Design, synthesis and biological evaluation of nitro oxide donating N-hydroxycinnamamide derivatives as histone deacetylase inhibitors

Article abstract of DOI:10.1248/cpb.c14-00449

Novel nitro oxide (NO)-donating N-hydroxycinnamamide derivatives 12a-j were designed and synthesized by coupling the carboxyl group of N-hydroxycinnamamides with phenylsulfonylfuroxan through various diols or alkylol amines, and their in vitro biological activities were evaluated. It was discovered that most of target compounds showed good histone deacetylases (HDACs) inhibition and anti-tumor activities, particularly for 12j, which had great HDACs inhibitory activities (IC₅₀s=0.15-0.26 μm) and antiproliferative effects (IC₅₀s=3.21-7.12 μm) comparable to suberoylanilide hydroxamic acid (SAHA) (IC₅₀s=0.16-1.41 μm for HDACs, IC₅₀s=3.15-7.45 μm for cancer cell inhibition). Furthermore, compound 12j with strong antitumor activities produced high levels of NO (up to 8.0 μm of nitrites/nitrates) in colon cancer cells, and its antiproliferative activity was nearly half-diminished by hemoglobin (10 μm), an NO scavenger. These results suggest that the strong antiproliferative activity of 12j could be attributed to the additive effects of high levels of NO production and inhibition of HDAC in the cancer cells.

Full text of DOI:10.1248/cpb.c14-00449

December 2014
Regular Article
Chem. Pharm. Bull. 62(12) 1185–1191 (2014)
1185
Design, Synthesis and Biological Evaluation of Nitro Oxide Donating
N-Hydroxycinnamamide Derivatives as Histone Deacetylase Inhibitors
,a
Shiliang Tu,* Hang Yuan,a Jie Hu,b Chengguang Zhao,b Rui Chai,a and Hongfeng Caoa
a
̕
Department of Coloproctology, Zhejiang Provincial Peoples Hospital; Hangzhou 310014, P.R. China: and
b Department of Pharmacy, Wenzhou Medical University; Wenzhou 325000, P.R. China.
Received June 17, 2014; accepted September 19, 2014
Novel nitro oxide (NO)-donating N-hydroxycinnamamide derivatives 12a–j were designed and syn-
thesized by coupling the carboxyl group of N-hydroxycinnamamides with phenylsulfonylfuroxan through
various diols or alkylol amines, and their in vitro biological activities were evaluated. It was discovered that
most of target compounds showed good histone deacetylases (HDACs) inhibition and anti-tumor activities,
particularly for 12j, which had great HDACs inhibitory activities (IC50s=0.15–0.26µM) and antiproliferative
effects (IC50s=3.21–7.12µM) comparable to suberoylanilide hydroxamic acid (SAHA) (IC50s=0.16–1.41 µM for
HDACs, IC50s=3.15–7.45µM for cancer cell inhibition). Furthermore, compound 12j with strong antitumor
activities produced high levels of NO (up to 8.0µM of nitrites/nitrates) in colon cancer cells, and its anti-
proliferative activity was nearly half-diminished by hemoglobin (10µM), an NO scavenger. These results sug-
gest that the strong antiproliferative activity of 12j could be attributed to the additive effects of high levels of
NO production and inhibition of HDAC in the cancer cells.
Key words nitro oxide; histone deacetylase inhibitor; furoxan; N-hydroxycinnamamide; anti-tumor agent
Pharmacological targeting of proteins that regulate epi- droxyacrylamide moiety exhibiting excellent HDAC inhibitory
genetics has emerged as a promising therapeutic area of activity,12–14) benzamides15,16) such as entinostat (MS-275), mo-
study.1,2) Epigenetic or chromatin modification is recognized cetinostat (MGCD-0103), and aliphatic acids, such as valproic
by nonhistone proteins and is a code of gene expression. acid.17) In addition, the selective isotype HDAC6 inhibitor,
Among the various histone modifiers, Histone acetyltransfer- ACY-1215, is currently undergoing human clinical trials for
ase (HAT) and histone deacetylase (HDAC) are two reversible the treatment of multiple myeloma.18) Among these approved
enzymes regulating histone acetylation status and executing or clinical HDACI, PXD101, LBH589, and SB-939 share a
the acetylation and deacetylation of the lysine residues at the common active fragment, N-hydroxycinnamamide. This frag-
amino terminal of histones.3) However, abnormal HDAC over- ment could not only form bichelation with the active site Zn2+
expression has been found to be involved in the development by its hydroxamic acid group but also form a sandwichlike
of several kinds of human cancers, including myeloid neopla- π–π interaction by inserting its vinyl benzene group into two
sia and solid tumors.4) Recent studies have shown that acetyla- parallel phenylalanine residues of HDAC.19,20)
tion of non-histone proteins is also relevant for tumorigenesis,
Nitro oxide (NO), which is naturally generated from L-argi-
cancer cell proliferation, and immune functions.5) Conse- nine by the action of NO synthase (NOS), is a key signaling
quently, histone deacetylases are considered to be important and effector molecule and plays a pivotal role in diverse physi-
targets in the development of anticancer agents, and in recent ological and pathophysiological processes.21) It is well-known
years considerable attention has been paid to HDAC inhibitors that high concentration of NO, generated from synthetic NO-
(HDACI) as anticancer agents.6–8)
releasing compounds, could not only have strong cytotoxic-
There has been a high level of interest in developing small- ity against human carcinoma cells and induce the apoptotic
molecule HDACI, and numerous structurally diverse HDACI of tumor cells, but also prevent tumors from metastasizing
have been developed as potential anticancer agents, which are and sensitize tumor cells to chemotherapy, radiation and im-
grouped chemically into four classes: hydroxamic acids, ben- munotherapy in vitro and in vivo.22) As an important class of
zamides, cyclic tetrapeptides, and short-chain fatty acids.9) The NO donors, furoxans such as phenylsulfonylfuroxans are ther-
common pharmacophore of these HDACI consists of three do- mally stable compounds and can produce high levels of NO in
mains: a zinc-binding group (ZBG), such as hydroxamic acid; vitro and inhibit the growth of tumors in vivo.23) Furthermore,
a cap group, generally a hydrophobic and aromatic group; a it was reported that tumor cells are more sensitive to relative
saturated or unsaturated linker domain, composed of linear high concentration of NO than normal cells and a variety of
or cyclic structures that connect the ZBG and the cap group. phenylsulfonylfuroxan-based NO releasing compounds have
Up to now, two of these HDACI, suberoylanilide hydroxamic been investigated to show selective antitumor activity in vitro
acid (SAHA, Fig. 1) and cyclic peptide Romidepsin (FK228), and in vivo.23–27)
have been approved by the U.S. Food and Drug Administra-
It is known that the Class I enzyme HDAC2 can be directly
tion (FDA) for the treatment of cutaneous T-cell lymphoma or indirectly modified by NO, undergoing either Tyr-nitration
(CTCL), validating HDACs’ potential as an important target or S-nitrosylation.28) One example of NO-donor HDACI,
for anticancer therapy.10,11) Other compounds in clinical trials which is able to simultaneously release NO and regulate
include hydroxamic acids such as belinostat (PXD101), pano- HDACs, affects a number of micro-RNAs and synergistically
binostat (LBH589), and pracinostat (SB-939) with an N-hy- promotes myogenic differentiation.29) It is therefore of high
interest to investigate whether introduction of phenylsulfonyl-
The authors declare no conflict of interest.
furoxan moiety to the N-hydroxycinnamamides would provide
© 2014 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: shiliang_tu@126.com

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Fig. 1. Structures of Representative HDAC Inhibitors
Reagents and conditions: a) 1. NaOH (aq.), 140°C, 2h; 2. conc. HCl; (b) 30% H2O2, AcOH, rt, 3h; c) fuming nitric acid, 90°C, 4h; d) diols or alkylol amines, THF, 30%
NaOH, rt, 4–8h; e) propanedioic acid, piperidine, pyridine, 85°C, 3h; f) 1. ethyl chloroformate, N-methylmorpholine, THF, 0°C to rt, 1h; 2. tetrahydro-2H-pyran-2-ol,
TEA, THF, 0°C, 1h; g) 1M NaOH (aq.), MeOH, rt, 2h; h) TFA, CH2Cl2, rt, 2–4h; i) 6a–j, EDCI, DMAP, CH2Cl2, rt, 10–15h.
Chart 1
a hitherto unknown class of NO donating HDAC inhibitors quence. Thiophenol 1 as the starting material was converted
that may effectively develop inhibitory effect of HDACs and to 2-(phenylthio)acetic acid 3 by treatment with chloroacetic
release high levels of NO with an additive effect for treat- acid 2 in 97% yeild. Oxidation of 3 using 30% H2O2 aque-
ment of cancer. As part of our ongoing research program, a ous solution in acetic acid afforded 2-(phenylsulfonyl)acetic
series of phenylsulfonylfuroxan-based N-hydroxycinnamamide acid 4, which was directly reacted with fuming nitric acid
derivatives 12a–j were designed and synthesized by coupling to form diphenylsulfonylfuroxan 5. Compound 5 was then
the carboxyl of N-hydroxycinnamamides with phenylsulfonyl- converted to various monophenylsulfonyl furoxans 6a–j by
furoxan through different carbochain length substituted diols treatment with corresponding diol or alkylol amines. On the
or alkylol amines.
other hand, methyl 4-formylbenzoate 7 was reacted with
propanedioic acid in the presence of piperidine to give (E)-3-
(4-(methoxycarbonyl)phenyl)acrylic acid 8, which was further
Chemistry
The synthesis of 12a–j was described in Chart 1. The treated with tetrahydro-2H-pyran-2-ol to form compound 9 in
substituted furoxans 6a–j were prepared in a four step se- the presence of N-methylmorpholine and ethyl chloroformate.

December 2014
1187
Table 1. Inhibition of HeLa Nuclear HDACs by 12a–j
Table 2. IC50 Value for the Inhibition of HDACs 2, 6, and 8 by 12f, g
and 12i, j
a)
a)
Compd.
IC50 (μM)
Compd.
IC50 (μM)
a)
IC50 (μM)
SAHA
12a
0.17
0.55
0.86
>1
12f
12g
12h
12i
0.18
0.21
0.59
0.25
0.15
Compd.
HDAC2
HDAC6
HDAC8
12b
12c
SAHA
6j
0.18
0.43
0.16
ND
0.16
NDb)
0.15
0.23
0.28
0.16
1.41
ND
12d
12e
>1
>1
12j
12f
0.29
0.46
0.42
0.26
12g
12i
a) The data are the mean values of IC50 from at least three independent experi-
ments.
ND
12j
ND
a) The data are the mean values of IC50 from at least three independent experi-
ments. b) Not detected.
Fig. 2. NO Release Behaviors of the Indicated Compounds and Their Correlation with in Vitro Antiproliferative Activities
(A) Quantitative measurement of intracellular NO production. HCT116 cells were treated with various compounds (50µM) for 6h, and the amounts of intracellular
nitrite/nitrate were determined using a colorimetric Griess reaction assay. Data are expressed as means S.D. from three separate experiments. (B) HCT116 cells were pre-
treated with, or without, the indicated concentrations of NO scavenger hemoglobin (Hb) for 1h and treated with 5 or 10µM of 12j for 24h. The cell growth was determined
by the MTT. Data are expressed as mean% of inhibition on the growth of HCT116 cells from three independent experiments. The cells treated with different concentra-
tions of hemoglobin alone did not affect their growth (data not shown).
Compound 9 was subsequently hydrolyzed to obtain interme- and isoform selectivity. Results listed in Table 2 showed that
diate 10, which were directly treated with 6a–j in CH2Cl2 in 12f and g and 12i and j exhibited significant HDACs inhibi-
the presence of 1-(3-dimethyl aminopropyl)-3-ethylcarbodi- tory activities against HDACs 2, 6, and 8. Both 12j and NO
imide hydrochloride (EDCI) and 4-dimethylamino pyridine donor moiety 6j demonstrated significant effects on HDAC2
(DMAP) to generate 11a–j, respectively. Finally, O-protected comparable to SAHA, which may partially be due to NO re-
groups of 10 and 11a–j were removed by CF3COOH to pro- lease from 12j or 6j inducing S-nitrosylation of HDAC2.29,30)
vide 10a and 12a–j. The products 12a–j were purified by col- Furthermore, compounds 12f and g and 12i and j also showed
umn chromatography, and their structures were characterized stronger inhibitory potency than SAHA for HDAC8. However,
1
by IR, H-NMR, MS, and elemental analyses.
the active compounds 12f and g and 12i and j did not exhibit
any isoform selectivity in the HDACs inhibitory assay.
Next, we tested whether the levels of NO production upon
Results and Discussion
All synthesized compounds 12a–j were evaluated for their treating HCT116 cells with target compounds were associated
ability to inhibit HeLa cell nuclear HDACs (which consist with their antitumor activities. The levels of nitrite/nitrate
of mostly HDAC1–3), with SAHA as a reference compound. produced in the cell lysates of different types of cells were
The values of half inhibitory concentration (IC50) about all characterized using the Griess assay. As can be seen in Fig.
the target compounds against HeLa cell nuclear HDACs were 2A, treatment with SAHA resulted in little nitrite/nitrate. In
measured and presented in Table 1. The results indicated the contrast, treatment with 12f–j produced high levels of nitrite/
HDACs inhibitory activities of ester derivatives 12a–e were nitrate in HCT116 cells. The most potent compound 12j re-
slightly weaker than those of amide compounds 12f–j, which leased the highest levels of NO, and the derivatives with lower
suggested the amide bond of 12f–j could be beneficial for antiproliferative activity released less NO under the same
them to bind to the active site. Moreover, among these amide conditions. These results indicate that the amounts of intra-
compounds, compound 12j possessed the strongest HeLa cellular NO release were well associated with their in vitro
nuclear HDACs inhibition comparable to that of SAHA.
antiproliferative activity. To further determine the contribution
Furthermore, the active compounds 12f and g, 12i and j, of NO to the inhibitory activities of NO-donating N-hydroxy-
and NO donor moiety 6j (the intermediate of 12j) was further cinnamamide derivatives, 12j was selected for testing its anti-
tested for their in vitro HDACs inhibitory activities toward proliferative activity in the presence or absence of NO scav-
HDAC2, HDAC6 and HDAC8 in order to know whether ac- enger hemoglobin (Hb). HCT116 cells were pre-treated with,
tive compounds possessed strong HDACs inhibitory effects or without, different concentrations of Hb for 1h and then

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Vol. 62, No. 12
treated with the indicated concentrations of 12j. The effects
of different treatments on the growth of HCT116 cells were
determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT) assays (Fig. 2B). Treatment with
12j alone greatly inhibited the growth of HCT116 cells, while
the inhibitory effects of 12j on the growth of HCT116 cells
were dramatically reduced by pre-treatment with Hb, which
appeared to be dose-dependent. Similar patterns of reduced
levels of nitrate/nitrite in the cell lysates were observed. These
data clearly demonstrated that high levels of NO production
by the compound contributed to its antiproliferative activity
against human cancer cells in vitro.
Next, synthetic compounds 11j, 12a–j were also assessed
for anti-proliferative activities against five human cancer cells
HCT116, SW620, Lovo (human colon carcinoma cells), MCF-7
(human breast adenocarcinoma cells), and Hela were evalu-
ated by MTT assays in vitro, with SAHA as positive control.
The values of half inhibitory concentration (IC50) about all
the target compounds against each tumor cell line were mea-
Fig. 3. Inhibitory Activity of 6j, 10a, 11j, and 12j against HCT116 Cells
HCT116 cells were incubated with the indicated compounds at 5.0 and 10µM for
24h, and cell proliferation was assessed by the MTT assay. Data are means S.D.
of the inhibition (%) from three independent experiments.
sured and presented in Table 3. For most of the compounds induced tumor cell antiproliferative effect comparable to 12j.
tested, the cell growth inhibition results were consistent with These results suggest that the antitumor activity of 12j may be
the HDAC inhibition profiles. Most of the target compounds attributed to the additive effects of phenylsulfonylfuroxan and
displayed good to moderate antiproliferative activities. The the N-hydroxycinnamamide moiety and that both of them may
antitumor activities of ester derivatives 12a–e were slightly play crucial roles in the antiproliferative activity of 12j.
weaker than those of amide compounds 12f–j. What’s more,
Structure–Activity Relationship (SAR) Analysis of
compound 12j exhibited strongest antiproliferative activities SAR revealed that 12a–j with different linkers displayed vari-
with IC50 values of 3.21–7.12µM against each tested cancer able antitumor activities. The compounds 12f–j linked with
cell, which were comparable to SAHA (IC50s=3.15–7.45 µM). alkanolamine exhibited stronger inhibitory effects than the
Interestingly, compound 11j (the intermediate of 12j, ester derivatives 12a–e linked with diol against human cancer
IC50s=9.12–11.4 µM) displayed weak cancer inhibitory activi- cells. It is likely that the ester compounds can usually be me-
ties, which were 2–3-fold less than those of 12j. Such phenom- tabolized into the corresponding acid and alcohol, which may
enon suggested that the hydroxamic acid moiety has a positive lead to the partial loss of their inhibitory activity. In addition,
influence on the cytotoxicity of this series of N-hydroxyferuli- variations in the carbochain lengths of linkers significantly af-
cylamide derivatives.
fected the in vitro anticancer activity of these derivatives. For
As active compound 12j consists of NO donor moiety (6j) example, the compounds 12a, 12f and 12j with a two-carbon
and the N-hydroxycinnamamide moiety (10a) (Chart 1), the diol or alkylol amine showed relatively stronger anticancer
inhibitory effects of these two moieties on HCT116 cells were activity than compounds with a three- or four-carbon linker.
examined at two different concentrations. As shown in Fig. 3, However, further investigation about the precise SAR of these
the NO-donating moiety 6j retained a certain degree of inhibi- compounds is ongoing.
tory potency, which was similar to the N-hydroxycinnamamide
moiety 10a. And both of them were obviously less potent than
12j. However, the combination of 10a with 6j significantly
Conclusion
In summary, a series of novel NO-donating HDAC in-
Table 3. The IC50 Values of 11j and 12a–j against Five Human Cancer Cell Lines
In vitro inhibition of human cancer cells proliferation (IC50a), μM)
Compd.
Hela
HCT116
SW620
Lovo
MCF-7
SAHA
11j
3.15
12.5
9.51
15.2
25.8
21.0
15.8
5.02
6.71
13.9
7.27
4.18
5.83
9.69
9.47
14.7
21.6
17.9
13.6
4.67
5.78
12.7
6.89
3.21
4.72
11.3
9.12
13.9
20.5
19.2
15.0
5.53
6.14
11.9
7.46
4.43
5.56
12.6
10.5
16.8
24.6
19.4
18.6
6.65
8.17
14.5
8.75
5.03
7.45
14.1
11.4
18.3
27.5
22.7
16.4
6.28
7.18
15.0
9.59
7.12
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
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.

December 2014
1189
hibitors 12a–j were designed and synthesized by coupling the completed, the resulting mixture was allowed to pour into
carboxyl group of N-hydroxycinnamamide with phenylsul- ice-water, and extracted with ethyl acetate (30mL×3). The or-
fonylfuroxan through various carbochain length substituted ganic phase was washed with water and brine, then dried over
diols or alkylol amines. The in vitro assay of their inhibitory anhydrous sodiumsulfate, filtered and evaporated to afford the
activities against five human cancer cell lines showed that the crude product, which was poured into 5mL methanol contain-
phenylsulfonyl- furoxan-based N-hydroxycinnamamide deriva- ing 1.5mL 2N NaOH and continuously stirred for 2h. After
tives displayed good to moderate antiproliferative activities. the mixture being cooled, the solvent was evaporated, and the
Especially, compound 12j had greater antiproliferation and residue was neutralized to pH=3 with 1M HCl. The precipitate
HDACs inhibitory activities, comparable to SAHA, in human was filtered, washed with water, and dried in vacuo to afford
carcinoma cells. What’s important, the compounds with strong the crude, which was purified by column chromatography on
antitumor activities against human cancer cells produced high silica gel to give compound 10 as pale yellow waxy solid,
levels of NO in colon cancer cells. In addition, the inhibi- yeild 56%, MS (ESI) m/z=292 [M+H]+.
tory effects of 12j were diminished by NO scavenger Hb in
a dose-dependent manner, suggesting that NO released by Acid (10a)
(E)-4-(3-(Hydroxyamino)-3-oxoprop-1-en-1-yl)benzoic
solution of 10 (0.23g, 0.80mmol) and
A
this compound may play an important role in antiproliferation CF3COOH (1mL) in 4mL dry CH2Cl2 was stirred at room
of HCT116 cells. Our findings suggest that the NO-donating temperature for 2h. The solvent was removed under reduced
HDAC inhibitors may hold greater promise as therapeutic pressure. The crude residue was dissolved in 15mL dichlo-
agents for the intervention of human cancers.
romethane and 2mL Et3N was slowly added to the solvent,
and the crude product was purified by column chromatogra-
phy (MeOH–CH2Cl2=1:6–1:10) to yield 10a as white waxy
Experimental
Melting points of individual compounds were determined solid, yeild 88%. Analytical data for 10a: MS (ESI) m/z=208
1
on a Mel-TEMP II melting point apparatus and uncorrected. [M+H]+; H-NMR (DMSO-d6, 300MHz, δ ppm): 10.33 (br,
Infrared (IR) spectra (KBr) were recorded on a Nicolet Im- 1H), 8.57 (br, 1H), 7.86 (d, J=7.8Hz, 2H, Ar-H), 7.66 (d,
pact 410 instrument (KBr pellet). 1H-NMR was recorded J=7.8Hz, 2H, Ar-H), 7.50 (d, J=16.2Hz, 1H, CH=), 6.55 (d,
with a Bruker Avance 300MHz spectrometer at 303K, with J=16.2Hz, 1H, CH=).
chemical shift in parts per million (ppm, δ) downfield from
General Procedure for the Synthesis of 12a–j Com-
tetramethylsilane, or trimethylsilyl (TMS) as an internal pound 10 (0.23g, 0.80mmol) was mixed with 6a–j
standard. MS spectra were recorded on a Mariner Mass Spec- (0.80mmol), EDCI (153mg, 0.8mmol), DMAP (97mg,
trum (electrospray ionization (ESI)) and HR-MS on Agilent 0.80mmol). The mixture was stirred at room temperature
technologies LC/MSD time-of-flight (TOF). Element analysis for 8–12h. After filtration, the filtrate was evaporated to
was performed on an Eager 300 instrument. All compounds dryness in vacuo, and poured into 4mL dry CH2Cl2 contain-
1
were routinely checked by TLC and H-NMR. TLCs and pre- ing 1mL CF3COOH in was stirred at room temperature for
parative thin-layer chromatography were performed on silica 1–2h. The solvent was removed under reduced pressure. The
gel GF/UV 254, and the chromatograms were conducted on crude residue was dissolved in 15mL dichloromethane and
silica gel (200–300 mesh, Merck) and visualized under UV 2mL Et3N was slowly added to the solvent, and the crude
light at 254 and 365nm. Thiophenol 1 and compounds 7 were product was purified by column chromatography (MeOH–
commercially available, and compounds 3–5 and 6a–j were CH2Cl2=1:6–1:10) to yield 12.
prepared according the literatures.24–26) All solvents were re-
(E)-4-(2-((4-(3-(Hydroxyamino)-3-oxoprop-1-en-1-yl)-
agent grade and, when necessary, were purified and dried by benzoyl)oxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole
standards methods. Solutions after reactions and extractions 2-Oxide (12a) The title compound was obtained starting
were concentrated using a rotary evaporator operating at a from 10 and 6a. White waxy solid; yield: 62%. Analytical
reduced pressure of ca. 20Torr. Organic solutions were dried data for 12a: IR (KBr, cm−1): 3433, 2925, 1746, 1622, 1446,
1
over anhydrous sodium sulfate.
1211, 1018; MS (ESI) m/z=476 [M+H]+; H-NMR (DMSO-
(E)-3-(4-(Methoxycarbonyl)phenyl)acrylic Acid 8 To a d6, 300MHz, δ ppm): 8.63 (br, 1H), 8.04 (d, 2H, J=7.8Hz,
flask was added methyl 4-formylbenzoate 7 (1.64g, 10mmol), Ar-H), 7.84 (d, J=7.8Hz, 2H, Ar-H), 7.73 (m, 1H, Ar-H),
propanedioic acid (1.56g, 15mmol), piperidine (0.08mL, 7.52–7.63 (m, 4H, Ar-H), 7.48 (d, J=16.2Hz, 1H, CH=), 6.55
0.81mmol), and pyridine (10mL) at room temperature. The re- (d, J=16.2Hz, 1H, CH=), 4.59 (t, 2H, J=4.8Hz, OCH2), 4.49
action mixture was heated to 85°C for 3h under a steady flow (t, 2H, J=4.8Hz, OCH2); ESI-HR-MS (m/z): [M+H]+ Calcd
of nitrogen gas, cooled to room temperature, and poured into for C20H18N3O9S: 476.0764; Obsd: 476.0770; Anal. Calcd for
2M aqueous HCl (100mL). The resulting mixture was cooled C20H17N3O9S: C, 50.53; H, 3.60; N, 8.84; Found: C, 50.36; H,
to 0°C and filtered. The filter cake washed with acetonitrile 3.69; N, 8.62.
(2×10mL), and dried in vacuo to afford 8 as white solid, yeild
85%, mp: 140–142°C.
(E)-4-(3-((4-(3-(Hydroxyamino)-3-oxoprop-1-en-1-yl)-
benzoyl)oxy)propoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole
(E)-4-(3-Oxo-3-(((tetrahydro-2H-pyran-2-yl)oxy)amino)- 2-Oxide (12b) The title compound was obtained starting
prop-1-en-1-yl)benzoic Acid 10 To a solution of 8 (1.65g, from 10 and 6b. White waxy solid, yield: 65%. Analytical
8mmol) in 50mL anhydrous tetrahydrofuran (THF) at 0°C data for 12b: IR (KBr, cm−1): 3441, 2930, 1732, 1615, 1450,
were added N-methylmorpholine (1.05g, 12mmol) and ethyl 1232, 1035; MS (ESI) m/z=490 [M+H]+; 1H-NMR (DMSO-d6,
carbonochloridate (0.87g, 8mmol), and the mixture was 300MHz, δ ppm): 10.27 (br, 1H), 8.56 (br, 1H), 8.00 (d, 2H,
stirred at room temperature for 1h, which was then added J=7.8Hz, Ar-H), 7.81 (d, J=7.8Hz, 2H, Ar-H), 7.70 (m, 1H,
dropwise to a solution of tetrahydro-2H-pyran-2-ol (0.94g, Ar-H), 7.51–7.65 (m, 4H, Ar-H), 7.46 (d, J=16.2Hz, 1H, CH=),
8mmol) in 10mL anhydrous THF. After the reaction was 6.52 (d, J=16.2Hz, 1H, CH=), 4.61 (t, 2H, J=4.8Hz, OCH2),

1190
Vol. 62, No. 12
4.50 (t, 2H, J=4.8Hz, OCH2), 2.39 (m, 2H, OCH2CH2); ESI-
(E)-4-(3-(4-(3-(Hydroxyamino)-3-oxoprop-1-en-1-yl)-
HR-MS (m/z): [M+H]+ Calcd for C21H20N3O9S: 490.0920; benzamido)propoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole
Found: 490.0915; Anal. Calcd for C21H19N3O9S: C, 51.53; H, 2-Oxide (12g) The title compound was obtained starting
3.91; N, 8.59; Found: C, 51.38; H, 4.02; N, 8.46.
from 10 and 6g. Pale yellow waxy solid, yield: 58%. Analyti-
(E)-4-(4-((4-(3-(Hydroxyamino)-3-oxoprop-1-en-1-yl)- cal data for 12g: IR (KBr, cm−1): 3425, 2946, 1718, 1617, 1431,
benzoyl)oxy)butoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 1234, 1165; MS (ESI) m/z=489 [M+H]+; H-NMR (DMSO-d6,
1
2-Oxide (12c) The title compound was obtained start- 300MHz, δ ppm): 10.33 (br, 1H), 8.52 (br, 1H), 8.02 (d, 2H,
ing from 10 and 6c. Pale yellow waxy solid, yield: 56%. J=7.8Hz, Ar-H), 7.83 (d, J=7.8Hz, 2H, Ar-H), 7.73 (m, 1H,
Analytical data for 12c: IR (KBr, cm−1): 3452, 2965, 1716, Ar-H), 7.58–7.66 (m, 4H, Ar-H), 7.46 (d, J=16.2Hz, 1H, CH=),
1611, 1444, 1242, 1168, 1052; MS (ESI) m/z=504 [M+H]+; 6.52 (d, J=16.2Hz, 1H, CH=), 4.59 (t, 2H, J=4.8Hz, OCH2),
1H-NMR (DMSO-d6, 300MHz, δ ppm): 8.47 (br, 1H), 8.02 3.55 (t, 2H, J=4.8Hz, NCH2), 2.32 (m, 2H, OCH2CH2); ESI-
(d, 2H, J=7.8Hz, Ar-H), 7.79 (d, J=7.8Hz, 2H, Ar-H), 7.71 HR-MS (m/z): [M+H]+ Calcd for C21H21N4O8S: 489.1080;
(m, 1H, Ar-H), 7.53–7.66 (m, 4H, Ar-H), 7.44 (d, J=16.2Hz, Found: 489.1077; Anal. Calcd for C21H20N4O8S: C, 51.64; H,
1H, CH=), 6.51 (d, J=16.2Hz, 1H, CH=), 4.63 (t, 2H, 4.13; N, 11.47; Found: C, 51.83; H, 3.98; N, 11.23.
J=4.8Hz, OCH2), 4.53 (t, 2H, J=4.8Hz, OCH2), 2.26–2.33
(E)-4-(4-(4-(3-(Hydroxyamino)-3-oxoprop-1-en-1-yl)-
(m, 2H, OCH2CH2CH2); ESI-HR-MS (m/z): [M+H]+ Calcd benzamido)butoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole
for C22H22N3O9S: 504.1077; Found: 504.1084; Anal. Calcd for 2-Oxide (12h) The title compound was obtained starting
C22H21N3O9S: C, 52.48; H, 4.20; N, 8.35; Found: C, 52.33; H, from 10 and 6h. Pale yellow waxy solid, yield: 52%. Ana-
4.38; N, 8.17.
lytical data for 12h: IR (KBr, cm−1): 3429, 2944, 1724, 1620,
(E)-4-((4-((4-(3-(Hydroxyamino)-3-oxoprop-1-en-1-yl)- 1441, 1220, 1032; MS (ESI) m/z=503 [M+H]+; 1H-NMR
benzoyl)oxy)but-2-yn-1-yl)oxy)-3-(phenylsulfonyl)-1,2,5- (DMSO-d6, 300MHz, δ ppm): 10.26 (br, 1H), 8.48 (br,
oxadiazole 2-Oxide (12d) The title compound was obtained 1H), 8.06 (d, 2H, J=7.8Hz, Ar-H), 7.87 (d, J=7.8Hz, 2H,
starting from 10 and 6d. Pale yellow waxy solid, yield: 53%. Ar-H), 7.77 (m, 1H, Ar-H), 7.56–7.64 (m, 4H, Ar-H), 7.48
Analytical data for 12d: IR (KBr, cm−1): 3420, 2931, 1710, (d, J=16.2Hz, 1H, CH=), 6.56 (d, J=16.2Hz, 1H, CH=),
1
1615, 1450, 1170, 1020; MS (ESI) m/z=500 [M+H]+; H-NMR 4.60 (t, 2H, J=4.8Hz, OCH2), 3.57 (t, 2H, J=4.8Hz, NCH2),
(DMSO-d6, 300MHz, δ ppm): 8.55 (br, 1H), 8.08 (d, 2H, 2.06–2.11 (m, 2H, OCH2CH2CH2); ESI-HR-MS (m/z): [M+H]+
J=7.8Hz, Ar-H), 7.83 (d, J=7.8Hz, 2H, Ar-H), 7.75 (m, 1H, Calcd for C22H23N4O8S: 503.1237; Found: 503.1241; Anal.
Ar-H), 7.55–7.67 (m, 4H, Ar-H), 7.51 (d, J=16.2Hz, 1H, CH=), Calcd for C22H22N4O8S: C, 52.58; H, 4.41; N, 11.15; Found: C,
6.57 (d, J=16.2Hz, 1H, CH=), 5.11 (s, 2H, OCH2), 5.03 (s, 2H, 52.43; H, 6.59; N, 11.06.
OCH2); ESI-HR-MS (m/z): [M+H]+ Calcd for C22H18N3O9S:
(E)-4-((1-(4-(3-(Hydroxyamino)-3-oxoprop-1-en-1-yl)-
500.0764; Found: 500.0758; Anal. Calcd for C22H17N3O9S: C, benzamido)propan-2-yl)oxy)-3-(phenylsulfonyl)-1,2,5-oxa-
52.91; H, 3.43; N, 8.41; Found: C, 53.12; H, 3.37; N, 8.55. diazole 2-Oxide (12i) The title compound was obtained
(E)-4-((4-((4-(3-(Hydroxyamino)-3-oxoprop-1-en-1-yl)- starting from 10 and 6i. Pale yellow waxy solid, yield:
benzoyl)oxy)butan-2-yl)oxy)-3-(phenylsulfonyl)-1,2,5-oxa- 55%. Analytical data for 12i: IR (KBr, cm−1): 3414, 2928,
diazole 2-Oxide (12e) The title compound was obtained 1717, 1615, 1425, 1235, 1052; MS (ESI) m/z=489 [M+H]+;
starting from 10 and 6e. Pale yellow waxy solid, yield: 1H-NMR (DMSO-d6, 300MHz, δ ppm): 10.31 (br, 1H), 8.45
61%. Analytical data for 12e: IR (KBr, cm−1): 3432, 2939, (br, 1H), 8.03 (d, 2H, J=7.8Hz, Ar-H), 7.81 (d, J=7.8Hz, 2H,
1728, 1622, 1458, 1210, 1021; MS (ESI) m/z=504 [M+H]+; Ar-H), 7.75 (m, 1H, Ar-H), 7.54–7.63 (m, 4H, Ar-H), 7.48 (d,
1H-NMR (DMSO-d6, 300MHz, δ ppm): 10.28 (br, 1H), 8.49 J=16.2Hz, 1H, CH=), 6.54 (d, J=16.2Hz, 1H, CH=), 4.53
(br, 1H), 8.06 (d, 2H, J=7.8Hz, Ar-H), 7.88 (d, J=7.8Hz, 2H, (m, 1H, OCH), 3.88 (m, 2H, NCH2), 1.48 (d, 3H, J=6.3Hz,
Ar-H), 7.76 (m, 1H, Ar-H), 7.56–7.68 (m, 4H, Ar-H), 7.50 (d, CHCH3); ESI-HR-MS (m/z): [M+H]+ Calcd for C21H21N4O8S:
J=16.2Hz, 1H, CH=), 6.56 (d, J=16.2Hz, 1H, CH=), 4.56 489.1080; Found: 489.1086; Anal. Calcd for C21H20N4O8S: C,
(t, 2H, J=4.8Hz, OCH2), 4.35 (m, 2H, OCH), 2.25–2.30 (m, 51.64; H, 4.13; N, 11.47; Found: C, 51.39; H, 4.31; N, 11.62.
2H, OCHCH2), 1.45 (d, 3H, J=6.3Hz, CHCH3); Anal. Calcd
(E)-4-(2-(4-(3-(Hydroxyamino)-3-oxoprop-1-en-1-yl)-N-
for C22H22N3O9S: 504.1077; Found: 504.1072; Anal. Calcd for methylbenzamido)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadi-
C22H21N3O9S: C, 52.48; H, 4.20; N, 8.35; Found: C, 52.61; H, azole 2-Oxide (12j) The title compound was obtained
4.33; N, 8.27.
starting from 10 and 6j. Pale yellow waxy solid, yield:
(E)-4-(2-(4-(3-(Hydroxyamino)-3-oxoprop-1-en-1-yl)- 60%. Analytical data for 12j: IR (KBr, cm−1): 3426, 2935,
benzamido)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 1719, 1626, 1440, 1243, 1162; MS (ESI) m/z=489 [M+H]+;
2-Oxide (12f) The title compound was obtained starting 1H-NMR (DMSO-d6, 300MHz, δ ppm): 10.23 (br, 1H), 8.38
from 10 and 6f. Pale yellow waxy solid, yield: 61%. Analyti- (br, 1H), 8.07 (d, 2H, J=7.8Hz, Ar-H), 7.87 (d, J=7.8Hz,
cal data for 12f: IR (KBr, cm−1): 3436, 2945, 1621, 1453, 1211, 2H, Ar-H), 7.78 (m, 1H, Ar-H), 7.57–7.66 (m, 4H, Ar-H),
1
1166, 1018; MS (ESI) m/z=477 [M+H]+; H-NMR (DMSO- 7.50 (d, J=16.2Hz, 1H, CH=), 6.57 (d, J=16.2Hz, 1H, CH=),
d6, 300MHz, δ ppm): 10.21 (br, 1H), 8.65 (br, 1H), 8.04 (d, 4.68 (t, 2H, J=4.8Hz, OCH2), 3.53 (t, 2H, J=4.8Hz, NCH2),
2H, J=7.8Hz, Ar-H), 7.85 (d, J=7.8Hz, 2H, Ar-H), 7.70 (m, 3.04 (s, 3H, NCH3); ESI-HR-MS (m/z): [M+H]+ Calcd for
1H, Ar-H), 7.63 (d, J=7.8Hz, 2H, Ar-H), 7.55 (m, 2H, Ar-H), C21H21N4O8S: 489.1080; Found: 489.1075; Anal. Calcd for
7.49 (d, J=16.2Hz, 1H, CH=), 6.55 (d, J=16.2Hz, 1H, CH=), C21H20N4O8S: C, 51.64; H, 4.13; N, 11.47; Found: C, 51.77; H,
4.60 (t, 2H, J=4.8Hz, OCH2), 3.92 (t, 2H, J=4.8Hz, NCH2); 4.22; N, 11.58.
ESI-HR-MS (m/z): [M+H]+ Calcd for C20H19N4O8S: 475.0924;
Cell Culture SW620, HCT116, Lovo (human colon car-
Found: 475.0919; Anal. Calcd for C20H18N4O8S: C, 50.63; H, cinoma cells), MCF-7 (human breast adenocarcinoma cells),
3.82; N, 11.81; Found: C, 50.45; H, 4.01; N, 11.64. and Hela cells were maintained in 10% fetal bovine serum

December 2014
1191
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overnight and treated with or without different concentrations
of test compounds for various periods of time. During the last
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the resulting formazan crystals were dissolved in 150µL of
DMSO and measured using a spectrophotometer (Tecan) at
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triplicate. Inhibition rate (%)=[(Acontrol−Atreated)/Acontrol]×100%.
In Vitro HDACs Inhibition Fluorescence Assay In vitro
HDACs inhibition assays were conducted according to the
literature.31) In brief, 10µL of enzyme solution (HeLa nuclear
extract, HDAC2, HDAC6, or HDAC8) was mixed with various
concentrations of tested compound. Five minutes later, fluo-
rogenic substrate Boc-Lys (acetyl)-AMC (40µL) was added,
and the mixture was incubated at 37°C for 30min and then
stopped by addition of 100µL of developer containing trypsin
and TSA. After incubation at 37°C for 20min, fluorescence
intensity was measured using a microplate reader at excitation
and emission wavelengths of 390 and 460nm, respectively.
The inhibition ratios were calculated from the fluorescence
intensity readings of tested wells relative to those of control
wells, and the IC50 values were calculated using a regression
analysis of the concentration/inhibition data.
Nitrate/Nitrite Measurement in Vitro The levels of
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were determined by the colorimetric assay using the nitrate/
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the manufacturer’s instructions. Briefly, HCT116 cells (1×106/
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were detected by the Griess assay. The absorbance was read
at 540nm on a spectrophotometer (Smart spec, Bio-Rad). The
cells treated with diluent were used as negative controls for
the background levels of nitrate/nitrite production, while with
sodium nitrate at different concentrations was used as positive
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