Synthesis of 1-benzyl-3-(5-hydroxymethyl-2-furyl)selenolo[3,2-c]pyrazole derivatives as new anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2009.12.039

As part of our continuing search for potential anticancer drug candidates among YC-1 analogs, 1, 3-disubstituted selenolo[3,2-c]pyrazole derivatives were synthesized and evaluated for their cytotoxicity against NCI-H226 non-small cell lung cancer and A-498 renal cancer cell lines. Significant cytotoxicity was observed in 3-(5-hydroxymethyl-2-furyl) derivatives (2, 33, 36 and 37). Among them, compound 2 was found to have the most potent activity. The mode of action of compound 2 seems to differ from those of the 175 anticancer agents listed in NCI's standard database and resembles that of YC-1. Thus, we recommend that compound 2 should be developed further as new drug candidate for treatment of non-small cell lung cancer and renal cancer.

Full text of DOI:10.1016/j.ejmech.2009.12.039

European Journal of Medicinal Chemistry 45 (2010) 1395–1402
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of 1-benzyl-3-(5-hydroxymethyl-2-furyl)selenolo[3,2-c]pyrazole
derivatives as new anticancer agents
Li-Chen Chou a, Li-Jiau Huang a, Mei-Hua Hsu a, Mei-Chi Fang a, Jai-Sing Yang b, Shi-Hong Zhuang a,
,
Hui-Yi Lin a, Fang-Yu Lee c, Che-Ming Teng d, Sheng-Chu Kuo a
*
a Graduate Institute of Pharmaceutical Chemistry, China Medical University, 91 Shueh Shih Road, North Section, Taichung 40402, Taiwan
b Departments of Pharmacology, China Medical University, 91 Shueh Shih Road, North Section, Taichung 40402, Taiwan
c Yung-Shin Pharmaceutical Industry Co., Ltd. No. 1191, Sec. 1, Chung Shan Rd., Tachia, Taichung, Taiwan
d Pharmacological Institute, College of Medicine, National Taiwan University, No. 1, Jen-Ai Road, Sec. 1, Taipei, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
As part of our continuing search for potential anticancer drug candidates among YC-1 analogs, 1,
3-disubstituted selenolo[3,2-c]pyrazole derivatives were synthesized and evaluated for their cytotoxicity
against NCI-H226 non-small cell lung cancer and A-498 renal cancer cell lines. Significant cytotoxicity
was observed in 3-(5-hydroxymethyl-2-furyl) derivatives (2, 33, 36 and 37). Among them, compound 2
was found to have the most potent activity. The mode of action of compound 2 seems to differ from those
of the 175 anticancer agents listed in NCI’s standard database and resembles that of YC-1. Thus, we
recommend that compound 2 should be developed further as new drug candidate for treatment of
non-small cell lung cancer and renal cancer.
Received 10 February 2009
Received in revised form
7 December 2009
Accepted 18 December 2009
Available online 4 January 2010
Keywords:
Selenolopyrazole
Thienopyrazole
Furopyrazole
Ó 2009 Elsevier Masson SAS. All rights reserved.
YC-1 analogs
Structure–activity relationships (SAR)
Cytotoxicity
1. Introduction
During our recent study, we found that the furo[3,2-c]pyrazole
isostere of YC-1 (1)(Fig. 1) exhibited greater cytotoxicity than YC-1
1-Benzyl-3-(5-hydroxymethyl-2-furyl)indazole (YC-1)(Fig. 1) is
an unigure activator of soluble quanyl cyclase (sGC) that not only
activates sGC in NO-independent manner, but also potentiates the
NO- and CO-dependent activation of sSC [1–3]. Through sGC/cGMP
pathway, YC-1 has demonstrated its multiple pharmacological
actions including antiplatelet, vascular relaxation, neuroprotection,
and so on [4]. Besides its role in these cGMP related pharmaco-
logical actions, YC-1 has also being studied for its high potential as
a new anticancer drug candidate. According to the literatures [5–9],
the anticancer effect of YC-1 may be attributable to its multiple
action including anti-angiogenesis, anti-inflammation, apoptosis-
induction and inhibition of matrix metalloproteinases (MMPs).
Results from animal studies revealed that the oral dosing of YC-1
exhibited excellent anti-tumor activity against non-small cell lung
cancer, hepatoma, prostate cancer, renal and breast cancers.
against HL-60 cells [10]. However, its action mechanism seemed to
differ from that of YC-1. This intrigued us into investigating the
anticancer activity of various YC-1 isosteres. Meanwhile, from the
chemical structure point of view, the core structure of YC-1 is an
indazole, and its furo[3,2-c]pyrazole isostere raised its cytotoxicity
considerably by introducing an oxygen heteroatom into it. There-
fore, we expected that the systematic introduction of other hetero-
atom from the same periodical group, namely the sulfur and the
selenium atoms, which yield thieno[3,2-c]pyrazole and sele-
nolo[3,2-c]pyrazole isosteres of YC-1, might also result in differ
physiochemical properties and biological activities. Since, the
synthesis and biological activities of both the furo[3,2-c]pyrazole
[10] and thieno[3,2-c]pyrazole analogs [11] of YC-1 were already
reported by us, this study focused on the selenolo[3,2-c]
pyrazole analogs of YC-1 having new heterocyclic core structure. At
first,1-benzyl-3-(5-hydroxymethyl-2-furyl)selenolo[3,2-c]pyrazole
(2)(Fig. 1), an isostere of YC-1, was prepared. And, during its evalu-
ation for cytotoxicity against a panel of 60 NCI human cancer cell
lines, compound 2 was found to demonstrate selective inhibition
toward NCI-H226 non-small cell lung cancer and A-498 renal cancer
* Corresponding author. Tel.: þ86 886
4
22053366x5608; fax: þ86 886
4
22030760.
E-mail address: sckuo@mail.cmu.edu.tw (S.-C. Kuo).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.12.039

1396
L.-C. Chou et al. / European Journal of Medicinal Chemistry 45 (2010) 1395–1402
hydrolyzed with NaOH into their corresponding carboxylic acid
derivatives (38–41).
OH
OH
OH
O
O
O
O
Se
3. Results and discussion
H3C
N
N
N
N
N
N
3.1. Cytotoxicity of compound 2 against human cancer cell line
panel
(1)
(2)
YC-1
Compound 2 was evaluated for cytotoxicity with a panel of NCI
human cancer cell lines [13,14]. As shown by the data in Fig. 2, its
mean graph midpoint (MID) values for log GI50, log TGI, log LC50
were ꢀ4.68, ꢀ4.12 and ꢀ4.06, respectively, indicating its poor
cytotoxicity toward human cancer cell lines. Nevertheless,
compound 2 demonstrated selective inhibition against NCI-H226
and A-498 human cancer cell lines. As comparison, its log GI50, log
TGI and log LC50 values against NCI-H226 non-small cell lung
cancer cells were ꢀ6.60, ꢀ6.01 and ꢀ5.19, respectively, indicating
about 20–100 times more potent than the MID values. Similarly, its
log GI50, log TGI, log LC50 values against A-498 renal cancer cells
were ꢀ6.79, ꢀ6.49 and ꢀ6.20 respectively, that were more than 100
times the values at the MID. We further utilized NCI’s COMPARE
Fig. 1. Structures of YC-1, 1-benzyl-3-(5-hydroxymethyl-2-furyl)-5-methylfuro[3,
2-c]pyrazole (1) and 1-benzyl-3-(5-hydroxymethyl-2-furyl)selenolo[3,2-c] pyrazole (2).
cell lines. Therefore, we synthesized a series of its analogs and
evaluated for their cytotoxicty against NCI-H226 and A-498 cancer
cell lines in this study.
2. Chemistry
The synthetic methods for target compounds 2 and 27–41 were
illustrated in Scheme 1. For the preparation of intermediates 10 and
11, the starting compounds 3 and 4 were prepared by published
methods [12] and were then treated with SOCl2 in CH2Cl2, to yield
the corresponding acid chlorides (5, 6) that were subjected to
Friedel–Craft’s reaction with 5-methyl furoate (8) to give the cor-
responding ketones (10, 11). Since the method for structural
confirmation of products 10 and 11 was similar, only that of the
latter was described here in detail. First, the molecular formula of
compound 11 was determined to be C12H10O4Se based on
elemental analysis and the detection of a molecular ion peak in
mass spectrum at m/z 298. Furthermore, its 1H NMR spectrum
program to compare the cancer-type-specific Fingerprint (log GI50
)
of compound 2 with NCI’s standard agent database which is con-
sisted of 175 drugs with cancer treatment New Drug Application
(NDA) or Investigational New Drug Application (IND) as well as
compounds that had reached a particular high stage of interest at
the NCI in recent years for the list. The result in Table 1, indicated
that the correlation coefficient is lower than 0.5 suggesting that the
mode of action of compound 2 might differ significantly from most
of the 175 drugs in the NCI database. On the other hand, the
fingerprint of compound 2 established in US NCI seemed to have
great degree of similarity with the fingerprint of YC-1 established in
the Japanese Foundation for Cancer Research (JFCR) [9]. Although
the panel of cancer cell line used in JFCR was somewhat different.
displayed two signals at
group of OCH3 and CH3, respectively. Two mutually coupled signals
at 7.30 (J ¼ 3.6 Hz) were assigned to the H-3
7.22 (J ¼ 3.6 Hz) and
and H-4 of the furan ring, and another signal at
7.06 (J ¼ 4.0,
1.0 Hz) indicated the coupling between H-3 and C-5–CH3, whereas
the signal at
d 3.92 and 2.60 which correlated with the
d
d
d
d
8.38 (J ¼ 4.0 Hz) were attributed to the H-3 of the
3.2. Cytotoxicity of 1, 3-disubstituted selenolo[3,2-c]pyrazoles (2,
27–41)
selenophene. Overall, the spectral and elemental analysis together
confirmed the structure of compound 11. Similary, compound 12
was prepared by Friedel–Crafts acylation of starting compound 7
with 4-methoxycarbonyl benzoyl choride (9).
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay was used to determine the cytotoxicity of target
compounds (2, 27–41) against NCI-H226 and A-498 cell lines, and
the result was summarized in Table 2.
As shown in Table 2, among the selenolo[3,2-c]pyrazoles (2,
27–41) tested, only compounds 2, 33, 36 and 37 demonstrated
significant cytotoxicity. These four compounds have the same
common structure (A) shown in the following.
The above synthesized intermediates 10, 11 and 12 were then
subjected to condensation reaction with phenylhydrazine (13) or
benzylhydrazine (14), in toluene, in the presence of acetic acid, to
afford the corresponding hydrazones (15–20). As expected from
our previous experience [12], both E- and Z-form isomers were
produced. However, they led to the same rarget compounds in
subsequent reactions and were not isolated. The above prepared
hydrazones (15–20) were then treated with Pb(OAc)4, in CH2Cl2, to
form intermediates 21–26 that were subsequently cyclized
by treatment with BF3$Et2O to yield the corresponding sele-
nolo[3,2-c]pyrazoles (27–32). The method for structural determi-
nation of these products was similar, and was exemplified by that of
product 32. First, its molecular formula was determined to be
C19H16N2O3Se based on elemental analysis and the detection of its
molecular ion peak in mass spectrum (m/z 400, Mþ). On its 1H NMR
OH
O
Se
R
N
N
(CH2)n
(A)
spectrum, the signals at
the CH3, OCH3 and –CH2– groups, respectively. And H-6 signal of
the selenolo[3,2-c]pyrazole ring was located at
6.62 (J ¼ 1.2 Hz),
whereas the H-3 signal of the furan ring was seen at 7.76
(J ¼ 3.6 Hz). Signals for the other 6 protons were located between
7.18 and 7.32. Based on the above spectral and elemental analysis,
its structure was confirmed as the expected 1-benzyl-3-(5-
methoxycarbonyl-2-furyl)-5-methylselenolo[3,2-c]pyrazole (32).
Subsequently, compounds 27–32 were reduced with Ca(BH4)2
into their corresponding carbinols (33, 2, 34–37)either, or
d 2.56, 3.89 and 5.46 were correlated with
Among them, compounds 2 and 37 with –CH2– link (n ¼ 1)
exhibited relatively greater cytotoxicity than those (33, 36) without
–CH2– link (n ¼ 0). Between two compounds with n ¼ 1, the one
with R]H showed greater cytotoxicity than the other with R]CH3
(37). Among these four, the cytotoxicity of compound 2 is the
greatest, which is comparable with the YC-1 control, and is much
greater than that of its furo[3,2-c]pyrazole (1) [10] and thieno[3,2-
c]pyrazole (42) [11] counterpart. Although the cytotoxicity of
compounds 33, 36 and 37 is relatively weaker than compound 2,
d
d
d

L.-C. Chou et al. / European Journal of Medicinal Chemistry 45 (2010) 1395–1402
1397
O
SOCl2
O
R
R
Se
Se
CH2Cl2
Cl
OH
5, R = H
3, R = H
4
6
, R = CH3
, R = CH3
Se
7
O
O
O
FeCl3 / CH2Cl2
Cl
O
9
AlCl3 / 1,2-dichloroethane
O
O
8
Ar
COOCH3
10
, R = H, Ar =
O
R
Se
11, R = CH3, Ar =
12, R = H, Ar =
O
O
10-12
(CH2)nNHNH2
AcOH/ toluene
13
, n = 0
14, n = 1
Ar
COOCH3
R
Se
N
N
H
(CH2)n
15-20
Compounds
15 21 27 33 38
R
H
Ar
n
0
1
0
1
0
1
O
O
,
,
,
,
O
O
Ar
COOCH3
16, 22, 28, 2, 39
H
R
Se
N
17 23 29 34
,
,
,
H
N
(CH2)n
18 24 30 35 40
,
,
,
,
H
21-26
19, 25, 31, 36, 41
CH3
CH3
O
O
BF3 Et2O
20 26 32 37
,
,
,
Ar
COOCH3
Se
R
N
N
(CH2)n
27-32
NaOH/H2O
Se
Ca(BH4)2 /THF
Ar
CH2OH
Ar
COOH
Se
R
R
N
N
N
N
(CH2)n
(CH2)n
33, 2, 34-37
38-41
Scheme 1.

1398
L.-C. Chou et al. / European Journal of Medicinal Chemistry 45 (2010) 1395–1402
Fig. 2. Differential activity patterns for compound 2 against 60 human cancer cell lines. MG-MID: mean of log X values (X ¼ GI50, TGI, and LC50). Delta: logarithm of the difference
between the MG-MID and the log X of the most sensitive cell line. Range: logarithm of the difference between the log X of the most resistant cell line and the log X of the most
sensitive cell line.
their exhibition of selective cytotoxicity toward A-498 cells
deserves further investigation.
5-substituted 1-benzyl or 1-phenyl-3-(5-hydroxymethyl-2-furyl)
selenolo[3,2-c]pyrazole structure (A). Themode of actionof compound
2, which demonstrated the greatest cytotoxicity, seems to differ from
that of the 175 anticancer agents listed in NCI’s standard database, and
resembles that of YC-1. Details of its action mechanism is currently
under investigation and will be published when available. Our
preliminary data suggest that cyclophlin A is an important target of
compound 2 cytotoxicity on A-498 cells. We thereby recommend that
compound 2 be further developed as the YC-1-derived, new drug
candidate for treatment of lung and renal cancers. On the other hand,
the structure–activity relationship established in the present study will
serve as guideline for future study.
4. Conclusion
A series of 1, 3-disubstituted selenolo[3,2-c]pyrazoles (2, 27–41)
were synthesized and evaluated for cytotoxicity against NCI-H226 and
A-498 cancer cell lines. Four compounds (2, 33, 36 and 37) that
demonstrated significant cytotoxicity were found to have the similar
Table 1
COMPARE correlation at GI50 level for compound 2.
5. Experimental
Rank
Compounds (NCI number)
ra
1
2
3
4
5
6
7
O6-methylguanine (S37364)
Bruceantin (S165563)
Pyrrolizine dicarbamate (S278214)
Cytembena (S104801)
Hydroxyurea (S32065)
Dihydro-5-azacytidine (S264880)
Morpholino-ADR (S354646)
0.354
0.296
0.254
0.246
0.221
0.218
0.215
5.1. Chemistry
All of the solvents and reagents were obtained commercially
and used without further purification. The progress of all reactions
was monitored by TLC on 2 ꢁ 6 cm pre-coated silica gel 60 F254
plates of thickness 0.25 mm (Merck). The chromatograms were
visualized under UV 254–366 nm. The following adsorbent was
a
r: correlation coefficient.

L.-C. Chou et al. / European Journal of Medicinal Chemistry 45 (2010) 1395–1402
1399
Table 2
Cytotoxicity of selenolo[3,2-c]pyrazoles.
R2
R2
O
Se
Se
R1
R1
N
N
N
N
R3
R3
27, 28, 31, 32,
33, 2, 36, 37,
38, 39, 41
29, 30, 34, 35,40
OH
OH
OH
O
O
O
S
O
N
N
N
N
N
N
42
1
YC-1
a
Comp’d
R1
R2
R3
IC50 (mM)
NCI-H226b
A498b
27
28
29
30
31
32
33
2
34
35
36
37
38
39
40
41
H
H
H
H
CH3
CH3
H
H
H
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
COOH
phenyl
benzyl
phenyl
benzyl
phenyl
benzyl
phenyl
benzyl
phenyl
benzyl
phenyl
benzyl
phenyl
benzyl
benzyl
phenyl
>50
>100
>100
>100
>100
>100
29.5
1.4
>50
>100
>100
>100
>100
>100
2.2
0.4
>100
>100
25
>100
>100
1.9
H
CH3
CH3
H
H
H
8.7
0.9
>100
>100
>100
>100
8.0
>100
>100
>100
>100
0.6
COOH
COOH
COOH
CH3
42 [11]
1 [10]
YC-1 [12]
2.0
1.9
0.4
0.3
Human tumor cells were treated with different concentrations of samples for 48 h.
a
Data was presented as IC50
Human lung cancer cell (NCI-H226), human renal cancer cell (A-498).
(
m
M, the concentration of 50% proliferation-inhibitory effect).
b
used for column chromatography: silica gel 60 (Merck, particle size
0.063–0.200 mm). Melting points were determined with a Yanaco
MP-500D melting point apparatus and are uncorrected. IR spectra
were recorded on Shimadzu IR-Prestige-21 spectrophotometers as
KBr pellets. NMR spectra were obtained on a Bruker Avance DPX-
200 FT-NMR spectrometer in CDCl3 or DMSO. The following
abbreviations are used: s, singlet; d, doublet; t, triplet; q, quartet;
dd, double doublet; and m, multiplet. MS spectra were measured
with an HP 5995 GC-MS instrument. Elemental analyses (C, H, and
N) were carried out at the instruments center of National Chung
Hsing University, Taichung, Taiwan and performed on a Perkin–
Elmer 2400 Series II CHNS/O analyzer or Elementar vario EL III
Heraeus CHNOS Rapid F002 and the results were within ꢂ0.4% of
the calculated values.
selenophene-2-carbonyl chloride (5) was given. Into the crude
product 5 was added dry CH2Cl2 (50 mL), then methyl furan-2-
carboxylate (8) (5.04 g, 0.04 mol) and anhydrous ferric chloride
(6.50 g, 0.04 mol) were added. The reaction mixture was then
heated under refluxing for 4 h, cooled and quenched with ice water.
The CH2Cl2 layer was sequentially washed with water, 5% NaHCO3
solution, and water, till neutral, then dried over MgSO4 and filtered.
The solvent of the filtrate was evaporated under reduced pressure,
the residue was recrystallized from n-hexane to afford compound
10 (7.0 g, 0.025 mol). Yield: 62%; yellow crystals; mp 116–118 ꢃC;
MS (EI, 70 eV): m/z 284 (Mþ); IR (KBr): 1618, 1717 (C]O) cmꢀ1; 1H
NMR (200 MHz, DMSO-d6):
7.52–7.57 (m, 2H), 8.41 (d, 1H, J ¼ 3.8 Hz); 8.76 (d, 1H, J ¼ 5.5 Hz);
13C NMR (50 MHz, DMSO-d6):
52.92, 119.58, 119.91, 132.12, 137.73,
d
3.84 (s, 3H), 7.43 (d, 1H, J ¼ 3.8 Hz),
d
143.86, 146.45, 147.74, 152.69, 158.40, 174.33; Anal. Calcd for
C11H8O4Se: C, 46.66; H, 2.85, Found: C, 46.76; H, 2.81.
5.1.1. General procedure for the synthesis of compounds 10–12
5.1.1.1. 5-Methoxycarbonyl-2-furyl 2-selenophenyl ketone (10). A
mixture of selenophene-2-carboxylic acid (3) (7.0 g, 0.04 mol) in
50 mL of dry CH2Cl2 and SOCl2 (9.52 g, 0.08 mol) was heated under
refluxing for 12 h. After evaporation, the crude product
5.1.1.2. 5-Methoxycarbonyl-2-furyl 5-methyl-2-selenophenyl ketone
(11). 5-Methylselenophene-2-carboxylic acid (4) (7.6 g, 0.04 mol)
and methyl furan-2-carboxylate (8) (5.04 g, 0.04 mol) were allowed

1400
L.-C. Chou et al. / European Journal of Medicinal Chemistry 45 (2010) 1395–1402
to react as in the preparation of compound 10 to afford 11 (5.94 g,
5.1.2.3. 1-Phenyl-3-(p-methoxycarbonylphenyl)selenolo[3,2-c]pyr-
azole (29). Compound 12 (5.86 g, 0.02 mol), phenylhydrazine (13)
(4.36 g, 0.04 mol) and acetic acid (1.5 mL) were allowed to react as
in the preparation of compound 27 to afford 29 (1.93 g, 5.06 mmol).
Yield: 25.3%; white solid; mp 165–166 ꢃC; MS (EI, 70 eV): m/z 382
0.02 mol). Yield 50%; yellow crystals; mp 101–104 ꢃC; MS (EI,
70 eV): m/z 298 (Mþ); IR (KBr): 1600, 1736 (C]O) cmꢀ1 1H NMR
;
(200 MHz, CDCl3):
d
2.60 (s, 3H), 3.92 (s, 3H), 7.06 (dd, 1H, J ¼ 4.0,
1.0 Hz); 7.22 (d, 1H, J ¼ 3.6 Hz), 7.30 (d, 1H, J ¼ 3.6 Hz); 8.38 (d, 1H,
J ¼ 4.0 Hz); 13C NMR (50 MHz, CDCl3):
d
18.92, 52.41, 118.18, 118.69,
(Mþ); IR (KBr): 1713 (C]O) cmꢀ1 1H NMR (200 MHz, DMSO-d6):
;
130.40, 138.33, 145.47, 146.01, 153.88, 158.61, 159.41, 173.87; Anal.
Calcd for C12H10O4Se: C, 48.50; H, 3.39, Found: C, 48.45; H, 3.42.
d
3.85 (d, 3H), 7.37 (t, 1H, J ¼ 7.2 Hz), 7.56 (t, 2H, J ¼ 8.2 Hz), 7.81–
7.89 (m, 3H), 7.95–8.11 (m, 4H), 8.40 (d, 1H, J ¼ 5.8 Hz); 13C NMR
(50 MHz, DMSO-d6):
d 52.67, 114.64, 119.47, 120.84, 125.99, 127.27,
5.1.1.3. p-Methoxycarbonylphenyl 2-selenophenyl ketone (12). Anhy-
drous AlCl3 (10.19 g, 0.076 mol) and, 4-methoxycarbonyl benzoyl
chloride (9) (8.34 g, 0.042 mol) were dissolved in 1,2-dichloro-
ethane (100 mL), then selenophene (7) (5.00 g, 0.038 mol) was
added dropwise. The reaction mixture was then heated under
refluxing for 30 min, cooled, and quenched with ice water. The
organic layer was sequentially washed with water, 5% NaHCO3
solution, and water, till neutral, then dried over MgSO4 and filtered.
The solvent of the filtrate was evaporated under reduced pressure,
and the residue was recrystallized from n-hexane to afford
compound 12 (4.78 g, 0.016 mol). Yield 4þ3%; yellow crystals; mp
124–127 ꢃC; MS (EI, 70 eV): m/z 294 (M ); IR (KBr): 1618, 1715
129.45, 130.23, 130.57, 136.57, 139.25, 140.13, 144.47, 149.93, 166.36;
Anal. Calcd for C19H14N2O2Se: C, 59.85; H, 3.70; N, 7.35. Found: C,
59.72; H, 3.64; N, 7.42.
5.1.2.4. 1-Benzyl-3-(p-methoxycarbonylphenyl)selenolo[3,2-c]pyr-
azole (30). Compound 12 (5.86 g, 0.02 mol), benzylhydrazine (14)
(4.88g,0.04mol)andaceticacid(1.5mL)wereallowedtoreactasinthe
preparation of compound 27 to afford 30 (2.49 g, 6.3 mmol). Yield:
31.4%; white solid; mp 150–153 ꢃC; MS (EI, 70 eV): m/z 396 (Mþ); IR
(KBr): 1717 (C]O) cmꢀ1; 1H NMR (200 MHz, DMSO-d6):
d 3.91 (s, 3H),
5.53 (s, 2H), 7.00 (d,1H, J¼ 5.8 Hz), 7.21–7.38 (m, 5H), 7.85–7.95 (m, 3H),
8.10 (d, 2H, J ¼ 8.6 Hz); 13C NMR (50 MHz, DMSO-d6):
d 52.12, 55.48,
(C]O) cmꢀ1; 1H NMR (200 MHz, CDCl3):
d
3.94 (s, 3H), 7.41 (dd, 1H,
112.51, 117.20, 125.41, 127.52, 128.13, 128.86, 130.16, 134.83, 136.12,
137.11,143.55,150.91,166.95; Anal. Calcd for C20H16N2O2Se: C, 60.77; H,
4.08; N, 7.09. Found: C, 60.79; H, 4.06; N, 7.11.
J ¼ 4.0, 5.4 Hz), 7.79 (d, 1H, J ¼ 4.0 Hz); 7.85 (d, 2H, J ¼ 8.6 Hz), 8.13
(d, 2H, J ¼ 8.6 Hz); 8.47 (d, 1H, J ¼ 5.4 Hz); 13C NMR (50 MHz,
CDCl3): d 52.46, 128.95, 129.63, 130.89, 133.06, 137.77, 141.23, 141.51,
150.01, 166.29, 188.77; Anal. Calcd for C13H10O3Se: C, 53.26; H, 3.44.
Found: C, 53.46; H, 3.41.
5.1.2.5. 1-Phenyl-3-(5-methoxycarbonyl-2-furyl)-5-methylselenolo
[3,2-c]pyrazole (31). Compound 11 (5.94 g, 0.02 mol), phenyl-
hydrazine (13) (4.36 g, 0.04 mol) and acetic acid (1.5 mL) were
allowed to react as in the preparation of compound 27 to afford 31
(2.86 g, 7.4 mmol). Yield: 37.2%; white solid; mp 156–158 ꢃC; MS (EI,
5.1.2. General procedure for the synthesis of compounds 27–32
5.1.2.1. 1-Phenyl-3-(5-methoxycarbonyl-2-furyl)selenolo[3,2-c]pyr-
azole (27). Into the solution of compound 10 (5.66 g, 0.02 mol) in
toluene (100 mL) was added phenylhydrazine (13) (4.36 g,
0.04 mol) and acetic acid (1.5 mL). The mixture was heated under
refluxing for 3 h. After cooling, the solvent was evaporated and the
residue was purified by column chromatography (silica gel,
toluene) to give 5-methoxycarbonyl-2-furyl 2-selenophenyl phe-
nylhydrazone (15). The crude product 15 was dissolved in CH2Cl2
(50 mL), then Pb(OAc)4 (26.60 g, 0.06 mol), followed with boron
trifluoride diethyl etherate (98% in ether, 100 mL). After shaking for
10 min, the reaction mixture was poured into ice water (200 mL)
and allowed to stand until two layers formed. The organic layer was
washed with water, then 10% NaOH solution till neutral, then dried
over MgSO4 and filtered. The solvent of the filtrate was evaporated
and the residue was purified by column chromatography (silica gel,
CH2Cl2) to afford compound 27 (2.74 g, 7.4 mmol). Yield 37%; white
solid; mp 145–146 ꢃC; MS (EI, 70 eV): m/z 372 (Mþ); IR (KBr): 1724
70 eV): m/z 386 (Mþ); IR (KBr): 1732 (C]O) cmꢀ1
;
1H NMR
(200 MHz, DMSO-d6): 2.64 (s, 3H), 3.82 (s, 3H), 7.00 (d, 1H,
d
J ¼ 3.8 Hz), 7.36–7.45 (m, 2H), 7.50–7.58 (m, 3H), 7.79 (d, 2H,
J ¼ 8.2 Hz); 13C NMR (50 MHz, DMSO-d6):
d 19.79, 52.36, 109.17,
112.72, 117.30, 120.77, 127.26, 130.12, 137.50, 139.95, 143.62, 148.19,
151.05, 154.29, 158.61; Anal. Calcd for C18H14N2O3Se: C, 56.11; H,
3.66; N, 7.27. Found: C, 56.15; H, 3.86; N, 7.32.
5.1.2.6. 1-Benzyl-3-(5-methoxycarbonyl-2-furyl)-5-methylselenolo
[3,2-c]pyrazole (32). Compound 11 (5.94 g, 0.02 mol), benzylhy-
drazine (14) (4.88 g, 0.04 mol) and acetic acid (1.5 mL) were allowed
to react as in the preparation of compound 27 to afford 32 (1.56 g,
3.9 mmol). Yield: 19.5%; white solid; mp 142–145 ꢃC; MS (EI,
70 eV): m/z 400 (Mþ); IR (KBr): 1730 (C]O) cmꢀ1 1H NMR
;
(200 MHz, CDCl3):
d 2.56 (s, 3H), 3.89 (s, 3H), 5.46 (s, 2H), 6.62 (d,
1H, J ¼ 1.2 Hz), 7.76 (d, 1H, J ¼ 3.6 Hz), 7.18–7.32 (m, 6H); 13C NMR
(C]O) cmꢀ1; 1H NMR (200 MHz, DMSO-d6):
d
3.82 (s, 3H), 7.04 (d,
(50 MHz, CDCl3): d 19.68, 51.84, 55.34, 107.46, 110.09, 115.72, 120.02,
1H, J ¼ 3.4 Hz), 7.21–7.58 (m, 5H), 7.75–8.85 (m, 2H), 8.38 (d, 1H,
127.43, 128.10, 128.84, 136.01, 136.46, 143.45, 149.57, 151.75, 152.52,
159.12; Anal. Calcd for C19H16N2O3Se: C, 57.15; H, 4.04; N, 7.02.
Found: C, 57.18; H, 4.07; N, 7.12.
J ¼ 5.8 Hz); 13C NMR (50 MHz, DMSO-d6):
d 52.42, 109.36, 113.99,
117.59, 120.81, 128.94, 129.54, 131.05, 131.40, 137.11, 137.94, 139.51,
143.71, 150.98, 152.05, 158.64; Anal. Calcd for C17H12N2O3Se: C,
55.00; H, 3.26; N, 7.55. Found: C, 55.04; H, 3.23; N, 7.59.
5.1.3. General procedure for the synthesis of compounds 33, 2,
34–37
5.1.2.2. 1-Benzyl-3-(5-methoxycarbonyl-2-furyl)selenolo[3,2-c]pyr-
azole (28). Compound 10 (5.66 g, 0.02 mol), benzylhydrazine (14)
(4.88 g, 0.04 mol) and acetic acid (1.5 mL) were allowed to react as
in the preparation of compound 27 to afford 28 (1.54 g, 4.0 mmol).
Yield: 20.1%; white solid; mp 130–131 ꢃC; MS (EI, 70 eV): m/z 386
5.1.3.1. 1-Phenyl-3-(5-hydroxymethyl-2-furyl)selenolo[3,2-c]pyrazole
(33). Compound 27 (744 mg, 2.0 mmol) was dissolved in
a homogenous solution of THF (50 mL) dispersed with Ca(BH4)2
(1.26 g, 0.018 mol). The mixture was heated under refluxing for 6 h
and then filtered. The solvent was evaporated and the residue was
recrystallized from n-hexane and then purified by column chro-
matography (silica gel, n-hexane: ethyl acetate ¼ 1:1) to afford
compound 33 (515 mg, 1.5 mmol). Yiþeld 75.1%; white solid; mp 54–
55 ꢃC; MS (EI, 70 eV): m/z 344 (M ); IR (KBr): 3200–3500 (OH)
(Mþ); IR (KBr): 1724 (C]O) cmꢀ1
;
1H NMR (200 MHz, DMSO-d6):
d
3.80 (s, 3H), 5.59 (s, 2H), 6.88 (d, 1H, J ¼ 3.8 Hz), 7.23–7.34 (m, 5H),
7.38 (d, 1H, J ¼ 3.8 Hz), 7.53 (d, 1H, J ¼ 5.7 Hz), 8.19 (d, 1H,
J ¼ 5.7 Hz); 13C NMR (50 MHz, DMSO-d6):
d 52.33, 54.73, 108.32,
113.54, 116.64, 120.85, 128.07, 128.31, 129.14, 136.02, 137.26, 137.90,
143.20, 151.60, 151.69, 158.68; Anal. Calcd for C18H14N2O3Se: C,
56.11; H, 3.66; N, 7.27. Found: C, 56.18; H, 3.62; N, 7.29.
cmꢀ1; 1H NMR (200 MHz, DMSO-d6):
d
4.46 (d, 2H, J ¼ 5.2 Hz), 5.35
(t, 1H, J ¼ 5.2 Hz), 6.46 (d, 1H, J ¼ 3.2 Hz), 6.79 (d, 1H, J ¼ 3.2 Hz),
7.33–7.37 (m, 1H), 7.53 (t, 2H, J ¼ 8.2 Hz), 7.77–7.83 (m, 3H), 8.36 (d,

L.-C. Chou et al. / European Journal of Medicinal Chemistry 45 (2010) 1395–1402
1401
1H, J ¼ 5.8 Hz); 13C NMR (50 MHz, DMSO-d6):
d
56.18, 108.48,
(50 MHz, DMSO-d6): d 19.78, 54.36, 56.09, 107.05, 109.45, 111.56,
109.72, 114.60, 118.63, 120.46, 126.88, 130.18, 139.10, 140.18, 146.62,
149.11, 151.99, 156.45; Anal. Calcd for C16H12N2O2Se: C, 59.89; H,
3.79; N, 7.91. Found: C, 59.87; H, 3.81; N, 7.88.
113.32, 127.90, 128.16, 129.09, 137.14, 137.66, 147.28, 149.90, 151.90,
155.59; Anal. Calcd for C18H16N2O2Se: C, 58.23; H, 4.34; N, 7.54.
Found: C, 58.18; H, 4.45; N, 7.53.
5.1.3.2. 1-Benzyl-3-(5-hydroxymethyl-2-furyl)selenolo[3,2-c]pyrazole
(2). Following the same synthetic procedure as for compound 33,
compound 2 (530 mg, 1.48 mmol) was prepared by reacting
compound 28 (772 mg, 2.0 mmol). Yield: 74.2%; white solid; mp
126–129 ꢃC; MS (EI, 70 eV): m/z 358 (Mþ); IR (KBr): 3200–3400
5.1.4. General procedure for the synthesis of compounds 38–41
5.1.4.1. 1-Phenyl-3-(5-hydroxycarbonyl-2-furyl)selenolo[3,2-c]pyr-
azole (38). Compound 27 (744 mg, 2.0 mmol) in 20 mL of 10%
NaOH solution was heated under refluxing for 2 h, cooled and
acidified with dilute HCl. The precipitates were collected, then
recrystallized from ethanol to afford compound 38 (557 mg,
1.56 mmol). Yield: 78.0%; white solid; mp 236–237 ꢃC; MS (EI,
70 eV): m/z 358 (Mþ); IR (KBr): 1697 (C]O), 2400–3300 (OH)
(OH) cmꢀ1; 1H NMR (200 MHz, DMSO-d6):
d
4.41 (d, 2H, J ¼ 5.6 Hz),
5.29 (t, 1H, J ¼ 5.6 Hz), 5.53 (s, 2H), 6.38 (d, 1H, J ¼ 3.0 Hz), 6.59 (d,
1H, J ¼ 3.0 Hz), 7.25–7.33 (m, 5H), 7.48 (d,1H, J ¼ 5.8 Hz), 8.11 (d,1H,
J ¼ 5.8 Hz); 13C NMR (50 MHz, DMSO-d6):
d
54.46, 56.11, 107.11,
cmꢀ1; 1H NMR (200 MHz, DMSO-d6):
d
7.01 (d, 1H, J ¼ 4.0 Hz), 7.32–
109.49, 113.53, 115.37, 127.99, 128.19, 129.09, 137.22, 137.62, 147.28,
151.24, 155.68; Anal. Calcd for C17H14N2O2Se: C, 57.15; H, 3.93; N,
7.80. Found: C, 56.25; H, 3.90; N, 7.86.
7.40 (m, 2H), 7.54 (t, 2H, J ¼ 7.8 Hz), 7.76–7.83 (m, 3H), 8.37 (d, 2H,
J ¼ 5.8 Hz); 13C NMR (50 MHz, DMSO-d6):
d 109.20, 114.52, 119.39,
120.21, 120.84, 127.35, 130.21, 137.87, 139.60, 139.98, 144.96, 149.50,
150.59, 159.61; Anal. Calcd for C16H10N2O3Se: C, 53.80; H, 2.82; N,
7.84. Found: C, 53.82; H, 2.85; N, 7.81.
5.1.3.3. 1-Phenyl-3-(p-hydroxymethylphenyl)selenolo[3,2-c]pyrazole
(34). Following the same synthetic procedure as for compound 33,
compound 34 (585 mg, 1.66 mmol) was prepared by reacting
compound 29 (764 mg, 2.0 mmol). Yield: 82.9%; white solid; mp
133–134 ꢃC; MS (EI, 70 eV): m/z 354 (Mþ); IR (KBr): 3100–3500
5.1.4.2. 1-Benzyl-3-(5-hydroxycarbonyl-2-furyl)selenolo[3,2-c]pyrazole
(39). Following the same synthetic procedure as for compound 38,
compound 39 (595 mg, 1.60 mmol) was prepared by reacting
compound 28 (772 mg, 2.0 mmol). Yþield: 80.2%; white solid; mp 243–
245 ꢃC; MS (EI, 70 eV): m/z 372 (M ); IR (KBr): 1697 (C]O), 2500–
(OH) cmꢀ1; 1H NMR (200 MHz, DMSO-d6):
d
4.53 (d, 2H, J ¼ 5.8 Hz),
5.28 (t, 1H, J ¼ 5.8 Hz), 7.34 (t, 1H, J ¼ 7.0 Hz), 7.45 (d, 2H, J ¼ 8.0 Hz),
7.55 (t, 2H, J ¼ 8.0 Hz), 7.79–7.86 (m, 5H), 8.37 (d, 1H, J ¼ 5.8 Hz); 13C
3400 (OH) cmꢀ1; 1H NMR (200 MHz, DMSO-d6):
d 5.58 (s, 2H), 6.82 (d,
NMR (50 MHz, DMSO-d6):
d
63.13, 114.70, 118.89, 120.53, 125.70,
1H, J ¼ 3.6 Hz), 7.18–7.35 (m, 6H), 7.52 (d, 1H, J ¼ 5.6 Hz), 8.18 (d, 1H,
126.84, 127.61, 130.17, 130.65, 138.72, 140.31, 143.34, 145.59, 149.55;
Anal. Calcd for C18H14N2OSe: C, 61.20; H, 3.99; N, 7.93. Found: C,
61.18; H, 3.96; N, 7.88.
J ¼ 5.6 Hz); 13C NMR (50 MHz, DMSO-d6):
d 54.69, 108.03, 113.52,
116.44, 119.55, 128.07, 128.29, 129.13, 136.37, 137.33, 137.75, 145.30,
150.86, 151.51, 160.03; Anal. Calcd for C17H12N2O3Se: C, 55.00; H, 3.26;
N, 7.55. Found: C, 54.96; H, 3.24; N, 7.57.
5.1.3.4. 1-Benzyl-3-(p-hydroxymethylphenyl)selenolo[3,2-c]pyrazole
(35). Following the same synthetic procedure as for compound 33,
compound 35 (564 mg, 1.54 mmol) was prepared by reacting
compound 30 (792 mg, 2.0 mmol). Yield: 76.8%; white solid; mp
158–160 ꢃC; MS (EI, 70 eV): m/z 368 (Mþ); IR (KBr): 3100–3500
5.1.4.3. 1-Benzyl-3-(p-hydroxycarbonylphenyl)selenolo[3,2-c]pyrazole
(40). Following the same synthetic procedure as for compound 38,
compound 40 (649 mg, 1.70 mmol) was prepared by reacting
compound 30 (792 mg, 2.0 mmol). Yield: 85.2%; white solid; mp
288–290 ꢃC; MS (EI, 70 eV): m/z 382 (Mþ); IR (KBr): 1684 (C]O),
(OH) cmꢀ1; 1H NMR (200 MHz, DMSO-d6):
d
4.49 (d, 2H, J ¼ 5.2 Hz),
5.21 (t, 1H, J ¼ 5.2 Hz), 5.56 (s, 2H), 7.20–7.33 (m, 5H), 7.39 (d, 2H,
J ¼ 8.0 Hz), 7.51 (d,1H, J ¼ 5.8 Hz), 7.68 (d, 2H, J ¼ 8.0 Hz), 8.16 (d,1H,
2400–3200 (OH) cmꢀ1; 1H NMR (200 MHz, DMSO-d6):
d 5.60 (s, 2H),
7.20–7.38 (m, 5H), 7.55 (d,1H, J¼ 5.8 Hz), 7.82 (d, 2H, J¼ 8.2Hz), 8.01(d,
J ¼ 5.8 Hz); 13C NMR (50 MHz, DMSO-d6):
d 54.49, 63.12, 113.68,
2H, J ¼ 8.2 Hz), 8.20 (d, 1H, J ¼ 5.8 Hz); 13C NMR (50 MHz, DMSO-d6):
115.57, 125.24, 127.49, 128.05, 128.16, 129.08, 131.27, 136.85, 137.70,
142.57, 143.91, 151.63; Anal. Calcd for C19H16N2OSe: C, 62.13; H,
4.39; N, 7.63. Found: C, 62.13; H, 4.27; N, 7.66.
d 54.66, 113.65, 116.42, 125.36, 128.08, 128.26, 129.13, 130.21, 130.64,
136.72, 137.38, 143.01, 151.91, 167.53; Anal. Calcd for C19H14N2O2Se: C,
59.85; H, 3.70; N, 7.35. Found: C, 59.81; H, 3.68; N, 7.36.
5.1.3.5. 1-Phenyl-3-(5-hydroxymethyl-2-furyl)-5-methylselenolo[3,2-c]
pyrazole (36). Following the same synthetic procedure as for
compound 33, compound 36 (613 mg, 1.7 mmol) was prepared by
reacting compound 31 (772 mg, 2.0 mmol). Yþield: 85.9%; white
solid; mp 67–68 ꢃC; MS (EI, 70 eV): m/z 358 (M ); IR (KBr): 3100–
5.1.4.4. 1-Phenyl-3-(5-hydroxycarbonyl-2-furyl)-5-methylselenolo[3,2-
c]pyrazole (41). Following the same synthetic procedure as for
compound 38, compound 41 (647 mg, 1.74 mmol) was prepared by
reacting compound 31 (772 mg, 2.0 mmol). þYield: 87.2%; white solid;
mp 238–239 ꢃC; MS (EI, 70 eV): m/z 372 (M ); IR (KBr): 1663 (C]O),
3500 (OH) cmꢀ1; 1H NMR (200 MHz, DMSO-d6):
d
2.62 (s, 3H), 4.45
2200–3300 (OH) cmꢀ1; 1H NMR (200 MHz, DMSO-d6):
d 2.63 (s, 3H),
(d, 2H, J ¼ 5.8 Hz), 5.33 (t, 1H, J ¼ 5.8 Hz), 6.44 (d, 1H, J ¼ 3.2 Hz),
6.96 (d, 1H, J ¼ 3.6 Hz), 7.31–7.42 (m, 2H), 7.45–7.57 (m, 3H), 7.77 (d,
6.73 (d, 1H, J ¼ 3.2 Hz), 7.28–7.36 (m, 1H), 7.47–7.56 (m, 3H), 7.76 (d,
2H, J ¼ 8.0 Hz); 13C NMR (50 MHz, DMSO-d6):
d 19.85, 109.14, 112.78,
2H, J ¼ 8.4 Hz); 13C NMR (50 MHz, DMSO-d6):
d
19.87, 56.16, 108.40,
117.20, 120.21, 120.80, 127.32, 130.19, 137.75, 139.96, 144.88, 148.22,
150.57, 154.40, 159.62; Anal. Calcd for C17H12N2O3Se: C, 55.00; H, 3.26;
N, 7.55. Found: C, 55.03; H, 3.24; N, 7.51.
109.66, 112.92, 116.41, 120.43, 126.81, 130.13, 138.77, 140.18, 146.62,
147.83, 153.80, 156.36; Anal. Calcd for C17H14N2O2Se: C, 60.22; H,
3.93; N, 7.80. Found: C, 60.18; H, 3.93; N, 7.78.
5.2. Biological evaluation
5.1.3.6. 1-Benzyl-3-(5-hydroxymethyl-2-furyl)-5-methylselenolo[3,2-
c]pyrazole (37). Following the same synthetic procedure as for
compound 33, compound 37 (612 mg, 1.65 mmol) was prepared by
reacting compound 32 (800 mg, 2.0 mmol). Yield: 82.5%; white
solid; mp 146–148 ꢃC; MS (EI, 70 eV): m/z 372 (Mþ); IR (KBr): 3200–
5.2.1. Cell culture and treatment
The human cancer cell line NCI-H226 and A498 were purchased
from the ATCC (Manassas, VA). In this study, the NCI-H226 and A498
cells were cultured in RPMI-1640 and DMEM medium (GIBCO)
respectively, supplemented with 10% FBS, penicillin (100 unit/mL)/
3400 (OH) cmꢀ1; 1H NMR (200 MHz, DMSO-d6):
d 2.54 (s, 3H), 4.39
(d, 2H, J ¼ 3.6 Hz), 5.27 (t, 1H, J ¼ 3.6 Hz), 5.46 (s, 2H), 6.36 (d, 1H,
streptomycin (100
mg/mL) and 1% L- glutamine. All cells were grown
J ¼ 3.0 Hz), 6.54 (d, 1H, J ¼ 3.0 Hz), 7.19–7.33 (m, 6H); 13C NMR
in a humidified atmosphere containing 5% CO2 at 37 ꢃC.

1402
L.-C. Chou et al. / European Journal of Medicinal Chemistry 45 (2010) 1395–1402
5.2.2. Cytotoxicity assay
References
The cytotoxicity was assessed using 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT) assay [15]. NCI-H226
and A498 cells were seeded at 1 ꢁ104 cells/well into 96-well plates.
After 24 h incubation to allow for cell attachment, cells were
treated with test compounds for 48 h. After treatment, cells were
washed once with PBS and incubated with 1 mg/ml MTT (Sigma, St.
Louis, MO, USA) for 2 h. Then the formazan precipitate was dis-
[1] F.N. Ko, C.C. Wu, S.C. Kuo, F.Y. Lee, C.M. Teng, Blood 84 (1994) 4226–4233.
[2] C.C. Wu, F.N. Ko, S.C. Kuo, F.Y. Lee, C.M. Teng, Br. J. Pharmacol. 116 (1995)
1973–1978.
[3] A. Friebe, D. Koesling, Mol. Pharmacol. 53 (1998) 123–127.
[4] Y.S. Chun, E.J. Yeo, J.W. Park, Cancer Lett. 207 (2004) 1–7.
[5] E.J. Yeo, Y.S. Chun, J. Kim, J.C. Lee, M.S. Kim, J.W. Park, J. Natl. Cancer Inst. 95
(2003) 516–525.
[6] B.J. Moeller, Y. Cao, C.Y. Li, M.W. Dewhirst, Cancer Cell 5 (2004) 429–441.
[7] S.L. Pan, J.H. Guh, C.Y. Peng, S.W. Wang, Y.L. Chang, F.C. Cheng, J.H. Chang,
S.C. Kuo, F.Y. Lee, C.M. Teng, J. Pharmacol. Exp. Ther. 314 (2005) 35–42.
[8] J.G. Chung, J.S. Yang, L.J. Huang, F.Y. Lee, C.M. Teng, S.C. Tsai, K.L. Lin, S.F. Wang,
S.C. Kuo, Proteomics 7 (2007) 3305–3317.
solved in 150 mL DMSO and the absorbance was measured on an
ELISA reader at a best wavelength of 570 nm.
[9] C.J. Chen, M.H. Hsu, L.J. Huang, T. Yamori, J.G. Chung, F.Y. Lee, C.M. Teng,
S.C. Kuo, Biochem. Pharmacol. 75 (2008) 360–368.
[10] L.C. Chou, L.J. Huang, J.S. Yang, F.Y. Lee, C.M. Teng, S.C. Kuo, Bioorg. Med. Chem.
15 (2007) 1732–1740.
[11] S.C. Kuo, F.Y. Lee, C.M. Teng, 1-(Substituted benzyl)-3-(substituted aryl)-
condensed pyrazole derivatives and processed of making the same, U.S. Patent
5574168, 1996.
5.2.3. Statistic evaluation
Values are expression as the mean ꢂ S.D. of three independent
experiments. Student’s t tests were used to assess the statistical
significance of the differences, with ‘‘P’’ values of less than 0.05
being considered statistically significant.
[12] F.Y. Lee, J.C. Lien, L.J. Huang, T.M. Huang, S.C. Tsai, C.M. Teng, C.C. Wu,
F.C. Cheng, S.C. Kuo, J. Med. Chem. 44 (2001) 3746–3749.
[13] M.R. Grever, S.A. Schepartz, B.A. Chabner, Semin. Oncol. 19 (1992) 622–638.
[14] A. Monks, D. Scudiero, P. Skehan, R. Shoemaker, K. Paull, D. Vistica, C. Hose,
J. Langley, P. Cronise, A. Vaigro-Wolff, M. Gray-Goodrich, H. Campbell, J. Mayo,
M. Boyd, J. Natl. Cancer Inst. 83 (1991) 757–766.
Acknowledgement
The investigation was supported by research grants from the
National Science Council of the Republic of China (NSC 96-2628-B-
039-001; NSC 97-2320-B-039-003-MY3) awarded to S. C. Kuo.
[15] M.H. Hsu, S.C. Kuo, C.J. Chen, J.G. Chung, Y.Y. Lai, L.J. Huang, Leuk. Res. 29
(2005) 1399–1406.