Hybrids from farnesylthiosalicylic acid and hydroxamic acid as dual ras-related signaling and histone deacetylase (HDAC) inhibitors: Design, synthesis and biological evaluation

Article abstract of DOI:10.1002/cmdc.201500019

Abstract A novel series of hybrids was designed and synthesized by combining key elements from farnesylthiosalicylic acid (FTS) and hydroxamic acid. Several 3,7,11-trimethyldodeca-2,6, 10-trien-1-yl) thio)benzamide derivatives, particularly those with bra

Full text of DOI:10.1002/cmdc.201500019

DOI: 10.1002/cmdc.201500019
Communications
Hybrids from Farnesylthiosalicylic Acid and Hydroxamic
Acid as Dual Ras-Related Signaling and Histone
Deacetylase (HDAC) Inhibitors: Design, Synthesis and
Biological Evaluation
Yong Ling,*[a, b] Xuemin Wang,[a] Chenniu Wang,[a] Chenjun Xu,[a] Wei Zhang,[a]
Yihua Zhang,[a, b] and Yanan Zhang*[a]
A novel series of hybrids was designed and synthesized by
combining key elements from farnesylthiosalicylic acid (FTS)
and hydroxamic acid. Several 3,7,11-trimethyldodeca-2,6,10-
trien-1-yl) thio)benzamide derivatives, particularly those with
branched and linear aliphatic linkers between the hydroxamic
zinc binding group (ZBG) and the benzamide core, not only
displayed significant antitumor activities against six human
cancer cells but also exhibited histone deacetylase (HDAC) in-
hibitory effects in vitro. Among them, N-(4-(hydroxyamino)-4-
oxobutyl)-2-(((2E,6E)-3,7,11-trimethyldodeca-2,6, 10-trien-1-yl)-
thio)benzamide (8d) was the most potent, with IC50 values of
4.9–7.6 mm; these activities are eight- to sixteen-fold more
potent than FTS and comparable to that of suberoylanilide hy-
droxamic acid (SAHA). Derivative 8d induced cell cycle arrest
in the G0/G1 phase, inhibited the acetylation of histone H3
and a-tubulin, and blocked Ras-related signaling pathways in
a dose-dependent manner. The improved tumor growth inhibi-
tion and cell-cycle arrest in vitro might result from the dual in-
hibition. These findings suggest dual inhibitors of Ras-related
signaling pathway and HDAC hold promise as therapeutic
agents for the treatment of cancer.
and has recently been evaluated in a phase II clinical study in
patients with solid tumors.[5] However, FTS by itself does not
induce differentiation or a complete cell-cycle arrest.[6] There-
fore, FTS as a single-target drug has exhibited limited thera-
peutic efficacy.
Histone acetyltransferases (HATs) and histone deacetylases
(HDACs) are enzymes that control reversible acetylation and
deacetylation of histone. In addition, these enzymes have been
shown to modulate other proteins such as transcription factors
NF-kB and p53.[7] It has been well documented that HDACs are
overexpressed in many cancers, such as human liver, colon,
breast, and others,[8] and aberrant HDAC pathways are believed
to promote cancer growth and metastasis.[9,10] HDAC inhibitors
(HDACis) are thought to represent a new class of potential
small-molecule cancer therapeutics.[11] A number of HDACis
have been developed to date that are generally grouped into
four chemical classes: hydroxamic acids, benzamides, cyclic tet-
rapeptides, and short-chain fatty acids. Suberoylanilide hy-
droxamic acid (SAHA, vorinostat, Figure 1) was the first HDACi
approved in 2006 by the US Food and Drug Administration
(FDA) for the treatment of cutaneous T-cell lymphoma
(CTCL).[12] FK228 (romidepsin), a cyclic tetrapeptide, was ap-
proved for CTCL and peripheral T-cell lymphoma (PTCL) thera-
py in 2009 and 2011, respectively.[13] Another hydroxamic acid,
PXD101 (belinostat), was approved for the treatment of PTCL
in 2014.[14] In addition, several hydroxamic acid HDACis, such
as PCI-24781 (abexinostat), ITF2357 (givinostat), BYK408740 (re-
sminostat), and LBH589 (panobinostat), are now at various
stages of clinical trials as drug candidates against various can-
cers.[15–17]
Ras proteins have long been considered drug targets for
cancer therapy since they were first identified and character-
ized 30 years ago.[1] A large percentage of all cancer types
either express one of the mutationally activated Ras isoforms
or harbor a chronically activated Ras isoform.[2,3] Therefore, the
inhibition of excessive activated Ras proteins or Ras signal
pathway could revert malignant cells to a nonmalignant phe-
notype and cause tumor regression both in vitro and in vivo.[4]
For instance, Ras inhibitor farnesylthiosalicylic acid (FTS, salira-
sib) exhibits antiproliferative effects in several cancer cell lines
Importantly, FTS combined with HDACi valproic acid (VPA)
has been shown to synergistically block Ras-related signal
pathways, inhibit histone deacetylation, and prevent prolifera-
tion of cancer cells.[6] In addition, recent studies have shown
that Ras protein inhibitor selumetinib in combination with
HDACi vorinostat exerts potent antitumor effects, including
synergistic inhibition of cell proliferation, G1 cell-cycle arrest,
and induction of apoptosis.[18] Moreover, the combination of
HDACi VPA and tyrosine kinase inhibitor imatinib (gleevec)
downregulates expression of Bcr-Abl and Bcl-2 in chronic mye-
loid leukemia and enhances apoptosis.[19] Since Bcr-Abl is
known to indirectly activate Ras proteins,[20] it is plausible that
the combined inhibitory effect of imatinib and VPA might op-
erate in part through inhibition of Ras pathways.[6,20] Therefore,
[a] Dr. Y. Ling, X. Wang, C. Wang, C. Xu, Prof. W. Zhang, Prof. Y. Zhang,
Prof. Y. Zhang
School of Pharmacy, Nantong University
Nantong 226001 (P. R. China)
E-mail: Lyyy111@sina.com
zhangyanan@gmail.com
[b] Dr. Y. Ling, Prof. Y. Zhang
State Key Laboratory of Natural Medicines
China Pharmaceutical University
Nanjing 210009 (P. R. China)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201500019.
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the synthesis and biological eval-
uation of these hybrids for their
inhibitory
activities
against
HDACs and in cancer cell lines,
the blockade effects of cell cycle,
the expression of Ras-related
signal pathways and the acetyla-
tion of histone in vitro.
The general route for the syn-
thesis of the target compounds
8a–j is shown in Scheme 1. As
we reported previously,[25] farne-
sol 2 was treated with phospho-
rus tribromide to form farnesyl
bromide 3, which was then re-
acted with methyl thiosalicylate
4 in the presence of potassium
carbonate to provide methyl
farnesylthiosalicylicate 5. The
Figure 1. Structures of clinically approved or representative hydroxamic acid-based HDAC inhibitors in clinical
trials.
development of novel antitumor agents with dual Ras-related
signaling and HDAC inhibitory effects could be of pharmaceut-
ical importance in the treatment of cancers. Indeed, a number
of dual-target inhibitors have recently seen success as an at-
tractive strategy of drug therapy,[21] including RTK/HDAC dual
inhibitors that have demonstrated potential ability to over-
come tumor recurrence and drug resistance.[22,23]
parent compound, FTS (1), was obtained via hydrolysis of 5 in
aqueous sodium hydroxide. Subsequently, FTS was treated
with oxalyl chloride to give (E,E)-farnesylthiosalicyl chloride 6,
which then reacted with differently substituted NH2RCOOMe in
the present of triethylamine to provide intermediates 7a–j.
Finally, esters 7a–j were converted to hydroxamic acid deriva-
tives 8a–j with NH2OK in methanol. Final products 8a–j were
purified by column chromatography, and their structures were
It is known that an HDACi consists of three domains: a zinc-
binding group (ZBG) that chelates with the zinc ion to form
a complex; a cap group, generally a hydrophobic and aromatic
group, linked to the ZBG that fits into the tubular pocket; a sa-
turated or unsaturated linker domain, composed of linear or
cyclic structures that connect the ZBG and the cap group.[24] In
an attempt to develop novel dual inhibitors as antitumor
agents, we have designed and synthesized a series of novel hy-
brids by introducing the ZBG group, the hydroxamic acid frag-
ment, into the carboxyl group of FTS via alkane or aromatic
ring linkers (Figure 2). We hypothesize that FTS/hydroxamic
acid hybrids might efficaciously inhibit both the Ras-signaling
pathway and the deacetylation of histone. Herein, we report
1
characterized by infrared and H NMR spectroscopy, mass spec-
trometry, and elemental analysis. All compounds were of deter-
mined to be >95% purity by high-performance liquid chroma-
tography (HPLC).
The inhibitory activities of synthesized compounds against
human hepatocellular carcinoma cells (SMMC-7721 and
HepG2), human breast cancer cells (MCF-7), human lung
cancer cells (H460), and human bladder carcinoma cells (EJ)
were evaluated in vitro in MTT assays. FTS and SAHA were
used as positive controls. The IC50 values of 7d, 8a–j against
the six human cancer cell lines are listed in Table 1. Most of
the target compounds displayed better antiproliferative activi-
ty against the tested cells than FTS. Compound 8d
(R=(CH2)3) was the most potent, exhibiting antiproli-
ferative activities with IC50 values of 4.93–7.61 mm,
which are comparable to those of SAHA (IC50 =5.46–
7.85 mm) and eight- to sixteen-fold more potent than
FTS (IC50 =48.6–98.5 mm) in vitro. All target com-
pounds were also tested against HeLa cell nuclear ex-
tract, a rich source of HDACs, for their HDAC inhibito-
ry potencies,[26] using SAHA as a positive control. Sim-
ilar to the antiproliferative activity, compound 8d
was the most active analogue, with an IC50 value of
0.48 mm against the nuclear extract.
The structure–activity relationship (SAR) studies of
the series confirm the importance of the hydroxamic
acid and also revealed that the length of the linker
has significant impacts on the inhibitory activities
against the cell growth and HDACs. For instance,
Figure 2. Design of hybrids 8a–j from farnesylthiosalicylic acid (FTS) and suberoylanilide
hydroxamic acid (SAHA).
compound 7d (the corresponding ester of 8d) only
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Scheme 1. Synthesis of the target compounds 8a–j. Reagents and conditions: a) PBr3, n-hexane, pyridine, ether, 08C!RT, 4 h, 76%; b) methyl 2-mercaptoben-
zoate (4), K2CO3, CH3CN, 508C, 6 h, 82%; c) aq NaOH (1n), MeOH, 608C, 10 h, 90%; d) oxalyl chloride, CH2Cl2, RT, 1 h; e) NH2RCOOMe, Et3N, CH2Cl2, 08C!RT,
2 h, 65–86%; f) NH2OK, MeOH, 5–10 h, RT, 65–75%.
Table 1. In vitro inhibitory activity of compounds 7d and 8a–j against five human cancer cell lines and
OH group of the ZBG hydroxa-
mic acid moiety with amino acid
residues near the HDAC zinc
binding pocket. In general, com-
pounds with aliphatic linker
chain showed slightly higher an-
titumor potency than com-
pounds with aryl linkers (except
8j). Further investigation about
the precise SAR of these com-
pounds is ongoing.
HDAC.[a]
Compd
IC50 [mm]
SMMC-7721
HepG2
MCF-7
H460
EJ
HDAC[b]
FTS
SAHA
7d
8a
8b
8c
8d
8e
8 f
66.7Æ5.49
5.46Æ0.66
>25
7.23Æ0.65
9.87Æ0.80
6.46Æ0.73
4.93Æ0.55
11.8Æ0.88
21.3Æ1.76
>25
98.5Æ5.10
6.17Æ0.81
22.4Æ2.16
8.50Æ1.02
9.34Æ0.86
5.79Æ0.65
5.89Æ0.81
16.2Æ1.62
48.6Æ3.42
7.85Æ0.89
51.9Æ4.38
49.2Æ3.85
ND
0.56Æ0.06
ND
ND
>25
>25
>25
>10
12.1Æ1.09
8.07Æ0.95
8.04Æ0.71
7.15Æ0.63
15.1Æ1.44
11.5Æ1.28
10.1Æ1.05
7.29Æ0.60
6.06Æ0.77
13.7Æ1.50
19.2Æ2.01
10.3Æ1.31
12.4Æ1.10
7.52Æ0.83
5.81Æ0.61
10.5Æ1.24
22.9Æ1.93
2.4Æ0.37
1.7Æ0.22
0.85Æ0.07
0.48Æ0.05
1.5Æ0.16
3.3Æ0.29
>25
>25
>25
>25
11.5Æ1.23
>25
10.5Æ1.16
Encouraged by the inhibitory
activity of this series of com-
pounds against HeLa cell nuclear
extracts, which mainly contains
HDAC1 and HDAC2, we further
tested a selected group of com-
pounds (8b–d,j) against several
HDAC enzymes. The in vitro in-
hibitory activities against HDAC
enzymes were assessed employ-
ing SAHA as the positive control.
8g
8h
8i
>25
>25
>10
10.3Æ1.06
>25
10.2Æ1.12
8.65Æ0.69
7.97Æ0.81
>25
9.02Æ1.62
9.49Æ1.00
>25
8.87Æ0.97
0.77Æ0.08
4.6Æ0.52
0.92Æ0.08
23.8Æ2.05
6.95Æ0.81
8j
[a] The inhibitory effects were determined by using an MTT assay; data are expressed as the meanÆSD of
three independent experiments performed in triplicate. Cell lines: hepatocellular carcinoma (SMMC-7721 and
HepG2), breast cancer (MCF-7), lung cancer (H460), and bladder carcinoma (EJ). [b] For HDAC inhibition, com-
pounds were evaluated against HeLa cell nuclear extract. Abbreviations: FTS: farnesylthiosalicylic acid; SAHA:
suberoylanilide hydroxamic acid; ND: not detected.
showed weak cell growth inhibitory activity, confirming that
the hydroxamic acid moiety plays an important role in the an-
titumor activity of this series of FTS derivatives. Interestingly,
among the alkyl linker series (8a–f), a trend was apparent
where the antiproliferative potency initially increased and then
deceased with the elongation of the linker. Compounds 8c
and 8d with the linker comprised of two and three carbon
units, respectively, showed greater growth inhibitory activity
against all five tumor cell lines than the other compounds,
with 8d exhibiting the most potent activity in the series. A
similar trend of linker-length-dependency was observed
against the HeLa cell nuclear extract (Table 1). Again, 8d
showed the highest potency within the series. This might be
because 8d, which has the appropriate linker chain, could be
more effective in forming hydrogen bonds between the NH or
The potency of these compounds reported herein were ob-
tained by measuring the fluorescence-based HDAC biochemi-
cal activity using recombinant human HDAC1, HDAC6, and
HDAC8 enzymes, and the IC50 values are summarized in
Table 2. Most of these compounds exhibited significant
HDAC1, HDAC6, and HDAC8 inhibition. In particular, com-
pound 8d exhibited the most potent inhibitory activity against
both HDAC1 and HDAC8, with IC50 values of 0.058 mm and
0.19 mm, respectively, which are similar to or even higher than
those of SAHA. Compound 8d is three-fold more potent than
SAHA against HDAC1. Since 8d showed the highest potency in
inhibition of both cell growth and HDACs, it was advanced for
further evaluation.
Cell-cycle checkpoints are check mechanisms that verify
whether the processes at each phase of the cell cycle have
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51.3%, respectively, and the percentages of cells in the S and
G0/G1 phases decreased concomitantly. This cell-cycle arrest at
the G0/G1 phase caused by treatment with 8d occurred in
a dose-dependent manner (Figure 3b).
Table 2. Inhibitory activities of compounds 8b–d,j against different
HDAC isoforms.[a]
Compd
IC50 [mm]
HDAC1
HDAC6
HDAC8
Given that the inhibition of HDACs by hybrid 8d enhanced
the tumor cell antiproliferative activity, the effects of 8d on
the levels of acetylation of histone H3 and tubulin were deter-
mined by immunoblotting assays using histone H3 and a-tu-
bulin as negative controls (Figure 4). SMMC-7721 cells were in-
cubated with the vehicle alone, FTS, SAHA, FTS+SAHA, or 8d
(5.0 and 10 mm). Similar to the control, FTS did not affect the
expression levels of Ac-histone H3 or Ac-a-tubulin. As expect-
ed, SAHA induced increases in the expression levels of Ac-H3
and Ac-a-tubulin without affecting the levels of non-acetylated
histone H3 and a-tubulin. The combination of FTS and SAHA
showed increased expression levels than SAHA alone, suggest-
ing inhibition of both pathways. Importantly, 8d at 5 mm signif-
icantly increased the amounts of Ac-a-tubulin and Ac-histone
H3, which were higher than SAHA alone and comparable to
FTS and SAHA together (Figure 4b). This effect was even more
pronounced at 10 mm, suggesting that 8d could dose-depend-
ently inhibit HDACs.
SAHA
8b
8c
8d
8j
0.19Æ0.03
0.18Æ0.02
0.11Æ0.01
0.058Æ0.008
0.27Æ0.03
0.076Æ0.01
0.41Æ0.05
0.20Æ0.03
0.23Æ0.02
0.36Æ0.04
0.33Æ0.05
0.52Æ0.06
0.35Æ0.04
0.19Æ0.02
0.22Æ0.03
[a] Inhibition was determined using a fluorescence-based assay with re-
combinant human enzymes; data are expressed as the meanÆSD of
three independent experiments performed in triplicate.
been precisely completed before entering into the next phase,
therefore ensuring the fidelity of cell division. The G1 phase
checkpoint, also called the restriction point, is susceptible to
arrest if environmental conditions, such as inhibition of HDAC
and cellular protein synthesis, cause cellular stress and make
division impossible.[27,28] Here, we investigated whether the
potent antiproliferative activity and HDAC inhibition by 8d re-
sulted from the induction of cell-cycle arrest. The cellular DNA
content was analyzed by flow cytometric analysis in propidium
iodide (PI) stained cells to detect changes in the cell-cycle dis-
tribution (Figure 3a). The analysis revealed a marked change in
the cell-cycle profile of 8d-treated SMMC-7721 cells (Fig-
ure 2b). Compared with the control cells treated with DMSO,
when SMMC-7721 cells were treated with increasing concen-
trations of 8d (1.0 and 5.0 mm), the mean percentage of cells
in the G0/G1 phase increased from 30.1% to 39.8% and
To investigate whether the antitumor activities of 8d result
from the function of the FTS moiety of the hybrids, we exam-
ined the inhibitory effects of 8d on the expression of Ras-relat-
ed signaling in SMMC-7721 cells. In this study, the cells were
incubated with the vehicle alone, SAHA, FTS, SAHA+FTS, or
8d (5.0 and 10 mm). The expression and phosphorylation levels
of the Ras-related signal events, Akt and ERK1/2, were deter-
mined by immunoblotting assays using b-actin as the negative
Figure 3. The effects of compound 8d on cell-cycle progression in SMMC-7721 cells. a) Cells were treated with vehicle (control), SAHA, or different doses of
8d and stained with propidium iodide, followed by flow cytometry analysis. b) Representative histograms; data are expressed as the meanÆSD of three
independent experiments. * P<0.01 versus control.
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Figure 4. Immunoblot analysis of the expression of the acetylation for histone H3 and tubulin in vitro. a) SMMC-7721 cells were treated with FTS, SAHA,
FTS+SAHA, or 8d for 48 h at the indicated concentrations. Cell lysates were prepared and subjected to immunoblot analysis using acetyl-histone H3 (Ac-H3),
histone H3, acetyl-a-tubulin, and a-tubulin antibodies. Histone H3 and a-tubulin were used as controls. b) Quantitative analysis; the relative levels of Ac-H3/
H3, Ac-a-tubulin/a-tubulin were determined by densimetric scanning. Data are expressed as the meanÆSD of three independent experiments. * P<0.01
versus control.
Figure 5. Immunoblot analysis of the expression of the Ras-related signal events in vitro. a) SMMC-7721 cells were treated with vehicle (control), SAHA,
SAHA+FTS, FTS, or 8d for 48 h at the indicated concentrations, and their lysates were subjected to immunoblot analysis using anti-Akt, antiphospho-Akt
(Ser473), anti-ERK1/2, antiphospho-ERK1/2 (Thr202/Tyr204), and anti-b-actin antibodies, respectively. b-Actin was used as the control. b) Quantitative analysis;
the relative levels of each signaling event to control b-actin were determined by densimetric scanning. Data are expressed as the meanÆSD of three inde-
pendent experiments. * P<0.01 versus control.
control. As shown in Figure 5, the expression of free Akt and
ERK1/2 remained at similar levels with these treatments. While
FTS treatment decreased the levels of phospho-Akt and phos-
pho-ERK1/2 in SMMC-7721 cells, SAHA also led to decreases,
although to a lesser extent. This result suggests that SAHA
might also modulate Ras-related signaling, possibly via an indi-
rect mechanism. The combination of these two compounds
further decreased the expression levels of phospho-Akt and
phospho-ERK1/2. Furthermore, the levels of phospho-Akt and
phospho-ERK1/2 under treatment with 8d at 5.0 mm resulted
in obvious decreases that were comparable to FTS and SAHA
combined but greater than FTS or SAHA alone. At 10 mm, com-
pound 8d caused an even more significant decrease than
SAHA and FTS together. These results suggest that 8d effec-
tively inhibits the Ras-related signaling pathway. Therefore, the
increased antitumor potency of 8d compared with FTS might
be attributed to the concomitant inhibition of both HDAC and
Ras-related signaling in a synergistic manner.
In conclusion, using a structure-based design strategy, we
have designed and synthesized a novel series of hybrids by
conjugating FTS and hydroxamic acid with different linkers as
dual Ras-related signaling pathway and HDAC inhibitors, and
evaluated their biological activities in vitro. It was found that
several hybrids, particularly 8b–e and 8j, not only displayed
significant antitumor activities against five human cancer cells,
but also showed HDAC inhibitory effects. Compound 8d dem-
onstrated the highest anticancer potency against cancer cell
lines with IC50 values of 4.93–7.61 mm, which are eight- to six-
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Acknowledgements
The authors gratefully acknowledge financial support by the Nat-
ural Science Foundation of China (Grant Nos. 81302628 and
81473089), the Project of “Jiangsu Six Peaks of Talent” (2014-
SWYY-044), China Pharmaceutical University for the Open Project
Program of State Key Laboratory of Natural Medicines
(SKLNMKF201415), and also thank a project funded by the Priori-
ty Academic Programs Development (PAPD) of Jiangsu Higher
Education Institutions.
Keywords: antitumor agents
histone deacetylases · hydroxamic acids · ras-related signaling
pathways
· farnesylthiosalicylic acids ·
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Published online on April 16, 2015
ChemMedChem 2015, 10, 971 – 976
www.chemmedchem.org
976
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