Synthesis, in vitro antiproliferative activity, and kinase inhibitory effects of pyrazole-containing diarylureas and diarylamides

Article abstract of DOI:10.1016/j.ejmech.2018.07.008

Twenty pyrazole-containing diarylureas and diarylamides were designed and synthesized. They were tested for in vitro antiproliferative activity over a 58-cancer cell line panel at the NCI, USA. The diarylurea derivatives 1b-e and 1g exerted the strongest

Full text of DOI:10.1016/j.ejmech.2018.07.008

Accepted Manuscript
Synthesis, in vitro antiproliferative activity, and kinase inhibitory effects of pyrazole-
containing diarylureas and diarylamides
Mohammed I. El-Gamal, Byung-Jun Park, Chang-Hyun Oh
PII:
S0223-5234(18)30564-6
10.1016/j.ejmech.2018.07.008
EJMECH 10543
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 March 2018
Revised Date: 23 June 2018
Accepted Date: 4 July 2018
Please cite this article as: M.I. El-Gamal, B.-J. Park, C.-H. Oh, Synthesis, in vitro antiproliferative activity,
and kinase inhibitory effects of pyrazole-containing diarylureas and diarylamides, European Journal of
Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.07.008.
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ACCEPTED MANUSCRIPT
Cl
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HN
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HN Ar
Ten urea target compounds
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Biological testing
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Cl
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CF3
CF3
HN
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Compound 1e (the most promising hit)
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Ten amide target compounds

ACCEPTED MANUSCRIPT
Synthesis, in vitro antiproliferative activity, and kinase inhibitory
effects of pyrazole-containing diarylureas and diarylamides
Mohammed I. El-Gamal1,2,3, Byung-Jun Park4, 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, Seou
l 130-650, Republic of Korea.
5
Department of Biomolecular Science, University of Science and Technology, 113 Gwahangno,
Yuseong-gu, Daejeon 305-333, Republic of Korea.
*
Corresponding author.
E-mail addresses: choh@kist.re.kr; Phone No.: +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].
Abstract
Twenty pyrazole-containing diarylureas and diarylamides were designed and
synthesized. They were tested for in vitro antiproliferative activity over a 58-cancer
cell line panel at the NCI, USA. The diarylurea derivatives 1b-e and 1g exerted the
strongest antiproliferative activity. Among them, compound 1e possessing 3,5-
bis(trifluoromethyl)phenyl terminal ring and 3`-methoxy-5`-chlorophenyl ring
attached to the central pyrazole ring was the most potent. Its IC50 values were in sub-
micromolar range against most of the tested cell lines. It showed superior potency
than sorafenib, a reference diarylurea drug, over all the tested cell lines. It was also
extremely selective towards cancer cells than non-cancerous cells (IC50 against RAW
264.7 macrophages was higher than 100 µM). At molecular level, compound 1e
selectively inhibited V600E mutated B-RAF kinase (IC50 = 0.39 µM). It also
stimulated caspase 3/7 enzymes in RPMI-8226 leukemia cells (2.79 fold increase at
10 µM concentration, EC50 = 1.52 µM). So compound 1e may kill cancer cells
through induction of apoptosis. This promising candidate can be considered further
for development of new efficient anticancer agents.
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Keywords: Amide; Antiproliferative; Caspase; Kinase, Pyrazole; Urea.
1. Introduction
Cancer is one of the major issues that requires every research efforts in order
to develop more efficient and safer drug candidates. It comes at the second rank after
cardiovascular diseases as a leading cause of death worldwide. The World Health
Organization (WHO) expects significant increase of the number of newly discovered
cancer cases to exceed 15 million new cases worldwide every year [1,2]. Kinases are
among the most interesting molecular targets for anticancer agents. They are over-
expressed in cancer cells. Kinase inhibition deprives the tumor cell from very
essential growth factors, and hence leads to cancer cell death.
Numerous pyrazole derivatives have been reported as anticancer agents [3-13].
Moreover, several diarylurea and diarylamide derivatives have been highlighted with
promising anticancer activity [6-8, 10, 14-30]. Sorafenib and imatinib are examples of
clinically-used diarylureas and diarylamides, respectively (Figure 1). Sorafenib has
been approved by the U.S. Food and Drug Administration (FDA) for treatment of
advanced renal cancer. It has also been under investigation in clinical trials against
other types of cancer such as hepatocellular carcinoma (HCC), Hodgkin’s lymphoma,
metastatic colorectal, esophageal/gastroesophageal, brain, glioblastoma, ovarian,
leukemia, metastatic breast, pancreatic, advanced gastric, thyroid, non-small cell lung
cancer (NSCLC), prostate, bladder, skin/ocular melanoma, and neuroendocrine
cancers [31,32]. Imatinib is used for treatment of chronic myeloid leukemia (CML)
with minimal adverse effects [33]. Imatinib has been evaluated in clinical trials for
treatment of thyroid cancer, breast cancer, gastrointestinal stromal tumors (GIST),
ovarian cancer, meningioma, and non-small cell lung cancer (NSCLC) in combination
with other drugs [34].
In this study, the target molecules were designed as hybrids of pyrazole and
diarylurea/diarylamide moieties. Twelve of them were selected for in vitro
antiproliferative screening over a panel of 58 human cancer cell lines of nine different
cancer types at the National Cancer Institute (NCI, Bethesda, Maryland, USA). The
most potent compound was further tested against a panel of kinases, and tested for
ability to induce apoptosis in cancer cells. The results are presented in details in the
next sections.
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[Figure 1]
2. Results and discussion
2.1. Chemistry
Scheme 1 shows the synthetic pathway utilized to prepare the target compounds
1a-j and 2a-j. 3,5-Dichlorobenzoic acid (3) was heated with sodium methoxide in
hexamethylphosphoric triamide (HMPA) to obtain 3-chloro-5-methoxybenzoic acid
(4). Treatment of the acid 4 with methyl alcohol in presence of acetyl chloride gave
the corresponding methyl ester 5 [35]. Compound 5 was treated with lithium
bis(trimethylsilyl)amide (LiHMDS) and 4-picoline to produce the ketone 6. The
product 6 was reacted with dimethylformamide dimethylacetal (DMF-DMA), and the
intermediate was treated with hydrazine to produce the 3,4-diarylpyrazole
intermediate 7 [3, 17, 36, 37]. Compound 7 was heated with 1-iodo-3-nitrobenzene
using cuprous iodide, potassium carbonate, and L-proline as catalysts to get the N-(m-
nitrophenyl)pyrazole intermediate 8, which was subsequently reduced to amino using
hydrogen gas in presence of palladium over carbon. Treatment of the aniline
derivative 9 with five different aryl isocyanate reagents yielded the corresponding
diarylurea derivatives 1a-e, while heating compound 9 with five different aryl
carboxylic acids in presence of 1-hydroxybenzotriazole (HOBt), 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDCI), and triethylamine (TEA) afforded the
corresponding diarylamide derivatives 2a-e. BBr3 was used further to demethylate the
methoxy compounds 1a-e and 2a-e to the corresponding hydroxyl derivatives 1f-j and
2f-j [3-12, 37]. Table 1 represents the exact structure of each target molecule and the
yield percentages.
[Scheme 1 & Table 1]
2.2.
Biological screening
2.2.1. In vitro antiproliferative activity
The NCI (Bethesda, Maryland, USA) selected twelve out of the twenty target
compounds for testing their in vitro antiproliferative activity against a panel 58 cancer
cell lines taken from 9 cancer types. They were initially tested at a single-dose
concentration of 10 µM to find the percentage of growth inhibition effect on the cell
lines. The mean inhibition percentages of the tested compounds over the NCI-58
panel are depicted in Figure 2.
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The results indicated that the urea derivatives 1b, 1c, and 1e were more active
than the corresponding amide analogues 2b, 2c, and 2e. This can be attributed to one
or both of the following effects; the terminal NH of the urea linker might contribute to
stronger affinity at the receptor site through additional hydrogen bond formation, and
the longer linker may allow better molecular fitting at the receptor site. Moreover,
three hydroxyl compounds (1g, 2i, and 2j) exerted better activity than the
corresponding methoxy analogues (1b, 2d, and 2e), but the methoxy compound 1d
showed higher activity compared to its hydroxyl analogue 1i. Upon investigating the
terminal ring effect on the activity, it was found that compounds 1d, 1e and 1g
possessing 4`-chloro-3`-(trifluoromethyl)phenyl, 3`,5`-bis(trifluoromethyl)phenyl, and
3`,4`-dichlorophenyl, respectively, were the most active. These terminal rings
carrying hydrophobic, bulky, and electron-withdrawing substituents may affect the
activity through stronger affinity at the receptor site, enhancing the ability to penetrate
the cancer cells, or through electronic effect. One or more of these effects can
rationalize the stronger activity of those derivatives. Compound 2i possessing 4`-
morpholino-3`-(trifluoromethyl)phenyl terminal ring was the most active among the
diarylamide derivatives. This can be rationalized that the steric and/or electronic
effect(s) of these substituents in addition to their ability to form additional hydrogen
bond(s) might enhance the potency.
[Figure 2]
The activities of each of the most active compounds 1d, 1e, and 1g against
each individual cell line of the tested panel are illustrated in Figure 3. The three
compounds showed broad-spectrum activity against all the nine tested cancer types.
At 10 µM, compounds 1d, 1e, and 1g demonstrated lethal effects (≥ 100% growth
inhibition) over 22, 30, and 35 cell lines, respectively.
[Figure 3]
Out of the twelve tested compounds at one-dose concentration, the most active
ten molecules 1b-e, 1g, 1i, 2c, 2d, 2i, and 2j were selected for further testing in a 5-
dose assay in order to investigate their efficacy and potency. The mean IC50 values
over the nine tested subpanels are summarized in Table 2. As discussed above, the
urea derivatives are significantly more potent than the amides. All the urea analogues
exerted sub-micromolar to one-digit micromolar mean IC50 values. Compound 1e
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with methoxy, urea, and bis(trifluoromethyl)phenyl moieties showed the strongest
potency. All its mean IC50 values were in the sub-micromolar range. Its dose-response
curves over all the tested cell lines of the nine different cancer types are shown in
Figure 4. It showed very promising antiproliferative activity to lethal effect and
broad-spectrum anticancer activity.
Upon comparison of the activity of compound 1e with its positional isomer,
4`-chloro-3`-methoxyphenyl analogue [8], it is found that compound 1e was more
potent against all the nine cancer subpanels. Moreover, compound 1e was more potent
against most of the individual cell lines. So the presence of chloro and methoxy
groups at positions 3` and 5` might be more optimal for stronger affinity at the
receptor site.
[Table 2 & Figure 4]
For more elaboration on the antiproliferative activity of the target compounds,
the IC50 values of the tested compounds over the most sensitive cell line of each
cancer type are depicted in Table 3. The results were compared with those of
sorafenib, diarylurea multikinase anticancer drug, as a reference standard. The urea
analogues are again much more potent than the amides, and showed superior
potencies than sorafenib. The strongest potency was given by compound 1e with
submicromolar IC50 values over the nine cell lines.
[Table 3]
The IC50 and TGI values of compound 1e and sorafenib are summarized in
Table 4. Compound 1e was more potent than sorafenib against all the 58 tested cell
lines. Most of its IC50 values were in the sub-micromolar scale (on 52 cell lines),
while sorafenib did not show any sub-micromolar IC50 value. In addition, the TGI
values of compound 1e were less than those of sorafenib against most of the 58 tested
cell lines. These findings indicate superior potency and efficacy of compound 1e over
the tested cell line panel. It is noteworthy that compound 1e is super-selective towards
cancer cells than non-cancerous cells. Its IC50 value against RAW 264.7 macrophages
was higher than 100 µM [37].
[Table 4]
2.2.2. Kinase profiling
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Due to the structural similarity between compound 1e and sorafenib (both are
diarylurea derivatives), and due to our previous reports of pyrazole derivatives as
kinase inhibitors [6, 9-13], we decided to test it over a panel of 27 kinases as a part of
our efforts to investigate its molecular mechanism of action. The inhibition percentage
values at a single dose of 10 µM are summarized in Table 5. The compound is
interestingly selective against V600E mutated B-RAF kinase with 94.72% inhibition.
Its IC50 value is 0.39 µM. So inhibition of V600E-B-RAF kinase could be, at least in
part, a mechanism of antiproliferative activity of compound 1e at molecular level.
Since V600E-B-RAF is over-expressed and is considered as an essential growth
factor in several types of cancer such as melanoma, colorectal cancer, lung cancer,
brain tumor, and many other types of cancer [10, 38], this can account for the broad-
spectrum antiproliferative activity of compound 1e.
[Table 5]
2.2.3. Caspase 3/7 assay
Compound 1e was further tested for apoptotic activity in RPMI-8226 leukemia
cells. We measured the caspase 3/7 enzyme levels in the cells after treatment with 10
different concentrations of the tested compound. The dose-response curve is
illustrated in Figure 5. It showed promising induction of caspase 3/7 enzymes (2.79-
fold increase at 10 µM concentration, EC50 = 1.52 µM). So caspase activity
stimulation and induction of apoptosis could be another mechanism of the
antiproliferative activity of compound 1e.
[Figure 5]
3. Conclusion
The present article reports the design, synthesis, and biological evaluation at
cellular and molecular levels of a series of triarylpyrazoles containing urea or amide
linker. We found that the urea linker and 3`-chloro-5`-methoxyphenyl moieties are
optimum for strong potency of this series of compounds. The results of this study led
to discovery of a very promising compound, 1e. It showed superior selectivity
towards cancer over non-cancer cells. It was much more potent than sorafenib against
the 58 tested cancer cell lines. Most of its IC50 values over the tested cancer cell lines
were in sub-micromolar scale, while all the results of sorafenib were in the
micromolar range. Furthermore, it is potent and selective against V600E-B-RAF
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kinase (IC50 = 0.39 µM), and induced apoptosis in the tested leukemia cells. This
compound is a promising candidate for future design and development of selective
kinase inhibitors as potential anticancer agents. Further structural modification and
optimization of this series of compounds is currently in progress.
4. Experimental
4.1. General
The target compounds 1a-j and 2a-j were purified by flash column
chromatography using silica gel (0.040-0.063 mm, 230-400 mesh) and technical grade
solvents. 1H NMR and 13C NMR analyses were conducted on a Bruker Avance 400 or
300 spectrometer using tetramethylsilane as an internal standard. Melting points were
measured on a Stuart melting point apparatus (Staffordshire, UK), 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. Elemental analysis was done using
FlashSmart elemental analyzer (Thermo Fisher Scientific, Waltham, MA, USA). 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 the amine intermediate 9
It was achieved through the synthetic pathway reported in the literature [37].
4.3. Synthesis of the diarylurea derivatives 1a-e
To a solution of compound 9 (50 mg, 0.1 mmol) in anhydrous THF (1 mL), a solution
of the appropriate aryl isocyanate (0.1 mmol) in THF (1 mL) was added dropwise at
room temperature under nitrogen atmosphere. The reaction mixture was stirred at
room temperature overnight. The solvent was evaporated under reduced pressure, and
the residue was purified by column chromatography (silica gel, hexane-ethyl acetate
mobile phase system) to yield the title compounds.
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Compound 1a: mp: 226-9 ºC; 1H NMR (400 MHz, DMSO-d6) δ 8.99 (s, 1H), 8.96 (s,
1H), 8.73 (s, 1H), 8.57 (d, 2H, J = 6.0 Hz), 8.17 (s, 1H), 7.53 (d, 1H, J = 7.6 Hz),
7.49-7.46 (m, 3H), 7.43 (d, 1H, J = 4.8 Hz), 7.38 (dd, 2H, J = 4.8 Hz, 1.2 Hz), 7.30 (t,
2H, J = 7.8 Hz), 7.11 (s, 1H), 7.11 (t, 1H, J = 2.0 Hz), 6.99 (d, 1H, J = 7.2 Hz), 6.97-
13
6.96 (m, 1H), 3.75 (s, 3H); C NMR (100 MHz, DMSO-d6) δ 160.6, 153.0, 150.4,
148.7, 141.5, 140.2, 139.9, 135.7, 134.5, 130.4, 129.7, 129.3, 123.2, 122.5, 120.6,
120.1, 118.9, 117.1, 114.4, 113.5, 112.3, 109.1, 56.1; LC-MS: 496.0 (M+ + 1);
elemental analysis: calculated C:67.81%, H:4.47%, N:14.12%; found: C:67.53%,
H:4.49%, N:14.18%..
Compound 1b: mp: 210-3 ºC; 1H NMR (400 MHz, DMSO-d6) δ 9.14 (s, 1H), 9.06 (s,
1H), 8.95 (s, 1H), 8.56 (d, 2H, J = 6.0 Hz), 8.16 (s, 1H), 7.90 (d, 1H, J = 2.4 Hz),
7.57-7.51 (m, 2H), 7.48-7.40 (m, 2H), 7.38-7.35 (m, 3H), 7.13-7.10 (m, 2H), 6.95 (dd,
1H, J = 2.0 Hz, 1.2 Hz), 3.74 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 160.6, 152.8,
150.4, 148.7, 141.1, 140.3, 140.2, 139.9, 135.6, 134.5, 131.5, 131.0, 130.5, 129.7,
123.8, 123.2, 120.6, 120.2, 120.0, 119.0, 117.4, 114.4, 113.6, 112.7, 109.4, 56.1; LC-
MS: 564.9 (M+ + 2), 563.9 (M+ + 1); elemental analysis: calculated C:59.54%,
H:3.57%, N:12.40%; found: C:59.70%, H:3.34%, N:12.33%.
1
Compound 1c: mp: 240-3 ºC; H NMR (400 MHz, DMSO-d6) δ 9.16 (brs, 2H), 8.97
(s, 1H), 8.57 (d, 2H, J = 6.0 Hz), 8.17 (s, 1H), 8.03 (s, 1H), 7.62 (d, 1H, J = 8.0 Hz),
7.57-7.47 (m, 3H), 7.45 (s, 1H), 7.38 (d, 2H, J = 5.6 Hz), 7.32 (d, 1H, J = 7.6 Hz),
7.14 (s, 1H), 7.11 (t, 1H, J = 1.8 Hz), 6.96 (s, 1H), 3.75 (s, 3H); 13C NMR (100 MHz,
DMSO-d6) δ 160.6, 153.0, 150.4, 148.7, 141.1, 140.9, 140.2, 139.9, 135.7, 134.5,
130.5, 130.4, 129.7, 123.2, 122.5, 120.6, 120.1, 117.4, 114.8, 114.4, 113.5, 112.7,
109.4, 56.1; LC-MS: 564.0 (M+ + 1); elemental analysis: calculated C:61.76%,
H:3.75%, N:12.42%; found: C:61.96%, H:3.62%, N:12.29%.
1
Compound 1d: mp: 213-5 ºC; H NMR (400 MHz, DMSO-d6) δ 9.68 (brs, 2H), 8.95
(s, 1H), 8.56 (d, 2H, J = 5.6 Hz), 8.18 (s, 1H), 8.13 (d, 1H, J = 2.4 Hz), 7.71 (dd, 1H,
J = 8.4 Hz, 1.6 Hz), 7.61 (d, 1H, J = 8.8 Hz), 7.55 (d, 1H, J = 4.0 Hz), 7.45 (d, 2H, J =
13
4.4 Hz), 7.38 (d, 2H, J = 6.0 Hz), 7.13-7.11 (m, 2H), 6.96 (s, 1H), 3.75 (s, 3H); C
NMR (100 MHz, DMSO-d6) δ 160.6, 153.1, 150.4, 148.7, 141.2, 140.2, 139.9, 135.7,
134.5, 132.4, 130.4, 129.7, 123.7, 123.2, 120.6, 120.1, 117.5, 117.4, 114.4, 113.5,
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112.7, 109.5, 56.1; LC-MS: 598.8 (M+ + 2), 597.8 (M+ + 1); elemental analysis:
calculated C:58.21%, H:3.37%, N:11.70%; found: C:58.24%, H:3.45%, N:11.45%.
Compound 1e: mp: 221-4 ºC; 1H NMR (400 MHz, DMSO-d6) δ 10.53 (brs, 2H), 8.90
(s, 1H), 8.56 (d, 2H, J = 6.0 Hz), 8.22-8.20 (m, 3H), 7.58 (s, 1H), 7.51 (d, 2H, J = 6.4
Hz), 7.43 (d, 1H, J = 8.0 Hz), 7.38 (d, 2H, J = 5.6 Hz), 7.13-7.10 (m, 2H), 6.96 (s,
1H), 3.74 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 160.6, 153.4, 150.3, 148.7,
141.4, 140.2, 139.8, 135.7, 134.5, 131.2, 130.9, 130.2, 129.6, 125.2, 123.1, 122.5,
120.6, 120.1, 118.3, 117.6, 114.4, 113.5, 112.7, 109.6, 56.1; LC-MS: 632.9 (M+ + 2),
631.9 (M+ + 1); elemental analysis: calculated C:57.02%, H:3.19%, N:11.08%; found:
C:56.94%, H:3.08%, N:11.22%.
4.4. Synthesis of the diarylamide derivatives 2a-e
A mixture of the amine compound 9 (50 mg, 0.1 mmol), appropriate benzoic acid
derivative (0.2 mmol), HOBt (36 mg, 0.3 mmol), and EDCI (38 mg, 0.2 mmol) in
DMF (1.0 mL) was cooled to 0 °C under nitrogen atmosphere. Triethylamine (0.03
mL, 0.2 mmol) was added to the reaction mixture at the same temperature. The
mixture was then stirred at 80 °C for 12 h. The reaction mixture was cooled and then
partitioned between saturated saline (10 mL) and ethyl acetate (10 mL). The organic
layer was separated, and the aqueous layer was extracted again with ethyl acetate (3 x
5 mL). The organic extract was washed with saturated saline (3 x 15 mL), and dried
using anhydrous sodium sulfate. The organic solvent was evaporated, and the crude
residue was purified by column chromatography (silica gel, hexane-ethyl acetate
mobile phase system) to yield the title products.
Compound 2a: mp: 173-5 ºC; 1H NMR (400 MHz, DMSO-d6) δ 10.48 (brs, 1H), 8.97
(s, 1H), 8.55 (dd, 2H, J = 4.4 Hz, 1.6 Hz), 8.45 (t, 1H, J = 2.0 Hz), 7.99 (d, 2H, J =
6.8 Hz), 7.83 (dd, 1H, J = 8.0 Hz, 1.2 Hz), 7.65 (dd, 1H, J = 8.0 Hz, 1.6 Hz), 7.62-
7.58 (m, 1H), 7.55-7.51 (m, 3H), 7.36 (dd, 2H, J = 4.4 Hz, 1.6 Hz), 6.94 (s, 1H), 3.72
13
(s, 3H); C NMR (100 MHz, DMSO-d6) δ 166.2, 160.6, 150.4, 148.8, 140.9, 140.2,
139.7, 135.6, 135.1, 134.5, 132.3, 130.3, 129.7, 128.9, 128.2, 123.1, 120.6, 120.2,
119.1, 114.4, 114.0, 113.6, 111.3, 56.1; LC-MS: 481.1 (M+ + 1); elemental analysis:
calculated C:69.92%, H:4.40%, N:11.65%; found: C:69.75%, H:4.31%, N:11.70%.
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Compound 2b: mp: 242-5 ºC; 1H NMR (400 MHz, DMSO-d6) δ 10.60 (s, 1H), 8.98 (s,
1H), 8.55 (d, 2H, J = 5.6 Hz), 8.40 (s, 1H), 8.25 (s, 1H), 7.96 (dd, 1H, J = 8.4 Hz, 2.0
Hz), 7.82 (d, 2H, J = 8.4 Hz), 7.67 (d, 1H, J = 8.0 Hz), 7.52 (t, 1H, J = 8.2 Hz), 7.36
13
(d, 2H, J = 5.6 Hz), 7.13 (s, 1H), 7.09 (s, 1H), 6.93 (s, 1H), 3.72 (s, 3H); C NMR
(100 MHz, DMSO-d6) δ 163.8, 160.6, 150.4, 140.5, 140.2, 139.7, 135.6, 135.3, 135.1,
131.8, 131.3, 130.4, 130.1, 129.7, 128.6, 123.1, 120.6, 120.2, 119.2, 114.4, 113.6,
111.3, 56.1; LC-MS: 549.0 (M+ + 1); elemental analysis: calculated C:61.16%,
H:3.48%, N:10.19%; found: C:61.05%, H:3.51%, N:10.10%.
Compound 2c: mp: 193-5 ºC; 1H NMR (400 MHz, DMSO-d6) δ 10.71 (brs, 1H), 9.01
(s, 1H), 8.57 (d, 2H, J = 4.8 Hz), 8.43 (s, 1H), 8.35 (s, 1H), 8.31 (d, 1H, J = 7.6 Hz),
7.99 (d, 1H, J = 7.6 Hz), 7.87 (d, 1H, J = 8.6 Hz), 7.81 (t, 1H, J = 8.0 Hz), 7.71 (d, 1H,
J = 8.0 Hz), 7.56 (t, 1H, 8.0 Hz), 7.38 (d, 2H, J = 4.8 Hz), 7.15 (d, 1H, J = 1.2 Hz),
7.11 (s, 1H), 6.96 (s, 1H), 3.74 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 164.7,
160.6, 150.4, 148.8, 140.5, 140.1, 139.8, 135.9, 135.6, 134.5, 132.4, 130.4, 130.3,
129.7, 124.8, 124.7, 123.1, 120.6, 120.2, 119.3, 114.4, 114.3, 113.6, 111.4, 56.1; LC-
MS: 549.1 (M+ + 1); elemental analysis: calculated C:63.45%, H:3.67%, N:10.21%;
found: C:63.41%, H:3.54%, N:10.33%.
Compound 2d: mp: 250-3 ºC; 1H NMR (400 MHz, DMSO-d6) δ 10.58 (brs, 1H), 8.99
(s, 1H), 8.55 (dd, 2H, J = 4.6 Hz, 1.4 Hz), 8.41 (t, 1H, 2.0 Hz), 8.28-8.26 (m, 2H),
7.83 (dd, 1H, J = 8.0 Hz, 1.2 Hz), 7.66 (t, 2H, J = 9.0 Hz), 7.53 (t, 1H, J = 8.2 Hz),
7.37 (dd, 2H, J = 4.4 Hz, 1.6 Hz), 7.13 (t, 1H, J = 1.6 Hz), 7.10 (t, 1H, 2.2 Hz), 6.94
13
(dd, 1H, J = 2.0 Hz, 1.2 Hz), 3.72-3.71 (m, 7H), 2.96 (t, 4H, J = 4.4 Hz); C NMR
(100 MHz, DMSO-d6) δ 164.5, 160.6, 150.4, 150.3, 148.8, 140.7, 140.2, 139.7, 135.6,
134.5, 133.5, 130.7, 130.4, 129.7, 124.3, 123.1, 120.6, 120.2, 114.4, 113.6, 111.3,
66.9, 56.1, 53.6; LC-MS: 634.1 (M+ + 1); elemental analysis: calculated C:62.51%,
H:4.29%, N:11.05%; found: C:62.33%, H:4.18%, N:11.06%.
Compound 2e: mp: 255-7 ºC; 1H NMR (400 MHz, DMSO-d6) δ 10.88 (s, 1H), 9.01 (s,
1H), 8.65 (s, 2H), 8.55 (d, 2H, J = 6.0 Hz), 8.40 (t, 1H, J = 1.8 Hz), 8.38 (s, 1H), 7.87
(d, 1H, J = 8.0 Hz), 7.72 (dd, 1H, J = 8.0 Hz, 1.6 Hz), 7.57 (t, 1H, J = 8.2 Hz), 7.37 (d,
2H, J = 6.0 Hz), 7.14 (s, 1H), 7.10 (t, 1H, J = 2.0 Hz), 6.94 (s, 1H), 3.73 (s, 3H); 13C
NMR (100 MHz, DMSO-d6) δ 163.2, 160.6, 150.4, 148.9, 140.2, 140.1, 139.8, 137.3,
10

ACCEPTED MANUSCRIPT
135.6, 134.5, 131.2, 130.8, 130.5, 129.7, 129.1, 124.9, 123.1, 122.2, 120.6, 120.3,
119.3, 114.5, 114.4, 113.6, 111.5, 56.1; LC-MS: 617.1 (M+ + 1); elemental analysis:
calculated C:58.40%, H:3.10%, N:9.08%; found: C:58.51%, H:3.13%, N:8.98%.
4.5. Synthesis of the demethylated derivatives 1f-j and 2f-j
The methoxy compound 1a-e or 2a-e (0.1 mmol) was dissolved in anhydrous
dichloromethane (1 mL), and boron tribromide (0.08 mL of a 1M solution in
methylene chloride, 1.2 mmol) was added thereto dropwise at -78 °C under nitrogen
atmosphere. The reaction mixture was stirred at the same temperature for 30 min, then
stirred overnight at room temperature. The mixture was quenched with saturated
aqueous sodium carbonate. Ethyl acetate (15 mL) was added, the mixture was stirred,
and the organic layer was separated. The aqueous layer was extracted again with ethyl
acetate (2 x 10 mL). The combined ethyl acetate extracts were washed with saturated
saline solution, and then dried with anhydrous sodium sulfate. The organic solvent
was evaporated under reduced pressure, and the remained crude product was purified
by short column chromatography (silica gel, hexane-ethyl acetate mobile phase
system) to yield the title compounds.
Compound 1f: mp: 233-5 ºC; 1H NMR (400 MHz, DMSO-d6) δ 10.10 (s, 1H), 8.98 (s,
1H), 8.95 (s, 1H), 8.71 (s, 1H), 8.57 (s, 2H), 8.18 (t, 1H, J = 2.0 Hz), 7.54-7.49 (m,
1H), 7.48 (d, 1H, J = 1.2 Hz), 7.46 (d, 1H, J = 0.8 Hz), 7.43 (d, 1H, J = 8.0 Hz), 7.39-
7.37 (m, 3H), 7.30 (t, 2H, J = 8.0 Hz), 6.99-6.98 (m, 2H), 6.87-6.85 (m, 2H); 13C
NMR (100 MHz, DMSO-d6) δ 159.0, 153.0, 150.2, 149.0, 141.5, 140.4, 140.0, 135.6,
134.2, 130.4, 129.7, 129.3, 123.2, 122.5, 120.0, 119.0, 118.9, 117.0, 115.8, 114.5,
112.3, 109.0; LC-MS: 482.0 (M+ + 1); elemental analysis: calculated C:67.29%,
H:4.18%, N:14.53%; found: C:67.10%, H:4.01%, N:14.65%.
Compound 1g: mp: 249-52 ºC; 1H NMR (400 MHz, DMSO-d6) δ 10.11 (s, 1H), 9.18
(s, 1H), 9.10 (s, 1H), 8.94 (s, 1H), 8.56 (dd, 2H, J = 4.4 Hz, 1.6 Hz), 8.18 (t, 1H, J =
2.0 Hz), 7.91 (d, 1H J = 2.4 Hz), 7.56-7.52 (m, 2H), 7.46 (t, 1H, J = 8.0 Hz), 7.40-
13
7.36 (m, 4H), 6.98 (t, 1H, J = 1.6 Hz), 6.87 (dt, 2H, J = 6.8 Hz, 1.9 Hz); C NMR
(100 MHz, DMSO-d6) δ 159.0, 152.8, 150.4, 149.0, 141.1, 140.3, 140.2, 140.0, 135.6,
134.1, 131.5, 131.0, 130.4, 129.7, 123.8, 123.1, 120.0, 119.9, 119.0, 118.9, 117.3,
115.8, 114.5, 112.6, 109.3; LC-MS: 551.6 (M+ + 2), 550.6 (M+ + 1); elemental
11

ACCEPTED MANUSCRIPT
analysis: calculated C:58.87%, H:3.29%, N:12.71%; found: C:58.81%, H:3.33%,
N:12.62%.
Compound 1h: mp: 205-6 ºC; 1H NMR (400 MHz, DMSO-d6) δ 10.10 (brs, 1H), 9.15
(d, 2H, J = 6.8 Hz), 8.95 (s, 1H), 8.57 (d, 2H, J = 5.6 Hz), 8.18 (s, 1H), 8.03 (s, 1H),
7.62 (d, 1H, J = 8.0 Hz), 7.55 (t 1H, J = 6.4 Hz), 7.51-7.40 (m, 3H), 7.38 (d, 2H, J =
5.6 Hz), 7.33 (d, 1H, J = 7.6 Hz), 6.98 (s, 1H), 6.86 (dd, 2H, J = 4.8 Hz, 2.0 Hz); 13C
NMR (100 MHz, DMSO-d6) δ 159.0, 153.0, 150.4, 149.0, 141.1, 140.9, 140.2, 140.0,
135.6, 134.2, 130.5, 130.4, 129.7, 123.1, 122.5, 120.0, 119.0, 118.8, 117.4, 115.8,
114.8, 114.5, 112.6, 109.3; LC-MS: 550.1 (M+ + 1); elemental analysis: calculated
C:61.15%, H:3.48%, N:12.73%; found: C:61.09%, H:3.45%, N:12.80%.
Compound 1i: mp: 225-8 ºC; 1H NMR (400 MHz, DMSO-d6) δ 10.32-10.28 (m, 3H),
8.90 (s, 1H), 8.53 (dd, 2H, J = 4.6 Hz, 1.4 Hz), 8.18-8.15 (m, 2H), 7.74 (d, 1H, J = 8.8
Hz), 7.59 (d, 1H, J = 8.8 Hz), 7.52-7.46 (m, 3H), 7.43 (d, 1H, J = 8.0 Hz), 7.38 (dd,
2H, J = 4.4 Hz, 1.6 Hz), 6.75-6.74 (m, 3H); LC-MS: 584.0 (M+ + 1); elemental
analysis: calculated C:57.55%, H:3.10%, N:11.98%; found: C:57.48%, H:3.02%,
N:12.15%.
Compound 1j: mp: 181-4 ºC; 1H NMR (400 MHz, DMSO-d6) δ 10.09 (brs, 1H), 9.49
(brs, 1H), 9.36 (brs, 1H), 8.96 (s, 1H), 8.57 (d, 2H, J = 4.8 Hz), 8.17 (brs, 2H), 7.67 (s,
1H), 7.58 (d, 1H, J = 7.2 Hz), 7.50-7.44 (m, 2H), 7.38 (d, 2H, J = 5.6 Hz), 6.98 (s,
1H), 6.87-6.84 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 159.0, 152.9, 150.4, 149.0,
142.2, 140.8, 140.2, 140.0, 135.6, 134.2, 131.4, 130.5, 129.7, 123.1, 122.4, 120.0,
119.0, 118.7, 117.8, 115.8, 114.5, 113.0, 109.7; LC-MS: 618.1 (M+ + 1); elemental
analysis: calculated C:56.37%, H:2.94%, N:11.33%; found: C:56.21%, H:2.80%,
N:11.55%.
Compound 2f: mp: ˃265 ºC; 1H NMR (400 MHz, DMSO-d6) δ 10.51 (s, 1H), 10.11 (s,
1H), 8.99 (s, 1H), 8.58 (dd, 2H, J = 4.8 Hz, 1.6 Hz), 8.49 (t, 1H, J = 2.0 Hz), 8.03-
8.01 (m, 2H), 7.85 (dd, 1H, J = 8.2 Hz, 1.0 Hz), 7.68 (dd, 1H, J = 8.2 Hz, 1.4 Hz),
7.64-7.53 (m, 4H), 7.40 (dd, 2H, J = 4.4 Hz, 1.6 Hz), 7.01 (t, 1H, J = 1.6 Hz), 6.88-
6.86 (m, 2H); 13C NMR (75 MHz, DMSO-d6) δ 166.4, 159.0, 149.3, 148.9, 140.8,
139.6, 135.4, 135.0, 134.2, 132.3, 130.4, 130.0, 128.9, 128.1, 123.4, 119.7, 119.3,
12

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119.1, 115.9, 114.6, 114.1, 111.4; LC-MS: 467.1 (M+ + 1); elemental analysis:
calculated C:69.45%, H:4.10%, N:12.00%; found: C:69.61%, H:3.95%, N:11.87%.
Compound 2g: mp: 234-6 ºC; 1H NMR (300 MHz, DMSO-d6) δ 10.62 (s, 1H), 8.98 (s,
1H), 8.56 (dd, 2H, J = 4.5 Hz, 1.6 Hz), 8.43 (d, 1H, J = 1.8 Hz), 8.27 (d, 1H, J = 2.0
Hz), 7.98 (dd, 1H, J = 2.0 Hz, 8.4 Hz), 7.84 (d, 2H, J = 8.4 Hz), 7.69 (d, 1H, J = 7.7
Hz), 7.54 (t, 1H, J = 8.1 Hz), 7.38 (dd, 2H, J = 4.5 Hz, 1.6 Hz), 6.99 (s, 1H), 6.87-
6.84 (m, 2H); 13C NMR (75 MHz, DMSO-d6) δ 163.8, 159.1, 150.4, 149.1, 140.5,
140.2, 139.8, 135.3, 134.1, 131.8, 131.3, 130.4, 130.1, 129.7, 128.6, 123.1, 120.1,
119.1, 118.9, 115.8, 114.6, 111.3; LC-MS: 535.0 (M+ + 1); elemental analysis:
calculated C:60.52%, H:3.20%, N:10.46%; found: C:60.41%, H:3.15%, N:10.57%.
1
Compound 2h: mp: 178-81 ºC; H NMR (300 MHz, DMSO-d6) δ 10.76 (brs, 1H),
8.98 (s, 1H), 8.55-8.53 (m, 2H), 8.43 (d, 1H, J = 1.8 Hz), 8.35-8.30 (m, 2H), 7.98 (1H,
d, J = 6.9 Hz), 7.88-7.77 (m, 2H), 7.69 (d, 1H, J = 7.5 Hz), 7.54 (t, 1H, J = 8.1 Hz),
7.38-7.36 (m, 2H), 6.89-6.80 (m, 3H); LC-MS: 535.0 (M+ + 1); elemental analysis:
calculated C:62.87%, H:3.39%, N:10.47%; found: C:62.95%, H:3.48%, N:10.26%.
Compound 2i: mp: 216-8 ºC; 1H NMR (400 MHz, DMSO-d6) δ 10.61 (s, 1H), 8.84 (s,
1H), 8.49 (dd, 2H, J = 4.6 Hz, 1.4 Hz), 8.34 (s, 1H), 8.24-8.19 (m, 2H), 7.71 (d, 1H, J
= 8.0 Hz), 7.63-7.50 (m, 3H), 7.35 (dd, 2H, J = 4.8 Hz, 1.6 Hz), 6.93 (t, 1H, J = 1.6
Hz), 6.86 (t, 1H, J = 2.0 Hz), 6.80 (t, 1H, J = 1.6 Hz), 3.74 (t, 4H, J = 4.2 Hz), 2.98 (t,
4H, J = 4.2 Hz); 13C NMR (100 MHz, DMSO-d6) δ 165.0, 158.8, 150.0, 149.2, 140.5,
140.2, 139.6, 135.4, 134.3, 133.4, 130.3, 129.5, 124.1, 123.3, 122.9, 120.0, 119.1,
115.9, 114.5, 111.5, 66.8, 53.4; LC-MS: 620.1 (M+ + 1); elemental analysis:
calculated C:61.99%, H:4.06%, N:11.30%; found: C:62.04%, H:4.01%, N:11.20%.
Compound 2j: mp: ˃265 ºC; 1H NMR (300 MHz, DMSO-d6) δ 10.87 (s, 1H), 9.00 (s,
1H), 8.67 (s, 2H), 8.57 (d, 2H, J = 4.8 Hz), 8.41 (d, 2H, J = 10.7 Hz), 7.88 (d, 1H, J =
7.9 Hz), 7.73 (d, 1H, J = 8.0 Hz), 7.57 (t, 1H, J = 8.1 Hz), 7.38 (d, 2H, J = 4.9 Hz),
6.97 (s, 1H), 6.86 (d, 2H, J = 4.9 Hz); 13C NMR (75 MHz, DMSO-d6) δ 163.2, 159.2,
150.4, 149.2, 140.2, 139.8, 137.3, 135.5, 134.1, 131.2, 130.8, 130.4, 129.6, 129.1,
125.4, 123.1, 121.8, 120.1, 119.2, 118.8, 115.9, 114.6, 114.4, 111.4; LC-MS: 603.0
13

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(M+ + 1); elemental analysis: calculated C:57.77%, H:2.84%, N:9.29%; found:
C:57.49%, H:2.74%, N:9.50%.
4.6. Antiproliferative screening
It was conducted at the National Cancer Institute (NCI, Bethesda, Maryland, USA)
following their standard protocol [https://dtp.cancer.gov/discovery_development/nci-
60/methodology.htm].
4.7. Kinase screening
It was carried out at Reaction Biology Corp. (Malvern, PA, USA)
[http://www.reactionbiology.com] following the procedure previously reported in our
published article [39].
4.8. Caspase 3/7 assay
It was conducted at Reaction Biology Corp. (Malvern, PA, USA)
[http://www.reactionbiology.com] following the procedure previously reported in our
published article [13].
Acknowledgment
This work was supported by Korea Institute of Science and Technology (KIST),
KIST Project (E26900).
Supplementary file
1H NMR, 13C NMR, and LC-MS spectra as well as the NCI result charts of the target
compounds can be found at
The authors have declared no conflict of interest
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Illustration captions:
Table 1. Structures of the target compounds and their yield percentages.
Table 2. Mean IC50 values (µM) of the tested compounds against NCI cell line
subpanels.
Table 3. IC50 values (µM) of the tested compounds and sorafenib against the most
sensitive cell line of each subpanel.
Table 4. IC50 and TGI values (µM) of compound 1e and sorafenib over the NCI
cancer cell line panel.
Table 5. Inhibitory effects of compound 1e against a panel of 27 kinases.
Figure 1. Design rationale of the target compounds 1a-j and 2a-j.
Figure 2. Mean inhibition percentage of the twelve tested compounds against the
NCI-58 cancer cell line panel.
Figure 3. %inhibition exerted 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
the NCI-58 cancer cell lines.
Figure 4. Dose-response curves of compound 1e over the NCI-58 cancer cell line
panel of the nine cancer types.
Figure 5. Caspase-3/7 activity in RPMI-8226 leukemia cells after treatment with
different concentrations of compound 1e.
Scheme 1. Reagents, reaction conditions, and yield percentages: a) 1) sodium
methoxide, HMPA, 115-120 °C, 15 h, 2) aq. HCl, 54%; b) acetyl chloride, CH3OH, rt,
15 h, 89%; c) 4-picoline, LiHMDS, THF, rt, overnight, 77%; d) DMF-DMA, rt, 18 h;
e) hydrazine monohydrate, C2H5OH, rt, overnight, 72%; f) 1-iodo-3-nitrobenzene,
K2CO3, CuI, -proline, DMSO, 90 °C, 8 h, 59%; g) H2 (g), 10% Pd/C, THF, rt, 2 h,
L
87%; h) appropriate aryl isocyanate, THF, rt, 12 h, 24~85%; i) BBr3, CH2Cl2, -78 °C,
30 min; rt, 1 h, 24~89% (1f-j) and 28~79% (2f-j); j) appropriate benzoic acid
derivative, HOBt, EDCI, TEA, DMF, 80 °C, 12 h, 28~79%.
18

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Table 1. Structures of the target compounds and their yield percentages.
Compound
R1
R2
Yield%
No.
1a
CH3
CH3
24%
60%
1b
1c
1d
CH3
CH3
62%
85%
CH3
82%
1e
H
H
24%
89%
1f
1g
H
H
62%
79%
1h
1i
H
72%
1j
CH3
CH3
65%
65%
2a
2b
Cl
Cl
CH3
59%
2c
CF3

ACCEPTED MANUSCRIPT
N
O
CH3
CH3
79%
58%
2d
2e
CF3
CF3
CF3
H
H
28%
65%
2f
Cl
2g
Cl
H
H
58%
79%
2h
2i
CF3
N
O
CF3
CF3
H
75%
2j
CF3

ACCEPTED MANUSCRIPT
Table 2. Mean IC50 values (µM) of the tested compounds against NCI cell line subpanelsa
No. of
cell line
in each
subpanel
6
Compound No.
1i
Cancer
Subpanel
1b
1c
1d
1e
1g
2c
2d
2i
2j
Leukemia
NSCLCb
Colon
1.67
2.48
2.46
2.49
2.23
3.05
2.48
2.49
2.58
1.11
1.64
1.35
1.78
1.69
2.20
1.67
1.51
1.94
2.84
2.08
2.20
2.06
1.77
2.65
1.89
2.25
2.20
2.36
2.50
2.62
2.34
1.98
3.26
2.64
2.69
2.43
10.56
45.36
22.11
52.68
49.12
59.99
22.06
51.36
35.75
3.54
2.48
6.47
2.89
4.47
3.53
6.70
3.64
3.74
3.57
0.92
1.60
1.27
1.68
1.66
1.89
1.77
1.10
1.89
0.32
0.49
0.44
0.66
0.61
0.69
0.55
0.38
0.86
0.63
5.99
4.13
2.67
1.64
20.43
4.67
3.37
3.14
8
7
6
8
7
8
2
6
39.85
11.49
35.51
19.86
48.26
19.50
51.65
21.59
CNS
Melanoma
Ovarian
Renal
Prostate
Breast
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.

ACCEPTED MANUSCRIPT
Table 3. IC50 values (µM) of the tested compounds and sorafenib against the most sensitive cell line of each subpanel.
Cancer Cell Lines
RPMI-
8226a
0.65
0.43
0.40
0.24
1.44
1.95
3.13
1.50
0.40
2.50
1.58
HCT-
116c
2.02
0.75
0.63
0.33
1.74
1.96
3.17
9.64
1.96
3.23
1.58
UACC- OVCAR-
MDA-
MB-468i
1.74
A549/ATCCb
U251d
UO-31g
PC-3h
62e
3f
2.40
1.47
1.27
0.36
1.69
2.08
8.04
9.10
1.72
3.79
1.58
1b
1.48
0.73
0.68
0.37
1.96
2.33
3.62
9.15
1.64
2.62
3.16
1.70
0.88
0.73
0.32
1.44
1.72
5.56
5.03
1.40
3.23
2.00
1.56
0.59
0.45
0.33
1.96
2.34
7.69
53.10
7.20
3.28
3.16
1.89
1.03
1.66
0.26
1.91
2.10
2.71
4.50
1.41
2.50
2.51
1.13
0.42
0.34
0.30
1.69
2.10
2.71
3.30
1.09
2.28
2.00
1c
1.05
1d
0.99
1e
0.34
1g
1.71
Compound
No.
2.08
1i
4.26
2c
12.10
3.42
2d
2i
2j
3.10
2.00
Sorafenib
a Leukemia cell line; b non-small cell lung cancer cell line; c colon cancer cell line; d CNS cancer cell line; e melanoma cell line; f ovarian cancer cell line; g renal cancer cell line; h
prostate cancer cell line; i breast cancer cell line.
Bold figures indicate superior potency than the reference compounds.

ACCEPTED MANUSCRIPT
Table 4. IC50 and TGI values (µM) of compound 1e and sorafenib over the NCI
cancer cell line panel
Comp. 1e
Sorafenib
Cancer
Type
Cell Line
a
a
IC50
TGIb
1.33
3.24
5.84
1.64
0.76
3.31
4.68
2.39
0.78
5.17
1.60
10.90
1.76
1.76
1.88
4.95
1.27
2.25
2.21
2.75
2.00
3.25
1.44
3.49
11.30
5.83
1.31
IC50
TGIb
5.01
100
0.27
0.33
0.35
0.31
0.24d
0.45
0.37d
0.65
0.23
0.70
0.33
0.93
0.38
0.35
0.45
0.62
0.33d
0.35
0.45
0.51
0.39
0.62
0.33
1.41
0.42
0.85
0.32d
2.00
1.58
3.16
3.16
1.58d
3.16
3.16d
2.00
1.58
2.00
2.00
2.51
2.51
2.00
2.00
3.16
1.58d
2.51
2.00
1.58
2.51
2.51
1.58
1.58
3.16
3.16
2.00d
CCRF-CEM
HL-60(TB)
K-562
NTc
Leukemia
100
MOLT-4
RPMI-8226
SR
3.16
63.10
7.94
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
HOP-62
HOP-92
NCI-H226
NSCLC
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
MALME-
3M
0.53
3.57
2.00
39.81
Melanoma

ACCEPTED MANUSCRIPT
0.44
0.43
2.16
2.03
2.00
1.58
3.16
5.01
M14
MDA-MB-
435
0.66
0.90
1.14
0.41
0.36d
0.57
0.33d
0.50
1.46
0.39
2.70
4.93
2.00
2.51
1.58
2.00
1.58d
2.51
3.16d
3.16
3.16
3.16
6.31
2.51
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROV1
2.39
2.51
1.84
5.01
1.50
2.51
12.60
1.41
10.00
3.98
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
21.40
12.80
2.19
25.12
6.31
Ovarian
Cancer
12.59
NCI/ADR-
RES
0.82
9.98
2.51
7.94
0.77
0.57
0.39
0.40
0.42
1.26
0.39
0.68
0.26d
0.30d
0.47
0.38
5.75
2.17
3.59
12.50
4.75
3.92
1.98
14.60
1.58
1.63
10.60
9.32
2.51
3.16
2.51
2.51
3.16
3.16
2.51
3.98
2.51d
2.00d
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
0.46
1.86
1.26
3.16
1.40
2.22
0.39
7.26
7.14
4.16
2.51
3.16
1.58
3.98
3.98
5.01
Breast
HS 578T
BT-549
T-47D
Cancer
0.34d
2.52
2.00d
5.01
MDA-MB-
468

ACCEPTED MANUSCRIPT
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.
d Data taken from Table 3.

ACCEPTED MANUSCRIPT
Table 5. Inhibitory effects of compound 1e against a panel of 27 kinases.
% inhibition at 10
Kinase
µM dosea
-12.53±0.37
-5.92±1.11
-3.77±0.09
49.22±1.10
-15.10±0.34
39.10±0.47
1.25±0.81
ABL1
AKT1
ALK
ARAF
Aurora A
B-RAF (wild type)
c-Kit
5.66±2.96
c-MET
57.77±1.36
3.50±0.40
-7.47±0.18
-14.82±1.23
0.90±0.77
-8.66±2.25
-1.71±1.06
-2.82±0.81
-0.94±1.71
10.70±0.20
-2.88±0.03
2.27±0.68
c-RAF
DNA-PK
EGFR
ERBB2/HER2
ERBB4/HER4
ERK2/MAPK1
FGFR1
FLT3
FMS
JAK1
JAK2
JNK1
-5.83±3.20
-7.99±0.30
-15.11±0.64
-5.41±5.28
-9.26±0.27
16.78±0.21
94.72±0.22
(IC50 = 0.39 µM)
KDR/VEGFR2
MEK1
mTOR/FRAP1
P38α/MAPK14
PDGFRb
PI3K (p110a/p85a)
V600E-B-RAF
a Inhibition percentage values expressed as a mean of duplicate measurements ±
S.E.M.

ACCEPTED MANUSCRIPT
O
CF3
Cl
O
H3C
O
N
HN
N
O
H
N
N
N
N
N
HN
H
H
N
N CH3
Sorafenib
Imatinib
Cl
Cl
A
R1
O
O
N
N
N
N
NH
O
B
O
HN
N
N
R2
R1 = H, CH3
R2 = Ar, ArNH
The target compounds 1a-j and 2a-j
A = H, CH3
B = Ar, ArNH
Figure 1. Design rationale of the target compounds 1a-j and 2a-j.

ACCEPTED MANUSCRIPT
Figure 2. Mean inhibition percentage of the twelve tested compounds against the NCI-58 cancer
cell line panel.