Design, synthesis, and biological evaluation of N-hydroxycinnamamide/ salicylic acid hybrids as histone deacetylase inhibitors

Article abstract of DOI:10.1016/j.cclet.2013.11.039

Novel histone deacetylase (HDAC) inhibitors 9a-l were designed and synthesized by coupling the carboxyl group of salicylic acid (SA) with N-hydroxycinnamamides through various alkylol amines, and their in vitro biological activities were evaluated. The N-hydroxycinnamamide/SA hybrids 9b-f and 9h showed good to moderate anti-tumor activities. Notably, compound 9e had a greater potency, comparable to vorinostat (SAHA), in human colon carcinoma cells, which was probably, or at least partially, attributable to the positive effects of the chain length noted in alkylol amines. Furthermore, the HDAC inhibitory activities of 9e against Hela cell nuclear were also similar to that of vorinostat (SAHA), while the tested compounds 9c-f did not exhibit any isoform selectivity in the inhibition of HDACs. In addition, compound 9e could selectively inhibit tumor cells, but not inhibit non-tumor cell proliferation in vitro. Our findings suggest that the N-hydroxycinnamamide/SA hybrids may hold significant promise as therapeutic agents for the intervention of human cancers.

Full text of DOI:10.1016/j.cclet.2013.11.039

Chinese Chemical Letters 25 (2014) 474–478
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Original article
Design, synthesis, and biological evaluation of
N-hydroxycinnamamide/salicylic acid hybrids as
histone deacetylase inhibitors
a
b
a
a
a
a
Tao You , Ke Chen , Fei-Hai Wang , Pi-Hong Li , Li-Yi Li , Zhi-Hao Wu ,
a
a,
Kong-Hai Ni , Zhi-Qiang Zheng *
a
Department of General Surgery, The Second Affiliated Hospital, Wenzhou Medical University, Wenzhou 325035, China
b
School of Clinical Medicine, Wenzhou Medical University, Wenzhou 325035, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Novel histone deacetylase (HDAC) inhibitors 9a–l were designed and synthesized by coupling the
carboxyl group of salicylic acid (SA) with N-hydroxycinnamamides through various alkylol amines, and
their in vitro biological activities were evaluated. The N-hydroxycinnamamide/SA hybrids 9b–f and 9h
showed good to moderate anti-tumor activities. Notably, compound 9e had a greater potency,
comparable to vorinostat (SAHA), in human colon carcinoma cells, which was probably, or at least
partially, attributable to the positive effects of the chain length noted in alkylol amines. Furthermore, the
HDAC inhibitory activities of 9e against Hela cell nuclear were also similar to that of vorinostat (SAHA),
while the tested compounds 9c–f did not exhibit any isoform selectivity in the inhibition of HDACs. In
addition, compound 9e could selectively inhibit tumor cells, but not inhibit non-tumor cell proliferation
in vitro. Our findings suggest that the N-hydroxycinnamamide/SA hybrids may hold significant promise
as therapeutic agents for the intervention of human cancers.
Received 28 August 2013
Received in revised form 29 October 2013
Accepted 7 November 2013
Available online 27 November 2013
Keywords:
Synthesis
Anti-tumor agents
Histone deacetylase inhibitors
N-Hydroxycinnamamides
Salicylic acid
ß 2013 Zhi-Qiang Zheng. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights
reserved.
1
. Introduction
Pharmacological targeting of proteins that regulate epigenetics
there are also isoform or class selective HDACi in the clinical
development stage [8,9]. More importantly, vorinostat (SAHA) and
romidepsin (FK228), as the verified HDACi, have been approved by
the FDA for the treatment of cutaneous T-cell lymphoma (CTCL)
[10,11]. These two approved drugs have validated the therapeutic
use of HDACi in cancer therapy. Other compounds in clinical trials
[12–17] for the treatment of hematological and solid tumors
include LBH589 (panobinostat, Novartis, phase III), PXD101
(belinostat, Topotarget, phase III), SB939 (pracinostat, S*BIO, phase
II), TSA (trichostatin A), MS-275 (entinostat, Syndax, phase II), and
MGCD-0103 (mocetinostat, Methylgene, phase II) (Fig. 1). Among
the approved HDACi or in clinical trials, LBH589, PXD101, and SB-
939 share a common active fragment, N-hydroxycinnamamide.
This fragment could not only undergo bichelation with the active
has emerged as a promising therapeutic area of study [1,2]. Histone
acetylation and deacetylation play a crucial role for regulating
protein function of eukaryotic cells, which is correlated with two
classes of enzymes: histone deacetylases (HDACs) and histone
acetyltransferases (HATs) [3]. The maintenance of equilibrium
between acetylation and deacetylation of histones and non-
histone substrates is essential for normal cell growth. However,
HDAC overexpression has been found in a variety of human
cancers, including myeloid neoplasia and solid tumors [4].
Therefore, HDACs represent a rational target for intervention of
a number of cancers, and the development of histone deacetylase
inhibitors (HDACi) has become an efficacious therapeutic strategy
in cancer treatment [5–7].
2
+
site Zn by its hydroxamic acid group, but also form a sandwich-
like interaction by inserting its vinyl benzene group into two
pÀp
There are a number of HDACi which are currently undergoing
clinical trials, either mono-therapy or combination therapy, for
cancer treatment (Fig. 1). Most of HDACi are pan-inhibitors, but
parallel phenylalanine residues of HDAC [9,18].
Ferulic acid (FA) and salicylic acid (SA) are both phenolic
compounds occurring in natural plant products, and they, or their
derivatives, are known to display antioxidant and antiproliferative
activities against some types of cancer cells [19–21]. Furthermore,
HDACi (SC-560 and NS-398) could interact synergistically with
aspirin, or its metabolite SA, to induce cancer cell death [22]. On the
*
Corresponding author.
E-mail address: zhe_zhi2000@aliyun.com (Z.-Q. Zheng).
1
001-8417/$ – see front matter ß 2013 Zhi-Qiang Zheng. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
http://dx.doi.org/10.1016/j.cclet.2013.11.039

T. You et al. / Chinese Chemical Letters 25 (2014) 474–478
475
Fig. 1. Approved and clinical trial HDAC inhibitors.
basis of the aforementioned information, we intended to couple FA
with hydroxylamine to form the N-hydroxycinnamamide frag-
ment, and hypothesized that the novel types of N-hydroxycinna-
mamide/SA hybrids may effectively develop an inhibitory effect of
HDACs and selectively inhibit tumor cell proliferation via a
synergistic effect for the treatment of cancer. Therefore, a series
of hybrid molecules were designed and synthesized by coupling
the carboxyl of SA with N-hydroxycinnamamides through
substituted alkylol amines with different carbochain lengths.
General synthetic procedure for 3a–c: A mixture of methyl
ferulicate 2 (2.1 g, 10 mmol), K CO (3.5 g, 25 mmol) and various
dibromoalkanes (30 mmol) in acetonitrile (80 mL) was refluxed for
4–6 h. The solvent was evaporated under vacuum and water was
added. The solution was neutralized with 2 mol/L HCl solution to
2
3
pH 7 ꢀ 8, and then extracted with CH
2
Cl
2
(60 mL Â 3). The organic
layer was concentrated in vacuo and the residue was purified by
column chromatography on silica gel (petroleum ether–ethyl
acetate = 10:1 ꢀ 6:1, v/v as the eluent) to yield 3a–c as yellowish
oil (63–77%).
2
. Experimental
General synthetic procedure for 4a–l: A mixture of 3a–c
(
2 3
2 mmol), K CO (0.7 g, 5 mmol), and excess of ammonium
Melting points were determined on a RDCSY-I capillary
apparatus and were uncorrected. The compounds synthesized
were purified by column chromatography using silica gel (200–
hydroxide or alkylamine (10 mmol) in THF (40 mL) was stirred
for 3–5 h at 40 8C. The solvent was evaporated under vacuum and
water was added. The solution was neutralized to pH 8, and then
extracted with ethyl acetate (40 mL Â 3). The organic layer was
3
00 mesh) except for recrystallization and thin-layer chromatog-
raphy (TLC) using silica gel 60 F254 plates (250 mm; Qingdao Ocean
2 4
combined, washed with brine, dried with anhydrous Na SO , and
1
Chemical Company, China). The H NMR spectra were recorded
concentrated in vacuo, affording 4a–l as pale yellow oil (71–80%).
General synthetic procedure for 7a–l: Acetylsalicylic acid 5
(1 mmol) was reacted with oxalyl chloride (3 mmol) for 4 h at
room temperature. The solvent was evaporated to obtain the
salicylicyl chloride 6, which was subsequently added to the
with a Bruker Avance 300 MHz spectrometer at 300 K, using TMS
as an internal standard. MS spectra were recorded on a Mariner
Mass Spectrum (ESI). Element analyses were performed on an
Eager 300 instrument.
The synthetic route of these target compounds is outlined in
Scheme 1. The carboxyl group of FA 1 was protected to form methyl
mixture of 4a–l (0.9 mmol) and TEA (1.1 mmol) in 30 mL CH
0 8C over 1 h. After stirring for another 0.5 h, the mixture was
poured into CH Cl . The solution was washed with 5% sodium
bicarbonate and brine, dried with anhydrous Na SO and
concentrated in vacuo to afford compound 7a–l as pale yellow
solid (66–75%).
2 2
Cl at
ferulicate 2 using SOCl
2
in MeOH solution. Then, compound 2 was
2
2
reacted with various dibromoalkanes to obtain compounds 3a–c.
Subsequently, 3a–c were ammoniated with ammonium hydroxide
or alkylamine to give key intermediates 4a–l. In addition,
acetylsalicylic acid 5 was treated with oxalylchloride to produce
acetylsalicylicyl chloride 6, which was treated without further
purification with 4a–l in the presence of triethylamine, respec-
tively, and then hydrolyzed with aqueous NaOH to gain
2
4
,
General synthetic procedure for 8a–l: To a solution of 7a–l
(0.5 mmol) in methanol (10 mL), 1 mol/L NaOH (1.5 mL) was
added drop wise at room temperature. The mixture was refluxed
for 2–4 h, and then cooled. The solvent was evaporated and the
residue was dissolved in water. The mixture was neutralized with
2 mol/L HCl solution to pH 6 ꢀ 7 and extracted with ethyl acetate
(30 mL Â 3). The organic layers were combined, washed with
2
intermediates 8a–l. Finally, 8a–l were further treated with NH OK
to get N-hydroxycinnamamide/SA hybrids 9a–l, which were
1
purified by column chromatography. The MS data, H NMR spectra
and elemental analysis (EA) of selected compounds are presented
in reference [23].
2 4
brine, dried with anhydrous Na SO , and concentrated in vacuo,
affording 8a–l as pale yellow waxy solid (85–92%).

4
76
T. You et al. / Chinese Chemical Letters 25 (2014) 474–478
Scheme 1. Reagents and conditions: (a) SOCl
CO , THF, 40 8C, 3–5 h, 71–80%; (d) oxalylchloride, CH
MeOH, 38– 56%.
2
, MeOH, 0 8C-reflux, 8 h, 82%; (b) dibromoalkane, K
2
CO
3
, CH
3
CN, reflux, 4–6 h, 63–77%; (c) ammonium hydroxide or alkylamine,
OK,
K
2
3
2
Cl , r.t., 4 h; (e) 4a–l, Et N, CH Cl , r.t., 4–8 h, 66–75%; (f) 1 mol/L NaOH (aq.), MeOH, r.t., 2–4 h, 85–92%; (g) NH
2
3
2
2
2
General synthetic procedure for 9a–l: To
compound 8a–l (0.4 mmol) in 5 mL of anhydrous methanol was
added a solution of NH OK (0.09 g, 4 mmol) in 3 mL of anhydrous
a
solution of
3. Results and discussion
2
The inhibitory activities of synthetic compounds 8e and 9a–l
against six human cancer cells SGC7901, HCT-15, HCT-116, MCF-7,
HeLa, and SKOV-3 were evaluated by MTT assays in vitro, with
SAHA used as positive control. The results (shown in Table 1)
illustrated the IC50 values of target compounds against each tumor
cell line. Compounds 9b–f and 9h showed good to moderate
cytotoxic activities. Among these compounds, compound 9e
methanol. The mixture was stirred for 0.5 h and the solvent was
evaporated under vacuum. The residue was acidified with 2 mol/L
HCl to pH 5–6, then extracted with ethyl acetate (10 mL Â 3). The
organic layers were combined, washed with brine (10 mL Â 3),
2 4
dried over anhydrous Na SO , and evaporated with the residue
being purified by column chromatography on silica gel (ethyl
acetate/methanol, 10:1 ꢀ 6:1, v/v as the eluent), yielding 9a–l as
yellowish solid (38–57%).
(IC50 = 4.11 À 7.57
mmol/L) exhibited more potent antiprolifera-
tive activities, which were comparable to, or slightly weaker than,
Table 1
The IC50 values of 8e and 9a–l against six human cancer cell lines.
a
Compd.
In vitro inhibition of human cancer cells proliferation (IC50 , mmol/L)
SGC-7901
HCT-15
HCT-116
MCF-7
HeLa
SKOV-3
SAHA
3.83
23.2
13.4
13.5
11.5
12.4
5.22
7.78
16.6
12.1
18.4
> 30
27.8
> 30
4.35
20.1
10.3
7.12
7.18
8.94
4.63
6.25
19.6
9.43
22.0
26.5
25.9
20.1
5.61
17.3
14.7
8.47
9.37
9.48
4.11
5.83
17.2
11.2
14.9
> 30
22.9
23.8
7.23
> 30
18.7
10.4
12.9
9.18
7.57
7.28
20.8
13.5
25.7
29.0
27.1
> 30
2.11
22.1
16.0
11.1
8.20
10.1
5.18
6.86
13.8
12.0
26.0
26.3
25.4
> 30
3.48
24.0
17.0
9.59
9.66
11.6
5.90
9.18
19.8
10.6
22.7
> 30
23.2
23.1
8
9
9
9
9
9
9
9
9
9
9
9
9
e
a
b
c
d
e
f
g
h
i
j
k
l
a
The inhibitory effects of individual compounds on the proliferation of cancer cell lines were determined by the MTT assay. The data are the mean values of IC50 from at
least three independent experiments.

T. You et al. / Chinese Chemical Letters 25 (2014) 474–478
477
Table 2
these derivatives. For example, the compounds 9b, 9e, and 9h with
a three-carbon alkylol amine showed relatively stronger antican-
cer activity than compounds with a two- or four-carbon linker. It is
likely that the compounds with three-carbon alkylol amines bind
relatively well to the receptor. Among the N-hydroxycinnama-
mide/SA hybrids 9b, 9e, 9h, and 9k with three-carbon alkylol
amines, compound 9e displayed the most potent activity against
tumor cells, suggesting that adding one atom to the chain length of
alkyl amides in N-hydroferulicylamide hybrids may be the optimal
modification, and extending or shortening the chain length leads to
the decrease of the anticancer activities in vitro. A further
investigation of the precise SAR of these compounds is ongoing.
The HDAC inhibitory activities of 9b–f.
a
Compd.
IC50
(
mmol/L)
HeLa nuclear extract
HDAC3
HDAC6
SAHA
0.19
0.37
0.22
0.24
0.18
0.22
0.033
0.68
0.41
0.38
0.29
0.26
0.23
0.55
0.36
0.43
0.35
0.38
9
9
9
9
9
b
c
d
e
f
a
The data are the mean values of IC50 from at least three independent
experiments.
4. Conclusion
SAHA (IC50 = 2.11 À 7.23
mmol/L). However, the intermediate 8e of
e displayed poor cancer inhibitory activities, which suggested
9
In summary, a series of novel HDAC inhibitors 9a–l as N-
that the hydroxamic acid moiety has a positive influence on the
cytotoxicity of this series of N-hydroxyferulicylamide derivatives.
Furthermore, the active compounds 9b–f were evaluated for
their in vitro HDAC inhibitory activities against HeLa cell nuclear
extract (containing primarily HDAC1 and HDAC2), in addition to
HDAC3 and HDAC6. Results, listed in Table 2, showed that 9c–f also
exhibited significant HDACs inhibition, which were comparable to
SAHA. As expected, the HDAC inhibitory effects of individual
compounds were associated with their cytotoxicities against these
cancer cells in vitro. In addition, the synthetic compounds 9c–f did
not exhibit any isoform selectivity in the HDAC inhibitory assay.
Given that most N-hydroxyferulicylamide derivatives exhibit
strong cancer and HDAC inhibition, we questioned (or desired to
confirm) whether those compounds were toxic to the normal cells.
So the cytotoxicities of 9e with the more prominent anticancer
activities were further determined by MTT assay against human
normal cells CCD-841, and human colon carcinoma cells HCT-116
hydroxycinnamamide/SA hybrids were designed and synthesized
by coupling the carboxyl group of SA with N-hydroxycinnama-
mides through various alkylol amines. The in vitro assay of their
inhibitory activities against six tumor cell lines showed that the N-
hydroxycinnamamide/SA hybrids displayed strong antitumor
activities. Especially, compound 9e had greater potency, compara-
ble to SAHA, in human colon carcinoma cells, which was probably,
or at least partially, attributable to the three-carbon chain length of
alkylol amines. Furthermore, the HDAC inhibitory activities of 9e
against HeLa cell nuclear demonstrated more potency than that of
SAHA, while compounds 9c–f did not exhibit any isoform
selectivity in the HDAC inhibitory assay. In addition, compound
9e could selectively inhibit tumor cells, but not human normal cell
proliferation in vitro. Further investigations are ongoing for more
precise SAR information and more potent agents.
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3.59 (t, 2H, J = 4.5 Hz), 1.75–1.98 (m, 4H); Anal. Calcd. for C21 : C, 62.99; H,
6.04; N, 7.00; Found: C, 62.85; H, 4.96; N, 7.13. 9d: Yield 38%, mp 119–122 8C; MS
4
24 2 6
H N O
+
1
6
(ESI): m/z 387 [M + H] ; H NMR (300 MHz, DMSO-d ): d 10.44 (s, 1H), 7.68–7.79
7
02.
(m, 2H), 7.45 (d, 1H, J = 16.2 Hz), 6.79–7.21 (m, 5H), 6.39 (d, 1H, J = 16.2 Hz), 4.15
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20 22 2 6
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+ 1
6
47%, mp 108–111 8C; MS (ESI): m/z 401 [M + H] ; H NMR (300 MHz, DMSO-d ): d
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(d, 1H, J = 16.2 Hz), 4.13 (t, 2H, J = 4.5 Hz), 3.81 (s, 3H), 3.64 (t, 2H, J = 4.5 Hz), 3.03
24 2 6
(s, 3H), 1.98–2.23 (m, 2H); Anal. Calcd. for C21H N O : C, 62.99; H, 6.04; N, 7.00;
1
4828–14844.
Found: C, 63.07; H, 6.26; N, 7.16. 9f: Yield 55%, mp 103–106 8C; MS (ESI): m/z 415
[M + H] ; H NMR (300 MHz, DMSO-d ): d 10.47 (s, 1H), 7.63–7.77 (m, 2H), 7.40
6
(d, 1H, J = 16.2 Hz), 6.73–7.17 (m, 5H), 6.34 (d, 1H, J = 16.2 Hz), 4.10 (t, 2H,
J = 4.5 Hz), 3.79 (s, 3H), 3.61 (t, 2H, J = 4.5 Hz), 3.02 (s, 3H), 1.78–2.02 (m, 4H);
+
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[
23] The data of selected compounds. 9b: Yield 46%, mp 111–114 8C; MS (ESI): m/z 387
Anal. Calcd. for C22
7.59. 9h: Yield 57%, mp 95–98 8C; MS (ESI): m/z 415 [M + H] ; H NMR (300 MHz,
DMSO-d ): d 10.39 (s, 1H), 7.59–7.75 (m, 2H), 7.38 (d, 1H, J = 16.2 Hz), 6.70–7.16
(m, 5H), 6.32 (d, 1H, J = 16.2 Hz), 4.07 (t, 2H, J = 4.5 Hz), 3.80 (s, 3H), 3.57 (t, 2H,
H N O : C, 63.76; H, 6.32; N, 6.76; Found: C, 63.57; H, 6.56; N,
26 2 6
+
1
+ 1
[
(
(
6
M + H] ; H NMR (300 MHz, DMSO-d ): d 10.45 (s, 1H), 8.96 (s, 1H), 7.66–7.78
m, 2H), 7.42 (d, 1H, J = 16.2 Hz), 6.78–7.12 (m, 5H), 6.38 (d, 1H, J = 16.2 Hz), 4.12
t, 2H, J = 4.5 Hz), 3.83 (s, 3H), 3.62 (t, 2H, J = 4.5 Hz), 1.96–2.21 (m, 2H); Anal.
6
Calcd. for C20
H
22
N
2
O
6
: C, 62.17; H, 5.74; N, 7.25; Found: C, 62.28; H, 5.56; N, 7.13.
26 2 6
J = 4.5 Hz), 3.00 (s, 3H), 1.95–2.22 (m, 2H); Anal. Calcd. for C22H N O : C, 63.76;
H, 6.32; N, 6.76; Found: C, 63.53; H, 6.48; N, 7.62.
+
1
9
c: Yield 52%, mp 102–104 8C; MS (ESI): m/z 401 [M + H] ; H NMR (300 MHz,
6
DMSO-d ): d 10.41 (s, 1H), 8.89 (s, 1H), 7.65–7.77 (m, 2H), 7.40 (d, 1H, J = 16.2 Hz),