Design and synthesis of new RAF kinase-inhibiting antiproliferative quinoline derivatives. Part 2: Diarylurea derivatives

Article abstract of DOI:10.1016/j.ejmech.2017.01.006

This article describes the design, synthesis, and biological screening of a new series of diarylurea derivatives possessing quinoline nucleus. Nine target compounds were selected by the National Cancer Institute (NCI, Bethesda, Maryland, USA) for in?vitro antiproliferative screening against a panel of 58 cancer cell lines of nine cancer types. Following one-dose initial screening, compounds 1d-g and 2b were selected for 5-dose screening in order to calculate their IC₅₀and total growth inhibition (TGI) values against the cell lines. Compounds 1e and 1g were the most promising analogues. Both compounds showed strong potency and broad-spectrum antiproliferative activity against the different tested cancer types. Their IC₅₀and TGI values were less than those of the reference drug, sorafenib, against most of the tested cell lines of the nine different cancer types. Furthermore, the most potent compounds 1d-g were tested against C-RAF kinase as a potential molecular target of this series of compounds. All of them showed high potency, and the most potent derivative was compound 1e (IC₅₀?=?0.10?μM). It was further tested against a panel of another twelve kinases, and it showed selectivity against C-RAF kinase. This could be, at least in part, the possible mechanism of antiproliferative action of this series of compounds at molecular level. The binding modes of compounds 1e and 1g were studied by docking studies, which highlighted the importance of the urea linker compared with the amide linker.

Full text of DOI:10.1016/j.ejmech.2017.01.006

Accepted Manuscript
Design and synthesis of new RAF kinase-inhibiting antiproliferative quinoline
derivatives. Part 2: Diarylurea derivatives
Mohammed I. El-Gamal, Mohammad Ashrafuddin Khan, Hamadeh Tarazi,
Mohammed S. Abdel-Maksoud, Mahmoud M. Gamal El-Din, Kyung Ho Yoo, Chang-
Hyun Oh
PII:
S0223-5234(17)30006-5
10.1016/j.ejmech.2017.01.006
EJMECH 9157
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 December 2016
Revised Date: 28 December 2016
Accepted Date: 4 January 2017
Please cite this article as: M.I. El-Gamal, M.A. Khan, H. Tarazi, M.S. Abdel-Maksoud, M.M. Gamal El-
Din, K.H. Yoo, C.-H. Oh, Design and synthesis of new RAF kinase-inhibiting antiproliferative quinoline
derivatives. Part 2: Diarylurea derivatives, European Journal of Medicinal Chemistry (2017), doi:
10.1016/j.ejmech.2017.01.006.
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ACCEPTED MANUSCRIPT
H
H
N
N
CF3
Cl
O
O
N
O
O
1e
C-RAF kinase; IC50 = 0.10 µM
RPMI-8226 cell line; IC50 = 0.64 µM
HOP-92 cell line; IC50 = 0.68 µM
COLO 205 cell line; IC50 = 0.66 µM
UACC-257 cell line; IC50 = 0.71 µM
MDA-MB-231 cell line; IC50 = 0.68 µM
HS 578T cell line; IC50 = 0.72 µM

ACCEPTED MANUSCRIPT
Design and synthesis of new RAF kinase-inhibiting antiproliferative
quinoline derivatives. Part 2: Diarylurea derivatives
Mohammed I. El-Gamal 1,2,3, Mohammad Ashrafuddin Khan 4,5, Hamadeh
Tarazi 1,2, Mohammed S. Abdel-Maksoud4,5,6, Mahmoud M. Gamal El-Din4,5,6
Kyung Ho Yoo 7, and Chang-Hyun Oh4,5,*
,
1 Department of Medicinal Chemistry, College of Pharmacy, University of Sharjah,
Sharjah 27272, United Arab Emirates.
2
Sharjah Institute for Medical Research, University of Sharjah, Sharjah 27272,
United Arab Emirates.
3
Department of Medicinal Chemistry, Faculty of Pharmacy, University of Mansoura,
Mansoura 35516, Egypt.
4 Center for Biomaterials, Korea Institute of Science and Technology, PO Box 131,
Cheongryang, Seoul 130-650, Republic of Korea.
5 Department of Biomolecular Science, Korea University of Science and Technology,
113 Gwahangno, Yuseong-gu, Daejeon 305-333, Republic of Korea.
6
Pharmaceutical and Drug Industries Research Division, National Research Centre,
Dokki-Giza 12622, Egypt.
7 Chemical Kinomics Research Center, Korea Institute of Science and Technology, PO
Box 131, Cheongryang, Seoul 130-650, Republic of Korea.
* Corresponding author: Tel.: +82 2 958 5160; fax: +82 2 958 5308.
Address: Center for Biomaterials, Korea Institute of Science and Technology, PO Box
131, Cheongryang, Seoul 130-650, Republic of Korea [C.-H. Oh].
E-mail address: choh@kist.re.kr [C.-H. Oh].
Abstract
This article describes the design, synthesis, and biological screening of a new
series of diarylurea derivatives possessing quinoline nucleus. Nine target compounds
were selected by the National Cancer Institute (NCI, Bethesda, Maryland, USA) for in
vitro antiproliferative screening against a panel of 58 cancer cell lines of nine cancer
types. Following one-dose initial screening, compounds 1d-g and 2b were selected for
5-dose screening in order to calculate their IC50 and total growth inhibition (TGI)
values against the cell lines. Compounds 1e and 1g were the most promising
analogues. Both compounds showed strong potency and broad-spectrum
antiproliferative activity against the different tested cancer types. Their IC50 and TGI
values were less than those of the reference drug, sorafenib, against most of the tested
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cell lines of the nine different cancer types. Furthermore, the most potent compounds
1d-g were tested against C-RAF kinase as a potential molecular target of this series of
compounds. All of them showed high potency, and the most potent derivative was
compound 1e (IC50 = 0.10 µM). It was further tested against a panel of another twelve
kinases, and it showed selectivity against C-RAF kinase. This could be, at least in
part, the possible mechanism of antiproliferative action of this series of compounds at
molecular level. The binding modes of compounds 1e and 1g were studied by docking
studies, which highlighted the importance of the urea linker compared with the amide
linker.
Keywords: Antiproliferative activity; C-RAF kinase; Diarylurea; Molecular Docking;
Quinoline.
1. Introduction
Cancer has become one of the major health challenges that require continuous
efforts to develop efficient drugs. It is the second leading cause of death globally after
the cardiovascular disorders. The World Health Organization (WHO) expects
increment of the number of newly discovered cancer cases to become 15 million cases
worldwide every year [1,2]. Kinases are potential therapeutic targets for treatment of
different types of cancers. Kinase inhibition leads to disturbance of different essential
biological processes of the cancer cells, and hence stopping cell proliferation and
growth.
Several antiproliferative agents possessing quinoline moiety have been
recently reported [3-12]. In addition, many diarylurea derivatives have exhibited
promising antiproliferative activity [13-33]. Sorafenib (Nexavar®, Fig. 1) is a
diarylurea drug approved by the U.S. Food and Drug Administration (FDA) for
treatment of advanced renal cancer. It has also been subjected to clinical trials for
treatment of other cancer types such as ovarian, metastatic colorectal,
esophageal/gastroesophageal, brain, glioblastoma, leukemia, metastatic breast,
Hodgkin’s lymphoma, hepatocellular carcinoma (HCC), pancreatic, advanced gastric,
thyroid, non-small cell lung cancer (NSCLC), prostate, skin/ocular melanoma,
bladder, and neuroendocrine cancers [34,35]. Regorafenib (BAY73-4506, Stivarga®,
Fig. 1) is the fluoro analogue of sorafenib (Fig. 1). It was approved by the FDA in
2012 for treatment of metastatic colorectal cancer [36]. In 2013, the FDA approved it
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for gastrointestinal stromal tumors (GIST) therapy [37]. Furthermore, many clinical
trials have been conducted for Regorafenib against NSCLC, biliary tract carcinoma,
renal cell carcinoma (RCC), angiosarcoma, liposarcoma, HCC (in combination with
Sorafenib), osteogenic sarcoma, and Ewing/Ewing-like sarcomas [38].
We have previously reported a series of antiproliferative diarylamide
derivatives possessing quinoline nucleus. The most potent compound shown in Fig. 1
exerted one-digit micromolar IC50 values over the most sensitive cell lines, and it
showed inhibitory effect on C-RAF kinase [7]. That compound was utilized as lead
compound for design of the target compounds 1a-i and 2a,b illustrated in Fig. 1 with
structural similarity to it but with urea linker instead of amide. The target diarylurea
compounds were synthesized and tested as in vitro cancer cell line growth inhibitors.
The most potent compounds were tested against C-RAF kinase and other off-target
kinases in order to investigate the molecular mechanism of action. Docking studies
were conducted to evaluate the binding mode and the contribution of the urea linker to
the antiproliferative activity.
[Figure 1]
2. Results and discussion
2.1. Chemistry
Scheme 1 illustrates the synthetic pathway via which the target compounds
1a-i and 2a,b were synthesized. The nitro group of 6-nitroveratraldehyde (3) was
reduced to the corresponding amino derivative 4 in hydrogen atmosphere in presence
of palladium over carbon as a catalyst [39]. Once the starting material 3 was
completely converted to 4 (confirmed by TLC), the reaction must be stopped to avoid
further reduction of the aldehyde group to primary alcohol. On the other hand, 4-
nitrophenol (5a) or 4-nitrothiophenol (5b) was refluxed with chloroacetone (6) in
acetone in presence of potassium carbonate as an acid-binding agent to form the
ether/thioether compounds 7a,b. The next step was Friedlander cyclization reaction
between the aminobenzaldehyde derivative 4 and compound 7a,b in presence of
piperidine in refluxing ethanol to yield the 3-(nitrophenyloxy/thio)quinoline
intermediates 8a,b. The conditions of this reaction were optimized and previously
reported [3]. The nitro compounds 8a,b were reduced to the corresponding amino
analogues 9a,b using Pd/C in hydrogen atmosphere [7]. The aniline intermediates
9a,b were then reacted with the appropriate aryl isocyanate in presence of potassium
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carbonate to produce the target diarylurea products 1a-i. Compounds 1c,f were
demethylated using boron tribromide to form the corresponding 6,7-
dihydroxyquinoline products 2a,b. The target compound structures are shown in
Table 1.
[Scheme 1 & Table 1]
2.2. In vitro antiproliferative activity against the NCI-58 cell line panel
2.2.1. One-dose testing
The target compounds shown in Fig. 2 were selected by the NCI, USA [40],
for in vitro antiproliferative screening against a panel of 58 cancer cell lines belonging
to 9 cancer types (leukemia, NSCLC, colon cancer, CNS cancer, melanoma, ovarian
cancer, renal cancer, prostate cancer, and breast cancer). The nine tested compounds
were initially tested over the cell line panel at a single dose of 10 µM in order to
determine the growth inhibition percentages. The mean %inhibition of each of the
tested compounds against the complete panel are shown in Fig. 2.
[Figure 2]
The results indicated that the 6,7-dimethoxyquinoline analogue 1f was more
active than the corresponding 6,7-dihydroxyquinoline derivative 2b. This could be
due the steric and/or electronic differences between the methoxy and hydroxyl groups.
In addition, the dimethoxy compounds are more lipophilic than the corresponding
dihydroxyl analogues. This could contribute to more penetration through the cancer
cell membrane, higher exposure of the intracellular components to the tested
compound, and hence stronger activity.
The oxygen linker-containing compounds 1f,g were more active than the
corresponding sulfur analogues 1h,i. So the oxygen linker is more optimal for
anticancer activity of this series of compounds.
Compounds 1d-g were the most active among this series of compounds. They
were also more active than the corresponding diarylamide derivatives previously
reported [7]. So the urea linker was found more favorable for activity than the amide
spacer. With urea linker, the compounds became longer and this might permit more
appropriate fitting at the receptor site, and/or the additional terminal NH group might
perform hydrogen bonding with the receptor site, which contributes to stronger
affinity and higher anticancer activity.
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The effects of the substituents on the terminal aryl group on the activity were
also investigated. Compounds 1c-g bearing electron-withdrawing groups were
generally more potent than compounds 1a and 1b possessing unsubstituted phenyl or
p-tolyl moiety, respectively. The electronic and/or steric effects of compounds 1c-g
substituents might enhance the activity. Moreover, compounds 1e and 1g containing
4-chloro-3-(trifluoromethyl)phenyl or 3,5-bis(trifluoromethyl)phenyl terminal rings
were more active than compound 1d possessing 3-(trifluoromethyl)phenyl moiety. So
insertion of p-chloro or m-CF3 groups on the 3-(trifluoromethyl)phenyl ring could
improve the activity. This can be attributed to enhanced bulkiness, hydrophobicity,
and/or electron-withdrawing effect.
Compounds 1d, 1e, and 1g were the most active amongst all the tested target
compounds. Their %inhibition values against each of the tested cell lines of the NCI-
58 panel are illustrated in Fig. 3. The three compounds showed strong inhibitory
effects over most of the tested cell lines at 10 µM concentration. So their IC50 values
were expected to come in the one-digit micromolar range or in the submicromolar
scale. Compounds 1d, 1e, and 1g also exerted broad-spectrum antiproliferative
activity against the nine tested cancer types.
[Figure 3]
2.2.2. Five-dose testing
The most promising compounds 1d-g and 2b in the one-dose testing were
selected for further 5-dose testing in order to determine their IC50 and Total Growth
Inhibition (TGI) values and compare their potencies. The mean IC50 values of those
five compounds over each cancer type subpanel are summarized in Table 2.
[Table 2]
The dimethoxyquinoline derivative 1f was more potent against all the nine
subpanels than the corresponding dihydroxyquinoline analogue 2b. And compounds
1e,g possessing 4-chloro-3-(trifluoromethyl)phenyl or 3,5-bis(trifluoromethyl)phenyl
terminal ring, respectively, were more potent than compound 1d with 3-
(trifluoromethyl)phenyl moiety. These results were similar to the one-dose findings.
Among the five tested compounds, 1e and 1g were the most potent. Their detailed
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IC50 and TGI values over each of the tested cell lines are shown in Table 3, and
compared with the results of sorafenib, a diarylurea drug, as a reference standard.
Each of compounds 1e and 1g demonstrated high potency and broad-spectrum
antiproliferative activity. The IC50 values of each of them were in submicromolar
scale against 18 cell lines. Both of them were more potent than the corresponding
amide analogues [7]. And they were generally more potent than sorafenib against
most of the tested cell lines. Furthermore, the TGI values of both of them were in one-
digit micromolar range against all the tested cell lines, and were generally less than
those of sorafenib (superior activity than sorafenib).
[Table 3]
2.3. Kinase assay
As the lead diarylamide compound (Fig. 1) was reported as C-RAF kinase
inhibitor (76.65% inhibition at 10 µM concentration), we decided to test the most
potent antiproliferative compounds 1d-g for inhibitory effect against C-RAF kinase.
The results are depicted in Table 4. Compound 1g possessing urea linker showed
stronger C-RAF inhibition than the corresponding diarylamide lead compound.
Compounds 1d-f also exerted stronger inhibition than that amide lead compound. All
the four compounds 1d-g showed submicromolar IC50 values against C-RAF kinase,
and compound 1e with 4-chloro-3-(trifluoromethyl)phenylurea moiety was the most
potent C-RAF kinase inhibitor.
C-RAF kinase is an essential growth factor for RCC [41], breast cancer [42],
and melanoma [43]. C-RAF is also related to poor prognosis in ovarian [44] and
androgen-insensitive prostate cancer [45]. Furthermore, C-RAF/MEK dual inhibition
has been reported to induce apoptosis in colorectal cancer cell lines such as HCT-116
[46]. So C-RAF kinase inhibition could be, at least in part, a molecular mechanism of
antiproliferative activity of this series of compounds contributing to their strong
potency and broad-spectrum activity.
[Table 4]
The most potent C-RAF inhibitor, compound 1e, was further tested against a
panel of another twelve kinases in order to investigate its selectivity and to discover
other molecular targets if any. The results are summarized in Table 5. In addition to
C-RAF kinase inhibition, compound 1e showed strong inhibitory effects against
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V600E-B-RAF kinase, but still 2.6-fold more selective for C-RAF. V600E-B-RAF
has been reported in different melanoma cell lines such as SK-MEL-5, SK-MEL-28,
and UACC-62 [47,48] as well as colon cancer cell lines such as COLO 205 and HT-
29 [48]. C-RAF over-expression has been also reported to enhance the resistance of
MCF-7 breast cancer cells to the standard chemotherapeutic agents [48]. Compound
1e demonstrated high potency against the cell lines in which V600E-B-RAF is an
essential growth factor. So V600E-B-RAF kinase inhibition also could contribute to
the high potency and broad-spectrum activity of compound 1e against the cancer cell
lines.
[Table 5]
2.4. Docking study
The superior inhibition pattern exhibited by the target diarylurea derivatives
compared to an earlier series of quinoline-based diarylamides [7], encouraged us to
explore their probable binding modes within the C-RAF kinase using molecular
docking technique, so that to provide a pleasant explanation for the differences in
activity. The lead diarylamide derivative as well as compounds 1e and 1g were
docked into the active site of C-RAF kinase (PDB ID: 3OMV), and the binding free
energies are shown in Table 6.
[Table 6]
The lead diarylamide derivative (Fig. 4) was able to form two H-bonds; one
H-bond formed between its amidic carbonyl oxygen and the corresponding gatekeeper
residue Thr421 (O…HO), while the second H-bond was established between its
quinolinyl nitrogen and the near C-terminus residue Asn472 (N…H-N) within the
CR3 domain.
[Figure 4]
On the other hand, compound 1g (Fig. 5) shared similar H-bond interaction
with the gatekeeper residue Thr421, while the second H-bond formed was between its
quinolinyl nitrogen and the residue Lys431 (N…H-N) within the CR3 domain. The
difference in the second H-bond could be attributed to structural elongation caused by
the urea appendage (-NH-CO-NH-) compared to the amide appendage (-NH-CO-).
[Figure 5]
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Our most potent compound 1e showed interesting binding profile within the
C-RAF kinase (Fig. 6). It was able to form three H-bonds with significant residues;
the first H-bond formed was between its urea carbonyl oxygen and the catalytic
Lys470 (O…H-N) of the K/D/D-motif; while the second H-bond was formed between
its urea terminal nitrogen and the DFG-residue Asp486 (NH…O). The third H-bond
was established between its quinolinyl nitrogen and the residue LYS431 (N…H-N) of
the CR3 domain. The additional hydrogen bond secured by the urea appendage
ensured strong interaction and an enhanced C-RAF inhibitory activity compared to
the amide-based derivatives. This could rationalize the stronger C-RAF kinase
inhibitory effect of this compound compared to the diarylamide lead compound.
[Figure 6]
3. Conclusion
This article reported the design, synthesis, and biological evaluation of new
diarylureas possessing 6,7-dimethoxy(dihydroxy)quinoline scaffold. The final
compounds were tested for antiproliferative activity against a panel of 58 cancer cell
lines of 9 cancer types at the NCI, Bethesda, Maryland, USA. Compounds 1d-g and
2b showed the most promising results in both single-dose and five-dose assays.
Among them, compounds 1e and 1g were the most potent. They were generally more
potent the sorafenib, the reference drug, in terms of IC50 and TGI values. Each of
them exerted submicromolar IC50 values against 18 cell lines of the nine different
tested cancer types. Their TGI values also were in one-digit micromolar scale over all
the tested 58 cell lines. The kinase screening results indicated that C-RAF and
V600E-B-RAF inhibition could be a possible mechanism of antiproliferative activity
of this series at molecular level. The highest potency against the kinases was
demonstrated by compound 1e possessing 4-chloro-3-(trifluoromethyl)phenyl
terminal moiety, similar to sorafenib.
The structure-activity relationship study of this series of compounds revealed
that 6,7-dimethoxyquinoline nucleus was more optimal for activity than 6,7-
dihydroxyquinoline. The O linker and the urea spacer were optimal for activity too.
And when the terminal aryl moiety contained electron-withdrawing groups such as
one or more of CF3 or chloro groups, the activity much increased. Taken together, it
can be concluded that a terminal aryl ring carrying 3-CF3, 4-Cl-3-CF3, 3,4-dichloro, or
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3,5-bis(CF3), together with 6,7-dimethoxyquinoline, oxygen linker, and urea spacer
can constitute the pharmacophore of this series of compounds.
Molecular docking study was carried out in order to study the binding mode of
compounds 1e and 1g with the most sensitive kinase, C-RAF. The results indicated
that compound 1e bound stronger to the kinase than compound 1g, in agreement with
the in vitro kinase assay results. The docking study showed the contribution of the
urea linker to stronger activity than the previously reported diarylamide lead
compound through the formation of hydrogen bond between the terminal NH moiety
of the urea linker with Asp486 in the C-RAF crystal structure.
4. Experimental
4.1. General
The target compounds 1a-i and 2a,b were purified by flash column
chromatography using silica gel (0.040-0.063 mm, 230-400 mesh) and technical grade
solvents. IR analyses (KBr disks) were done using a Bruker FT-IR instrument (Bruker
Bioscience, Billerica, MA, USA). 1H NMR and 13C NMR analyses were carried out on a
Bruker Avance 400 or 300 spectrometer using tetramethylsilane as an internal
standard. Melting points were measured on a Walden Precision Apparatus
Electrothermal 9300 apparatus and are uncorrected. LC-MS analysis was conducted
using the following system: Waters 2998 photodiode array detector, Waters 3100
mass detector, Waters SFO system fluidics organizer, Waters 2545 binary gradient
module, Waters reagent manager, Waters 2767 sample manager, SunfireTM C18
column (4.6 x 50 mm, 5 µm particle size); Solvent gradient = 95% A at 0 min, 1% A
at 5 min; solvent A: 0.035% trifluoroacetic acid (TFA) in water; solvent B: 0.035%
TFA in CH3OH; flow rate = 3.0 mL/min; the AUC was calculated using Waters
MassLynx 4.1 software. The solvents and liquid reagents were transferred using
hypodermic syringes. All the solvents and reagents were purchased from commercial
companies, and used as such.
4.2. Synthesis of 4-(6,7-dimethoxy-2-methylquinolin-3-yloxy)benzenamine (9a) and 4-
(6,7-dimethoxy-2-methylquinolin-3-ylthio)benzenamine (9b)
They were carried out utilizing the 4-step procedure previously reported in the
literature [3,7].
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4.3. Synthesis of 1-(4-(6,7-dimethoxy-2-methylquinolin-3-yloxy)phenyl)-3-phenylurea
(1a) [3]
To a solution of compound 9a (31 mg, 0.1 mmol) in anhydrous THF (5 mL), the
phenyl isocyanate (12 mg, 0.1 mmol) and anhydrous potassium carbonate (28 mg, 0.2
mmol) were added. The reaction mixture was stirred at room temperature under
nitrogen atmosphere for 12 h. After reaction completion, the inorganic material was
filtered off and washed with THF, the organic solvent was evaporated under reduced
pressure, and the residue was purified by column chromatography (silica gel, using
the proper ratio of hexane and ethyl acetate) to get the compound 1a as a pure
product.
1
Yield: 52%; m.p.: 220-2 ºC; IR (KBr) [cm-1]: 3298, 2920, 1646, 1505; H NMR
(Acetone-d6, 400 MHz) δ 8.04 (brs, 1H, NH), 7.98 (brs, 1H, NH), 7.48-7.41 (m, 5H,
Ar-H), 7.24 (s, 1H, Ar-H), 7.17-7.13 (m, 3H, Ar-H), 7.03 (s, 1H, Ar-H), 6.90-6.84 (m,
2H, Ar-H), 3.83 (s, 3H, OCH3), 3.76 (s, 3H, OCH3), 2.45 (s, 3H, CH3); 13C NMR
(Acetone-d6, 100 MHz) δ 153.6 (C=O), 152.8, 151.4, 150.5, 149.5, 148.3, 141.1,
141.0, 131.8, 126.2, 123.3, 123.0, 120.6, 118.9, 107.1, 104.3 (Ar-C), 56.1, 56.0 (2
OCH3), 19.9 (CH3); LC-MS m/z: 430.0 (M+ + 1); elemental analysis: calculated
C:69.92%, H:5.40%, N:9.78%; found: C:69.67%, H:5.48%, N:9.87%.
Compound 1b-i were synthesized by the same procedure using the appropriate aryl
isocyanate derivative.
4.3.1. 1-(4-(6,7-Dimethoxy-2-methylquinolin-3-yloxy)phenyl)-3-p-tolylurea (1b)
Yield: 40%; m.p.: 227-8 ºC (dec.); IR (KBr) [cm-1]: 3296, 2919, 1645, 1505; 1H NMR
(Acetone-d6, 400 MHz) δ 8.05 (brs, 1H, NH), 7.93 (brs, 1H, NH), 7.46 (d, J = 7.1 Hz,
2H, Ar-H), 7.28-7.24 (m, 3H, Ar-H), 7.16 (s, 1H, Ar-H), 7.02 (s, 1H, Ar-H), 6.95 (d, J
= 6.4 Hz, 2H, Ar-H), 6.88 (d, J = 8.5 Hz, 2H, Ar-H), 3.83 (s, 3H, OCH3), 3.76 (s, 3H,
13
OCH3), 2.45 (s, 3H, quinolinyl-CH3), 2.13 (s, 3H, tolyl-CH3); C NMR (Acetone-d6,
100 MHz) δ 153.5 (C=O), 152.7, 151.4, 150.3, 149.4, 148.2, 141.0, 139.9, 131.6,
126.0, 123.2, 123.0, 120.5, 118.8, 107.0, 104.1 (Ar-C), 56.1, 56.0 (2 OCH3), 21.2
(tolyl- CH3), 19.9 (quinolinyl-CH3); LC-MS m/z: 444.1 (M+ + 1); elemental analysis:
calculated C:70.41%, H:5.68%, N:9.47%; found: C:70.15%, H:5.80%, N:9.30%.
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4.3.2.
1-(4-((6,7-Dimethoxy-2-methylquinolin-3-yl)oxy)phenyl)-3-(4-
(trifluoromethyl)phenyl)urea (1c)
Yield: 70%; m.p.: 231-2 ºC; IR (KBr) [cm-1]: 3304, 2961, 1651, 1506; 1H NMR (400
MHz, CDCl3) δ 7.53 (d, J = 8.3 Hz, 2H, Ar-H), 7.48 (d, J = 8.3 Hz, 2H, Ar-H), 7.38-
7.36 (m, 3H), 7.33 (d, J = 8.6 Hz, 2H, Ar-H), 7.04 (brs, 1H, NH), 6.98 (d, J = 8.4 Hz,
2H, Ar-H), 6.89 (s, 1H, Ar-H), 3.99 (s, 3H, OCH3), 3.96 (s, 3H, OCH3), 2.64 (s, 3H,
CH3); 13C NMR (CDCl3, 100 MHz) δ 153.9 (C=O), 153.0, 151.5, 150.8, 149.7, 148.6,
141.5, 141.2, 131.9, 126.4, 123.6, 123.3, 120.9, 119.2, 119.1, 107.2, 104.5 (Ar-C and
CF3), 56.1, 56.0 (2 OCH3), 20.1 (CH3); LC-MS m/z: 498.1 (M+ + 1); elemental
analysis: calculated C:62.77%, H:4.46%, N:8.45%; found: C:62.64%, H:4.62%,
N:8.52%.
4.3.3.
1-(4-((6,7-Dimethoxy-2-methylquinolin-3-yl)oxy)phenyl)-3-(3-
(trifluoromethyl)phenyl)urea (1d)
Yield: 78%; m.p.: 234-7 ºC; IR (KBr) [cm-1]: 3339, 2964, 1654, 1507; 1H NMR (400
MHz, CDCl3) δ 7.69 (brs, 1H, NH), 7.60 (brs, 1H, NH), 7.51 (d, J = 8.5 Hz, 2H, Ar-
H), 7.36 (s, 1H, Ar-H), 7.34-7.31 (m, 3H, Ar-H), 7.28 (s, 1H, Ar-H), 7.27 (s, 1H, Ar-
H), 6.93 (d, J = 8.9 Hz, 2H, Ar-H), 6.86 (s, 1H, Ar-H), 3.97 (s, 3H, OCH3), 3.93 (s,
13
3H, OCH3), 2.62 (s, 3H, CH3); C NMR (CDCl3, 100 MHz) δ 153.6 (C=O), 153.5,
151.4, 150.8, 149.7, 148.8, 141.1, 138.9, 133.3, 129.6, 123.3, 123.1 (2C), 122.8,
120.6, 120.0, 119.2 (2C), 116.4, 107.1, 104.6 (Ar-C and CF3), 56.1, 56.0 (2 OCH3),
20.0 (CH3); LC-MS m/z: 498.1 (M+ + 1); elemental analysis: calculated C:62.77%,
H:4.46%, N:8.45%; found: C:62.57%, H:4.39%, N:8.53%.
4.3.4. 1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(4-((6,7-dimethoxy-2-methylquinolin-
3-yl)oxy)phenyl)urea (1e)
Yield: 66%; m.p.: 267-70 ºC; IR (KBr) [cm-1]: 3338, 2963, 1654, 1506; 1H NMR (400
MHz, CDCl3) δ 7.66 (brs, 1H, NH), 7.54 (d, J = 8.7 Hz, 1H, Ar-H), 7.46 (brs, 1H,
NH), 7.38-7.36 (m, 3H, Ar-H), 7.30 (d, J = 8.7 Hz, 2H, Ar-H), 7.21 (s, 1H, Ar-H),
6.96 (d, J = 8.7 Hz, 2H, Ar-H), 6.89 (s, 1H, Ar-H), 3.99 (s, 3H, OCH3), 3.95 (s, 3H,
OCH3), 2.63 (s, 3H, CH3); 13C NMR (CDCl3, 100 MHz) δ 153.9 (C=O), 153.2, 151.5,
150.8, 149.2, 148.6, 141.1, 137.4, 132.9, 132.1, 128.6, 126.0, 123.5 (2C), 121.0, 119.1
(2C), 118.5, 107.0, 104.6 (Ar-C and CF3), 56.1, 56.0 (2 OCH3), 19.9 (CH3); LC-MS
11

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m/z: 532.1 (M+ + 1); elemental analysis: calculated C:58.71%, H:3.98%, N:7.90%;
found: C:58.65%, H:4.16%, N:7.82%.
4.3.5.
1-(3,4-Dichlorophenyl)-3-(4-(6,7-dimethoxy-2-methylquinolin-3-
yloxy)phenyl)urea (1f)
1
Yield: 76%; m.p.: 241-4 ºC; IR (KBr) [cm-1]: 3337, 2961, 1655, 1507; H NMR
(CDCl3, 300 MHz) δ 7.98 (brs, 1H, NH), 7.83 (brs, 1H, NH), 7.35 (d, J = 7.0 Hz, 1H,
Ar-H), 7.24 (s, 1H, Ar-H), 7.19-7.09 (m, 4H, Ar-H), 6.99 (d, J = 8.5 Hz, 1H, Ar-H),
6.83-6.76 (m, 3H, Ar-H), 3.89 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 2.53 (s, 3H, CH3);
13C NMR (CDCl3, 75 MHz) δ 162.3 (C=O), 153.8, 153.2, 151.6, 150.4, 149.9, 148.7,
140.5, 138.0, 133.5, 132.7, 130.5, 126.5, 123.4, 122.8, 121.3, 121.0, 119.1, 106.6,
104.6 (Ar-C), 56.1 (2 OCH3), 19.7 (CH3); LC-MS m/z: 498.2 (M+ + 1); elemental
analysis: calculated C:60.25%, H:4.25%, N:8.43%; found: C:60.13%, H:4.43%,
N:8.66%.
4.3.6.
1-(3,5-Bis(trifluoromethyl)phenyl)-3-(4-((6,7-dimethoxy-2-methylquinolin-3-
yl)oxy)phenyl)urea (1g)
1
Yield: 78%; m.p.: >280 ºC; IR (KBr) [cm-1]: 3339, 2962, 1654, 1506; H NMR
(CDCl3, 400 MHz) δ 7.85 (brs, 1H, NH), 7.81 (s, 2H, Ar-H), 7.43 (s, 2H, Ar-H), 7.32
(s, 2H, Ar-H), 7.29 (s, 1H, Ar-H), 7.19 (s, 1H, Ar-H), 6.89 (d, J = 8.6 Hz, 2H, Ar-H),
13
6.84 (s, 1H, Ar-H), 3.91 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 2.58 (s, 3H, CH3); C
NMR (CDCl3, 75 MHz) δ 162.3 (C=O), 152.4, 152.1, 151.7, 150.1, 149.5, 149.2,
142.2, 140.5, 135.3, 131.8, 131.4, 123.5, 121.0, 119.7, 119.2, 118.1, 106.9, 104.9 (Ar-
C), 55.2 (2 OCH3), 19.2 (CH3); LC-MS m/z: 566.1 (M+ + 1); elemental analysis:
calculated C:57.35%, H:3.74%, N:7.43%; found: C:57.06%, H:4.02%, N:7.34%.
4.3.7.
1-(3,4-Dichlorophenyl)-3-(4-((6,7-dimethoxy-2-methylquinolin-3-
yl)thio)phenyl)urea (1h)
Yield: 58%; m.p.: 249-52 ºC; IR (KBr) [cm-1]: 3336, 2959, 1655, 1504; 1H NMR (300
MHz, CDCl3) δ 10.27 (brs, 1H, NH), 9.10 (d, J = 15.5 Hz, 1H, Ar-H), 8.15-8.10 (m,
3H, Ar-H and NH), 7.86 (s, 1H, Ar-H), 7.60 (s, 1H, Ar-H), 7.39-7.31 (m, 3H, Ar-H),
6.85 (d, J = 8.2 Hz, 1H, Ar-H), 6.76 (s, 1H, Ar-H), 3.91 (s, 3H, OCH3), 3.85 (s, 3H,
OCH3), 2.60 (s, 3H, CH3); LC-MS m/z: 514.0 (M+ + 1); elemental analysis: calculated
C:58.37%, H:4.11%, N:8.17%; found: C:58.20%, H:4.20%, N:8.06%.
12

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4.3.8.
1-(3,5-Bis(trifluoromethyl)phenyl)-3-(4-((6,7-dimethoxy-2-methylquinolin-3-
yl)thio)phenyl)urea (1i)
1
Yield: 46%; m.p.: >280 ºC; IR (KBr) [cm-1]: 3338, 2960, 1654, 1504; H NMR (300
MHz, CDCl3) δ 8.11-7.98 (m, 3H, NH and Ar-H), 7.74-7.28 (m, 3H, Ar-H), 7.45-7.30
(m, 3H, Ar-H), 7.02 (s, 1H, Ar-H), 6.88 (s, 1H, Ar-H), 6.78 (s, 1H, Ar-H), 3.96 (s,
3H, OCH3), 3.88 (s, 3H, OCH3), 2.60 (s, 3H, CH3); 13C NMR (CDCl3, 75 MHz) δ
162.3 (C=O), 152.4, 152.1, 151.7, 150.1, 149.5, 149.2, 142.2, 140.4, 135.5, 131.7,
131.5, 123.4, 121.0, 119.8, 119.3, 118.3, 106.9, 104.9 (Ar-C), 55.2 (2 OCH3), 19.3
(CH3); LC-MS m/z: 581.82 (M+ + 1); elemental analysis: calculated C:55.77%,
H:3.64%, N:7.23%; found: C:55.54%, H:4.50%, N:7.42%.
4.4. Synthesis of the dihydroxyquinoline derivatives 2a,b
To a solution of compound 1c,f (0.1 mmol) in anhydrous dichloromethane (3 mL),
BBr3 (0.16 mL of a 1M solution in methylene chloride, 2.4 mmol) was added
dropwise at -78 °C under nitrogen atmosphere. The reaction mixture was stirred at the
same temperature for 1 h. The mixture was allowed to warm to room temperature and
then stirred for 4 h. The mixture was quenched with saturated aqueous NaHCO3, and
ethyl acetate (5 mL) was added. The organic layer was separated, and the aqueous
layer was extracted with ethyl acetate (3 X 2 mL). The combined organic layer
extracts were washed with saturated saline and dried over anhydrous Na2SO4. After
evaporation of the organic solvent, the residue was purified by short flash column
chromatography (silica gel, ethyl acetate then the proper ratio of ethyl acetate and
methanol) to yield compound 2a,b.
4.4.1.
1-(4-((6,7-dihydroxy-2-methylquinolin-3-yl)oxy)phenyl)-3-(4-
(trifluoromethyl)phenyl)urea (2a)
Yield: 32%; m.p.: 243-4 ºC; 1H NMR (CD3OD, 400 MHz) δ 7.37-7.32 (m, 5H), 7.30-
7.27 (m, 3H), 7.22-7.13 (m, 3H), 6.89-6.82 (m, 4H), 2.48 (s, 3H, CH3); LC-MS m/z:
470.1 (M+ + 1); elemental analysis: calculated C:61.41%, H:3.87%, N:8.95%; found:
C:61.29%, H:4.10%, N:8.90%.
4.4.2.
1-(3,4-Dichlorophenyl)-3-(4-(6,7-dihydroxy-2-methylquinolin-3-
yloxy)phenyl)urea (2b)
13

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Yield: 25%; m.p.: 254-6 ºC; 1H NMR (CD3OD, 300 MHz) δ 7.71 (s, 1H), 7.37 (s, 1H),
7.34 (d, J = 5.0 Hz, 1H, Ar-H), 7.29 (d, J = 7.6 Hz, 2H, Ar-H), 7.21 (s, 1H), 7.17 (s,
1H), 7.15 (s, 1H), 6.90-6.85 (m, 4H, Ar-H), 2.48 (s, 3H, CH3); 13C NMR (CD3OD, 75
MHz) δ 162.3 (C=O), 153.7, 153.1, 151.5, 150.4, 149.8, 148.6, 140.3, 138.1, 133.2,
132.5, 130.4, 126.5, 123.6, 122.7, 121.3, 121.1, 119.1, 106.4, 104.5 (Ar-C), 19.8
(CH3); LC-MS m/z: 470.0 (M+ + 1); elemental analysis: calculated C: 58.74%, H:
3.64%, N: 8.93%; found: C: 59.01%, H: 3.54%, N: 8.65%.
4.5. Antiproliferative screening against the NCI-58 cancer cell line panel
Screening against the cancer cell lines was carried out at the National Cancer
Institute (NCI), Bethesda, Maryland, USA, applying the standard protocol of the NCI
[49].
4.6. Kinase screening
Reaction Biology Corp. Kinase HotSpotSM service [50] was used for screening
of compounds 1d-g. Assay protocol: as reported on Reaction Biology Corp. website
[51] using 1 µM concentration of ATP.
4.7. Molecular docking
Molecular docking study was performed employing the program Autodock
Vina [52]. An X-ray crystal structure for the C-RAF (PDB ID: 3OMV) was obtained
from the RCSB protein data bank (http://www.rcsb.org). The complexed inhibitor
(coded as SM5) was extracted from the initial X-ray structure followed by removal of
water molecules. Polar hydrogens and Gastieger charges were added and the
corresponding charge files were generated using the MGL Tools. The compounds
under study were drawn using the software ChemDraw Ultra 8.0 (Cambridge Soft
Corporation, USA), and were optimised for energy and geometry using MMFF94
force field. All the compounds were treated employing the preparation procedure
mentioned earlier. Later, a grid box of the size (22 Å3) was established to cover the
kinase site of the C-RAF, with a spacing of 1.0 Å between the grid points. The box
was centered toward the coordinates of (28.6×39.17×39.09). The exhaustiveness and
the number of poses were set to 12 and 10, respectively. The 3D-best docked poses
were visualized using PyMOL molecular viewer (Schrödinger Inc., New York, NY,
14

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USA), and the 2D-interaction maps were illustrated using the LigPlot+ graphical
system [53].
Acknowledgments
This work was supported by Korea Institute of Science and Technology (KIST), KIST
Project (2E24680). We are grateful to the National Cancer Institute (NCI), Bethesda,
Maryland, USA, for testing the antiproliferative activity of the target compounds
against 58 cancer cell lines of nine different cancer types.
Appendix A. Supplementary data
Supplementary data related to this article containing charts of the NCI-58 cell line
screening results, and IR, NMR, and mass spectra can be found at
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Illustration captions:
Table 1. Structures of the target compounds 1a-i and 2a,b.
Table 2. Mean IC50 values (µM) of the tested compounds over in vitro subpanel
cancer cell lines.
Table 3. IC50 and TGI values in µM of compounds 1e, 1g, and sorafenib over the NCI
cancer cell line panel.
Table 4. Inhibitory effects of the target compounds 1d-g against C-RAF kinase.
Table 5. Kinase inhibitory effects of compound 1e.
Figure 1. Structures of sorafenib, regorafenib, the diarylamide lead compound, and
the target compounds 1a-i and 2a,b.
Figure 2. Mean inhibition percentages expressed by the target compounds tested
against the NCI-58 cancer cell line panel at a single-dose of 10 µM. The mean %
inhibition values were calculated by subtracting the growth percentages from 100.
Figure 3. % Inhibition produced by a single-dose concentration of 10 µM of each of
compound 1d (Fig. 3a), compound 1e (Fig. 3b), and compound 1g (Fig. 3c) against all
cell lines of the NCI cancer cell line panel of nine different cancer types.
Figure 4. Best docked pose for the lead quinoline-based diarylamide derivative within
the active site of C-RAF kinase. Hydrogen bond interactions are shown as green
dashed lines (left) and its corresponding detailed interactions map (right).
Figure 5. Best docked pose of compound 1g within the active site of C-RAF kinase.
Hydrogen bond interactions are shown as green dashed lines (left) and its
corresponding detailed interactions map (right).
Figure 6. Best docked pose of compound 1e within the active site of C-RAF kinase.
Hydrogen bond interactions are shown as green dashed lines (left) and its
corresponding detailed interactions map (right).
Scheme 1. Reagents and conditions: (a) H2, Pd/C, rt, 5 min, 99%; (b) K2CO3, acetone,
reflux, 5 h, 90% (7a), 92% (7b); (c) piperidine, ethanol, reflux, 24 h, 85% (8a), 75%
(8b); (d) H2, Pd/C, methanol, rt, 4 h, 90% (9a), 70% (9b); (e) ArNCO, K2CO3, THF,
rt, overnight, 40~78%; (f) BBr3, CH2Cl2, -78 0C, 1 h; rt, 4 h. 32% (2a), 25% (2b).
19

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Table 1. Structures of the target compounds 1a-i and 2a,b.
H
H
N
N
O
O
N
Ar
R
R
O
X
Compound
R
X
Ar
No.
CH3
CH3
O
O
Ph
1a
1b
1c
CH3
CH3
O
O
1d
1e
1f
CH3
CH3
O
O
CH3
CH3
CH3
O
S
S
1g
1h
1i
H
H
O
O
2a
2b

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Table 2. Mean IC50 values (µM) of the tested compounds over in vitro subpanel
cancer cell linesa
No. of
cell line
in each
subpanel
6
Compound No.
Cancer
Subpanel
Lead
1d
1e
1f
1g
2b
diarylamide
compoundc
Leukemia
NSCLCb
Colon
1.41
1.76
1.82
1.85
1.57
2.56
1.74
2.09
1.39
0.98
1.13
1.16
1.31
1.09
1.46
1.08
1.01
0.93
2.70
3.21
3.42
4.03
3.01
4.92
3.16
2.90
2.80
0.99
1.19
1.28
1.17
1.45
1.25
1.28
1.40
1.43
6.70
8.09
7.29
8.85
7.28
9.77
10.99
5.81
8.35
3.04
13.10
16.36
29.16
27.86
18.96
2.53
9
7
6
9
7
8
2
5
CNS
Melanoma
Ovarian
Renal
Prostate
Breast
3.22
3.77
a The mean IC50 values were calculated by dividing the summation of IC50 values of
the compound over cell lines of the same cancer type by the number of cell lines in
the subpanel.
b Non-Small Cell Lung Cancer.
c The structure of the lead diarylamide compound is shown in Figure 1.

ACCEPTED MANUSCRIPT
Table 3. IC50 and TGI values in µM of compounds 1e, 1g, and sorafenib over the NCI
cancer cell line panel
Comp. 1e
Comp. 1g
Sorafenib
Cancer
Type
Cell Line
a
a
a
IC50
TGIb
3.09
3.12
4.81
5.49
1.65
2.65
4.16
3.75
5.32
2.05
5.01
3.79
6.28
3.57
2.65
1.31
5.48
3.84
4.86
3.98
3.85
4.57
6.34
2.91
8.39
5.31
4.90
3.78
IC50
TGIb
3.47
4.37
2.53
4.04
5.51
5.97
4.75
2.98
2.88
3.90
3.91
3.55
2.57
2.72
2.95
3.58
3.15
1.92
2.04
3.46
2.46
5.71
3.68
1.94
3.21
2.16
5.05
2.47
IC50
TGIb
5.01
100
1.01
1.13
1.11
1.14
0.64
0.83
1.00
1.01
1.30
0.68
1.27
0.99
2.05
1.07
0.77
0.66
1.77
0.99
1.15
1.20
1.07
1.25
1.55
0.90
1.96
1.41
1.03
1.03
0.64
1.58
0.57
1.13
0.41
1.62
1.42
1.00
1.35
1.12
1.80
0.64
1.16
0.96
1.25
1.79
1.53
0.59
0.67
1.52
0.92
1.94
1.62
0.48
1.45
0.65
1.73
1.08
2.00
1.58
3.16
3.16
1.58
3.16
3.16
2.51
2.00
1.58
2.00
2.00
2.51
2.51
2.00
2.00
3.16
1.58
2.51
2.00
1.58
2.51
2.51
1.58
1.58
3.16
3.16
2.00
CCRF-CEM
HL-60(TB)
K-562
NTc
Leukemia
100
MOLT-4
RPMI-8226
SR
3.16
63.10
7.94
19.95
3.16
5.01
3.16
5.01
5.01
5.01
5.01
3.16
NTc
2.51
3.16
5.01
2.51
5.01
3.98
6.31
2.51
3.98
3.98
3.16
A549/ATCC
EKVX
HOP-62
HOP-92
NSCLC
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
Colon
Cancer
KM12
SW-620
SF-268
SF-295
CNS
SF-539
Cancer
SNB-19
SNB-75
U251

ACCEPTED MANUSCRIPT
1.22
1.33
1.25
1.03
4.16
5.71
5.25
4.71
1.44
1.10
1.00
1.80
2.96
3.46
2.88
4.28
1.58
2.00
2.00
1.58
2.51
39.81
3.16
LOX IMVI
MALME-
3M
M14
MDA-MB-
435
5.01
Melanoma
0.80
1.41
0.79
0.71
1.24
2.34
1.16
1.37
1.93
1.04
1.94
5.45
2.17
2.36
6.01
9.83
4.45
6.10
6.22
4.55
1.82
1.19
1.62
2.48
0.56
0.51
0.49
1.22
1.93
1.63
4.24
3.09
3.28
5.49
2.09
2.30
1.70
4.44
4.14
4.22
2.00
2.51
1.58
2.00
1.58
2.51
3.16
3.16
3.16
3.16
6.31
2.51
2.51
5.01
2.51
10.00
3.98
25.12
6.31
12.59
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
Ovarian
Cancer
NCI/ADR-
RES
1.06
4.91
1.27
3.44
2.51
7.94
1.35
1.17
0.75
1.07
0.78
1.33
1.31
1.32
0.92
0.78
1.24
1.08
5.79
4.97
3.32
4.00
2.54
4.21
6.12
6.19
2.38
2.96
5.11
3.88
1.71
1.61
1.60
0.39
1.41
2.06
0.59
2.32
0.27
0.42
2.38
0.54
3.47
3.09
4.29
1.69
5.10
4.78
2.09
9.18
1.65
2.48
9.36
2.36
2.51
3.16
2.51
2.51
3.16
3.16
2.51
3.98
2.51
2.00
3.16
2.51
5.01
5.01
3.16
10.00
12.59
5.01
3.16
10.00
6.31
5.01
7.94
7.94
SK-OV-3
786-0
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
Renal
Cancer
UO-31
PC-3
Prostate
Cancer
DU-145
MCF7
MDA-MB-
231/ATCC
0.68
1.84
1.08
2.49
1.26
3.16
Breast
0.72
0.98
1.21
2.31
4.59
7.90
2.65
1.75
1.48
6.94
3.76
5.67
2.51
3.16
1.58
3.98
3.98
5.01
HS 578T
BT-549
T-47D
Cancer

ACCEPTED MANUSCRIPT
NTc
NTc
1.08
3.96
2.00
5.01
MDA-MB-
468
a IC50 is the concentration producing 50% inhibition.
b TGI is the concentration producing 100% inhibition.
c NT means that the compound was not tested by the NCI against this cell line.

ACCEPTED MANUSCRIPT
Table 4. Inhibitory effects of the target compounds 1d-g and the lead diarylamide
compound against C-RAF kinase
%Inhibition at 10
Compound No.
IC50 (µM)
µM concentration
99.24%
0.15
0.10
0.22
0.37
1d
99.67%
1e
97.81%
1f
1g
92.37%
Lead diarylamide
compounda
76.65%
NTb
a Its structure is shown in Figure 1. And its inhibition percentage value has been
reported in reference [7].
b Not tested.

ACCEPTED MANUSCRIPT
Table 5. Kinase inhibitory effects of compound 1e
%Inhibition at 10 µM
concentration
-6.78%
Kinase
IC50 (µM)
ABL1
AKT1
-
-
0.06%
B-RAF (wild-
type)
74.25%
1.33
C-RAF
99.67%a
-0.30%
1.04%
0.10a
EGFR
-
ERK2/MAPK1
FLT3
-
33.49%
-7.21%
0.83%
-
HER2/ERBB2
KDR/VEGFR2
MEK1
-
-
-.25.75%
56.42%
29.48%
88.90%
-
P38α/MAPK14
PDGFRb
-
-
V600E-B-RAF
a Datum taken from Table 4.
0.26

ACCEPTED MANUSCRIPT
Table 6. Binding free energy in kcal/mol for the docked compounds against C-RAF kinase
active site.
Compound
E (kcal/mol)
Diarylamide lead
compound
1e
-10.5
-9.3
-10.5
1g

ACCEPTED MANUSCRIPT
Figure 1. Structures of sorafenib, regorafenib, the diarylamide lead compound, and the target
compounds 1a-i and 2a,b.