Design, synthesis and biological evaluation of novel benzoylimidazole derivatives as RAF and histone deacetylases dual inhibitors

Article abstract of DOI:10.1248/cpb.c19-00425

In recent studies, combinations of histone deacetylases (HDACs) inhibitor with kinase inhibitor showed additive and synergistic effects. BRaf^V600E as an attractive target in many diseases treatments has been studied extensively. Herein, we pres

Full text of DOI:10.1248/cpb.c19-00425

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Chem. Pharm. Bull. 67, 1116–1122 (2019)
Vol. 67, No. 10
Regular Article
Design, Synthesis and Biological Evaluation of Novel Benzoylimidazole
Derivatives as Raf and Histone Deacetylases Dual Inhibitors
,a
Xin Chen,* Guoliang Gong,a Xinyang Chen,a Ruihu Song,a Mei Duan,a Ruizhi Qiao,a Yu Jiao,b
Jianzhao Qi,a Yadong Chen,b and Yong Zhub
a Shaanxi Key Labotory of Natural Products & Chemical Biology, College of Chemistry & Pharmacy, Northwest
b
A&F University; Yangling 712100, P. R. China: and School of Science, China Pharmaceutical University; Nanjing,
210009, P. R. China.
Received May 20, 2019; accepted July 18, 2019
In recent studies, combinations of histone deacetylases (HDACs) inhibitor with kinase inhibitor showed
additive and synergistic effects. BRafV600E as an attractive target in many diseases treatments has been stud-
ied extensively. Herein, we present a novel design approach though incorporating the pharmacophores of
BRafV600E inhibitor and HDACs inhibitor in one molecule. Several synthesized compounds exhibited distinct
BRafV600E and HDAC1 inhibitory activities. The representative dual Raf/HDAC inhibitor, 7a, showed better
antiproliferative activities against A549 and SK-Mel-2 in cellular assay than SAHA and sorafenib, with IC50
values of 9.11µM and 5.40µM, respectively. This work may lay the foundation for the further development of
dual Raf/HDAC inhibitors as potential anticancer agents.
Key words BRafV600E; histone deacetylase 1 (HDAC1); antiproliferation; dual inbihitor; synthesis
cancers. Up to data, a lot of BRafV600E inhibitors have been
Introduction
During the last few decades, developing mechanism-based developed such as sorafenib,5) dabrafenib, and vemurafenib
targeted anticancer drugs has made great achievement. How- (Fig. 1). They effectively block the MAPK signaling pathway
ever, unsustainable clinical effectiveness and acquired drug and inhibit proliferation of tumor cells expressing BRafV600E
.
resistance always limits the use of these agents.1) Since cancer Histone deacetylases (HDACs) are important targets for tumor
is a disease involving complex signaling networks, blocking a therapy.6–9) The HDACs family containing 18 isoforms is cate-
single biological target may not completely shut off the core gorized into Class I (HDAC1, 2, 3, and 8), Class IIa (HDAC4,
hallmark capability, allowing residual cancer cells to go on 5, 7, and 9), Class IIb (HDAC6 and 10), Class III (sirtuin1–7),
working. To address these problems, one particularly promis- and Class IV (HDAC11). Class I, II, and IV HDACs are all
ing approach is incorporating the elements that simultaneously zinc-dependent deacetylases, and Class III HDACs are mecha-
tackle multiple cancer-fighting targets into one molecule to nistically distinct from other HDACs, which require nicotine
obtain new chemical entity. Compared to single-target treat- adenine dinucleotide as a cofactor. The pharmacophore of
ment, this kind of therapeutic regimens have superior efficacy HDACs inhibitors were generally composed of three parts:
and fewer side effects.2)
cap, linker, and zinc binding group (ZBG). Hydroxamic acid
Protein kinases are important participants in the processes is the most frequently used ZBG group, three of which (i.e.,
of governing cellular proliferation, differentiation and evasion vorinostat,10) belinostat,11) panobinostat12) have gained U.S.
from apoptosis. BRaf, as one member of the Raf family, plays Food and Drug Administration approvals for clinical treat-
a crucial role in the Ras/Raf/Mek/Erk (mitogen-activated ment.
protein kinase, MAPK) signaling pathway.3,4) The Mutation
A lot of literatures have described the synergistic and ad-
of BRaf, especially BRafV600E, is the most common in human ditive effects by the joint use of HDACs inhibitors and vari-
Fig. 1. Representative BRafV600E and HDACs Inhibitors
*To whom correspondence should be addressed. e-mail: chenxin1888@nwsuaf.edu.cn
© 2019 The Pharmaceutical Society of Japan

Vol. 67, No. 10 (2019)
Chem. Pharm. Bull.
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Fig. 2. Design of Representative Raf/HDAC Dual Inhibitor 7a
Reagents and conditions: (a) 1-fluoro-4-nitrobenzene, K2CO3, CH3CN, Reflux, 4h; (b) dimethylamine hydrochloride, CH2O, H2O, HCl, r.t., 48h; (c) n-BuLi, THF,
−78°C, 4h; (d) iron powder, NH4Cl, H2O, C2H5OH, reflux, 4h; (e) CDI, CH2Cl2, r.t. 12h, then R-NH2, overnight.
Chart 1.
ous antitumor agents.13–15) Recently, Emmons et al. revealed various arylamines in the presence of 1,1′-carbonyldiimidazole
that HDACs inhibitor could enhance the durability of BRaf at room temperature gave the target compounds 7a–h (Chart
inhibitor therapy.16) Hence, the development of BRafV600E and 1).
HDACs dual inhibitors maybe a valuable strategy to circum-
vent resistance. Our group had previously reported a 2-(1H-
imidazol-2-yl) pyridine derivative CLW27 possessing compa-
Results and Discussion
Enzymatic Activities and Structure–Activity Relation-
rable antiproliferative activities to sorafenib in vitro or in vivo, ship (SAR) Study of Target Compounds The antitumor
and it showed inhibitory effect on BRaf and KDR-VEGFR2 effect of the inhibition of the class I HDAC isoforms, es-
kinases.17) In that compound, imidazole motief in the hinge pecially HDAC1, was well confirmed.21–23) Therefore, all
region played a beneficial role for antitumor potency. For an- prepared derivatives were tested for their inhibitory ability
other, considering the genotoxicity of hydroxamic acid group against HDAC1, using SAHA as the reference compound. As
and generated chromosomal aberrations in many cases,18,19) an shown in Table 1, IC50 values illustrated that compound 7c
alternative to hydroxamate as ZBG was tried in our design of bearing a terminal 4-methoxylphenyl, exhibited the best in-
novel Raf/HDAC dual inhibitors. Vasudevan et al. reported hibitory activity against HDAC1 (IC50 =635nM). However,
the use of acyl imidazole as ZBG in HDAC inhibitors, but it it was much weaker than that of SAHA. While changing me-
did not show potent inhibitory activity.20) However, the HDAC thoxyl from para-position to meta-position, compound 7f also
activity is not be determined only by the ZBG group, the spa- showed a comparative activity (IC50 =679nM). Additionally,
tial structures of cap and linker parts also have influence on compounds with groups such as –Me (7b, 7g), –Cl or –CF3
activity. In this study, we incorporated acyl imidazole motif (7a, 7d, and 7e) on phenyl inhibited HDAC1 with IC50 values
as ZBG and retained key urea group in sorafenib, then synthe- in submicromolar or micromolar range. The activities against
sized a series of novel derivatives (Fig. 2).
BRafV600E of these derivatives were also evaluated. 7a, which
reserved the same 4-chloro-3-trifluoromethyl substituent as
sorafenib, had the strongest activity (IC50 =86nM), although
Chemistry
Compounds 7a–h were synthesized as follows. Nucleophilic it was slightly less potent than sorafenib (IC50 =38nM).
reaction of ethyl 4-hydroxybenzoate (1) with 1-fluoro-4-ni- Compound 7d and 7e with electron-withdrawing substitutions
trobenzene yielded intermediate 2 using potassium carbonate also showed moderate inhibitory activities. However, electron-
with acetonitrile as solvent. Compound 4 was obtained by donating substitutions such as -Me or -OMe, were detrimental
mannich reaction with imidazole (3), formaldehyde and di- to BRafV600E inhibition, exemplified by 7b, 7c, 7f, and 7g, with
methylamine hydrochloride as materials. Nucleophilic addition IC50 values ranging from 0.840 to 4.727µM. When the phenyl
of 2 and 4 in tetrahydrofuran yielded key intermediate 5 with was replaced with pyridyl, obvious declines on both enzy-
n-BuLi as the base. Subsequently, reduction of 5 with iron matic activities were observed.
powder as catalyst in ethanol afforded 6. Reaction of 6 with
Raf and HDAC Isoforms Selectivity of Representative

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Vol. 67, No. 10 (2019)
Table 1. Enzymatic Inhibitory Activities of Target Compounds 7a–h (IC50a, µM)
Table 2. Raf and HDAC Isoforms Selectivity of Compounds 7a and 7c (IC50a), µM)
Cpd.
ARaf
BRafV600E
BRafWT
CRaf
HDAC1
HDAC6
HDAC8
7a
0.045
1.103
0.010
/
0.086
1.853
0.038
/
0.134
0.226
0.024
/
0.102
2.374
0.041
/
1.710
0.635
/
NAb)
NA
NA
NA
/
7c
Sorafenib
SAHA
/
0.037
0.026
3.21
a) We ran experiments in duplicate, S.D. <15%. b) NA: no activity was observed at 50µM concentration.
Compounds Based on the enzymatic results, representative
Table 3. Kinase Inhibitory Effects of Compound 7a
compounds 7a and 7c with better HDAC1 or BRafV600E activ-
Kinase
Inhibition rate%
IC50 (µM)
ity were further evaluated against Raf and HDAC isoforms.
As shown in Table 2, 7a also exhibited potent ARaf, BRafWT
,
KDR-VEGFR2
CDK4/cyclin D1
CDK6/cyclin D1
FLT3
41.75%
0.08%
19.210
and CRaf inhibitions. However, 7a and 7c showed no activities
against HDAC6 and HDAC8.
/
−2.05%
9.20%
/
The most potent compound 7a was further tested against
another six kinases which were frequently used in our lab
to evaluate its selectivity and to discovery potential other
molecular targets. The initial screening of 7a was conducted
at 10µM concentration for the inhibitory rates. As sum-
/
BCR-ABL
−0.13%
38.60%
/
EGFR
35.470
marized in Table 3, compound 7a showed weak inhibitory other two tumor cells SK-Mel-2 (malignant melanoma) and
effects against KDR-VEGFR2 and EGFR with IC50 values of MV4-11 (leukemia) which had extraordinary expression of
19.210 µM and 35.470µM, respectively, and had no obvious BRafV600E or BRafWT. 7a displayed submicromolar inhibiton
inhibition against CDK4/6, FLT3, and BCR/ABL kinases.
toward MV4-11 with IC50 value of 0.38µM. Especially, 7a had
Cell Proliferation Inhibition We chose compounds 7a, more potent activity against SK-Mel-2 than those of SAHA
7b, 7c, and reference compounds sorafenib and SAHA to eval- and sorafenib.
uate antiproliferative activities against tumor cell lines K562
Molecular Docking Study To further understand the in-
(leukemia), HCT116 (colon cancer) and A549 (non-small cell teraction between the inhibitors and two proteins, we docked
lung cancer). As shown in Table 4, all three compounds dem- 7c in the active site of HDAC1 (PDB code: 5ICN) and 7a in
onstrated obvious antiproliferative activities against all tumor BRafV600E (PDB code: 1UWJ) respectively. As shown in Fig.
cells, especially 7a, although it had the weakest HDAC1 inhi- 3A, 7c exhibited excellent shape complementarity with the
bition. Compared to sorafenib, 7a showed overall more potent binding pocket of HDAC1. The acyl imidazole moiety could
antiproliferative activity. 7a also exhibited superior activity in form bidentate coordination with zinc ion at the bottom of the
solid tumor cell line A549 (IC50 =9.11 µM) to that of SAHA pocket, with O-Zn2+ distance of 2.510Å for C=O and N-Zn2+
(IC50 =18.13 µM). Besides, 7a was further tested against an- distance of 2.731Å for imidazole –NH–, respectively. Addi-

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Table 4. Antiproliferative Activities of 7a, 7b, and 7c (IC50a), µM)
Cpd.
K562
HCT116
A549
SK-Mel-2
MV4-11
7a
9.13
12.09
10.10
0.65
10.87
14.24
11.26
6.21
9.11
16.25
13.37
18.13
10.63
5.40
/
0.38
/
7b
7c
/
/
SAHA
Sorafenib
8.20
9.30
0.11
0.42
11.20
12.25
a) IC50 values are averages of three independent experiments, S.D.<15%.
Fig. 3. (A) Docking Model of 7c (Yellow) in the Catalytic Pocket of HDAC1, with Key Residues in the Hydrophobic Channel Labeled in Green
The zinc ion appears as a large gray sphere. Metal coordination between inhibitor and the catalytic zinc ion were shown in solid black lines. The H-bonding interactions
with residues were labeled in dash line; (B) Docked position obtained for 7c in HDAC1. The HDAC1 protein was shown as a surface representation in gray. (C) The bind-
ing mode of sorafenib (wathet) in BRafV600E; (D) The binding mode of 7a in BRafV600E. For clarity, all hydrogen atoms were hidden.
tionally, the O atom of carbonyl and the N atom of imidazole tory activities. Among these, compounds 7a and 7c were the
generated H-bond interactions with His178 and Cys151. More- most potent compounds against BRafV600E and HDAC1, re-
over, the phenyl linkage connected to acyl imidazole formed spectively. Then 7a and 7c were submitted to evaluate the
stacking π–π interactions with the residues of Phe150 and isoforms selectivities. The results showed these derivatives to
Phe205. The urea group and two adjacent phenyls occupied be pan-Raf and selective HDAC1 inhibitors. Further in vitro
the surface groove and came into close contact with the resi- antiproliferative assay showed that 7a, 7b, and 7c had inhibi-
dues (Gln26, Gly27, and His28) at the rim region. Another H- tory effects against several tumor cell lines including K562,
bond was formed between NH of urea and Glu98 to enhance HCT116, and A549. Moreover, 7a exhibited more potent anti-
their binding (Fig. 3B). Comparing the binding modes of proliferative activities against A549 and SK-Mel-2 cell lines
sorafenib (Fig. 3C) and 7a (Fig. 3D) in BRafV600E, 7a reserved than sorafenib and SAHA. Subsequently, molecular docking
three key H-bond interactions between the urea group and the was performed to explain the structure–activity relationship.
residues of Glu500 and Asp593, while the H-bond in the hinge The demonstration of dual Raf/HDAC inhibitors in this paper
was missed. This might be responsible for reduced potency provided useful tool compounds for further studies of multiple
against BRafV600E of 7a.
pathway inhibition achieved with a single molecule.
Conclusion
Experimental
Chemistry All of the starting materials were obtained
In summary, we designed a series of novel Raf/HDAC
dual inhibitors bearing acyl imidazole as ZBG. Eight target commercially and were used without further purification. All
compounds were synthesized and tested against BRafV600E of the reported yields were for isolated products and were not
and HDAC1, and most compounds exhibited obvious inhibi- optimized. Melting points were determined in open capillaries

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Chem. Pharm. Bull.
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on a WRS-1A digital melting point apparatus (Shenguang). was refluxed for 4h, then filtered. The mother liquid was ex-
1H-NMR spectra was recorded in dimethyl sulfoxide (DMSO)- tracted with ethyl acetate (20mL×3). The combined organic
d6 on a Bruker DRX-500 (500MHz) using TMS as internal extracts were dried over anhydrous MgSO4 and concentrated
standard. The chemical shifts were reported in ppm (δ) and under reduced pressure. The product was obtained as a white
coupling constants (J) values were given in Hertz (Hz). Mass solid by chromatography on a silica gel column (1.66g,
1
spectra were obtained from Agilent 1100 LC/MSD (Agilent, 85.0%); mp: 192–194°C; H-NMR (500MHz, DMSO-d6) δ:
U.S.A.) or Q-tof micro MS (Micromass, U.S.A.). The IR spec- 13.36 (br, 1H), 8.53 (dd, J=8.8, 2.7Hz, 2H), 7.48 (s, 1H), 7.27
tra were performed on a FTIR-8400S (Shimadzu, Japan) in (s, 1H), 6.97 (d, J=8.9Hz, 2H), 6.85 (d, J=8.7Hz, 2H), 6.63
KBr pellets; the frequencies are expressed in cm−1. The puri- (d, J=8.7Hz, 2H), 5.08 (s, 2H).
ties of all tested compounds were established by HPLC to be
>95.0%. HPLC analysis was performed at room temperature 7a–h
using an Agilent Eclipse XDB-C18 (250×4.6mm) and 20%
General Procedure for the Preparation of Target Compounds
To a solution of 6 (3mmol) in anhydrous dichloromethane
MeOH/H2O as a mobile phase and plotted at 254nm.
(5mL) was added CDI (0.58g, 3.6mmol) at room temperature.
After the mixture was stirred for 12h, different arylamine
Ethyl 4-(4-Nitrophenoxy)benzoate (2)
To a solution of ethyl 4-hydroxybenzoate (16.6g, 0.1mol), (3.6mmol) was added. The mixture was stirred for overnight.
potassium carbonate (55.2g, 0.4mol) in anhydrous aceto- After completion of the reaction as monitored by TLC, the
nitrile (300mL) under nitrogen atmosphere was added fluoro- reaction mixture was filtered. The filter cake was washed with
4-nitrobenzene (15.5g, 0.11mol). The mixture was refluxed CH2Cl2 (2mL×2) and dried in vacuo. The crude product was
for 4h, then filtered. The mixture was concentrated in vacuo, recrystallizated in mixed solvent of ethyl acetate and tetra-
then washed with water. The mother liquid was extracted hydrofuran (v/v=1:1) to give the purified target compound.
with ethyl acetate (30mL×2). The combined organic extracts
1-(4-(4-(1H-Imidazole-2-carbonyl)phenoxy)phenyl)-3-(4-
were dried over anhydrous MgSO4 and concentrated under chloro-3-(trifluoromethyl)phenyl)urea (7a)
reduced pressure. The product was obtained as a white solid
White solid; yield 73.6%; mp: 235–237°C; 1H-NMR
by chromatography on a silica gel column (24.3g, 84.4%); (500MHz, DMSO-d6) δ: 13.37 (s, 1H), 9.16 (s, 1H), 8.91 (s,
1
mp: 110–112°C. H-NMR (500MHz, DMSO-d6) δ: 8.57 (dd, 1H), 8.58 (d, J=9.0Hz, 2H), 8.11 (d, J=2.4Hz, 1H), 7.66
J=7.0, 2.5Hz, 2H), 8.31 (dd, J=7.0, 2.3Hz, 2H), 7.25–7.89 (dd, J=8.8, 2.4Hz, 1H), 7.61 (d, J=8.8Hz, 1H), 7.55 (d,
(m, 4H), 4.23 (q, J=7.2Hz, 2H), 1.25 (t, J=7.2Hz, 3H);
1-(1H-Imidazol-1-yl)-N,N-dimethylmethanamine (4)
J=8.9Hz, 2H), 7.49 (d, J=2.4Hz, 1H), 7.28 (s, 1H), 7.12 (d,
J=8.9Hz, 2H), 7.06 (d, J=9.0Hz, 2H); ESI-MS m/z: 501.1
Imidazole (20.4g, 0.3mol) and dimethylamine hydrochlo- ([M+H]+), 523.1 ([M+Na]+); IR (KBr, cm−1): 3344, 1674,
ride (26.0g, 0.3mol) were added in 50mL water at 0°C, then 163, 1597, 1541, 1489, 1323, 906, 775, 611.
conc. hydrochloric acid was added to adjust pH to 4. Then
1-(4-(4-(1H-Imidazole-2-carbonyl)phenoxy)phenyl)-3-(p-
resulting mixture was stirred for 10min, and formalin (37%, tolyl)urea (7b)
27g, 0.33mol) was added. The resulting mixture was stirred
White solid; yield 75.7%; mp: 246–247°C; 1H-NMR
for additional 48h at room temperature, then 20% KOH solu- (500MHz, DMSO-d6) δ: 13.39 (s, 1H), 8.70 (s, 1H), 8.58 (d,
tion was added to adjust pH to 10. The reaction mixture was J=8.9Hz, 2H), 8.55 (s, 1H), 7.53 (d, J=8.9Hz, 2H), 7.50 (d,
extracted with chloroform (20mL×3). The combined organic J=2.4Hz, 1H), 7.35 (d, J=8.3Hz, 2H), 7.28 (s, 1H), 7.12-7.04
extracts were dried over anhydrous Na2SO4 and concentrated (m, 6H), 2.25 (s, 3H); ESI-MS m/z: 413.3 ([M+H]+); IR (KBr,
under reduced pressure. The product was obtained as a liquid cm−1): 3304, 1635, 1603, 1553, 1501, 1302, 1217, 823, 744, 665.
(28.4g, 75.6%). 1H-NMR (500MHz, DMSO-d6) δ: 8.03 (s,
1-(4-(4-(1H-Imidazole-2-carbonyl)phenoxy)phenyl)-3-(4-
1H), 7.53 (d, J=1.9Hz, 1H), 7.14 (d, J=1.8Hz, 1H), 4.82 (s, methoxyphenyl)urea (7c)
2H), 2.51 (s, 6H).
White solid; yield 76.1%; mp: 239–240°C; 1H-NMR
(1H-Imidazol-2-yl)(4-(4-nitrophenoxy)phenyl)methanone (5) (500MHz, DMSO-d6) δ: 13.39 (s, 1H), 8.67 (s, 1H), 8.57 (d,
Under nitrogen atmosphere, to a solution of 4 (1.25g, J=8.9Hz, 2H), 8.47 (s, 1H), 7.54 (d, J=9.0Hz, 2H), 7.50 (s,
10mmol) in THF (40mL) at −78°C was added n-BuLi 2.5M 1H), 7.36 (d, J=8.3Hz, 2H), 7.28 (s, 1H), 7.10 (s, 2H), 7.05 (s,
solution in n-hexane (4.2mL), then the resulting mixture was 2H), 6.88 (d, J=8.9Hz, 2H), 3.71 (s, 3H); ESI-MS m/z: 429.3
stirred for 1h at −78°C. A solution of 2 (3.01g, 10.5mmol) in ([M+H]+); IR (KBr, cm−1): 3348, 1634, 1591, 1501, 1475,
THF (10mL) was added. The mixture was stirred for 4h at 1389, 1225, 903, 773, 662.
−78°C and monitored end by TLC. The mixture was acidi-
1-(4-(4-(1H-Imidazole-2-carbonyl)phenoxy)phenyl)-3-(4-
fied with 2N HCl (80mL) to adjust pH to 6–7 and extracted chlorophenyl)urea (7d)
with ethyl acetate (40mL×3). The combined organic extracts
White solid; yield 70.1%; mp: 268–270°C; 1H-NMR
were dried over anhydrous Na2SO4 and concentrated under (500MHz, DMSO-d6) δ: 13.40 (s, 1H), 8.82 (s, 1H), 8.80
reduced pressure. The product was obtained as a white solid (s, 1H), 8.56 (d, J=9.0Hz, 2H), 7.54 (d, J=8.9Hz, 2H),
by chromatography on a silica gel column (2.20g, 71.2%); mp: 7.51–7.47 (m, 3H), 7.33 (d, J=8.9Hz, 2H), 7.28 (s, 1H), 7.10
1
177–179°C; H-NMR (500MHz, DMSO-d6) δ: 13.46 (br, 1H), (d, J=8.9Hz, 2H), 7.06 (d, J=8.9Hz, 2H); ESI-MS m/z:
8.65 (dd, J=7.0, 2.5Hz, 2H), 8.31 (dd, J=7.0, 2.3Hz, 2H), 433.2 ([M+H]+); IR (KBr, cm−1): 3360, 1686, 1595, 1497,
7.53 (d, J=1.9Hz, 1H), 7.34–7.29 (m, 5H).
(4-(4-Aminophenoxy)phenyl)(1H-imidazol-2-yl)methanone
(6)
1389, 1229, 905, 839, 768, 598.
1-(4-(4-(1H-Imidazole-2-carbonyl)phenoxy)phenyl)-3-(3,4-
dichlorophenyl)urea (7e)
To a solution of 5 (2.00g, 7mmol), ammonium chloride
White solid; yield: 78.1%; mp: 263–265°C; 1H-NMR
(0.37g, 7mmol) in water (20mL) and ethanol (1mL) was (500MHz, DMSO-d6) δ: 13.38 (s, 1H), 9.00 (s, 1H), 8.88 (s,
added iron powder (3.70g, 70mmol) in batches. The mixture 1H), 8.57 (d, J=9.0Hz, 2H), 7.88 (d, J=2.4Hz, 1H), 7.53 (d,

Vol. 67, No. 10 (2019)
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1121
J=9.0Hz, 2H), 7.51 (d, J=9.0Hz, 1H), 7.49 (d, J=2.4Hz, 10 µCi/µL) (Perkin Elmer, NEG302H001 MC) was added to
1H), 7.35 (dd, J=8.9, 2.5Hz, 1H), 7.28 (s, 1H), 7.12 (d, initiate the reaction, the reactions were carried out at 25°C
J=8.9Hz, 2H), 7.06 (d, J=9.0Hz, 2H); ESI-MS m/z: 467.2 for 2h. The kinase activities were detected by filter-binding
([M+H]+); IR (KBr, cm−1): 3348, 1634, 1593, 1543, 1386, method. IC50 values and curve fits were obtained by Prism
1225, 903, 847, 771, 662.
(GraphPad Software).
1-(4-(4-(1H-Imidazole-2-carbonyl)phenoxy)phenyl)-3-(3-
methoxyphenyl)urea (7f)
The Pan-Raf and other kinases inhibitory assay were in a
manner same as BRafV600E
.
White solid; yield: 70.7%; mp: 219–221°C; 1H-NMR
Cell Culture and Cytotoxicity/Proliferation Assay The
(500MHz, DMSO-d6) δ: 13.36 (s, 1H), 8.72 (s, 1H), 8.66 (s, antiproliferative activitives of compounds 7a, 7b, and 7c were
1H), 8.58 (d, J=9.0Hz, 2H), 7.55 (d, J=9.0Hz, 2H), 7.52 evaluated against different tumor cell lines by the standard
(s, 1H), 7.28 (s, 1H), 7.20–7.19 (m, 2H), 7.12 (d, J=8.9Hz, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
2H), 7.07 (d, J=9.0Hz, 2H), 6.94 (d, J=8.0Hz, 1H), 6.55 (d, (MTT) assay in vitro, with SAHA and sorafenib as the posi-
J=7.5Hz, 1H), 3.29 (s, 3H); ESI-MS m/z: 429.3 ([M+H]+); tive control. The cancer cell lines were cultured in RPMI 1640
IR (KBr, cm−1): 3265, 1695, 1572, 1499, 1420, 1393, 1232, 901, medium with 10% fetal bovine serum (FBS). Approximate
771, 656.
1-(4-(4-(1H-Imidazole-2-carbonyl)phenoxy)phenyl)-3-(m- plated into each well of a 96-well plate and incubated in 5%
tolyl)urea (7g)
CO2 at 37°C for 24h. The tested compounds at the indicated
2.5×103 cells, suspended in RPMI 1640 medium, were
White solid; yield: 70.2%; mp: 233–235°C; 1H-NMR final concentrations were added to the culture medium and
(500MHz, DMSO-d6) δ: 13.38 (s, 1H), 8.72 (s, 1H), 8.58 (s, incubated for 48h. Fresh MTT was added to each well at the
1H), 8.56 (d, J=9.0Hz, 2H), 7.53 (d, J=9.0Hz, 2H), 7.49 (d, terminal concentration of 0.5mg/mL, and incubated with cells
J=2.4Hz, 1H), 7.30 (s, 1H), 7.28 (s, 1H), 7.23 (d, J=9.0Hz, at 37°C for 4h. After the supernatant was discarded, 150µL
1H), 7.16 (s, J=8.0Hz, 1H), 7.12 (d, J=8.9Hz, 2H), 7.05 (d, DMSO was added to each well, and the absorbance values
J=9.0Hz, 2H), 6.79 (d, J=7.5Hz, 1H), 2.28 (s, 3H); ESI-MS were determined by a microplate reader (Bio-Rad, Hercules,
m/z: 413.3 ([M+H]+); IR (KBr, cm−1): 3300, 1639, 1568, 1398, CA, U.S.A.) at 490nm.
1240, 906, 847, 775, 658, 517.
1-(4-(4-(1H-Imidazole-2-carbonyl)phenoxy)phenyl)-3- performed in Discovery Studio 3.0 software (BIOVIA, 5005
(pyridin-2-yl)urea (7h)
Wateridge Vista Drive, San Diego, CA92121 U.S.A.). Docking
Computational Methods All computational work was
White solid; yield: 73.9%; mp: 230–232°C; 1H-NMR was conducted using Cdocker module based on the cocrystal
(500MHz, DMSO-d6) δ: 13.40 (s, 1H), 10.61 (s, 1H), 9.47 (s, of BRafV600E (PDB: 1UWJ) and HDAC1 (PDB: 5ICN). Water
1H), 8.58 (d, J=8.8Hz, 2H), 8.29 (d, J=8.0Hz, 1H), 7.76 (d, molecules outside the binding pockets were excluded. The en-
J=8.5Hz, 1H), 7.62 (d, J=8.8Hz, 2H), 7.49 (m, 2H), 7.28 (s, ergy minimization for compounds was performed by Powell’s
1H), 7.14 (s, 2H), 7.07 (d, J=8.9Hz, 2H), 7.02 (t, J=6.9Hz, method for 1000 iterations using tripos force field and with
1H); ESI-MS m/z: 400.3 ([M+H]+); IR (KBr, cm−1): 3275, Gasteiger–Hückel charge. The other docking parameters were
1639, 1603, 1558, 1402, 1234, 1159, 908, 849, 773.
kept as default.
In Vitro HDAC Enzyme Assay The HDAC activity was
determined using the HDAC fluorimetric activity assay kit
Acknowledgments This work was supported by Chinese
(Biomol, Plymouth Meeting, PA, U.S.A.). Briefly, recombi- Universities Scientific Fund (Grant No. 2452017174), the Sci-
nant proteins of HDAC1, 6 and 8 was incubated with test entific Startup Foundation for Doctors of Northwest A&F Uni-
compounds, and HDAC reaction was initiated by addition of versity (Grant No. 2452016146) and National Natural Science
Fluor-de-Lys substrate. Samples were incubated for 10min at Foundation of China (Grant No. 31800031).
room temperature, followed by adding developer to stop the
reaction. Fluorescence was measured by a fluorimetric reader
Conflict of Interest The authors declare no conflict of
with excitation at 360nm and emission at 460nm. The HDAC interest.
activity was expressed as arbitrary fluorescence units (AFU).
The HDAC activity was calculated as a percentage of activity
Supplementary Materials The online version of this ar-
compared with the control group. The IC50 values for the test ticle contains supplementary materials.
compounds were calculated using SigmaPlot software.
In Vitro Kinase Enzyme Assay Activity of full length
BRafV600E was determined using Hot-SpotSM kinase assay.
5nM of human GST-tagged BRafV600E protein (AA416–766)
(Invitrogen, Cat# PV3894) was mixed with 20µM of the
substrate His 6-Tagged Full-length Human MEK1 (K97R) in
reaction buffer (20mM Hepes pH 7.5, 10mM MgCl2, 1mM
ethylene glycol bis(2-aminoethyl ether)-N,N,N′,N′-tetra acetic
acid (EGTA), 0.02% Brij35, 0.02mg/mL BSA (albumin from
bovine serum), 0.1mM Na3VO4, 2mM DTT, 1% DMSO at
room temperature, the compounds dissolved in 100% DMSO
at indicated doses (starting at 30µM with 3-fold dilution)
was delivered into the kinase reaction mixture by Acoustic
technology (Echo550; nanoliter range), incubate for 20min at
room temperature. After 10µM 33P-γ-ATP (specific activity
References
1) Hanahan D., Weinberg R. A., Cell, 144, 646–674 (2011).
2) Guerrant W., Patil V., Canzoneri J. C., Oyelere A. K., J. Med.
Chem., 55, 1465–1477 (2012).
3) Ribas A., Flaherty K. T., Nat. Rev. Clin. Oncol., 8, 426–433 (2011).
4) Rebocho A. P., Marais R., Oncogene, 32, 3207–3212 (2013).
5) Agianian B., Gavathiotis E., J. Med. Chem., 61, 5775–5793 (2018).
6) Falkenberg K. J., Johnstone R. W., Nat. Rev. Drug Discov., 13,
673–691 (2014).
7) Weichert W., Cancer Lett., 280, 168–176 (2009).
8) Khan O., La Thangue N. B., Immunol. Cell Biol., 90, 85–94 (2012).
9) Gammoh N., Lam D., Puente C., Ganley I., Marks P. A., Jiang X.,
Proc. Natl. Acad. Sci. U.S.A., 109, 6561–6565 (2012).
10) Marks P. A., Breslow R., Nat. Biotechnol., 25, 84–90 (2007).
11) Sawas A., Radeski D., O’Connor O. A., Ther. Adv. Hematol., 6,

1122
Chem. Pharm. Bull.
Vol. 67, No. 10 (2019)
202–208 (2015).
18) Olaharski A. J., Ji Z., Woo J. Y., Lim S., Hubbard A. E., Zhang L.,
Smith M. T., Toxicol. Sci., 93, 341–347 (2006).
12) Laubach J. P., Moreau P., San-Miguel J. F., Richardson P. G., Clin.
Cancer Res., 21, 4767–4773 (2015).
13) Zhu Y., Ran T., Chen X., Niu J., Zhao S., Lu T., Tang W., Chem.
Pharm. Bull., 64, 1136–1141 (2016).
14) Chen X., Zhao S., Wu Y., Chen Y., Lu T., Zhu Y., RSC Adv., 6,
103178–103184 (2016).
19) Johnson D., Walmsley R., Mutat. Res., 751, 96–100 (2013).
20) Vasudevan A., Ji Z., Frey R. R., Wada C. K., Steinman D., Heyman
H. R., Guo Y., Curtin M. L., Guo J., Li J., Pease L., Glaser K. B.,
Marcotte P. A., Bouska J. J., Davidsen S. K., Michaelides M. R.,
Bioorg. Med. Chem. Lett., 13, 3909–3913 (2003).
15) Liang X., Zang J., Li X., Tang S., Huang M., Geng M., Chou C. J.,
Li C., Cao Y., Xu W., Liu H., Zhang Y., J. Med. Chem., 62, 3898–
3923 (2019).
21) Weichert W., Roske A., Gekeler V., Beckers T., Stephan C., Jung K.,
Fritzsche F. R., Niesporek S., Denkert C., Dietel M., Kristiansen G.,
Br. J. Cancer, 98, 604–610 (2008).
16) Emmons M. F., Faião-Flores F., Sharma R., Thapa R., Messina J.
L., Becker J. C., Schadendorf D., Seto E., Sondak V. K., Koomen J.
M., Chen Y. A., Lau E. K., Wan L., Licht J. D., Smalley K. S. M.,
Cancer Res., 79, 2947–2961 (2019).
17) Jiao Y., Xin B. T., Zhang Y., Wu J., Lu X., Zheng Y., Tang W., Zhou
X., Eur. J. Med. Chem., 90, 170–183 (2015).
22) Weichert W., Roske A., Niesporek S., Noske A., Buckendahl A. C.,
Dietel M., Gekeler V., Boehm M., Beckers T., Denkert C., Clin.
Cancer Res., 14, 1669–1677 (2008).
23) Chen X., Zhao S., Li H., Wang X., Geng A., Cui H., Lu T., Chen Y.,
Zhu Y., Eur. J. Med. Chem., 168, 110–122 (2019).