Pyrimidinedione-mediated selective histone deacetylase 6 inhibitors with antitumor activity in colorectal cancer HCT116 cells

Article abstract of DOI:10.1039/c5ob01509j

We synthesized a series of pyrimidinedione derivatives and evaluated their activities. The results indicate that compound 6, 4-[5-fluoro-2,6-dioxo-3-(tetrahydro-furan-2-yl)-3,6-dihydro-2H-pyrimidin-1-ylmethyl]-N-hydroxy-benzamide, exhibits potent antiproliferative activity, apoptosis induction with cleavage of caspase and PARP, and enhanced tendency to inhibit HDAC6 (IC₅₀ = 12.4 nM) activity over HDAC1 (IC₅₀ = 1710 nM) and HDAC2 (IC₅₀ = 5500 nM). Compound 6 also inhibits tumor growth and is less toxic than parent 4in vivo. These data provide compelling evidence that compound 6 is a potential antitumor compound with HDAC6 targeted inhibitory activity and may be tested for preclinical investigation for cancer treatment.

Full text of DOI:10.1039/c5ob01509j

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Pyrimidinedione-mediated selective histone
deacetylase 6 inhibitors with antitumor activity in
Cite this: DOI: 10.1039/c5ob01509j
colorectal cancer HCT116 cells†
Yi-Min Liu,a Hsueh-Yun Lee,a Mei-Jung Lai,b Shiow-Lin Pan,c Hsiang-Ling Huang,a
Fei-Chiao Kuo,a Mei-Chuan Chen*d and Jing-Ping Liou*a
We synthesized a series of pyrimidinedione derivatives and evaluated their activities. The results indicate
that compound 6, 4-[5-fluoro-2,6-dioxo-3-(tetrahydro-furan-2-yl)-3,6-dihydro-2H-pyrimidin-1-
ylmethyl]-N-hydroxy-benzamide, exhibits potent antiproliferative activity, apoptosis induction with clea-
vage of caspase and PARP, and enhanced tendency to inhibit HDAC6 (IC50 = 12.4 nM) activity over
HDAC1 (IC50 = 1710 nM) and HDAC2 (IC50 = 5500 nM). Compound 6 also inhibits tumor growth and is
less toxic than parent 4 in vivo. These data provide compelling evidence that compound 6 is a potential
antitumor compound with HDAC6 targeted inhibitory activity and may be tested for preclinical investi-
gation for cancer treatment.
Received 22nd July 2015,
Accepted 17th August 2015
DOI: 10.1039/c5ob01509j
www.rsc.org/obc
tuins), and IV (HDAC11).5 Class I and II HDACs, which are
called classical HDACs, require zinc ions to carry out the de-
Introduction
The battle against colorectal cancer (CRC) has lasted for many acetylation of lysine residues. Some groups such as hydroxa-
years due to the severe threat it poses to human life worldwide. mic acid, 2-aminophenylamide, and thiol in HDAC inhibitors
CRC caused an estimated 1 361 000 new cases and 694 000 are able to chelate the zinc ion in the active site, which results
deaths in 2012, which is the third leading cause of death by in the shutdown of enzymatic function.6 To date, three hydro-
cancer.1 This adenocarcinoma is generated from normal epi- xamic acid-containing HDAC inhibitors have been approved by
thelial cells due to continuous genetic and epigenetic the US FDA: SAHA (1),7 PXD101 (2),8 and LBH589 (3)9 (Fig. 1),
changes;2 therefore, a bioactive agent able to restore the epige- which makes hydroxamic acid a good starting point for the
netic process becomes a potential strategy for the treatment of development of HDAC inhibitors. In addition to the zinc-
CRC. Epigenetics regulate gene expression through modifi- binding domain, the pharmacophore of HDAC inhibitors also
cations of DNA, histones, and chromatin, without changing consists of linker and cap parts. Our previous study using
the DNA sequence.3 The deacetylation of histones at the azaindole as a cap provided a series of azaindolylsulfonamides
N-terminal lysine residues is a crucial epigenetic modification as potent and selective HDAC6 inhibitors, drawing our atten-
which is mediated by histone deacetylase (HDAC). The deregu- tion to this moiety.10 Tegafur (4), a prodrug of 5-fluorouracil
lated function of HDAC is likely related to oncogenesis; as a (5-FU, 5) is metabolized by CYP2A6 to release 5-FU.11 It
result, HDAC becomes an attractive target to reverse the aber-
rant regulation.4 HDAC is comprised of four classes: I (HDAC
1, 2, 3, and 8), II (HDAC 4, 5, 6, 7, 9, and 10), III HDAC (sir-
aSchool of Pharmacy, College of Pharmacy, Taipei Medical University, 250 Wuxing
Street, Taipei 11031, Taiwan. E-mail: jpl@tmu.edu.tw;
Tel: +886-2-27361661 ext 6130
bCenter for Translational Medicine, Taipei Medical University, Taiwan
cThe Ph.D program for Cancer Biology and Drug Discovery, College of Medical
Science and Technology, Taipei Medical University, Taipei, Taiwan
dPh.D Program for the Clinical Drug Discovery from Botanical Herbs, College of
Pharmacy, Taipei Medical University, Taiwan. E-mail: mcchen1250@tmu.edu.tw;
Tel: +886-2-27361661 ext 6184
†Electronic supplementary information (ESI) available: The HPLC purity results
of target compounds 6–11, 1H-NMR and 13C-NMR spectra of compounds 6–11.
See DOI: 10.1039/c5ob01509j
Fig. 1 FDA-approved hydroxamic acids.
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attracted much attention and entered phase I clinical trials in the reaction of 4 with 4-formylbenzyl chloride. This is followed
1985. The clinical tests were however discontinued in the by Wittig olefination with methyl(triphenylphosphoranylidene)
United States due to CNS toxicity.12 The usage of tegafur in acetate, and ester hydrolysis to obtain the corresponding cin-
combination with uracil and folinic acid is currently a first-line namic acid 15. This compound is allowed to react with
treatment for metastatic colorectal cancer. In an attempt to NH2OTHP to form a N-protected N-hydroxamide which is
explore the cap effect on the biological activity, this study aims hydrolyzed by 10% TFA(aq) to give compound 8. Compound 9
to link the pyrimidinedione moiety in 4 and 5 with hyrdro- is prepared by the same procedure used to synthesize 7 from
xamic acid and 2-aminobenzamide groups using various 15. To synthesize compounds 10 and 11 containing a long ali-
linkers. Compounds 6–11 were synthesized and the relevant phatic chain, compound 4 is reacted with various nitrobenzyl
biological assays are discussed below.
chlorides, and this is followed by reduction of the nitro group
to afford the corresponding amine 16. Subsequent amidation
of 16 with monomethyl suberate followed by hydrolysis of the
ester provides carboxylic acid 17, which is converted to hydro-
xamates 10 and 11 by the same procedure used to generate 6
from 13 (Fig. 2).
Chemistry
Scheme 1 shows the synthesis of designed compounds 6–11.
Compound 4 reacts with methyl 4-chloromethylbenzoate in
the presence of K2CO3, yielding compound 12. The ester group
in 12 is hydrolyzed by LiOH to afford the corresponding car-
boxylic acid 13 which is amidated by reaction with NH2OBn in
the presence of coupling reagents EDC·HCl and HOBt. The
Biological evaluation
In vitro cell growth inhibition
crude product is debenzylated using hydrogen and catalytic Table 1 shows the antiproliferative activities of compounds
Pd/C to provide the hydroxamic acid (6). Compound 13 is 6–11 and reference compounds, 4 and 5, against three human
made to react with o-phenylenediamine, EDC·HCl, and HOBt cancer cell lines, colorectal carcinoma HCT116 cells, non-
to furnish the corresponding benzamide 7. Preparation of small cell lung carcinoma A549 cells, and human prostate
compounds 8 and 9, which have an ethylene group between cancer PC3 cells. Compared to compound 5, five compounds
the hydroxamic acid moiety and the benzene ring, starts from (6–8 and 10–11) show comparable or slightly enhanced activity
Scheme 1 Reagents and conditions: (a) methyl 4-(chloromethyl)benzoate, K2CO3, DMF, rt; (b) 1 M LiOH(aq), p-dioxane, 40 °C; (c) for 6, 10, and 11:
i. NH2OBn, EDC·HCl, HOBt, NMM, DMF, rt; ii. 10% Pd/C, H2, MeOH, rt; (d) for 7 and 9: o-phenylenediamine, EDC·HCl, HOBt, NMM, DMF, rt; (e)
i. 4-formylbenzyl chloride, K2CO3, DMF, rt; ii. methyl (triphenylphosphoranylidene)acetate, DCM, rt; (f) for 8: i. NH2OTHP, EDC·HCl, HOBt, NMM,
DMF, rt; ii. 10% TFA(aq), MeOH, rt; (g) i. 4-nitrobenzyl bromide or 3-nitrobenzyl chloride, K2CO3, DMF, rt; ii. 10% Pd/C, H2, MeOH, rt; (h)
i. monomethyl suberate, EDC·HCl, HOBt, NMM, DMF, rt; ii. 1 M LiOH(aq), p-dioxane, 40 °C.
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against HCT116 cells and, in addition, these compounds show
remarkable enhancement in PC3 inhibitory activity when com-
pared with 5. For the inhibitory activity against A549 cells, 6, 7,
8, and 10 show a 2- to 3-fold decrease of potency, while 9 and
11 have negligible activity. The presence of a CvC double
bond in compound 9 results in a remarkable decrease of
activity in A549 and PC3 cells, as compared with compound 7.
In brief, the combination of tegafur (4) with various HDACi-
specific side chains leads to compounds that retain tegafur’s
antiproliferative activity.
HDAC isoform inhibition
The synthetic compounds (6, 7, 8, 10, and 11) and reference
compounds suberoylanilide hydroxamic acid (SAHA) and Tri-
chostatin A (TSA) were assayed for HDAC isoform inhibitory
activity (Table 2). Compounds 10 and 11 show potent HDAC6
activity at the low nM level, with IC50 values of 5.1 and 7.3 nM,
respectively. Comparison of compounds 10 and 11 indicate
that the N-hydroxysuberamide group favors the meta position
and contributes the selective HDAC6 inhibitory activity over
other isoforms. Notably, compound 6, with IC50 of 12.4 nM in
HDAC6, shows ∼137- and ∼443-fold increases in HDAC6
inhibitory activity over HDAC1 and HDAC2, respectively. To
further confirm the HDAC isozyme selectivity, we performed
western blot analysis to evaluate the protein levels of HDAC
inhibition biomarker acetyl-histone H3, which results mostly
from class I HDAC inhibition and acetyl-tubulin, resulting
from HDAC6 inhibition in treated cells. As shown in Fig. 3,
compounds 8, 10, and 11, with low selectivity between human
HDAC isoforms, affects both acetyl-histone H3 and acetyl-
tubulin protein expression levels. Compound 7 exhibits stron-
ger inhibition of HDAC1 and HDAC2 enzymes, resulting in
upregulation of acetyl-histone H3 without significant pro-
duction of acetyl-tubulin.
Fig. 2 The design of pyrimidinedione-containing derivatives.
Table 1 Antiproliferative activity (IC50) of compounds 6–11
IC50 SDa (μM)
Compd
HCT116 cells
A549 cells
PC3 cells
6
7
8
9
10
11
4
8.08 0.79
4.70 0.91
4.20 0.58
22.97 1.04
4.59 0.23
6.48 0.73
>50
34.94 1.21
38.96 1.95
27.06 2.38
>60
42.31 1.93
>60
11.6 0.87
12.25 1.36
12.07 0.26
>60
10.46 1.72
16.42 2.23
>50
Colony formation ability of HCT116 colorectal cancer cells
treated with compound 6
>50
5
10.98 0.92
19.76 2.9
>30
To determine the colony forming ability of cells treated with
compound 6, a long-term clonogenicity assay was carried out
to assess the compound’s capacity to cause irreversible growth
a SD: standard deviation, all experiments were independently
performed at least three times.
Table 2 HDAC inhibition activity and isoform selectivity of tested compounds
IC50a (μM)
Selective ratio
Compd
HDAC1
HDAC2
HDAC6
HDAC8
HDAC1/6
HDAC2/6
HDAC8/6
6
7
8
1.71
0.258
1.48
0.0761
0.234
0.286
0.00827
5.50
1.06
4.24
0.257
0.791
1.01
0.0124
—
0.16
—
∼137
∼443
∼12
0.0373
0.00514
0.00735
0.0281
0.00199
0.552
0.331
0.548
1.09
0.551
∼39
∼14
∼30
∼10
∼4
∼113
∼5
∼107
∼35
∼8
∼14
∼64
∼74
∼38
∼276
10
11
SAHA
TSA
0.0174
a These assays were conducted by the Reaction Biology Corporation, Malvern, PA. All compounds were dissolved in DMSO and tested in 10-dose
IC50 mode with 3-fold serial dilution starting at 10 μM.
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Fig. 3 The protein expression levels of acetyl-histone H3 and acetyl-tubulin by tested compounds. Cells were treated with indicated compounds
with various concentrations for 24 h then subjected to western blot analysis.
Fig. 4 Colony formation ability of compound 6 in HCT116 cells. (A) Compound 6 suppressed colony formation and the numbers of colonies were
determined and are shown in (B). Clonogenic survival was assessed as described in the Experimental section.
arrest in HCT116 cells. HCT116 cells were exposed to various thymidylate synthase to form an additional higher molecular
concentrations of compound 6 or DMSO vehicle in 10% FBS- weight band in treated cells, which is the ternary complex
supplemented medium for 24 h, washed with fresh medium, formed by FdUMP-thymidylate synthase and CH2-THF (methyl-
trypsinized and plated for clonogenic cell survival. Concen- enetetrahydrofolate).13 However, compound 6 does not induce
tration-dependent decreases in the colony-forming capacities the upper band in drug-treated HCT116 cells, suggesting com-
of the drug-treated cells are observed (Fig. 4A and B), pound 6 does not have the ability to interfere with thymidylate
suggesting that irreversible growth arrest and reproductive cell synthase in cells.
death is caused by compound 6.
Suppression of survival signaling of compound 6 in HCT116
cells
The effects of compound 6 on cell cycle distribution in
HCT116 cells
Previous reports show epidermal growth factor receptor
(EGFR) can be a promising therapeutic target for colorectal
cancer since its expression level is associated with aggressive
disease and poor prognosis.14 To assess the protein expression
of EGFR and the activation of its downstream signaling pro-
teins, AKT and extracellular signal-regulated kinase (ERK) were
analyzed by western blotting. Treatment of compound 6
reduces the levels of EGFR, phosphorylated-EGFR, and the
downstream signaling proteins (Fig. 7). These findings show
that compound 6 exhibits promising results in triggering cell
death and, in parallel, the reduction of the survival signaling
networks in HCT116 cells.
To investigate the underlying mechanism of cell growth repres-
sion by compound 6, the effects of compound 6 on cell cycle
progression were assessed by flow cytometry. Treatment with
compound 6 induces concentration-dependent subG1 phase
accumulation (Fig. 5A and B), suggesting that cell apoptosis
may follow from treatment with compound 6. We next exam-
ined some apoptosis related proteins in HCT116 cells treated
with compound 6. As shown in Fig. 6A, apoptotic markers
such as PARP and caspases 3, 8, and 9 are activated by com-
pound 6. It is noteworthy that compound 6 exerts more potent
anticancer activity than the compound 4, which fails to cause
obvious cell apoptotic death (PARP and caspase 3 activation)
in HCT116 cells (Fig. 6B). Furthermore, we also investigated if
In vivo animal model for evaluation of antitumor activity
compound 6 has any inhibitory effect against thymidylate To further evaluate the in vivo efficacy of compound 6, thymic
synthase (TS) by western blot assay. As shown in Fig. 6C, 5-Fu nude mice bearing established HCT116 tumor xenografts were
(5) and Tegafur (4) were included as positive control to inhibit treated orally with compound 6, compound 4, and SAHA. The
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Fig. 5 The effects of compound 6 on cell cycle distribution in HCT116 cells. (A) Concentration-dependent effects of compound 6 on cell cycle pro-
gression. (B) Data shown are the means values of at least three independent experiments. Cells were treated with compound 6 with indicated con-
centrations for 48 h and assessed by flow cytometry.
Fig. 6 Compound 6 induces apoptosis in HCT116 cells. (A) Compound 6 induces apoptosis by activating PARP, and concentration-dependent clea-
vage of caspase 3, -8, and -9. (B) Compound 4 induces no significant apoptosis in cells compared with compound 6. Cells were treated with indi-
cated concentrations of indicated drugs for 48 h and the cell lysates were immunoblotted. (C) Compound 6 does not have the ability to interfere
with thymidylate synthase in cells. Cells were treated with indicated concentrations of indicated drugs for 12 h and the cell lysates were
immunoblotted.
FDA-approved pan-HDAC inhibitor SAHA, which has been ment with 100 and 200 mg kg−1 of compound 6 inhibited
demonstrated to exhibit antitumor effects in various cancer tumor growth dose-dependently. Notably, toxicity was observed
types, was used as a reference drug. As shown in Fig. 8, treat- by server body weight loss in the early phases of compound
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compound 6 has stronger inhibitory activity towards HDAC6,
and is 137- and 443-times more selective over HDAC1 and
HDAC2, respectively. Moreover, in vivo suppression of tumor
growth by compound 6 and induction of severe toxicity by 4
were observed in a HCT116 tumor xenograft model in nude
mice. As shown in the ESI (Fig. S2†), compound 1 exhibits
better potency than compound 6 in cell models. However,
compound 6 shows better potency than SAHA in xenograft
animal study (Fig. 8), suggesting the solubility, pharmaco-
dynamic properties, or even oral bioavailability between 1 and
6 may play crucial roles in this disparity. Previous reports have
shown that compound 1 has short elimination half-life15 and
poor bioavailability.16 These data suggest that the PK para-
meters may cause marked differences between in vitro and
in vivo potency of compound 1. In conclusion, our data offer
evidence for the safety and efficacy of compound 6, which dis-
Fig. 7 Significant suppression of EGFR and downstream signaling
molecules following treatment with compound 6. Cells were treated
with the indicated concentrations of compound 6 for 48 h and the cell
lysates were immunoblotted with the indicated antibodies.
plays antitumor activity in HCT116 cells and reveals
a
better therapeutic outcome than 4 or the FDA-approved HDAC
inhibitor 1.
Experimental section
General information
Chemistry. Nuclear magnetic resonance (1H NMR) spectra
were recorded with Bruker Fourier 300 and DRX-500 spectro-
meters (operating at 300 MHz and 500 MHz), with chemical
shifts in parts per million (δ) downfield from TMS, the internal
standard. High-resolution mass spectra (HRMS) were recorded
with a JEOL (JMS-700) electron impact (EI) mass spectrometer.
Elemental analyses were performed on a Heraeus varioIII-NCH
instrument. The purities of the final compounds were deter-
mined using an Agilent 1100 series HPLC system using a C-18
column (Agilent ZORBAX Eclipse XDB-C18 5 μm, 4.6 mm ×
150 mm) and were found to be ≥95%. Flash column chromato-
graphy was conducted using silica gel (Merck Kieselgel 60, no.
9385, 230–400 mesh ASTM). All reactions were conducted
under an atmosphere of dry nitrogen.
4-((5-Fluoro-2,6-dioxo-3-(tetrahydrofuran-2-yl)-2,3-dihydropyri-
midin-1(6H)-yl)methyl)-N-hydroxybenzamide (6). A mixture of
13 (0.13 g, 0.39 mmol), EDC·HCl (0.11 g, 0.59 mmol), HOBt
(0.06 g, 0.47 mmol), NMM (0.10 mL, 0.94 mmol) and DMF
(1.5 mL) was stirred at room temperature for 10 min. o-Benzyl-
hydroxylamine hydrochloride (0.08 g, 0.47 mmol) was added
to the mixture which was stirred at room temperature over-
night. The reaction mixture was purified by flash column
chromatography over silica gel (1 : 1 EtOAc/n-hexane; Rf = 0.30)
to afford a yellow oil. The resulting product was dissolved in
MeOH (10 mL), a catalytic amount of 10% palladium on
carbon was added, and the mixture was stirred at room
temperature under hydrogen for 3 h. The reaction mixture was
Fig. 8 Compound 6 suppresses tumor growth in HCT116 xenograft
tumor model. Drugs were suspended in 1% carboxymethyl cellulose and
0.5% Tween 80 as described in Experimental section. Tumor growth is
tracked by the mean tumor volume (mm3)
SE. Tumor volume was
determined using caliper measurements and was calculated as the
product of (w2 × l)/2, where w = width and l = length in mm of the
tumor.
4-treated mice (ESI, Fig. S1†). This caused the death of
animals and led to discontinuation of the study on day 11
(Fig. 8). Together, these findings showed that mice tolerated
treatment with compound 6 well and, compared with tegafur,
without overt signs of toxicity.
Conclusions
In this study, we synthesized some pyrimidinedione-contain- filtered and the filtrate was dried in vacuum to afford 6 (0.98 g,
ing hydroxamates and 2-aminobenzamides (6–11). Our results 27%) as a white solid. 1H NMR (300 MHz, DMSO-d6)
show that compound 6 potently induces apoptosis and δ 1.91–2.07 (m, 3H), 2.18–2.30 (m, 1H), 3.77–3.84 (m, 1H),
reduces endogenous levels of EGFR downstream signaling in 4.22–4.28 (m, 1H), 5.00 (s, 2H), 5.92–5.96 (m, 1H), 7.32 (d, J =
colorectal cancer HCT116 cells. Evidence is also provided that 8.1 Hz, 2H), 7.67 (d, J = 8.1 Hz, 2H), 7.99 (d, J = 6.6 Hz, 1H).
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13C NMR (125 MHz, DMSO-d6) δ 23.42, 31.59, 43.79, 69.54, 7.31–7.36 (m, 3H), 7.48–7.57 (m, 3H), 7.99 (d, J = 6.6 Hz, 1H),
87.46, 123.93, 124.20, 126.93, 127.20, 127.35, 127.51, 131.80, 9.40 (s, 1H). 13C NMR (125 MHz, DMSO-d6) δ 23.43, 31.61,
138.46, 139.51, 140.28, 148.79, 156.47, 156.68, 163.93; HRMS 43.85, 69.54, 87.46, 115.95, 116.21, 122.16, 123.44, 123.90,
(ESI) for C16H17FN3O5 (M + H+) calcd 350.1152, found 124.17, 124.68, 125.72, 127.64, 128.19, 133.94, 137.94, 138.48,
350.1147.
139.11, 140.30, 141.58, 148.82, 156.50, 156.70, 163.40; HRMS
N-(2-Aminophenyl)-4-((5-fluoro-2,6-dioxo-3-(tetrahydrofuran-2-yl)- (ESI) for C24H24FN4O4 (M + H+) calcd, 451.1782, found
2,3-dihydropyrimidin-1(6H)-yl)methyl)benzamide (7). A mixture of 451.1776.
13 (0.74 g, 2.21 mmol), EDC·HCl (0.64 g, 3.32 mmol), HOBt
N1-(3-((5-Fluoro-2,6-dioxo-3-(tetrahydrofuran-2-yl)-2,3-dihydro-
(0.36 g, 2.65 mmol), NMM (0.58 mL, 5.30 mmol) and DMF pyrimidin-1(6H)-yl)methyl)phenyl)-N8-hydroxyoctanediamide (10).
(2.5 mL) was stirred at room temperature for 10 min. o-Phenyl- The title compound was obtained in 69% overall yield from
enediamine (0.24 g, 2.65 mmol) was added and the mixture compound 17a in a manner similar to that described for the
1
was stirred at room temperature overnight. The reaction preparation of 6: H NMR (500 MHz, CD3OD) δ 1.38 (br, 4H),
mixture was purified by flash column chromatography over 1.61–1.68 (m, 2H), 1.94–1.97 (m, 2H), 1.98–2.02 (m, 2H),
silica gel (2 : 1 EtOAc/n-hexane; Rf = 0.25) to afford 7 (0.16 g, 2.07–2.11 (m, 3H), 2.32–2.37 (m, 3H), 3.91–3.96 (m, 1H),
1
17%) as a yellow solid. H NMR (500 MHz, CDCl3) δ 1.92–1.96 4.25–4.29 (m, 1H), 5.07 (s, 2H), 5.98 (s, 1H), 7.10 (d, J = 7.5 Hz,
(m, 1H), 2.03–2.08 (m, 2H), 2.38–2.43 (m, 1H), 3.73 (s, 2H), 1H), 7.24 (t, J = 8.0 Hz, 1H), 7.48–7.51 (m, 2H), 7.79 (d, J = 6.5
4.01 (q, J = 6.5 Hz, 1H), 4.20–4.24 (m, 1H), 5.17 (dd, J = 14.0, Hz, 1H). 13C NMR (125 MHz, CD3OD) δ 24.85, 26.71, 26.81,
15.5 Hz, 2H), 5.97–5.99 (m, 1H), 6.84–6.86 (m, 2H), 7.09 (t, J = 29.94, 30.00, 30.79, 33.71, 33.83, 37.98, 45.66, 71.46, 89.86,
7.5 Hz, 1H), 7.32 (d, J = 7.5 Hz, 1H), 7.41 (d, J = 6.0 Hz, 1H), 120.63, 120.93, 124.58, 124.85, 125.11, 130.01, 138.60, 140.24,
7.57 (d, J = 8.0 Hz, 2H), 7.84 (d, J = 8.0 Hz, 3H). 13C NMR 140.48, 142.32, 150.77, 159.10, 159.30, 173.10, 174.80; HRMS
(125 MHz, DMSO-d6) δ 23.47, 31.66, 43.88, 62.79, 69.61, 87.54, (ESI) for C23H30FN4O6 (M + H+) calcd 477.2149, found
116.08, 116.22, 123.26, 123.98, 124.25, 126.46, 126.62, 127.32, 477.2144.
127.87, 133.72, 138.52, 139.84, 140.34, 143.07, 148.84, 156.54,
156.74, 165.05; HRMS (ESI) for C22H22FN4O4 (M + H+) calcd pyrimidin-1(6H)-yl)methyl)phenyl)-N8-hydroxyoctanediamide (11).
425.1625, found 425.1620. The title compound was obtained in 41% overall yield from
N1-(4-((5-Fluoro-2,6-dioxo-3-(tetrahydrofuran-2-yl)-2,3-dihydro-
(E)-3-(4-((5-Fluoro-2,6-dioxo-3-(tetrahydrofuran-2-yl)-2,3-di- compound 17b in a manner similar to that described for the
hydropyrimidin-1(6H)-yl)methyl)phenyl)-N-hydroxyacrylamide (8). preparation of 6: 1H NMR (300 MHz, DMSO-d6) δ 1.25 (br, 4H),
A mixture of 15 (0.50 g, 1.39 mmol), EDC·HCl (0.4 g, 1.46–1.54 (m, 4H), 1.89–1.93 (m, 4H), 2.02–2.03 (m, 1H),
2.09 mmol), HOBt (0.23 g, 1.67 mmol), NMM (0.37 mL, 2.22–2.27 (m, 3H), 3.76–3.83 (m, 1H), 4.23–4.27 (m, 1H), 4.90
3.34 mmol) and DMF (7.0 mL) was stirred at room temperature (s, 2H), 5.95 (s, 1H), 7.21 (d, J = 8.4 Hz, 2H), 7.49 (t, J = 8.4 Hz,
for 10 min. NH2OTHP (0.20 g, 1.67 mmol) was added to the 2H), 7.95 (d, J = 6.7 Hz, 1H), 8.62 (s, 1H), 9.83 (s, 1H), 10.30 (s,
mixture which was then stirred at room temperature overnight. 1H). 13C NMR (125 MHz, DMSO-d6) δ 23.44, 24.97, 28.34,
The reaction mixture was purified by flash column chromato- 31.61, 32.19, 36.26, 43.62, 69.52, 87.41, 118.95, 123.75, 124.02,
graphy over silica gel (2 : 1 EtOAc/n-hexane; Rf = 0.43) to afford 128.28, 130.88, 138.47, 138.51, 140.28, 148.78, 156.44, 156.64,
a yellow oil. The resulting product was dissolved in MeOH 169.05, 171.13; HRMS (ESI) for C23H30FN4O6 (M + H+) calcd
(4 mL), 10% TFA(aq) (4 mL) was added and the mixture was 477.2149, found 477.2144.
stirred at room temperature overnight. The reaction mixture
Methyl
4-((5-fluoro-2,6-dioxo-3-(tetrahydrofuran-2-yl)-2,3-di-
was purified by flash column over silica gel (15 : 1 CH2Cl2/ hydropyrimidin-1(6H)-yl)methyl)-benzoate (12). A mixture of 4
1
MeOH; Rf = 0.20) to afford 8 (0.12 g, 23%) as a pink solid. H (0.15 g, 0.75 mmol), K2CO3 (0.16 g, 1.13 mmol) and DMF
NMR (300 MHz, DMSO-d6) δ 1.87–2.08 (m, 3H), 3.83 (dd, J = (1.5 mL) was stirred for 10 min. Methyl 4-(chloromethyl)benzo-
7.2, 7.8 Hz, 1H), 4.25 (dd, J = 6.3, 7.5 Hz, 1H), 4.93–5.03 (m, ate (0.21 g, 1.13 mmol) was added and the mixture was stirred
2H), 5.93–5.96 (m, 1H), 6.41 (d, J = 15.9 Hz, 1H), 7.30 (d, J = at room temperature overnight. The residue was purified by
8.1 Hz, 2H), 7.39 (d, J = 15.9 Hz, 1H), 7.49 (d, J = 8.1 Hz, 2H), flash column chromatography over silica gel (1 : 2 EtOAc/n-
7.98 (d, J = 6.6 Hz, 1H). 13C NMR (125 MHz, CD3OD) δ 24.84, hexane; Rf = 0.25) to afford 12 (0.31 g, 88%) as a white solid.
33.65, 45.52, 71.43, 89.83, 118.75, 124.63, 124.90, 128.98, 1H NMR (300 MHz, CDCl3) δ 1.89–1.99 (m, 1H), 2.01–2.12 (m,
130.10, 135.79, 139.67, 140.45, 141.18, 142.28, 150.74, 159.07, 2H), 2.37–2.46 (m, 1H), 3.91 (s, 3H), 3.97–4.04 (m, 1H),
159.28, 166.37; HRMS (ESI) for C18H19FN3O5 (M + H+) calcd 4.20–4.27 (m, 1H), 5.11–5.23 (m, 2H), 5.97–6.00 (m, 1H), 7.42
376.1309, found 376.1303.
(d, J = 5.7 Hz, 1H), 7.54 (d, J = 5.7 Hz, 1H), 7.99 (d, J = 8.4 Hz,
(E)-N-(2-Aminophenyl)-3-(4-((5-fluoro-2,6-dioxo-3-(tetrahydro- 2H). 13C NMR (125 MHz, CDCl3) δ 23.86, 33.11, 44.52, 52.19,
furan-2-yl)-2,3-dihydropyrimidin-1(6H)-yl)methyl)phenyl)acrylamide 70.43, 88.27, 121.97, 122.24, 129.09, 129.79, 129.89, 139.12,
(9). The title compound was obtained in 40% overall yield 140.98, 141.16, 149.10, 157.04, 157.25, 166.85. HRMS (ESI) for
from compound 15 in a manner similar to that described for C17H18FN2O5 (M + H+) calcd 349.1200, found 349.1200.
the preparation of 7: 1H NMR (300 MHz, DMSO-d6) δ 1.87–2.09
4-((5-Fluoro-2,6-dioxo-3-(tetrahydrofuran-2-yl)-2,3-dihydropyrimi-
(m, 3H), 2.21–2.28 (m, 1H), 3.77–3.85 (m, 1H), 4.22–4.29 (m, din-1(6H)-yl)methyl)benzoic acid (13). A mixture of 12 (0.23 g,
1H), 4.93 (s, 2H), 5.00 (s, 2H), 5.94–5.97 (m, 1H), 6.56 (td, J = 0.66 mmol), 1 M LiOH (1.32 mL, 1.32 mmol), and p-dioxane
1.5, 7.8 Hz, 1H), 6.72–6.75 (m, 1H), 6.84–6.91 (m, 2H), (5 mL) was stirred at 40 °C overnight. The reaction mixture was
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extracted with EtOAc (30 mL × 3) and the organic layer was gen at room temperature for 3 h. The reaction mixture was fil-
1
dried in vacuo to afford 13 (0.13 g, 59%) as a white solid. H tered and the filtrate was dried in vacuo to afford 16a (0.27 g,
NMR (300 MHz, CD3OD) δ 2.01–2.11 (m, 1H), 2.34–2.39 (m, 81%) as a gray solid. 1H NMR (300 MHz, CDCl3) δ 1.81–2.07
1H), 3.92–3.97 (m, 1H), 4.28–4.30 (m, 1H), 4.39–4.46 (m, 2H), (m, 3H), 2.31–2.38 (m, 1H), 3.49 (s, 2H), 3.89–3.97 (m, 1H),
5.17 (s, 2H), 6.00 (s, 1H), 7.83 (d, J = 6.0 Hz, 1H), 7.98 (d, J = 4.13–4.20 (m, 1H), 4.98 (dd, J = 9.9, 13.5 Hz, 2H), 5.92–5.95 (m,
7.5 Hz, 4H). 13C NMR (125 MHz, DMSO-d6) δ 23.33, 31.54, 1H), 6.52–6.56 (m, 1H), 6.76 (s, 1H), 6.82 (d, J = 7.5 Hz, 1H),
43.79, 69.48, 87.42, 123.82, 124.09, 126.78, 127.42, 129.18, 7.03 (t, J = 7.8 Hz, 1H), 7.25 (d, J = 5.7 Hz, 1H). 13C NMR
129.30, 129.35, 129.66, 138.39, 140.21, 141.29, 148.71, 156.40, (125 MHz, CDCl3) δ 23.88, 33.12, 44.53, 51.80, 70.42, 88.24,
156.61, 166.92. HRMS (ESI) for C16H14FN2O5 (M − H+) calcd 118.13, 121.90, 122.17, 128.31, 129.84, 134.13, 138.47, 139.17,
333.0887, found 333.0885.
(E)-Methyl 3-(4-((5-fluoro-2,6-dioxo-3-(tetrahydrofuran-2-yl)-2,3- C15H17FN3O3 (M + H+) calcd 306.1254, found 306.1252.
dihydropyrimidin-1(6H)-yl)-methyl)phenyl)acrylate (14). A 3-(4-Aminobenzyl)-5-fluoro-1-(tetrahydrofuran-2-yl)pyrimidine-
141.03, 144.41, 149.15, 157.07, 157.28, 167.45. HRMS (ESI) for
mixture of 4 (0.69 g, 3.46 mmol), K2CO3 (0.57 g, 4.15 mmol) 2,4(1H,3H)-dione (16b). The title compound was obtained in
and DMF (2.5 mL) was stirred for 10 min. 4-Formylbenzyl 98% overall yield from compound 4 in a manner similar to
1
chloride (0.8 g, 5.19 mmol) was added to the mixture which that described for the preparation of 16a: H NMR (300 MHz,
was then stirred at room temperature overnight. The residue CDCl3) δ 1.90–2.08 (m, 3H), 2.35–2.42 (m, 1H), 3.92–4.01 (m,
was purified by flash column chromatography over silica gel 1H), 4.15–4.23 (m, 1H), 5.00 (dd, J = 12.0, 13.5 Hz, 2H),
(1 : 4 EtOAc/n-hexane; Rf = 0.50) to afford a pale yellow solid. 5.96–5.98 (m, 1H), 6.61 (d, J = 8.4 Hz, 2H), 7.32–7.35 (m, 3H).
The resulting product was dissolved in DCM (20 mL), methyl 13C NMR (125 MHz, DMSO-d6) δ 23.48, 31.58, 43.73, 69.46,
(triphenylphosphoranylidene)acetate (1.74 g, 5.19 mmol) was 87.31, 113.47, 123.41, 123.48, 123.75, 129.35, 138.48, 140.30,
added and the mixture was stirred at room temperature over- 148.08, 148.78, 156.40, 156.60. HRMS (ESI) for C15H16FN3O3Na
night. The residue was purified by flash column chromato- (M + Na+) calcd 328.1073, found 328.1075.
graphy over silica gel (1 : 1 EtOAc/n-hexane; Rf = 0.50) to afford
8-((3-((5-Fluoro-2,6-dioxo-3-(tetrahydrofuran-2-yl)-2,3-dihydro-
acid
14 (1.25 g, 68%) as a pale yellow solid. 1H NMR (300 MHz, pyrimidin-1(6H)-yl)methyl)phenyl)amino)-8-oxooctanoic
CDCl3) δ 1.90–1.99 (m, 1H), 2.00–2.09 (m, 2H), 2.35–2.44 (m, (17a). A mixture of monomethyl suberate (0.11 mL,
1H), 3.79 (s, 3H), 3.94–4.02 (m, 1H), 4.18–4.24 (m, 1H), 5.11 0.60 mmol), EDC·HCl (0.17 g, 0.90 mmol), HOBt (0.10 g,
(dd, J = 11.7, 13.8 Hz, 2H), 6.40 (d, J = 15.9 Hz, 1H), 7.39 (d, J = 0.72 mmol), NMM (0.16 mL, 1.44 mmol) and DMF (2 mL) was
5.7 Hz, 1H), 7.44–7.51 (m, 2H), 7.64 (d, J = 15.9 Hz, 1H). stirred for 10 min. 16a (0.22 g, 0.72 mmol) was added to the
13C NMR (125 MHz, CDCl3) δ 23.83, 33.03, 44.77, 88.14, mixture which was then stirred at room temperature overnight.
114.80, 115.91, 119.52, 121.70, 121.97, 129.42, 137.29, 139.18, The reaction mixture was purified by flash column chromato-
141.04, 146.49, 149.14, 157.16, 157.36. HRMS (ESI) for graphy over silica gel (3 : 1 EtOAc/n-hexane; Rf = 0.18) to afford
C19H20FN2O5 (M + H+) calcd 375.1356, found 375.1356.
a pale yellow oil. The resulting product was dissolved in
(E)-3-(4-((5-Fluoro-2,6-dioxo-3-(tetrahydrofuran-2-yl)-2,3-dihydro- p-dioxane (5 mL), 1 M LiOH (1.18 mL, 1.18 mmol) was added
pyrimidin-1(6H)-yl)methyl)-phenyl)acrylic acid (15). The title and the mixture was stirred at 40 °C overnight. The reaction
compound was obtained in 93% overall yield from compound was extracted with EtOAc (30 mL × 3) and the organic layer was
1
14 in a manner similar to that described for the preparation of dried in vacuo to afford 17a (0.27 g, 79%) as a colorless oil. H
13: 1H NMR (300 MHz, DMSO-d6) δ 1.89–2.08 (m, 3H), NMR (300 MHz, CD3OD) δ 1.39 (br, 3H), 1.61–1.69 (m, 5H),
2.20–2.25 (m, 1H), 3.77–3.84 (m, 1H), 4.17–4.28 (m, 2H), 4.98 1.94–2.00 (m, 3H), 2.26–2.37 (m, 5H), 3.53–3.94 (m, 2H), 4.27
(s, 2H), 5.93–5.95 (m, 1H), 6.48 (d, J = 15.9 Hz, 1H), 7.28–7.32 (s, 2H), 5.94–5.98 (m, 1H), 7.00–7.10 (m, 1H), 7.24 (t, J =
(m, 3H), 7.54 (d, J = 15.9 Hz, 1H), 7.61–7.63 (m, 3H), 7.98 (d, 7.5 Hz, 1H), 7.42–7.51 (m, 2H) 7.73–7.81 (m, 1H). 13C NMR
J = 6.6 Hz, 1H), 12.39 (br, 1H). 13C NMR (125 MHz, DMSO-d6) (125 MHz, DMSO-d6) δ 23.48, 24.38, 24.96, 28.33, 28.40, 31.68,
δ 23.66, 31.86, 44.09, 69.82, 87.76, 119.00, 119.33, 124.08, 33.63, 36.34, 44.01, 69.59, 87.48, 117.96, 118.02, 122.28,
124.35, 127.65, 127.85, 128.30, 128.43, 133.11, 133.54, 138.69, 123.85, 124.12, 128.65, 136.92, 138.49, 139.45, 140.30, 148.79,
138.81, 140.51, 142.09, 143.67, 143.86, 149.04, 153.87, 156.76, 156.48, 156.68, 171.26, 174.47. HRMS (ESI) for C23H27FN3O6
156.96, 167.80, 167.85. HRMS (ESI) for C18H16FN2O5 (M − H+) (M − H+) calcd 460.1884, found 460.1885.
calcd 359.1043, found 359.1039.
8-((4-((5-Fluoro-2,6-dioxo-3-(tetrahydrofuran-2-yl)-2,3-dihydro-
3-(3-Aminobenzyl)-5-fluoro-1-(tetrahydrofuran-2-yl)pyrimidine- pyrimidin-1(6H)-yl)methyl)phenyl)amino)-8-oxooctanoic
acid
2,4(1H,3H)-dione (16a). A mixture of 4 (0.20 g, 1.00 mmol), (17b). The title compound was obtained in 88% overall yield
K2CO3 (0.18 g, 1.30 mmol) and DMF (2 mL) was stirred for from compound 16b in a manner similar to that described for
1
5 min and 3-nitrobenzyl chloride (0.22 g, 1.30 mmol) was the preparation of 17a: H NMR (300 MHz, CD3OD) δ 1.37 (br,
added to the mixture which was then stirred at room tempera- 3H), 1.60–1.69 (m, 5H), 1.97–2.13 (m, 3H), 2.24–2.35 (m, 5H),
ture overnight. The residue was purified by flash column 3.90–3.93 (m, 2H), 4.23–4.26 (m, 2H), 5.95–5.98 (m, 1H),
chromatography over silica gel (1 : 2 EtOAc/n-hexane; Rf = 0.35) 7.20–7.22 (m, 1H), 7.31–7.34 (m, 1H), 7.45–7.48 (m, 2H)
to afford a white solid. The resulting product was dissolved in 7.75–7.77 (m, 1H). 13C NMR (125 MHz, DMSO-d6) δ 23.46,
MeOH (10 mL), a catalytic amount of 10% palladium on 24.36, 24.94, 28.29, 28.34, 31.63, 33.59, 36.28, 43.64, 69.54,
carbon was added, and the mixture was stirred under hydro- 87.44, 118.97, 119.04, 123.73, 124.00, 127.32, 127.33, 127.60,
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128.31, 130.90, 138.49, 138.54, 140.30, 148.79, 156.45, 156.66, fuged at 13 000 rpm for 30 min. Cell lysates were then collected
171.16, 174.43. HRMS (ESI) for C23H27FN3O6 (M − H+) calcd and analyzed by immunoblotting for specific antibodies. The
460.1884, found 460.1885.
signals were visualized by enhanced chemiluminescence (ECL)
Biology
detection system (Amersham, Buckinghamshire, UK).
HDAC enzymes inhibition assays. Enzyme inhibition assays
Antitumor activity in vivo. Eight-week-old female nude
were conducted by the Reaction Biology Corporation, Malvern, athymic mice were housed in groups under conditions of a
PA. (http://www.reactionbiology.com) The substrate for constant photoperiod (12 h light/12 h dark at 21–23 °C and
HDAC-1, -2, and -6 is a fluorogenic peptide derived from p53 60–85% humidity) with ad libitum access to sterilized food and
residues 379–382 [RHKK(Ac)]. Compounds were dissolved in water. All animal experiments followed ethical standards, and
DMSO and tested in 10-dose IC50 mode with 3-fold serial protocols have been reviewed and approved by Animal Use and
dilution starting at 10 μM. HDAC Control Compound Tricho- Management Committee of Taipei Medical University. Each
statin A (TSA) was tested in a 10-dose IC50 with 3-fold serial mouse was inoculated s.c. with 1 × 106 HCT116 cells in a total
dilution starting at 10 μM as shown in ESI.†
volume of 0.1 mL serum-free medium. As tumors became
In vitro cell growth inhibitory activity. Culture medium and established at ∼100 mm3 in volume, mice were randomized
culture condition of cell lines. Human lung adenocarcinoma into five groups (n = 7–8) that received the following treat-
A549, colorectal carcinoma HCT116 cells, and prostatic adeno- ments: (a) control: 1% carboxymethyl cellulose/0.5% Tween 80
carcinoma PC-3 cells were maintained in RPMI 1640 medium vehicle, (b) SAHA at 200 mg per kg per day, (c) compound 6 at
supplied with 10% fetal bovine serum. Cells were cultured at 100 mg per kg per day, (d) compound 6 at 200 mg per kg per
37 °C in a humidified atmosphere with 5% CO2. Cell growth day, and (e) Tegafur at 200 mg per kg per day. Mice received
inhibitory assay. Cells in their logarithmic phase were cultured treatments by gavage for the duration of the study. Tumors
in completed medium in 96-well plates at 37 °C with 5% CO2 were measured weekly using calipers. Tumor size, in mm3, was
for overnight incubation, and treated with various concen- calculated as volume = (w2 × l)/2, where w = width and l =
trations of test compounds for 72 h. Cells were then incubated length in mm of the tumor.
with a serum free medium containing MTT at a final concen-
Statistics and data analysis. Results are expressed as the
tration of 0.5 mg mM−1 for 4 h. The conversion of MTT to for- mean S.E. for the indicated number of separate experiments.
mazan by metabolically viable cells was solubilized using Means were assessed for significant differences using t-test
DMSO. Absorbance was measured at 570 nm using a spectra- and P-values <0.05 were considered significant.
max M5 microplate reader (Molecular Devices, UK). Colony for-
mation assay. Cells were treated with various concentrations of
compound 6 or DMSO vehicle for 24 h. The drugs were washed
out and the cells were trypsinized and plated at 500 cells per
plate. After 14 days, the colonies were fixed and stained with
Acknowledgements
crystal violet (0.5% in 70% EtOH). Colonies containing more
This research were supported by the Ministry of Science and
than 50 cells were scored. The survival fraction was calculated
Technology of the Republic of China (Grant no. MOST 103-
based on the number of colonies formed in drug-treated cells
2320-B-038-030-MY3 and MOST 103-2113-M-038-001-MY3),
relative to that of the untreated control. Each dose was
and TMU102-AE1-B43 by Taipei Medical University.
carried out in triplicate, and the experiments were repeated at
least twice.
Effect on cell cycle progression. Flow cytometric analysis. Cell
cycle progression was determined by flow cytometric analysis.
Cells were plated onto 6-well plates at a final density of 250 000
cells per well and treated with test compounds, then trypsi-
Notes and references
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−20 °C. After centrifugation, the fixed cells were rinsed with
ice-cold phosphate-buffered saline (PBS) and incubated with
0.1 M phosphate/citric acid buffer (0.2 M Na2HPO4, 0.1 M
citric acid, pH 7.8) at room temperature for 30 min. The cells
were then centrifugated and resuspended in the dark with pro-
pidium iodide (PI) staining buffer containing RNase (100 μg
mL−1), PI (80 μg mL−1), and 0.1% Triton X-100 (v/v). The DNA
content was analyzed by FACScan flow cytometer with Cell-
Quest software (Becton Dickinson, San Jose, CA). SDS-PAGE
and western blot analysis. Cells were harvested by lysis buffer
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150 mM NaCl, 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF, 0.1%
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