6154
B. P. Bandgar et al. / Bioorg. Med. Chem. 18 (2010) 6149–6155
4.2.8. 2-[5-(2,4-Dimethoxy-phenyl)-1H-pyrazol-3-yl]-6-(2-hydroxy-
methyl-1-methyl-pyrrolidin-3-yl)-3,5-dimethoxy-phenol (2h)
Mp: 88–90 °C; IR cmꢀ1: 3428, 1656, 1600; 1H NMR (300 MHz,
CDCl3): d 7.7 (d, 1H, J = 8.7), 7.62 (d, 1H, J = 8.4), 7.42 (s, 1H), 7.30
(s, 1H), 6.1 (s, 1H), 4.04 (m, 1H), 3.95 (s, 3H), 3.94 (s, 3H), 3.9 (s,
3H), 3.86 (s, 3H), 3.66 (m, 1H), 3.46 (m, 1H), 3.24 (m, 1H), 2.90
(m, 1H), 2.5 (s, 3H), 2.46 (m, 1H), 2.04 (m, 2H); MS: m/z 470 (M+).
4.3. Anticancer activity
Cytotoxic assay is performed on MCF-7 (breast cancer cell line),
PC-3 (prostate cancer cell line), HL-60 (promyelocytic leukemia
cell line), H-460 (lung cancer cell line) and HCT-116 (colon cancer
cell line) cell lines using propidium iodide fluorescence assay.37
Dyes such as propidium iodide (PI), which bind DNA, provide a ra-
pid and accurate means for quantitating total nuclear DNA. The
fluorescence signal intensity of the PI is directly proportional to
the amount of DNA in each cell, PI is not able to penetrate an intact
membrane, and so cells must first be permeabilized. Seed cells of
4.2.9. 2-[5-(3-Chloro-phenyl)-1H-pyrazol-3-yl]-6-(2-hydroxy-
methyl-1-methyl-pyrrolidin-3-yl)-3,5-dimethoxy-phenol (2i)
:
Mp: 143–148 °C; IR cmꢀ1 3420, 1646, 1597; 1H NMR
(300 MHz, CDCl3): d 7.78 (s, 1H), 7.66 (d, 1H, J = 7.8), 7.44 (d, 1H,
J = 7.8), 7.32 (s, 1H), 6.0 (s, 1H), 4.06 (m, 1H), 3.97 (s, 3H), 3.94 (s,
3H), 3.67 (m, 1H), 3.47 (m, 1H), 3.24 (m, 1H), 2.91 (m, 1H), 2.48
(s, 3H), 2.41 (m, 1H), 2.07 (m, 2H); MS: m/z 444 (M+).
3000–7500 cells/well were placed in 2000 ll of tissue culture
grade 96 well plates and allowed them to recover for 24 h in
humidified 5% CO2 incubator at 37 °C. After culturing for 24 h com-
pounds (in 0.1% DMSO) were added onto triplicate wells with
10
48 h in humidified 5% CO2 incubator at 37 °C condition, the med-
ium was removed and treated with 25 l of propidium iodide
(50 g/mL in water/medium) per well. The plates were frozen at
lM concentrations. 0.1% DMSO alone was used as control. After
4.2.10. 2-[5-(3-Bromo-phenyl)-1H-pyrazol-3-yl]-6-(2-hydroxy-
methyl-1-methyl-pyrrolidin-3-yl)-3,5-dimethoxy-phenol (2j)
l
Mp: 157–163 °C; IR cmꢀ1
:
3421, 1646, 1598; 1H NMR
l
(300 MHz, CDCl3): d 7.79 (s, 1H), 7.6 (d, 1H, J = 7.6), 7.48 (d, 1H,
J = 7.8), 7.30 (s, 1H), 6.0 (s, 1H), 4.04 (m, 1H), 3.96 (s, 3H), 3.92 (s,
3H), 3.66 (m, 1H), 3.42 (m, 1H), 3.24 (m, 1H), 2.90 (m, 1H), 2.48
(s, 3H), 2.41 (m, 1H), 2.06 (m, 2H); MS: m/z 488/490 (M+).
ꢀ80 °C for 24 h then thawed and allowed it to come at room tem-
perature and the plate absorbance was read on a fluorometer (Po-
lar-Star BMG Tech), using 530 nm excitation and 620 nm emission
wavelength. Lastly, percent cytotoxicity of the compounds was cal-
culated by using following formula:
4.2.11. 2-(2-Hydroxymethyl-1-methyl-pyrrolidin-3-yl)-3,5-di-
methoxy-6-(5-p-tolyl-1H-pyrazol-3-yl)-phenol (2k)
Reading of control ꢀ Reading of treated cells
% Cytotoxicity ¼
Reading of control
Mp: 89–94 °C; IR cmꢀ1: 3420, 1646, 1592; 1H NMR (300 MHz,
CDCl3): d 7.58 (d, 2H, J = 8), 7.24 (d, 2H, J = 7.8), 7.3 (s, 1H), 6.04
(s, 1H), 4.06 (m, 1H), 3.94 (s, 3H), 3.92 (s, 3H), 3.65 (m, 1H), 3.47
(m, 1H), 3.24 (m, 1H), 2.91 (m, 1H), 2.42 (s, 3H), 2.43 (m, 1H), 2.3
(s, 3H), 2.08 (m, 2H); MS: m/z 424 (M+).
ꢁ 100
The results were compared with the standard drug inhibitors
flavopiridol (0.5 lM).
4.4. Anti-inflammatory and cytotoxicity assay
4.2.12. 2-[5-(4-Fluoro-phenyl)-1H-pyrazol-3-yl]-6-(2-hydroxy–
methyl-1-methyl-pyrrolidin-3-yl)-3,5-dimethoxy-phenol (2l)
Proinflammatory cytokine production by lipopolysaccharide
(LPS) in THP-1 cells was measured according to the method de-
scribed by Hwang et al.38 During the assay, THP-1 cells were cul-
tured in the RPMI 1640 culture media (Gibco BRL, Pasley, UK)
containing 100 U/mL penicillin and 100 mg/mL streptomycin con-
taining 10% fetal bovine serum (FBS, JRH). Cells were differentiated
with phorbol myristate acetate (PMA, Sigma). Following cell plat-
ing, the test compounds in 0.5% DMSO was added to each well
and the plate was incubated for 30 min at 37 °C. Finally, LPS (Esch-
erichia coli 0127: B8, Sigma Chemical Co., St. Louis, MO) was added,
Mp: 146–150 °C; IR cmꢀ1
:
3423, 1649, 1596; 1H NMR
(300 MHz, CDCl3): d 7.68 (t, 2H, J = 6.6), 7.42 (t, 2H, J = 8.7), 7.3
(s, 1H), 6.06 (s, 1H), 4.04 (m, 1H), 3.96 (s, 3H), 3.94 (s, 3H), 3.66
(m, 1H), 3.48 (m, 1H), 3.25 (m, 1H), 2.91 (m, 1H), 2.49 (s, 3H),
2.42 (m, 1H), 2.06 (m, 2H). MS: m/z 428 (M+).
4.2.13. 2-(2-Hydroxymethyl-1-methyl-pyrrolidin-3-yl)-3,5-di-
methoxy-6-[5-(4-methoxy-phenyl)-1H-pyrazol-3-yl]-phenol
(2m)
Mp: 108–114 °C; IR cmꢀ1
:
3421, 1651, 1594; 1H NMR
at a final concentration of 1
incubated at 37 °C for 24 h in 5% CO2. After incubation, superna-
tants were harvested, and assayed for TNF- and IL-6 by ELISA as
lg/mL in each well. Plates were further
(300 MHz, CDCl3): d 7.61 (d, 2H, J = 8.2), 7.30 (d, 2H, J = 8.2), 7.30
(s, 1H), 6 (s, 1H), 4.06 (m, 1H), 3.92 (s, 3H), 3.90 (s, 3H), 3.86 (s,
3H), 3.66 (m, 1H), 3.48 (m, 1H), 3.25 (m, 1H), 2.92 (m, 1H), 2.46
(s, 3H), 2.41 (m, 1H), 2.04 (m, 2H); MS: m/z 440 (M+).
a
described by the manufacturer (BD Biosciences). The cells were
simultaneously evaluated for cytotoxicity using CCK-8 from Dojin-
do Laboratories. Percent inhibition of cytokine release in compari-
son to the control was calculated. The 50% inhibitory concentration
(IC50) values were calculated by a nonlinear regression method.
4.2.14. 2-[5-(4-Chloro-phenyl)-1H-pyrazol-3-yl]-6-(2-hydroxy-
methyl-1-methyl-pyrrolidin-3-yl)-3,5-dimethoxy-phenol (2n)
:
Mp: 112–117 °C; IR cmꢀ1 3420, 1646, 1595; 1H NMR
4.5. Assay of tyrosinase activity
(300 MHz, CDCl3): d 7.78 (d, 2H), 7.64 (d, 2H), 7.32(s, 1H), 6.1 (s,
1H), 4.08 (m, 1H), 3.96 (s, 3H), 3.94 (s, 3H), 3.66 (m, 1H), 3.48
(m, 1H), 3.24 (m, 1H), 2.91 (m, 1H), 2.48 (s, 3H), 2.41 (m, 1H),
2.06 (m, 2H); MS: m/z 444 (M+).
Mushroom tyrosinase (EC 1.14.18.1) (Sigma Chemical Co.) was
used as described previously with some modifications, using
either, L-DOPA (diphenolase) or L-tyrosine (monophenolase) as
substrate.39 In spectrophotometric experiments, enzyme activity
was monitored by observing dopachrome formation at 475 nm
with a UV–vis spectrophotometer (Spectro UV–vis Double beam;
Shimadzu, Inc.) at 30 °C. All samples were first dissolved in 0.1%
4.2.15. 2-[5-(4-Bromo-phenyl)-1H-pyrazol-3-yl]-6-(2-hydroxy-
methyl-1-methyl-pyrrolidin-3-yl)-3,5-dimethoxy-phenol (2o)
Mp: 165–171 °C; IR cmꢀ1
:
3424, 1651, 1596; 1H NMR
DMSO at 10
(Km = 180 M) or 5.4 mM
was mixed with 2687 L of 0.25 M phosphate buffer (pH 6.8).
Then, 100 L of the sample solution and 13 L of the same
l
M–50
l
M. First, 200
l
L of a 2.7 mM
L-tyrosine
(300 MHz, CDCl3): d 7.8 (d, 2H), 7.66 (d, 2H), 7.31 (s, 1H), 6.1 (s,
1H), 4.06 (m, 1H), 3.95 (s, 3H), 3.92 (s, 3H), 3.68 (m, 1H), 3.46
(m, 1H), 3.26 (m, 1H), 2.90 (m, 1H), 2.47 (s, 3H), 2.4 (m, 1H), 2.07
(m, 2H); MS: m/z 488/490 (M+).
l
L
-DOPA (Km = 360
lM) aqueous solution
l
l
l