Design, synthesis, and cytostatic activity of novel pyrazine sorafenib analogs

Article abstract of DOI:10.1007/s00044-016-1667-y

The current study is focused on a series of sorafenib analogs as potential antitumor agents. We have designed and synthesized nine novel pyrazine analogs 6a–i differing in amide and/or urea regions. Two alternative strategies for the preparation of title

Full text of DOI:10.1007/s00044-016-1667-y

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1667-y
ORIGINAL RESEARCH
Design, synthesis, and cytostatic activity of novel pyrazine sorafenib
analogs
Zrinka Rajić Džolić1 Ivana Perković1 Sandra Kraljević Pavelić2 Mirela Sedić2
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Nataša Ilić2 Dominique Schols3 Branka Zorc1
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Received: 24 September 2015 / Accepted: 4 July 2016
© The Author(s) 2016; This article is published with open access at Springerlink.com
Abstract The current study is focused on a series of sor-
afenib analogs as potential antitumor agents. We have
designed and synthesized nine novel pyrazine analogs 6a–i
differing in amide and/or urea regions. Two alternative
strategies for the preparation of title compounds were
applied. The first strategy involved ether formation between
4-hydroxyphenyl urea 3 and 5-chloro-pyrazine-2-carbox-
amides 4. In the second strategy, ether functionality was
introduced in the molecule before urea moiety and included
preparation of 5-(4-aminophenoxy)-N-alkylpyrazine-2-car-
boxamides 5 and their reaction with 4-chloro-3-(fluor-
omethyl)phenyl isocyanate. Cytostatic activity of the title
compounds was evaluated in vitro against a panel of cancer
cell lines. Most of the tested compounds showed strong
antiproliferative activity in the low micromolar range.
5-/4-[3-(4-chloro-3-trifluoromethyl-phenyl)-ureido]-phenoxy/-
pyrazine-2-carboxylic acid (4-chloro-3-trifluoromethylphenyl)-
amide (6g) was the most active compound (IC50 0.9–7.5 μM)
and showed comparable or stronger activity than sorafenib,
but also similar cytotoxicity to normal human fibroblast
cells. Two compounds, namely, 5-/4-[3-(4-bromophenyl)-
ureido]-phenyloxy/-pyrazine-2-carboxylic acid cyclopenty-
lamide (6c) and 5-/4-[3-(4-chloro-3-trifluoromethyl-phenyl)-
ureido]-phenoxy/-pyrazine-2-carboxylic acid cyclopentyla-
mide (6h), exerted cytostatic activities that surpassed the
effects observed with sorafenib in three cancer cell lines
(HepG2, HeLa, A549, IC50 0.6–0.9 μM). Similar to sor-
afenib, compound 6h proved to be cytotoxic to normal
human fibroblast cells, whereas compound 6c did
not diminish proliferative capacity of these cells and could
be regarded as the most promising derivative. Additional
biological studies on the c-Raf activity using Western blot
method revealed that antiproliferative activity of 6h could
be at least partially attributed to its inhibitory effect on
c-Raf activation similar to sorafenib. In contrast, 6c did not
inhibit the activity of c-Raf, which implies that other cell
signaling pathways govern its antiproliferative effects.
Taking into account structural differences between com-
pounds 6c and 6h, it is plausible to believe that the sub-
stituent in urea part of the molecule is essential for the
interaction with c-Raf.
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Keywords Sorafenib Pyrazine Synthesis Cytostatic
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activity c-Raf Western blotting
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-016-1667-y) contains supplementary material,
which is available to authorized users.
Abbreviations
A549
BSA
lung adenocarcinoma
bovine serum albumin
* Branka Zorc
BtcCl
BtH
1-benzotriazole carboxylic acid chloride
1H-benzo[d][1,2,3]triazole
acute lymphoblastic leukemia
Dulbecco’s modified Eagle’s medium
ethylenediamine tetraacetic acid
ethyleneglycol tetraacetic acid
foetal bovine serum
bzorc@pharma.hr
1
CEM
DMEM
EDTA
EGTA
FBS
Faculty of Pharmacy and Biochemistry, University of Zagreb,
A. Kovačića 1, HR-10 000 Zagreb, Croatia
2
Department of Biotechnology, University of Rijeka, Radmile
Matejčić 2, HR-51 000 Rijeka, Croatia
3
Rega Institute for Medical Research, Katholieke Universiteit
HeLa
cervical carcinoma
Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium

Med Chem Res
HepG2
IC50
hepatocellular carcinoma
the concentration that causes 50 % growth
inhibition
currently approved for the treatment of metastatic colorectal
cancer and advanced gastrointestinal stromal tumors (Crona
et al., 2013).
KOtBu
L1210
MCF-7
potassium t-butoxide
murine lymphocytic leukemia
breast carcinoma
The widespread use of pyrazine heterocycle as an
important pharmacophore in medicinal chemistry estab-
lishes this moiety as a member of a privileged structures
class. As a part of ongoing studies focused on the devel-
opment of new heterocycle anticancer agents, novel sor-
afenib analogs bearing pyrazine scaffold were prepared.
Herein, we report their synthesis, chemical characterization,
in vitro screening, and the effects on the activity of known
sorafenib target, namely, c-Raf kinase.
Molt4/C8 acute lymphoblastic leukemia
MR
MTT
molecular refractivity
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide
percentage growth
polar surface area
polyvinylidene fluoride
RAF proto-oncogene serine/threonine-protein
kinase
radioimmunoprecipitation assay buffer
metastatic colorectal adenocarcinoma
Tris-buffered saline with Tween 20
triethylamine
PG
PSA
PVDF
c-Raf
Results and discussion
Chemistry
RIPA
SW620
TBST
TEA
Novel compounds 6a–i described in this paper are derived
from the cytostatic drug sorafenib. Crucial parts of the
structure (diarylurea, ether, heterocycle, amide) are pre-
served, but a different heterocycle (pyrazine instead of
pyridine) and various substituents in urea and/or amide
regions were introduced. Comparison of the title sorafenib
analogs and the parent molecule is given in Fig. 1.
A pyridine heterocycle was replaced by its isoster pyr-
azine in the hope that the additional nitrogen atom would
increase the binding interactions between the drug and
c-Raf kinase. The central bis-aryl urea and the amide
functionalities were left intact, since it was shown that they
played key roles in the interaction with Raf (Wan et al.,
2004), but various substituents in these two regions were
introduced.
Majority of the pyrazine sorafenib analogs are fully in
agreement with the Lipinski’s and Gelovani’s rules for
prospective small molecular drugs (MW ≤ 500, log P ≤ 5,
number of H-bond donors ≤ 5, number of H-bond accep-
tors ≤ 10, molecular polar surface area < 140 Å2, molar
refractivity within the range of 40 and 130 cm3/mol, the
number of atoms 20–70) and some analogs showed minimal
aberrations of the rules (Luzina and Popov, 2012). The
parameters are calculated with Chemicalize.org program
and presented in Table 1.
WI-38
normal human diploid fibroblasts
Introduction
Cancer is one of the major health concerns in the world that
represents the leading cause of death worldwide as evi-
denced by 8.2 million cancer-related deaths in 2012 (WHO,
2015). Because of high cancer morbidity and mortality, new
approaches in cancer therapy are needed that require
development of novel, more effective antitumor agents
(Chong and Jänne, 2013; Holohan et al., 2013). Modifica-
tion of known, clinically approved drugs, is one of the
possible strategies in drug discovery. In the present study,
we have directed our attention to sorafenib, the first multi-
kinase inhibitor approved in 2005 for the therapy of
advanced renal cell carcinoma and hepatocellular carci-
noma. Sorafenib supresses tumor growth through the inhi-
bition of C-RAF and B-RAF serine/threonine kinase
activities and tumor angiogenesis by abrogating vascular
endothelial growth factor receptor (VEGFR) and platelet-
derived growth factor receptors signaling (Wilhelm et al.,
2006, 2008). Because of its multi-antitumoral mechanisms,
a broad spectrum of anticancer activity, good results in
combination trials, poor physicochemical properties, inac-
tivity against mutated BRAF, toxicity and drug resistance
(Schutz et al., 2011; Villaneuva and Llovet, 2012), many
research efforts have been focused on the optimization of
sorafenib molecule (Sun et al., 2010; Yao et al., 2012; Babić
et al., 2012; Zhan et al., 2012; Lu et al., 2014; Qin et al.,
2014; Jiao et al., 2015). As a result, a closely related analog
of sorafenib, regorafenib, was registered in 2012, and is
Two alternative strategies for the preparation of the title
compounds 6a–i were applied (Scheme 1). The first strategy
involved ether formation between 4-hydroxyphenyl urea
3a–c and 5-chloro-pyrazine-2-carboxamides 4a–f. The
reactions were performed in dimethylformamide (DMF) in
the presence of K2CO3. Ureas 3 were prepared from N-(4-
hydroxyphenyl)-1H-benzo[d][1,2,3]triazole-1-carboxamide
(2) and the corresponding amine [cyclopentylamine,
4-bromoaniline or 4-chloro-3-(trifluoromethyl)aniline], while
compound
2 was synthesized from BtcCl (1) and

Med Chem Res
Fig. 1 Comparison of sorafenib
analogs 6a–i and the parent
molecule. The regions exposed
to changes are cycled
CF3
O
Cl
O
CH3
O
N
H
N
N
H
N
H
sorafenib
O
N
N
O
H
N
R2
N
H
N
H
R1
6a-i
O
CF3
N
O
F3C
Cl
N
Cl
O
H
O
NCO
N
H
R1
N
5a,b
H2N
N
R1
CH2Cl2
N
N
N
O
H
H
O
6h,i
H2N
OH
K2CO3, KOtBu, DMF
Cl
N
HO
N
N
1. SOCl2, DMF, toluene
2. R1NH2, TEA, CH2Cl2
H
N
R1
O
N
N
COOH
O
O
H
K2CO3
DMF
O
R2
N
4a-f
R1
N
N
N
H
H
OH
BtcCl (1)
OH
OH
6a-i
O
R2NH2
O
R2
OH
R1
R2
=
=
TEA, dioxane
DMF
Cl
CF3
CH3
H2N
N
H
N
N
H
O
Bt
H
2
3a-c
Cl
CF3
Br
O
N
N
N
Btc =
Scheme 1 Synthetic pathways of intermediates 2–5 and target compounds 6a–i
Table 1 Properties of pyrazine sorafenib analogs 6a–i: the Lipinski’s and Gelovani’s parameters
Compounds Molecular formula Number of
atoms
MW
log P H-bond
H-bond
acceptor
Lipinski
scorea
MR cm3/mol PSA (Å2)
donor
6a
6b
6c
6d
6e
6f
C22H27N5O3
57
44
54
409.48 2.93
442.27 3.01
496.36 3.62
465.81 3.72
495.84 3.03
557.91 5.60
630.33 7.22
519.90 5.07
533.93 5.52
3
3
3
3
4
3
3
3
3
4
4
4
4
5
5
4
4
4
4
4
4
4
4
3
3
2b
3
113.164
109.519
125.932
112.675
118.967
138.638
145.012
129.088
133.689
105.24
105.24
105.24
105.24
125.47
114.47
105.24
105.24
105.24
C
C
C
C
C
C
C
19H16BrN5O3
23H22BrN5O3
20H15ClF3N5O3 47
21H17ClF3N5O4 51
26H19ClF3N5O4 58
26H15Cl2F6N5O3 57
24H21ClF3N5O3 57
6g
6h
6i
C25H23ClF3N5O3 60
Calculated with Chemicalize.org program
a
Out of four
b
Minimal aberrations of two rules; MR molecular refractivity, PSA polar surface area

Med Chem Res
4-aminophenol according to our slightly modified procedure
(Butula et al., 1978). On the other hand, amides 4 were
obtained by the reaction of the corresponding amine [methy-
lamine, ethanolamine, cyclopentylamine, cyclohexylamine, O-
benzylhydroxylamine, or 4-chloro-3-(trifluoromethyl)aniline]
with 5-chloropyrazine-2-carboxylic chloride, prepared from
5-hydroxy-pyrazine-2-carboxylic acid and thionyl chloride.
The amidation step was performed at room temperature in the
presence of triethylamine (TEA) as HCl acceptor.
In the second strategy, leading to sorafenib pyrazine
analogs 6, ether functionality was introduced in the mole-
cule before the urea moiety. Amides 4 were first converted
to 5-(4-aminophenoxy)-N-alkylpyrazine-2-carboxamides 5.
Ether bond formation was performed by the reaction of
precursors 4 with p-aminophenol in the presence of K2CO3
and potassium t-butoxyde. Under the basic conditions
employed, the alkoxyde derived from 4-aminophenol was a
stronger nucleophile than the amino group; therefore the
main product was the ether, rather than the secondary
amine. The last step in the preparation of products 6
involved urea formation between the amino group of
compounds 5 and 4-chloro-3-(fluoromethyl)phenyl iso-
cyanate. Urea bond formation was performed at room
temperature, using a slight excess of isocyanate.
Preparation of compounds 3b and 3c has been previously
published (Winum et al., 2012; Yang et al., 2013), but their
analytical and spectral data were not given. Compound 4a is
commercially available and all other compounds are new
compounds. They are isolated and fully characterized by the
usual spectroscopic methods [infrared (IR), 1H-, 13C nuclear
magnetic resonance (NMR), and MS] and elemental ana-
lyses. Spectral data are consistent with the proposed struc-
tures and are given in short in the Experimental section and
in detail in Supporting information. As could be expected,
the signals of carbon atoms bearing three fluoro atoms and
aromatic carbon atoms adjacent to CF3 group split in 13C
NMR spectra and appeared as quartets.
by 5-/4-[3-(4-chloro-3-trifluoromethyl-phenyl)-ureido]-
phenoxy/-pyrazine-2-carboxylic
acid
(4-chloro-3-tri-
fluoromethylphenyl)-amide (6g), which showed comparable
or even more potent activity than sorafenib (IC50 0.9–7.5
μM). 6e, 6f, and 6i were also very potent against all tested
cell lines, including WI-38, like sorafenib itself. The com-
mon structural feature for these compounds is 4-chloro-3-
trifluoromethylphenyl moiety in urea part. The most active
compound 6g have 4-chloro-3-trifluoromethylphenyl resi-
due on both wings of the molecule, e.g., both in urea and
amide region. This compound was also the most liphophilic,
suggesting that lipohilicity is important for anticancer
activity.
Two compounds, namely, 5-/4-[3-(4-bromophenyl)-
ureido]-phenyloxy/-pyrazine-2-carboxylic acid cyclopenty-
lamide (6c) and 5-/4-[3-(4-chloro-3-trifluoromethyl-
phenyl)-ureido]-phenoxy/-pyrazine-2-carboxylic acid cyclo-
pentylamide (6h), exerted cytostatic activities (IC50 0.6–0.9
μM) that significantly surpassed the effects observed with
sorafenib in three cancer cell lines (HepG2, HeLa, A549).
Similar to sorafenib, compound 6h proved to be cytotoxic to
normal human fibroblast cells, whereas compound 6c did not
diminish proliferative capacity of these cells (IC50 > 100
μM). Compounds 6a and 6b showed also high cytostatic
activity against the same cancer cell lines (6a: HeLa and
HepG2; 6b: HeLa and A549). All these compounds have
either cyclopentyl residue in amide part or bromophenyl
group in urea part or both these structural features (com-
pound 6c).
All compounds exerted cytostatic activity against HeLa
cell line. However, most of the compounds were active
toward WI-38 as well and showed comparable activity to
that observed for sorafenib. Compounds 6b–d were
exceptions: 6b and 6d showed low toxicity toward fibro-
blast WI-38 (IC50 58.1 and 21.9 μM, respectively) and 6c
no toxicity. It is interesting to note that compound 6d, the
most similar compound to sorafenib, with the same urea
and amide part, but different heterocycle, showed much
higher selectivity than sorafenib itself: it was very active
against three cancer cell lines (HepG2, HeLa, and A549
again) and practically inactive against other five. It also
showed lower toxicity to fibroblast WI-38 than sorafenib.
The observed selectivity could be fully attributed to pyr-
azine moiety.
In conclusion, all the tested compounds showed strong
antiproliferative activity in the low micromolar range or
high selectivity against the selected cancer cell lines.
Compound 6c may be considered as the most promising
pyrazine sorafenib analog, due to more potent anticancer
activity against three cancer cell lines, reduced toxicity in
comparison with sorafenib, and full agreement with the
Lipinski’s and Gelovani’s rules for prospective small
molecular drugs. It could be used as a scaffold for
Biological evaluation
Cytostatic activity
Eight human tumor cell lines derived from seven human
cancer types: acute lymphoblastic leukemia (CEM, Molt4/
C8), cervical carcinoma (HeLa), lung adenocarcinoma
(A549), hepatocellular carcinoma (HepG2), metastatic
colorectal adenocarcinoma (SW620), breast carcinoma
(MCF-7), murine cancer cell line (L1210), and normal
human diploid fibroblasts (WI-38) derived from embryonic
lung tissue were used in antiproliferative evaluation
assay. The results of in vitro screening of sorafenib
analogs 6a–i against selected cell lines are presented
in Table 2. The strongest cytostatic activity was exerted

Med Chem Res

Med Chem Res
Perkin Elmer Paragon 500 spectrometer (Perkin-Elmer,
UK). H and 13C NMR spectra were recorded on a Bruker
1
75 kDa
c-Raf
AV-600 spectrometer (Bruker, USA) operating at 300 and
600 MHz for 1H and 75 and 150 MHz for 13C nuclei.
Samples were measured in dimethyl sulfoxide (DMSO)-d6
solutions at 20 °C in 5-mm NMR tubes. Chemical shifts (δ)
in ppm were referred to tetramethylsilane. Mass spectra
were taken on a high-performance liquid chromatography–
mass spectrometry (HPLC, Agilent Technologies 1200
Series; MS, Agilent Technologies 6410 Triple Quad). Ele-
mental analyses were performed on a CHNS LECO ana-
lyzer (LECO Corporation, USA). Analyses indicated by the
symbols of the elements were within 0.4 % of the theo-
retical values. All compounds are routinely checked by thin-
layer chromatography (TLC) with Merck silica gel 60F-254
glass plates using the following solvent systems: cyclo-
hexane/ethyl acetate/methanol 3:1:0.5, cyclohexane/
ethyl acetate 3:1, 1:1, and 1:2, ethyl acetate/methanol
2:0.1, dichloromethane/methanol/cyclohexane/ethyl acetate
9:1:5:5, dichloromethane/methanol 9.5:0.5, ethyl acetate/
petrolether/methanol 2:2:0.1 and 3:1:0.1, petrolether/ethyl
acetate 1:1, and ethyl acetate. Spots were visualized by
short-wave UV light and iodine vapour. Column chroma-
tography was performed on silica gel (0.063−0.200 mm),
with the same eluents used in TLC. 1H-benzo[d][1,2,3]
triazole (BtH), 5-hydroxy-pyrazine-2-carboxylic acid,
methylamine hydrochloride, cyclopentylamine, cyclohex-
ylamine, ethanolamine, 4-bromoaniline, 4-chloro-3-(fluor-
omethyl)aniline, O-benzylhydroxylamine hydrochloride,
and potassium t-butoxide (KOtBu) were purchased from
Aldrich (Germany), 4-aminophenol and 4-chloro-3-(fluor-
omethyl)phenyl isocyanate from Acros Organics (Belgium),
triphosgene from Alpha Aesar (USA), and TEA from Sigma
Chemical Co. (USA). 1-benzotriazole carboxylic acid
chloride (BtcCl, 1) was prepared from BtH and triphosgene
following our published procedure (Butula et al., 1977;
Butula and Jadrijević-Mladar Takač, 2000).
6h
6h
6c
control control
control
Fig. 2 Representative Western blots for expression of phospho-c-Raf
(Ser259), 75kDa in cancer cell lines treated with compounds 6h and 6c
after 24h. Downregulation of phospho-c-Raf is visible in A549 and
SW 620 cells treated with 6h
development of new, more effective, and safer drugs for
treating hepatocellular, lung, and cervical carcinoma.
c-Raf kinase inhibition
In vitro antiproliferative screening identified two com-
pounds, 6h and 6c, with potent cytostatic activity that sur-
passed the effects observed with sorafenib in three cancer
cell lines (HepG2, HeLa, A549). To investigate if com-
pounds 6c and 6h share the common cytostatic mechanism
with sorafenib, we analyzed the effects of these compounds
on the activity of c-Raf kinase, known sorafenib target,
using Western blot method with specific phospho-c-Raf
(Ser259) antibody.
The method detects endogenous levels of c-Raf only
when phosphorylated at Ser259, indicating its activation.
Western blot analysis clearly showed marked down-
regulation of phospho-c-Raf in A549 and SW620 cell lines
treated with 6h at its IC50 value for 48 h, which suggests
that 6h acts as c-Raf inhibitor. On contrary, compound 6c
did not elicit significant change in the expression level of
phospho-c-Raf in HepG2 cells, which suggests that other
mechanism than c-Raf inhibition may account for its
observed cytostatic activity in hepatocellular carcinoma
cells. This striking difference between compounds 6h and
6c in the mechanism underlying their antiproliferative
effects as well as opposing cytotoxic effects in normal
human fibroblasts could be attributed to their structural
differences. Both compounds possess the same cyclopentyl
substituent in amide region and differ only in the urea part
of the molecules: 6h has trifluoromethyl and chloro-
substituted benzene ring as sorafenib, while 6c is a bro-
mophenyl derivative. The results suggest that the urea part
of the molecule is essential for the interaction between 6h
and c-Raf (Fig. 2).
N-(4-hydroxyphenyl)-1H-benzo[d][1,2,3]triazole-1-carbox-
amide (2) A solution of 1.086 g (6.0 mmol) BtcCl (1) in
25 ml of dry dioxane was added dropwise to a solution of
equimolar amount of p-aminophenol (0.655 g) and TEA
(0.606 g) in 30 ml of dry dioxane. Reaction mixture was
stirred 30 min at room temperature. TEA × HCl was filtered
off and the mother liquor was evaporated under reduced
pressure. After purification by column chromatography
(mobile phase dichloromethane/methanol 9.5:0.5) and
crystallization from toluene and acetone/water, 0.915 g
(60 %) of 2 was obtained; mp. 240 °C (decomp.); IR (KBr):
νmax 3456, 3252, 1726, 1544, 1616, 1544, 1618, 1488,
1446, 1370, 1236, 1212, 1060, 926, 825, 146, 583 cm−1; 1H
NMR (DMSO, 300 MHz): δ = 10.89 (s, 1H, 1), 9.41 (s, 1H,
8), 8.24 (d, J = 9.12, 2H, arom.), 7.76–7.73 (m, 1H, arom.),
Experimental
Chemistry
Melting points were determined on a Stuart Melting Point
Apparatus SMP3 and were uncorrected. IR spectra were
recorded on a Fourier transform-infrared spectroscopy

Med Chem Res
7.60–7.57 (m, 3H, arom.), 6.83–6.81 (m, 2H, arom.);
13C NMR (DMSO, 75 MHz): δ = 154.61 (9), 146.99 (2),
145.50 (15), 131.47 (10), 129.94 (13), 128.40 (5), 125.62
(12), 123.19 (4, 6), 119.18 (14), 115.18 (3, 7), 113.69 (11);
ESIMS m/z (pos): 277.1, C13H10N4NaO2 (calcd. 277.2).
1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-hydroxyphenyl)
urea (3c) This compound was prepared by the reaction of
0.280 g (1.1 mmol) amide 2 and 0.430 g (2.2 mmol) 4-
chloro-3-(trifluoromethyl)aniline. After purification by col-
umn chromatography (mobile phase dichloromethane/
methanol 9.5:0.5), 0.109 g (30 %) of a white solid was
obtained; mp. 210–215 °C; IR (KBr): νmax 3305, 3112,
2930, 1673, 1625, 1591, 1560, 1482, 1436, 1328, 1268,
General procedure for preparation of 4-hydroxyphenyl
ureas 3a–c Method A: A suspension of 0.280 g (1.1
mmol) benzotriazole-1-carboxylic acid (4-hydroxyphenyl)-
amide (2) and corresponding amine (2.2 mmol) in 20 ml of
ethanol was heated 1 h at 50 °C. The reaction mixture was
evaporated under reduced pressure. Method B: A suspen-
sion of 0.280 g (1.1 mmol) benzotriazole-1-carboxylic acid
(4-hydroxyphenyl)-amide (2) and corresponding amine (2.2
mmol) in 25 ml of DMF was heated 3 h at 115 °C. The
reaction mixture was evaporated under reduced pressure.
Products 3a–c were purified by column chromatography or
by crystallization.
1
1185, 1147, 1126, 832, 684 cm–1; H NMR (DMSO, 300
MHz): δ = 9.09 (s, 1H, 1), 9.00 (s, 1H, 8), 8.46 (s, 1H, 10),
8.09 (d, J = 2.13, 1H, 16), 7.64–7.56 (m, 2H, 12, 13),
7.24–7.19 (m, 2H, 3, 7), 6.72–6.67 (m, 2H, 4, 6); 13C NMR
(DMSO, 75 MHz): δ = 152.95 (1), 152.60 (9), 139.66 (11),
131.88 (13), 130.45 (5), 126.93–126.32 (q, J = 30.57, 15),
125.53–120.10 (q, J = 272.90, 17), 122.77 (12), 121.84 (14),
120.99 (4, 6), 116.57–116.45 (q, J = 5.72, 16), 115.17 (3, 7);
ESIMS m/z (pos): 330.7, C14H10ClF3N2O2 (calcd. 330.7);
Anal. Calcd for C14H10ClF3N2O2: C, 50.85; H, 3.05; N,
8.47. Found: C, 50.49, H, 3.22; N, 8.88.
1-cyclopentyl-3-(4-hydroxyphenyl)urea (3a) This com-
pound was prepared by the reaction of 0.280 g (1.1 mmol)
amide 2 and 0.113 g (2.2 mmol) cyclopentylamine. After
purification by column chromatography (mobile phase
petrolether/etyl acetate 1:1), 0.208 g (86 %) of a white solid
was obtained; mp. 175–177 °C; IR (KBr): νmax 3243, 2960,
2870, 1608, 1574, 1517, 1442, 1309, 1259, 1220, 1163,
835, 805, 727, 667, 523 cm−1; 1H NMR (DMSO, 300
MHz): δ = 8.88 (s, 1H, 1), 7.86 (s, 1H, 8), 7.13–7.11 (m,
2H, arom.), 6.63–6.60 (m, 2H, arom.), 5.94 (d, J = 7.20,
1H, 10), 3.93–3.88 (m, 1H, 11), 1.84–1.78 (m, 2H, 12),
1.64–1.59 (m, 2H, 15), 1.55–1.50 (m, 2H, 13), 1.36–1.30
(m, 2H, 12); 13C NMR (DMSO, 75 MHz): δ = 155.11 (9),
151.79 (2), 132.07 (5), 119.60 (4, 6), 115.02 (3, 7), 50.82
(11), 32.86 (12, 15), 23.10 (13, 14); ESIMS m/z (pos):
221.1, C12H17N2O2 (calcd. 221.3); Anal. Calcd for
C12H16N2O2: C, 65.43; H, 7.32; N, 12.72. Found: C, 65.16,
H, 6.96; N, 12.35.
General procedure for preparation of 5-chloro-pyrazine-2-
carboxamides 4a–f A suspension of 0.308 g (2.2 mmol) 5-
hydroxy-2-pyrazine carboxylic acid, 1.666 g (14.0 mmol)
thionyl chloride, and 2 drops of DMF in 30 ml of dry
toluene was heated at 100 °C 70 min. The reaction mixture
was cooled, filtrated, and evaporated under reduced pres-
sure. The red oil residue was dissolved in 10 ml of dry
dichloromethane and added dropwise to a cold solution
(0 °C) of corresponding amine (1.7 mmol) and 0.404 g
(4.0 mmol) TEA in 20 ml of dry dichloromethane. The
reaction mixture was stirred 30 min and evaporated under
reduced pressure. Products 4a–f were purified by column
chromatography or by crystallization.
5-chloro-N-methylpyrazine-2-carboxamide
(4a) This
compound was prepared by the reaction of 0.308 g (2.2
mmol) 5-hydroxy-2-pyrazine carboxylic acid, 1.666 g (14.0
mmol) thionyl chloride, 0.144 g (1.7 mmol) methylamine
hydrochloride, and 0.606 g (6.0 mmol) TEA. After pur-
ification by column chromatography (mobile phase ethyl
acetate/petrolether/methanol 3:1:0.1), 0.198 g (68 %) of a
white solid was obtained; mp. 153–156 °C; IR (KBr): νmax
3369, 3078, 2944, 1660, 1573, 1536, 1517, 1456, 1412,
1278, 1181, 1126, 1032, 1003, 932, 852, 664, 632, 522,
1-(4-bromophenyl)-3-(4-hydroxyphenyl)urea
(3b) This
compound was prepared by the reaction of 0.280 g (1.1
mmol) amide 2 and 0.378 g (2.2 mmol) 4-bromoaniline.
After crystallization from dichloromethane, 0.301 g (89 %)
of a white solid was obtained; mp. 244–245 °C (decomp.);
IR (KBr): νmax 3302, 1637, 1590, 1565, 1509, 1488, 1464,
1393, 1302, 1224, 1102, 1071, 1010, 853, 824, 793, 649
1
491 cm–1; H NMR (DMSO, 300 MHz): δ = 8.99 (d, J =
1
cm–1; H NMR (DMSO, 300 MHz): δ = 9.05 (s, 1H, 1),
1.32, 1H, 5), 8.89–8.86 (m, 2H, 3, 1′), 2.83 (d, 3H, J = 4.83,
2′); 13C NMR (DMSO, 75 MHz): δ = 162.40 (1), 150.54
(4), 143.59 (2), 143.17 (5), 143.00 (3), 25.98 (2′); ESIMS
m/z (pos): 172.1, C6H7ClN3O (calcd. 171.6); Anal. Calcd
for C6H6ClN3O: C, 42.00; H, 3.52; N, 24.49. Found: C,
42.33, H, 3.00; N, 24.11.
8.65 (s, 1H, 8), 8.33 (s, 1H, 10), 7.41 (s, 4H, 12, 13, 15, 16),
7.23–7.18 (m, 2H, 3, 7), 6.71–6.66 (m, 2H, 4, 6); 13C NMR
(DMSO, 75 MHz): δ = 152.68 (2), 152.61 (9), 139.42 (11),
131.39 (12, 16), 130.82 (5), 120.58 (4, 6), 119.89 (13, 15),
115.16 (3, 7), 112.77 (14); ESIMS m/z (pos): 309.1,
C13H1281BrN2O2 (calcd. 309.1); 307.2, C13H1279BrN2O2
(calcd. 307.1); Anal. Calcd for C13H11BrN2O2: C, 50.85; H,
3.61; N, 9.12. Found: C, 50.65, H, 3.99; N, 9.02.
N-(2-hydroxyethyl)-5-chloropyrazine-2-carboxamide (4b)
This compound was prepared by the reaction of 0.308 g

Med Chem Res
(2.2 mmol) 5-hydroxy-2-pyrazine carboxylic acid, 1.666 g
(14.0 mmol) thionyl chloride, and 0.267 g (4.3 mmol)
ethanolamine. After purification by column chromatography
(mobile phase ethyl acetate/petrolether/methanol 3:1:0.1),
0.218 g (59 %) of a white solid was obtained; mp. 94–96 °C;
IR (KBr): νmax 3356, 3079, 2942, 2879, 1665, 1573, 1529,
1459, 1439, 1365, 1302, 1278, 1210, 1182, 1127, 1055,
1031, 872, 794, 742, 692, 519 cm–1; 1H NMR (DMSO, 300
MHz): δ = 9.00 (d, J = 1.32, 1H, 5), 8.87 (d, J = 1.32, 1H,
3), 8.76 (t, J = 4.63, 1H, 1′), 4.77 (t, J = 5.51, 1H, 4′), 3.54
(q, J = 5.90, 2H, 3′), 3.39 (q, J = 5.96, 2H, 2′); 13C NMR
(DMSO, 75 MHz): δ = 161.97 (1), 150.64 (4), 143.46 (2),
143.27 (5), 143.00 (3), 59.41 (3′), 41.67 (2′); ESIMS m/z
(pos): 224.2, C7H9ClN3NaO2 (calcd. 224.6); 202.2,
C7H9ClN3O2 (calcd. 201.6); Anal. Calcd for C7H8ClN3O2:
C, 41.70; H, 4.00; N, 20.84. Found: C, 41.99, H, 3.68; N,
20.61.
C11H15ClN3O (calcd. 240.7); Anal. Calcd for
C11H14ClN3O: C, 55.12; H, 5.89; N, 17.53. Found: C,
54.96, H, 5.56; N, 17.77.
5-chloro-N-(4-chloro-3-(trifluoromethyl)phenyl)pyrazine-2-
carboxamide (4e) This compound was prepared by the
reaction of 0.308 g (2.2 mmol) 5-hydroxy-2-pyrazine car-
boxylic acid, 1.666 g (14.0 mmol) thionyl chloride, 0.332 g
(1.7 mmol) 4-chloro-3-trifluormethylaniline, and 0.444 g
(4.4 mmol) TEA. After crystallization with ether and
recrystallization from acetone/water, 0.371 g (65 %) of a
white solid was obtained; mp. 149–151 °C; IR (KBr): νmax
3376, 3360, 3122, 3083, 1693, 1590, 1530, 1423, 1332,
1
1262, 1251, 1180, 1136, 1116, 1023, 830, 663 cm–1; H
NMR (DMSO, 300 MHz): δ = 11.22 (s, 1H, 1′), 9.13 (d, J
= 1.20, 1H, 5), 8.96 (d, J = 1.26, 1H, 3), 8.52 (d, J = 2.37,
1H, 3′), 8.23 (dd, J = 2.31, J = 6.51, 1H, 7′), 7.74 (d, J =
8.79, 1H, 6′); 13C NMR (DMSO, 75 MHz): δ = 161.53 (1),
151.24 (4), 144.19 (5), 143.22 (2), 142.99 (3), 137.66 (2′),
132.01 (7′), 126.97–126.36 (q, J = 30.94, 4′), 125.43
(6′), 125.09 (5′), 125.40–119.97 (q, J = 273.67, 8′),
119.56–119.45 (q, J = 5.58, 3′); ESIMS m/z (pos):
336.2, C12H6Cl2F3N3O (calcd. 336.1); Anal. Calcd for
C12H6Cl2F3N3O: C, 42.88; H, 1.80; N, 12.50. Found: C,
42.65, H, 2.06; N, 12.09.
N-cyclopentyl-5-chloropyrazine-2-carboxamide (4c) This
compound was prepared by the reaction of 0.308 g (2.2
mmol) 5-hydroxy-2-pyrazine carboxylic acid, 1.666 g (14.0
mmol) thionyl chloride, 0.145 g (1.7 mmol) cyclopentyla-
mine, and 0.404 g (4.0 mmol) TEA. After purification by
column chromatography (mobile phase dichloromethane/
methanol 9.5:0.5), 0.384 g (89 %) of a white solid was
obtained; mp. 107–108 °C; IR (KBr): νmax 3373, 3079,
2951, 2870, 1660, 1571, 1528, 1466, 1295, 1176, 1161,
N-(benzyloxy)-5-chloropyrazine-2-carboxamide (4f) This
compound was prepared by the reaction of 0.308 g (2.2
mmol) 5-hydroxy-2-pyrazine carboxylic acid, 1.666 g (14.0
mmol) thionyl chloride, 0.271 g (1.7 mmol) O-benzylhy-
droxylamine hydrochloride, and 0.444 g (4.4 mmol) TEA.
After crystallization with ether and recrystallization from
acetone/water, 0.319 g (67 %) of a white solid was obtained;
mp. 124–125 °C; IR (KBr): νmax 3246, 3089, 3062, 3032,
2963, 1678, 1491, 1452, 1276, 1121, 1016, 906, 751, 699,
1
1125, 1027, 927, 632, 526, 492 cm–1; H NMR (DMSO,
300 MHz): δ = 8.97 (d, J = 1.32, 1H, 5), 8.84 (d, J = 1.52,
1H, 3), 8.79 (d, J = 7.56, 1H, 1′), 4.29–4.23 (m, 1H, 2′),
1.91–1.86 (m, 2H, 3′), 1.72–1.67 (m, 2H, 6′), 1.63–1.51 (m,
4H, 4′, 5′); 13C NMR (DMSO, 75 MHz): δ = 161.56 (1),
150.45 (4), 143.80 (2), 143.37 (5), 142.84 (3), 50.68 (2′),
31.85 (3′, 6′), 23.52 (4′, 5′); ESIMS m/z (pos): 248.1,
C10H12ClN3NaO (calcd. 248.7); 226.1, C10H13ClN3O
(calcd. 226.7); Anal. Calcd for C10H12ClN3O: C, 53.22; H,
5.36; N, 18.62. Found: C, 53.67, H, 5.02; N, 18.33.
1
505 cm–1; H NMR (DMSO, 300 MHz): δ = 12.33 (s, 1H,
1′), 8.98 (d, J = 0.99, 1H, 5), 8.85 (d, J = 1.23, 1H, 3),
7.49–7.37 (m, 5H, 4′–8′), 4.96 (s, 2H, 2′); 13C NMR
(DMSO, 75 MHz): δ = 160.05 (1), 151.42 (4), 143.83 (5),
143.72 (3), 143.54 (2), 136.04 (3′), 129.30 (5′, 7′), 128.83
(4′), 128.78 (6′, 8′), 77.18 (2′); ESIMS m/z (pos): 286.2,
C12H10ClN3NaO2 (calcd. 286.7); 264.2, C12H11ClN3O2
(calcd. 264.7); Anal. Calcd for C12H10ClN3O2: C, 54.66; H,
3.82; N, 15.94. Found: C, 54.29, H, 3.80; N, 16.03.
N-cyclohexyl-5-chloropyrazine-2-carboxamide (4d) This
compound was prepared by the reaction of 0.308 g (2.2
mmol) 5-hydroxy-2-pyrazine carboxylic acid, 1.666 g (14.0
mmol) thionyl chloride, 0.169 g (1.7 mmol) cyclohex-
ylamine, and 0.404 g (4.0 mmol) TEA. After two crystal-
lization from ether, 0.277 g (68 %) of a white solid was
obtained; mp. 153–154 °C; IR (KBr): νmax 3377, 3081,
2923, 2851, 1657, 1570, 1530, 1465, 1454, 1310, 1169,
General procedure for preparation of 5-(4-aminophenoxy)-
N-alkylpyrazine-2-carboxamides 5a, b A suspension of
0.112 g (1 mmol) KOtBu and 0.11 g (1 mmol) p-amino-
phenol in 10 ml of dry DMF was stirred at room tempera-
ture for 30 min. Amide 4 (1 mmol) and 0.07 g (0.5 mmol),
K2CO3 was added and the reaction mixture was stirred at
80 °C for 2 h. DMF was evaporated under reduced pressure.
Products 5 were purified by column chromatography and
crystallization from ether.
1
1124, 1079, 1026, 925, 895, 861, 633, 524, 439 cm–1; H
NMR (DMSO, 300 MHz): δ = 8.99 (d, J = 1.35, 1H, 5),
8.84 (d, J = 1.32, 1H, 3), 8.60 (d, J = 8.37, 1H, 1′),
3.85–3.74 (m, 1H, 2′), 1.81–1.69, 1.63–1.58, 1.48–1.24,
1.20–1.06 (m, 10H, 3′–7′); 13C NMR (DMSO, 75 MHz): δ
= 160.96 (1), 150.49 (4), 143.77 (2), 143.38 (5), 142.84 (3),
48.19 (2′), 31.93 (3′, 7′), 25.01 (5′), 24.76 (4′, 6′); ESIMS
m/z (pos): 262.1, C11H14ClN3NaO (calcd. 262.7); 240.1,

Med Chem Res
5-(4-aminophenoxy)-N-cyclopentylpyrazine-2-carbox-
amide (5a) This compound was prepared by the reaction
of 0.226 g (1 mmol) amide 4c, 0.112 g (1 mmol) KOtBu,
and 0.11 g (1 mmol) p-aminophenol. After purification by
column chromatography (mobile phase dichloromethane/
methanol 95:5) and crystallization from ether, 0.176 g (59
%) of a white solid was obtained; mp. 142–144 °C; IR
(KBr): νmax 3458, 3380, 3079, 3020, 2938, 2864, 1652,
1619, 1587, 1506, 1470, 1352, 1326, 1298, 1268, 1196,
petrolether/ethyl acetate/methanol (2:2:01, v/v) (6d and 6f),
dichloromethane/methanol (9.5:0.5, v/v) (6g) or by crystal-
lization from ether (6a, 6h–i) or methanol (6e). Method B:
A suspension of 5-(4-aminophenoxy)-N-alkylpyrazine-2-
carboxamide 5 (0.2 mmol) and 0.066 g (0.3 mmol) 4-
chloro-3-(trifluoromethyl)phenylisocyanate in 3 ml of dry
dichloromethane was stirred at room temperature for 2 h.
The precipitated products 6h or 6i were filtered off and
crystallized from ether.
1
1025, 836, 594 cm–1; H NMR (DMSO, 300 MHz): δ =
8.67 (d, J = 1.29, 1H, 3), 8.46 (d, J = 7.95, 1H, 1′), 8.40 (d,
J = 1.29, 1H, 5), 6.91–6.86 (m, 2H, 7, 11), 6.63–6.58 (m,
2H, 8, 10), 5.09 (s, 2H, 12), 4.31–4.19 (m, 1H, 2′),
1.93–1.81 (m, 2H, 3′), 1.75–1.65 (m, 2H, 6′), 1.63–1.51 (m,
4H, 4′, 5′); 13C NMR (DMSO, 75 MHz): δ = 162.11 (4),
161.72 (1), 146.54 (6), 142.44 (9), 140.93 (5), 139.22 (2),
133.13 (3), 121.69 (7, 11), 114.41 (8, 10), 50.48 (2′), 32.02
(3′, 6′), 23.53 (4′, 5′); ESIMS m/z (pos): 299.3,
C16H19N4O2 (calcd. 299.3); Anal. Calcd for C16H18N4O2:
C, 64.411; H, 6.08; N, 18.78. Found: C, 64.86, H, 5.66; N,
18.53.
5-[4-(3-cyclopentyl-ureido)-phenyloxy]-pyrazine-2-car-
bocylic acid cyclopentylamide (6a) This compound was
prepared by the reaction of 0.090 g (0.4 mmol) amide 4c
and 0.088 g (0.4 mmol) urea 3a (Method A). After crys-
tallization from ether, 0.067 g (41 %) of a white solid was
obtained; mp. 235–237 °C; IR (KBr): νmax 3341, 3070,
3052, 2952, 2867, 1660, 1606, 1571, 1505, 1461, 1408,
1349, 1316, 1284, 1242, 1194, 1023, 897, 835, 626, 527,
1
502 cm–1; H NMR (DMSO, 300 MHz): δ = 8.67 (d, J =
1.29, 1H, 3), 8.48 (d, J = 1.32, 1H, 5), 8.43 (d, J = 2.88, 1H,
1′), 8.32 (s, 1H, 12), 7.46–7.41 (m, 2H, 8, 10), 7.12–7.07
(m, 2H, 7, 11), 6.15 (d, J = 7.23, 1H, 14), 4.31–4.19 (m,
1H, 2′), 4.00–3.89 (m, 1H, 15), 1.93–1.79, 1.72–1.50,
1.42–1.32 (3m, 16H, 3′–6′, 16–19); 13C NMR (DMSO, 75
MHz): δ = 162.01 (4), 161.21 (1), 154.78 (13), 146.18 (6),
140.82 (5), 139.57 (2), 138.06 (9), 133.87 (3), 121.45 (8,
10), 118.70 (7, 11), 50.86 (15), 50.48 (2′), 32.78 (3′, 6′),
31.99 (16, 19), 23.49 (4′, 5′), 23.62 (17, 18); ESIMS m/z
(pos): 432.2, C22H27N5NaO3 (calcd. 432.5), 410.2,
C22H28N5O3 (calcd. 410.5); Anal. Calcd for C22H27N5O3:
C, 64.53; H, 6.65; N, 17.10. Found: C, 64.72, H, 6.60; N,
16.82.
5-(4-aminophenoxy)-N-cyclohexylpyrazine-2-carboxamide
(5b) This compound was prepared by the reaction of
0.240 g (1 mmol) amide 4d, 0.112 g (1 mmol) KOtBu, and
0.11 g (1 mmol) p-aminophenol. After purification by col-
umn chromatography (mobile phase cyclohexane/ethyl
acetate 1:2) and crystallization from ether, 0.140 g (45 %) of
a white solid was obtained; mp. 167–170 °C; IR (KBr): νmax
3404, 3336, 3018, 2929, 2855, 1661, 1609, 1579, 1522,
1504, 1459, 1344, 1326, 1262, 1181, 1022, 890, 834, 593,
1
509 cm–1; H NMR (DMSO, 300 MHz): δ = 8.67 (d, J =
1.29, 1H, 3), 8.39 (d, J = 1.32, 1H, 5), 8.32 (d, J = 8.52, 1H,
1′), 6.91–6.86 (m, 2H, 7, 11), 6.63–6.58 (m, 2H, 8, 10),
5.06 (s, 2H, 12), 3.84–3.72 (m, 1H, 2′), 1.80–1.70,
1.62–1.56, 1.47–1.25, 1.20–1.11 (m, 10H, 3′–7′); 13C NMR
(DMSO, 75 MHz): δ = 161.70 (4), 161.47 (1), 146.48 (6),
142.45 (9), 140.92 (5), 139.17 (2), 133.09 (3), 121.60 (7,
11), 114.38 (8, 10), 47.85 (2′), 32.10 (3′, 7′), 25.04 (5′),
24.76 (4′, 6′); ESIMS m/z (pos): 313.3, C17H21N4O2 (calcd.
313.4); Anal. Calcd for C17H20N4O2: C, 65.37; H, 6.45; N,
17.94. Found: C, 65.29, H, 6.56; N, 17.61.
5-/4-[3-(4-bomophenyl)-ureido]-phenoxy/-pyrazine-2-car-
boxylic acid methylamide (6b) This compound was pre-
pared by the reaction of 0.069 g (0.4 mmol) amide 4a and
0.123 g (0.4 mmol) urea 3b (Method A). After purification
by column chromatography (mobile phase petrolether/ethyl
acetate/methanol 3:1:0.5) and crystallization from ether,
0.058 g (33 %) of a white solid was obtained; IR (KBr):
νmax 3311, 1662, 1646, 1591, 1551, 1505, 1457, 1348,
1309, 1271, 1236, 1194, 1008, 831 cm–1; 1H NMR (DMSO,
300 MHz): δ = 8.85 (s, 1H, 14), 8.80 (s, 1H, 12), 8.73–8.69
(m, 2H, 3, 1′), 8.53 (d, J = 1.25, 1H, 5), 7.55–7.49 (m, 2H,
8, 10), 7.45 (s, 4H, 16, 17, 19, 20), 7.21–7.16 (m, 2H, 7,
11), 2.82 (d, J = 4.87, 3H, 2′); 13C NMR (DMSO, 75 MHz):
δ = 162.95 (4), 161.21 (1), 152.45 (13), 146.98 (6), 140.66
(5), 139.58 (15), 139.11 (2), 137.01 (9), 133.63 (3), 131.50
(17, 19), 121.76 (16, 20), 120.13 (8, 10), 119.68 (7, 11),
113.21 (18), 25.91 (2′); ESIMS m/z (neg): 442.1,
C19H1581BrN5O3 (calcd. 442.3), 440.0, C19H1579BrN5O3
(calcd. 440.3); Anal. Calcd for C19H16BrN5O3: C, 51.60; H,
3.65; N, 15.84. Found: C, 51.99, H, 3.36; N, 15.72.
General procedure for preparation of pyrazine sorafenib
analogs 6a–i Method A: A suspension of 5-chloro-pyr-
azine-2-carboxamide 4 (0.4 mmol), urea 3 (0.4 mmol), and
0.111 g (0.8 mmol) potassium carbonate in 4 ml of dry
DMF was heated at 100 °C for 3–4.5 h. The reaction mix-
ture was cooled to room temperature, diluted with 30 ml
ethyl acetate, and extracted three times with brine. Organic
layer was dried over anhydrous sodium sulfate, filtered, and
evaporated under reduced pressure. The crude products
were purified by column chromatography on silica gel using
petrolether/ethyl acetate/methanol (3:1:0.5, v/v) (6b),

Med Chem Res
5-/4-[3-(4-bromophenyl)-ureido]-phenyloxy/-pyrazine-2-
carboxylic acid cyclopentylamide (6c) This compound
was prepared by the reaction of 0.090 g (0.4 mmol) amide
4c and 0.123 g (0.4 mmol) urea 3b (Method A). After
crystallization from ether/petrolether and recrystallization
from acetone/water, 0.107 g (54 %) of a white solid was
obtained; mp. 238–241 °C (decomp.); IR (KBr): νmax 3306,
2951, 2870, 1660, 1640, 1591, 1533, 1505, 1469, 1348,
1302, 1277, 1240, 1198, 1071, 1024, 1011, 903, 831, 657
crystallization from methanol, 0.070 g (35 %) of a white
solid was obtained; mp. 177–180 °C; IR (KBr): νmax 3437,
3352, 3140, 2929, 1712, 1647, 1608, 1561, 1509, 1486,
1462, 1420, 1354, 1329, 1304, 1279, 1258, 1225, 1192,
1143, 1076, 1032, 1019, 915, 894, 845, 688, 664, 641, 513
1
cm–1; H NMR (DMSO, 600 MHz): δ = 9.26 (s, 1H, 14),
9.00 (s, 1H, 12), 8.70 (d, J = 1.30, 1H, 3), 8.56 (t, J = 5.85,
1H, 1′), 8.54 (d, J = 1.31, 1H, 5), 8.12 (d, J = 2.32, 1H, 20),
7.68–7.59 (m, 2H, 16, 17), 7.56–7.51 (m, 2H, 8, 10),
7.22–7.17 (m, 2H, 7, 11), 4.77 (t, J = 5.47, 1H, 4′),
3.56–3.50 (q, J = 5.85, 2H, 3′), 3.41–3.35 (q, J = 5.74, 2H,
2′); 13C NMR (DMSO, 150 MHz): δ = 162.39 (4), 161.17
(1), 152.42 (13), 147.24 (6), 140.73 (5), 139.40 (15), 139.30
(2), 136.63 (9), 133.58 (3), 131.91 (17), 126.55 (t, 19, J =
30.33), 125.46–120.03 (q, 21, J = 272.95), 123.01 (16),
122.23 (18), 121.63 (8, 10), 120.01 (7, 11), 116.80–116.68
(q, 20, J = 5.68), 59.55 (3′), 41.48 (2′); ESIMS m/z (pos):
518.2, C21H17ClF3N5NaO4 (calcd. 518.8), 496.8,
C21H18ClF3N5O4 (calcd. 496.8); Anal. Calcd for
C21H17ClF3N5O4: C, 50.87; H, 3.46; N, 14.12. Found: C,
50.50, H, 3.41; N, 13.78.
1
cm–1; H NMR (DMSO, 300 MHz): δ = 8.84 (s, 1H, 14),
8.79 (s, 1H, 12), 8.69 (d, J = 1.21, 1H, 3), 8.51 (d, J = 1.17,
1H, 5), 8.47 (d, J = 7.93, 1H, 1′), 7.55–7.51 (m, 2H, 8, 10),
7.45 (s, 4H, 16, 17, 19, 20), 7.19–7.17 (m, 2H, 7, 11),
4.28–4.22 (m, 1H, 2′), 1.91–1.87 (m, 2H, 3′), 1.73–1.67 (m,
2H, 6′), 1.61–1.51 (m, 4H, 4′, 5′); 13C NMR (DMSO, 75
MHz): δ = 162.01 (4), 161.14 (1), 152.43 (13), 147.02 (6),
140.83 (5), 139.66 (15), 139.10 (2), 136.97 (9), 133.47 (3),
131.47 (17, 19), 121.67 (16, 20), 120.13 (8, 10), 119.67 (7,
11), 113.20 (18), 50.49 (2′), 32.00 (3′, 6′), 23.51 (4′, 5′);
ESIMS m/z (neg): 496.1, C23H2181BrN5O3 (calcd. 496.4),
494.1, C23H2179BrN5O3 (calcd. 494.4); Anal. Calcd for
C23H22BrN5O3: C, 55.65; H, 4.47; N, 14.11. Found: C,
55.29, H, 4.05; N, 14.55.
5-/4-[3-(4-chloro-3-trifluoromethylphenyl)-ureido]-phe-
noxy/-pyrazine-2-carboxylic acid benzyloxy-amide (6f)
This compound was prepared by the reaction of 0.112 g
(0.4 mmol) amide 4f and 0.132 g (0.4 mmol) urea 3c
(Method A). After purification by column chromatography
(mobile phase petrolether/ethyl acetate/methanol 2:2:0.1)
and crystallization from ether/petrolether, 0.040 g (18 %) of
a white solid was obtained; mp. 208–212 °C; IR (KBr): νmax
3351, 3073, 1678, 1595, 1547, 1506, 1486, 1460, 1420,
1349, 1330, 1280, 1230, 1194, 1137, 1033, 1017, 912, 839,
753, 701, 664, 636, 532, 516 cm–1; 1H NMR (DMSO, 600
MHz): δ = 12.10 (s, 1H, 1′), 9.20 (s, 1H, 14), 8.94 (s, 1H,
12), 8.68 (d, J = 1.23, 1H, 3), 8.52 (d, J = 1.29, 1H, 5), 8.12
(d, J = 2.28, 1H, 20), 7.68–7.60 (m, 2H, 16, 17), 7.57–7.51
(m, 2H, 8, 10), 7.49–7.45, 7.42–7.35 (2m, 5H, 4′–8′),
7.22–7.17 (m, 2H, 7, 11), 4.94 (s, 2H, 2′); 13C NMR
(DMSO, 150 MHz): δ = 161.35 (4), 160.17 (1), 152.48
(13), 147.15 (6), 140.93 (5), 139.35 (15), 138.83 (2), 136.74
(9), 135.73 (3′), 133.92 (3), 131.99 (17), 128.78 (4′, 8′),
128.28 (5′–7′), 128.24–117.38 (q, 18, J = 272.87),
127.30–126.09 (q, 19, J = 30.54), 123.07 (16), 121.78 (8,
10), 120.05 (7, 11), 116.88–116.66 (q, 20, J = 5.53), 77.11
(2′); ESIMS m/z (pos): 558.2, C26H20ClF3N5O4 (calcd.
558.1); Anal. Calcd for C26H19ClF3N5O4: C, 55.97; H,
3.43; N, 12.55. Found: C, 56.14, H, 3.40; N, 12.17.
5-/4-[3-chloro-3-trifluoromethyl-phenyl)-ureido]-phenoxy/-
pyrazine 2-carboxylic acid methylamide (6d) This com-
pound was prepared by the reaction of 0.068 g (0.4 mmol)
amide 4a and 0.132 g (0.4 mmol) urea 3c (Method A). After
purification by column chromatography (mobile phase
petrolether/ethyl acetate/methanol 2:2:0.1) and crystal-
lization from ether, 0.093 g (50 %) of a white solid was
obtained; mp. 233–239 °C; IR (KBr): νmax 3415, 3289,
3134, 3103, 1710, 1662, 1608, 1586, 1550, 1510, 1487,
1460, 1402, 1301, 1176, 1125, 1029, 837, 664, 509 cm–1;
1H NMR (DMSO, 600 MHz): δ = 9.18 (s, 1H, 14), 8.91 (s,
1H, 12), 8.69 (s, 2H, 3, 1′), 8.53 (s, 1H, 5), 8.12 (s, 1H, 20),
7.68–7.60 (m, 2H, 16, 17), 7.54 (d, J = 8.73, 2H, 8, 10),
7.19 (d, J = 8.70, 2H, 7, 11), 2.82 (d, J = 4.56, 3H, 2′); 13
C
NMR (DMSO, 150 MHz): δ = 162.94 (4), 161.16 (1),
152.47 (13), 147.25 (6), 140.64 (5), 139.61 (15), 139.35 (2),
136.68 (9), 133.63 (3), 131.96 (17), 126.99–126.38 (q, 19,
J = 30.48), 125.51–120.28 (q, 21, J = 273.27), 123.10 (16),
122.27 (18), 121.75 (8, 10), 120.04 (7, 11), 116.82–116.71
(q, 20, J = 5.44), 25.89 (2′); ESIMS m/z (pos): 466.1,
C20H16ClF3N5O3 (calcd. 466.1); Anal. Calcd for
C20H15ClF3N5O3: C, 51.57; H, 3.25; N, 15.03. Found: C,
51.22, H, 3.11; N, 15.44.
5-/4-[3-(4-chloro-3-trifluoromethyl-phenyl)-ureido]-phe-
noxy/-pyrazine-2-carboxylic acid (2-hydroxyethyl)amide
(6e) This compound was prepared by the reaction of
0.087 g (0.4 mmol) amide 4b and 0.132 g (0.4 mmol) urea
3c (Method A). After purification by column chromato-
graphy (mobile phase ethyl acetate/methanol 2:0.1) and
5-/4-[3-(4-chloro-3-trifluoromethyl-phenyl)-ureido]-phenoxy/-
pyrazine-2-carboxylic acid (4-chloro-3-trifluoromethylphenyl)-
amide (6g) This compound was prepared by the reaction of
0.134 g (0.4 mmol) amide 4e and 0.132 g (0.4 mmol) urea 3c
(Method A). After purification by column chromatography
(mobile phase dichloromethane/methanol 9.5:0.5) and

Med Chem Res
crystallization from ether/petrolether, 0.068 g (27 %) of a
white solid was obtained; 141−144 °C; IR (KBr): νmax 3353,
3123, 1689, 1593, 1532, 1506, 1482, 1463, 1421, 1353, 1321,
1274, 1261, 1230, 1185, 1138, 1113, 1034, 1020, 835, 665
(Method A). After several crystallizations from ether, 0.135
g (63 %) of a white solid was obtained. Product 6i was also
prepared by the reaction of 0.062 g (0.2 mmol) compound
5b and 0.066 g (0.3 mmol) 4-chloro-3-(trifluoromethyl)
phenylisocyanate (Method B). After crystallization from
ether, 0.065 g (61 %) of pure product was obtained; mp.
217–220 °C; IR (KBr): νmax 3394, 3322, 3122, 3082, 2928,
2857, 1711, 1660, 1595, 1513, 1481, 1421, 1310, 1227,
1
cm–1; H NMR (DMSO, 600 MHz): δ = 11.09 (s, 1H, 1′),
9.20 (s, 1H, 14) 8.95 (s, 1H, 12), 8.86 (d, J = 1.26, 1H, 3),
8.65 (d, J = 2.46, 1H, 5), 8.24 (dd, J = 2.40, J = 6.42, 1H, 3′),
8.12 (d, J = 2.22, 1H, 7′), 7.73 (d, J = 8.89, 1H, 6′), 7.68−7.60
(m, 2H, 16, 17), 7.58−7.53 (m, 2H, 8, 10), 7.26−7.21 (m, 2H,
7, 11); 13C NMR (DMSO, 150 MHz): δ = 161.97 (4) 161.60
(1) 152.47 (13), 147.10 (6) 142.02 (5), 139.34 (15), 138.91
(2), 137.97 (2′), 136.83 (9), 133.76 (3), 131.98 (17), 131.96
(6′), 128.23−117.37 (q, 21, J = 273.33), 128.18−117.32 (q, J
= 273.33, 8′), 127.30−126.08 (q, J = 30.59, 19), 127.24
−126.02 (q, J = 30.77, 4′), 125.26 (16) 124.71 (d, J = 1.66, 5′)
123.06 (7′), 122.29 (d, J = 1.65, 18), 121.79 (8, 10), 120.04 (7,
11) 119.44−119.21 (q, J = 5.71, 20), 116.88−116.65 (q, 3′, J
= 5.71); ESIMS m/z (pos): 630.2, C26H15Cl2F6N5O3 (calcd.
630.3); Anal. Calcd for C26H15Cl2F6N5O3: C, 49.54; H, 2.40;
N, 11.11. Found: C, 49.75, H, 2.76; N, 10.83.
1
1143, 1033, 844, 576, 515 cm–1; H NMR (DMSO, 600
MHz): δ = 9.19 (s, 1H, 14), 8.93 (s, 1H, 12), 8.69 (d, J =
1.20, 1H, 3), 8.52 (d, J = 1.20, 1H, 5), 8.40 (d, J = 8.52, 1H,
1′), 8.12 (d, J = 2.13, 1H, 20), 7.68–7.60 (m, 1H, 16, 17),
7.56–7.51 (m, 2H, 8, 10), 7.22–7.17 (m, 2H, 7, 11),
3.84–3.73 (m, 1H, 2′), 1.80–1.70, 1.62–1.58, 1.48–1.25,
1.19–1.07 (4m, 10H, 3′–7′); 13C NMR (DMSO, 150 MHz):
δ = 161.43 (4), 161.17 (1), 152.48 (13), 147.30 (6) 140.90
(5) 139.67 (15) 139.36 (2), 136.68 (9) 133.55 (3), 131.99
(17) 128.25–117.41 (q, J = 273.26, 21) 127.31–126.09 (q,
J = 30.29, 19), 123.54 (16), 122.27 (18), 122.18 (8, 10),
120.53 (7, 11) 116.88–116.65 (q, J = 5.82, 20), 47.93 (2′),
32.13 (3′, 7′), 25.08 (5′), 24.82 (4′, 6′); ESIMS m/z (pos):
534.4, C25H24ClF3N5O3 (calcd. 534.9); Anal. Calcd for
C25H23ClF3N5O3: C, 56.24; H, 4.34; N, 13.12. Found: C,
56.65, H, 4.01; N, 12.74.
5-/4-[3-(4-chloro-3-trifluoromethyl-phenyl)-ureido]-phe-
noxy/-pyrazine-2-carboxylic acid cyclopentylamide (6h)
This compound was prepared by the reaction of 0.090 g
(0.4 mmol) amide 4c and 0.132 g (0.4 mmol) urea 3c
(Method A). After crystallization from ether, 0.094 g (45 %)
of a white solid was obtained. Product 6h was also prepared
by the reaction of 0.060 g (0.2 mmol) compound 5a and
0.066 g (0.3 mmol) 4-chloro-3-(trifluoromethyl)phenyliso-
cyanate (Method B). After crystallization from ether 0.051 g
(49 %) of pure product was obtained; mp. 231–233 °C; IR
(KBr): νmax 3382, 3294, 3115, 3081, 2963, 2875, 1711,
1658, 1604, 1549, 1509, 1485, 1460, 1327, 1195, 1133,
1023, 840, 661, 513 cm–1; 1H NMR (DMSO, 600 MHz): δ
= 9.20 (s, 1H, 14), 8.95 (s, 1H, 12), 8.69 (d, J = 1.20, 1H,
3), 8.53 (d, J = 1.26, 1H, 5), 8.49 (d, J = 7.98, 1H, 1′), 8.12
(d, J = 2.46, 1H, 20), 7.67–7.65 (m, 1H, 16), 7.63–7.61 (m,
1H, 17), 7.55–7.53 (m, 2H, 8, 10), 7.21–7.18 (m, 2H, 7,
11), 4.27–4.22 (m, 1H, 2′), 1.91–1.85 (m, 2H, 3′),
1.72–1.68 (m, 2H, 6′), 1.62–1.53 (m, 4H, 4′, 5′); 13C NMR
(DMSO, 150 MHz): δ = 162.02 (4), 161.14 (1), 152.48
(13), 147.28 (6), 140.84 (5), 139.70 (15), 139.35 (2), 136.67
(9), 133.51 (3), 131.97 (17), 126.99-126.39 (q, J = 30.50,
19), 125.52–120.09 (q, J = 273.44, 21), 123.05 (16), 122.28
(18), 121.71 (8, 10), 120.04 (7, 11), 116.81–116.70 (q, J =
4.39, 20), 50.50 (2′), 32.01 (3′, 6′), 23.52 (4′, 5′); ESIMS
m/z (pos): 520.1, C24H22ClF3N5O3 (calcd. 520.1); Anal.
Calcd for C24H21ClF3N5O3: C, 55.44; H, 4.07; N, 13.47.
Found: C, 55.10, H, 3.77; N, 13.10.
Biological evaluation
Antiproliferative evaluation assay
The cell lines (CEM, HeLa, A549, HepG2, SW620, MCF-
7, Molt4/C8, L1210,WI-38) were cultured as monolayers
and maintained in Dulbecco’s modified Eagle medium
(DMEM) supplemented with 10 % fetal bovine serum
(FBS), 2 mM L-glutamine, 100 U/mL penicillin, and 100
μg/mL streptomycin in a humidified atmosphere with 5 %
CO2 at 37 °C. The cell lines were inoculated onto a series of
standard 96-well microtiter plates on day 0 at seeding
density of 3000–6000 cells per well depending upon their
specific doubling times. Freshly prepared dilutions of test
compounds in culture medium in the concentration range
from 10−8 to 10−4 M were added to the microtiter plates, and
the cells were grown for further 3–4 days. Working dilu-
tions of the compounds were freshly prepared on the day of
testing. The solvent (DMSO) was also tested for its poten-
tial inhibitory activity by adjusting its concentration to the
values used for the preparation of the working concentra-
tions (DMSO concentration never exceeded 0.1 %). After
3–4 days of incubation, the cell growth rate was evaluated
first light microscopically, then by performing the MTT [3-
5-/4-[3-(4-chloro-3-trifluoromethyl-phenyl)-ureido]-phe-
noxy/-pyrazine-2-carboxylic acid cyclohexylamide (6i)
This compound was prepared by the reaction of 0.096 g
(0.4 mmol) amide 4d and 0.132 g (0.4 mmol) urea 3c
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bro-
mide] assay. Experimentally determined absorbance values
were transformed into the cell percentage of growth (PG)
using the formulas proposed by NIH and described

Med Chem Res
previously (Gazivoda et al., 2008). This method directly
relies on control cells behaving normally at the day of assay
commencement because it compares the growth of treated
cells with that of untreated cells in control wells on the same
plate. The results are therefore a percentile difference from
the calculated expected value. The IC50 values for each
compound are calculated from dose-response curves using
linear regression analysis by fitting the mean test con-
centrations that give PG values above and below the
reference value. However, if all of the tested concentrations
produce PGs exceeding the respective reference level of
effect (e.g., PG value of 50) for a given cell line, then the
highest tested concentration is assigned as the default value
(in the screening data report that default value is preceded
by a “>” sign). Each test point was performed in quad-
ruplicate in three individual experiments. The results were
statistically analyzed (Analysis of variance, Tukey post hoc
test at p < 0.05).
antiproliferative activity, as well as cytotoxicity to normal
human fibroblasts cells, was comparable to sorafenib.
Compounds 6c and 6h exerted cytostatic activities that
surpassed the effects observed with sorafenib in three can-
cer cell lines (HepG2, HeLa, A549). Similar to sorafenib,
compound 6h proved to be cytotoxic to normal human
fibroblast cells, whereas compound 6c did not diminish
proliferative capacity of these cells. Antiproliferative
activity of 6h could be, at least partially, attributed to its
inhibitory effect on c-Raf activation. On contrary, 6c did not
inhibit the activity of c-Raf, which implies that other cell
signaling pathways govern its antiproliferative effects. In
conclusion, due to its reduced adverse effects on the growth
of normal cells and more potent anticancer activity in par-
ticular cancer cell lines in comparison with sorafenib,
compound 6c, e.g., 5-/4-[3-(4-bromophenyl)-ureido]-phe-
nyloxy/-pyrazine-2-carboxylic acid cyclopentylamide, may
be considered a potential scaffold for development of new,
more effective, and safer drugs for treating hepatocellular,
lung, and cervical carcinoma.
Determination of c-Raf activity using Western blotting
Acknowledgments Support for this study by Croatian Science
Foundation (project number IP-2014-09-1501), University of Zagreb
and University of Rijeka (Projects numbers BM1.9, 13.11.2.1.12 and
13.11.1.1.11), Republic of Croatia and the KU Leuven, Belgium
(GOA 10/014), are gratefully acknowledged. We sincerely thank
Lizette Van Berckelaer for performing the cytostatic in vitro
experiments.
In order to study the effects of selected compounds on c-Raf
activity, known protein target of multikinase inhibitor sor-
afenib, cells were cultured in 6-well plates at seeding den-
sity of 2 × 105 cells/well and subjected to treatment for
indicated time points. Cells were lysed in RIPA buffer
containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM
Na2EDTA, 1 mM ethyleneglycol tetraacetic acid (EGTA),
1 % NP-40, and 1 % sodium deoxycholate supplemented
with protease inhibitor cocktail (Roche). Total proteins (50
μg) were resolved on 12 % Tris-glycine polyacrylamide gels
and transferred to polyvinylidene fluoride (PVDF) mem-
branes. Subsequently, membranes were blocked for 1 h at
room temperature with 4 % bovine serum albumin (BSA) in
TBST [50 mmol/L Tris base, 150 mM NaCl, 0.1 % Tween
20 (pH 7.5)] and probed overnight at 4 °C with primary
antibody against Phospho-c-Raf (Ser259) (Cell Signaling
Technology; dilution 1:500). Membranes were washed with
TBST and incubated with an anti-rabbit (DakoCytomation)
horseradish peroxidase-conjugated secondary antibody at
room temperature for 1 h. Individual proteins were visua-
lized by the BM Chemiluminescence Western Blotting
Substrate (POD) (Roche, Switzerland).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Open Access This article is distributed under the terms of the
Creative Commons Attribution
4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrest-
ricted use, distribution, and reproduction in any medium, provided you
give appropriate credit to the original author(s) and the source, provide
a link to the Creative Commons license, and indicate if changes were
made.
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