Discovery of novel phenoxybenzamide analogues as Raf/HDAC dual inhibitors

Article abstract of DOI:10.1016/j.bmcl.2019.04.047

Histone deacetylase (HDAC)inhibitors as an important epigenetic therapeutic strategy affect signaling networks and act synergistically with kinase inhibitors for the treatment of cancer. Herein we presented a series of novel phenoxybenzamide analogues with inhibition of Raf and HDAC. Among them, compound 10e showed potent antiproliferative activities against Hepg2 and MDA-MB-468 in cellular assays. This work may lay the foundation for developing novel dual Raf/HDAC inhibitors as potential anticancer therapeutics.

Full text of DOI:10.1016/j.bmcl.2019.04.047

Accepted Manuscript
Discovery of novel phenoxybenzamide analogues as Raf/HDAC dual inhibitors
Aixin Geng, Hao Cui, Liyuan Zhang, Xin Chen, Hongmei Li, Tao Lu, Yong
Zhu
PII:
S0960-894X(19)30280-X
https://doi.org/10.1016/j.bmcl.2019.04.047
BMCL 26414
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
1 February 2019
22 April 2019
27 April 2019
Please cite this article as: Geng, A., Cui, H., Zhang, L., Chen, X., Li, H., Lu, T., Zhu, Y., Discovery of novel
phenoxybenzamide analogues as Raf/HDAC dual inhibitors, Bioorganic & Medicinal Chemistry Letters (2019),
doi: https://doi.org/10.1016/j.bmcl.2019.04.047
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Discovery of novel phenoxybenzamide analogues as Raf/HDAC dual
inhibitors
Aixin Genga, Hao Cui a, Liyuan Zhanga, Xin Chena,d, Hongmei Lia, Tao Lub,c,*, Yong Zhua,*
aDepartment of Organic chemistry, School of Science, China Pharmaceutical University,639
Longmian Avenue, Nanjing 211198, China.
bLaboratory of Molecular Design and Drug Discovery, School of Science, China Pharmaceutical
University, 639 Longmian Avenue, Nanjing 211198, China.
cState Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang,
Nanjing 210009, China
dShanxi Key Laboratory of Natural Products & Chemical Biology, College of Science, Northwest
A&F University, Yangling 712100, China
*Corresponding authors. Tel.: +86 25 86185180; fax: +86 25 86185179 (T.L.); tel.: +86 25
86185173; fax: +86 25 86185182 (Y.Z.).
E-mail addresses: lutao@cpu.edu.cn (T. Lu), zhuyong@cpu.edu.cn (Y. Zhu).
Abstract Histone deacetylase (HDAC) inhibitors as an important epigenetic
therapeutic strategy affect signaling networks and act synergistically with kinase
inhibitors for the treatment of cancer. Herein we presented a series of novel
phenoxybenzamide analogues with inhibition of Raf and HDAC. Among them,
compound 10e showed potent antiproliferative activities against Hepg2 and
MDA-MB-468 in cellular assays. This work may lay the foundation for developing
novel dual Raf/HDAC inhibitors as potential anticancer therapeutics.
Keywords Raf, HDAC, dual inhibitor, antiproliferative activity, phenoxybenzamide
Cancer has been a significant public health problem in the world with more patients
being diagnosed every year.1 However, chemotherapy from non-specific cytotoxic
agents that as an effective means of cancer treatment is limited by severe side effects
and poor selectivity.2 Thus, the development of mechanism-based targeted antitumor

drugs has made significant progress in the past few years. Unfortunately, their clinical
effectiveness is generally unsustainable in long-term treatment and the drug resistance
always limits the use of these agents based on complex signaling networks of
cancers.3 Nowadays, new treatment strategies, such as combined, multi-targeted
therapies or recently, immunotherapy have been advocated.4-7
Protein kinases in different cell signaling pathways play key roles in the processes
of governing cellular proliferation, differentiation and evasion from apoptosis.8-10
Targeting kinases like Raf and PI3K have become one of the most pursued classes of
anticancer agents.11-13 The approval of Raf kinase inhibitor such as sorafenib (1),
PI3K inhibitor such as pictilisib has been adopted to produce higher potency and
selectivity.14, 15 In addition, targeting epigenetic modifiers particularly histone
deacetylases (HDACs) have recently set the foundation for a new generation of
anti-cancer drugs.16, 17 Several small molecule HDAC inhibitors have been approved
by FDA or NMPA for the treatment of various haematological and solid tumors, such
as vorinostat and chidamide (2).18, 19 The therapeutic combination of an HDAC
inhibitor and a kinase inhibitor, such as epidermal growth factor receptor (EGFR)
tyrosine kinase inhibitors, Raf or PI3K inhibitors, can overcome kinase inhibitor
resistance and induce apoptosis in human solid cancers in a synergistic manner.20
However, combination therapy may be associated with unwanted drug-drug
interactions, inappropriate pharmacokinetics, toxicity and scarce patient compliance.
In order to overcome the problems related to the polypharmacological approach, the
development of dual inhibition of HDAC and the tyrosine kinase signaling pathways
has been recently proposed as a promising strategy which could provide novel drug
candidates for the treatment of multifactorial diseases, such as CUDC-907,
FK228.21-24
Our group has also explored HDAC inhibitors based on a high-throughput
screening campaign and found a lead compound 10a with IC50 of HDAC1 5.23 μM.25
Interestingly, compound 10a possessed the o-aminobenzamide structure similar to
chidamide and acylaminophenoxybenzamide scaffold similar to sorafenib (Fig. 1).
Based on the screening of kinase inhibitory activity assay in vitro, compound 10a also

showed an modest inhibition against BRafV600E with IC50 = 1.78 nM. In this report, we
describe the identification of a novel series of phenoxybenzamide dual Raf/HDAC
inhibitors, which were obtained by lead optimization of 10a through a
structure–activity relationship (SAR) study. These studies led to the discovery of a
novel scaffold and potental dual Raf/HDAC inhibitors.
Fig. 1 Structures of representative Raf inhibitor 1, HDAC inhibitor 2 and the lead compound 10a.
Our initial efforts on the lead compound 10a focused on converting the pyridyl
moiety of 10a to various aryl group (Table 1). Compound 10a and its derivatives
(10b-j) were prepared as outlined in Scheme 1. Acid 3 was esterified to give methyl
ester 4, which was then reacted with methylamine to afford N-methyl amide 5. Then
compound 5 was converted to the ether 6 via nucleophilic substitution by using
p-aminophenol. Furthermore, substituted aromatic carboxylic acids 7a-j were treated
with SOCl2 to generate acyl chlorides 8a-j, which were reacted with 6 to produce
amides 9a-j. Finally, these amides were reduced with iron powder in water/ethanol to
afford final amines 10a–j. On the other hand, we probed the effect of different
substituents on amide (Table 1). Similar chemistry was applied to the synthesis of
compounds 16a-c in Scheme 2.

Scheme 1 Reagents and conditions: (a) SOCl2, MeOH, reflux, 4 h; (b) methylamine,
rt, 4 h; (c) p-aminophenol, t-BuOK, K2CO3, DMF, 80 ℃, 4 h; (d) SOCl2, DMF, reflux,
6 h; (e) Et3N, CH2Cl2, 0 ℃, 4 h. (f) Fe, HOAc, 95% EtOH, reflux, 4 h.
Scheme 2 Reagents and conditions: (a) p-aminophenol, t-BuOK, K2CO3, DMF, 80 ℃,
4 h; (b) 4-chloro-3-(trifluoromethyl)- benzoyl chloride, Et3N, CH2Cl2, 0 ℃, 4 h. (c)
NaOH, MeOH, H2O, reflux, 2 h; (d) SOCl2, DMF, reflux, 6 h; (e) amine, rt, 4 h; (f) Fe,
HOAc, 95% EtOH, reflux, 4 h.

To study the biological activity, compounds 10a–j and 16a-c were evaluated
against BRafV600E activity assays with sorafenib as a positive control and HDAC1
enzyme assay with chidamide as a positive control. As shown in Table 1, most
compounds exhibited moderate inhibitory activity against both BRafV600E and HDAC1.
Instead of pyridine with benzene and naphthalene, compound 10b and 10j showed
about 8-fold and 6-fold increase against BRafV600E activity. Hydrophilic groups at
terminal phenyl couldn’t enhance the enzyme activity but could enhance HDAC1
activity, such as compounds 10g, 10h and 10i. Interestingly, compound 10e with Cl
and CF3 still had the higher activity against HDAC1. Different types of amides had
relatively significant effect on both BRafV600E and HDAC1 inhibition. The potency at
enzymatic level of compound 16a with primary amine was significantly decreased
compared to 10e. One tertiary amine was introduced at the carbonyl group (16c),
which resulted in loss of HDAC1 and reduction of BRafV600E activity. However,
another secondary amine 16b could sustain the HDAC1 activity compared to 10e.
Among the compounds tested, compound 10e showed powerful inhibitory activity
against HDAC1 (IC50 = 1.17 μM) and BRafV600E (IC50 = 0.073 μM).
Table 1
IC50 values of compounds 10a-j and 16a-c against BRafV600E and HDAC1
Compd.
Ar
R1
R2
IC50 (μM)a
BRafV600E HDAC1
3-pyridyl
H
H
H
H
CH3 1.780
CH3 0.342
CH3 0.303
CH3 0.165
CH3 0.073
CH3 0.187
5.23
6.19
7.65
7.11
1.17
2.46
10a
10b
10c
10d
10e
10f
phenyl
4-fluorophenyl
4-chlorophenyl
4-chloro-3-(trifluoromethyl)phenyl H
4-methoxyphenyl
H

4-aminophenyl
3-aminophenyl
3-hydroxyphenyl
2-naphthyl
H
H
H
H
CH3 0.942
CH3 3.260
CH3 4.060
CH3 0.237
2.27
1.03
1.95
8.21
2.79
1.62
NAc
NDb
0.167
10g
10h
10i
10j
4-chloro-3-(trifluoromethyl)phenyl H
4-chloro-3-(trifluoromethyl)phenyl H
H
0.688
16a
C2H5 0.354
16b
4-chloro-3-(trifluoromethyl)phenyl CH3 CH3 4.170
16c
0.065
NDb
sorafenib
chidamide
a Values were the average of three experiments, SD < 10%. b ND = not determined.
c NA = no activity observed at 50 μM concentration tested.
For further understanding of the interaction between these inhibitors and proteins
(BRafV600E and HDAC1) for guiding the SAR, compound 10e, the most potent
compound of this series, was chosen as a representative for docking into the
BRafV600E (PDB code: 1UWJ) and HDAC1 (PDB code: 1C3R), revealing excellent
shape complementarity between ligand and the binding pocket. As shown in Fig. 2,
10e binds to the BRaf ATP binding pocket, interacting with three aromatic residues:
Trp530 of the hinge region, Phe582 at the end of the catalytic loop, and Phe594 of the
DFG motif, which also contacts the central phenyl ring of the inhibitor. At one end of
the inhibitor, the lipophilic trifluoromethyl phenyl forms hydrophobic interactions
with the surrounding amino acid residues (Val503, Ile571, Ile591). The aliphatic side
chains of Lys482, Leu513, and Thr528 contact the central phenyl ring of the inhibitor.
In addition to the van der Waals interactions, the phenylamine of compound 10e
forms one essential hydrogen bond with the hinge region Cys531. The amide group
forms two hydrogen bonds with Asp593 and Lys482. In addition, the methyl amide
side group directs to the solvent accessible region of the enzyme. On the other hand,
the docking mode of 10e in complex with HDAC1 reveals that this compound binds
in the HDAC1 pocket with the similar manner to chidamide. The

4-chloro-3-(trifluoromethyl)phenyl moiety of 10e projects outside into the solvent,
weakly contacting hydrophobic residues on the protein surface. Phenoxyphenyl
moiety exits the reaction center along the long tubular channel and is stacked between
Phe198 and Phe141. The o-aminobenzamide group entered into the active site by
chelating the essential catalytic zinc ion, and forms three hydrogen bonds with His131,
Tyr297 and Gly140, respectively. However, the only one chelating bond derived from
the amino group may be an important cause of its reduced activity.
Fig. 2 The docking model of 10e bound to BRafV600E (PDB entry 1UWJ, A) and
HDAC1 (PDB entry 1C3R, B). The protein is shown as ribbons. The key amino acids
forming the pocket are represented by stick with carbon atoms colored in green,
oxygen atoms in red, nitrogen atoms in blue and zinc in grey. 10e is shown in stick
with carbon atoms colored in yellow, oxygen atoms in red and nitrogen atoms in blue.
The hydrogen bond and chelating bond were denoted by red and black dash lines
respectively.
In order to further ascertain the pan-inhibition of Raf kinases and HDACs, two
representative compounds 10e and 10f with better HDAC1 and BRafV600E inhibition
were selected and evaluated against recombinant human HDAC1, HDAC6, HDAC8
enzymes and ARaf, BRafV600E and CRaf, using chidamide and sorafenib as the
positive control compounds. As shown on Table 2, it is noteworthy that this set of
structures show promising pan-inhibition for Raf and selective activity against
HDAC1.

Table 2
Inhibition of Raf and HDAC isoforms by compound 10e and 10f
Compd.
IC50(μM)a
ARaf
BRafV600E CRaf
HDAC1
1.17
HDAC6
NAc
HDAC8
NAc
0.183
0.211
0.073
0.187
0.065
NDb
0.092
0.652
0.055
NDb
10e
2.46
NAc
NAc
10f
0.012
NDb
NDb
NDb
NDb
sorafenib
chidamide
0.167
NAc
NAc
a Values were the average of three experiments, SD < 10%. b ND = not determined.
c NA = no activity observed at 50 μM concentration tested.
To test the anticancer activities of the synthesized compounds, we evaluated
antiproliferative activities of representative compounds 10c, 10d, 10e, 10f and 10j
against human chronic myelogenous leukemia cell line K562, human acute
myelogenous leukemia cell line MV4-11, human hepatocellular carcinoma cell line
Hepg2 and breast cancer cell line MDA-MB-468 by applying the MTT colorimetric
assay. The IC50 values were summarized on Table 3. According to the inhibition data,
all tested compounds showed obvious antiproliferative activities. Consistent with this
observation of HDAC1 and BRafV600E inhibition, we found that 10e and 10f potently
inhibited the growth of those cancer cells derived from both hematologic and solid
tumors. Especially, compound 10e exhibited the better anti-proliferative activity in
Hepg2 and MDA-MB-468 cell lines than sorafenib and chidamide.
Table 3
Antiproliferative activities of compounds 10c, 10d, 10e, 10f and 10j
IC50 (μM)a
Compd
K562
6.43
MV4-11
5.32
Hepg2
7.56
MDA-MB-468
5.76
10c

7.12
2.73
5.22
10.32
17.23
0.87
9.23
1.12
1.87
8.21
0.51
3.73
5.13
1.33
6.65
7.28
3.04
4.89
4.24
0.57
0.95
3.91
12.17
2.65
10d
10e
10f
10j
sorafenib
chidamide
a Values were the average of two experiments, SD < 10%.
In conclusion, a series of phenoxybenzamide analogues have been prepared and
assessed for their Raf and HDAC inhibitory activity and cytotoxicity against the K562,
MV4-11, Hepg2 and MDA-MB-468 cell lines. Moreover, the selectivity assay of the
respective compounds showed that they were not only excellent selective inhibition of
HDAC1, but also pan-Raf inhibitors. Preliminary SAR data and docking mode
revealed the o-aminobenzamide moieties were essential for binding to the proteins
BRafV600E and HDAC1. In addition, compound 10e exhibited higher potency against
Hepg2 and MDA-MB-468 cell lines than sorafenib and chidamide. In summary, we
have demonstrated the example of dual-action inhibitors targeting the Raf and HDAC
as a promising approach to search for efficient anticancer multi-target agents.
Acknowledgements
This work was financially supported by the National Natural Science Foundation of
China (Grant No. 81803033) and the “Double First-Class” University project
(CPU2018GF02).
Supplementary data
Supplementary data associated with this article can be found, in the online version，
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Graphical abstract