The synthesis and biologic evaluation of anti-platelet and cytotoxic β-nitrostyrenes

Article abstract of DOI:10.1016/j.bmc.2010.08.039

Our previous studies demonstrated that two cytotoxic β-nitrostyrene derivatives, 3,4-methylenedioxy-β-nitrostyrene (MNS) and 4-O-benzoyl-3-methoxy-β-nitrostyrene (BMNS) exhibit potent anti-platelet activities. In this study, a series of β-nitrostyrenes we

Full text of DOI:10.1016/j.bmc.2010.08.039

Bioorganic & Medicinal Chemistry 18 (2010) 7621–7627
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
The synthesis and biologic evaluation of anti-platelet and cytotoxic
b-nitrostyrenes
Pei-Wen Hsieh a, , Yu-Ting Chang a, Wen Yin Chuang b, Hsin-Chu Shih b,
⇑
Shin-Zan Chiang a, Chin-Chung Wu b,
⇑
a Graduate Institute of Natural Products, Chang Gung University, Taoyuan 333, Taiwan
b Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Our previous studies demonstrated that two cytotoxic b-nitrostyrene derivatives, 3,4-methylenedioxy-b-
nitrostyrene (MNS) and 4-O-benzoyl-3-methoxy-b-nitrostyrene (BMNS) exhibit potent anti-platelet activ-
ities. In this study, a series of b-nitrostyrenes were synthesized and subjected to anti-platelet aggregation
assay and cytotoxicity assay. The mono- and di-substitutions on the B ring of BMNS tended to increase
the anti-platelet activity and decrease the cytotoxic activity. Of these, compounds 19 and 24 exhibited
Received 12 June 2010
Revised 17 August 2010
Accepted 18 August 2010
Available online 24 August 2010
the most potent inhibitory effects on thrombin- and collagen-induced platelet aggregation (IC50 6 0.7
lM)
Keywords:
Anti-platelet aggregation
Cytotoxicity
b-Nitrostyrenes
Structure–activity relationships (SAR)
without significant cytotoxicity on a human cancer cell line (up to 20 M). Further studies indicated that
l
compounds 19 and 24 inhibited platelet aggregation via prevention of glycoprotein IIb/IIIa activation.
The potent and novel effects of BMNS derivatives make them attractive candidates for the development
of new anti-platelet agents.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
search and development of new generation anti-platelet agents
are needed and are now in progress.
Platelets play a crucial role in the pathogenesis of athero-
thrombosis, which accounts for approximately 40% of all deaths
in developed countries.1 In clinical practice, aspirin, a cyclooxy-
genase inhibitor, in combination with clopidogrel, an ADP recep-
tor antagonist, is the current ‘gold standard’ for preventing
platelet aggregation.2 However, aspirin and clopidogrel still have
considerable limitations in their mode of action and efficacy.3 In
addition, some patients are refractory to these drugs. For exam-
ple, patients with at least one variant CYP2C19 allele have
reduced clopidogrel responsiveness and an increased risk of car-
diovascular events due to decreased formation of the active
metabolite.4 Glycoprotein (GP) IIb/IIIa is a major integrin in plate-
lets. On platelet stimulation, GPIIb/IIIa undergoes a conforma-
tional change that allows it to bind to fibrinogen with high
affinity, leading to platelet aggregation. GPIIb/IIIa activation thus
represents the final common pathway for platelet aggregation
regardless of the stimulus used.5 It has been proved that intrave-
nous GPIIb/IIIa antagonists can improve the outcome of patients
undergoing percutaneous interventions (PCI);6 however, the use
of oral GPIIb/IIIa antagonists has been unsuccessful. Therefore, re-
Compounds with a b-nitrostyrene moiety have been recognized
to have diverse biological activities, particularly antimicrobial and
anticancer effects.7–11 Furthermore, b-nitrostyrenes also exhibited
inhibitory effects on protein phosphatases, human telomerase,
and snake venom phospholipase.7 Our previous studies demon-
strated that two b-nitrostyrene derivatives, 3,4-methylenedioxy-
b-nitrostyrene (MNS) and 4-O-benzoyl-3-methoxy-b-nitrostyrene
(BMNS) (Fig. 1), potently prevented platelet aggregation by inhib-
iting the function of GPIIb/IIIa.12,13 Unlike GPIIb/IIIa antagonists,
these compounds inhibit GPIIb/IIIa activation rather than directly
blocking GPIIb/IIIa. This feature may provide a new strategy for
the treatment of platelet-dependent thrombosis. In this study,
BMNS and MNS were chosen as model structures in an attempt
to obtain more potent anti-platelet agents. In particular, the route
of the test structures in the: (1) mono- or di-substitution effect on
the B ring were determined; (2) substitution effect on the A ring of
the C-3 and C-4 position; (3) the effect of the b-methyl fragments
on bioactivity;8 and (4) replacement of the A ring with a five or six
member aromatic ring. All synthetic and commercial compounds
(Tables 1 and 2) were assayed for in vitro inhibitory effects on
platelet aggregation induced by collagen and thrombin, as well
as cytotoxicity against a human breast cancer cell line (Tables 1
and 2). Herein, we describe the synthesis, pharmacologic data,
and structure–activity relationship (SAR) of the anti-platelet and
cytotoxic effects related to b-nitrostyrenes.
⇑
Corresponding authors. Tel.: +886 3 211 8800x3105; fax: +886 3 211 8643
(P.W.H.); tel.: +886 7 3121101x2669; fax: +886 7 311 4773 (C.C.W.).
E-mail addresses: pewehs@mail.cgu.edu.tw (P.-W. Hsieh), ccwu@kmu.edu.tw
(C.-C. Wu).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.08.039

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P.-W. Hsieh et al. / Bioorg. Med. Chem. 18 (2010) 7621–7627
Table 1
The anti-platelet and cytotoxic activities of BMNS derivatives with substituted in B ring
H3CO
NO2
A
O
O
B
R
Compd
R
Aggregation IC50
(l
M)a,b
Collagen
Cytotoxicity IC50
MDA-MB-231
(l
M) or %Inhb,c
HBL-100
Thrombin
BMNS
1
2
3
4
5
6
7
8
H
2.7
4.7
1.0
5.4
2.5
1.5
2.1
2.6
1.2
1.3
3.3
1.7
2.9
0.9
1.5
1.2
6.2
2.7
3.5
0.6
3.3
3.0
2.9
3.4
0.7
0.7
1.0
1.9
23.0
1.4
1.1
1.1
1.6
0.6
2.0
1.3
1.2
0.8
1.4
0.5
0.9
1.7
0.9
0.7
0.8
0.7
0.6
8.3
2.0
0.8
0.6
1.6
2.0
1.1
1.4
0.6
0.7
1.8
1.4
2.7
0.9
1.1
13.1
>20 (0%)
20-F
>20 (26.5%)
>20 (7.0%)
>20 (8.3%)
>20 (20.4%)
>20 (36.3%)
19.4
>20 (44.9%)
18.9
>20 (19.1%)
>20 (34.9%)
13.4
>20 (4.9%)
>20 (8.2%)
>20 (10.0%)
>20 (2.4%)
>20 (1.8%)
>10 (8.8%)
>20 (38.2%)
>20 (23.2%)
>20 (18.2%)
>20 (12.5%)
4.1
20-Cl
20-Br
20-CF3
20-CH3
20-OCH3
30-F
30-Cl
30-Br
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
30-CF3
30-CH3
30-OCH3
40-F
>20 (28.9%)
9.8
12.1
>20 (25.1%)
>20 (7.3%)
>20 (6.3%)
>20 (ꢀ7.3%)
>20 (8.0%)
>20 (22.7%)
>20 (ꢀ2.1%)
>20 (4.1%)
>20 (16.0%)
>20 (0.5%)
>20 (19.0%)
>20 (11.5%)
>20 (9.9%)
>20 (15.2%)
>20 (15.6%)
>20 (1.1%)
>20 (16.6%)
>20 (47.4%)
40-Cl
40-Br
>20 (33.2%)
>20 (9.1%)
>20 (ꢀ4.9%)
>20 (7.9%)
18.1
>20 (2.5%)
>20 (25.5%)
>20 (18.5%)
>20 (22.1%)
>20 (24.7%)
>20 (ꢀ4.9%)
>20 (ꢀ0.1%)
>20 (9.4%)
>20 (20.0%)
>20 (3.7%)
12.4
40-CF3
40-CH3
40-OCH3
40-tert-Butyl
20,30-F
20,40-F
30,40-F
30,50-F
20,30-Cl
20,40-Cl
30,40-Cl
30,50-Cl
20,30,40-F
30,40,50-F
30,40,50-OCH3
13.9
a
b
c
Platelets were pre-incubated with DMSO (0.5%, control) or test compounds at 37 °C for 3 min before addition of the inducers.
IC50 represents the 50% inhibitory concentration. Results represent the means from two independent experiments.
Compounds were tested against the human breast cancer cell line, MDA-MB-231 or the non-malignant breast epithelial cell line, HBL-100 using the MTT method. The
percentage of inhibition obtained at a test concentration of 20 lM is given in parentheses (%Inh).
2.2. Cytotoxic activity
2. Results and discussion
2.1. Chemistry
The cytotoxic properties of b-nitrostyrenes have been known
for some time: these compounds induce antiproliferative and proa-
poptotic effects in several cancer cell lines.7,9–11 The unsaturated
nitroolefinic bond in b-nitrostyrenes has been recognized as an
essential fragment in its bioactivity, as it can interact with cellular
protein cysteine sulfhydryls via the Michael addition mecha-
nism.14,15 The reactivity of b-nitrostyrenes following the Michael
addition is dependent on the position of the withdrawing group
in the A ring. In contrast, methyl substitution in the b position re-
sulted in a reduced reaction rate.14 In the present study, the syn-
thetic b-nitrostyrene derivatives were tested in the human breast
cancer cell line MDA-MB-231 to evaluate their cytotoxicity. By
comparing BMNS with MNS, it was found that the unsubstituted
4-O-benzoyl group did not significantly affect the cytotoxic po-
tency. However, substitution at the benzoyl ring of BMNS resulted
in a loss of cytotoxic activity (Table 1).
The BMNS derivatives (1–30) were prepared by acid halide
esterification of trans-4-hydroxy-3-methoxy-b-nitrostyrene and
were reacted with corresponding substituted benzoyl chlorides
in a solution of dichloromethane (DCM)/ pyridine (10:1) (Scheme
1). 3-Ethoxyl-4-methoxy-b-nitrostyrene (33), 3-hydroxy-4-meth-
oxy-b-nitrostyrene (34), and their b-methyl derivatives (37–38)
were synthesized according to the method of Milhazes et al.
(Scheme 2).8 The benzoyl esters (31, 32, 35, and 36) of com-
pounds 33, 34, 37, and 38 were prepared by acid halide esterifi-
cation as described before (Scheme 2). Compounds 39–44, 48,
and 49 were obtained commercially. Accordingly, compounds
1–49 (Tables 1 and 2) were fully characterized using spectro-
scopic data. The spectroscopic data of the new compounds are
shown in the Section 4.

P.-W. Hsieh et al. / Bioorg. Med. Chem. 18 (2010) 7621–7627
7623
Table 2
The anti-platelet and cytotoxic activities of BMNS/MNS analogs modifying in the A ring of C-3 and C-4 position, and in the b-carbon of the vinyl side chain
R1O
NO2
R3
R2O
M) or %Inhb,c
HBL-100
a,b
R1
R2
R3
Aggregation IC50
(
l
M)
Cytotoxicity IC50
MDA-MB-231
(l
Thrombin
Collagen
BMNS
MNS
31
32
33
34
35
36
37
CH3
–OCH2O–
CH2CH3
Benzoyl
CH2CH3
H
CH2CH3
Benzoyl
CH2CH3
H
H
H
H
H
H
H
Benzoyl
H
H
H
H
H
H
CH3
CH3
CH3
CH3
H
H
H
H
OH
NO2
H
H
H
2.7
14.0
1.2
1.1
7.2
1.0
13.1
14.0
15.3
7.7
>20 (34.4%)
18.9
12.1
6.6
5.3
7.8
>20 (38.3%)
16.3
>20 (36.6%)
>20 (21.2%)
13.9
>20 (0%)
>20 (39.2%)
11.6
<10 (79.8%)
>20 (35.2%)
12.8
14.6
7.3
4.5
6.6
Benzoyl
CH3
H
CH3
Benzoyl
CH3
H
CH3
OH
OCH3
CH3
Cl
H
H
OH
OCH3
H
1.9
0.8
78.1
28.5
0.8
16.2
7.2
1.1
1.0
5.8
11.0
18.4
3.5
5.9
3.5
7.8
3.0
16.4
7.6
4.9
7.7
6.8
2.9
82.7
61.0
45.1
12.1
8.9
7.5
35.5
5.7
66.5
12.8
17.8
29.8
22.9
38
39
40
41
42
43
44
16.3
13.0
15.0
15.7
>20 (34.9%)
>20 (34.2%)
NTe
9.9
45d
46d
47d
48
OCH3
OCH3
H
—
—
NTe
NTe
NTe
NTe
NTe
—
—
—
—
>20 (9.9%)
>20 (33.3%)
>20 (28.5%)
17.9
49
a
b
c
Platelets were pre-incubated with DMSO (0.5%, control) or test compounds at 37 °C for 3 min before addition of the inducers.
IC50 represents the 50% inhibitory concentration. Results represent the means from two independent experiments.
Compounds were tested against the human breast cancer cell line, MDA-MB-231 or the non-malignant breast epithelial cell line, HBL-100 using the MTT method. The
percentage of inhibition obtained at a test concentration of 20 lM is given in parentheses (%Inh).
d
The biological data of compounds 45–47 were came from literature directly.12
None test.
e
Scheme 1. The straightforward synthesis of BMNS analogues (1–30).
Scheme 2. Reagents and conditions: (a) nitromethane, ammonium acetate, reflux, 24 h; (b) nitroethane, ammonium acetate, reflux, 24 h; (c) benzoyl chloride, DCM/pyridine
(10:1), 24 h.

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P.-W. Hsieh et al. / Bioorg. Med. Chem. 18 (2010) 7621–7627
Figure 1. The chemical structures of MNS and BMNS.
Table 2 shows that b-methyl substituted nitrostyrenes (35, 36,
induce platelet aggregation without receptor activation. Both 19
and 24 completely prevented A23187-induced platelet aggregation
37, and 38) were more potent than b-nitrostyrenes (31, 32, 33,
and 34) in inhibiting the viability of MDA-MB-231 cells. These
findings conflict with those report where b-methyl substitution
reduced the reactivity of the nitroolefinic group with sulfhydryl
and, suggests another mechanism for the action of b-methyl
substituted nitrostyrenes. In addition, we also tested the cytotoxic-
ity of the b-nitrostyrenes in a non-malignant breast epithelial cell
line HBL-100. As shown in Tables 1 and 2, the cytotoxic activities of
most of these compounds in HBL-100 epithelial cells were less
than or equal to that observed in MDA-MB-231 cancer cells, except
compounds 11, 12, 39, 41, 42, and 49.
with IC50 values of 1.1 0.2 and 3.3 0.1
In a similar manner, 19 and 24 also abolished TPA-induced platelet
aggregation with IC50 values of 3.5 0.6 and 5.3 0.4 M, respec-
tively (n = 3). These results suggested that 19 and 24 inhibited
platelet aggregation by acting at sites downstream of the Ca2+
lM, respectively (n = 3).
l
/
PKC pathway. We thus examined the effects of 19 and 24 on the
activation of GPIIb/IIIa, which represents the final common path-
way for platelet aggregation regardless of the stimulus used. In
the present study, we used flow cytometry and FITC-conjugated
PAC-1 monoclonal antibody, which only binds to the activated
form of GPIIb/IIIa, to determine whether the b-nitrostyrene deriv-
atives were able to prevent GPIIb/IIIa activation. As shown in
Figure 2, BMNS, 19, and 24 inhibited thrombin-induced PAC-1
binding in a concentration-dependent manner. Furthermore, the
rank order of potency of these compounds in preventing GPIIb/IIIa
activation (19 > 24 > BMNS) was correlated with their ability to
inhibit platelet aggregation, suggesting that the b-nitrostyrene
derivatives inhibited platelet aggregation by preventing GPIIb/IIIa
activation.
2.3. Anti-platelet activity
All b-nitrostyrenes (1–49) were subjected to an anti-platelet
aggregation assay with collagen (Col) and thrombin (Thr) as induc-
ers. The results showed that in most cases, the mono- and di-sub-
stitutions on the B ring of BMNS led to increased anti-platelet
activity and decreased cytotoxic activity (Table 1). Of these, com-
pounds 2, 13, 19, 24, and 25 exhibited dual inhibitory effects on
Thr- and Col-induced platelet aggregation with IC50 values of 1.0
and 0.6, 0.9 and 0.8, 0.6 and 0.6, 0.7 and 0.6, as well as 0.7 and
3. Conclusion
0.7
lM, respectively.
The anti-platelet aggregation assay data and SAR analysis con-
In this study, we synthesized approximately forty b-nitrosty-
rene derivatives and compared their anti-platelet and cytotoxic
activities. The analysis of structure–activity relationship revealed
that substitutions on ring B of BMNS improved the anti-platelet
activity and decreased the cytotoxic activity. Alteration of the
firmed that activity depended on the presence of the B ring. Fur-
thermore, most chloro-substitution(s) at the B ring were more
favorable than others. However, difluoro derivatives were unfavor-
able compared with mono-fluoro substituted derivatives (Table 1).
Conversely, exchanging the substituents at C-3 and C-4, or replac-
ing methoxyl with an ethoxyl group at C-3 slightly enhanced the
anti-platelet aggregation activity. In addition, substitution at the
b position with a methyl group did not affect anti-platelet activity
(Table 2). On the other hand, analysis of the anti-platelet data of
MNS analogs (Table 2) showed that activity depended on the
hydrophobicity of the substitution at C-4. Compared with the data
of compounds 33, 34, and 39, with a hydroxyl at C-4, more hydro-
phobic groups at C-3 showed decreased activities, however, this
phenomenon was reversed by replacing a hydroxyl with a methox-
yl group at C-4 (34 and 46). Moreover, when the A ring was mod-
ified with a five member ring, for example, furan or thiophene,
activity was also diminished (Table 2).
Because compounds 19 and 24 exhibited potent dual inhibitory
effects on platelet aggregation induced by Thr and Col, which both
are critical for thrombosis, and no cytotoxicity was found, these
compounds were chosen for further studies on the mechanisms in-
volved in the anti-platelet effect. Protein kinase C activation and
[Ca2+]i rise are the major signaling pathways involved in the induc-
tion of platelet aggregation. In the present study, the calcium ion-
ophore A23187 (500 nM) and the protein kinase C (PKC) activator
tetradecanoyl phorbol acetate (TPA, 200 nM), were used to directly
100
80
60
40
20
0
0.2 0.4
1
2
0.2 0.4
1
2 0.2 0.4
1
2 (µM)
BMNS
19
24
Figure 2. Effects of BMNS analogs on platelet GPIIb/IIIa activation analyzed by flow
cytometry as described in Materials and Methods. Results are presented as
*
** ***
mean S.E.M. (n = 3). P <0.05, P <0.01, P <0.001 as compared with the control.

P.-W. Hsieh et al. / Bioorg. Med. Chem. 18 (2010) 7621–7627
7625
bioactivity of BMNS derivatives implied that a more specific
mechanism might be involved in the anti-platelet effect. Our study
has demonstrated that the BMNS derivatives 19 and 24 inhibited
platelet aggregation via prevention of GPIIb/IIIa activation. The po-
tent and novel effects of BMNS derivatives make them attractive
candidates for the development of new anti-platelet agents.
m, H-40, H0-5), 8.12 (1H, m, H-60), 3.91 (3H, s, OCH3). 13C NMR
(CDCl3) d 138.7 (d, C- ), 137.8 (d, C-b), 129.7 (s, C-1), 112.7 (d,
a
C-2), 143.0 (s, C-3), 152.4 (s, C-4), 124.1 (d, C-5), 122.9 (d, C-6),
127.3 (s, JC–F = 5 Hz, C-10), 129.4 (s, JC–F = 33 Hz, C-20), 127.4 (d,
JC–F = 5 Hz, C-30), 132.4 (d, C-40), 132.3 (d, C-50), 131.3 (d, C-60),
164.2 (s, C@O), 56.5 (q, OCH3), 123.7 (s, JC–F = 272 Hz, CF3). EI-MS
(m/z, %): 367 [M]+ (8), 173 (100), 145 (96). HRESI-MS m/z
368.0748 [M+H]+ (calcd for C17H13F3NO5 368.0746).
4. Experimental
4.6. (E)-2-Methoxy-4-(2-nitrovinyl)phenyl 3-bromobenzoate (9)
4.1. General
Yield (89%) from trans-4-hydroxy-3-methoxy-b-nitrostyrene
(Aldrich) and 3-bromobenzoyl chloride (Aldrich). Mp 170–172 °C;
The NMR spectra using CDCl3 or DMSO-d6 as solvents were ob-
1H NMR (CDCl3) d 8.00 (1H, d, J = 14.0 Hz, H-
a), 7.58 (1H, d,
tained on
a Bruker AVANCE-400 MHz FT-NMR spectrometer.
Chemical shifts were internally referenced to the solvent signals
in TMS. Low-resolution EI-MS were recorded on a Quattro GC/MS
spectrometer having a direct inlet system, low-resolution and
high-resolution ESI-MS spectra on a Bruker Daltonics APEX II 30e
spectrometer.
J = 14.0 Hz, H-b), 7.14 (1H, br s, H-2), 7.25 (1H, J = 8.8 Hz, H-5),
7.21 (1H, dd, J = 1.6, 8.8 Hz, H-6), 8.34 (1H, t, J = 1.6 Hz, H-20),
7.78 (1H, br.d, J = 8.0 Hz, H-40), 7.40 (1H, t, J = 8.0 Hz, H0-5), 8.13
(1H, br.d, J = 8.0 Hz, H-60), 3.87 (3H, s, OCH3). 13C NMR (CDCl3) d
138.8 (d, C-a), 137.7 (d, C-b), 129.4 (s, C-1), 112.6 (d, C-2), 143.3
(s, C-3), 152.3 (s, C-4), 124.3 (d, C-5), 122.9 (d, C-6), 131.2 (s, C-
10), 133.7 (d, C-20), 123.1 (s, C-30), 137.2 (d, C-40), 130.6 (d, C-50),
129.6 (d, C-60), 163.4 (s, C@O), 56.5 (q, OCH3). ESI-MS (m/z, %):
400 [M+Na]+ (25), 381 (100). HRESI-MS m/z 399.9798 [M+Na]+,
(calcd for C16H12BrNO5Na 399.9797).
4.2. General procedure for synthesis of b-nitrostyrene benzoyl
esters (1–32, and 35–36)
To a mixture solution of b-nitrostyrenes (1.0 mmol) in DCM/
pyridine (10:1) was added the suitable benzoyl chlorides
(2.0 mmol). The reaction mixture was stirred at room temperature
for 24 h. The solvent was evaporated at reduced pressure. The res-
idue was purified by flash column chromatography (Si-Gel) to af-
ford the products.
4.7. (E)-2-Methoxy-4-(2-nitrovinyl)phenyl 3-(trifluoromethyl)-
benzoate (10)
Yield (67%) from trans-4-hydroxy-3-methoxy-b-nitrostyrene
(Aldrich) and 3-(trifluoromethyl)benzoyl chloride (Alfa Aesar).
Mp 155–157 °C; 1H NMR (CDCl3) d 8.00 (1H, d, J = 13.6 Hz, H-
a),
4.3. General procedure for synthesis of compounds 33–34 and
37–38
7.59 (1H, d, J = 13.6 Hz, H-b), 7.15 (1H, d, J = 1.6 Hz, H-2), 7.26
(1H, J = 8.0 Hz, H-5), 7.23 (1H, dd, J = 1.6, 8.0 Hz, H-6), 8.47 (1H,
br s, H-20), 7.92 (1H, br.d, J = 8.0 Hz, H-40), 7.68 (1H, t, J = 8.0 Hz,
H-50), 8.39 (1H, br.d, J = 8.0 Hz, H-60), 3.88 (3H, s, OCH3). 13C NMR
The synthetic method as described by Milhazes et al.8 Briefly,
compound 3-hydroxy-4-methoxybenzaldehyde or 3-ethoxy-4-
hydroxybenzaldehyde (1.0 mmol) dissolved in nitromethane or
nitroethane (3.0 ml), respectively, were added ammonium acetate
(1.0 mmol). The reaction mixture was reflux for 24 h. The solvent
was evaporated at reduced pressure. The residue was purified by
column chromatography (Si-Gel) using solution of CHCl3/hexane
(3:4) or acetone/hexane (6:1) mixture to afford the products.
(CDCl3) d 138.7 (d, C-a), 137.8 (d, C-b), 129.8 (s, C-1), 112.7 (d, C-
2), 143.2 (s, C-3), 152.3 (s, C-4), 124.3 (d, C-5), 122.9 (d, C-6),
130.2 (s, C-10), 127.7 (d, JC–F = 4 Hz, C-20), 131.8 (s, JC–F = 33 Hz, C-
30), 130.7 (d, JC–F = 3 Hz, C-40), 129.7 (d, C-50), 134.0 (d, C-60),
163.4 (s, C@O), 56.5 (q, OCH3), 124.0 (s, JC–F = 270 Hz, CF3). EI-MS
(m/z, %): 367 [M]+ (19), 173 (100), 145 (95). HRESI-MS m/z
368.0750 [M+H]+ (calcd for C17H13F3NO5 368.0746).
4.4. (E)-2-Methoxy-4-(2-nitrovinyl)phenyl 2-fluorobenzoate (1)
4.8. (E)-2-Methoxy-4-(2-nitrovinyl)phenyl 4-fluorobenzoate (13)
Yield (17%) from trans-4-hydroxy-3-methoxy-b-nitrostyrene
(Aldrich) and 2-fluorobenzoyl chloride (Aldrich). Mp 120–122 °C;
Yield (37%) from trans-4-hydroxy-3-methoxy-b-nitrostyrene
(Aldrich) and 4-fluorobenzoyl chloride (Aldrich) Mp 186–188 °C;
1H NMR (CDCl3) d 7.98 (1H, d, J = 13.6 Hz, H-
a), 7.58 (1H, d,
1H NMR (CDCl3) d 8.03 (1H, d, J = 13.6 Hz, H-
a), 7.60 (1H, d,
J = 13.6 Hz, H-b), 7.14 (1H, br s, H-2), 7.25 (1H, J = 8.4 Hz, H-5),
7.21 (1H, dd, J = 1.6, 8.4 Hz, H-6), 7.21 (1H, m, H-30), 7.62 (1H, m,
H-40), 7.29 (1H, t, J = 8.0 Hz, H-50), 8.12 (1H, td, J = 1.2, 8.0 Hz, H-
J = 13.6 Hz, H-b), 7.16 (1H, br s, H-2), 7.25 (1H, d, J = 8.8 Hz, H-5),
7.21 (1H, br.d, J = 8.8 Hz, H-6), 8.25 (2H, dd, J = 5.6, 8.8 Hz, H-
20,60), 7.23 (2H, m, H-30,50), 3.90 (3H, s, OCH3). 13C NMR (CDCl3) d
60), 3.87 (3H, s, OCH3). 13C NMR (CDCl3) d 138.9 (d, C-
a), 137.7
(d, C-b), 129.5 (s, C-1), 112.6 (d, C-2), 143.3 (s, C-3), 152.3 (s, C-
4), 124.4 (d, C-5), 123.0 (d, C-6), 117.8 (s, JC–F = 13 Hz, C-10),
162.8 (s, JC–F = 160 Hz, C-20), 117.7 (d, JC–F = 22 Hz, C-30), 136.0 (d,
JC–F = 9 Hz, C-40), 133.1 (d, C-50), 124.7 (d, JC–F = 4 Hz, C-60), 162.1
(s, C@O), 56.5 (q, OCH3). ESI-MS (m/z, %): 340 [M+Na]+ (100). HRES-
I-MS m/z 340.0596 [M+Na]+ (calcd for C16H12FNO5Na 340.0597).
138.9 (d, C-a), 137.7 (d, C-b), 129.4 (s, C-1), 112.6 (d, C-2), 143.5
(s, C-3), 152.4 (s, C-4), 124.4 (d, C-5), 123.0 (d, C-6), 118.5 (s, C-
10), 133.5 (d, JC–F = 11 Hz, C-20, 60), 116.3 (d, JC–F = 11 Hz, C-30, 50),
166.3 (s, JC–F = 210 Hz, C-40), 163.8 (s, C@O), 56.5 (q, OCH3). ESI-
MS (m/z, %): 340 [M+Na]+ (100). HRESI-MS m/z 340.0599
[M+Na]+ (calcd for C16H12FNO5Na 340.0597).
4.5. (E)-2-Methoxy-4-(2-nitrovinyl)phenyl 2-(trifluoromethyl)-
4.9. (E)-2-Methoxy-4-(2-nitrovinyl)phenyl 4-(trifluoromethyl)-
benzoate (4)
benzoate (16)
Yield (77%) from trans-4-hydroxy-3-methoxy-b-nitrostyrene
Yield (79%) from trans-4-hydroxy-3-methoxy-b-nitrostyrene
(Aldrich) and 2-(trifluoromethyl)benzoyl chloride (Aldrich). Mp
(Aldrich) and 4-(trifluoromethyl)benzoyl chloride (Alfa Aesar).
158–160 °C; 1H NMR (CDCl3) d 7.99 (1H, d, J = 13.6 Hz, H-
a), 7.57
Mp 197–199 °C; 1H NMR (CDCl3) d 8.01 (1H, d, J = 14.0 Hz, H-
a),
(1H, d, J = 13.6 Hz, H-b), 7.14 (1H, br s, H-2), 7.27 (1H, J = 8.0 Hz,
7.59 (1H, d, J = 14.0 Hz, H-b), 7.15 (1H, d, J = 1.2 Hz, H-2), 7.27
(1H, d, J = 8.0 Hz, H-5), 7.23 (1H, dd, J = 1.2, 8.0 Hz, H-6), 8.33
H-5), 7.22 (1H, br.d, J = 8.0 Hz, H-6), 7.84 (1H, m, H-30), 7.70 (2H,

7626
P.-W. Hsieh et al. / Bioorg. Med. Chem. 18 (2010) 7621–7627
(2H, d, J = 8.0 Hz, H-20,60), 7.80 (2H, d, J = 8.0 Hz, H-30,50), 3.88 (3H,
s, OCH3). 13C NMR (CDCl3) d 138.7 (d, C-
), 137.8 (d, C-b), 129.7 (s,
C-1), 112.6 (d, C-2), 143.2 (s, C-3), 152.3 (s, C-4), 124.3 (d, C-5),
122.9 (d, C-6), 118.5 (s, C-10), 131.2 (d, C-20, 60), 126.1 (d, JC–F
C-10), 113.1 (d, JC–F = 24 Hz, C-20, 60), 162.4 (s, JC–F = 247 Hz, C-30),
109.9 (d, JC–F = 26 Hz, C-40), 162.3 (s, JC–F = 248 Hz, C-50), 161.6 (s,
C@O), 56.3 (q, OCH3). EI-MS (m/z, %): 335 [M]+ (44), 141 (100),
113 (84). HRESI-MS m/z 336.0682 [M+H]+ (calcd for C16H12F2NO5
336.0684).
a
=
3 Hz, C-30, 50), 135.7 (s, JC–F = 32 Hz, C-40), 163.5 (s, C@O), 56.5 (q,
OCH3), 123.9 (s, JC–F = 271 Hz, CF3). EI-MS (m/z, %): 367 [M]+ (4),
337 (5), 173 (100), 145 (61). HREI-MS m/z 368.0748 [M+H]+ (calcd
for C17H13F3NO5 368.0746).
4.14. (E)-2-Methoxy-4-(2-nitrovinyl)phenyl 2,3-dichlorobenzoate
(24)
4.10. (E)-2-Methoxy-4-(2-nitrovinyl)phenyl 2,3-difluorobenzoate
Yield (85%) from trans-4-hydroxy-3-methoxy-b-nitrostyrene
(20)
(Aldrich) and 2,3-dichlorobenzoyl chloride (Alfa Aesar). Mp 132–
134 °C; 1H NMR (CDCl3) d 7.99 (1H, d, J = 13.6 Hz, H-
a), 7.55 (1H,
Yield (50%) from trans-4-hydroxy-3-methoxy-b-nitrostyrene
d, J = 13.6 Hz, H-b), 7.15 (1H, br s, H-2), 7.27 (1H, d, J = 8.0 Hz, H-
5), 7.21 (1H, br.d, J = 8.0 Hz, H-6), 7.69 (1H, d, J = 8.0 Hz, H-40),
7.35 (1H, t, J = 8.0 Hz, H-50), 7.88 (1H, d, J = 8.0 Hz, H-60), 3.90
(Aldrich) and 2,3-difluorobenzoyl chloride (Aldrich). Mp 167–
169 °C; 1H NMR (CDCl3) d 7.99 (1H, d, J = 13.6 Hz, H-
a), 7.55 (1H, d,
J = 13.6 Hz, H-b), 7.15 (1H, br s, H-2), 7.25 (1H, d, J = 8.0 Hz, H-5),
7.22 (1H, br.d, J = 8.0 Hz, H-6), 7.25 (1H, m, H-40), 7.46 (1H, br.q,
J = 8.0 Hz, H-50), 7.88 (1H, br.t, J = 8.0 Hz, H-60), 3.88 (3H, s, OCH3).
(3H, s, OCH3). 13C NMR (CDCl3) d 138.2 (d, C-
a), 137.3 (d, C-b),
129.2 (s, C-1), 112.2 (d, C-2), 142.6 (s, C-3), 151.7 (s, C-4), 123.7
(d, C-5), 122.5 (d, C-6), 132.4 (s, C-10), 131.1 (s, C-20), 134.9 (s,
C-30), 134.0 (d, C-40), 127.2 (d, C-50), 129.8 (d, C-60), 162.5 (s,
C@O), 56.1 (q, OCH3). ESI-MS (m/z, %): 390 [M+Na]+ (93), 368
[M+H]+ (64). HRESI-MS m/z 389.9914 [M+Na]+ (calcd for
13C NMR (CDCl3) d 138.3 (d, C-
a), 137.3 (d, C-b), 129.3 (s, C-1),
112.3 (d, C-2), 142.6 (s, C-3), 151.8 (s, C-4), 123.8 (d, C-5), 122.5 (d,
C-6), 119.7 (s, JC–F = 40 Hz, C-10), 151.1 (s, JC–F = 228 Hz, C-20, 60),
151.3 (s, JC–F = 249 Hz, C-30), 122.4 (d, JC–F = 21 Hz, C-40), 124.0 (d,
JC–F = 5 Hz, C-50), 127.2 (d, JC–F = 3 Hz, C-60), 160.9 (s, C@O), 56.1 (q,
OCH3). EI-MS (m/z, %): 335 [M]+ (44), 141 (100), 113 (94). HRESI-
MS m/z 336.0683 [M+H]+ (calcd for C16H12F2NO5 336.0684).
C16H11Cl2NO5Na 389.9912).
4.15. (E)-2-Methoxy-4-(2-nitrovinyl)phenyl 3,5-dichlorobenzoate
(27)
4.11. (E)-2-Methoxy-4-(2-nitrovinyl)phenyl 2,4-difluorobenzoate
Yield (68%) from trans-4-hydroxy-3-methoxy-b-nitrostyrene
(21)
(Aldrich) and 3,5-dichlorobenzoyl chloride (Aldrich). Mp 244–
245 °C; 1H NMR (CDCl3) d 8.00 (1H, d, J = 13.6 Hz, H-
a), 7.57 (1H,
Yield (28%) from trans-4-hydroxy-3-methoxy-b-nitrostyrene
d, J = 13.6 Hz, H-b), 7.14 (1H, br s, H-2), 7.25 (2H, br.d, J = 10.8 Hz,
H-5, 6), 8.07 (2H, br s, H-20, 60), 7.64 (1H, s, H-40), 3.88 (3H, s,
OCH3). ESI-MS (m/z, %): 390 [M+Na]+ (25). HRESI-MS m/z
389.9909 [M+Na]+ (calcd for C16H11Cl2NO5Na 389.9912).
(Aldrich) and 2,4-difluorobenzoyl chloride (Aldrich). Mp 170–
172 °C; 1H NMR (CDCl3) d 7.99 (1H, d, J = 13.6 Hz, H-
a), 7.57 (1H, d,
J = 13.6 Hz, H-b), 7.14 (1H, d, J = 1.2 Hz, H-2), 7.25 (1H, d, J = 8.0 Hz,
H-5), 7.21 (1H, dd, J = 1.2, 8.0 Hz, H-6), 7.00 (1H, m, H-30), 6.96 (1H,
m, H-50), 8.16 (1H, br.t, J = 8.4 Hz, H-60), 3.88 (3H, s, OCH3). 13C
4.16. (E)-2-Methoxy-4-(2-nitrovinyl)phenyl 2,4,5-trifluoroben-
NMR (CDCl3) d 138.3 (d, C-
a
), 137.3 (d, C-b), 129.2 (s, C-1), 112.3
zoate (28)
(d, C-2), 142.8 (s, C-3), 151.9 (s, C-4), 123.9 (d, C-5), 122.5 (d, C-6),
112.2 (s, JC–F = 24 Hz, C-10), 162.0 (s, JC–F = 210 Hz, C-20, 60), 105.6
Yield (42%) from trans-4-hydroxy-3-methoxy-b-nitrostyrene (Al-
drich) and 2,4,5-trifluorobenzoyl chloride (Aldrich). Mp 229–231 °C;
(d, JC–F = 26 Hz, C-30), 165.2 (d, JC–F = 280 Hz, C-40), 111.9 (d, JC–F
=
25 Hz, C-50), 134.6 (d, JC–F = 10 Hz, C-60), 160.8 (s, C@O), 56.1 (q,
OCH3). EI-MS (m/z, %): 335 [M]+ (14), 141 (100), 113 (80). HRESI-
MS m/z 336.0682 [M+H]+ (calcd for C16H12F2NO5 336.0684).
1H NMR (CDCl3) d 8.00 (1H, d, J = 13.6 Hz, H-
a), 7.58 (1H, d,
J = 13.6 Hz, H-b), 7.14 (1H, br s, H-2), 7.25 (1H, d, J = 8.8 Hz, H-5),
7.22 (1H, dd, J = 1.2, 8.8 Hz, H-6), 7.85 (2H, br.t, J = 6.8 Hz, H-30, 60),
3.88 (3H, s, OCH3). 13C NMR (DMSO-d6) d 138.5 (d, C-
a), 138.5 (d, C-
4.12. (E)-2-Methoxy-4-(2-nitrovinyl)phenyl 3,4-difluorobenzoate
(22)
b), 129.8 (s, C-1), 113.2 (d, C-2), 141.8 (s, C-3), 151.1 (s, C-4), 123.6
(d, C-5), 123.5 (d, C-6), 124.7 (s, C-10), 150.4 (s, JC–F = 236 Hz, C-20),
103.8 (d, C-30), 150.4 (s, JC–F = 236 Hz, C-40), 139.8 (s, JC–F = 278 Hz,
C-50), 115.1 (d, JC–F = 23 Hz, C-60), 161.2 (s, C@O), 56.3 (q, OCH3). EI-
MS (m/z, %): 353 [M]+ (32), 159 (100), 131 (86). HRESI-MS m/z
354.0587 [M+H]+ (calcd for C16H11F3NO5Na 354.0589).
Yield (48%) from trans-4-hydroxy-3-methoxy-b-nitrostyrene
(Aldrich) and 3,4-difluorobenzoyl chloride (Aldrich). Mp 207–
209 °C; 1H NMR (CDCl3) d 8.00 (1H, d, J = 13.6 Hz, H-
a), 7.58 (1H, d,
J = 13.6 Hz, H-b), 7.14 (1H, br s, H-2), 7.25 (1H, d, J = 8.4 Hz, H-5),
7.22 (1H, dd, J = 1.6, 8.0 Hz, H-6), 7.99 (1H, m, H-20), 7.31 (1H, br.q,
J = 8.4 Hz, H-50), 8.02 (1H, m, H-60), 3.88 (3H, s, OCH3). EI-MS (m/z,
%): 335 [M]+ (37), 141 (100), 113 (97). HRESI-MS m/z 336.0683
[M+H]+ (calcd for C16H12F2NO5 336.0684).
4.17. (E)-2-Methoxy-4-(2-nitrovinyl)phenyl 3,4,5-trifluoroben-
zoate (29)
Yield (73%) from trans-4-hydroxy-3-methoxy-b-nitrostyrene
(Aldrich) and 3,4,5-trifluorobenzoyl chloride (Aldrich). Mp 183–
4.13. (E)-2-Methoxy-4-(2-nitrovinyl)phenyl 3,5-difluorobenzoate
185 °C; 1H NMR (CDCl3) d 8.00 (1H, d, J = 13.6 Hz, H-
a), 7.58 (1H,
(23)
d, J = 13.6 Hz, H-b), 7.14 (1H, d, J = 1.2 Hz, H-2), 7.25 (1H, d,
J = 8.8 Hz, H-5), 7.22 (1H, dd, J = 1.2, 8.8 Hz, H-6), 7.96 (1H, m, H-
20), 7.08 (1H, m, H-60), 3.89 (3H, s, OCH3). 13C NMR (CDCl3) d
Yield (47%) from trans-4-hydroxy-3-methoxy-b-nitrostyrene
(Aldrich) and 3,5-difluorobenzoyl chloride (Alfa Aesar). Mp 206–
138.2 (d, C-
a), 137.4 (d, C-b), 129.4 (s, C-1), 112.2 (d, C-2), 142.4
208 °C; 1H NMR (CDCl3) d 8.00 (1H, d, J = 13.6 Hz, H-
a), 7.58 (1H,
(s, C-3), 152.6 (s, C-4), 123.7 (d, C-5), 122.5 (d, C-6), 120.5 (s, JC–F =
d, J = 13.6 Hz, H-b), 7.14 (1H, br s, H-2), 7.25 (1H, d, J = 8.0 Hz, H-
5), 7.22 (1H, dd, J = 1.6, 8.0 Hz, H-6), 7.73 (2H, m, H-20, 60), 7.09
(1H, m, H-40), 3.88 (3H, s, OCH3). 13C NMR (DMSO-d6) d 138.5 (d,
28 Hz, C-10), 107.4 (d, JC–F = 38 Hz, C-20), 151.1 (s, JC–F = 278 Hz,
C-30, 50), 143.9 (s, JC–F = 298 Hz, C-40), 107.4 (d, JC–F = 38 Hz, C-60),
159.9 (s, C@O), 56.1 (q, OCH3). EI-MS (m/z, %): 353 [M]+ (22), 159
(100), 131 (73). HRESI-MS m/z 354.0586 [M+H]+ (calcd for
C-a), 138.4 (d, C-b), 129.8 (s, C-1), 113.2 (d, C-2), 141.8 (s, C-3),
151.1 (s, C-4), 123.6 (d, C-5), 123.5 (d, C-6), 131.7 (s, JC–F = 2 Hz,
C16H11F3NO5 354.0589).

P.-W. Hsieh et al. / Bioorg. Med. Chem. 18 (2010) 7621–7627
7627
4.18. (E)-2-Ethoxy-4-(2-nitrovinyl)phenyl benzoate (31)
Yield (55%) from 33 and benzoyl chloride (Aldrich). Mp 134–
metric analysis was performed on a Beckman Coulter EPICS XL flow
cytometer with EXPO32 ADC software. Platelets were identified by
logarithmic signal amplification for forward and side scatter. The
levels of PAC-1 binding were expressed as the percentages of cells
positive for PAC-1.
135 °C; 1H NMR (CDCl3) d 7.96 (1H, d, J = 13.6 Hz, H-
a), 7.56 (1H,
d, J = 13.6 Hz, H-b), 7.12 (1H, d, J = 1.2 Hz, H-2), 7.23 (1H, d,
J = 8.0 Hz, H-5), 7.17 (1H, dd, J = 1.2, 8.0 Hz, H-6), 8.12 (2H, d,
J = 7.6 Hz, H-20, 60), 7.51 (2H, t, J = 7.6 Hz, H-30, 50), 7.65 (1H, t,
J = 7.6 Hz, H-40), 4.07 (2H, q, J = 6.8 Hz, OCH2CH3), 1.31 (3H, t,
4.23. Cell viability assays17
J = 6.8 Hz, OCH2CH3). 13C NMR (CDCl3) d 138.5 (d, C-
a), 137.0 (d,
Compounds were tested against MDA-MB-231 cells (a human
breast cancer cell line) and HBL-100 cells (a non-malignant breast
epithelial cell line) using the MTT method as described before.17
Freshly trypsinized cell suspensions were seeded in 96 well micro-
titer plates at densities of 8000 cells per well with tested com-
pounds added from DMSO-diluted stock. After 72 h in culture,
attached cells were incubated with MTT (0.5 mg/mL, 1 h) and sub-
sequently solubilized in DMSO for determining of IC50. The cyto-
toxic activity of compounds was expressed as the concentration
inhibiting cell growth by 50% (IC50) calculated from the survival
curves.
C-b), 128.9 (s, C-1), 113.3 (d, C-2), 143.5 (s, C-3), 151.3 (s, C-4),
123.8 (d, C-5), 122.4 (d, C-6), 128.7 (s, C-10), 130.2 (d, C-20, 60),
128.5 (d, C-30, 50), 133.7 (d, C-40), 164.3 (s, C@O), 64.7 (t, OCH2CH3),
14.5 (q, OCH2CH3). ESI-MS (m/z, %): 336 [M+Na]+ (100), 331 (85),
314 [M+H]+ (19). HRESI-MS m/z 336.0847 [M+Na]+ (calcd for
C17H15NO5Na 336.08448).
4.19. (E)-2-Ethoxy-4-(2-nitroprop-1-enyl)phenyl benzoate (35)
Yield (12%) from 37 and benzoyl chloride (Aldrich). Mp 85–
87 °C; 1H NMR (CDCl3) d 8.07 (1H, s, H-
a), 7.04 (1H, br s, H-2),
7.25 (1H, d, J = 8.4 Hz, H-5), 7.07 (1H, d, J = 8.4 Hz, H-6), 8.21 (2H,
d, J = 7.6 Hz, H-20, 60), 7.52 (2H, t, J = 7.6 Hz, H-30, 50), 7.65 (1H, t,
J = 7.6 Hz, H-40), 4.07 (2H, q, J = 6.8 Hz, OCH2CH3), 1.32 (3H, t,
J = 6.8 Hz, OCH2CH3), 2.48 (3H, b-CH3). 13C NMR (CDCl3) d 133.6
Acknowledgments
The investigation was supported by research grants to C. C. Wu
and P. W. Hsieh from the National Science Council of the Republic
of China in Taiwan.
(d, C-a), 147.7 (d, C-b), 129.1 (s, C-1), 115.2 (d, C-2), 141.6 (s, C-
3), 150.9 (s, C-4), 123.4 (d, C-5), 122.5 (d, C-6), 131.1 (s, C-10),
130.2 (d, C-20, 60), 128.5 (d, C-30, 50), 133.0 (d, C-40), 164.5 (s,
C@O), 64.7 (t, OCH2CH3), 14.5 (q, OCH2CH3), 14.0 (q, b-CH3). ESI-
MS (m/z, %): 350 [M+Na]+ (66), 328 [M+H]+ (9). HRESI-MS m/z
350.1002 [M+Na]+ (calcd for C18H17NO5Na 350.1004).
Supplementary data
Supplementary data (additional information on compound pur-
ity, including HPLC analysis of the target compounds) associated
with this article can be found, in the online version, at doi:10.1016/
j.bmc.2010.08.039.
4.20. Preparation of washed human platelets
Human blood anticoagulated with acid citrate dextrose (ACD)
was obtained from healthy human volunteers who had not taken
any drugs within the last two weeks. The platelet suspension
was then prepared according to the washing procedure previously
described.15 Platelets were finally suspended in Tyrode0s solution
containing Ca2+ (2 mM), glucose (11.1 mM) and bovine serum
albumin (3.5 mg/ml) at a concentration of 3 ꢁ 108 platelets/ml.
References and notes
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Washed human platelets (3 ꢁ 107 platelets/ml) were pre-incu-
bated with DMSO or test compounds for 3 min, and then treated
with or without thrombin in the presence of FITC-conjugated
PAC-1 monoclonal antibody for 15 min at room temperature. The
samples were then fixed with 1% paraformaldehyde. Flow cyto-