Synthesis and antiproliferative evaluations of certain 2-phenylvinylquinoline (2-styrylquinoline) and 2-furanylvinylquinoline derivatives

Article abstract of DOI:10.1016/j.bmc.2009.11.012

The present study describes the synthesis of 2-phenylvinylquinoline (styrylquinoline) and 2-furanylvinylquinoline derivatives and evaluation for their antiproliferative activities. (E)-2-Styrylquinolin-8-ol (14a) was inactive against a 3-cell line panel consisting of MCF-7 (Breast), NCI-H460 (Lung), and SF-268 (CNS). Replacement of the phenyl ring with 5-nitrofuran-2-yl group significantly enhanced antiproliferative activity in which (E)-2-(2-(5-nitrofuran-2-yl)vinyl)quinolin-8-ol (14i) and its 4-substituted derivatives 15-19 exhibited strong inhibitory effects against the growth of all three cancer cells. These compounds were further evaluated for their IC₅₀ against the growth of MCF-7, LNCaP, and PC3. Results indicated that a hydrogen bond donating oxime derivative 19a was more active than its hydrogen bond accepting methyloxime derivative 19b. For the inhibition of LNCaP, the potency decreased in an order 14i > 19a > 19b > 15 > 18 > 16. Compound 14i is the most active with an IC₅₀ value of 0.35 and 0.14 μM, respectively, against the growth of LNCaP and PC3 cancer cells. Therefore, compound 14i was evaluated by flow cytometric analysis for its effects on cell cycle distributions. Results indicated that 14i effectively induced cell cycle arrest at S phase for both cell lines, which consequently trigger late apoptosis for both LNCaP and PC3 cells.

Full text of DOI:10.1016/j.bmc.2009.11.012

Bioorganic & Medicinal Chemistry 18 (2010) 124–133
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antiproliferative evaluations of certain
2-phenylvinylquinoline (2-styrylquinoline) and 2-furanylvinylquinoline
derivatives
a
a,
Feng-Shuo Chang a, Weichung Chen b, Chihuei Wang b, , Cherng-Chyi Tzeng , Yeh-Long Chen
*
*
a Department of Medicinal and Applied Chemistry, College of Life Science, Kaohsiung Medical University, Kaohsiung City 807, Taiwan
b Department of Biotechnology, College of Life Science, Kaohsiung Medical University, Kaohsiung City 807, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
The present study describes the synthesis of 2-phenylvinylquinoline (styrylquinoline) and 2-furanylvi-
nylquinoline derivatives and evaluation for their antiproliferative activities. (E)-2-Styrylquinolin-8-ol
(14a) was inactive against a 3-cell line panel consisting of MCF-7 (Breast), NCI-H460 (Lung), and SF-
268 (CNS). Replacement of the phenyl ring with 5-nitrofuran-2-yl group significantly enhanced antipro-
liferative activity in which (E)-2-(2-(5-nitrofuran-2-yl)vinyl)quinolin-8-ol (14i) and its 4-substituted
derivatives 15–19 exhibited strong inhibitory effects against the growth of all three cancer cells. These
compounds were further evaluated for their IC50 against the growth of MCF-7, LNCaP, and PC3. Results
indicated that a hydrogen bond donating oxime derivative 19a was more active than its hydrogen bond
accepting methyloxime derivative 19b. For the inhibition of LNCaP, the potency decreased in an order
14i > 19a > 19b > 15 > 18 > 16. Compound 14i is the most active with an IC50 value of 0.35 and
Received 10 September 2009
Revised 4 November 2009
Accepted 5 November 2009
Available online 11 November 2009
Keywords:
Antiproliferative activity
2-Phenylvinylquinoline (styrylquinoline)
2-Furanylvinylquinoline
Apoptosis
0.14 lM, respectively, against the growth of LNCaP and PC3 cancer cells. Therefore, compound 14i was
evaluated by flow cytometric analysis for its effects on cell cycle distributions. Results indicated that
14i effectively induced cell cycle arrest at S phase for both cell lines, which consequently trigger late
apoptosis for both LNCaP and PC3 cells.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
because 4-anilinofuro[2,3-b]quinoline derivatives, if proved to be
active, could be developed as a new structural type of potential
We have synthesized certain furo[2,3-b]quinoline derivatives
and evaluated for their antiproliferative activities on the ground
that this tricyclic furo[2,3-b]quinoline moiety is an isosteric isomer
of the versatile acridine ring.1–6 Among them, 1-[4-(furo[2,3-
anti-microtubule drug candidates.
Quinoline moiety is present in many classes of biologically
active compounds.7–11 The biological activities of these quinoline
derivatives depends not only on the bicyclic heteroaromatic phar-
macophore but also on the nature of the peripheral substituents
and their spatial relationship. Recently, we have synthesized
2-phenylquinoline and 2-furanylquinoline derivatives which can
be considered as the isomers of acridine and furo[2,3-b]quinoline,
respectively, in which the phenyl and furanyl moiety, respectively,
is appended on the C-2 position instead of fused with bicyclic quin-
oline.3,12–14 The 2-phenylquinoline derivatives can also be consid-
ered as aza-analogues of 2-phenylnaphthalene skeleton which
consists of a large number of antitumor compounds.15–18 In contin-
uation of our search for potential drug candidates, the present
study describes the synthesis and antiproliferative evaluations of
2-phenylvinylquinoline (styrylquinoline) and 2-furanylvinylquino-
line derivatives, the insertion of a vinyl bridge between the 2-aryl
moiety and the bicyclic quinoline ring. Styrylquinoline derivatives
have gained strong attention recently due to their extensive bio-
logical activities.19–23
b]quinolin-4-ylamino)phenyl]ethanone (CIL-102) (mean GI50
0.025 M) was more active than both clinically used anticancer
drugs, m-AMSA (mean GI50 = 0.44 M) and Daunomycin (mean
GI50 = 0.044 M) against the full panel of 60 human tumor cell
=
l
l
l
lines derived from nine cancer cell types.1,2 CIL-102 had been se-
lected as a lead compound and was evaluated by flow cytometric
analysis for its effects on cell cycle distributions. Results indicated
that CIL-102 inhibits cell proliferation by the alteration of cell divi-
sion, accumulation of cells in the G2/M phase followed by the cell
apoptosis which is similar to microtubule-targeting agents such as
paclitaxel but distinct from that of amsacrine.4 This is interesting
* Corresponding authors. Tel.: +886 7 3121101x2699; fax: +886 7 3125339
(C.W.); tel.: +886 7 3121101x2684; fax: +886 7 3125339 (Y.-L.C.).
E-mail addresses: chwang@kmu.edu.tw (C. Wang), yeloch@kmu.edu.tw (Y.-L.
Chen).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.11.012

F.-S. Chang et al. / Bioorg. Med. Chem. 18 (2010) 124–133
125
2. Chemistry
(i)
Ar
N
Me
N
Reaction of 2-methoxyaniline (2) and ethyl acetoacetate gave 4-
hydroxy-2-methyl-8-methoxyquinoline (4)24 as described in
Scheme 1. Condensation of 4 with benzaldehyde in acetic anhy-
dride gave (E)-4-hydroxy-8-methoxy-2-styrylquinoline (6) in a
good overall yield. The known (E)-4-hydroxy-2-styrylquinoline
(5)25 was prepared under the same reaction conditions from com-
mercially available 4-hydroxy-2-methylquinoline (3) and benzal-
OH
OH
14
13
b:
f:
c:
a:
d:
h:
CN
OH
F
dehyde. Treatment of
6 with POCl3 afforded (E)-4-chloro-8-
methoxy-2-styrylquinoline (8) which was reacted with 4-amino-
acetophenone to give (E)-1-(4-(8-methoxy-2-styrylquinolin-4-yla-
mino)phenyl)ethanone (10) in a good overall yield. Accordingly,
compound 9 was obtained from (E)-4-chloro-2-styrylquinoline
(7) and 4-aminoacetophenone. Treatment of 9 and 10 with NH2OH
in EtOH gave (E)-1-{4-[(E)-2-styrylquinolin-4-ylamino]phenyl}-
ethanone oxime (11a) and its derivative 12a, respectively. The con-
figuration of the oxime moiety was confirmed by the chemical shift
of 13C NMR spectra in which the Me moiety (d 11.53 ppm for 11a
and 11.45 ppm for 12a) of the E-form isomer shifted more upfield
(d approximately at 11.50 ppm) than its Z-form counterpart (d
approximately at 18.80 ppm).26 Accordingly, compounds 11b and
12b were synthesized from their respective ketone precursors 9
and 10 with NH2OMe under the same reaction conditions.
We have also prepared certain 8-hydroxy-2-styrylquinoline
derivatives in which the styryl moiety was substituted or replaced
with various heterocycles as described in Scheme 2. Reaction of 8-
hydroxy-2-methylquinoline (13) with benzaldehyde in acetic
anhydride afforded 2-styrylquinolin-8-ol (14a)20 in 45% yield.
Accordingly, compounds 14b–i were obtained from 13 and various
aldehydes in a fairly good yield. (E)-1-{4-{2-[(E)-2-(5-Nitrofuran-
2-yl)vinyl]quinolin-4-ylamino}phenyl}ethanone oxime (19a) and
its methyloxime derivative 19b were prepared from 3 and 5-ni-
tro-2-furaldehyde (Scheme 3) by the same reaction sequences for
the preparation of 11a and 11b.
OH
O
OH
N
g:
e:
i:
NO2
O
NO2
Scheme 2. Reagents and conditions: (i) aldehyde and Ac2O, 150 °C, 30 h.
(CNS). In this protocol, each cell line is inoculated and preincubated
on a microtiter plate. Test agents are then added at a concentration
of 4.0 lg/mL and the culture incubated for 48 h. End-point deter-
minations are made with sulforhodamine B, a protein-binding
dye. Results for each test agent are reported as the percent of
growth of the treated cells when compared to the untreated con-
trol cells and results are summarized in Tables 1 and 2. With an
exception of (E)-1-{4-[8-methoxy-(E)-2-styrylquinolin-4-ylami-
no]phenyl}ethanone oxime (12a) which exhibited a marginal anti-
proliferative activity, all the 2-styrylquinolines 5–10, 11a, 11b, and
12b are inactive at a concentration of 4.0 lM (Table 1). (E)-2-Sty-
rylquinolin-8-ol (14a) was inactive although 8-hydroxyquinoline
constitutes a number of biologically active compounds.10,23 Intro-
duction of an electron-donating group such as OH or an electron-
withdrawing group such as CN, F, or NO2 at the 2-styryl moiety
of 14a did not improve antiproliferative activity. Replacement of
the phenyl ring with its heterocyclic isosteric isomers such as pyr-
idine or furan ring proved to be no improvement in which com-
pounds 14g and 14h were inactive. However, replacement of the
phenyl ring with 5-nitrofuran-2-yl group significantly enhanced
3. Results and discussion
All compounds were evaluated in vitro against a 3-cell line pa-
nel consisting of MCF-7 (Breast), NCI-H460 (Lung), and SF-268
OH
OH
ii
iii
i
N
N
Me
NH2
R
R
R
5: R = H
6: R = OMe
3: R = H
4: R = OMe
1: R = H
2: R = OMe
OR1
N
O
Me
Me
HN
N
HN
Cl
N
v
iv
N
R
R
R
7: R = H
8: R = OMe
9: R = H
10: R = OMe
11a: R = H, R1 = H
11b: R = H, R1 = Me
12a: R = OMe, R1 = H
12b: R = OMe, R1 = Me
Scheme 1. Reagents and conditions: (i) (a) ethyl acetoacetate, AcOH, benzene, 80 °C, 24 h; (b) phenyl ether, 250 °C, 1 h; (ii) benzaldehyde, Ac2O, 150 °C, 30 h; (iii) POCl3, 80–
90 °C, 1 h; (iv) 4-aminoacetophenone, EtOH, reflux, 2 h; (v) hydroxyamine or methylhydroxyamine, K2CO3, EtOH, rt, 1 h.

126
F.-S. Chang et al. / Bioorg. Med. Chem. 18 (2010) 124–133
OH
N
OAc
OH
N
(i)
(ii)
O
O
N
NO2
CH3
NO2
15
3
16
O
Me
O
Cl
HN
(v)
(iv)
(iii)
O
N
NO2
N
NO2
17
OR
18
N
Me
HN
N
O
NO2
19a: R = H
19b: R = Me
Scheme 3. Reagents and conditions: (i) (5-nitrofuran-2-yl)methylene diacetate, Ac2O, 150 °C, 30 h; (ii) pyridine/H2O (v/v 4:1), 100 °C, 1 h; (iii) POCl3, 80–90 °C, 1 h; (iv) 4-
aminoacetophenone, EtOH, reflux, 2 h; (v) hydroxyamine or methylhydroxyamine, K2CO3, EtOH, rt, 1 h.
Table 1
Table 2
Preliminary antiproliferative activities of 2-phenylvinylquinoline derivatives in vitroa
Preliminary antiproliferative activities of 2-arylvinylquinoline derivatives in vitroa
X
R
Me
O
N
R
NO2
HN
N
X
OH
15, 16
14
N
N
X
R
R
Me
O
9-12
5-8
HN
N
R
X
Survival (% control)
NCI-H460
MCF-7
SF-268
NO2
5
6
7
8
H
OMe
H
OMe
H
OMe
H
H
OMe
OMe
OH
OH
Cl
Cl
O
O
NOH
NOMe
NOH
NOMe
119
104
120
99
83
95
54
108
40
118
111
109
109
52
75
67
79
<20
87
120
119
118
81
107
114
90
112
63
97
18, 19
9
R
X
Survival (% control)
NCI-H460
10
11a
11b
12a
12b
MCF-7
SF-268
14a
14b
14c
14d
14e
14f
14g
14h
14i
15
Ph
—
—
—
—
—
—
—
—
—
—
—
O
110
108
107
103
89
55
52
66
66
52
53
63
70
<20
<20
<20
<20
<20
<20
72
78
90
82
77
52
89
76
<20
<20
<20
<20
<20
<20
Ph-4-OH
Ph-4-CN
Ph-4-F
97
a
Cells were cultured with agents at a concentration of 4.0
growth and viability were assessed using the MTS assay.
l
g/mL for 72 h before
Ph-4-NO2
Ph-2,3-diOH
Pyridin-3-yl
Furan-2-yl
5-NO2-furan-2-yl
OAc
95
115
110
21
<20
59
<20
<20
<20
antiproliferative activity in which (E)-2-(2-(5-nitrofuran-2-
yl)vinyl)quinolin-8-ol (14i) exhibited a strong inhibitory effect
against the growth of all three cancer cells (Table 2). These results
indicated that the substituent of 2-(5-nitrofuran-2-yl)vinyl moiety
is crucial for antiproliferative activity. Therefore, a number of (E)-
2-(2-(5-nitrofuran-2-yl)vinyl)quinoline derivatives 15–19 had
been synthesized and evaluated. Among them, (E)-2-(2-(5-nitrofu-
16
18
19a
19b
OH
—
—
—
NOH
NOMe
a
Cells were cultured with agents at a concentration of 4.0
growth and viability were assessed using the MTS assay.
l
g/mL for 72 h before

F.-S. Chang et al. / Bioorg. Med. Chem. 18 (2010) 124–133
127
ran-2-yl)vinyl)quinolin-4-ol (16) was less active than its 4-acetyl
derivative 15 against the growth of MCF-7 which implied that a
bulky group at C-4 position is favorable. This SAR can also be ap-
plied in which significant antiproliferative activities were observed
for C-4 bulky derivatives 18, 19a, and 19b.
(E)-2-(2-(5-Nitrofuran-2-yl)vinyl)quinolin-8-ol (14i) and its 4-
substituted derivatives 15–19 which exhibited potent antiprolifer-
ative effects were further evaluated for their inhibitory activity
against the growth of MCF-7, LNCaP, and PC3 and the IC50 values
are given in Table 3. A hydrogen bond donating oxime derivative
19a was found to be more active than its methyloxime derivative
19b. For the inhibition of LNCaP, the potency decreased in an order
14i > 19a > 19b > 15 > 18 > 16. Compound 14i is the most active
with an IC50 value of 0.35 and 0.14 lM, respectively, against the
growth of LNCaP and PC3 cancer cells. Therefore, compound 14i
was selected as a lead compound and was evaluated by flow cyto-
metric analysis for its effects on cell cycle distributions. Results
indicated that 14i effectively induced cell cycle arrest of LNCaP at
S phase, about 37% at a concentration of 0.5
in Figure 1. For the PC3 cells, compound 14i induced cell cycle ar-
rest at S phase, about 44% at a concentration of 0.2 M for 24 h. Our
results indicated that 14i induce higher percentage of PC3 cells
than that of LNCaP cells arrested at S phase by using the respective
dose near IC50 and the respective time course related to doubling
time of cell cycle.27,28
lM for 48 h as shown
Figure 1. Compound 14i induced cell cycle arrest at S phase for LNCaP and PC3
cells. LNCaP and PC3 cells were incubated in the presence or absence of 14i for 48
and 24 h, respectively, and then the cells were harvested for flow cytometry
analysis of cell cycle. The experimental details were described in Section 5.4.
Representative cell cycle distribution of LNCaP and PC3 cells after treatment of 14i
was shown (n = 3).
l
We further asked if 14i-induced cell cycle arrest at S phase
might trigger apoptosis for LNCaP and PC3 cells by Annexin-V
and propidium iodide (PI) staining with flow cytometry. The
FITC-conjugated Annexin-V staining can detect apoptotic cells by
binding to their exposed phosphatidylserine, and PI is included
with Annexin-V because dead cells or cells in the late stage of
apoptosis will allow PI to diffuse into the cell and bind to
DNA.29,30 Apoptotic cells can be sorted into two quadrants, the
upper right representing late apoptosis (both Annexin-V and PI po-
sitive) and the lower right representing early apoptosis (Annexin
positive and PI negative), among four quadrants in this dual dot
plot by flow cytometry. As shown in Figure 2A, treatment of LNCaP
of LNCaP cells with the compoundfor 48 h significantly increases the
cells in this quadrant up to 24%. In contrast, treatment of PC3 cells
with 0.2 lM of 14i for either 24 h or 48 h induces the significant
amounts of cells toward the upper right quadrant (Fig. 2B). This dif-
ferent outcome between LNCaP and PC3 cells is simply just due to
the different doubling time of them. Since PC3 cells have a short dou-
bling time of about 24 h, the treatment of 14i for 24 h might induce
the most cells arrested at S phase, resulting in entering into apopto-
sis. However, LNCaP cells have long doubling time of about 60 h,
reflecting that late apoptosis appears after 48 h of treatment. Thus,
we concluded that compound 14i can induce cell cycle arrest at S
phase for both cell lines, which consequently trigger late apoptosis
for both LNCaP and PC3 cells. Moreover, we also examined the cleav-
age pattern of poly(ADP-ribose) polymerase (PARP) to confirm that
14i induced an apoptotic response in PC3 cells by western blotting.
Our results indicated that the native 116 kDa PARP protein is cleaved
into its characteristic 85 kDa fragment upon treatment with 0.4 and
cells with 0.5 lM of 14i for 24 h, we observed that only 1% increase
of cells appears in the upper right quadrant and prolong treatment
Table 3
Antiproliferative activities (IC50) of 5-nitrofuran-2-ylvinylquinoline derivativesa
R
0.6 lM of 14i in PC3 cells (Fig. 3).
O
O
NO2
NO2
4. Conclusion
N
N
OH
We have synthesized and evaluated antiproliferative activities
of certain 2-phenylvinylquinoline (styrylquinoline) and 2-furanyl-
vinylquinoline derivatives. Among them, (E)-2-(2-(5-nitrofuran-2-
yl)vinyl)quinolin-8-ol (14i) exhibited a strong inhibitory effect
against the growth of MCF-7 (Breast), NCI-H460 (Lung), and SF-
268 (CNS). Compound 14i was evaluated by flow cytometric anal-
ysis for its effects on cell cycle distributions of LNCaP and PC3 cells.
Results indicated that 14i effectively induced cell cycle arrest at S
phase for both cell lines, which consequently trigger late apoptosis.
Structural optimization and mechanism studies of 14i are cur-
rently under investigation.
15, 16
14i
X
Me
O
HN
N
NO2
18, 19
R
X
MCF-7
LNCaP
PC3
5. Experimental
5.1. General
14i
15
16
18
19a
19b
—
—
—
—
O
NOH
NOMe
0.30 0.04
0.71 0.17
2.21 0.54
0.43 0.10
0.25 0.03
0.40 0.04
0.35 0.06
1.61 0.33
5.69 2.75
4.12 1.13
0.95 0.08
1.04 0.08
0.14 0.02
0.86 0.10
1.37 0.35
0.45 0.10
0.19 0.03
0.52 0.06
OAc
OH
—
—
—
Melting points were determined on a Electrothermal IA9100
melting point apparatus and are uncorrected. IR Spectra: Nicolet
Magana-IR 550 infrared spectrophotometer using KBr pellets
a
Values are given in lM and are means of three experiments.

128
F.-S. Chang et al. / Bioorg. Med. Chem. 18 (2010) 124–133
Figure 2. Compound 14i possesses both necrosis- and apoptosis-inducing effects on (A) LNCaP and (B) PC3 cells. LNCaP or PC3 cells were incubated in the presence or
absence of 14i for 24 and 48 h and then the cells were harvested for flow cytometry analysis of Annexin-V staining. The experimental details were described in Section 5.5.
Representative cell distribution by PI and Annexin-V staining were shown (n = 3).
(solid), and reported on wavenumber (cmÀ1). Nuclear magnetic
resonance (1H and 13C) spectra were recorded on a Varian Gemini
200 spectrometer or Varian-Unity-400 spectrometer. Chemical
shifts were expressed in parts per million (d) with tetramethylsil-
ane (TMS) as an internal standard. Thin-layer chromatography
was performed on Silica Gel 60 F-254 plates purchased from E.
Merck and Co. The low resolution EIMS data was collected on a
Bruker APEX II mass spectrometer. The elemental analyses were
performed in the Instrument Center of National Science Council
at National Cheng-Kung University and National Taiwan University
using Heraeus CHN-O Rapid EA, and all values are within 0.4% of
the theoretical compositions.
5.1.1. 8-Methoxy-2-methylquinoline-4(1H)-one (4)
A mixture of o-anisidine (2.46 g, 20 mmol), ethyl acetoacetate
(2.60 g, 20 mmol), acetic acid (0.1 mL) and benzene (150 mL) was
heated at 80 °C for 24 h (TLC monitoring). After cooling, the solvent
was removed in vacuo, and the residue was heated at 250 °C for 1 h
(TLC monitoring) in diphenyl ether (80 mL). After cooling, the sol-
vent was added hexane and the resulting precipitate was collected,

F.-S. Chang et al. / Bioorg. Med. Chem. 18 (2010) 124–133
129
1496, 1338, 1197. 1H NMR (400 Hz, DMSO-d6): 7.44–7.51 (m, 3H,
Ar-H), 7.63 (d, 1H, J = 16.4 Hz, @CH), 7.74–7.84 (m, 3H, Ar-H),
8.02 (m, 1H, Ar-H), 8.16 (d, 1H, J = 16.4 Hz, @CH), 8.26 (m, 2H,
Ar-H) 8.45 (s, 1H, 3-H). 13C NMR (100 MHz, DMSO-d6): 119.84,
123.64, 124.19, 124.75, 125.86, 127.87 (2C), 128.15, 128.75,
129.18 (2C), 129.25, 130.11, 132.79, 135.35, 139.47, 154.64. Anal.
Calcd for C17H12ClNÁ1.7H2OÁ1.0HCl: C, 61.35; H, 4.97; N, 4.21.
Found: C, 61.21; H, 5.03; N, 4.23.
5.1.5. (E)-4-Chloro-8-methoxy-2-styrylquinoline-4-ol
hydrochloride (8)
This compound was obtained from 6 and POCl3 as described for
7 and was crystallized from EtOH in 90% yield. Mp 129–131 °C. IR
(KBr): 1631, 1584, 1550, 1489, 1455, 1405, 1326, 1257, 1201, 1112,
1014. 1H NMR (400 Hz, DMSO-d6): 4.08 (s, 3H, OCH3), 7.42–7.52
(m, 4H, Ar-H), 7.71–7.78 (m, 5H, Ar-H), 8.08 (d, 1H, J = 16.4 Hz,
@CH), 8.49 (s, 1H, 3-H). 13C NMR (100 MHz, DMSO-d6): 56.46,
111.50, 115.25, 119.90, 124.64, 125.68, 127.78 (2C), 129.03,
129.16 (2C), 129.29, 129.88, 135.60, 138.79, 144.47, 153.31,
153.79. Anal. Calcd for C18H14ClNOÁ1.2H2OÁ1.0HCl: C, 61.10; H,
4.96; N, 3.96. Found: C, 61.02; H, 4.91; N, 3.89.
Figure 3. Compound 14i induced apoptosis in PC3 cells as assessed by Western
blot. PC3 cells were seeded onto 10-cm dish, cultured for 12 h and then treated with
0, 0.1, 0.2, 0.4, and 0.6 lM of 14i, respectively, for 24 h. Then the cells were
harvested for Western blot. The experimental details were described in Section 5.6.
Actin was used as a control of constant protein loading of sampling. Representative
immunoblots for PARP, its degradation product and actin loading controls were
shown (n = 3).
washed with hexane, and crystallized from EtOH to give 4 (1.68 g,
45%). Mp 238–239 °C (lit.: 230–232 °C).24 IR (KBr): 3366, 1659,
1635, 1594, 1545, 1518, 1424, 1265, 1198, 1065. 1H NMR
(400 MHz, DMSO-d6): 2.37 (s, 3H, Me), 3.99 (s, 3H, OMe), 5.92 (d,
1H, J = 1.2 Hz, 3-H), 7.20–7.22 (m, 2H, 5- and 7-H), 7.61 (m, 1H,
6-H), 11.01 (s, 1H, OH). 13C NMR (100 MHz, DMSO-d6): 19.52,
56.10, 109.09, 110.98, 116.09, 122.43, 125.47, 130.86, 148.21,
149.58, 176.47. Anal. Calcd for C11H11NO2Á1.0H2O: C, 63.74; H,
6.33; N, 6.75. Found: C, 63.77; H, 6.39; N, 6.79.
5.1.6. (E)-1-[4-(2-Styrylquinolin-4-ylamino)phenyl]ethanone
hydrochloride (9)
A mixture of 7 (0.54 g, 2 mmol), 4-aminoacetophenone (0.27 g,
2 mmol) and EtOH (30 mL) was refluxed for 2 h (TLC monitoring).
The mixture was then cooled and evaporated in vacuo to yield a
yellow residue, treated with H2O (50 mL), and the resulting precip-
itate was filtered and washed with H2O. The crude product was
crystallized from EtOH to give 9 (0.67 g, 93%). Mp 298–300 °C. IR
(KBr): 3311, 1674, 1637, 1585, 1547, 1493, 1442, 1357, 1268,
1183, 1183. 1H NMR (400 Hz, DMSO-d6): 2.64 (s, 3H, CH3), 7.35
(s, 1H, 3-H), 7.44–7.49 (m, 3H, Ar-H), 7.54 (d, 1H, J = 16.4 Hz,
@CH), 7.70–7.79 (m, 5H, Ar-H), 8.02 (m, 1H, 7-H), 8.13–8.18 (m,
3H, Ar-H), 8.36 (d, 1H, J = 8.8 Hz, 5-H), 8.79 (d, 1H, J = 8.4 Hz, 8-
H), 10.98 (br s, 1H, HCl), 14.65 (br s, 1H, NH). 13C NMR
(100 MHz, DMSO-d6): 26.72, 99.64, 112.55, 117.23, 120.17,
123.61, 124.06 (2C), 126.83, 127.97 (2C), 129.11 (2C), 130.02
(2C), 130.42, 134.01, 134.47, 134.85, 138.87, 140.47, 142.15,
150.56, 153.52, 196.87. Anal. Calcd for C25H20N2OÁ1.5H2OÁ1.0HCl:
C, 70.15; H, 5.66; N, 6.54. Found:C, 70.06; H, 5.73; N 6.58.
5.1.2. (E)-2-Styryl-1-4-dihydroquinoline-4-ol (5)
A mixture of 4-hydroxy-2-methylquinoline (0.32 g, 2 mmol),
benzaldehyde (0.85 g, 8 mmol) and acetic anhydride (100 mL) was
heated at 150 °C for 30 h (TLC monitoring). After cooling, the solvent
was removed in vacuo, and the residue was heated at 100 °C for 1 h
(TLC monitoring) in pyridine/water (v/v = 4:1) (100 mL). After cool-
ing, the solvent was removed in vacuo to provide the crude product,
which was purified by FC (MeOH/CH2Cl2 = 1:30) to give 5 (0.23 g,
48%). Mp 220–223 °C (lit.: 279 °C).25 IR (KBr): 3141, 1655, 1555,
1506, 1433, 1393, 1261, 1207, 1071, 1034. 1H NMR (400 Hz,
DMSO-d6): 7.14 (s, 1H, 3-H), 7.43 (d, 1H, J = 16.4 Hz, @CH), 7.46–
7.54 (m, 3H, Ar-H), 7.65 (m, 1H, Ar-H), 7.77 (m, 2H, Ar-H), 7.90–
8.00 (m, 3H, Ar-H), 8.24 (m, 1H, Ar-H). 13C NMR (100 MHz, DMSO-
d6): 104.02, 119.41, 120.42, 121.25, 123.83, 126.27, 128.00 (2C),
129.24 (2C), 129.29, 130.40, 132.92, 133.93, 134.84, 139.55,
171.25. Anal. Calcd for C17H13NOÁ0.2H2O: C, 81.37; H, 5.39; N,
5.58. Found: C, 81.54; H, 5.29; N 5.61.
5.1.7. (E)-1-[4-(8-Methoxy-2-styrylquinolin-4-
ylamino)phenyl]ethanone hydrochloride (10)
This compound was obtained from 8 and 4-aminoacetophenone
as described for 9 and was crystallized from EtOH in 90% yield. Mp
179–181 °C. IR (KBr): 3343, 1677, 1625, 1586, 1550, 1492, 1432,
1266, 1160, 1067. 1H NMR (400 Hz, DMSO-d6): 2.63 (s, 3H, CH3),
4.13 (s, 3H, OCH3), 7.43–7.50 (m, 4H, Ar-H), 7.59 (d, 1H,
J = 8.0 Hz, Ar-H), 7.67–7.75 (m, 5H, Ar-H), 7.89 (d, 1H, J = 16.4 Hz,
@CH), 8.13 (m, 2H, Ar-H), 8.46 (d, 1H, J = 8.4 Hz, Ar-H), 11.00 (br
s, 1H, HCl), 13.35 (br s, 1H, NH). 13C NMR (100 MHz, DMSO-d6):
26.72, 56.87, 97.46, 113.18, 114.86, 118.46, 120.14, 123.74 (2C),
127.30, 127.95 (2C), 129.11 (2C), 129.64, 130.05 (2C), 130.36,
134.32, 135.02, 140.38, 142.26, 149.24, 150.72, 153.01, 196.90.
Anal. Calcd for C26H22N2O2Á1.2H2OÁ1.5HCl: C, 66.31; H, 5.56; N,
5.95. Found: C, 66.48; H, 5.89; N, 5.98.
5.1.3. (E)-8-Methoxy-2-styrylquinoline-4-ol (6)
This compound was obtained from 4 and benzaldehyde as de-
scribed for 5 and was crystallized from EtOH in 55% yield. Mp
238–240 °C. IR (KBr): 3348, 1677, 1626, 1587, 1551, 1492, 1432,
1266, 1064. 1H NMR (400 Hz, DMSO-d6): 4.03 (s, 3H, OCH3), 6.58
(d, 1H, J = 1.2 Hz, 3-H), 7.23–7.26 (m, 2H, 5- and 7-H), 7.36–7.51
(m, 4H, Ar-H), 7.61–7.66 (m, 4H, Ar-H), 10.97 (s, 1H, OH). 13C
NMR (100 MHz, DMSO-d6): 56.17, 104.22, 111.26, 112.50, 112.76,
116.04, 119.33, 122.79, 122.87, 126.27 (2C), 130.75 (2C), 144.68,
147.02, 148.52, 151.66, 176.35. Anal. Calcd for C18H15NO2Á0.3H2O:
C, 76.46; H, 5.57; N, 4.95. Found: C, 76.46; H, 5.44; N, 4.88.
5.1.8. (E)-1-{4-[(E)-2-Styrylquinolin-4-
ylamino]phenyl}ethanone oxime (11a)
5.1.4. (E)-4-Chloro-2-styrylquinoline hydrochloride (7)
A mixture of 9 (0.36 g, 1 mmol), NH2OHÁHCl (0.34 g, 5.0 mmol),
and K2CO3 (0.21 g, 1.5 mmol) in EtOH (30 mL) was stirred at room
temperature for 1 h (TLC monitoring). The mixture was evaporated
under reduced pressure and then H2O (80 mL) was added. The
resulting precipitate was collected, washed with H2O, and crystal-
lized from EtOH to give 11a (0.30 g, 85%). Mp 268–270 °C. IR (KBr):
A mixture of 5 (0.50 g, 2 mmol) and POCl3 (20 mL) was heated
at 80–90 °C for 1 h (TLC monitoring). After cooling, the mixture
was slowly poured into ice water (150 mL). The precipitate was
collected, washed with H2O, and then crystallized from EtOH to
give 7 (0.47 g, 90%). Mp 180–182 °C. IR (KBr): 1655, 1629, 1585,

130
F.-S. Chang et al. / Bioorg. Med. Chem. 18 (2010) 124–133
3253, 1579, 1520, 1430, 1385, 1301, 1273, 1178, 1004. 1H NMR
(400 Hz, DMSO-d6): 2.23 (s, 3H, CH3), 7.17 (s, 1H, 3-H), 7.37–7.60
(m, 5H, Ar-H), 7.70–7.77 (m, 4H, Ar-H), 8.01 (m, 1H, 7-H), 8.22
(d, 1H, J = 16.8 Hz, @CH), 8.42 (d, 1H, J = 8.4 Hz, 5-H), 8.83 (d, 1H,
J = 8.0 Hz, 8-H), 10.98 (s, 1H, NOH), 11.36 (br, 1H, NH). 13C NMR
(100 MHz, DMSO-d6): 11.53, 99.03, 116.81, 120.26, 122.81,
123.59, 124.91 (2C), 126.79, 126.94 (2C), 127.98 (2C), 129.09
(2C), 130.32, 133.88, 134.94, 135.42, 137.71, 138.88, 140.13,
150.22, 152.28, 154.09. Anal. Calcd for C25H21N3OÁ0.4H2O: C,
77.66; H, 5.68; N, 10.87. Found: C, 77.56; H, 5.59; N, 10.63.
(dd, 1H, J = 7.6, 1.2 Hz, 7-H), 7.30 (dd, 1H, J = 8.4, 1.2 Hz, 5-H),
7.32–7.43 (m, 5H, Ar-H), 7.62–7.65 (m, 3H, Ar-H), 7.71 (d, 1H,
J = 16.4 Hz, @CH), 8.11 (d, 1H, J = 8.8 Hz, 4-H). 13C NMR
(100 MHz, DMSO-d6): 110.19, 117.64, 120.29, 127.23 (2C),
127.29, 127.44, 128.02, 128.75, 128.81 (2C), 134.39, 136.32,
136.43, 137.92, 152.00, 153.57. Anal. Calcd for C17H13NOÁ0.2H2O:
C, 80.81; H, 5.42; N, 5.54. Found: C, 80.89; H, 5.36; N, 5.40.
5.1.13. 2-[2-(4-Hydroxyphenyl)vinyl]quinolin-8-ol (14b)
This compound was obtained from 8-hydroxy-2-methylquino-
line and 4-hydroxybenzaldehyde as described for 5 and was crys-
tallized from EtOH in 48% yield. Mp 125–126 °C (lit.: 235 °C).31 IR
(KBr): 3399, 1588, 1562, 1510. 1H NMR (400 MHz, DMSO-d6):
6.89 (m, 2H, Ar-H), 7.15–7.29 (m, 3H, Ar-H), 7.38 (m, 1H, 6-H),
7.53 (m, 2H, Ar-H), 7.59 (d, 1H, J = 8.4 Hz, 3-H), 7.65 (d, 1H,
J = 16.4 Hz, @CH), 8.08 (d, 1H, J = 8.4 Hz, 4-H). 13C NMR
(100 MHz, DMSO-d6): 110.10, 115.82 (2C), 117.70, 120.25, 125.
91, 127.01, 127.29, 128.85 (2C), 129.27, 133.94, 136.32, 137.95,
151.85, 153.94, 156.33. Anal. Calcd for C17H13NO2Á0.1H2O: C,
77.01; H, 5.02; N, 5.28. Found: C, 76.63; H, 5.14; N, 5.18.
5.1.9. (E)-1-{4-[2-(E)-Styrylquinolin-4-
ylamino]phenyl}ethanoneomethyl oxime (11b)
A
mixture of
9
(0.36 g, 1 mmol), NH2OCH3ÁHCl (0.42 g,
5.0 mmol), and K2CO3 (0.21 g, 1.5 mmol) in EtOH (30 mL) was stir-
red at room temperature for 1 h (TLC monitoring). The mixture was
evaporated under reduced pressure and then H2O (80 mL) was
added. The resulting precipitate was collected, washed with H2O,
and crystallized from EtOH to give 11b (0.30 g, 80%). Mp 146–
147 °C. IR (KBr): 3421, 1572, 1534, 1440, 1389, 1321, 1255, 1050.
1H NMR (400 Hz, DMSO-d6): 2.21 (s, 3H, CH3), 3.93 (s, 3H, OMe),
7.26 (s, 1H, 3-H), 7.31–7.51 (m, 6H, Ar-H), 7.63 (m, 1H, Ar-H),
7.69–7.88 (m, 6H, Ar-H), 8.07 (d, 1H, J = 7.6 Hz, 5-H), 8.51 (d, 1H,
J = 8.0 Hz, 8-H), 9.91 (br, 1H, NH). 13C NMR (100 MHz, DMSO-d6):
12.22, 61.64, 100.75, 118.30, 121.72, 122.74 (2C), 125.00, 125.24,
125.52, 127.17 (2C), 127.59 (2C), 128.96 (2C), 129.35, 129.77,
131.74, 132.06, 135.69, 136.10, 140.30, 150.44, 153.06, 153.52.
Anal. Calcd for C26H23N3OÁ0.1H2O: C, 78.98; H, 5.92; N, 10.63.
Found: C, 78.80; H, 5.92; N, 10.54.
5.1.14. 4-[2-(8-Hydroxyquinolin-2-yl)vinyl]benzonitrile (14c)
This compound was obtained from 8-hydroxy-2-methylquino-
line and 4-cyanobenzaldehyde as described for 5 and was crystal-
lized from EtOH in 62% yield. Mp 235–236 °C. IR (KBr): 3347, 2220,
1598, 1555, 1506. 1H NMR (400 MHz, CDCl3): 7.20 (dd, 1H, J = 7.6,
1.2 Hz, 7-H), 7.33 (dd, 1H, J = 8.4, 1.2 Hz, 5-H), 7.40–7.46 (m, 2H,
Ar-H), 7.65 (d, 1H, J = 8.4 Hz, 3-H), 7.67–7.72 (m, 5H, Ar-H), 8.17
(d, 1H, J = 8.4 Hz, 4-H). 13C NMR (100 MHz, CDCl3): 110.42,
111.68, 117.74, 118.79, 120.60, 127.55 (2C), 127.76, 127.92,
131.47, 132.05, 132.59 (2C), 136.74, 138. 00, 140.78, 152.09,
152.41. Anal. Calcd for C18H12N2O: C, 79.39; H, 4.44; N, 10.29.
Found: C, 79.50; H, 4.53; N, 10.22.
5.1.10. (E)-1-{4-[8-Methoxy-(E)-2-styrylquinolin-4-
ylamino]phenyl}ethanone oxime hydrochloride (12a)
This compound was obtained from 10 and NH2OHÁHCl as de-
scribed for 11a and was crystallized from EtOH in 90% yield. Mp
303–304 °C. IR (KBr): 3230, 1611, 1583, 1525, 1460, 1427, 1389,
1312, 1258, 1062. 1H NMR (400 Hz, DMSO-d6): 2.21 (s, 3H, CH3),
4.10 (s, 3H, OCH3), 7.33 (s, 1H, 3-H), 7.39–7.56 (m, 7H, Ar-H),
7.62–7.70 (m, 4H, Ar-H), 7.82 (m, 2H, Ar-H), 8.25 (d, 1H,
J = 8.8 Hz, Ar-H), 10.44 (br s, 1H, NH), 11.30 (s, 1H, NOH). 13C
NMR (100 MHz, DMSO-d6): 11.45, 56.65, 97.55, 112.33, 114.39,
118.45, 122.01, 123.91 (2C), 126.67, 126.91 (2C), 127.77 (2C),
129.04 (2C), 129.92, 130.9, 131.66, 134.61, 135.28, 138.42,
150.39, 151.04, 152.35. Anal. Calcd for C25H21N3OÁ2.4H2OÁ1.0HCl:
C, 65.40; H, 5.88; N, 9.15. Found: C, 65.36; H, 5.69; N, 8.81.
5.1.15. 2-[2-(4-Fluorophenyl)vinyl]quinolin-8-ol (14d)
This compound was obtained from 8-hydroxy-2-methylquino-
line and 4-fluorobenzaldehyde as described for 5 and was crystal-
lized from EtOH in 59% yield. Mp 107–108 °C. IR (KBr): 3398, 2364,
1630, 1594, 1561, 1510. 1H NMR (400 MHz, CDCl3): 7.07–7.13 (m,
2H, Ar-H), 7.18 (dd, 1H, J = 7.6, 1.2 Hz, 7-H), 7.27 (d, 1H, J = 16.0 Hz,
@CH), 7.30 (dd, 1H, J = 8.4, 1.2 Hz, 5-H), 7.40 (dd, 1H, J = 8.4, 7.6 Hz,
6-H), 7.57–7.62 (m, 3H, Ar-H), 7.69 (d, 1H, J = 16.0 Hz, @CH), 8.11
(d, 1H, J = 8.8 Hz, 4-H). 13C NMR (100 MHz, CDCl3): 110.16,
115.86 (2C, J = 21.3 Hz), 117.62, 120.33, 127.31, 127.44, 127.81
(J = 2.3 Hz), 128.85 (2C, J = 8.4 Hz), 130.54 (J = 3.0 Hz), 133.03,
136.44, 137.97, 151.99, 153.39, 162.98 (J = 247.0 Hz). Anal. Calcd
for C17H12FNO: C, 76.97; H, 4.56; N, 5.28. Found: C, 76.99; H,
4.63; N, 5.26.
5.1.11. (E)-1-{4-[8-Methoxy-(E)-2-styrylquinolin-4-
ylamino]phenyl}ethanoneomethyl oxime hydrochloride (12b)
This compound was obtained from 10 and NH2OCH3ÁHCl as de-
scribed for 11b and was crystallized from EtOH in 80% yield. Mp
154–156 °C. IR (KBr): 3362, 1624, 1591, 1554, 1490, 1435, 1278,
1049. 1H NMR (400 Hz, DMSO-d6): 2.24 (s, 3H, CH3), 3.95 (s, 3H,
OCH3), 4.13 (s, 3H, OCH3), 7.34 (s, 1H, 3-H), 7.42–7.46 (m, 3H,
Ar-H), 7.55–7.48 (m, 3H, Ar-H), 7.66–7.71 (m, 4H Ar-H), 7.81 (d,
1H, J = 16.4 Hz, @CH), 7.85 (m, 2H, Ar-H), 8.38 (d, 1H, J = 8.8 Hz,
Ar-H), 10.85 (br s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): 12.22,
56.81, 61.71, 96.72, 113.01, 114.67, 118.12, 120.48, 124.40 (2C),
127.07, 127.28 (2C), 127.90 (2C), 129.07 (2C), 129.79, 130.23,
133.90, 135.06, 138.43, 139.80, 149.34, 150.51, 153.23, 153.44.
Anal. Calcd for C27H25N3O2Á2.0H2OÁ1.0HCl: C, 65.38; H, 6.10; N,
8.47. Found: C, 65.22; H, 6.11; N, 8.41.
5.1.16. 2-[2-(4-Nitrophenyl)vinyl]quinolin-8-ol (14e)
This compound was obtained from 8-hydroxy-2-methylquino-
line and 4-nitrobenzaldehyde as described for 5 and was crystal-
lized from EtOH in 80% yield. Mp 200–201 °C. IR (KBr): 3342,
1591, 1508. 1H NMR (400 MHz, CDCl3): 7.21 (dd, 1H, J = 7.6,
1.2 Hz, 7-H), 7.34 (dd, 1H, J = 8.4, 1.2 Hz, 5-H), 7.43–7.50 (m, 2H,
Ar-H), 7.67 (d, 1H, J = 8.4 Hz, 3-H), 7.75–7.78 (m, 3H, Ar-H), 8.19
(d, 1H, J = 8.4 Hz, 4-H), 8.28 (m, 2H, Ar-H). 13C NMR (100 MHz,
CDCl3): 110.52, 117.76, 120.65, 124.20 (2C), 127.65, 127.82 (2C),
128.05, 131.59, 132.20, 136.82, 138.00, 142.71, 147.43, 152.11,
152.27. Anal. Calcd for C17H12N2O3: C, 69.86; H, 4.14; N, 9.58.
Found: C, 69.89; H, 4.17; N, 9.46.
5.1.12. 2-Styrylquinolin-8-ol (14a)
This compound was obtained from 8-hydroxy-2-methylquino-
line and benzaldehyde as described for 5 and was crystallized from
EtOH in 45% yield. Mp 104–105 °C (lit.: 96–100 °C).26 IR (KBr):
3399, 2362, 1594, 1559, 1506. 1H NMR (400 Hz, DMSO-d6): 7.18
5.1.17. 3-[2-(8-Hydroxyquinolin-2-yl)vinyl]benzene-1,2-diol
(14f)
This compound was obtained from 8-hydroxy-2-methylquino-
line and 2,3-duhydroxybenzaldehyde as described for 5 and was

F.-S. Chang et al. / Bioorg. Med. Chem. 18 (2010) 124–133
131
crystallized from EtOH in 41% yield. Mp 173–174 °C. IR (KBr):
3342, 1600, 1505. 1H NMR (400 Hz, DMSO-d6): 6.68–6.80 (m, 2H,
Ar-H), 7.06–7.12 (m, 2H, Ar-H), 7.32–7.38 (m, 2H, Ar-H), 7.47 (d,
1H, J = 16.4 Hz, @CH), 7.80 (d, 1H, J = 8.8 Hz, 3-H), 8.12 (d, 1H,
J = 16.4 Hz, @CH), 8.26 (d, 1H, J = 8.8 Hz, 4-H), 8.92 (br s, 1H, OH),
9.53 (br s, 2H, OH). 13C NMR (100 MHz, DMSO-d6): 111.27,
115.23, 117.58, 118.16, 119.12, 120.16, 123.67, 126.78, 127.55,
128.14, 130.36, 136.41, 138.20, 144.36, 145.60, 152.87, 154.27.
Anal. Calcd for C17H13NO3Á0.5H2O: C, 70.81; H, 4.90; N, 4.85.
Found: C, 70.46; H, 5.10; N, 4.82.
@CH), 7.66 (m, 1H, Ar-H), 7.78–7.87 (m, 3H, Ar-H), 8.01 (d, 1H,
J = 8.4 Hz, Ar-H), 8.07 (d, 1H, J = 8.8 Hz, Ar-H). 13C NMR
(100 MHz, DMSO-d6): 20.78, 113.12, 114.50, 115.33, 120.43,
121.60, 127.45, 129.05, 130.91, 132.51, 149.15, 151.44, 154.53,
154.79, 154.86, 168.65. Anal. Calcd for C17H12N2O5: C, 62.97; H,
3.73; N, 8.64. Found: C, 62.98; H, 4.12; N, 8.46.
5.1.22. (E)-2-[2-(5-Nitrofuran-2-yl)vinyl]quinolin-4-ol (16)
A mixture of 15 (0.97 g, 3 mmol) was heated at 100 °C for 1 h
(TLC monitoring) in pyridine/water (v/v = 4:1) (50 mL). After cool-
ing, the solvent was removed in vacuo to provide the crude prod-
uct, which was purified by FC (MeOH/CH2Cl2 = 1:20) to give 16
(0.44 g, 52%). Mp 305–307 °C. IR (KBr): 3452, 1626, 1594, 1538,
1508, 1470, 1389, 1352, 1242, 1017. 1H NMR (400 MHz, DMSO-
d6): 6.47 (d, 1H, J = 1.6 Hz, 3-H), 7.15 (d, 1H, J = 3.6 Hz, furanyl-3-
H), 7.25 (d, 1H, J = 16.4 Hz, @CH), 7.32 (m, 1H, Ar-H), 7.61–7.70
(m, 3H, Ar-H), 7.81 (d, 1H, J = 4.0 Hz, furanyl-4-H), 8.05 (dd, 1H,
J = 8.0, 1.2 Hz, 5-H), 11.69 (br s, 1H, 4-OH). 13C NMR (100 MHz,
DMSO-d6): 107.93, 114.75, 115.22, 118.34, 120.79, 123.37,
124.79, 125.31, 126.49, 132.22, 140.17, 145.25, 151.64, 153.88,
177.07. Anal. Calcd for C15H10N2O4: C, 63.83; H, 3.57; N, 9.92.
Found: C, 63.47; H, 3.69; N, 9.73.
5.1.18. 2-(2-Pyridin-3-ylvinyl)quinolin-8-ol (14g)
This compound was obtained from 8-hydroxy-2-methylquino-
line and pyridine-3-carboxaldehyde as described for 5 and was
crystallized from EtOH in 51% yield. Mp 138–139 °C. IR (KBr):
3041, 2363, 1721, 1562, 1502. 1H NMR (400 MHz, CDCl3): 7.18
(dd, 1H, J = 7.2, 1.2 Hz, 7-H), 7.32 (dd, 1H, J = 8.4, 1.2 Hz, 5-H),
7.33–7.44 (m, 3H, Ar-H), 7.63 (d, 1H, J = 8.8 Hz, 3-H), 7.69 (d, 1H,
J = 16.4 Hz, @CH), 7.95 (m, 1H, pyridinyl-H), 8.16 (d, 1H,
J = 8.8 Hz, 4-H), 8.57 (dd, 1H, J = 4.8, 1.6 Hz, pyridinyl-H), 8.84 (d,
1H, J = 2.4 Hz, pyridinyl-H). 13C NMR (100 MHz, CDCl3): 110.33,
117.69, 120.39, 123.69, 127.66 (2C), 130.06, 130.40, 132.08,
133.34, 136.62, 137.98, 149.07, 149.48, 152.08, 152.76. Anal. Calcd
for C16H12N2O: C, 77.40; H, 4.87; N, 11.28. Found: C, 77.12; H, 4.90;
N, 11.15.
5.1.23. (E)-4-Chloro-2-[2-(5-nitrofuran-2-yl)vinyl]quinoline (17)
A mixture of 16 (1.41 g, 5 mmol), POCl3 (100 mL) was heated at
80–90 °C for 1 h (TLC monitoring). After cooling, the mixture was
slowly poured into ice water (200 mL). The precipitate was col-
lected, washed with H2O, and then crystallized from EtOH to give
17 (1.42 g, 95%). Mp 206–207 °C (lit.: 205 °C).33 IR (KBr): 1637,
1581, 1515, 1486, 1450, 1391, 1350, 1332. 1249, 1041. 1H NMR
(400 MHz, DMSO-d6): 7.24 (d, 1H, J = 4.0 Hz, furanyl-3-H), 7.57
(d, 1H, J = 16.4 Hz, @CH), 7.74–7.92 (m, 4H, Ar-H), 8.07 (d, 1H,
J = 8.4 Hz, 8-H), 8.18 (d, 1H, J = 8.4 Hz, 5-H), 8.22 (s, 1H, 3-H). 13C
NMR (100 MHz, DMSO-d6): 114.73, 115.31, 120.90, 120.93,
123.63, 124.96, 128.39, 129.50, 131.37, 131.77, 141.89, 148.29,
151.50, 154.16, 154.70. Anal. Calcd for C15H9ClN2O3: C, 59.92; H,
3.02; N, 9.32. Found: C, 59.58; H, 3.40; N, 9.23.
5.1.19. 2-(2-Furan-2-ylvinyl)quinolin-8-ol (14h)
This compound was obtained from 8-hydroxy-2-methylquino-
line and furfural as described for 5 and was crystallized from EtOH
in 63% yield. Mp 173–174 °C. IR (KBr): 3336, 1736, 1641, 1591,
1552, 1504. 1H NMR (400 MHz, CDCl3): 6.48 (dd, 1H, J = 3.2,
1.6 Hz, furanyl-H), 6.55 (d, 1H, J = 3.6 Hz, furanyl-H), 7.15 (dd,
1H, J = 7.2, 1.6 Hz, 7-H), 7.22 (d, 1H, J = 16.0 Hz, @CH), 7.27 (dd,
1H, J = 8.0, 1.2 Hz, 5-H), 7.38 (dd, 1H, J = 8.0, 7.2 Hz, 6-H), 7.48 (d,
1H, J = 1.6 Hz, furanyl-H), 7.51 (d, 1H, J = 8.8 Hz, 3-H), 7.55 (d, 1H,
J = 16.0 Hz, @CH), 8.07 (d, 1H, J = 8.8 Hz, 4-H). 13C NMR
(100 MHz, CDCl3): 110.06, 111.43, 112.06, 117.63, 120.85, 121.53,
125.88, 127.15, 127.38, 136.36, 138.00, 143.28, 151.92, 152.62,
153.21. ESIMS (m/z): 238 [M+H]+. Anal. Calcd for C15H11NO2: C,
75.94; H, 4.67; N, 5.90. Found: C, 75.62; H, 4.70; N, 5.78.
5.1.24. (E)-1-{4-{2-[2-(5-Nitrofuran-2-yl)vinyl]quinolin-4-
ylamino}phenyl}ethanone hydrochloride (18)
A mixture of 17 (0.60 g, 2 mmol) and 4-aminoacetophenone
(0.27 g, 2 mmol) in EtOH/HCl (30:1 mL) was refluxed for 2 h (TLC
monitoring). The mixture was then cooled and evaporated in vacuo
to yield a yellow residue, treated with H2O (50 mL), and the result-
ing precipitate was filtered and washed with H2O. The crude prod-
uct was crystallized from EtOH to give 17a (0.64 g, 80%). Mp 275–
276 °C. IR (KBr): 3432, 1675, 1587, 1548, 1518, 1464, 1357, 1274,
1244, 1197, 1026. 1H NMR (400 MHz, DMSO-d6): 2.60 (s, 3H,
Me), 7.46 (s, 1H, 3-H), 7.51 (d, 1H, J = 16.0 Hz, @CH), 7.55 (d, 1H,
J = 4.0 Hz, furanyl-3-H), 7.59 (m, 2H, Ar-H), 7.68 (m, 1H, Ar-H),
7.90 (m, 1H, Ar-H), 8.01 (m, 2H, Ar-H), 8.08 (m, 2H, Ar-H), 8.12
(d, 1H, J = 4.0 Hz, furanyl-4-H), 8.48 (m, 1H, Ar-H), 10.12 (br s,
1H, NH). 13C NMR (100 MHz, DMSO-d6): 26.60, 102.69, 115.22,
115.56, 118.77, 121.57, 122.87 (2C), 124.89, 126.35, 127.90,
128.87, 129.18, 130.14 (2C), 130.96, 132.34, 132.74, 143.97,
148.21, 150.25, 151.45, 196.56. ESIMS (m/z): 400 [M+H]+. Anal.
Calcd for C23H17N3O4Á1.0HCl: C, 63.38; H, 4.16; N, 9.64. Found: C,
63.58; H, 4.25; N, 9.39.
5.1.20. 2-[2-(5-Nitrofuran-2-yl)vinyl]quinolin-8-ol (14i)
This compound was obtained from 8-hydroxy-2-methylquino-
line and 5-nitro-2-furaldehyde diacetate as described for 5 and
was crystallized from EtOH in 51% yield. Mp 179–180 °C (lit.:
192 °C).32 IR (KBr): 3423, 2363, 1554, 1503. 1H NMR (400 MHz,
CDCl3): 6.72 (d, 1H, J = 4.0 Hz, furanyl-H), 7.20 (dd, 1H, J = 8.0,
1.2 Hz, 7-H), 7.33 (dd, 1H, J = 8.0, 1.2 Hz, 5-H), 7.40 (d, 1H,
J = 3.6 Hz, furanyl-H), 7.45 (dd, 1H, J = 8.0, 8.0 Hz, 6-H), 7.52–7.61
(m, 3H, Ar-H), 8.16 (d, 1H, J = 8.4 Hz, 4-H). 13C NMR (100 MHz,
CDCl3): 110.52, 112.68, 113.84, 117.80, 119.00, 121.56, 128.03,
128.26, 132.60, 136.92 (2C), 138.11, 151.25, 152.11, 154.91. ESIMS
(m/z): 283 [M+H]+. Anal. Calcd for C15H10N2O4: C, 63.83; H, 3.57; N,
9.92. Found: C, 63.58; H, 3.70; N, 9.83.
5.1.21. (E)-2-[2-(5-Nitrofuran-2-yl)vinyl]quinolin-4-yl acetate
(15)
A mixture of 2-methylquinoline (0.72 g, 5 mmol), (5-nitrofuran-
2-yl)methylene diacetate (4.87 g, 20 mmol) and acetic anhydride
(150 mL) was heated at 150 °C for 30 h (TLC monitoring). After
cooling, the solvent was removed in vacuo to provide the crude
product, which was purified by flash column chromatography
(FC, silica gel use CH2Cl2 as eluent) to give 15 (0.64 g, 45%). Mp
>320 °C. IR (KBr): 1770, 1594, 1567, 1540, 1508, 1469, 1354,
1238, 1168, 1069. 1H NMR (400 MHz, DMSO-d6): 2.53 (s, 3H,
CH3), 7.23 (d, 1H, J = 4.0 Hz, furanyl-3-H), 7.57 (d, 1H, J = 16.4 Hz,
5.1.25. (E)-1-{4-{2-[(E)-2-(5-Nitrofuran-2-yl)vinyl]quinolin-4-
ylamino}phenyl}ethanone oxime hydrochloride (19a)
A
mixture of 18 (0.40 g, 1 mmol), NH2OHÁHCl (0.34 g,
5.0 mmol), and K2CO3 (0.21 g, 1.5 mmol) in EtOH (30 mL) was stir-
red at room temperature for 1 h (TLC monitoring). The mixture was
evaporated under reduced pressure and then H2O (80 mL) was
added. The resulting precipitate was collected, washed with H2O,

132
F.-S. Chang et al. / Bioorg. Med. Chem. 18 (2010) 124–133
and crystallized from EtOH to give 19a (0.35 g, 85%). Mp 289–
290 °C. IR (KBr): 3255, 1628, 1589, 1529, 1474, 1441, 1375, 1341,
1244, 1014. 1H NMR (400 MHz, DMSO-d6): 2.21 (s, 3H, Me), 7.22
(d, 1H, J = 3.2 Hz, furanyl-3-H), 7.29 (s, 1H, 3-H), 7.48 (d, 1H,
J = 16.4 Hz, @CH), 7.50 (m, 2H, Ar-H), 7.72–8.00 (m, 6H, Ar-H),
8.11 (d, 1H, J = 8.4 Hz, 5-H), 8.67 (d, 1H, J = 8.4 Hz, 8-H), 10.60 (br
s, 1H, NH), 11.32 (s, 1H, NOH). 13C NMR (100 MHz, DMSO-d6):
11.48, 99.63, 100.87, 115.09 (2C), 115.42, 116.23, 117.41, 121.64,
123.22, 124.32, 126.19, 126.86, 127.01 (3C), 133.69, 135.04,
138.06, 149.32, 151.92, 152.37, 153.42. ESIMS (m/z): 415 [M+H]+.
Anal. Calcd for C23H18N4O4Á0.2H2OÁ1.0HCl: C, 60.79; H, 4.30; N,
12.33. Found: C, 60.86; H, 4.59; N, 12.20.
14i for 48 and 24 h, respectively. The cells were harvested by tryp-
sinization, centrifuged, washed with PBS and collected by centrifu-
gation. The cells were fixed with ice-cold 70% ethanol for 30 min,
washed with PBS and centrifuged to remove supernatant. The cells
were re-suspended by PBS containing contain 0.05% Triton X-100
and RNAase A (40
dium iodide (PI) was added into the cell suspension to final con-
centration 50 g/mL for another 1 h incubation. The cells were
lg/mL) and incubated at 37 °C for 1 h and propi-
l
harvested by centrifugation, washed with PBS and centrifuged to
remove supernatant. Finally, the cells were re-suspended by PBS
and analyzed by Coulter EPICS XL Flow Cytometry (Beckman Coul-
ter) with Win cycle software (Beckman Coulter).
5.1.26. (E)-1-{4-{2-[(E)-2-(5-Nitrofuran-2-yl)vinyl]quinolin-4-
ylamino}phenyl}ethanone O-methyl oxime hydrochloride (19b)
5.5. Flow cytometric analysis of Annexin-V staining
A
mixture of 18 (0.40 g, 1 mmol), NH2OCH3ÁHCl (0.41 g,
About 5 Â 105 cells of LNCaP and PC3 were seeded onto 10-cm
5.0 mmol), and K2CO3 (0.21 g, 1.5 mmol) in EtOH (30 mL) was stir-
red at room temperature for 1 h (TLC monitoring). The mixture was
evaporated under reduced pressure and then H2O (80 mL) was
added. The resulting precipitate was collected, washed with H2O,
and crystallized from EtOH to give 19b (0.34 g, 80%). Mp 230–
231 °C. IR (KBr): 3384, 1633, 1592, 1547, 1521, 1473, 1448, 1355,
1246, 1055. 1H NMR (400 MHz, DMSO-d6): 2.20 (s, 3H, Me), 3.92
(s, 3H, OMe), 7.12 (d, 1H, J = 3.6 Hz, furanyl-3-H), 7.23 (s, 1H, 3-
H), 7.34 (d, 1H, J = 16.4 Hz, @CH), 7.51 (d, 2H, J = 8.8 Hz, Ar-H),
7.65–7.72 (m, 3H, Ar-H), 7.79 (d, 2H, J = 8.8 Hz), 7.89–7.97 (m,
2H, Ar-H), 8.52 (d, 1H, J = 8.4 Hz, 8-H), 10.42 (s, 1H, NH). 13C
NMR (100 MHz, DMSO-d6): 12.67, 62.11, 99.95, 115.45, 116.53,
117.86, 122.51, 123.27, 124.07, 124.35 (2C), 126.27, 127.29,
127.76 (2C), 133.90, 134.00, 139.10, 141.14, 149.98, 152.15,
153.10, 153.79, 154.06. ESIMS (m/z): 429 [M+H]+. Anal. Calcd for
C24H20N4O4Á0.6H2OÁ1.0HCl: C, 60.60; H, 4.70; N, 11.78. Found: C,
60.70; H, 5.04; N, 11.77.
dish, cultured for 12 h and then treated with 0.5 lM and 0.2 lM of
14i, respectively, for 24 and 48 h. The cell media were removed
into a centrifugation tube. The attached cells were washed by
PBS and harvested by trypsinization. The washed PBS and har-
vested cells were collected into the above centrifugation tube,
which contained the cell media. The tubes were centrifuged to re-
move supernatant and the cell pellets were washed by PBS twice.
After centrifugation, the cell pellets were re-suspended with
200
Annexin-V (1
room temperature for 15 min. Finally Annexin-V buffer were
added into this cell suspension to final 1000 L and analyzed by
lL Annexin-V binding buffer (Strong Biotech), which contain
l
g/mL) and PI (2.5 g/mL), and then incubated at
l
l
Coulter EPICS XL Flow Cytometry (Beckman Coulter) with XL SYS-
TEM II software (Beckman Coulter).
5.6. Analysis of the cleavage of poly (ADP-ribose) polymerase
(PARP) in PC3 cells by Western blot
About 5 Â 105 cells of PC3 were seeded onto 10-cm dish, cul-
5.2. Cell Growth Inhibitory Assay
tured for 12 h and then treated with 0, 0.1, 0.2, 0.4 and 0.6 lM of
Human breast carcinoma MCF-7 cells, nonsmall cell lung carci-
noma H460 cells, and human glioma (SF-268) cells were main-
tained in RPMI-1640 medium supplied with 5% fetal bovine
serum. Cell in logarithmic phase were cultured at a density of
5000 cells/mL/well in a 24-well plate. The cells were exposed in
14i, respectively, for 24 h. Then the cells were subjected to sonifi-
cation and centrifuged to remove the cell debris, and the superna-
tants were collected. Protein concentration was determined by a
BCA Protein Assay Kit (PIERCE). About 60 lg of protein/per well
were resolved in NuPAGE Novex Tris–Acetate Mini Gel (Invitro-
gen). After electrophoresis, the proteins were transferred to a
nitrocellulose membrane. The transferred membranes were
blocked in 5% (w/v) nonfat dry milk in TBST (0.5 M NaCl, 20 mM
Tris–HCl, 0.05% (v/v) Tween 20, pH 7.4) and probed for the respec-
tive anti-PARP antibody (Santa Crutz) and anti-actin antibody
(Santa Crutz), followed by incubation with a secondary antibody
containing horseradish peroxidase (anti-mouse, anti-goat; Jackson
ImmunoResearch) and visualization with a SuperSignal West Pico
Rabbit IgG Detection kit (PIERCE) by the ChemiDoc XRS system
(Bio-Rad).
4.0 lg/mL of the tested drugs for 72 h. The MTS (3-(4,5-dimethyl-
thiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-
tetrazolium, inner salt) assay was used to evaluate the effect of the
tested compounds on cell growth as described previously.34
5.3. Measurements of IC50 in LNCAP and PC3 cells
LNCaP and PC3 cells were obtained from the American Type
Culture Collection. These cells were maintained in RPMI-1640 sup-
plemented with 10% nonheat inactivated fetal bovine serum (FBS)
(Invitrogen). Exponentially growing cells (5 Â 103 cells) were pla-
ted in 96-well plates and incubated with various concentrations
of compounds through a series of 2Â fold-dilution from 10 to
Acknowledgments
0.02 lM. Each concentration was tetra-plicate. Incubation was car-
Financial support of this work by the National Science Council
of the Republic of China is gratefully acknowledged. We also thank
National Cancer Institute (NCI) of the United States for the antican-
cer screenings and the National Center for High-Performance Com-
puting for providing computer resources and chemical database
services.
ried out at 37 °C for 48 h. ATPLite assay reagents (Perkin-Elmer)
were added and luminescence measured by TopCounter (Perkin-
Elmer). The concentration that killed 50% of cells (IC50) was deter-
mined from the curve by calculating the concentration of agent
that reduced the readout of luciferase activity in treated cells, com-
pared to control cells by SigmaPlot (SYSTAT).
References and notes
5.4. Flow cytometric analysis of cell cycle
About 5 Â 105 cells of LNCaP and PC3 were seeded onto 10-cm
1. Chen, I. L.; Chen, Y. L.; Tzeng, C. C.; Chen, I. S. Helv. Chim. Acta 2002, 85, 2214.
2. Chen, I. L.; Chen, Y. L.; Tzeng, C. C. Chin. Pharm. J. 2003, 55, 49.
dish, cultured for 12 h and then treated with 0.5 lM and 0.2 lM of

F.-S. Chang et al. / Bioorg. Med. Chem. 18 (2010) 124–133
133
3. Zhao, Y. L.; Chen, Y. L.; Tzeng, C. C.; Chen, I. L.; Wang, T. C.; Han, C. H. Chem.
Biodiv. 2005, 2, 205.
19. Jiang, J. B.; Hesson, D. P.; Dusak, B. A.; Dexter, D. L.; Kang, G. J.; Hamel, E. J. Med.
Chem. 1990, 33, 1721.
4. Huang, Y. T.; Huang, D. M.; Guh, J. H.; Chen, I. L.; Tzeng, C. C.; Teng, C. M. J. Biol.
Chem. 2005, 280, 2771.
20. Mekouar, K.; Mouscadet, J. F.; Desmaele, D.; Subra, F.; Leh, H.; Savoure, D.;
Auclair, C.; d’Angelo, J. J. Med. Chem. 1998, 41, 2846.
5. Chen, Y. L.; Chen, I. L.; Wang, T. C.; Han, C. H.; Tzeng, C. C. Eur. J. Med. Chem.
2005, 40, 928.
6. Chen, Y. L.; Lin, H. C.; Yang, C. N.; Lu, P. J.; Tzeng, C. C. Chem. Biodiv. 2008, 4, 267.
7. Ramesh, R. D.; Manian, R. S.; Raghunathan, R.; Sainath, S.; Raghunathan, M.
Bioorg. Med. Chem. 2009, 17, 660.
8. Vazquez, M. T.; Romero, M.; Pujol, M. D. Bioorg. Med. Chem. 2004, 12, 949.
9. Musiol, R.; Jampilek, J.; Kralova, K.; Richardson, D. R.; Kalinowski, D.; Podeszwa,
B.; Finster, J.; Niedbala, H.; Palka, A.; Polanski, J. Bioorg. Med. Chem. 2007, 15,
1280.
21. Zouhiri, F.; Danet, M.; Bernard, C.; Normand-Bayle, M.; Mouscadet, J. F.; Leh, H.;
Thomas, C. M.; Mbemba, G.; d’Angelo, J.; Desmaele, D. Tetrahedron Lett. 2005,
46, 2201.
22. Pommier, Y.; Johnson, A. A.; Marchand, C. Nat. Rev. Drug Disc. 2005, 4, 236.
23. Polanski, J.; Zouhiri, F.; Jeanson, L.; Desmaele, D.; d’Angelo, J.; Mouscadet, J.;
Gieleciak, R.; Gasteiger, J.; Bret, M. L. J. Med. Chem. 2002, 45, 4647.
24. Sawada, Y.; Kayakiri, H.; Abe, Y.; Imai, K.; Mizutani, T.; Inamura, N.; Asano, M.;
Aramori, I.; Hatori, C.; Katayama, A.; Oku, T.; Tanaka, H. J. Med. Chem. 2004, 47,
1617.
10. Musiol, R.; Jampilek, J.; Buchta, V.; Silva, L.; Niedbala, H.; Podeszwa, B.; Palka,
A.; Majerz-Maniecka, K.; Oleksyn, B.; Polanski, J. Bioorg. Med. Chem. 2006, 14,
3592.
11. Righi, G.; Ciambrone, S.; Bonini, C.; Campaner, P. Bioorg. Med. Chem. 2008, 16,
902.
12. Zhao, Y. L.; Chen, Y. L.; Chang, F. S.; Tzeng, C. C. Eur. J. Med. Chem. 2005, 40, 792.
13. Chen, Y. L.; Huang, C. J.; Huang, Z. Y.; Tseng, C. H.; Chang, F. S.; Yang, S. H.; Lin, S.
R.; Tzeng, C. C. Bioorg. Med. Chem. 2006, 14, 3098.
25. Nakajima, J. Jpn. Biochem. Soc. 1957, 29, 129.
26. Silverstein, R. M.; Webster, F. X. In Spectrometric Identification of Organic
Compounds, 7th ed.; Rose, N., Ed.; John Wiley and Sons: New York, pp 227–228.
27. Ghosh, A. K.; Steele, R.; Ray, R. B. J. Biol. Chem. 2006, 281, 23652.
28. Horoszewicz, J. S.; Leong, S. S.; Kawinski, E.; Karr, J. P.; Rosenthal, H.; Chu, T. M.;
Mirand, E. A.; Murphy, G. P. Cancer Res. 1983, 43, 1809.
29. Pigault, C.; Follenius-Wund, A.; Schmutz, M.; Freyssinet, J. M.; Brisson, A. J. Mol.
Biol. 1994, 236, 199.
14. Chen, Y. L.; Zhao, Y. L.; Lu, C. M.; Tzeng, C. C.; Wang, J. P. Bioorg. Med. Chem.
2006, 14, 4373.
30. Koopman, G.; Reutelingsperger, C. P.; Kuijten, G. A.; Keehnen, R. M.; Pals, S. T.;
van Oers, M. H. Blood 1994, 84, 1415.
15. Cheng, C. C. Med. Hypothesis 1986, 20, 157.
31. Phillips, J. P.; Breese, R.; Barrall, E. M. J. Org. Chem. 1959, 24, 1104.
32. Ujiie, T. Chem. Pharm. Bull. 1966, 14, 461.
33. Giller, S. A.; Lidak, M. Y.; Sukhova, N. M.; Venter, K. K. Chem. Heterocycl. Compd.
1976, 12, 217.
16. Atwell, G. J.; Baguley, B. C.; Denny, W. A. J. Med. Chem. 1989, 32, 396.
17. Kuo, S. C.; Lee, H. Z.; Juang, J. P.; Lin, Y. T.; Wu, T. S.; Chang, J. J.; Lednicer, D.;
Paull, K. D.; Lin, C. M.; Hamel, E.; Lee, K. H. J. Med. Chem. 1993, 36, 1146.
18. Xia, Y.; Yang, Z. Y.; Xia, P.; Bastow, K. F.; Tachibana, Y.; Kuo, S. C.; Hamel, E.;
Hackl, T.; Lee, K. H. J. Med. Chem. 1998, 41, 1155.
34. Finlay, G. J.; Baguley, B. C.; Wilson, W. R. Anal. Biochem. 1984, 139, 272.