Design, synthesis and biological evaluation of 4-piperazinyl-containing Chidamide derivatives as HDACs inhibitors

Article abstract of DOI:10.1016/j.bmcl.2017.05.026

The synthesis and biological evaluation of a variety of 4-piperazinyl-containing Chidamide derivatives is described. Some of these compounds were shown to inhibit HDAC1 with IC₅₀ values below micromolar range, and inhibited proliferation of sev

Full text of DOI:10.1016/j.bmcl.2017.05.026

Accepted Manuscript
Design, synthesis and biological evaluation of 4-piperazinyl-containing Chida-
mide derivatives as HDACs inhibitors
Qingwei Zhang, Bingliu Lu, Jianqi Li
PII:
S0960-894X(17)30508-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.05.026
BMCL 24971
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
8 February 2017
22 April 2017
9 May 2017
Please cite this article as: Zhang, Q., Lu, B., Li, J., Design, synthesis and biological evaluation of 4-piperazinyl-
containing Chidamide derivatives as HDACs inhibitors, Bioorganic & Medicinal Chemistry Letters (2017), doi:
http://dx.doi.org/10.1016/j.bmcl.2017.05.026
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Design, synthesis and biological evaluation of
-piperazinyl-containing Chidamide derivatives as
HDACs inhibitors
4
a,b
Qingwei Zhang ,Bingliu Lu , Jianqi Li
a,b
a,b,
*
a
(
Novel Technology Center of Pharmaceutical Chemistry, Shanghai Institute of Pharmaceutical Industry,
Shanghai 201203)
b
(
Shanghai Engineering Research Center of Pharmaceutical Process, Shanghai 201203)
Abstract:
The synthesis and biological evaluation of a variety of 4-piperazinyl-containing
Chidamide derivatives is described. Some of these compounds were shown to inhibit
HDAC1 with IC50 values below micromolar range, and inhibited proliferation of
several human cancer cells, not possessing toxicity to human normal cells and hERG
+
K ion channels. Compound 9g, proved to be the most potent and efficacious
derivative in this series, was orally active in an HCT116 xenograft model in vivo.
Epigenetic mechanisms are essential for normal development and maintenance
of tissue-specific gene expression patterns in mammals. Epigenetic defects can lead to
alter gene function and malignant cellular transformation and have been linked to the
1, 2
progression of a number of diseases, such as cancer. The reversible acetylation of
histones, which is controlled by histone acetyltransferases (HATs) and histone
deacetylases (HDACs), has emerged as an important epigenetic modification
implicated in cell proliferation and has been identified as a valuable target for
3
anticancer drug design. HDACs comprise a family of 18 genes, which are grouped
-5
2
into “classical HDACs” (Zn for Classes I, II and IV) and sirtuins ( NAD for Class
+
+
6
, 7
III) based on their catalytic mechanisms. While the biological functions of the many
HDACs subtypes are still being defined, there is compelling evidence that class I
HDACs (HDAC1, 2, 3, 8) in particular are overexpressed in human cancers and
8
-11
therefore are viable targets for cancer therapeutics.
Up to date, four HDACs inhibitors (HDACIs）,Vorinostat (Zolinza; 2006),
Romidepsin (Istodax; 2009), Belinostat (Beleodaq; 2014) and Panobinostat (Farydak;
1
2
2
015) have been approved by the US FDA. However, it turned out that most of the

currently used HDACIs inhibited either all or at least several members of the HDACs
1
3,
family, which may suffer from numerous side effects, low potenty, or low stability
1
4
Meanwhile,
a
number of benzamide HDACIs, such as Entinostat
1
5
16
17
MS-275), Mocetinostat (MGCD0103) and Chidamide (CS055), are currently
(
undergoing clinical evaluation in solid tumors and hematological malignancies, which
can selectively inhibit HDACs 1-3 but does not significantly inhibit the other HDACs
isoforms. Recently, Chidamide was approved by the China FDA for the treatment of
advanced peripheral T-cell lymphoma (PTCL) as the first orally available benzamide
1
8
class of HDACIs .
Despite the variety of structural characteristics, most HDACIs can be broadly
described by a common pharmacophore according to the HDACs active site, as
shown in Figure 1. This model is composed of a zinc binding group (ZBG), attached
to a linker chain occupying the hydrophobic tunnel of the active site. This chain is
terminated by a functional“cap”group, mainly aromatic,interacting with the HDACs
external surface. The connecting unit between the cap and the linker chain can be
modulated to improve interactions.

Figure 1. Chemical structures of HDACIs
In our previous study, we prepared the Chidamide analog 4k which had a
N-(3-Aminopyridin-4-yl)-substituted residue as the ZBG. 4k exhibited comparable
HDAC1 inhibitory activity (IC50=3.3 μM) as Chidamide (IC50= 2.7 μM), though it
had a problem with moderate antitumor activity against PC-3 cell xenografts in nude
1
9
mice (TGI=39%, at 100 mg/kg). Several efforts have been made to replace the linker
chain with alternative chemical moieties, such as N-substituted piperazine ,which
could lead to a significant enhancement in HDACs inhibitory activity over
2
0-22
analogs.
Inspired by the structure-activity relationship (SAR) , herein we reported
the design, synthesis, and preliminary bioactivity evaluation of
4
-piperazinyl-containing Chidamide derivatives as HDACIs.
Scheme 1. General synthesis of 4-piperazinyl-containing Chidamide derivatives 9a-9m
The preparation of target compounds was shown in Scheme 1. The intermediate
methyl 4-(piperazin-1-yl)benzoate 3 could be obtained via the phenyl fluoride
displacement of commercially available methyl 4-fluorobenzoate with excess
3
piperazine (5.4 equiv) at 80
in CH CN, which was subsequently reacted with
(
E)-3-(pyridin-3-yl)acrylic acid or cinnamic acid analogs in the presence of HBTU to
afford the key intermediates 5a–5i. 5a–5i were then converted by hydrolysis of the
methyl ester with NaOH to intermediates 6a–6i and further by HBTU coupling in the
presence of excess phenylenediamine or 4-fluorophenylenediamine to obtain the
desired compounds 9a-9m.

Table 1. The chemical structures and HDAC1 inhibitory activities of 4-piperazin-containing
derivatives 9a-9m
a
%
Inhibition
Compound
Ar
R’
1
µM
10 µM
9
9
a
H
34.30
18.11
52.02
66.82
b
H
H
53.82
79.05
N
9
c
9
d
H
46.52
79.24
9
e
H
H
H
50.18
49.63
56.30
87.82
75.50
94.22
9
f
9
g
9
h
H
H
45.33
21.82
66.89
60.54
OCH3
9
i

9
j
F
F
F
F
4.71
19.22
58.83
64.31
9
k
36.03
17.43
9
l
9
m
18.01
55.12
53.37
81.27
Chidamide
a
23
Assays done in replicates (n≥3) as described in reference. Mean values are shown, and the standard
deviations are <30% of the mean.
All of the target compounds were initially screened for their ability to inhibit
recombinant human HDAC1 at two concentrations (1 µM and 10 µM). The
preliminary results showed that most of 4-piperazinyl-containing derivatives showed
potent HDAC1 inhibitory activity but not signficiantly superior to Chidamide. As
presented in Table 1, analogs containing 2-amino-4-fluorophenyl group in the ZBG
positions, such as 9j, 9k, 9l and 9m, were typically less potent than derivatives (9b,9c,
9g and 9f) with 2-aminophenyl substitution at the same positions. Interestingly,
introduction of more polar groups such as 3-pyridyl in the“cap”domain (9b and 9j)
led to reduce binding affinity to HDAC1 versus phenyl substituted analogues,
indicating that the electrostatic properties of the substitutions on the“cap”group may
show critical effect on inhibitory activities. Three compounds (9c, 9e, 9g) were further
evaluated for the IC50 of HDAC1 inhibition and showed promising activities with IC50
values in the nanomolar range (Table 2). The IC50 values of 9c, 9e and 9g are 0.983,
0.903 and 0.798 µM, respectively, whereas Chidamide exhibited IC50 values of 0.864
µM.
Table 2. Profiles of selected compounds 9c, 9e and 9g in vitro
a
IC50 （µM）
a
b
IC50 （µM）
IC50 （µM）
Compound
cell lines
K562
HDAC1
hERG
HCT116
A549
PC-3
MRC-5

9
9
9
c
e
g
0.983
0.903
0.798
0.864
1.977
0.765
0.069
0.202
0.811
0.530
0.135
0.665
1.102
2.963
4.856
0.975
2.142
8.706
3.964
2.093
>30.0
>30.0
>30.0
>30.0
32.6
25.1
27.1
32.0
Chidamide
a
Assays done in replicates (n≥3). Mean values are shown, and the standard deviations are <30% of
the mean.
b
24
hERG Patch clamp screen as described in reference. IC50 values represent the concentration to
inhibit 50% of hERG current (IKr). Numbers represent IC50 values generated from 3-point concentration
response relationships in duplicate.
Encouraged by their potent anti-HDAC1 profiles, anti-proliferative effects of
more potent compounds (9c, 9e and 9g) against HCT116 cells, A549 cells, K562 cells
2
5
and PC-3 cells were tested using MTT assay as described in reference. As shown in
Table 2, three selected compounds exhibited good antiproliferative activities against
all the tested cancer cells with IC50 values in the micromolar or sub-micromolar range.
Compound 9g showed significant growth inhibition with IC50 values of 0.069 and
0.135µM against HCT116 and A549 cells respectively, which was obviously superior
to Chidamide. To investigate the selective cytotoxicity of this new set of compounds,
9
c, 9e and 9g were tested over the human fetal lung fibroblast (MRC-5) normal cell
lines (Table 2). Interestingly, all the tested compounds including Chidamide exhibited
high IC50 values (>30.0 µM), which indicated their differential growth inhibitory
activities towards human cancer cells rather normal cell lines.
+
In vitro cardiovascular safety was assessed in a patch-clamp hERG K channels
screen (Table 2). Compounds 9c, 9e and 9g were inactive in hERG binding assay
with a IC50 > 20.0 µM (reducing incidence of QT interval prolongation).
In order to gain insights into the possible interactions of 9g with the active site of
HDAC1(PDB code: 4BKX), the molecular docking was performed using MOE
26
software (Molecular Operating Environment, Version 2015.1001) (Figure 2).

Cys
51
1
Leu
39
Met
30
1
His
41
1
Gly
00
3
Phe
50
Phe
05
1
Asn
95
2
Gly
49
1
Gly
01
3
Gly
38
1
H
N
His
40
H
1
N
N
Glu
NH2
O
O
9
8
Asp
9
9
Gln
60
2
Asp
76
1
ZN
00
Tyr
303
6
Leu
71
2
Asp
64
His
2
1
78
Figure 2. Binding modes of 9g at the active site of HDAC1
From the docking result, we found that compound 9g could insert into HDAC1
pocket, and the 2-aminophenyl moiety anchored onto the bottom of the pocket, which
was coordinated to the zinc ion, and formed hydrogen bonds with Tyr303 and Gly149.
Meanwhile, the phenyl ring formed a H-π interaction with residues of Asp99 in the
cap region, which could help to establish electrostatic interaction with the malleable
protein surface. Interestingly, according to our docking modes, the introduction of
piperazine group in the linker chain may not be favorable due to the lack of
interaction with residues of the tunnel of the HDAC1 active site. This was proved to
be consistent with experimental datas as shown that most 4-piperazinyl-containing
derivatives exhibited moderate HDAC1 inhibitory activity compared to Chidamide.
We could presume that the size and position of the substituents on the piperazine ring
may play an important role in enhancing the binding affinity, which could provide
invaluable information for further design of novel piperazin-containing derivatives.
Despite the sub-optimal potency in vitro, 9g was demonstrated to be the most
potent compound in this study and was selected for further biological evaluation.
The pharmacokinetic parameters of compound 9g were evaluated in female
Sprague Dawley rats after single iv (2 mg/kg) and po (10 mg/kg) administration. The
terminal phase half-life after iv dosing was 1.07 h in rats. The clearance was 0.45

L/h/kg and the steady state volume of distribution was 0.11 L/kg. 9g was quickly
absorbed after oral dosing, with a Tmax of 0.43 h. The oral bioavailability was found to
be 36.3% in rats.
a
Table 3. Pharmacokinetic evaluation of compound 9g
T
1/2
CL
iv (L/h/kg)
0.45
V
ss iv
T
max
C
max
AUC
po(h*µg/L)
8907
F
Compound
iv (h)
1.07
(L/kg)
0.11
po (h)
0.43
po(µg/L)
(%)
36.3
9
g
2509
a
For pharmacokinetic study, blood was collected from rats at various time points up to 6 h, and plasma
samples were analyzed using an Agilent 1200 HPLC system coupled with Agilent 6410 B triple quadruple mass
spectrometer. A solution of 0.05 N HCl in saline was used as the vehicle for both iv and po dosing.
9g was further tested in a mouse xenograft model (HCT116 colon) at daily oral
doses of 25 mg/kg, 50 mg/kg and 100mg/kg for 21 days (Fig. 3), and dose-dependent
inhibition of tumor growth was observed. At a dose of 25 mg/kg administered po the
inhibitor was tolerated and a 42.4% tumor growth inhibition(TGI) was observed
without significant weight loss (less than 5% body weight loss). The 50 and 100
mg/kg groups gave a 55.1% and 76.3% TGI, respectively. Unfortunately, mice treated
with 9g at 50mg/kg and 100 mg/kg oral dosage experienced significant and dose
proportionate weight loss (11.3% loss in the 50 mg/kg ,19.2% loss in the 100
mg/kg).Adverse effects caused termination of the 100 mg/kg study by 12 days.
Despite having a unsatisfactory therapeutic margin, compound 9g represents a
promising discovery lead. Inhibitors possessing an improved therapeutic index will be
the focus of future disclosures.

1
100
000
1
Vehicle
900
800
700
600
500
400
300
200
100
0
25 mg/kg,p.o.
50 mg/kg,p.o.
100 mg/kg,p.o.
0
3
6
9
12
15
18
21
Days after Treatment
Figure 3. Effects of 9g on HCT116 cell-inoculated xenografts in vivo
In summary, we designed and synthesized a series of novel HDACIs with
4
-piperazine group in the linker chain of Chidamide. Among them, 9c, 9e and 9g
exhibited similar or higher inhibitory activities in both enzymatic inhibitory activity
and cellular anti-proliferative activity assay compared with Chidamide, not possessing
+
significant toxicity to primary human cells and the patch clamp hERG K ion channel.
Molecular docking showed that optimisation of the linker remained an important
challenge. Compound 9g displayed good profiles in vitro , possessed favorable
pharmacokinetic characteristics and exhibited potent orally antitumor activity in an
HCT116 human colon carcinoma mouse xenograft model study. However, intolerable
body weight loss of experimental mice at high doses has limited the further
development of 9g . Efforts to identify a safe, orally efficacious alternative of 9g will
be the subject of future communications.
Acknowledgement
This work was financially supported by the National Natural Science Foundation
of China (No. 81273373), and Science and Technology Commission of Shanghai
Municipality (No.14YF1412800、14QB1404400).
Supplementary data
Supplementary data associated with this article can be found, in the online
version.

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Highlights
ꢀ
A series of 4-piperazinyl-containing Chidamide derivatives were
synthesized.
ꢀ
ꢀ
Target compounds were screened for their HDAC1 inhibitory activity.
The promising compound 9g displayed good profiles in vitro and had
low affinities for hERG channels.
ꢀ
ꢀ
9g possessed favorable pharmacokinetic characteristics.
9g exhibited potent orally antitumor activity in vivo.