Synthesis of furopyrazole analogs of 1-benzyl-3-(5-hydroxymethyl-2-furyl)indazole (YC-1) as novel anti-leukemia agents

Article abstract of DOI:10.1016/j.bmc.2006.12.001

As part of our continuing search for potential anticancer drug candidates in YC-1 analogs, several 1-benzyl-3-(substituted aryl)-5-methylfuro[3,2-c]pyrazoles were synthesized and evaluated for their cytotoxicity against HL-60 cell line. Among these compou

Full text of DOI:10.1016/j.bmc.2006.12.001

Bioorganic & Medicinal Chemistry 15 (2007) 1732–1740
Synthesis of furopyrazole analogs of 1-benzyl-3-(5-hydroxymethyl-
2-furyl)indazole (YC-1) as novel anti-leukemia agents
Li-Chen Chou,a Li-Jiau Huang,a Jai-Sing Yang,a Fang-Yu Lee,b
Che-Ming Tengc and Sheng-Chu Kuoa,*
aGraduate Institute of Pharmaceutical Chemistry, China Medical University, Taichung, Taiwan
bYung-Shin Pharmaceutical Industry Co., Ltd Tachia, Taichung, Taiwan
cPharmacological Institute, College of Medicine, National Taiwan University, Taiwan
Received 1 September 2006; revised 1 December 2006; accepted 2 December 2006
Available online 6 December 2006
Abstract—As part of our continuing search for potential anticancer drug candidates in YC-1 analogs, several 1-benzyl-3-(substituted
aryl)-5-methylfuro[3,2-c]pyrazoles were synthesized and evaluated for their cytotoxicity against HL-60 cell line. Among these com-
pounds, 1-benzyl-3-(5-hydroxymethyl-2-furyl)-5-methylfuro[3,2-c]pyrazole (1) showed more potency than YC-1. Through investiga-
tion of action mechanism, it was found that compound 1 induced terminal differentiation of HL-60 cells toward granulocyte lineage
and promoted HL-60 cell differentiation by regulation of Bcl-2 and c-Myc proteins. Meanwhile, compound 1 also demonstrated
apoptosis-inducing effect. Such anti-leukemia mechanism of action is apparently different from that of YC-1 which mainly works
by inducing apoptosis, but not cell differentiation. Therefore, compound 1 is identified here as a new lead compound of cell differ-
entiating agent and apoptosis inducer for further development of new anti-leukemia agents.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
dose of YC-1 suppressed tumor growth and prolonged
the medium survival time in xenograft animal models
carrying various human cancers, including the non-
small cell lung cancer,13 hepatoma,9 and prostate
cancer.11
Previously, we have synthesized a series of 1-arylmethyl-
3-arylimidazole derivatives (A) and found that 1-benzyl-
3-(5-hydroxymethyl-2-furyl)indazole (YC-1) was the
most promising anti-platelet agent.1,2 Subsequent inves-
tigation of its action mechanism revealed that YC-1 is a
unique NO-independent, and NO-enhancing, activator
of soluble guaunylyl cyclase (sGC).3,4 Due to such un-
ique action mechanism of YC-1,5–8 and the fact that
sGC is associated with a lot of physiological functions,
more than 200 papers deal with the biological functions
and pharmacological action of YC-1, and related inda-
zole derivatives, have been published during last decade.
In some of these literatures, YC-1 was reported to exhib-
it excellent anti-cancer activity. The anti-cancer effect of
YC-1 seemed to result from its multiple action, including
apoptosis induction,9–11 anti-angiogenesis,12,13 anti-in-
flammation,14 and inhibition of matrix metalloprotein-
ases (MMPs).15 Animal studies indicated that an oral
We recently found that YC-1 acted against U937 leuke-
mia cell in vitro via an intrinsic mitochondrial-depen-
dent apoptosis pathway, and that YC-1 induced
apoptosis in a cGMP-independent pathway. The in vivo
anti-leukemia effects of YC-1, were evaluated in BALB/c
mice inoculated with WEHI-3B cells. The result indicat-
ed that YC-1 enhanced survival rate and prevented the
body weight loss in mice tested. This suggested that
YC-1 is an excellent lead compound for further develop-
ment of anti-leukemia agents.16
In the course of our search for novel anti-leukemia drug
candidates, we tried to replace the indazole skeleton of
YC-1 with a bioisosteric furo[3,2-c]pyrazole chromo-
phore to give 1-benzyl-3-(5-hydroxymethyl-2-furyl)-5-
methylfuro[3,2-c]pyrazole (1) which was further derived
into a series of furo[3,2-c]pyrazoles that were tested
against HL-60 cells for anti-leukemia activity. It was
found that the anti-leukemia mechanism of compound
Keywords: Furopyrazole; YC-1; Cell differentiation; Anti-leukemia
agent.
*
Corresponding author. Tel.: +886 4 22053366x5608; fax: +886 4
22030760; e-mail: sckuo@mail.cmu.edu.tw
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.12.001

L.-C. Chou et al. / Bioorg. Med. Chem. 15 (2007) 1732–1740
1733
OH
OH
R'
Ar1
O
O
O
N
N
H3C
N
R
N
N
N
R"
Ar2
(A)
YC-1
Ar1, Ar2 = benzene, thiophene, furan
(1)
1, comprising of inducing both cell differentiation and
apoptosis, differed from that of YC-1. Therefore, the
synthesis and anti-leukemia activities of these novel
furo[3,2-c]pyrazoles are reported here.
2.3. Compound 1 induced G0/G1 and sub G1 arrest in
HL-60 cells
The cell cycle analysis was performed to determine
whether compound 1 could influence the progression
of cell cycle. As shown in Figure 2, the exposure of
HL-60 cells to compound 1 for the duration of 24,
48, and 72 h resulted in significant increase in the per-
centage of cells accumulated in G0/G1 phase, accom-
panied by the slow increase in the percentage of cells
in the sub G1 nuclei population. This finding suggest-
ed that compound 1 is effective in inducing differenti-
ation and apoptosis of HL-60 cells. Similar
phenomenon, however, was not observed when com-
pound 1 was tested in other leukemia cell lines
(U937 and K562).
2. Results and discussion
2.1. Synthesis of 1,3-disubstituted-5-methylfuro[3,2-c]pyr-
azole (1, 14–24)
Target compounds 1 and 14–24 were synthesized follow-
ing similar procedure for the synthesis of YC-1 as shown
in Scheme 1.2 The starting diaryl ketones (6 and 7) were
prepared by reacting 5-methyl furan-2-carbonyl chloride
(2), or 4-methoxycarbonyl benzoyl chloride (5), with
substituted furans (3 and 4), respectively, according to
Friedel–Craft’s acylation condition. The resulted diaryl
ketones (6 and 7) were then treated with hydrazines (8
and 9), respectively, to yield the corresponding hydraz-
ones (10–13) which were subsequently treated with
mixed reagents of Pb(OAc)4 and BF3ÆEt2O to undergo
oxidation and cyclization to give the desired 1,3-disub-
stituted furo[3,2-c]pyrazoles (14–17). The ester forms
of compounds 14–17 were either hydrolyzed into their
corresponding carboxylic acids (18–21) or reduced with
Ca(BH4)2 to afford their corresponding carbinol deriva-
tives (1, 22–24).
2.4. Compound 1 induces differentiation of HL-60 cells
toward granulocytic lineage
HL-60 cells are said to have dual potential because they
are competent to differentiate into monocytes, in re-
sponse to vitamin D3 and 12-O-tetradecanoylphorbol-
13-acetate (TPA), or differentiate into granulocytes, in
response to retinoic acid or DMSO.17 To explore the
ability of compound 1 in inducing differentiation of
HL-60, Nitroblue Tetrazolium (NBT) reduction activity
was assessed in HL-60 cells treated with 5 lM dose of
compound 1 for 72 h.
2.2. Cytotoxic effects of compounds 1, 14–24 and YC-1 on
HL-60 cells
Compared with the control cells, the results from NBT-
reduction assay indicated that the number of differenti-
ated cells increased significantly after 72 h of cultivation
with compound 1 (Fig. 3A). On the contrary, the per-
centages of NBT-positive cells were 3.56% after treat-
ment with YC-1 (26 lM) (Fig. 3A), which indicated
that YC-1 can not induce differentiation in HL-60 cells.
Meanwhile, after treatment with compound 1, the flow
cytometric diagrams exhibited significant elevation of
surface CD11b expression (Fig. 3C), while the presence
of monocyte-specific CD14 surface antigen is almost
negligible if compared with that of CD11b. These find-
ings together demonstrated that compound 1 is an effec-
tive inducer for differentiation of HL-60 cells in vitro,
and is able to commit leukemia cells toward terminal
maturation. Not only could compound 1 induce the dif-
ferentiation of HL-60 cells into granulocyte-like cells, it
also enhances the all-trans retinoic acid (ATRA)-in-
duced differentiation (Figs. 3B and C). However, we
All of the synthesized furo[3,2-c]pyrazoles (1, 14–24)
and positive control, YC-1 were evaluated for their
cytotoxic activities against HL-60 cell line by using
propidium iodide (PI)-exclusion assay. As shown in
Table 1, compound 1 was the only one exhibiting con-
siderable cytotoxicity, whereas YC-1 and all other
furopyrazoles (14–24) resulted in ED50 values close
to or greater than 20 lM. Further investigation of
the concentration- and time-dependent viability of
HL-60 cells exposed to compound 1 was conducted.
As indicated by the result in Figure 1, compound 1
inhibited HL-60 cell proliferation by a dose- and
time-dependent manner. To reconfirm the inhibitory
effect in compound 1-treated HL-60 cell, the mito-
chondrial reduction activity of compound 1 was mea-
sured by MTT assay, and the result agreed nicely with
that of PI-exclusion method.

1734
L.-C. Chou et al. / Bioorg. Med. Chem. 15 (2007) 1732–1740
O
O
O
O
O
+
+
H3C
H3C
O
O
O
Cl
CH3
Cl
O
CH3
2
3
4
5
(a)
(a)
Ar
H3C
COOCH3
(CH2)n
O
O
6, 7
(b)
NH2
HN
8, 9
6
7
8
9
Ar =
O
Ar
H3C
Ar =
n =0
n =1
COOCH3
O
N
HN
(CH2)n
10, 14, 18, 22 n =0, Ar =
11, 15, 19, 23 n =0, Ar =
O
O
10, 11, 12, 13
12, 16, 20, 1 n =1, Ar =
13, 17, 21, 24 n =1, Ar =
(c)
COOCH3
Ar
O
H3C
N
N
(CH2)n
14, 15, 16, 17
(d)
(e)
CH2OH
Ar
COOH
Ar
O
O
H3C
H3C
N
N
N
N
(CH2)n
(CH2)n
1, 22, 23, 24
18, 19, 20, 21
Scheme 1. Reagents and conditions: (a) Lewis acid/CH2Cl2; (b) AcOH/toluene; (c) Pb(OAc)4/BF3ÆEt2O; (d) NaOH/H3O+; (e) Ca(BH4)2/THF.
Table 1. Cytotoxicity data for compounds 1, 14–24 and YC-1 against HL-60 cell line
Cell line
Cytotoxicity ED50 (lM)
14
15
16
17
18
19
20
21
22
23
1
24
YC-1
HL-60
>20
>20
>20
19.36
17.22
>20
>20
>20
18.25
>20
5.12
19.25
25.27

L.-C. Chou et al. / Bioorg. Med. Chem. 15 (2007) 1732–1740
1735
tion of c-Myc could inhibit the proliferation of normal
and leukemia myeloid cells.19 The expression levels of
these Bcl-2 family proteins and transcription factor
c-Myc were examined by Western blotting. The result
shown in Figure 4 indicated that Bcl-2 and c-Myc pro-
tein levels decreased simultaneously during the differen-
tiation of HL-60 cells into granulocytes that
subsequently undergo compound 1-induced apoptosis
for 72–96 h. Our results indicated compound 1-induced
HL-60 cell differentiation may be associated with Bcl-2
and c-Myc protein levels.
2.6. Compound 1 and YC-1 induce apoptosis of HL-60
cells
Figure 1. Effects of compound 1 on viability of HL-60 cells. Cells were
treated with various concentrations of compound 1 for indicated
duration. Viable cells were measured by PI exclusion and immediately
analyzed by flow cytometry. The percentage of cell viability was
calculated as a ratio between drug-treated cells and control cells. Each
value represents mean SD from three independent experiments.
The morphology of cells treated with compound 1 was
studied to verify whether cell death was a result of apop-
tosis in HL-60 cells. The nuclear morphological changes
of the cells were assessed by staining the cell with 40-6-
diamidino-2-phenylindole (DAPI). After 72-h culture
with compound 1, the cells exhibited nuclear shrinkage
and chromatin condensation (Fig. 5B). Such morpho-
logical changes were not apparent in the control cells
(Fig. 5A). Our results indicated that compound 1 in-
duced HL-60 cell apoptosis. As was mentioned briefly
in the introduction, we have previously observed YC-1
inducing apoptosis of U937 cells. In this study, DAPI
staining was assessed in HL-60 cells treated with YC-1
(26 lM). After 48-h cultivation with YC-1, the cells
exhibited nuclear shrinkage and chromatin condensa-
tion (Fig. 5C). This result indicated that YC-1 induced
HL-60 cell apoptosis at high dose.
did not detect significant NBT-reduction in compound
1-treated U937 and K562 cells (Table 2).
2.5. Compound 1 promotes HL-60 cell differentiation by
regulation of Bcl-2 and cMyc proteins
Regulation of the relative levels of Bcl-2 protein may
play an important role in modulating the susceptibility
of cells to differentiation.18 Intriguingly, this raises the
possibility that c-Myc may be a key contributor to dis-
ease by deregulating cell proliferation, and by virtue of
its opposing role in engendering apoptosis. Downregula-
0 h
24 h
48 h
72 h
Sub-G1
Time
0 h
G0/G1
0.41
S
G2/M
39.24
23.84
3.61 0.45
11.11 0.68*
12.62 0.30*
16.43 0.19*
0.58
1.61
10.48 0.19
8.09 0.22
50.28
68.07 1.40*
24 h
48 h
72 h
0.76*
69.94
22.89 1.03
13.78
0.30
0.51
7.17
6.29
0.68*
0.87
79.93
Figure 2. Cell cycle progression of HL-60 cells after treatment with compound 1. HL-60 cells were treated with compound 1 for the indicated
incubation times, then stained for DNA with PI, and analyzed for cell cycle progression or apoptosis by flow cytometry. Cell cycle analysis showed
that compound 1 induced a prominent G0/G1 population arrest in HL-60 cells. The percentage of positive cells was analyzed using a flow cytometer,
and presented as mean SD. Three separate experiments were each tested in duplicate. Values significantly different from the 0 h-treated group are
marked by *P < 0.01.

1736
L.-C. Chou et al. / Bioorg. Med. Chem. 15 (2007) 1732–1740
Figure 4. Compound 1-induced Bcl-2, c-Myc, and a-tubulin protein
levels’ change in HL-60 cells assayed by Western blot analysis. Cells
were treated with compound 1 for the indicated incubation times.
Total protein extracts were analyzed by immunoblotting with anti-
bodies specific to Bcl-2, c-Myc and a-tubulin.
Figure 3. Compound 1-induced HL-60 cell’s differentiation presented
by (A and B) NBT reduction and (C) CD11b and CD14 differentiation
marker expression. NBT reduction was assay in HL-60 cells after 72-;h
treatment with compound 1 (5 lM), YC-1 (26 lM), and ATRA
(1 lM). The percentage of formazan-containing NBT reduction cells
was assessed microscopically. Expression of CD11b and CD14 in
compound 1- and/or ATRA-treated HL-60 cells. After 72 h-treatment
of compound 1 and/or ATRA, HL-60 cells were stained with FITC-
conjugated anti-CD11b and anti-CD14 antibodies. The percentage of
positive cells was analyzed using a flow cytometer and presented as
mean SD. Three separate experiments were each tested in duplicate.
Values significantly different from the control group are marked by
*P < 0.01.
Figure 5. Compound 1-induced apoptosis in HL-60 cells measured by
DAPI staining (400·) followed by microscopic analysis. (A) 0.1% (v/v)
DMSO-treated cells (control). (B) Compound 1-treated cells. (C) YC-
1-treated cells.
Table 2. NBT reduction in compound 1-treated human leukemia cells
Compound 1
NBT (%)
U937
3. Conclusions
HL-60
K562
0 lM
5 lM
*** P < 0.01.
3.77 0.25
53.41 2.38***
3.01 0.92
4.57 0.39
2.57 1.38
3.11 1.64
We have synthesized a series of furo[3,2-c]pyrazole ana-
logs of YC-1. Through anti-leukemia activity evalua-
tion, it was found that compound 1 demonstrated an

L.-C. Chou et al. / Bioorg. Med. Chem. 15 (2007) 1732–1740
1737
anti-leukemia mechanism of action different from that
of YC-1 which mainly works by inducing apoptosis.
Compound 1 induced differentiation of HL-60 cells to-
ward granulocyte lineage and promoted differentiation
of HL-60 cells by regulation of the dual effects of Bcl-
2 and c-Myc proteins. Meanwhile, compound 1 was also
observed to induce apoptosis of HL-60 cells. Since cell
differentiation and apoptosis are both considered impor-
tant action mechanism for anti-leukemia therapy, the
development of novel cell differentiation agents that also
induce apoptosis would be the keystone for successful
development of anti-leukemia agent. Thus, compound
1 is identified here as a new lead compound of cell differ-
entiation agent and apoptosis inducer that will be fur-
ther optimized for development of new anti-leukemia
drug candidate.
6.28–6.30 (m, 1H), 7.26–7.28 (m, 1H), 7.44 (d, 1H,
J = 3.7 Hz); 7.74 (d, 1H, J = 3.5 Hz); 13C NMR
(50 MHz, CDCl3, d): 13.82, 100.13, 108.73, 121.43,
141.95, 158.32, 163.13.
4.1.2.2. p-Methoxycarbonylphenyl 5-methyl-2-furyl
ketone (7). Anhydrous AlCl3 (26.70 g, 0.20 mol) and
4-methoxycarbonyl benzoyl chloride (5) (9.93 g,
0.10 mol) were dissolved in CH2Cl2 (100 mL), then
2-methylfuran (4) (8.21 g, 0.10 mol) was added drop-
wise. The reaction mixture was then heated under reflux-
ing for 30 min, cooled, and quenched with ice water. The
CH2Cl2 layer was sequentially washed with water, 5%
NaHCO3 solution, and water, until 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 7
(14.20 g, 0.058 mol). Yield: 58.2%; mp 93–96 ꢁC; MS
(EI, 70 eV): m/z 244 (M+); found: C, 68.87; H, 4.94.
C14H12O4 requires: C, 68.85; H, 4.95; IR (KBr): 1623,
4. Experimental
4.1. Chemistry
1723 (C@O) cmꢀ1 1H NMR (200 MHz, CDCl3, d):
;
2.45 (s, 3H), 3.95 (s, 3H), 6.22–6.24 (m, 1H), 7.12 (d,
1H, J = 3.5 Hz), 7.92–7.97 (m, 2H), 8.11–8.15 (m, 2H);
13C NMR (50 MHz, CDCl3, d): 13.94, 52.17, 109.11,
123.14, 128.72, 129.30, 132.87, 141.07, 150.49, 159.06,
166.06, 181.05.
4.1.1. General methods. All of the solvents and reagents
were obtained commercially and used without further
purification. Reactions were monitored by thin-layer
chromatography, using Merck plates with fluorescent
indicator. Column chromatography was performed on
silica gel. Melting points were determined with a Yanaco
MP-500D melting point apparatus and are uncorrected.
IR spectra were recorded on Shimadzu IRPrestige-21
spectrophotometers as KBr pellets. NMR spectra were
obtained on a Bruker Avance DPX-200 FT-NMR spec-
trometer in CDCl3 or DMSO. The following abbrevia-
tions are used: s, singlet; d, doublet; t, triplet; dd,
double doublet; and m, multiplet. MS spectra were mea-
sured with an HP 5995 GC–MS instrument. Elemental
analyses (C, H, and N) were performed on a Perkin-El-
mer 2400 Series II CHNS/O analyzer and the results
were within 0.4% of the calculated values.
4.1.2.3. 1-Phenyl-3-(5-methoxycarbonyl-2-furyl)-5-meth-
ylfuro[3,2-c]pyrazole (14). Into the solution of com-
pound 6 (4.68 g, 0.02 mol) in toluene (100 mL) were
added phenylhydrazine (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 (sil-
ica gel, toluene) to give 5-methoxycarbonyl-2-furyl
5-methyl-2-furyl phenylhydrazone (10). The crude prod-
uct 10 was dissolved in CH2Cl2 (50 mL), then Pb(OAc)4
(26.60 g, 0.06 mol), followed with BF3ÆEt2O (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 until neu-
tral, then dried over MgSO4 and filtered. The solvent of
the filtrate was evaporated and the residue was purified
by column chromatography (silica gel, CHCl3) to afford
compound 14 (1.81 g, 0.0056 mol). Yield: 28.1%; mp
130–131 ꢁC; MS (EI, 70 eV): m/z 322 (M+); found: C,
67.05; H, 4.39; N, 8.68. C18H14N2O4 requires: C,
4.1.2. Synthesis of substituted furo[3,2-c]pyrazoles (1,
14–24)
4.1.2.1. 5-Methoxycarbonyl-2-furyl 5-methyl-2-furyl
ketone (6). A mixture of 5-methylfuran-2-carboxylic
acid (7.56 g, 0.06 mol) in 50 mL of dry CH2Cl2 and
SOCl2 (8.33 g, 0.07 mol) was heated under refluxing
for 4–5 h. After evaporation, the crude product 5-
methylfuran-2-carbonyl chloride (2) was obtained. Into
the crude product 2 was added dry CH2Cl2 (50 mL),
then methyl furan-2-carboxylate (3) (7.56 g, 0.06 mol)
and anhydrous ferric chloride (9.75 g, 0.06 mol) were
added. The reaction mixture was then heated under
refluxing for 30 min, cooled and quenched with ice
water. The CH2Cl2 layer was sequentially washed with
water, 5% NaHCO3 solution, and water, until 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 com-
pound 6 (8.70 g, 0.037 mol). Yield: 62%; mp 122–
125 ꢁC; MS (EI, 70 eV): m/z 234 (M+); found: C,
61.53; H, 4.29. C12H10O5 requires: C, 61.54; H, 4.30;
67.07; H, 4.38; N, 8.69; IR (KBr): 1711 (C@O) cmꢀ1
;
1H NMR (200 MHz, CDCl3, d): 2.53 (d, 3H,
J = 0.8 Hz), 3.95 (s, 3H), 6.49 (d, 1H, J = 0.9 Hz), 6.96
(d, 1H, J = 3.6 Hz), 7.27–7.34 (m, 2H), 7.49 (t, 2H,
J = 8.3 Hz), 7.77–7.81 (m, 2H); 13C NMR (50 MHz,
CDCl3, d): 14.92, 51.71, 96.04, 109.01, 117.75, 119.61,
124.21, 125.47, 129.21, 136.85, 139.84, 143.67, 149.81,
158.87, 162.00.
4.1.2.4. 1-Phenyl-3-(p-methoxycarbonylphenyl)-5-meth-
ylfuro[3,2-c]pyrazole (15). Compound
7
(4.48 g,
0.02 mol), phenylhydrazine (4.36 g, 0.04 mol), and acetic
acid (1.5 mL) were allowed to react as in the preparation
of compound 14 to afford 15 (2.13 g, 0.0064 mol). Yield:
32.1%; mp 156–159 ꢁC; MS (EI, 70 eV): m/z 332 (M+);
IR (KBr): 1635, 1728 (C@O) cmꢀ1
;
1H NMR
(200 MHz, CDCl3, d): 2.47 (s, 3H), 3.96 (s, 3H),

1738
L.-C. Chou et al. / Bioorg. Med. Chem. 15 (2007) 1732–1740
found: C, 72.31; H, 4.86; N, 8.42. C20H16N2O3 requires:
(EI, 70 eV): m/z 318 (M+); found: C, 71.66; H, 4.44;
N, 8.81. C19H14N2O3 requires: C, 71.69; H, 4.43; N,
C, 72.28; H, 4.85; N, 8.43; IR (KBr): 1713 (C@O) cmꢀ1
;
1H NMR (200 MHz, CDCl3, d): 2.53 (d, 3H,
J = 0.9 Hz), 3.95 (s, 3H), 6.48 (d, 1H, J = 1.0 Hz),
7.25–7.27 (m, 1H), 7.49 (t, 2H, J = 8.4 Hz), 7.78–7.82
(m, 2H), 8.14 (s, 4H); 13C NMR (50 MHz, CDCl3, d):
14.91, 51.88, 96.05, 117.52, 125.18, 125.64, 128.96,
129.21, 129.83, 131.66, 135.44, 137.01, 140.09, 145.35,
161.47, 166.77.
8.80; IR (KBr): 1691 (C@O), 2500–3200 (OH) cmꢀ1
;
1H NMR (200 MHz, DMSO-d6, d): 2.50 (s, 3H), 7.06
(d, 1H, J = 0.9 Hz), 7.30 (t, 1H, J = 7.3 Hz), 7.54 (t,
2H, J = 8.4 Hz), 7.87 (d, 1H, J = 7.8 Hz), 8.02–8.12
(m, 4H); 13C NMR (50 MHz, DMSO-d6, d): 15.06,
97.26, 117.42, 125.74, 129.99, 130.42, 131.15, 134.75,
137.38, 139.69, 144.76, 162.57, 167.23.
4.1.2.5. 1-Benzyl-3-(5-methoxycarbonyl-2-furyl)-5-meth-
ylfuro[3,2-c]pyrazole (16). Compound
4.1.2.9. 1-Benzyl-3-(5-hydroxycarbonyl-2-furyl)-5-meth-
ylfuro[3,2-c]pyrazole (20). Following the same synthetic
procedure for compound 18, compound 20 (482 mg,
1.5 mmol) was prepared by reacting compound 16
(676 mg, 2.0 mmol). Yield: 74.8%; mp 231–234 ꢁC; MS
(EI, 70 eV): m/z 322 (M+); found: C, 67.09; H, 4.40;
N, 8.67. C18H14N2O4 requires: C, 67.07; H, 4.38; N,
6
(4.68 g,
0.02 mol), benzylhydrazine (4.88 g, 0.04 mol), and acetic
acid (1.5 mL) were allowed to react as in the preparation
of compound 14 to afford 16 (1.76 g, 0.0052 mol). Yield:
26.2%; mp 114–117 ꢁC; MS (EI, 70 eV): m/z 336 (M+);
found: C, 67.87; H, 4.78; N, 8.34. C19H16N2O4 requires:
C, 67.85; H, 4.79; N, 8.33; IR (KBr): 1724 (C@O) cmꢀ1
;
8.69; IR (KBr): 1681 (C@O), 2500–3100 (OH) cmꢀ1
;
1H NMR (200 MHz, DMSO-d6, d): 2.38 (d, 3H,
J = 0.4 Hz), 3.83 (s, 3H), 5.41 (s, 2H), 6.28 (d, 1H,
J = 0.9 Hz), 6.85 (d, 1H, J = 3.6 Hz), 7.28–7.36 (m,
5H), 7.40 (d, 1H, J = 3.7 Hz); 13C NMR (50 MHz,
DMSO-d6, d): 14.89, 52.04, 54.84, 95.41, 108.55,
120.45, 121.55, 128.18, 128.91, 136.63, 139.35, 142.32,
142.92, 150.23, 158.45, 161.94.
1H NMR (200 MHz, DMSO-d6, d): 2.37 (s, 3H), 5.39
(s, 2H), 6.28 (s, 1H), 6.82 (d, 1H, J = 3.6 Hz), 7.32–
7.38 (m, 6H); 13C NMR (50 MHz, DMSO-d6, d):
14.89, 54.84, 95.40, 108.39, 119.63, 121.82, 128.19,
128.91, 136.67, 139.33, 142.33, 144.33, 149.72, 159.45,
161.88.
4.1.2.10. 1-Benzyl-3-(p-hydroxycarbonylphenyl)-5-meth-
ylfuro[3,2-c]pyrazole (21). Following the same synthetic
procedure for compound 18, compound 21 (504 mg,
1.5 mmol) was prepared by reacting compound 17
(692 mg, 2.0 mmol). Yield: 75.9%; mp 211–214 ꢁC; MS
(EI, 70 eV): m/z 332 (M+); found: C, 72.30; H, 4.84;
N, 8.45. C20H16N2O3 requires: C, 72.28; H, 4.85; N,
4.1.2.6. 1-Benzyl-3-(p-methoxycarbonylphenyl)-5-meth-
ylfuro[3,2-c]pyrazole (17). Compound
7
(4.88 g,
0.02 mol), benzylhydrazine (4.88 g, 0.04 mol), and acetic
acid (1.5 mL) were allowed to react as in the preparation
of compound 14 to afford 17 (2.22 g, 0.0064 mol). Yield:
32.1%; mp 122–125 ꢁC; MS (EI, 70 eV): m/z 346 (M+);
found: C, 72.85; H, 5.21; N, 8.11. C21H18N2O3 requires:
8.43; IR (KBr): 1712 (C@O), 2500–3400 (OH) cmꢀ1
;
C, 72.82; H, 5.24; N, 8.09; IR (KBr): 1712 (C@O) cmꢀ1
;
1H NMR (200 MHz, DMSO-d6, d): 2.39 (s, 3H), 5.41
(s, 2H), 6.27 (s, 1H), 7.26–7.44 (m, 5H), 7.97 (dd, 4H,
J = 8.2, 15.1 Hz); 13C NMR (50 MHz, DMSO-d6, d):
14.94, 54.78, 95.41, 125.14, 128.16, 128.90, 129.80,
130.34, 135.54, 136.86, 139.57, 143.72, 161.55, 167.30.
1H NMR (200 MHz, DMSO-d6, d): 2.38 (d, 3H,
J = 0.9 Hz), 3.84 (s, 3H), 5.40 (s, 2H), 6.25 (d, 1H,
J = 1.1 Hz), 7.32–7.34 (m, 5H), 7.90–8.06 (m, 4H); 13C
NMR (50 MHz, DMSO-d6, d): 14.90, 52.29, 54.82,
95.37, 125.28, 128.16, 128.39, 128.87, 130.17, 136.01,
136.80, 139.60, 143.76, 161.57, 166.18.
4.1.2.11. 1-Phenyl-3-(5-hydroxymethyl-2-furyl)-5-meth-
ylfuro[3,2-c]pyrazole (22). Compound 14 (644 mg,
2.0 mmol) was dissolved in a homogeneous solution of
THF (50 mL) dispersed with CaBH4 (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 chromatography (silica gel, n-
hexane–ethyl acetate) to afford compound 22 (512 mg,
1.7 mmol). Yield: 87.1%; mp 127–130 ꢁC; MS (EI,
70 eV): m/z 294 (M+); found: C, 69.35; H, 4.78; N,
9.50. C17H14N2O3 requires: C, 69.38; H, 4.79; N, 9.52;
4.1.2.7. 1-Phenyl-3-(5-hydroxycarbonyl-2-furyl)-5-meth-
ylfuro[3,2-c]pyrazole (18). Compound 14 (644 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 18 (492 mg,
1.6 mmol). Yield: 79.9%; mp 247–250 ꢁC; MS (EI,
70 eV): m/z 308 (M+); found: C, 66.13; H, 4.01; N,
9.11. C17H12N2O4 requires: C, 66.23; H, 3.92; N, 9.09;
IR (KBr): 1700 (C@O), 2500–3200 (OH) cmꢀ1 1H
;
1
NMR (200 MHz, DMSO-d6, d): 2.49 (s, 3H), 7.00 (d,
1H, J = 3.6 Hz), 7.04 (s, 1H), 7.27–7.37 (m, 2H), 7.53
(t, 2H, J = 8.0 Hz), 7.82 (d, 2H, J = 8.1 Hz); 13C NMR
(50 MHz, DMSO-d6, d): 15.02, 97.21, 109.87, 117.58,
119.69, 123.98, 126.08, 130.00, 137.16, 139.49, 143.32,
144.90, 148.86, 159.41, 162.88.
IR (KBr): 3200–3500 (OH) cmꢀ1; H NMR (200 MHz,
CDCl3, d): 2.49 (d, 3H, J = 0.9 Hz), 4.73 (s, 2H), 6.43–
6.45 (m, 2H), 6.82 (d, 1H, J = 3.3 Hz), 7.19–7.27 (m,
1H), 7.42–7.50 (m, 2H), 7.72–7.77 (m, 2H); 13C NMR
(50 MHz, CDCl3, d): 14.85, 57.34, 96.04, 108.84,
109.38, 117.59, 125.05, 125.31, 129.15, 136.61, 139.99,
143.59, 145.82, 154.04, 161.56.
4.1.2.8. 1-Phenyl-3-(p-hydroxycarbonylphenyl)-5-meth-
ylfuro[3,2-c]pyrazole (19). Following the same synthetic
procedure for compound 18, compound 19 (548 mg,
1.7 mmol) was prepared by reacting compound 15
(664 mg, 2.0 mmol). Yield: 86.2%; mp 285–288 ꢁC; MS
4.1.2.12. 1-Phenyl-3-(p-hydroxymethylphenyl)-5-meth-
ylfuro[3,2-c]pyrazole (23). Following the same synthetic
procedure for compound 22, compound 23 (526 mg,
1.7 mmol) was prepared by reacting compound 15

L.-C. Chou et al. / Bioorg. Med. Chem. 15 (2007) 1732–1740
1739
(664 mg, 2.0 mmol). Yield: 86.5%; mp 114–116 ꢁC; MS
(EI, 70 eV): m/z 304 (M+); found: C, 74.95; H, 5.31;
N, 9.18. C19H16N2O2 requires: C, 74.98; H, 5.30; N,
After washing with PBS, low-molecular-weight frag-
ments of DNA were extracted for 10 min in citrate buff-
er (Na2HPO4, C6H3O7, pH 7.8), RNA was removed by
ribonuclease A (5 mg/mL), and DNA was stained with
propidium iodide (20 lg/mL PBS) for 30 min in the
dark. Fluorescence was measured using a flow cytometer
(FACSCalibur, Becton–Dickinson, San Jose, CA)
equipped with an argon laser at 488 nm wavelength
for excitation.
9.20; IR (KBr): 3100–3400 (OH) cmꢀ1 1H NMR
;
(200 MHz, CDCl3, d): 2.51 (s, 3H), 4.74 (s, 2H), 6.46
(d, 1H, J = 0.9 Hz), 7.20–7.27 (m, 1H), 7.44–7.52 (m,
4H), 7.77–7.81(m, 2H), 8.08 (d, 2H, J = 8.1 Hz); 13C
NMR (50 MHz, CDCl3, d): 14.90, 64.99, 96.01,
117.38, 124.83, 126.10, 127.09, 129.15, 130.36, 132.51,
136.84, 140.21, 140.40, 145.14, 161.23.
4.2.3. NBT assay. Cells were suspended in Hanks’ bal-
anced salt solution (HBSS) at a concentration of
1 · 106 cells/mL and then incubated for 30 min at
37 ꢁC with an equal volume of NBT test stock solution
(0.2 mL of HBSS containing 1 mg/ml NBT and 5 lg/mL
TPA) (Sigma, MO, USA). After incubation, the positive
cells reduced NBT to give intracellular black-blue for-
mazan deposit. The percentage of differentiated cells
was assessed microscopically by counting a minimum
of 200 cells. Three separate experiments were each tested
in duplicate.
4.1.2.13. 1-Benzyl-3-(5-hydroxymethyl-2-furyl)-5-meth-
ylfuro[3,2-c]pyrazole (1). Following the same synthetic
procedure for compound 22, compound 1 (480 mg,
1.6 mmol) was prepared by reacting compound 16
(676 mg, 2.0 mmol). Yield: 77.9%; mp 117–120 ꢁC; MS
(EI, 70 eV): m/z 308 (M+); found: C, 70.10; H, 5.25;
N, 9.10. C18H16N2O3 requires: C, 70.12; H, 5.23; N,
9.09; IR (KBr): 3200–3500 (OH) cmꢀ1 1H NMR
;
(200 MHz, DMSO-d6, d): 2.36 (s, 3H), 4.43 (d, 2H,
J = 5.7 Hz), 5.33 (t, 1H, J = 5.8 Hz), 5.35 (s, 2H), 6.25
(d, 1H, J = 1.0 Hz), 6.40 (d, 1H, J = 3.3 Hz), 6.60 (d,
1H, J = 3.2 Hz), 7.29–7.33 (m, 5H); 13C NMR
(50 MHz, DMSO-d6, d): 14.91, 54.55, 55.82, 95.30,
107.25, 109.04, 122.92, 128.06, 128.86, 137.03, 138.97,
141.89, 145.69, 155.31, 161.36.
4.2.4. Analysis of cellular surface antigen CD11b/CD14
expression. A direct immuno-fluorescence method was
employed to detect the CD11b/CD14 expression of
cellular surface antigens on HL-60 cells after differen-
tiation induction.20 Cells collected from 72 h cultures
were treated with monoclonal antibodies anti-
CD11b-FITC and anti-CD14-PE (Pharmingen, CA,
USA) at 37 ꢁC for 30 min, then washed with PBS.
The percentage of positive cells was analyzed using
flow cytometry (FACS Calibur, Becton–Dickinson,
San Jose, CA).
4.1.2.14. 1-Benzyl-3-(p-hydroxymethylphenyl)-5-meth-
ylfuro[3,2-c]pyrazole (24). Following the same synthetic
procedure for compound 22, compound 24 (504 mg,
1.6 mmol) was prepared by reacting compound 17
(692 mg, 2.0 mmol). Yield: 79.2%; mp 118–121 ꢁC; MS
(EI, 70 eV): m/z 318 (M+); found: C, 75.41; H, 5.71;
N, 8.78. C20H18N2O2 requires: C, 75.45; H, 5.70; N,
8.80; IR (KBr): 3200–3500 (OH) cmꢀ1
;
1H NMR
4.2.5. Western blot analysis. Total protein extracts were
prepared with RIPA protein lysis buffer (50 mM Tris
(pH 6.8), 5 lg/mL leupeptin, 5 lg/mL aprotinin, and
10 lM aqueous PMSF). The concentration of protein
was determined by the Bradford method using the
Bio-Rad protein assay dye reagent (Amresco, SO,
USA).21 The lysates containing 30 lg of protein were
separated by SDS–PAGE and transferred onto polyvi-
nylidene fluoride (PVDF) membrane (Millipore, MA,
USA). Nonspecific binding sites were blocked with 5%
non-fat milk in PBST buffer (0.05% Triton X-100 in
PBS) for 1 h. The PVDF membrane was incubated over-
night at 4 ꢁC with specific primary antibodies against
Bcl-2 (Upstate Biochemistry, NY, USA), c-Myc
(Pharmingen, CA, USA), and a-Tubulin (Santa Cruz
Biotechnology, CA, USA). After washing with PBST
buffer, the membrane was incubated with horseradish
peroxidase (HRP)-conjugated secondary antibodies
(Santa Cruz Biotechnology, CA, USA). Immunoreac-
tive proteins were detected by using Western Blotting
Chemiluminescence Reagent Plus kit (NENTM Life Sci-
ence, MA, USA) and exposed to X-ray films.
(200 MHz, CDCl3, d): 2.36 (s, 3H), 4.71 (s, 2H), 5.35
(s, 2H), 5.61 (d, 1H, J = 0.9 Hz), 7.26–7.38 (m, 5H),
7.42 (d, 2H, J = 8.1 Hz), 7.96 (d, 2H, J = 8.2 Hz); 13C
NMR (50 MHz, CDCl3, d): 14.78, 55.28, 64.99, 94.29,
125.73, 127.12, 127.92, 128.01, 128.52, 130.55, 130.83,
135.70, 138.83, 139.86, 144.05, 160.63.
4.2. Biological evaluation
4.2.1. Cell culture and viability assay. Leukemia cells
were obtained from the Culture Collection and Research
Center (CCRC, Taiwan, ROC), originally from the
American Type Culture Collection (ATCC, USA). Cells
were cultured in RPMI-1640 cell culture medium (Gibco
BRL, Life Technologies, MD, USA) supplemented with
10% heat-inactivated fetal bovine serum (HyClone, UT,
USA), 2 mM L-glutamine, penicillin (100 U/mL), and
streptomycin (100 mg/mL) (Gibco BRL), and incubated
at 37 ꢁC in humidified 5% CO2 atmosphere. For cell via-
bility and cell cycle analysis, cells [2.5 · 105 cells/mL]
were seeded in a 24-well culture plate (Falcon, CA,
USA). Compound was added to each well and the plates
were incubated at 37 ꢁC for 24, 48, and 72 h. Cell viabil-
ity was estimated by propidium iodide (PI)-exclusion
assay.
4.2.6. Statistical analysis. Results in this report are pre-
sented as means SD. Differences between the different
treatment groups, which consisted of matched samples,
were assessed by Student’s t test. A confidence level of
1% (P value of less than 0.01) was considered to be
significant.
4.2.2. Cell cycle analysis. Cells were harvested together,
washed twice in PBS, and fixed in 70% ethanol at 4 ꢁC.

1740
L.-C. Chou et al. / Bioorg. Med. Chem. 15 (2007) 1732–1740
9. Wang, S. W.; Pan, S. L.; Guh, J. H.; Chen, H. L.; Huang,
Acknowledgment
D. M.; Chang, Y. L.; Kuo, S. C.; Lee, F. Y.; Teng, C. M.
J. Pharmacol. Exp. Ther. 2005, 312, 917.
10. Wu, S. Y.; Pan, S. L.; Guh, J. H.; Huang, D. Y.; Chang,
Y. L.; Kuo, S. C.; Lee, F. Y.; Teng, C. M. YC-1 induces
apoptosis of human renal carcinoma A498 cells through
activating JNK pathway. Unpublished data.
The investigation was supported by research grant from
the National Science Council of the Republic of China
(NSC 95-2320-B-039-010) awarded to S. C. Kuo.
11. Huang, Y. T.; Pan, S. L.; Guh, J. H.; Chang, Y. L.; Lee, F.
Y.; Kuo, S. C.; Teng, C. Mol. Cancer Ther. 2005, 4, 1628.
12. Hsu, H. K.; Juan, S. H.; Ho, P. Y.; Liang, Y. C.; Lin, C.
H.; Teng, C. M.; Lee, W. S. Biochem. Pharmacol. 2003, 66,
263.
13. Pan, S. L.; Guh, J. H.; Peng, C. Y.; Wang, S. W.; Chang,
Y. L.; Cheng, F. C.; Chang, J. H.; Kuo, S. C.; Lee, F. Y.;
Teng, C. M. J. Pharmacol. Exp. Ther. 2005, 314, 35.
14. Pan, S. L.; Guh, J. H.; Peng, C. Y.; Chang, Y. L.; Cheng,
F. C.; Chang, J. H.; Kuo, S. C.; Lee, F. Y.; Teng, C. M.
Thromb. Haemost. 2005, 93, 940.
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