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Vol. 59, No. 8
7-Methoxy-3-p-tolyl-1H-benzofuro[3,2-c]pyrazole 2g: (89%) having 1H-
NMR (400 MHz, DMSO-d6) d: 2.36 (s, 3H, Me), 3.86 (s, 3H, MeO), 6.96—
7.00 (m, 1H, H-6ꢆ), 7.33—7.37 (m, 3H, Ar-H)), 7.37—7.86 (m, 3H, Ar-H),
13.08 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6) d: 20.8 (Me), 55.6
(MeO), 98.5 (C-11ꢆ), 110.7 (C-8ꢆ), 112.1 (C-5ꢆ), 119.5, 120.6, 124.5 (C-
13ꢆ), 128.7, 129.4, 130.8, 136.9 (C-3ꢆ, C-16ꢆ), 143.8 (C-10ꢆ), 158.4 (C-7ꢆ).
ESI-MS m/z: 279.07 ([MꢀH]ꢀ), 301.12 ([MꢀNa]ꢀ). HR-ESI-MS for
C17H15N2O2 ([MꢀH]ꢀ) Calcd: 279.1134; Found: 279.1091.
lighted the facts that the ability to form hydrogen bonding,
for example by pyrazole unit and methoxy group on the ring
A, might play a crucial role in modulating inhibitory activity.
In addition, compounds 1e and 2e were capable of forming
stable complexes with CT DNA, possibly via an intercalative
mode.
Experimental
3-(4-Chlorophenyl)-7-methoxy-1H-benzofuro[3,2-c]pyrazole 2h: (87%)
having 1H-NMR (400 MHz, DMSO-d6) d: 3.86 (s, 3H, MeO), 6.96—7.02
(m, 1H, H-6ꢆ), 7.34 (d, Jꢂ13.2 Hz, 1H, H-5ꢆ), 7.60—7.93 (m, 5H, Ar-H),
13.24 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6) d: 55.6 (MeO), 98.5
(C-11ꢆ), 110.6, 111.0, 120.0, 120.1, 126.4, 129.1 (C-16ꢆ), 131.9 (C-3ꢆ),
158.7 (C-10ꢆ), 163.2 (C-7ꢆ). ESI-MS m/z: 299.05 ([MꢀH]ꢀ). HR-ESI-MS
for C16H12ClN2O2 ([MꢀH]ꢀ) Calcd: 299.0587; Found: 299.0541.
General Electrospray ionization (ESI)-MS and HR-ESI-MS spectra
were measured on Waters UPLC/Quattro Premier XE and Agilent 6460
Triple Quadrupole mass spectrometers, respectively. 1H- and 13C-NMR spec-
tra were recorded in DMSO-d6 using Varian Mercury 400 spectrometers and
TMS as an internal reference. UV–Vis absorption measurements were con-
ducted by using a TU-1901 spectrophotometer (Purkinje Instrument Co.,
China) equipped with quartz cells. Viscosity experiments were carried out
on an Ubbelohde viscometer. CT DNA was obtained from Sigma Chemical
Co. (St. Louis, MO, U.S.A.), and its concentration was determined spec-
trophotometrically using the molar extinction coefficient of 6600 molꢄ1
cmꢄ1/base at 260 nm. All the other reagents and chemicals were obtained
from commercial sources and used as received unless otherwise stated.
Synthesis of Compounds 2a—i. General Procedures Hydrazine hy-
drate (0.65 g, 10 mmol) was added to a solution of 1a—i (5.0 mmol) in
acetic acid (25 ml) and the resulting mixture was refluxed for 24 h. Acetic
acid was removed in vacuum and the obtained residue was purified by chro-
matography on a silica-gel column (petroleum ether/acetone, 10/1 by vol-
ume) to give compounds 2a—i.
5-Nitro-3-p-tolyl-1H-benzofuro[3,2-c]pyrazole 2i: (10%) having 1H-NMR
(400 MHz, DMSO-d6) d: 2.37 (s, 3H, Me), 7.38—7.39 (m, 2H, Ar-H),
7.58—7.59 (m, 1H, Ar-H), 7.78—7.85 (m, 2H, Ar-H), 8.21—8.37 (m, 2H,
Ar-H), 13.48 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6) d: 20.8 (Me),
98.5 (C-11ꢆ), 122.2, 123.4, 124.8 (C-6ꢆ), 126.8, 129.7, 134.1 (C-5ꢆ), 137.6,
137.7, 153.7 (C-10ꢆ). ESI-MS m/z: 294.01 ([MꢀH]ꢀ). HR-ESI-MS for
C16H12N3O3 ([MꢀH]ꢀ) Calcd: 294.0879; Found: 294.0849.
Biological Measurements. Cell Culture MKN45, HepG2 and MCF-7
cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with
10% fetal bovine serum (FBS), whereas A549 cells were cultured in 1640
with 10% FBS. Cells were passaged at 70—80% confluence, about twice a
week by trypsinization.
3-Phenyl-1H-benzofuro[3,2-c]pyrazole 2a: (92%) having 1H-NMR (400
MHz, DMSO-d6) d: 7.38—7.42 (m, 2H, H-6ꢆ, H-8ꢆ), 7.50 (t, Jꢂ7.8 Hz, 1H,
H-7ꢆ), 7.56—7.95 (m, 6 H, Ar-H), 13.38 (s, 1H, NH). 13C-NMR (100 MHz,
DMSO-d6) d: 112.9 (C-11ꢆ), 119.3, 120.4, 123.3 (C-8ꢆ), 125.1, 126.0, 126.9,
127.7, 128.9, 129.2, 131.4 (C-12ꢆ), 143.9 (C-3ꢆ), 162.38 (C-10ꢆ). ESI-MS
m/z: 234.78 ([MꢀH]ꢀ). HR-ESI-MS for C15H11N2O ([MꢀH]ꢀ) Calcd:
235.0871; Found: 235.0844.
Cell Proliferation Assay Cell proliferation was determined using an
MTT assay.15) Exponentially growing cells were plated in 96-well plates
(16000 cells/well for MKN45 and MCF-7 cells, 10000 cells/well for HepG2
cells and 18000 cells/well for A549 cells) and incubated at 37 °C for 24 h for
attachment. Test compounds were prepared by dissolving in dimethyl sulfox-
ide (DMSO) at 100 mM and diluted with the medium. Then, culture medium
was changed, and cells grew in medium with the test compounds. DMSO
(0.1%) was used as negative control. Cells were incubated at 37 °C for 48 h.
Then the medium was replaced with MTT solution (5 mg/ml, 100 ml) fol-
lowed by incubation for another 3 h. The medium was then aspirated and
formazan crystals were dissolved in DMSO (100 ml) for about 10 min. The
absorbance at 570 nm (Abs) of the suspension was measured by an enzyme-
linked immunosorbent assay (ELISA) reader. The inhibition percentage
was calculated using the following formula: % inhibitionꢂ(Abscontrolꢄ
Abscompound)/Abscontrolꢅ100%.
3-(4-Methoxyphenyl)-1H-benzofuro[3,2-c]pyrazole 2b: (80%) having 1H-
NMR (400 MHz, DMSO-d6) d: 3.82 (s, 3H, MeO), 7.13 (d, Jꢂ7.6 Hz, 2H,
H-17ꢆ, H-15ꢆ), 7.38 (t, Jꢂ7.2 Hz, 1H, H-6ꢆ), 7.47 (t, Jꢂ7.2 Hz, 1H, H-7ꢆ),
7.71—7.91 (m, 4 H, Ar-H), 13.38 (s, 1H, NH). 13C-NMR (100 MHz,
DMSO-d6) d: 55.2 (MeO), 113.0, 113.7, 114.6, 119.9 (C-9ꢆ), 120.0 (C-8ꢆ),
126.3, 126.4, 131.1 (C-15ꢆ, C-17ꢆ), 143.7 (C-3ꢆ), 158.8 (C-10ꢆ), 161.9 (C-
16ꢆ). ESI-MS m/z: 264.84 ([MꢀH]ꢀ). HR-ESI-MS for C16H13N2O2
([MꢀH]ꢀ) Calcd: 265.0977; Found: 265.0940.
3-p-Tolyl-1H-benzofuro[3,2-c]pyrazole 2c: (84%) having 1H-NMR
(400 MHz, DMSO-d6) d: 2.36 (s, 3H, Me), 7.36—7.38 (m, 3H, Ar-H)), 7.48
(t, Jꢂ7.6 Hz, 1H, H-7ꢆ), 7.69—7.95 (m, 4H, Ar-H), 13.26 (s, 1H, NH). 13C-
NMR (100 MHz, DMSO-d6) d: 20.8 (Me), 98.3 (C-11ꢆ), 123.3 (C-5ꢆ), 124.9
(C-9ꢆ), 125.1, 126.0, 126.8, 127.7 (C-13ꢆ), 128.9, 129.2, 129.7, 137.3 (C-
12ꢆ), 143.8 (C-3ꢆ), 162.3 (C-10ꢆ). ESI-MS m/z: 248.70 ([MꢀH]ꢀ). HR-ESI-
MS for C16H13N2O ([MꢀH]ꢀ) Calcd: 249.1028; Found: 249.1002.
3-(4-Chlorophenyl)-1H-benzofuro[3,2-c]pyrazole 2d: (68%) having 1H-
NMR (400 MHz, DMSO-d6) d: 7.37—7.43 (m, 1H, H-6ꢆ), 7.46—7.51 (m,
1H, H-7ꢆ), 7.58 (d, Jꢂ8.4 Hz, 1H, H-8ꢆ), 7.64 (d, Jꢂ8.4 Hz, 1H, H-5ꢆ),
7.72—7.83 (m, 2H, H-17ꢆ, H-15ꢆ), 7.90—7.99 (m, 2H, H-14ꢆ, H-18ꢆ), 13.43
(s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6) d: 112.9, 113.3, 116.1 (C-9ꢆ),
119.4, 120.5, 123.4 (C-6ꢆ), 126.7 (C-13ꢆ), 129.0, 129.3, 130.2 (C-16ꢆ), 144.0
(C-12ꢆ), 145.2 (C-3ꢆ), 161.5 (C-10ꢆ). ESI-MS m/z: 268.70 ([M]ꢀ). HR-ESI-
MS for C15H10ClN2O ([MꢀH]ꢀ) Calcd: 269.0482; Found: 269.0442.
7-Methoxy-3-phenyl-1H-benzofuro[3,2-c]pyrazole 2e: (88%) having 1H-
NMR (400 MHz, DMSO-d6) d: 3.86 (s, 3H, MeO), 6.96—7.01 (m, 1H, H-
6ꢆ), 7.33—7.40 (m, 2H, H-5ꢆ and H-8ꢆ), 7.50—7.58 (m, 2H, H-15ꢆ, H-17ꢆ),
7.67—7.95 (m, 3H, Ar-H), 13.15 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-
d6) d: 55.6 (MeO), 98.5 (C-11ꢆ), 112.9 (C-5ꢆ), 120.4 (C-9ꢆ), 123.3 (C-8ꢆ),
124.6 (C-12ꢆ), 126.8, 127.6, 128.9, 129.5, 137.2 (C-16ꢆ), 142.7 (C-3ꢆ), 150.4
(C-10ꢆ), 162.3 (C-7ꢆ). ESI-MS m/z: 265.09 ([MꢀH]ꢀ), 287.07 ([MꢀNa]ꢀ).
HR-ESI-MS for C16H13N2O2 ([MꢀH]ꢀ) Calcd: 265.0977; Found: 265.0939.
7-Methoxy-3-(4-methoxyphenyl)-1H-benzofuro[3,2-c]pyrazole 2f: (82%)
The IC50 values of the test compounds and doxorubicin were measured by
treating cells with drugs of varying concentrations, and analyzing by use of
the prism statistical package (GraphPad Software, San Diego, CA, U.S.A.).
DNA-Binding Study. Spectrophotometric Titrations Spectrophoto-
metric titrations were performed at room temperature by fixing the concen-
trations of compounds 1e and 2e, while gradually increasing the concentra-
tion of CT DNA.20) Typically, to a solution of 1e (3.0ꢅ10ꢄ5 M) in 5 mM
Tris–HCl buffer (5 mM NaCl, pH 7.0) were added aliquots of CT DNA
(3.1ꢅ10ꢄ3 M) solution containing 1e (3.0ꢅ10ꢄ5 M) in 5 mM Tris–HCl buffer
(5 mM NaCl, pH 7.0). The mixing was achieved by stirring for 2 min. Then,
the corresponding absorption spectra were measured. The operations re-
peated until saturation reached. The spectrophotometric titrations of 2e were
conducted in a similar way.
Viscosity Measurements Viscosity experiments were carried out in
5 mM Tris–HCl buffer (5 mM NaCl, pH 7.0) at 20 °C. Thus, the solution of
compound 1e (or 2e) (1.0ꢅ10ꢄ5—1.0ꢅ10ꢄ4 M) was added to the solution of
CT DNA (1.0ꢅ10ꢄ4 M). Flow time (t) of each sample was measured three
times, and the average flow time was calculated. Data were presented as
(h/h0)1/3 versus the concentration ratios of compound 1e (or 2e) to CT
DNA, where h is the relative viscosity of CT DNA in the presence of com-
pound 1e (or 2e), and h0 is the relative viscosity of CT DNA alone. Relative
viscosity was calculated according to hꢂtꢄt0,19) where t0 is the flow time of
buffer solution.
1
having H-NMR (400 MHz, DMSO-d6) d: 3.82 (s, 3H, MeO), 3.86 (s, 3H,
Acknowledgment This work was financially supported by the Natural
Science Foundation (9451051501002541) and the Department of Education
of Guangdong Province, Southern Medical University and the National Nat-
ural Science Foundation of China (No. 21002048).
MeO), 6.95 (d, Jꢂ8.0 Hz, 1H, H-6ꢆ), 7.12 (d, Jꢂ8.6 Hz, 2H, H-17ꢆ, H-15ꢆ),
7.32—7.37 (m, 1H, H-5ꢆ), 7.66—7.88 (m, 3H, H-8ꢆ, H-14ꢆ, H-18ꢆ), 13.15
(s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6) d: 55.1 (MeO), 55.6 (MeO),
98.5 (C-11ꢆ), 110.8 (C-8ꢆ), 120.3, 120.5, 120.6, 121.4, 126.1 (C-13ꢆ), 139.4,
140.1, 142.3 (C-10ꢆ), 158.8 (C-7ꢆ), 163.7 (C-16ꢆ). ESI-MS m/z: 295.11
([MꢀH]ꢀ), 317.14 ([MꢀNa]ꢀ). HR-ESI-MS for C17H15N2O3 ([MꢀH]ꢀ)
Calcd: 295.1083; Found: 295.1041.
References and Notes
1) Henderson I. C., Curr. Prob. Cancer, 14, 165—230 (1990).
2) Lown J. W., Anticancer Drug Des., 3, 25—40 (1988).