for treatment. Pharmaceuticals 2010;3:2238.
15. Saito Y, Haendeler J, Hojo Y, et al. Receptor heterodimeriza- 25. Zhang W, Peyton M, Xie Y, et al. Histone deacetylase inhibi-
tion: essential mechanism for platelet-derived growth factor-
induced epidermal growth factor receptor transactivation.
Mol Cell Biol 2001;21:6387.
tor romidepsin enhances anti-tumor effect of erlotinib in
non-small cell lung cancer (NSCLC) cell lines. J Thorac Oncol
2009;4:161.
16. Black PC, Brown GA, Dinney CP, et al. Receptor heterodimeri- 26. Fischel J-L, Ciccolini J, Formento P, Ferrero J-M, Milano G.
zation: a new mechanism for platelet-derived growth factor
induced resistance to anti-epidermal growth factor receptor
therapy for bladder cancer. J Urol 2011;185:693.
Synergistic cytotoxic interaction in hormone-refractory pros-
tate cancer with the triple combination docetaxel-erlotinib
and 5-fluoro-50-deoxyuridine. Anti-Cancer Drugs 2006;17:807.