Synthesis and biological evaluation of novel pyrido[2,3-b]pyrazines inhibiting both erlotinib-sensitive and erlotinib-resistant cell lines

Article abstract of DOI:10.1016/j.bmcl.2013.09.005

A series of novel pyrido[2,3-b]pyrazines were synthesized as potential antitumor agents for erlotinib-resistant tumors. Known signal inhibitor compounds from our Nested Chemical Library were tested in phenotypic assays on erlotinib-sensitive PC9 and erlotinib-resistant PC9-ER cell lines to find a compound class to be active on erlotinib resistant cell lines. Based on the screening data, novel pyrido[2,3-b]pyrazines were designed and synthesized. The effect of the substituent position of the heteroaromatic moiety in position 7 and the importance of unsubstituted position 2 of the pyridopyrazine core were explored. Compound 7n had an IC₅₀ value of 0.09 μM for the inhibition of PC9 and 0.15 μM for the inhibition of PC9-ER. We found that some lead compounds of these structures overcome erlotinib-resistance which might become promising drug candidates to fight against NSCLC with EGFR T790M mutation. The signaling network(s) involved in the mechanism(s) of action of these novel compounds in overcoming erlotinib resistance remain to be elucidated.

Full text of DOI:10.1016/j.bmcl.2013.09.005

Bioorganic & Medicinal Chemistry Letters 23 (2013) 6152–6155
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of novel pyrido[2,3-b]pyrazines
inhibiting both erlotinib-sensitive and erlotinib-resistant cell lines
László Kékesi a,b, Anna Sipos a,b, Gábor Németh a,b, János Pató , Nóra Breza , Ferenc Baska , László Orfi a,b,c,
György Kéri
a
a
a,b
}
a,b,d,
⇑
a
Vichem Chemie Research Ltd., Herman Ottó u. 15., H-1022 Budapest, Hungary
b
c
Rational Drug Design Laboratory CRC, Semmelweis University, POB 131, H-1367 Budapest 5., Hungary
Department of Pharmaceutical Chemistry, Semmelweis University, H o} gyes Endre u. 9., H-1092 Budapest, Hungary
Pathobiochemistry Research Group of Hungarian Academy of Sciences in Department of Medical Chemistry, Semmelweis University School of Medicine, H-1085, T u} zoltó u.
d
37-47., Budapest, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 July 2013
Revised 1 September 2013
Accepted 3 September 2013
Available online 8 September 2013
A series of novel pyrido[2,3-b]pyrazines were synthesized as potential antitumor agents for erlotinib-
resistant tumors. Known signal inhibitor compounds from our Nested Chemical Library were tested in
phenotypic assays on erlotinib-sensitive PC9 and erlotinib-resistant PC9-ER cell lines to find a compound
class to be active on erlotinib resistant cell lines. Based on the screening data, novel pyrido[2,3-b]pyra-
zines were designed and synthesized. The effect of the substituent position of the heteroaromatic moiety
in position 7 and the importance of unsubstituted position 2 of the pyridopyrazine core were explored.
Keywords:
Compound 7n had an IC50 value of 0.09 lM for the inhibition of PC9 and 0.15 lM for the inhibition of
Pyrido[2,3-b]pyrazine
Erlotinib resistance
Phenotypic screen
PC9
PC9-ER. We found that some lead compounds of these structures overcome erlotinib-resistance which
might become promising drug candidates to fight against NSCLC with EGFR T790M mutation. The signal-
ing network(s) involved in the mechanism(s) of action of these novel compounds in overcoming erlotinib
resistance remain to be elucidated.
PC9-ER
Inhibitor
Ó 2013 Elsevier Ltd. All rights reserved.
Erlotinib is a well-known Epidermal Growth Factor Receptor
tyrosine kinase inhibitor (EGFR TKI) showing strong antitumor
activity against certain EGFR-dependent tumors such as EGFR-dri-
ven non-small cell lung cancer (NSCLC) and colon cancer. Tumor
samples from patients with NSCLC who are responsive to erlotinib
usually have sensitizing mutations of the epidermal growth factor
but several not yet identified mechanisms can undoubtedly cause
resistance as well other than EGFR gene alterations. Our compound
library containing known inhibitors (and their derivatives) of dif-
ferent signaling-related cancer targets were tested on these two
cell lines. From this medium throughput screening the pyr-
ido[2,3-b]pyrazine core emerged as one candidate. In this paper
we describe the development process resulting in effective inhibi-
tors derived from the pyrido[2,3-b]pyrazine hit compound. This
novel, biologically active compound family is the object of our re-
1
,2
receptor (EGFR) gene. Despite the prominent responses, most
patients ultimately have a relapse that is considered to be a conse-
quence of a later acquired, second mutation that cause resistance
3
6
to EGFR TKIs. EGFR inhibitors—such as erlotinib—are effective on
cent patent application.
EGFR-sensitive mutant NSCLC, but not on EGFR wild type (or KRAS
mutant) tumors. In our study our aim was to find an inhibitor
Compounds with pyrido[2,3-b]pyrazine core are described in
the literature having various biological effects including an iso-
zyme-selective inhibitor of PI3K, as a drug candidate for myocar-
4
which is effective not only on an EGFR mutant, erlotinib-sensitive
cell line, but also on a resistant variant NSCLC cell line containing
acquired EGFR mutation responsible for erlotinib resistance. In a
phenotypic screen two NSCLC cancer cell lines were used: PC9
and PC9-ER. PC9-ER cells were made erlotinib-insensitive by pro-
longed incubation with erlotinib. Cell lines were kindly provided
by the Cancer Research UK London Research Institute, Signal Trans-
duction Laboratory. One identified genotypic change responsible
7
8
dial infarction, TRPV1 antagonist in pain treatment, dipeptidyl
9
peptidase IV inhibitor in type 2 diabetes mellitus, Mycobacterium
1
0,11
3
12
tuberculosis growth inhibitor,
fungicide, human neurokinin-3
1
14
(hNK-3) receptor antagonist, GnRH antagonist, Wnt/b-catenin
1
5
16
17
inhibitor, PKB inhibitor, ALK-inhibitor, p38
inhibitor and BRAF inhibitor in cancer treatment.
a MAP kinase
1
8,19
Traditional synthetic methods of the pyrido[2,3-b]pyrazine
5
for erlotinib resistance in the PC9-ER cells is the T790M mutation,
cores apply the condensation of pyridine-2,3-diamine and a vicinal
1
8
13
20
dioxo compound, 2,3-dihydroxy-1,4-dioxane, oxalate ester or
2
1
⇑
Corresponding author. Tel.: +36 1 487 2080; fax: +36 1 799 9990.
E-mail address: keri@vichem.hu (G. Kéri).
hydroxyacetone. Pyrido[2,3-b]pyrazine can be prepared from
pyridine-monoamine as well, when there is only one amine
0
960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.09.005

L. Kékesi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6152–6155
6153
Table 2
Cellular assay results for compounds 1, 5a–d
S
S
S
N
HN
N
N
N
N
N
O
NH2
N
1
N
N
HAr
N
2
N
Ar
N
N
N
H
Compound
Ar1
H
Ar2
IC50
PC9
(lM)
N
H
PC9-ER
2
,3,7-tri-2-thienyl-
1
A-674563
pyrido[2,3-b]pyrazine
1
Phenyl
Phenyl
1.86
>30
2.34
5a
Phenyl
21.97
18.27
5.74
Figure 1. Structure of 1 derived from structures of known Akt inhibitors.
5b
4-Methylphenyl
4-Fluorophenyl
2-Thienyl
4-Methylphenyl
4-Fluorophenyl
2-Thienyl
11.60
6.90
10.77
5c
5d
11.01
substituent in either positions 2 or 3. In that case, a suitable func-
tional group (e.g., halogen, nitro, carboxylic acid, oxo, nitrile) has to
be placed in the other position. The reaction partner has to contain
an amine and/or one of these prior functional groups. The prepara-
tion of the ring system can thus be carried out in one or two
ity could be achieved by our method, using trifluoroacetic acid as
solvent. This way the condensation yielded mostly the 3-substi-
tuted pyridopyrazine and less than 5% of the 2-substituted iso-
31
2
2–25
steps.
Pyrido[2,3-b]pyrazine can also be obtained by conden-
mer. Moreover, the product could be purified easily by
crystallization from ethanol. In the second step we applied the Su-
zuki coupling reaction to substitute the position 7 bromine with
heteroaromatic rings, resulting in novel compounds (Scheme 1).
sation of (hydroxyimino)malononitrile and two malononitrile mol-
2
6
ecules.
In the presence of strong bases, 3-aminopyrazine
derivatives—like 2-carboxylates or 2-aldehydes—yield pyrido[2,3-
b]pyrazine with the appropriate aldehyde.27,28
32
33
Some of the planned oxo-acetaldehydes and boronic acids are
not commercially available, so we prepared them.
In TKI-resistant cell lines we have to consider alternative path-
ways responsible for the proliferation. Our compound library con-
tains known inhibitors (and their derivatives) of such targets, for
example Akt (Table 1).29 A-674563 and 2,3,7-tri-2-thienylpyri-
Biological results showed that compounds bearing substituents
both in 2 and 3-position exhibit a week effect (Table 2). In the
development process we selected a compound with a phenyl group
in position 3 and hydrogen in position 2 (1) as a potential lead
compound. We have studied the role of different substituents in
position 7. There are many known structures with carbocyclic
1
6,30
do[2,3-b]pyrazine compounds are known as Akt inhibitors.
As a combination of these structures, pyrido[2,3-b]pyrazine
with substituents of A-674563 was synthesised (Fig. 1). This novel
structure (1) and some of its derivatives were included in our com-
pound library, and they were tested on PC9 and PC9-ER cancer cell
lines. Since 1 showed promising biological effects, it was selected
as a developmental candidate.
Four derivatives of 1 were the members of the compound li-
brary tested in the first round. In contrast to the structure of 1,
these compounds have a substituent in position 2. The pyrido-pyr-
azine ring system was synthesized via condensation of 2 and dioxo
compounds (Scheme 1). The condensation reaction of 2 and
oxo-acetaldehydes can yield two regioisomers. High regioselectiv-
16
and sulfur containing heteroaromatic moieties; therefore, we
prepared nitrogen containing heteroaromatic rings to obtain novel
derivatives.
It can be seen in Table 3 that pyridine, quinolone and isoquino-
line derivatives are inactive, while 1H-Indol-4-yl, 1H-indol-5-yl
and 1H-indazol-5-yl rings appear as the requisite moiety in 7-posi-
tion of pyridopyrazine (6i, 6j, 6m). The position of carbon–carbon
bond between the [5 + 6] ring system and the pyrido–pyrazine ring
has a crucial role (6k, 6l, 6n). Replacing the nitrogen atom by a car-
0
bon atom in position 2 of the heterocyclic substituent enhanced
Table 1
Cellular assay results for reference compounds
Compound
IC50
PC9
(
l
M)
Table 3
Cellular assay results for compounds 6a–n
PC9-ER
3
Ar
N
N
Erlotinib
A-674563
0.005
0.91
5.65
0.82
2
,3,7-tri-2-thienylpyrido[2,3-b]pyrazine
>30
15.64
N
1
2
Compound
Ar3
IC50
PC9
(lM)
1
HAr
Br
Ar
N
N
HAr
Br
NH2
NH2
O
O
a
PC9-ER
+
2
N
HAr
N
2
HAr
6a
6b
Pyridin-3-yl
Pyridin-4-yl
6-Fluoropyridin-3-yl
2-Piperazin-1-ylpyridin-4-yl
Isoquinolin-5-yl
Quinolin-3-yl
Quinolin-6-yl
1-Methyl-1H-indol-5-yl
1H-Indol-4-yl
1H-Indol-5-yl
1H-Indol-6-yl
1H-Indol-7-yl
1H-Indazol-5-yl
1H-Indazol-6-yl
11.30
>30
>30
6.29
>30
>30
>30
>30
0.78
0.36
>30
>30
1.28
>30
11.11
>30
>30
8.96
30.00
>30
>30
>30
2.56
4.43
>30
4
3
6c
b
6d
6e
3
1
2
6f
N
HAr
6g
6h
N
N
, 5a-d
HAr
6i
1
6j
6
a-n, 7a-x
6k
6l
>30
1.39
>30
Scheme 1. Synthesis of pyrido[2,3-b]pyrazines. Ar1: aromatic ring or H, Ar2:
aromatic ring or H, Ar : heteroaromatic ring. Reagents and conditions: (a) rt, 5 h,
6m
3
6n
3
TFA; (b) Ar -boronic acid, Pd(PPh
)
3 4
, Na
2
CO
3
, 75–80 °C, 3 h, DME/water.

6
154
L. Kékesi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6152–6155
Table 4
Table 5
Cellular assay results for compounds 7a–w
Cellular assay results for compounds 8a–h
3
Ar
N
N
HN
N
1
N
N
R
N
H
N
Compound Ar3 Substituent position R1
IC50
PC9
(
l
M)
PC9-ER
2
R
(
)
n
O
0
7
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
b
b
c
c
c
c
d
e
f
4
4
4
4
4
4
4
4
4
4
4
3
2
F
Cl
CH
CF
OCH
OH
NH
COOH
NO
0.94
>30
>30
7.89
1.11
4.31
10.33 5.53
>30
>30
0.32
0.14
0.87
0.10
1.02
>30
>30
10.01
9.40
9.10
0
0
0
0
0
0
0
0
0
0
0
0
0
R2
7
b
Compound
n
IC50
PC9
(lM)
7
c
3
PC9-ER
7
d
3
7
7
e
3
8a
8b
8c
8d
8e
8f
2
2
2
3
2
3
2
2
Piperidin-1-yl
Morpholin-4-yl
1-Methylpyrrolidin-2-yl
4-Methylpiperazin-1-yl
NH2
3.19
3.62
2.87
2.72
27.72
2.93
6.91
4.13
3.97
11.34
5.99
4.71
19.65
4.54
24.29
>30
f
3
4
7
g
2
7
h
>30
>30
7
7
i
j
2
Morpholinyl
Piperidin-1-yl
Cl
0.59
0.20
1.29
0.36
0.15
>30
N(CH3)2
7
k
8g
8h
tert-Buthylcarbamate
Acetamide
7
l
7
m
OCH
3
0
7
n
3 ,4
Ethylenedioxy 0.09
0
7
7
7
o
p
q
4
F
Cl
>30
0.25
2.33
0
3
0.30
17.95
0
0
0
0
0
HN
4
4
4
3
4
Morpholinyl
F
7
7
7
r
s
t
25.03 27.58
HN
a
N
OCH
Cl
F
3
0.53
>30
>30
0.47
>30
>30
N
N
N
7
u
0
0
0
OH
N
N
7
v
3 ,4
Ethylenedioxy 10.55 13.45
Ethylenedioxy 0.49 0.57
H
2
0
3 ,4
N
R
7
w
O
( )
n
7
h
8
a-h
3
O
Ar : a: 1H-indol-5-yl, b: 1H-indazol-5-yl, c: 3-methyl-1H-indazol-5-yl, d: 1H-
Indazol-6-yl, e: 1H-Indol-6-yl, f: 1-Methyl-1H-indol-5-yl
0
Scheme 2. Synthesis of carboxamide derivatives. Reagents and conditions: (a) 1,1 -
Carbonylbis(1H-imidazole), corresponding amine, reflux, 24 h, CH Cl , THF.
2
2
In order to improve solubility properties, we prepared deriva-
tives containing an unsaturated, nitrogen containing, heterocyclic,
carboxamide- or amine group connected through an alkyl chain
linker. These moieties were connected through a carboxamide
bond. The coupling between the carboxyl group of 7h and the pri-
HN
N
N
N
N
0
mary amine function of the linker was performed with 1,1 -carbon-
ylbis(1H-imidazole) (Table 5) (Scheme 2).
7x
Figure 2. Structure of 7x that is a regioisomer of 7k.
In order to exclude toxic molecules from further investigations
two assays were used for quantitating cytotoxicity/cytolysis. The
fraction of membrane-disrupted PC9 cells after compound treat-
ment was assessed both by measuring lactate dehydrogenase
the activity (6j, 6m). Methylation of the indole nitrogen of the
effective 6j compound resulted in loss of effect (6h).
(
LDH) activity released from membrane-damaged PC9 NSCLC cell
3
5
We chose 5-indolyl-, and 5-indazolyl- substituted derivatives for
further development. Substituted derivatives of the 3-phenyl ring
were prepared and the condensation was performed with different
oxo(phenyl)acetaldehyde derivatives. To ensure regioselectivity
the synthesis was carried out in trifluoroacetic acid as solvent. The
g amino-derivative was prepared via the reduction of the 7i ni-
tro-derivative with sodium sulfide. Substituted 1H-indol-6-yl and
H-indazol-6-yl derivatives were also studied in order to validate
line(cytotoxic%, CT%) and flowcytometricanalysisafterPI staining
of PC9 NSCLC cell line (necrosis specific%, NSP%) (Table 6). According
to these results we can conclude that the molecules are not
cytotoxic; therefore, the mechanism of action is not necrosis.
Investigatingthemolecularmechanismof actionof ourbestcom-
pounds, we found that although this series derived from two differ-
ent Akt inhibitors, Akt kinase inhibition of the compounds was less
7
1
than 32% at 10
lM. While one of the reference Akt inhibitors, A-
the role of 1H-nitrogen in the structure-activity relationships. The
change of substituent position from 5 to 6 decreased the biological
effect (7n and 7v). Methylation of the indole nitrogen of 7n also re-
sulted in reduction of the biological effect (7w) (Table 4).
6
74563 shows good inhibition on both cell lines, the other reference
Akt inhibitor has no effect, and our compounds do not show Akt
inhibitory effect either. This indicates that the mechanism of action
of these compounds is not associated with Akt inhibition, and A-
Both regioisomers of 7-bromo-2- or 3-(4-piperidin-1-ylphe-
nyl)pyrido[2,3-b]pyrazine were prepared.31 Isomers were sepa-
rated with TLC (eluent chloroform: methanol 100:1) and 7k and
6
74563 is supposed to have other targets in addition to Akt.
Although the two cell lines are EGFR-driven cancer cell lines,
our best 15 compounds are ineffective in enzymatic EGFR assays.
All the compounds showed less than 41% inhibition in EGFRwt,
EGFR-L858R and EGFR-L858R T790M mutant assays at 10
these assays A-674563 showed 19% inhibition on EGFR-L858R
and 69% inhibition on EGFR-L858R T790M mutant. Effects on nei-
ther cell lines are associated with inhibition of sensitizing mutant
and T790M mutant EGFR.
7
x were prepared via Suzuki reaction (Scheme 1). 7k had an IC50
value of 0.14 M for the inhibition of PC9 and 0.20 M for the inhi-
bition of PC9-ER while with 7x the IC50 reached 9.03 M for PC9
and 12.88 M for PC9-ER. These biological results demonstrated
l
l
lM. In
l
l
that substitution in position 3 favors activity in contrast with
substitution in position 2 of the pyrido/pyrazine core (Fig. 2).

L. Kékesi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6152–6155
6155
Table 6
2. Wu, J.-Y.; Wu, S.-G.; Yang, C.-H.; Chang, Y.-L.; Chang, Y.-C.; Hsu, Y.-C.; Shih, J.-
Quantification of cytolysis
Y.; Yang, P.-C. Lung Cancer 2011, 72, 205.
Kobayashi, S.; Boggon, T. J.; Dayaram, T.; Jänne, P. A.; Kocher, O.; Meyerson, M.;
Johnson, B. E.; Eck, M. J.; Tenen, D. G.; Halmos, B. N. Eng. J. Med. 2005, 352, 786.
3
.
Cell line
method
incubation time (h)
PC9
LDH
24
PC9
FACS
24
4. Mok, T. S.; Wu, Y.-L.; Thongprasert, S.; Yang, C.-H.; Chu, D.-T.; Saijo, N.;
Sunpaweravong, P.; Han, B.; Margono, B.; Ichinose, Y.; Nishiwaki, Y.; Ohe, Y.;
Yang, J.-J.; Chewaskulyong, B.; Jiang, H.; Duffield, E. L.; Watkins, C. L.; Armour,
A. A.; Fukuoka, M. N. Eng. J. Med. 2009, 361, 947.
Compound
Concentration (IC50 10;
l
M)
CT%
NSP%
Erlotinib
1
0.05
18.57
7.75
3.60
12.75
9.45
3.20
1.44
8.74
1.02
0.88
2.54
5.33
3.44
3.35
2.28
1.20
2.57
1.96
2.27
2.90
2.22
3.63
1.99
1.94
2.04
2.58
5.
Ogino, A.; Kitao, H.; Hirano, S.; Uchida, A.; Ishiai, M.; Kozuki, T.; Takigawa, N.;
Takata, M.; Kiura, K.; Tanimoto, M. Cancer Res. 2007, 67, 7807.
Kékesi, L.; Sipos, A.; Németh, G.; Pató, J.; Kéri, Gy.; Orfi, L. Hugarian Patent
Application P1300006, 2013.
6.10
6.82
3.87
ND
6
6
i
j
6.
}
6
m
7. Palanki, M. S. S.; Dneprovskaia, E.; Doukas, J.; Fine, R. M.; Hood, J.; Kang, X.;
Lohse, D.; Martin, M.; Noronha, G.; Soll, R. M.; Wrasidlo, W.; Yee, S.; Zhu, H. J.
Med. Chem. 2007, 50, 4279.
7
a
ND
7
j
4.23
ꢀ4.01
6.19
3.12
3.94
4.10
5.84
8.
Blum, C. A.; Caldwell, T.; Zheng, X.; Bakthavatchalam, R.; Capitosti, S.;
Brielmann, H.; De Lombaert, S.; Kershaw, M. T.; Matson, D.; Krause, J. E.;
Cortright, D.; Crandall, M.; Martin, W. J.; Murphy, B. A.; Boyce, S.; Jones, A. B.;
Mason, G.; Rycroft, W.; Perrett, H.; Conley, R.; Burnaby-Davies, N.; Chenard, B.
L.; Hodgetts, K. J. J. Med. Chem. 2010, 53, 3330.
Cox, J. M.; Harper, B.; Mastracchio, A.; Leiting, B.; Sinha Roy, R.; Patel, R. A.; Wu,
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Edmondson, S. D. Bioorg. Med. Chem. Lett. 2007, 17, 4579.
7
k
7
l
7
m
7
n
7
p
9.
7
s
1
1
0. White, E. L.; Suling, W. J.; Ross, L. J.; Seitz, L. E.; Reynolds, R. C. J. Antimicrob.
Chemother. 2002, 50, 111.
1. Mathew, B.; Srivastava, S.; Ross, L. J.; Suling, W. J.; White, E. L.; Woolhiser, L. K.;
Lenaerts, A. J.; Reynolds, R. C. Bioorg. Med. Chem. 2011, 19, 7120.
Compound 7m was tested in an active site-directed competi-
tion binding assay on 456 kinases and disease relevant mutant
3
6
12. Crowley, P. J.; Lamberth, C.; Müller, U.; Wendeborn, S.; Nebel, K.; Williams, J.;
Sageot, O.-A.; Carter, N.; Mathie, T.; Kempf, H.-J.; Godwin, J.; Schneiter, P.;
Dobler, M. R. Pest Manag. Sci. 2010, 66, 178.
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L. F.; Rigolio, R.; Sarau, H. M.; Schmidt, D. B.; Zanoni, G.; Farina, C. Farmaco
variants. Inhibition at 1 lM was less than 54% on each kinase.
Although the structures of these compounds suggest that they
might be kinase inhibitors, relevant kinase inhibition of these com-
pounds was not found.
As we have described before, one identified genotypic change
1999, 54, 364.
responsible for erlotinib resistance in the PC9-ER NSCLC cell line
14. Young, J. R.; Huang, S. X.; Walsh, T. F.; Wyvratt, M. J.; Yang, Y. T., Jr.; Yudkovitz,
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is the T790M mutation.5 Several not yet identified mechanisms
3
7,38
such as a type of unidentified signaling pathway switch,
or
inhibition of some protein-protein interaction(s)3
9–41
can also be
responsible for the resistance of PC9-ER cell line other than EGFR
gene alterations. While Akt and EGFR inhibition and kinase profil-
ing did not give insight to the molecular mechanism, target identi-
fication of these compounds is still an on-going effort.
16. Kawakami, J.; Duncton, M.; Sherman, D.; He, H.-Y.; Kiselyov, A.; Pytowski, B.
PTC Patent WO 2005 007 099, 2005.
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1
1
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Whittaker, S.; Niculescu-Duvaz, D.; Nourry, A.; Davies, L.; Manne, H. A.;
Lopes, F.; Preece, N.; Hedley, D.; Ogilvie, L. M.; Kirk, R.; Marais, R.; Springer, C. J.
J. Med. Chem. 2010, 53, 5639.
In summary, in this paper we have described the synthesis and
biological evaluation of a series of novel pyrido[2,3-b]pyrazines
substituted in position 7 with nitrogen heterocycles as inhibitors
of erlotinib-sensitive PC9 and erlotinib-resistant PC9-ER NSCLC
cancer cells. The development process resulted in several very
effective compounds—7n, 7k, 7m, 7p, 7s, 7j—which are potent
inhibitors of both cell lines, in contrast with the reference com-
pound erlotinib that is an effective inhibitor of PC9 cell line only.
SAR studies clearly demonstrated that substitution in position 2
of the core is unfavorable even in case of mono- or disubstituted
1
20. Charlton, S. J.; LeBlanc, C.; McKeown, S. C. PCT Patent WO 2012 007 539, 2012.
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2
2
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0
0
0
derivatives; while substituent positions 4 and 5 (preferably 5 )
of 1H-indol- or 1H-indazol favors the inhibitory effect. Methylation
of the 1H nitrogen of the heterocycle is unfavorable also. The fur-
ther aim of our on-going efforts is to improve physicochemical
properties (such as solubility) and drug-like properties, and clarify
the signaling network that is involved in the mechanisms of action
of these novel compounds.
27. Crowley, P. J.; Müller, U.; Dobler, M.; Williams, J. PTC Patent WO 2005 123 733,
005.
2
2
2
8. Lippmann, E.; Teichert, J. Zeitschrift für Chemie 1979, 19, 422.
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30. Thomas, S. A.; Li, T.; Woods, K. W.; Song, X.; Packard, G.; Fischer, J. P.; Diebold,
R. B.; Liu, X.; Shi, Y.; Klinghofer, V.; Johnson, E. F.; Bouska, J. J.; Olson, A.; Guan,
R.; Magnone, S. R.; Marsh, K.; Luo, Y.; Rosenberg, S. H.; Giranda, V. L.; Li, Q.
Bioorg. Med. Chem. Lett. 2006, 16, 3740.
31. Condensation-reaction of 2 and oxo(4-piperidin-1-ylphenyl)acetaldehyde
Acknowledgments
monohydrate was examined. At ambient temperature in DMF the isomer
ratio of 2- and 3- substituted derivatives was 42:58. This ratio was 2:98 at 0 °C,
in TFA.
The research leading to these results received funding from the
European Commission’s Seventh Framework Programme (FP7/
3
2. Corresponding acetophenone, 48% HBr, 50–60 °C, 3 h, DMSO.
33. Corresponding bromo-compound, bis(pinacolato)diboran, Pd(PPh
CH COOK, 88–95 °C, 20 h, abs. DMF.
4. Synthesis reagents and conditions: 7i, Na
5. Cytotoxicity Detection Kit PLUS™ (Roche).
3 2 2
) Cl ,
3
2
2
007-2013) under the Lungtarget Grant agreement (HEALTH-F2-
010-259770). Authors are grateful to National Innovation Office
3
3
2
S, 80–85 °C, 6 h, DMF, ethanol.
(
NIH, Hungary) for financial support (TECH_08_A2-STEMKILL).
36. KINOMEscan™ Profiling Service (Discoverx).
37. Thomson, S.; Petti, F.; Sujka-Kwok, I.; Epstein, D.; Haley, J. D. Clin. Exp.
Metastasis 2008, 25, 843.
8. Guille, A.; Chaffanet, M.; Birnbaum, D. Cancer Cell Int. 2013, 13, 66.
9. Ivanov, A. A.; Khuri, F. R.; Fu, H. Trends Pharmacol. Sci. 2013, 34, 393.
40. Harris, C. C. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 1659.
41. White, A. W.; Westwell, A. D.; Brahemi, G. Expert Rev. Mol. Med. 2008, 10, e8.
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