Design, synthesis and biological evaluation of novel compounds with conjugated structure as anti-tumor agents

Article abstract of DOI:10.1016/j.bmc.2008.07.066

A series of hydroxamic acids with conjugated structure was designed and synthesized to explore the possible HDAC subtype selectivity by testing these compounds against recombinant human HDAC1 and HDAC4. The most selective compound resulted 5a, with a SI of 11.9. The enzymatic inhibitory activity of these conjugated compounds was relatively weak; however, some of these compounds showed significant effect in inducing apoptosis. Moreover, the anti-proliferative activity in cancer cells resulted quite promising, especially in the HCT119 cell line.

Full text of DOI:10.1016/j.bmc.2008.07.066

Bioorganic & Medicinal Chemistry 16 (2008) 7992–8002
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of novel compounds with
conjugated structure as anti-tumor agents
a,
Hong Su a, Angela Nebbioso b, Vincenzo Carafa b, Yadong Chen a, Bo Yang c, Lucia Altucci b, , Qidong You
*
*
a Department of Medicinal Chemistry, China Pharmaceutical University, People’s Republic of China
b Dipartimento di Patologia generale, Seconda Università degli Studi di Napoli, Italy
c Department of Pharmacology, School of Pharmaceutical Science, Zhejiang University, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of hydroxamic acids with conjugated structure was designed and synthesized to explore the pos-
sible HDAC subtype selectivity by testing these compounds against recombinant human HDAC1 and
HDAC4. The most selective compound resulted 5a, with a SI of 11.9. The enzymatic inhibitory activity
of these conjugated compounds was relatively weak; however, some of these compounds showed signif-
icant effect in inducing apoptosis. Moreover, the anti-proliferative activity in cancer cells resulted quite
promising, especially in the HCT119 cell line.
Received 12 June 2008
Revised 22 July 2008
Accepted 23 July 2008
Available online 29 July 2008
Keywords:
Ó 2008 Elsevier Ltd. All rights reserved.
Histone deacetylase
HDAC inhibitor
Hydroxamic acids
Anti-tumor
1. Introduction
metabolic and pharmacokinetic problems such as glucoronidation
and sulfation that may result in a short half-life in vivo.8
The correlation between histones acetylation and gene tran-
scription has been recognized for many years.1 The acetylation sta-
tus of histones is determined by histone deacetylases (HDACs) and
histone acetyltransferases (HATs). HATs transfer acetyl groups to
amino-terminal lysine residues on histones, leading to local expan-
sion of chromatin and increased accessibility of regulatory proteins
to DNA,2 whereas HDACs catalyze the removal of acetyl groups,
resulting in chromatin condensation and transcriptional repres-
sion.3 Recently, many non-histone proteins including transcription
factors, transcription regulators, signal transduction mediators,
DNA repair enzymes have been identified to be subject to acetyla-
tion,4 further suggesting the importance of acetylation in the up-
stream regulation of gene transcription. HDACs are widely
recognized as promising targets for therapeutic intervention in
cancer.3,5,6 More than 80 clinical trials are underway, testing differ-
ent HDAC inhibitors in malignant diseases from rare leukemias and
lymphomas to breast, prostate and ovarian cancers.7 Half of these
inhibitors have the hydroxamic acid moiety, a typical zinc-binding
group. One of them, SAHA, is the first HDAC inhibitor that has been
approved by the FDA for the second line treatment of cutaneous T-
cell lymphoma (CTCL). However, hydroxamic acids usually have
Oxamflatin (1, Fig. 1) was originally identified as a compound
inducing the morphological reversion of v-K-ras-transformed
NIH3T3 cells from a chemical library. Only later it was shown to
be a potent HDAC inhibitor (IC50 = 15.7 nM).9 CG1521 (2, Fig. 1),
which is orally available, was reported to have anti-proliferative ef-
fects in cancer cell lines, displaying also an in vivo activity, causing
a 50% reduction in mean tumor volume after intraperitoneal
administration. The HDAC inhibitory activity of CG1521 was mod-
* Corresponding authors. Tel.: +39 081 566 7569; fax: +39 081 2144840 (L.A.);
tel./fax: +86 025 83141351 (Q.Y.).
E-mail addresses: lucia.altucci@unina2.it (L. Altucci), youqidong@gmail.com (Q.
You).
Figure 1. Pharmacophore model of HDAC inhibitors and HDAC inhibitors with
stereodefined structures.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.07.066

H. Su et al. / Bioorg. Med. Chem. 16 (2008) 7992–8002
7993
erate (IC50 = 1.8
l
M). The structure of (2E,4E)-5-arylpenta-2,4-die-
furfural 7 with triethyl phosphonacetate in absolute ethanol con-
taining anhydrous potassium carbonate. (E)-Ethyl 3-(5-formylfu-
ran-2-yl)acrylate 9 was prepared by Vilsmeier-Hacck reaction.
Aldol condensation with acetophenone or substituted acetophe-
none gave the corresponding highly conjugated (E)-Ethyl 3-(5-
(un)substituted-styrylfuran-2-yl)acrylates, which were hydrolyzed
without separation and purification to give 10a–j. Further reaction
with oxalyl chloride and hydroxylamine gave the desired hydroxa-
mates 5a–j.
For compounds 6a–h, the starting material was Methyl furan-2-
carboxylate 12, which was chloromethylated. The resulted methyl
5-(chloromethyl) furan-2-carboxylate 13 was converted into the
corresponding triphenyl phosphonium salt 14, which was treated
with benzaldehyde or substituted benzaldehyde to give the corre-
sponding (E)-methyl 5-(un)substituted-styrylfuran-2-carboxylates
15a–h. The Methyl esters were hydrolyzed; further reaction with
oxalyl chloride and hydroxylamine gave the desired hydroxamates
6a–h (Scheme 2).
noic acid hydroxyamides (3, Fig. 1) is similar to 2, and they act as
HDAC inhibitors with moderate inhibitory potency.10 These find-
ings confirmed that the stereochemical rigidity seems to be advan-
tageous for the enzyme inhibition.
On the other hand, Mai and co-workers have disclosed a series
of aroyl-pyrrolyl-hydroxy-amides (APHAs).11 Some APHAs showed
distinct selectivity against HD1-B and HD1-A, two maize deacety-
lases that are homologous of mammalian class I and class IIa
HDACs, respectively. The most selective APHAs is 4 (Fig. 1), and
its selective ratio is 176.4. As hydroxamates generally lack selectiv-
ity due to their strong zinc-binding ability, and also because of the
rarity of class II selective HDAC inhibitors reported so far, we got
interested in the selectivity of APHAs. We speculated that the con-
jugated structure may contribute to the selectivity for its rigid
structure directing the ‘cap’ group of the inhibitor to interact with
the rim of the catalytic pocket of the HDAC enzymes. To further
exploration the inhibitory activity of hydroxamic acids character-
ized with conjugated stereodefined structure against mammalian
HDACs, as well as their anti-proliferative activity at the cellular le-
vel, we designed and synthesized compounds 5a–j and 6a–h.
The structures of compounds 5a–j are quite similar to APHAs,
the main difference lying in the aromatic linker group. The furan
ring in 5a–j is smaller than N-methyl pyrrole ring, and most impor-
tantly, the substitutes on the furan ring are at 2,5-position, while
the substitutes on the pyrrole ring are at 2,4-position. Thus, the ri-
gid backbones in compound 4 and compounds 5a–j could direct
the cap group of these compounds to interact with the rim of the
catalytic pocket of the enzyme in a relative different configuration.
The structures of 6a–h are similar to that of 3, with a furan ring
connecting the hydroxamic acid group. We presumed that 6a–h
with more bulky furan ring could make more favored contacts with
the stacked phenyl groups of the two Phe residues than 3.
We initially carried out plasma stability in vitro studies, validat-
ing the positive effect of conjugated structures on the stability of
the hydroxamic acid group. The cellular effects of these com-
pounds on apoptosis, granulocytic differentiation, and cell cycle
progression in human acute myeloid leukemia (AML) U937 cells
have also been assessed. The inhibitory activity of these com-
pounds against human HDAC1 and HDAC4, considered as repre-
sentative of class I and class IIa HDACs, respectively, was further
tested to evaluate their selectivity profile. As direct measure of
the anti-proliferative effects in vitro, as well as indirect measure
of cell permeability, the anti-proliferative cytotoxic activity was
also evaluated by using four different tumor cell lines. Finally,
the most effective compounds were tested for their ability to affect
acetylation levels and expression of p21WAF1/CIP1 taken as histone
and non-histone targets.
3. Results and discussion
We chose compounds 5j and 6a as representatives to study.
Both 5j and 6a have neither electro-withdrawing nor electro-
donating substitutes, thereby eliminating the possible influence
of substitutes on stability.
As shown in Figures 2A and B, both 5j and 6a show significant
increase in plasma stability. 5j seems more stable than 6a for its
highly conjugated structure.
Then, we analyzed the effect of 5a–j and 6a–h on cell cycle, dif-
ferentiation, and apoptosis in the human acute myeloid leukemia
U937 cell line.
As shown in Figure 3, 5a, 5e, 5h, and 5j significantly induced
accumulation of the cells in the S phase, whereas 5b–d, 5f, 5g,
and 5i induced accumulation of the cells in the G1 phase. 5a, 5c,
5e, 5f, 5h, and 5i were able to induce apoptosis. The effect of 5a–
j on differentiation could not be evaluated for the interference of
their fluorescence with CD11c FACS analysis.
Compounds 6a–c induce higher percentage of apoptosis (Fig.
4B), whereas they only moderately increase differentiation after
30 h of treatment (Fig. 4A), as assessed by the analysis of the
expression of CD11c, a marker for granulocytic maturation; the
expression of the CD14, a marker for monocytic differentiation
was also measured but did not show signal (data not shown) thus
indicating that these compounds did not induce monocytic differ-
entiation in these experimental conditions.
Compound 6f, 6d, 6e, 6g and 6h induced high toxicity in the
cells at the concentration of 5 lM, thereby the concentration of
these compounds was diluted to 0.5 lM and the effects were eval-
2. Chemistry
As shown in the Scheme 1, synthesis of compounds 5a–j started
from preparation of (E)-ethyl 3-(furan-2-yl)acrylate 8 by reacting
a
a
Scheme 1. Synthesis of compounds 5a–j . Reagents and conditions: (a)
Scheme 2. Synthesis of compounds 6a–h . Reagents and conditions: (a) parafor-
maldehyde, ZnCl2, HCl, CH2Cl2, rt; (b) PPh3, CH2Cl2, reflux; (c) Et3N, CH2Cl2; (d) 2 N
NaOH, EtOH, H2O; (e) 1—(COCl)2, DMF, CH2Cl2, 0 °C; 2—NH2OHÁHCl, Et3N, THF, H2O.
(C2H5)2P(O)CH2CO2C2H5, K2CO3, EtOH; (b) POCl3, DMF; (c) KOH, EtOH, H2O; (d)
1—(COCl)2, DMF, CH2Cl2, 0 °C; 2—NH2OHÁHCl, Et3N, THF, H2O.

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H. Su et al. / Bioorg. Med. Chem. 16 (2008) 7992–8002
Figure 2. (A) Stability comparison of the indicated compounds in rat plasma at 25 lg/mL. (B) Stability comparison of the indicated compounds in plasma at 100 lg/mL.
Figure 3. Analysis of cell cycle and apoptosis in U937 cells treated with the indicated compounds at 5 lM for 24 h. The data represent the media of independent duplicates.
uated. Compounds 6d and 6e showed no effect on cell cycle, differ-
entiation and apoptosis after dilution. Compounds 6g and 6h were
able to increase differentiation significantly after 28 h of treatment
tor, with IC50 of 1.10
inhibitor, with IC50 of 22.78
l
M, whereas compound 5i is the weakest
M. The only structural difference be-
l
tween 5a and 5j is the substitute in position C4 of the benzene ring,
and the inhibitory potency of compounds 5a, 5d, 5i, and 5j changed
regularly. Their inhibitory potency against HDAC4 increased when
the substitutes (H, F, Cl and methyl) in position C4 of the benzene
ring changed bigger, indicating a shallow groove may exist around
the rim of the catalytic pocket of HDAC4 and the substitutes in
position C4 of the benzene ring should fit into this groove. The
inhibitory potency of compound 5b with a much bigger substitute
(iso-propyl) in position C4 of the benzene ring decreased slightly
at the concentration of 0.5 lM (Fig. 5A), and compound 6h could
also induce apoptosis after 28 h of treatment (Fig. 5B). Note that
in all differentiation assays PI positive cells have been excluded
from the analysis as specified.
We further performed enzymatic assays to evaluate the activity
on human recombinant HDAC1 and HDAC4, taken as markers for
class I and class II HDACs, respectively. As to the inhibitory potency
against HDAC4 (Table 1), compound 5a is the most potent inhibi-

H. Su et al. / Bioorg. Med. Chem. 16 (2008) 7992–8002
7995
Figure 4. (A) Differentiation analysis in U937 cells after 30 h of treatment with the indicated compounds at 5
quadruplicates. (B) Analysis of cell cycle and apoptosis in U937 cells treated with the indicated compounds at 5
duplicates.
l
M. The data shown represent the media of independent
lM for 30 h. The data represent the media of independent
against HDAC4, compared with compound 5a, suggesting a strict
size restriction of the groove. The inhibitory potency of compounds
5a, 5d, 5i, and 5j against HDAC1 did not change regularly, verifying
the structural difference around the rim of the catalytic pockets be-
tween HDAC1 and HDAC4. Compounds 5g and 5h bearing two
chlorine substitutes in the benzene ring had better inhibitory po-
tency than both 5c and 5d bearing only one chlorine substituted
in the benzene ring both against HDAC1 and HDAC4. Compound
5e bearing substituted naphthyl ring was more selective than com-
pound 5f bearing un-substituted naphthyl ring.
that improved stability of hydroxamic acids may contribute to bio-
logical effects of the molecules. In general, these compounds
showed higher anti-proliferative activities against PC3 and
HCT116 than against A549 and HEPG2 cells.
Tubulin acetylation was taken as potential marker of HDAC6
inhibition. As shown in Figure 6, 5b–j showed significant effect
on inducing the tubulin acetylation level, whereas 6a–c, 6f–h
had no effect.
Class I- and pan-HDAC inhibitors usually have the capacity to
induce over-expression of p21WAF1/CIP1. As shown in Figure 5, 5b–
The enzymatic inhibitory potency and selectivity of 6a–h were
weak (Table 2); 6h bearing two bulky substitutes on the benzene
ring was the most potent of this compound series, in correspon-
dence with the SAR of 3.10
j and 6g were able to induce the expression of p21WAF1/CIP1
whereas 5a and, 6a–f and 6h did not show any effect (Fig. 7).
,
4. Conclusion
All compounds were evaluated for their anti-proliferative cyto-
toxic activity using the PC3 (prostate carcinoma), HCT116 (colorec-
tal carcinoma), A549 (lung carcinoma) and HEPG2 (hepatocellular
carcinoma) (Table 3) tumor cell lines. The majority of the com-
pounds displayed excellent anti-proliferative profiles, showing
We have synthesized and evaluated a series of hydroxamic
acids with stereodefined-conjugated structures. 5a–j, 6a–c, 6f,
and 6h showed significant effect on arresting cell cycle progres-
sion. Most of these compounds could induce apoptosis. 6a–c, 6f,

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H. Su et al. / Bioorg. Med. Chem. 16 (2008) 7992–8002
Figure 5. (A) CD11c analysis in U937 cells after 28 h treatment with the indicated compounds. 6d, 6e, 6g and 6h were analyzed at 0.5
lM while SAHA and MS-275 were at
5l
M. The data shown represent the media of independent quadruplicates. (B) Analysis of cell cycle and apoptosis in U937 cells treated with the indicated compounds for 28 h.
6d, 6e, 6g and 6h were analyzed at 0.5 lM, while SAHA and MS-275 at 5lM. The data represent the media of independent duplicates.
Table 1
Table 2
HDAC inhibitory activities of compounds 5a–ja
HDAC inhibitory activities of compounds 6a–ha
Compound
Ar
IC50 SD (lM)
SIb
Compound
Ar
IC50 SD (
l
M)
SIb
HDAC1
HDAC4
HDAC1
HDAC4
5a
5b
5c
5d
5e
5f
5g
5h
5i
4-Methyl-Ph
4-iso-Propyl-Ph
3-Chloro-Ph
4-Chloro-Ph
2-(6-Methyloxy)-naphthyl
2-Naphthyl
2,5-Dichloro-Ph
2,4-Dichloro-Ph
4-Fluro-Ph
13.20
24.10
12.60
15.70
11.04
3.77
4.72
4.29
14.98
11.55
0.59
1.10
4.89
12.91
6.03
4.04
4.38
4.31
8.19
7.68
22.78
0.55
11.9
4.9
1.0
2.6
2.7
0.9
1.1
0.5
0.5
2.0
6a
6b
6c
6d
6e
6f
6g
6h
SAHA
Ph
49.15
43.1
47.07
29.6
25.31
50.14
49.10
13.41
0.59
47.17
57.06
74.24
85.6
49.28
42.81
50.58
10.25
0.55
1.04
0.76
0.63
0.34
0.51
1.17
0.97
1.30
1.01
4-Hydroxyl-Ph
2-Hydroxyl-Ph
[3,4]Dioxol-Ph
2,3-Dichloro-Ph
2-Hydroxyl,3-methyloxy-Ph
4-(N,N-Dimethyl)-Ph
3-Methyloxy,4-benzyloxy-Ph
—
5j
SAHA
Ph
—
a
Data represent mean values of at least three separate experiments.
SI, selective index, calculated as IC50 (HDAC1)/IC50 (HDAC4).
1.01
b
a
Data represent mean values of at least three separate experiments.
SI, selective index, calculated as IC50 (HDAC1)/IC50 (HDAC4).
b
limited. We also tested the relatively potent compounds 5f and 6h
and 6h also showed promising profile in induction of differentia-
tion. However, the selectivity as well as the inhibitory potency of
these compounds against mammalian HDAC1 and HDAC4 resulted
using the commercially available kit AK-500 (BIOMOL), the IC50 of
5f and 6h are 0.63
lM and 1.89
l
M, respectively. In the same as-
M and
say, the inhibitory IC50 of SAHA and MS-275 are 0.042
l

H. Su et al. / Bioorg. Med. Chem. 16 (2008) 7992–8002
7997
Table 3
activity, while straight shape molecular skeleton leads to selective
HD1-B compounds.12 Thus, the shape of the stereodefined struc-
ture is also crucial in subtype selectivity. From our enzyme inhib-
itory data we could deduce that the substitutes on the ‘cap’ region
might also influence the subtype selectivity, high-lightened by the
structure and SI relationship of 5a and 5j. Although the HDAC
inhibitory activity of these compounds is weaker than SAHA, many
of these compounds had anti-proliferative activity in several tumor
cell lines. This finding might be due to the improved stability of
hydroxamic acids, or to off-target effects of these compounds.
The existence of ‘off-target’ effect of 5a–h in tumor cell is compat-
ible with the observation that these compounds had no effects in
increasing tubulin acetylation and/or p21WAF1/CIP1 levels. Further
biological study of these compounds is underway.
Anti-proliferative activities of the indicated compounds
Compound
PC3
IC50
HCT116
IC50 M)
A549
IC50
HEPG2
IC50 M)
(lM)
(l
(l
M)
(l
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
6a
6b
6c
6d
6e
6f
16.01
2.27
9.60
0.54
0.49
1.51
2.01
2.06
2.22
4.12
6.73
2.82
2.89
2.49
2.73
5.10
4.98
3.39
48.64
56.68
18.92
98.11
—
29.37
27.06
88.11
—
20.15
32.54
86.37
79.15
63.61
23.58
—
46.31
50.28
—
16.04
35.90
54.48
6.74
6.22
9.93
1.35
36.27
15.09
24.70
16.24
40.14
13.28
40.40
21.78
91.30
45.93
39.25
35.13
3.20
48.27
13.52
12.00
12.12
12.78
20.92
54.03
33.56
37.20
—
31.69
30.93
63.48
60.12
0.62
5. Experimental
5.1. General
—
—
—
2.69
27.47
6.18
6g
6h
SAHA
MS-275
Taxol
1.20
8.17
59.06
2.73
Melting points were determined on a Mel-TEMP II melting point
apparatus and are uncorrected. Infrared (IR) spectra (KBr) were re-
corded on a Nicolet Impact 410 instrument (KBr pellet). 1H NMR
spectra were recorded with a Bruker Avance 300 MHz spectrome-
ter at 300 K, using TMS as an internal standard. MS spectra were
recorded on a Shimadzu GC–MS 2010 (EI) or a Mariner Mass Spec-
trum (ESI), or a LC/MSD TOF HR-MS Spectrum. Compound purity
was assessed by the reverse phase HPLC (Waters 510, Shimadzu
—
13.05
SPD-10A VP, using Hanbang C18 4.6 Â 150 mm, 5
lm). All com-
pounds were routinely checked by TLC and 1H NMR. TLCs and pre-
parative thin-layer chromatography were performed on silica gel
GF/UV 254, and the chromatograms were performed on silica gel
(200–300 mesh) visualized under UV light at 254 and 365 nm. All
solvents were reagent grade and, when necessary, were purified
and dried by standards methods. Concentration of solutions after
reactions and extractions involved the use of a rotary evaporator
operating at a reduced pressure of ca. 20 Torr. Organic solutions
were dried over anhydrous sodium sulfate. Analytical results are
within (0.40% of the theoretical values).
Figure 6. Western blot analyses of a-tubulin acetylation carried out in U937 cells
after 24 h of treatment with the indicated compounds. Total ERK levels as account
for equal loading control.
5.1.1. (E)-Ethyl 3-(furan-2-yl)acrylate (8)
Furfural (7, 0.1 mol, 8.28 mL) in absolute ethanol (10 mL) was
added in one portion to a mixture of triethyl 2-phosphonobutyrate
(0.12 mol, 23.8 mL) and anhydrous potassium carbonate (0.3 mol,
41.4 g) in absolute ethanol (100 mL). After being stirred at 70 °C
for 2 h, the reaction mixture was cooled to room temperature, fil-
trated and washed with ethanol (2Â 20 mL). The filtrate was con-
centrated and distilled under reduced pressure to give pure
yellowish solid (13.6 g) in 82% yield; mp 24–26 °C [lit.13 24 °C].
5.1.2. (E)-Ethyl 3-(5-formylfuran-2-yl)acrylate (9)
POCl3 (0.13 mol, 11.9 mL) was added to a cooled (0 °C) mixture
of (E)-ethyl 3-(furan-2-yl)acrylate (0.12 mol, 19.6 g) and DMF
(0.13 mol, 10.0 mL) over a period of 20 min. After stirring at 60 °C
for 2 h, the mixture was cooled to room temperature and poured
onto crushed ice (200 g). The pH of the mixture was adjusted to 7
with 10% NaOH. The resulted precipitate was filtered and purified
to give brown solid (19.2 g) in 82.5% yield by crystallization with
a mixture of ethanol and water; mp 73.4–74 °C [lit.14 74 °C].
Figure 7. Western blot analysis of p21WAF1/CIP1 expression in U937 cells after 24 h
treatment with the indicated compounds. Tubulin is used as equal loading control.
2.76 lM, respectively. Thus, the general inhibitory activity of our
compounds against HDACs is acceptable. The relative weak inhib-
itory activities of our compounds were likely due to the unfavor-
able binding of the stereodefined structure with enzyme catalytic
pocket. Comparing the structure of oxamflatin, we could conclude
that the shape of the stereodefined structure is very important in
HDAC inhibition. Alignment-independent GRIND 3D-QSAR and
docking studies on APHAs carried out on homology modeling of
maize HD1-A and HD1-B, revealed that in general a bent molecular
shape structure is a prerequisite for HD1-A selective inhibitory
5.1.3. Representative procedure for aldol condensation
5.1.3.1. (E)-3-(5-((E)-3-Oxo-3-p-tolylprop-1-enyl)furan-2-yl)acry
lic acid (10a). A mixture of 9 (5.0 mmol, 0.97 g), 1-p-tolyletha-
none (5.0 mmol, 0.67 mL), and 2 N KOH (20.0 mmol, 10.0 mL) in
ethanol (10 mL)/water (10 mL) was stirred at room temperature
for 24 h. Afterward, the solution was poured into water (60 mL)
and was made acid with 2 N HCl. The obtained precipitate was fil-

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H. Su et al. / Bioorg. Med. Chem. 16 (2008) 7992–8002
tered and recrystallized to give the pure acid (0.97 g) in 68.5%
yield; mp 154–155 °C; 1H NMR (DMSO-d6, 300 MHz, d, ppm) 2.41
(s, 3H), 6.53 (d, 1H, J = 15.5), 6.95 (d, 1H, J = 3.3), 7.19 (d, 1H,
J = 3.3), 7.32 (d, 1H, J = 15.5), 7.39 (d, 2H, J = 8.0), 7.52 (d, 1H,
J = 15.4), 7.60 (d, 1H, J = 15.4), 7.98 (d, 2H, J = 8.0), 12.4 (s, 1H);
MS (ESI) m/z 281 (MÀ1); Anal. (C17H14O4) C, H.
the analogous literature.15 Oxalyl chloride (0.65 mL, 7.54 mmol)
was added slowly to a cooled (0 °C) solution of 10a (0.95 g,
3.36 mmol) and DMF (0.26 mL, 3.36 mmol) in CH2Cl2 (50 mL).
Gas evolution was noted during this addition. After being stirred
for 40 min, this solution was added to a solution of hydroxylamine
hydrochloride (0.94 g, 13.5 mmol) and triethylamine (2.8 mL,
20.2 mmol) in THF (20 mL)/water (10 mL). After being stirred for
an additional 30 min, the mixture was poured into 2 N HCl and ex-
tracted with CH2Cl2. The organic phase was concentrated and puri-
fied by column chromatography on silica gel by eluting with a
mixture of CH2Cl2 and methanol (1:10) to give the desired product
(0.36 g) as yellowish solid in 36.5% yield; mp 145–146 °C; 1H NMR
(DMSO-d6, 300 MHz, d ppm) 2.41 (s, 3H, CH3-Ar), 6.53 (d, 1H,
J = 15.6, vinyl-H), 6.95 (d, 1H, J = 3.4, furan-H), 7.19 (d, 1H, J = 3.5,
furan-H), 7.31 (d, 1H, J = 15.6, vinyl-H), 7.39 (d, 2H, J = 8.0, Ar-H),
7.52 (d, 1H, J = 15.4, vinyl-H), 7.60 (d, 1H, J = 15.4, vinyl-H), 7.98
(d, 2H, J = 8.0, Ar-H), 9.11 (s, 1H, CONHOH), 10.84 (s, 1H, CONHOH);
MS (ESI) m/z 298 (M+H); HRMS calcd for C17H15NO4 [MH]+:
5.1.3.2. (E)-3-(5-((E )-3-(4-Isopropylphenyl)-3-oxoprop-1-enyl)-
furan-2-yl)acrylic acid (10b). Compound 10b was synthesized
from 9 (1.94 g, 0.01 mol), 1-(4-isopropylphenyl)ethanone (1.62 g,
0.01 mol) and 2 N KOH according to the procedure used to synthe-
size 10a in 19.8% yield (g); mp 160–162 °C; MS (EI) m/z 310 (M+).
5.1.3.3. (E)-3-(5-((E)-3-(3-Chlorophenyl)-3-oxoprop-1-enyl)furan-
2-yl)acrylic acid (10c). Compound 10c was synthesized from 9
(1.94 g, 0.01 mol), 1-(3-chlorophenyl)ethanone (1.3 mL, 0.01 mol)
and 2 N KOH according to the procedure used to synthesize 10a
in 66.9% yield; mp 132–134 °C; MS (EI) m/z 302 (M+).
298.1073; found, 298.1079; IR (KBr)
1612.
m
(cmÀ1) = 3451, 2911, 1648,
5.1.3.4. (E)-3-(5-((E)-3-(4-Chlorophenyl)-3-oxoprop-1-enyl)furan-
2-yl)acrylic acid (10d). Compound 10d was synthesized from 9
(1.94 g, 0.01 mol), 1-(4-chlorophenyl)ethanone (1.3 mL, 0.01 mol)
and 2 N KOH according to the procedure used to synthesize 10a
in 29.8% yield; mp 132–134 °C; MS (EI) m/z 302 (M+).
5.1.4.2. (E)-N-Hydroxy-3-(5-((E)-3-(4-isopropylphenyl)-3-oxoprop-
1-enyl)furan-2-yl)acrylamide (5b). Compound 5b was synthe-
sized from 10b according to the procedure used to synthesize 5a
in 39.2% yield; mp 118–120 °C; 1H NMR (DMSO-d6, 300 MHz, d
ppm) 1.24 (d, 6H, J = 3.0, CH(CH3)2), 2.30 (m, 1H, CH(CH3)2), 6.56
(d, 1H, J = 14.9, vinyl-H), 6.96 (d, 1H, J = 3.4, furan-H), 7.18 (d, 1H,
J = 3.4, furan-H), 7.30 (d, 1H, J = 14.9, vinyl-H), 7.45 (d, 2H, J = 7.9,
Ar-H), 7.52 (d, 1H, J = 15.4, vinyl-H), 7.58 (d, 1H, J = 15.4, vinyl-
H), 7.90 (d, 2H, J = 8.0, Ar-H), 9.1 (s, 1H, CONHOH), 10.8 (s, 1H,
CONHOH); MS (ESI) m/z 324 (MÀH); HRMS calcd for C19H19NO4
5.1.3.5. (E)-3-(5-((E)-3-(6-Methoxynaphthalen-2-yl)-3-oxoprop-
1-enyl)furan-2-yl)acrylic acid (10e). Compound 10e was syn-
thesized from 9 (1.94 g, 0.01 mol), 1-(6-methoxynaphthalen-2-
yl)ethanone (2.0 g, 0.01 mol) and 2 N KOH according to the proce-
dure used to synthesize 10a in 17.8% yield; mp 200–202 °C; MS (EI)
m/z 348 (M+).
[MH]+: 326.1386; found, 326.1382; IR (KBr)
3453, 1797, 1708, 1644.
m
(cmÀ1) = 3777,
5.1.3.6. (E)-3-(5-((E)-3-(Naphthalen-2-yl)-3-oxoprop-1-enyl)furan-
2-yl)acrylic acid (10f). Compound 10f was synthesized from 9
(1.94 g, 0.01 mol), 1-(naphthalen-2-yl)ethanone (1.70 g, 0.01 mol)
and 2 N KOH according to the procedure used to synthesize 10a in
50.0% yield; mp 190–191 °C; MS (EI) m/z 318 (M+).
5.1.4.3. (E)-3-(5-((E)-3-(3-Chlorophenyl)-3-oxoprop-1-enyl)furan-
2-yl)-N-hydroxyacrylamide (5c). Compound 5c was synthesized
from 10c according to the procedure used to synthesize 5a in
37.3% yield; mp 155–157 °C; 1H NMR (DMSO-d6, 300 MHz, d
ppm) 6.53 (d, 1H, J = 15.6, vinyl-H), 6.97 (d, 1H, J = 3.6, furan-H),
7.23 (d, 1H, J = 3.6, furan-H), 7.32 (d, 1H, J = 15.6, vinyl-H), 7.60
(d, 1H, J = 7.9, vinyl-H), 7.62 (d, 1H, J = 7.9, vinyl-H), 7.57–8.1 (m,
4H, Ar-H), 9.1 (s, 1H, CONHOH), 10.8 (s, 1H, CONHOH); MS (ESI)
m/z 316 (MÀH); HRMS calcd for C16H12NO4Cl [MH]+: 318.0527;
5.1.3.7. (E)-3-(5-((E)-3-(2,5-Dichlorophenyl)-3-oxoprop-1-enyl)-
furan-2-yl)acrylic acid (10g). Compound 10g was synthesized
from 9 (1.94 g, 0.01 mol), 1-(2,5-dichlorophenyl)ethanone (1.41
mL, 0.01 mol) and 2 N KOH according to the procedure used to syn-
thesize 10a in 65.5% yield; mp 188–190 °C; MS (EI) m/z 337 (M+).
found, 318.0528; IR (KBr)
m
(cmÀ1) = 3779, 3459, 1818, 1644.
5.1.3.8. (E)-3-(5-((E)-3-(2,4-Dichlorophenyl)-3-oxoprop-1-enyl)-
furan-2-yl)acrylic acid (10h). Compound 10h was synthesized
from 9 (1.94 g, 0.01 mol), 1-(2,4-dichlorophenyl)ethanone (1.43
mL, 0.01 mol) and 2 N KOH according to the procedure used to syn-
thesize 10a in 56.9% yield; mp 193–194 °C; MS (EI) m/z 337 (M+).
5.1.4.4. (E)-3-(5-((E)-3-(4-Chlorophenyl)-3-oxoprop-1-enyl)furan-
2-yl)-N-hydroxyacrylamide (5d). Compound 5d was synthesized
from 10d according to the procedure used to synthesize 5a in
37.7% yield; mp 150–151 °C; 1H NMR (DMSO-d6, 300 MHz, d
ppm) 6.53 (d, 1H, J = 15.2, vinyl-H), 6.97 (d, 1H, J = 3.4, furan-H),
7.23 (d, 1H, J = 3.4, furan-H), 7.26 (d, 1H, J = 15.2, vinyl-H), 7.62
(d, 1H, J = 7.9, vinyl-H), 7.63 (d, 2H, J = 7.1, Ar-H), 7.74 (d, 2H,
J = 7.1, Ar-H), 8.04 (d, 1H, J = 7.9, vinyl-H), 9.1 (s, 1H, CONHOH),
10.8 (s, 1H, CONHOH); MS (ESI) m/z 318 (M+H); HRMS calcd for
5.1.3.9. (E)-3-(5-((E)-3-(4-Flurophenyl)-3-oxoprop-1-enyl)furan-
2-yl)acrylic acid (10i). Compound 10i was synthesized from 9
(1.94 g, 0.01 mol), 1-(4-fluorophenyl)ethanone (1.23 mL, 0.01
mol), and 2 N KOH according to the procedure used to synthesize
10a in 55.1% yield; mp 196–198 °C; MS (EI) m/z 286 (M+).
C
16H12NO4Cl [MH]+: 318.0527; found, 318.0527; IR (KBr)
(cmÀ1) = 3444, 2069, 1904, 1644.
m
5.1.3.10. (E)-3-(5-((E)-3-Oxo-3-phenylprop-1-enyl)furan-2-yl)-
acrylic acid (10j). Compound 10j was synthesized from 9 (1.94 g,
0.01 mol), acetophenone (1.2 mL, 0.01 mol) and 2 N KOH according
to the procedure used to synthesize 10a in 39.2% yield (1.89 g); mp
177–178 °C; MS (EI) m/z 268 (M+).
5.1.4.5. (E)-N-Hydroxy-3-(5-((E)-3-(6-methoxynaphthalen-2-yl)-
3-oxoprop-1-enyl)furan-2-yl)acrylamide (5e). Compound 5e
was synthesized from 10e according to the procedure used to syn-
thesize 5a in 38.2% yield; mp 199–200 °C; 1H NMR (DMSO-d6,
300 MHz, d ppm) 3.93 (s, 3H, OCH3), 6.56 (d, 1H, J = 15.6, vinyl-
H), 6.97 (d, 1H, J = 3.4, furan-H), 7.20 (d, 1H, J = 3.4, furan-H),
7.33 (d, 1H, J = 15.4, vinyl-H), 7.28–8.73 (m, 8H, naphthalene-H
and vinyl-H), 9.1 (s, 1H, CONHOH), 10.8 (s, 1H, CONHOH); MS
5.1.4. Representative procedure for conversion of carboxylic
acid to hydroxamic acid
5.1.4.1. (E)-N-Hydroxy-3-(5-((E)-3-oxo-3-p-tolylprop-1-enyl)furan-
2-yl)acrylamide (5a). The compounds were prepared according to
(ESI) m/z 364 (M+H); HRMS calcd for
C
21H17NO5 [MH]+:

H. Su et al. / Bioorg. Med. Chem. 16 (2008) 7992–8002
7999
364.1179; found, 364.1176; IR (KBr)
1623, 1548.
m
(cmÀ1) = 3587, 3466, 1648,
at 30–35 °C. The reaction mixture was poured in 80 mL water.
The mixture was extracted with CH2Cl2 (3Â 30 mL). The organic
phase was combined and dried with anhydrous Na2SO4. The solu-
tion was evaporated to dryness. The remaining residue was dis-
tilled and collected the distillate as colorless oil in 32.3% yield
(11.3 g); 29–30 °C [lit.15 31–33 °C].
5.1.4.6. (E)-N-Hydroxy-3-(5-((E)-3-(naphthalen-2-yl)-3-oxoprop-
1-enyl)furan-2-yl)acrylamide (5f). Compound 5f was synthe-
sized from 10f according to the procedure used to synthesize 5a
in 35.2% yield; mp 190–191 °C; 1H NMR (DMSO-d6, 300 MHz, d
ppm) 6.49 (d, 1H, J = 15.5, vinyl-H), 6.94 (d, 1H, J = 3.5, furan-H),
7.15 (d, 1H, J = 3.5, furan-H), 7.22 (d, 1H, J = 15.6, vinyl-H), 7.28
(d, 1H, J = 15.5, vinyl-H), 7.40 (d, 1H, J = 15.6, vinyl-H), 7.58–8.31
(m, 7H, naphthalene-H), 9.1 (s, 1H, CONHOH), 10.8 (s, 1H, CON-
HOH); MS (ESI) m/z 334 (M+H); HRMS calcd for C20H15NO4
5.1.6. ((5-(Methoxycarbonyl)furan-2-yl)methyl)triphenyl-
phosphonium chloride (13)
Methyl 5-(chloromethyl)furan-2-carboxylate (3.48 g, 20 mmol),
and triphenyl phosphine (4.76 g, 20 mmol) were dissolved in anhy-
drous toluene (20 mL), refluxed for 2 h. The mixture was cooled to
room temperature. The precipitate was filtrated, washed with
anhydrous toluene (2 mL), and dried to give white powder in
87.5% yield (7.64 g); mp 223–225 °C.
[MH]+: 334.1073; found, 334.1073; IR (KBr)
3571, 3455, 1637, 1552.
m
(cmÀ1) = 3623,
5.1.4.7. (E)-3-(5-((E)-3-(2,5-Dichlorophenyl)-3-oxoprop-1-enyl)-
furan-2-yl)-N-hydroxyacrylamide (5g). Compound 5g was syn-
thesized from 10g according to the procedure used to synthesize
5a in 39.3% yield; mp 158–159 °C; 1H NMR (DMSO-d textsub-
script6, 300 MHz, d ppm) 6.49 (d, 1H, J = 15.6, vinyl-H), 6.89 (d,
1H, J = 15.9, vinyl-H), 6.94 (d, 1H, J = 3.5, furan-H), 7.15 (d, 1H,
J = 3.5, furan-H), 7.25 (d, 1H, J = 15.9, vinyl-H), 7.30 (d, 1H,
J = 15.6, vinyl-H), 7.56–7.78 (m, 3H, Ar-H), 9.11 (s, 1H, CONHOH),
10.8 (s, 1H, CONHOH); MS (ESI) m/z 352 (M+H); HRMS calcd for
5.1.7. Representative procedure for the Wittig reaction
5.1.7.1. (E)-Methyl 5-styrylfuran-2-carboxylate (14a). Benzal-
dehyde (1.02 mL, 10 mmol) diluted in CH2Cl2 (2 mL) was slowly
added to the solution of 13 (4.37 g, 10 mmol) and Et3N (1.66 mL,
12 mmol) in CH2Cl2 (10 mL). After stirring under reflux for 2 h,
the mixture was cooled to room temperature and added in 30 mL
petroleum ether. The resulted precipitate was filtered. The filtrate
was concentrated and purified by column chromatography on sil-
ica gel by eluting with a mixture of ether acetate and petroleum
ether (1:100) to give the desired product in 52.6% yield (1.09 g);
mp 102–104 °C; MS (EI) m/z 228 (M+).
C
16H11NO4Cl2 [MH]+: 352.0137; found, 352.0132; IR (KBr)
(cmÀ1) = 3672, 3629, 3459, 1637.
m
5.1.4.8. (E)-3-(5-((E)-3-(2,4-Dichlorophenyl)-3-oxoprop-1-enyl)-
furan-2-yl)-N-hydroxyacrylamide (5h). Compound 5h was syn-
thesized from 10h according to the procedure used to synthesize
5a in 42.3% yield; mp 162–164 °C; 1H NMR (DMSO-d6, 300 MHz,
d ppm) 6.49 (d, 1H, J = 15.6, vinyl-H), 6.89 (d, 1H, J = 15.9, vinyl-
H), 6.94 (d, 1H, J = 3.4, furan-H), 7.16 (d, 1H, J = 3.4, furan-H),
7.25 (d, 1H, J = 15.9, vinyl-H), 7.29 (d, 1H, J = 15.6, vinyl-H), 7.57–
7.79 (m, 3H, Ar-H), 9.11 (s, 1H, CONHOH), 10.8 (s, 1H, CONHOH);
MS (ESI) m/z 352 (M+H); HRMS calcd for C16H11NO4Cl2 [MH]+:
5.1.7.2. (E)-Methyl 5-(4-hydroxystyryl)furan-2-carboxylate
(14b). Compound 14b was synthesized from 13 (4.37 g, 10
mmol), 4-hydroxybenzaldehyde (1.22 g, 10 mmol) and Et3N
(1.66 mL, 12 mmol) according to the procedure used to synthesize
14a in 66.3% yield (1.62 g); mp 180 °C; MS (EI) m/z 244 (M+).
5.1.7.3. (E)-Methyl 5-(2-hydroxystyryl)furan-2-carboxylate
(14c). Compound 14c was synthesized from 13 (4.37 g, 10
mmol), 2-hydroxybenzaldehyde (1.1 mL, 10 mmol) and Et3N
(1.66 mL, 12 mmol) according to the procedure used to synthe-
size 14a in 86.8% yield (2.12 g); mp 129–130 °C; MS (EI) m/z 244
(M+).
352.0137; found, 352.0130; IR (KBr)
1651.
m
(cmÀ1) = 3637, 3580, 3451,
5.1.4.9. (E)-3-(5-((E)-3-(4-Fluorophenyl)-3-oxoprop-1-enyl)furan-
2-yl)-N-hydroxyacrylamide (5i). Compound 5i was synthesized
from 10i according to the procedure used to synthesize 5a in
37.1% yield; mp 160–161 °C; 1H NMR (DMSO-d6, 300 MHz, d
ppm) 6.53 (d, 1H, J = 15.5, vinyl-H), 6.96 (d, 1H, J = 3.3, furan-H),
7.20 (d, 1H, J = 3.3, furan-H), 7.31 (d, 1H, J = 15.5, vinyl-H), 7.40
(d, 2H, J = 8.6, Ar-H), 7.54 (d, 2H, J = 15.3, vinyl-H), 7.59 (d, 2H,
J = 15.3, vinyl-H), 8.15 (d, 2H, J = 8.6, Ar-H), 9.1 (s, 1H, CONHOH),
10.8 (s, 1H, CONHOH); MS (ESI) m/z 300 (MÀH); HRMS calcd for
5.1.7.4. (E)-Methyl 5-(2-(benzo[d][1,3]dioxol-5-yl)vinyl)furan-
2-carboxylate (14d). Compound 14d was synthesized from 13
(4.37 g, 10 mmol), benzo[d][1,3]dioxole -5-carbaldehyde (1.5 g,
10 mmol) and Et3N (1.66 mL, 12 mmol) according to the procedure
used to synthesize 14a in 60.1% yield (1.63 g); mp 120–121 °C; MS
(EI) m/z 272 (M+).
C
16H12NO4F [MH]+: 302.0823; found, 302.0822; IR (KBr)
m
5.1.7.5. (E)-Methyl 5-(2,3-dichlorostyryl)furan-2-carboxylate
(14e). Compound 14e was synthesized from 13 (4.37 g, 10
mmol), 2,3-dichlorobenzaldehyde (1.75 g, 10 mmol) and Et3N
(1.66 mL, 12 mmol) according to the procedure used to synthesize
14a in 30.6% yield (0.9 g); mp 87–88 °C; MS (EI) m/z 297 (M+).
(cmÀ1) = 3637, 3594, 3459, 2832, 1669.
5.1.4.10. (E)-N-Hydroxy-3-(5-((E)-3-oxo-3-phenylprop-1-enyl)-
furan-2-yl)acrylamide (5j). Compound 5j was synthesized from
10j according to the procedure used to synthesize 5a in 40.6%
yield; mp 172–174 °C; 1H NMR (DMSO-d6, 300 MHz, d ppm) 6.53
(d, 1H, J = 15.5, vinyl-H), 6.95 (d, 1H, J = 3.3, furan-H), 7.19 (d, 1H,
J = 3.3, furan-H), 7.32 (d, 1H, J = 15.5, vinyl-H), 7.51–7.70 (m, 6H,
benzene-H and vinyl-H), 8.06 (d, 1H, J = 7.2, vinyl-H), 9.10 (s, 1H,
CONHOH), 10.8 (s, 1H, CONHOH); MS (ESI) m/z 282 (MÀH); HRMS
5.1.7.6. (E)-Methyl 5-(2-hydroxy-3-methoxystyryl)furan-2-car-
boxylate (14f). Compound 14f was synthesized from 13 (4.37 g,
10 mmol), 2-hydroxy-3-methoxybenzaldehyde (1.52 g, 10 mmol)
and Et3N (1.66 mL, 12 mmol) according to the procedure used to
synthesize 14a in 53.2% yield (1.46 g); mp 103–105 °C; MS (EI)
m/z 274 (M+).
calcd for C16H13NO4 [MH]+: 284.0917; found, 284.0909; IR (KBr)
(cmÀ1) = 3651, 3615, 3580, 3437, 2918, 1626.
m
5.1.7.7. (E)-Methyl 5-(4-(dimethylamino)styryl)furan-2-carbox-
ylate (14g). Compound 14g was synthesized from 13 (4.37 g,
10 mmol), 4-(dimethylamino)benzaldehyde (1.49 g, 10 mmol) and
Et3N (1.66 mL, 12 mmol) according to the procedure used to synthe-
size 14a in 65.1% yield (1.76 g); mp 97–99 °C; MS (EI) m/z 271 (M+).
5.1.5. Methyl 5-(chloromethyl)furan-2-carboxylate (12)
Dry HCl gas was induced into the stirred mixture of methyl fur-
an-2-carboxylate (25.2 g, 0.2 mol), paraformaldehyde (6.0 g,
0.2 mol), and ZnCl2 (30.0 g, 0.22 mol) in CH2Cl2 (100 mL) for 3 h

8000
H. Su et al. / Bioorg. Med. Chem. 16 (2008) 7992–8002
5.1.7.8. (E )-Methyl 5-(4-(benzyloxy)-3-methoxystyryl)furan-2-
carboxylate (14h). Compound 14h was synthesized from 13
5.1.9. Representative procedure for conversion of carboxylic
acids to hydroxamates
(4.37 g,
10 mmol),
4-(benzyloxy)-3-methoxybenzaldehyde
5.1.9.1. (E)-N-Hydroxy-5-styrylfuran-2-carboxamide (6a). Com-
pound 6a was synthesized from 15a according to the procedure
used to synthesize 5a in 37.4% yield; mp 177–178 °C; 1H NMR
(DMSO-d6, 300 MHz, d ppm) 6.64 (1H, d, J = 3.6, furan-H), 7.12 (1H,
d, J = 3.6, furan-H), 7.17 (1H, d, J = 8.0, vinyl-H), 7.35 (1H, d, J = 8.0,
vinyl-H), 7.34–7.63 (5H, m, Ar-H), 9.18 (1H, s, CONHOH), 11.25
(1H, s, CONHOH); MS (EI) m/z 229 (M+); HRMS calcd for C13H12NO3
(2.42 g, 10 mmol) and Et3N (1.66 mL, 12 mmol) according to the
procedure used to synthesize 14a in 78.4% yield (2.85 g); mp 93–
95 °C; MS (EI) m/z 364 (M+).
5.1.8. Representative procedure for conversion of methyl ester
to carboxylic acid
[MH]+: 230.0811; found, 230.0809; IR (KBr)
2897, 2384, 2342, 1627, 1584.
m
(cmÀ1) = 3452, 3174,
5.1.8.1. (E)-5-Styrylfuran-2-carboxylic acid (15a). A mixture of
14a (0.9 g, 3.95 mmol), 2 N KOH (4.0 mL, 7.9 mmol) and EtOH
(5 mL) was stirred at 60 °C for 3 h. The solution was poured into
water (30 mL) and extracted with ethyl acetate (2Â 20 mL). The
pH of the aqueous layer was adjusted to 5 by adding 2 N HCl.
The precipitate was filtered and recrystallized to give a white solid
(0.82 g) in 97.6% yield; mp 211–212 °C; 1H NMR (DMSO-d6,
300 MHz, d, ppm) 6.64 (1H, d, J = 3.6), 7.12 (1H, d, J = 3.6), 7.17
(1H, d, J = 8.0), 7.35 (1H, d, J = 8.0), 7.34–7.63 (5H, m), 10.2 (1H,
s); MS (EI) m/z 214 (M+).
5.1.9.2. (E)-N-Hydroxy-5-(4-hydroxystyryl)furan-2-carboxam-
ide (6b). Compound 6b was synthesized from 15b according
to the procedure used to synthesize 6a in 41.4% yield; mp 178–
180 °C; 1H NMR (DMSO-d6, 300 MHz, d ppm) 6.58 (1H, d, J = 3.6,
furan-H), 6.84 (2H, d, J = 8.4, Ar-H), 6.94 (1H, d, J = 16.5, vinyl-H),
7.11 (1H, d, J = 3.6, furan-H), 7.26 (1H, d, J = 16.5, vinyl-H), 7.46
(2H, d, J = 8.4, Ar-H), 9.15 (1H, s, CONHOH), 9.74 (1H, s, HO-Ar),
11.20 (1H, s, CONHOH); MS (ESI) m/z 244 (MÀH); HRMS calcd
for C13H12NO4 [MH]+: 246.0760; found, 246.0756; IR (KBr)
(cmÀ1) = 3814, 3444, 2840, 1644, 1601.
m
5.1.8.2. (E)-5-(4-Hydroxystyryl)furan-2-carboxylic acid (15b). Com-
pound 15b was synthesized from 14b (1.95 g, 8 mmol) and 2 N
KOH (16 mL, 32 mmol) according to the procedure used to syn-
thesize 15a in 66.3% yield (1.22 g); mp 180 °C; 1H NMR
(DMSO-d6, 300 MHz, d ppm) 6.61 (1H, d, J = 3.6, furan-H), 6.74
(2H, d, J = 8.3, Ar-H), 6.76 (1H, d, J = 16.9, vinyl-H), 7.16 (1H, d,
J = 3.6, furan-H), 7.11 (1H, d, J = 16.4, vinyl-H), 7.45 (2H, d,
J = 8.6, Ar-H), 9.72 (1H, s, HO-Ar), 12.96 (1H, s, COOH); MS (EI)
m/z 230 (M+).
5.1.9.3. (E)-N-Hydroxy-5-(2-hydroxystyryl)furan-2-carboxam-
ide (6c). Compound 6c was synthesized from 15c according
to the procedure used to synthesize 6a in 42.2% yield; mp 132–
134 °C; 1H NMR (DMSO-d6, 300 MHz, d ppm) 6.58 (1H, d, J = 3.5,
furan-H), 6.82 (1H, m, Ar-H), 6.88 (1H, m, Ar-H), 7.06 (1H, d,
J = 3.5, furan-H), 7.11 (1H, m, Ar-H), 7.13 (1H, d, J = 16.5, vinyl-H),
7.44 (1H, d, J = 16.5, vinyl-H), 7.49 (1H, m, Ar-H), 9.07 (1H, s, CON-
HOH), 9.91 (1H, s, HO-Ar), 11.16 (1H, s, CONHOH); MS (ESI) m/z
244 (MÀH); HRMS calcd for C13H12NO4 [MH]+: 246.0760; found,
5.1.8.3. (E)-5-(2-Hydroxystyryl)furan-2-carboxylic acid (15c). Com-
pound 15c was synthesized from 14c (1.95 g, 8 mmol) and 2 N
KOH (16 mL, 32 mmol) according to the procedure used to syn-
thesize 15a in 89% yield (1.64 g); mp 180–182 °C; MS (EI) m/z
230 (M+).
246.0764; IR (KBr)
m
(cmÀ1) = 3430, 2355, 2840, 1623, 1584.
5.1.9.4. (E)-5-(2-(Benzo[d][1,3]dioxol-5-yl)vinyl)-N-hydroxyfu-
ran-2-carboxamide (6d). Compound 6d was synthesized
from 15d according to the procedure used to synthesize 6a in
39.3% yield; mp 170–171 °C; 1H NMR (DMSO-d6, 300 MHz, d
ppm) 6.04 (2H, s, OCH2O), 6.54 (1H, d, J = 3.5, furan-H), 6.98 (1H,
d, J = 16.3, vinyl-H), 6.91-7.06 (2H, m, Ar-H), 7.06 (1H, d, J = 3.5, fur-
an-H), 7.21 (1H, d, J = 16.3, vinyl-H), 7.24 (1H, m, Ar-H), 9.09 (1H, s,
CONHOH), 11.13 (1H, s, CONHOH); MS (EI) m/z 273 (M+); HRMS
calcd for C14H11NO5 [MH]+: 274.2740; found, 274.2734; IR (KBr)
5.1.8.4. (E)-5-(2-(Benzo[d][1,3]dioxol-5-yl)vinyl)furan-2-carbox-
ylic acid (15d). Compound 15d was synthesized from 14d (1.33 g,
4.9 mmol) and 2 N KOH (9.8 mL, 19.6 mmol) according to the pro-
cedure used to synthesize 15a in 99.8% yield (2.85 g); mp 230–
231 °C; MS (EI) m/z 258 (M+).
5.1.8.5. (E)-5-(2,3-Dichlorostyryl)furan-2-carboxylic acid
(15e). Compound 15e was synthesized from 14e (0.72 g,
2.4 mmol) and 2 N KOH (4.8 mL, 9.6 mmol) according to the proce-
dure used to synthesize 15a in 66.1% yield (0.45 g); mp 226–
228 °C; MS (EI) m/z 283 (M+).
m
(cmÀ1) = 3437, 3288, 3124, 2790, 1662.
5.1.9.5. (E)-5-(2,3-Dichlorostyryl)-N-hydroxyfuran-2-carbox-
amide (6e). Compound 6e was synthesized from 15e accord-
ing to the procedure used to synthesize 6a in 36.8% yield; mp 180–
181 °C; 1H NMR (DMSO-d6, 300 MHz, d ppm) 6.74 (1H, d, J = 3.6,
furan-H), 7.11 (1H, d, J = 3.6, furan-H), 7.24 (1H, d, J = 16.2, vinyl-
H), 7.39 (1H, t, Ar-H), 7.55 (1H, d, J = 16.2, vinyl-H), 7.57–7.84
(2H, m, Ar-H), 9.17 (1H, s, CONHOH), 11.37 (1H, s, CONHOH); MS
5.1.8.6. (E)-5-(2-Hydroxy-3-methoxystyryl)furan-2-carboxylic
acid (15f). Compound 15f was synthesized from 14f (1.46 g,
5.3 mmol) and 2 N KOH (10.6 mL, 21.3 mmol) according to the
procedure used to synthesize 15a in 79.6% yield (1.09 g); mp
217–219 °C; MS (EI) m/z 260 (M+).
(ESI) m/z 296 (MÀH); HRMS calcd for
C
13H9Cl2NO3 [MH]+:
m
(cmÀ1) = 3460, 2356, 1630,
298.0032; found, 298.0026; IR (KBr)
1503.
5.1.8.7. (E)-5-(4-(Dimethylamino)styryl)furan-2-carboxylic acid
(15g). Compound 15g was synthesized from 14g (1.63 g, 6 mmol)
and 2 N KOH (12 mL, 24 mmol) according to the procedure used to
synthesize 15a in 77.1% yield (1.19 g); mp 204–205 °C; MS (EI) m/z
257 (M+).
5.1.9.6. (E)-N-Hydroxy-5-(2-hydroxy-3-methoxystyryl)furan-2-
carboxamide (6f). Compound 6f was synthesized from 15f
according to the procedure used to synthesize 6a in 40.2% yield;
mp 122–124 °C; 1H NMR (DMSO-d6, 300 MHz, d ppm) 3.81 (3H,
s, CH3O), 6.26 (1H, d, J = 3.6, furan-H), 6.42 (1H, d, J = 12.3, vinyl-
H), 6.73 (1H, d, J = 12.3, vinyl-H), 6.96 (1H, d, J = 3.6, furan-H),
6.67–6.93 (3H, m, Ar-H), 8.80 (1H, s, HO-Ar), 9.05 (1H, s, CONHOH),
10.87 (1H, s, CONHOH); MS (ESI) m/z 274 (MÀH); HRMS calcd for
5.1.8.8. (E)-5-(4-(Benzyloxy)-3-methoxystyryl)furan-2-carbox-
ylic acid (15h).
Compound 15h was synthesized from 14h
(1.82 g, 5 mmol) and 2 N KOH (10 mL, 20 mmol) according to the
procedure used to synthesize 15a in 68.3% yield (1.2 g); mp
>250 °C; MS (EI) m/z 350 (M+).
C
14H13NO5 [MH]+: 276.0102; found, 276.0106; IR (KBr)
m
(cmÀ1) = 3409, 2932, 2355, 1637.

H. Su et al. / Bioorg. Med. Chem. 16 (2008) 7992–8002
8001
5.1.9.7. (E)-5-(4-(Dimethylamino)styryl)-N-hydroxyfuran-2-car-
boxamide (6g). Compound 6g was synthesized from 15g
flow cytometer using the Cell Quest software (Becton–Dickinson)
and analyzed with standard procedures using the Cell Quest soft-
ware (Becton–Dickinson) and the ModFit LT version 3 Software
(Verity) as previously reported.17 All of the experiments were per-
formed at least two times.
according to the procedure used to synthesize 6a in 41.3% yield;
mp 116–117 °C; 1H NMR (DMSO-d6, 300 MHz, d ppm) 2.94 (6H,
s, (CH3)2N), 6.46 (1H, d, J = 3.3, furan-H), 6.72 (2H, d, J = 8.7, Ar-
H), 6.81 (1H, d, J = 16.3, vinyl-H), 7.01 (1H, d, J = 3.3, furan-H),
7.17 (1H, d, J = 16.3, vinyl-H), 7.39 (2H, d, J = 8.7, Ar-H), 9.07 (1H,
s, CONHOH), 11.1 (1H, s, CONHOH); MS (ESI) m/z 273 (M+H);
HRMS calcd for C15H16N2O3 [MH]+: 273.1233; found, 273.1233;
5.2.5. FACS analysis of apoptosis in U937 cells
Apoptosis was measured with the active caspase 3 detection (B-
Bridge) and samples were analyzed by FACS with Cell Quest tech-
nology (Becton–Dickinson) as previously reported.18
IR (KBr)
m
(cmÀ1) = 3451, 2903, 2355, 1633.
5.1.9.8. (E)-5-(4-(Benzyloxy)-3-methoxystyryl)-N-hydroxyfuran-
2-carboxamide (6h). Compound 6h was synthesized from
5.2.6. Granulocytic differentiation
Granulocytic differentiation was carried out according to Alt-
15h according to the procedure used to synthesize 6a in 43.6%
yield; mp 180–181 °C; 1H NMR (DMSO-d6, 300 MHz, d ppm) 3.83
(3H, s, CH3O), 5.11 (2H, s, CH2O), 6.54 (1H, d, J = 3.5, furan-H),
7.03 (1H, d, J = 16.5, vinyl-H), 7.06 (1H, d, J = 3.5, furan-H), 7.42
(1H, d, J = 16.5, vinyl-H), 7.20–7.46 (8H, m, Ar-H), 9.08 (1H, s, CON-
HOH), 11.13 (1H, s, CONHOH); MS (ESI) m/z 364 (MÀH); HRMS
calcd for C21H19NO5 [MH]+: 366.1335; found, 366.1333; IR (KBr)
ucci et al.19 Briefly, U937 cells were harvested and resuspended
in 10
samples were incubated with 10
incubated for 30 min at 4 °C in the dark, washed in PBS, and resus-
pended in 500 L of PBS containing propidium iodide (0.25 g/mL).
lL of phycoerythrine-conjugated CD11c (CD11c-PE). Control
lL of PE-conjugated mouse IgG1,
l
l
Samples were analyzed by FACS with Cell Quest technology (Bec-
ton–Dickinson). Propidium iodide positive cells have been ex-
cluded from the analysis.
m
(cmÀ1) = 3430, 2882, 2362, 1634.
5.2. Bioactivity test
5.2.7. Determination of
For -tubulin, an amount of 50
separated on 10% polyacrylamide gels and blotted. Western blots
were shown for acetylated -tubulin (Sigma, dilution of 1:500),
a
-tubulin acetylation
a
l
g of total protein extracts was
5.2.1. General
5.2.1.1. Cell lines and cultures.
The U937 cell line was cul-
a
tured in RPMI with 10% fetal calf serum, 100 U/mL of penicillin,
100 g/mL of streptomycin, and 250 ng/mL of amphotericin-B,
and total ERKs (Santa Cruz) were used to normalize for equal
loading.
l
10 mM Hepes and 2 mM glutamine. U937 cells were kept at the
constant concentration of 200,000 cells per milliliter of culture
medium.
5.2.8. Determination of p21WAF1/CIP1 induction in U937 cells
Total protein extracts (50 lg) were separated on a 15% poly-
acrylamide gel and blotted as previously described.17 Western
blots were shown for p21 (Transduction Laboratories, dilution
1:500), and tubulin (Sigma) were used to normalize for equal
loading.
5.2.1.2. Ligands and materials.
SAHA (kind gift of Merck)
M. MS-275 (kind gift from
M. All other
was dissolved in DMSO and used at 5
l
Schering AG) was dissolved in ethanol and used at 5
l
compounds were dissolved in DMSO and used at the indicated
concentrations.
5.2.9. Cellular proliferation assay
All compounds were dissolved in DMSO with the stock concen-
tration of 10 mg/mL, and diluted with medium freshly before drug
administration. Cell lines were seeded into 96-well flat bottom
plates at density of 4000 cells/well. Twenty-four hours after seed-
ing, each compound dilution was added in duplicate and incuba-
tion continued at 37 °C in a humidified atmosphere containing
5.2.2. Stability studies in rat plasma
The stock standard solutions of SAHA, 5j and 6a were prepared
by dissolving the respective compound in fresh blank rat plasma to
yield a concentration of 1 mg/mL. The working standard solutions
were made by a serial dilution of the stock solution with blank
plasma. Aliquots of 1 mL plasma samples were incubated at 37 °C
5% CO2. After 48 h, add 20 lL MTT at 5 mg/mL in PBS (filter steril-
and retrieved at different times. Aliquots of 100
were then treated with two volumes of acetonitrile to precipitate
plasma proteins. The resulting samples were centrifuged at
l
L plasma samples
ized, light protected, and stored at 4 °C) per well, and after 4 h of
incubation at 37 °C, the fluorescence was measured at 570 nm
using Thermo Multiskan Spectrum.
16,000 rpm for 5 min. Aliquots of 20
taken for HPLC analysis.16
lL supernatant solutions were
Acknowledgements
5.2.3. Fluorimetric HDAC1 and 4 assays
The authors thank Dr. Yulan Rao and Teng Wang (Department
of Pharmaceutical Analysis, China Pharmaceutical University) for
their efforts in stability studies in rat plasma. This work was sup-
ported by the Natural Science Foundation of Jiangsu Province
(No. BK2007171), and by grants from Ministero dell’Istruzione Uni-
versità e Ricerca, PRIN 2006 (L.A.), European Union (APO-SYS Pro-
ject No. 200767) (L.A.), Regione Campania, L.5 annualità 2005
(L.A.).
The HDAC Fluorescent Activity Assay is based on the Fluor de
Lys Substrate and Developer combination (BioMol) and has been
carried out according to supplier’s instructions. First, the Fluor de
Lys Substrate, which comprises an acetylated lysine side chain,
has been incubated with purified recombinant HDAC1, 4 enzymes
in presence or absence of the inhibitors. Deacetylation of the sub-
strate sensitizes the substrate so that, in the second step, treatment
with the Developer produces a fluorophore. Fluorescence has been
quantified with a TECAN inphinite M200 station.
References and notes
5.2.4. Cell cycle analysis in U937 cells
1. Allfrey, V. G.; Faulkner, R.; Mirsky, A. E. Proc. Natl. Acad. Sci. U.S.A. 1964, 51, 786.
2. Roth, S. Y.; Denu, J. M.; Allis, C. D. Annu. Rev. Biochem. 2001, 70, 81.
3. Bolden, J. E.; Peart, M. J.; Johnstone, R. W. Nat. Rev. Drug Disc. 2006, 5, 769.
4. Xu, W. S.; Parmigiani, R. B.; Marks, P. A. Oncogene 2007, 26, 5541.
5. Altucci, L.; Clarke, N.; Nebbioso, A.; Scognamiglio, A.; Gronemeyer, H. Int. J.
Biochem. Cell Biol. 2005, 37, 1752.
Cells (2.5 Â 105) were collected and resuspended in 500
l
L of
hypotonic buffer (0.1%Triton X-100, 0.1% sodium citrate, 50
lg/
mL of propidium iodide (PI), and RNAse A). Cells were incubated
in the dark for 30 min. Samples were acquired on a FACS-Calibur

8002
H. Su et al. / Bioorg. Med. Chem. 16 (2008) 7992–8002
14. Brochet, C.; Syssa, J. L.; Mouloungui, Z.; Delmas, M.; Gaset, A. Synth. Commun.
6. Minucci, S.; Pelicci, P. G. Nat. Rev. Cancer 2006, 6, 38.
7. Garber, K. Nat. Biotechnol. 2007, 25, 17.
1991, 21, 1735.
8. Mulder, G. J.; Meerman, J. H. Environ. Health Perspect 1983, 49, 27.
9. Wiech, N. L.-H. H.; Lea, M.; Jung, M. AACR 93rd Annual Meeting Abstract, 2002.
10. Marson, C. M.; Serradji, N.; Rioja, A. S.; Gastaud, S. P.; Alao, J. P.; Coombes, R. C.;
Vigushin, D. M. Bioorg. Med. Chem. Lett. 2004, 14, 2477.
11. Mai, A.; Massa, S.; Pezzi, R.; Simeoni, S.; Rotili, D.; Nebbioso, A.; Scognamiglio,
A.; Altucci, L.; Loidl, P.; Brosch, G. J. Med. Chem. 2005, 48, 3344.
12. Ragno, R.; Simeoni, S.; Rotili, D.; Caroli, A.; Botta, G.; Brosch, G.; Massa, S.; Mai,
A. Eur. J. Med. Chem. 2008, 48, 621.
15. Finan, P. A. J. Chem. Soc. 1963, 3917.
16. Du, L.; Musson, D. G.; Wang, A. Q. J. Pharm. Biomed. Anal. 2006, 42, 556.
17. Nebbioso, A.; Clarke, N.; Voltz, E.; Germain, E.; Ambrosino, C.; Bontempo, P.;
Alvarez, R.; Schiavone, E. M.; Ferrara, F.; Bresciani, F.; Weisz, A.; de Lera, A. R.;
Gronemeyer, H.; Altucci, L. Nat. Med. 2005, 11, 77.
18. Mai, A.; Rotili, D.; Tarantino, D.; Ornaghi, P.; Tosi, F.; Vicidomini, C.; Sbardella,
G.; Nebbioso, A.; Miceli, M.; Altucci, L.; Filetici, P. J. Med. Chem. 2006, 49, 6897.
19. Altucci, L.; Rossin, A.; Raffelsberger, W.; Reitmair, A.; Chomienne, C.;
Gronemeyer, H. Nat. Med. 2001, 7, 680.
13. Haru, O.; Haruo, S. Synthesis 1972, 138.