Discovery of multi-target anticancer agents based on HDAC inhibitor MS-275 and 5-FU

Article abstract of DOI:10.2174/1573406411666150714111045

Histone deacetylases (HDACs) inhibitors have multiple effects targeting the cancer cells and have become one of the promising cancer therapeutics with possibly broad applicability. Combination of HDAC inhibitors with the cytotoxic fluorouracil (5-FU) show

Full text of DOI:10.2174/1573406411666150714111045

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30
Medicinal Chemistry, 2016, 12, 30-36
Discovery of Multi-target Anticancer Agents Based on HDAC Inhibitor
MS-275 and 5-FU
Yuqi Jiang, Xiaoguang Li, Xiaoyang Li, Jinning Hou, Yongzheng Ding, Jian Zhang, Wenfang Xu*
and Yingjie Zhang*
Department of Medicinal Chemistry, School of Pharmaceutical Sciences,
Shandong University, 44 West Wenhua Road, 250012 Ji’nan, Shandong, PR
China
Abstract: Histone deacetylases (HDACs) inhibitors have multiple effects targeting the
cancer cells and have become one of the promising cancer therapeutics with possibly
broad applicability. Combination of HDAC inhibitors with the cytotoxic fluorouracil
(5-FU) showed additive and synergistic effects both in vitro and in vivo. To explore
the possibility in cancer therapy of a bivalent agent that combines two bioactive
groups within a single molecular architecture, we designed and synthesized
new dual-acting compounds by combining the bioactive fragment of MS-275, a clini-
cal HDACs inhibitor, with cytotoxic agent 5-FU. The target compounds 9a and 9b
showed comparable HDACs inhibition with MS-275 and moderate antiproliferative
acitivities against six cancer cells lines.
Keywords: Anticancer, HDAC, MS-275, Multitarget, 5-Fluorouracil.
5-Fluorouracil (5-FU) is one of the clinical antitumor
drugs most frequently used for treating a wide range of solid
tumors, such as colorectal cancer, stomach and breast cancer.
However, the clinical applications of 5-FU are subjected to
great limitations because of its short plasma half-life, poor
tumor selectivity and high incidences of toxicity in gastroin-
testinal tract, the bone marrow central, nerve system, skin
and so on [21]. Therefore, to overcome these problems, a lot
of novel 5-FU derivatives have been developed with high
efficiency and much less toxicity, such as Floxuridine®,
Carmofur®, Doxifluridine®, Capecitabine®, Atofluding® and
so on [22, 23]. The common feature of these derivatives is
that they are all N1-modi ed or N3-modi ed derivatives
through different biodegradable linkers [24].
1. INTRODUCTION
Histone deacetylases (HDACs) are a family of hydrolytic
enzymes that catalyze the removal of acetyl groups from the
side chain of lysines in histone. Recent studies have reported
that HDACs play a significant role in epigenetic control of
gene expression. Aberrant histone acetylation is associated
with the development of numerous malignancies [1-3].
HDACs are well conserved enzymes and there are 18 mem-
bers of human HDACs which are classified into 4 classes
according to their homology to yeast prototypes, subcellular
localization and function: Class I (HDACs 1, 2, 3, and 8), II
(HDACs 4, 5, 6, 7, 9, and 10), IV (HDAC 11), Class III
HDACs (SIRT 1-7). All the members of Classes I, II, and IV
HDACs are zinc2+-dependent enzymes, whereas class III
HDACs are NAD+-dependent [4].
In recent study, Sylwia Flis, et al. have con rmed that
combination of 5-FU with MS-275 could induce cell cycle
perturbation and caspase-dependent apoptosis of colorectal
carcinoma (CRC) cells [20]. Additionally, they also indi-
cated that MS-275 synergistically potentiated cytotoxic ef-
fects of 5-FU in SW48, HT-29 and Colo-205 cell lines [20].
Over the past few years, over 490 clinical trials of more
than 20 HDAC inhibitors (HDACIs) have been initiated,
among which, MS-275 is an orally active synthetic ben-
zamide derivative that functions as a selective inhibitor of
primary class I (HDACs 1 and 3) [5-7]. MS-275 has been
evaluated in multiple Phase I and II trials as therapy for ad-
vanced and/or refractory solid tumors and hematologic ma-
lignancies [8-13]. In the meanwhile, combinational therapy
of MS-275 with other agents is now being further explored
in preclinical and clinical trials, such as, exemestane [14],
erlotinib [15], 5-azacitidine [16, 17], 13-cis-retinoic acid [18,
19], 5-fluorouracil [20] and so on.
On the basis of these premises, following multi-target
approach [25, 26], we designed and synthesized a novel
multi-target antitumor agent 9a by replacing the pyridine cap
group of MS-275 with its bioisostere, the cytotoxic agent 5-
FU (Fig. 1). Compared with the parent compound MS-275,
incorporation of the more hydrophilic 5-FU group might
dramatically increase the solubility of compound 9a. Moreo-
ver, the carbamate linker has been successfully used for pro-
drugs of norfloxacin [27], entacaponeo [28], pseudomycins
[29] and so on, as this linker was labile and could be cleaved
by the enzyme in vivo. Therefore, we assumed that com-
pound 9a could not only perform HDACs inhibition as a
single molecule, but also act as a prodrug, of which the car-
bamate bond cleavage in vivo could release 5-FU and an-
*Address correspondence to these authors at the Department of Medicinal
Chemistry, School of Pharmaceutical Sciences, Shandong University, 44
West Wenhua Road, 250012 Ji’nan, Shandong, PR China; Tel: 86-531-
88382264; 86-531-88382009; Fax: 86-531-88382264;
E-mails: wenfxu@163.com; zhangyingjie@sdu.edu.cn
1875-6638/16 $58.00+.00
© 2016 Bentham Science Publishers

Discovery of Multi-target Anticancer Agents
Medicinal Chemistry, 2016, Vol. 12, No. 1
31
Fig. (1). Schematic representation of the design and chemical structure of 9a, 9b. To create 9a, 9b we introduced 5-FU with a flexible side
chain onto the backbone of the HDAC inhibitor (MS-275).
aReagents and conditions: (a) TFAA, 25°C, 2h; (b) TBTU, TEA, DCM. ; (c) (Boc)2O, TEA, THF; (d) K2CO3, aqueous MeOH, 25°C, 10h ; (e) NaHCO3, BTC,
2h; (f) 37% oxymethylene; (g) TEA, 1,4-dioxane; (h) dry HCl in EtOAc.
Scheme 1. Synthesis of Compound 9a, 9ba.
other HDACs inhibitor 9am to exert synergistic antitumor
effects (Fig. 1). In order to investigate the effects of different
linker length on compound property, compound 9b was de-
signed and synthesized.
accomplished using 5-FU in the presence of 37% oxymeth-
ylene. Reaction of 7 with 6a and 6b in the presence of TEA
gave the compounds 8a and 8b. Subsequent deprotection
gave the target products 9a and 9b.
2. RESULTS AND DISCUSSION
2.1. Chemistry
2.2. HeLa Cell Nuclear Extract Inhibition of the Target
Compounds 9a, 9b
We used HeLa cell nuclear extract as the HDACs en-
zyme source to efficiently screen our compounds 9a and 9b.
In this assay, MS-275 was used as positive control. The IC50
values (μmol/L) towards HeLa extract are shown in Table 1.
The results showed that the inhibitory activities of 9a and 9b
were comparable with that of MS-275. The IC50 values were
5.92 ± 0.75, 2.31 ± 0.24 and 2.09 ± 0.11 μM for 9a, 9b and
MS-275, respectively, which indicated that replacement of
the pyridine ring of MS-275 with 5-FU almost had no effect
to its inhibitory potency against HDACs.
Compounds 9a and 9b were prepared following the syn-
thetic route illustrated in Scheme 1. The starting materials 1a
(p-aminomethylbenzoic acid) and 1b (p-aminobenzoic acid),
were protected by trifluoroacetic anhydride (TFAA), fol-
lowed by condensation reaction to give compounds 3a and
3b, respectively. Boc-protected products 4a and 4b were
treated with K2CO3 in aqueous MeOH to afford the interme-
diates 5a and 5b, respectively. The isocyanates 6a and 6b
were synthesized from 5a and 5b by reaction with triphos-
gene (BTC) in the presence of NaHCO3. Synthesis of 7 was

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Medicinal Chemistry, 2016, Vol. 12, No. 1
Jiang et al.
Table 1. Inhibitory activities of 9a, 9b, and MS-275 against
HDACs.
ferent in the pyrimidine ring and pyridine ring of cap group.
Detailed interactions between compounds and HDAC3 were
shown in Fig. (2c) (9a) and (2d) (MS-275). Comparing the
two figures, we could find in the cap group, both 9a and MS-
275 could not form hydrogen bonds with HDAC3. However,
in the linker and ZBG fragment, 9a could form two hydrogen
bonds with amide NꢀH of Asp104 and amide NꢀH of
Gly154, while MS-275 could form three hydrogen bonds
with amide NꢀH of Asp104, amide NꢀH of Gly154 and am-
ide NꢀH of Asp181. Therefore, we postulated that the lost
hydrogen bond with Asp81 might be the reason why 9a was
less potent than MS-275.
Compound
IC50 against HeLa Extract (μM)a
9a
9b
5.92±0.75
2.31±0.24
2.09±0.11
MS-275
aAssays were performed in three times; values are shown as mean ± SD.
2.3. Molecular Docking
2.4. Antiproliferative Activity Assay
In order to explore the interaction between our target
compounds and HDAC, 9a and MS-275 [30, 31] were cho-
sen to be constructed using a Sybyl/Sketch module. Fig. (2)
showed the docked conformation of compounds 9a and MS-
275. The conformations showed in Fig. (2a and 2b) demon-
strated the docking modes of the two compounds were simi-
lar in the linker and ZBG fragment, while they seemed dif-
Aiming to investigate the antiproliferative activities of 9a
and 9b, we selected 6 types of hematological or solid tumor
cell lines which were most frequently used in evaluating
HDACs to test our compounds (Table 2 and Fig. 3). Overall,
9a and 9b were less potent than MS-275 in the examined cell
lines, which did not meet our expectations. We inferred that
Fig. (2). (a-d) Proposed binding mode of compounds 9a (a,c) and MS-275 (b,d) with HDAC3. The green sphere is zinc ion, and the dashed
lines represent the hydrogen bonds (atom types: H = white; N = blue; O = red). (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this paper).
Table 2. Antiproliferative activities of 9a, 9b, and MS-275 against six tumor cell lines (IC50 in μMa).
Cell Lines
9a
9b
MS-275
K562
A549
5.70±1.06
16.50±1.43
7.48±1.01
7.83±0.64
19.99±2.59
8.54±0.87
15.49±1.20
17.30±1.91
15.47±2.43
NDb
1.02±0.25
4.45±0.92
1.54±0.63
6.18±1.02
16.34±2.03
4.98±0.89
30.43±3.86
U266
PC-3
26.54±1.07
23.79±5.51
53.74±5.71
100.20±16.32
HCT-116
ES-2
HL-7702
aAssays were performed in three times; values are shown as mean ± SD. bNot determined.

Discovery of Multi-target Anticancer Agents
Medicinal Chemistry, 2016, Vol. 12, No. 1
33
Fig. (3). Antiproliferative activities of 9a, 9b, and MS-275 against six tumor cell lines.
Fig. (4). Stability of compound 9a in vitro. Points were achieved after 0, 0.5, 1, 2, 4, 8, and 24 h, respectively, and values are shown as mean
+ SD.
the modest potency of these two compounds only came from
their HDACs inhibition as single molecules, and their car-
bamate bonds could not been hydrolyzed to release 5-FU and
another HDACs inhibitor. However, MS-275 inhibited non-
malignant HL-7702 cell, but 9a had relatively lower cytotox-
icity (Table 2).
mixture was precipitated, extracted and analyzed by HPLC.
Unfortunately, 9a was very stable in arti cial gastric juice,
arti cial intestinal juice, and human plasma (Fig. 4). This
may explain why compounds 9a and 9b displayed less po-
tent activities than MS-275 in in vitro antiproliferative assay.
However, the stability in vitro could not be the evidence that
9a would not release 5-FU in vivo, the pharmacokinetic
study of 9a in vivo is underway in our lab.
2.5. Stability of Compound 9a in vitro.
To validate our aforementioned inference, the stability
study of 9a in arti cial gastric juice, arti cial intestinal juice,
and human plasma was studied using a HPLC method.
Brie y, the hybrid 9a was incubated into each condition at
37°C for 24 h. At predetermined time points, a sample of the
CONCLUSION
In this study, we designed and synthesized two dual-
acting compounds 9a and 9b following multi-target ap-
proach. In HDAC inhibitory assay, 9a and 9b showed similar

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Medicinal Chemistry, 2016, Vol. 12, No. 1
Jiang et al.
HDAC inhibitory activity with MS-275. Though their in
vitro antiproliferative activities were disappointing due to
their unexpected in vitro stability and disability of releasing
5-FU, their detailed in vivo pharmacokinetic profiles deserve
further investigation. Moreover, considering there were
many successful examples of carbamate-based prodrugs, we
hoped that structural modification of our compounds 9a and
9b using medicinal chemistry methods could lead to promis-
ing analogues with ideal stability which could release 5-FU
in vitro and in vivo. The proof of concept described in this
research could also be used in other compound design and
synthesis.
3.1.3. General Procedure for the Preparation of 4a and 4b
tert-butyl-(2-(4-((2,2,2-trifluoroacetamido)methyl)ben-
zamido)phenyl)carbamate(4a). The solution of 3a (1.8 g, 5
mmol) in CH2Cl2 was added TEA (2.1 mL, 15 mmol) for
neutralization, followed by the addition of (Boc)2O (2.2 g, 10
mmol) under ice bath. After 0.5h, the reaction mixture was
stirred at 25ºC for another 12h. The resulting solution was
washed by 1 M aqueous citric acid (3 ꢁ 30 mL), 10% Na-
HCO3 solution (3 ꢁ 30 mL), and saturated brine (3 ꢁ 30 mL)
and dried over anhydrous sodium sulfate overnight. After
evaporating the solvent, the crude material 4a was puri ed
by ash chromatography to afford a white solid (1.7 g, 78%
yield). mp: 162-164ºC, 1H-NMR(400 MHz DMSO-d6): ꢀ
1.44 (s, 9H), 4.45-4.49 (m, 2H), 7.15-7.20 (m, 2H), 7.40-
7.45 (m, 2H), 7.53-7.55 (m, 2H), 7.91-7.95 (m, 2H), 8.67 (s,
1H), 9.81 (s, 1H), 10.03-10.09 (m, 1H). ESI-MS m/z: 438.5
[M + H]+.
3. EXPERIMENTAL SECTION
3.1. Chemistry
All materials, reagents and solvents were purchased from
commercial suppliers and used without further purification
unless otherwise stated. All reactions were monitored via
thin-layer chromatography with 0.25 mm silica gel plates
(60GF-254), while the UV light was used to visualize the
spots. The compounds were puri ed via column chromatog-
raphy which was performed using silica gel or C18 silica gel.
NMR spectra were recorded with a Bruker DRX spectrome-
ter at 400 MHz, which use the TMS as an internal standard.
High-resolution mass spectra were performed by Shandong
Analysis and Test Center in Ji’nan, China. ESI-MS spectra
were determined on an API 4000 spectrometer. Melting
points were determined with an electrothermal melting point
apparatus and were uncorrected.
3.1.4. General Procedure for the Preparation of 5a and 5b
tert-butyl-(2-(4-(aminomethyl)benzamido)phenyl)carba-
mate (5a). A solution of 4a (1.3 g, 3 mmol) and K2CO3 (1.1
g, 7.5 mmol) in MeOH-H2O (1:1) was maintained at 25°C
for 10h. The solution was concentrated under reduced pres-
sure. The EtOAc (3 ꢁ 40 mL) was added to the residue. The
solution of EtOAc was washed with saturated NaHCO3 solu-
tion (3 ꢁ 40 mL), and saturated brine (3ꢁ 40 mL), dried with
anhydrous sodium sulfate over night, and evaporated under
vacuum to obtain 0.8 g of white solid powder 5a (0.8 g,
yield: 78%).
3.1.1. General Procedure for the Preparation of 2a and 2b
[32]
3.1.5. General Procedure for the Preparation of 6a and 6b
tert-butyl-(2-(4-(isocyanatomethyl)benzamido)phenyl)
carbamate (6a). To a solution of NaHCO3 (0.4 g, 4.8 mmol)
in H2O was added CH2Cl2 and 5a (0.7 g, 1.9 mmol), then the
solution was cooled to 0°C. Triphosgene (0.3 g, 1.0 mmol)
was charged in one portion. After stirred at room tempera-
ture for 1h, the organic phase was separated and the aqueous
phase was extracted with CH2Cl2. The combined organic
phase were washed with water and saturated brine, dried
with anhydrous sodium sulfate, and evaporated under vac-
uum to give the 6a as a liquid, which was used for next step
directly.
4-((2,2,2-trifluoroacetamido)methyl)benzoic acid (2a).
Tri uoroacetic anhydride (5.9 mL, 41.3 mmol) was added in
small portions to solid 4-(aminomethyl) benzoic acid (2.5 g,
16.5 mmol) at 4°C. Upon completion of addition, the reac-
tion mixture was homogeneous. Stirring was continued at
room temperature for 2 h, and then ice water was added to
precipitate the product. The white solid 2a was precipitated
and collected by ltration. (3.3 g, 81% yield). mp:214-
215ºC, 1H-NMR(400 MHz DMSO-d6): ꢀ 4.47 (d, J = 6.0 Hz,
2H), 7.40 (d, J = 8.2 Hz, 2H), 7.94 (d, J = 8.2 Hz, 2H), 10.07
(s, 1H), 12.94 (s, 1H). ESI-MS m/z: 248.5 [M + H]+.
3.1.6. General Procedure for the Preparation of 7 [33]
3.1.2. General Procedure for the Preparation of 3a and 3b
5-fluoro-1-(hydroxymethyl)pyrimidine-2,4(1H,3H)-
dione (7). 5-FU (1.1 g, 8.5 mmol) was dissolved in 37% for-
maldehyde solution (1.53 g, 18.9 mmol) and the reaction
mixture was kept at 60°C for 2 h. After evaporating the sol-
vent, the residue was dried under vacuum to get colorless oil
7 which was directly used without further purification.
N-(2-aminophenyl)-4-((2,2,2-trifluoroacetamido)meth-
yl)benzamide (3a). To a solution of 2a (2.5 g, 10 mmol) in
anhydrous THF was added 2-(1H-benzotriazole-1-yl)-
1,1,3,3-tetramethyluronium tet-ra uoroborate (TBTU, 3.5 g,
11 mmol), followed by TEA (1.5 mL, 11 mmol). After 30
min, 1,2-diaminobenzene (1.0 g, 9 mmol) was added. After
5h, the solution of THF was evaporated with the residue
taken up in EtOAc. The organic phase was washed with 10%
NaHCO3 solution (3 ꢁ 30 mL), and brine (3 ꢁ 30 mL), dried
over anhydrous sodium sulfate overnight, and the solvent
was evaporated under vacuum. The crude product 3a was
puri ed by recrystallization with saturated chloride hydrogen
in dry ethyl acetate to get a white pure hydrochloride solid
(1.9 g, 56% yield).
3.1.7. General Procedure for the Preparation of 8a and 8b
(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)me-
thyl-4-((2-tert-butyl-aminop-henyl)carbamoyl) benzylcar-
bamate (8a). The solution of 7 (0.5 g, 3 mmol) in 1,4-
dioxane was added TEA (0.4 mL, 3 mmol) followed by the
addition of 6a (0.7 g, 2 mmol) under ice bath. The ice bath
was removed. After stirred at 30°C for 5h, 1,4-dioxane was
evaporated under vacuum and the residue was dissolve in

Discovery of Multi-target Anticancer Agents
Medicinal Chemistry, 2016, Vol. 12, No. 1
35
EtOAc (3 ꢀ 40 mL). The organic phase was washed with 1
M aqueous citric acid, saturated NaHCO3 solution and satu-
rated brine for 3 times, dried with anhydrous sodium sulfate
over night, evaporated and the residue chromatographed,
using CH2Cl2/EtOAc(1:3) as the mobile phase, to obtain 0.4
g of white solid 8a (0.4 g, yield: 38%).
structures were constructed using the Sybyl/Sketch module
and optimized via Powell’s method by the Tripos force field
with convergence criterion set at 0.05 kcal/ (Å mol), and
assigned charges with the Gasteiger–Hückel method.
3.4. In Vitro Antiproliferative Assay
All cell lines were grown in medium (RPMI1640) con-
taining 10% FBS at 37°C in a 5% CO2 humidified incubator.
Cell proliferation assay was studied using the MTT ((3-[4,
5-dimethyl-2-thiazolyl]-2,5-diphe-nyl-2H-tetrazolium bro-
mide)) method. Briefly, cells were seeded into a 96-well cell
plate. After incubation for 12 h, different concentrations of
compound sample were added in complete medium and in-
cubated for a further 48 h. Then, a 0.5% MTT solution was
added to each well and cultured for 4 h. After the media were
removed, formazan formed from MTT were dissolved in 150
μL of DMSO. Absorbance was then measured using an
ELISA reader at 570 nm.
3.1.8. General Procedure for the Preparation of 9a and 9b
(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)me-
thyl-4-((2-aminopheny- l)carbamoyl)benzylcarbamate hy-
drochloride (9a). 8a (0.4 g, 0.7 mmol)was dissolved in 20
mL saturated chloride hydrogen in dry ethyl acetate. Let the
solution stirring at room temperature overnight. 0.3 g white
powder 9a was obtained after removing the solvent under
1
vacuum. (0.3g, yield: 92%). mp:160-162ºC, H-NMR(300
MHz DMSO-d6): ꢁ 4.29 (d, J = 6.0 Hz, 2H), 5.54 (s, 2H),
5.76 (s, 1H), 6.68 (t, J = 7.2 Hz, 1H), 6.84 (d, J = 7.2 Hz,
1H), 7.03(dt, J1 = 1.2 Hz, J2 = 8.1 Hz, 1H), 7.20 (d, J = 7.5
Hz, 1H), 7.39 (d, J = 8.1 Hz, 2H), 7.95 (d, J = 8.1 Hz, 2H),
8.12 (d, J = 6.6 Hz, 1H), 8.19 (d, J = 6.0 Hz, 1H), 9.68 (s,
1H), 11.98 (d, J = 4.2 Hz,1H). HRMS (AP-ESI) m/z calcd
for C20H18FN5O5 [M + H]+ 428.1685, found 428.1688.
3.5. Stability of Test Compound in vitro
Preparation of artificial gastric juice, artificial intestinal
juice, and Human plasma were conducted as previously de-
scribed [30]. Artificial gastric juice, artificial intestinal juice,
and human plasma added to the stock solution of 9a (4
mg/mL in CH3CN) and incubated at 37°C for 24 h. At
scheduled times sample aliquots were collected and the en-
zymatic reaction was quenched by adding acetonitrile. The
samples were extracted with 600 μL acetonitrile and were
filtered (0.22 ꢂm) after shocking 30s and centrifugation at
12,000 rpm for 10 min. Analytical HPLC was performed on
Agilent 1200 HPLC instrument using a ODS HYPERSIL
column (5 μm, 4.6 mm ꢀ 250 mm), compound was eluted
with 22% acetonitrile/78% Phosphate Buffered Saline
(PH3.0) over 20min. The absorbance was measured at 233
nm, the flow rate was 1 mL/min and the quantity of injection
was 40 μM.
Compound 2b-9b was prepared following the general
procedure as described above.
(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)
methyl-(4-((2-aminopheny-l)carbamoyl)phenyl)carba-
mate hydrochloride (9b) White powder, 87% yield mp:179-
1
181ºC, H-NMR(400 MHz DMSO-d6): ꢁ 5.68 (s, 2H), 7.35
(t, J = 7.6 Hz, 1H), 7.41 (t, J = 7.6 Hz, 1H), 7.47 (d, J = 7.6
Hz, 1H), 7.57 (d, J = 7.6 Hz, 1H), 7.63 (d, J = 8.6 Hz, 2H),
8.11 (d, J = 8.7 Hz, 2H), 8.21 (d, J = 6.5 Hz, 1H), 10.39 (s,
1H), 10.45 (s, 1H), 12.04 (d, J = 4.9 Hz, 1H). HRMS (AP-
ESI) m/z calcd for C19H16FN5O5 [M + H]+ 414.1359, found
414.1358.
3.2. In Vitro HDAC Inhibition Assay
In particular, 10 μL of enzyme solution (HeLa nuclear
extract) was added to different concentrations of test com-
pounds (50 μL) and incubated for 5 min at 37°C, then the
specific fluorogenic substrate (Boc-Lys-(acetyl)-AMC) was
used at 40 μL. Samples were incubated for 1 h at 37°C and
stopped by the addition of 100 μL of 2 ꢀ HDAC developer in
present of trypsin and TSA. After incubation for 20 min, the
fluorescence intensity was detected with excitation-emission
wavelengths of 390-460 nm, respectively. The HDAC inhi-
bition ratios were calculated as a percentage of activity com-
pared with the control group and the IC50 values for the test
compounds were calculated using a regression analysis of
the concentration/inhibition data.
CONFLICT OF INTEREST
The authors confirm that this article content has no con-
flict of interest.
ACKNOWLEDGEMENTS
This work was supported by National Scientificand
Technological Major Project of Ministry of Science and
Technology of China (Grant no. 2011ZX09401-015), Na-
tional Natural Science Foundation of China (Grant no.
21302111 and 21172134), Postdoctoral Innovation Project
Foundation of Shandong Province (Grant No.201303090),
China Postdoctoral Science Foundation funded project
(Grant no. 2014T70654), Independent Innovation Founda-
tion of Shandong University, IIFSDU (Grant no.
2013GN013), and National High-tech R&D Program of
China, 863 Program (Grant No.2014AA020523).
3.3. Molecular Docking Analysis
The docking study of Compounds and the active site of
HDAC3 were performed using Sybyl/FlexX module. Other
docking parameters used in the program were remained the
default values. The protein structure utilized was PDB code
4A69. During the first step, the protein structure was treated
by removing water molecules, adding hydrogen atoms, regu-
lating atom types, and assigning AMBER7 FF99 charges.
Then, the protein structure was further optimized by per-
forming a 100-step minimization process. The molecular
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Received: November 14, 2014
Revised: July 08, 2015
Accepted: July 08, 2015