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W. Liu et al.
Arch. Pharm. Chem. Life Sci. 2011, 344, 487–493
a cell suspension in culture medium at 7500 cells/mL (MCF-7 and
MDA-MB–231) or 3000 cells/mL (HT-29) were plated into each
well and were incubated for three days under culture conditions.
After the addition of various concentrations of the test com-
pounds, cells were incubated for up to 144 h. Then the medium
was removed, the cells were fixed with glutardialdehyde solution
1% and stored under phosphate buffered saline (PBS) at 48C.
Cell biomass was determined by a crystal violet staining assay,
followed by extracting of the bound dye with ethanol and
a photometric measurement at 590 nm. Mean values were
calculated and the effects of the compounds were expressed
as % Treated/Controlcorr values according to the following
equation:
2-(Nitrooxy)ethyl- 2-(6-(4-chlorophenyl)-2,2-dimethyl-7-
phenyl-2,3-dihydro-1H-pyrrolizin-5-yl)acetate 6a
Yield 61.3%; MS (m/z): 468 [M]þ; 1H-NMR (CDCl3): d 1.30 (s, 6H,
2 –CH3), 2.85 (s, 2H, –CH2–), 3.58 (s, 2H, –CH2COO–), 3.73 (s, 2H,
–CH2N–), 4.41 (t, 2H, J ¼ 4.4 Hz, –CH2O–), 4.68 (t, 2H, J ¼ 6.0 Hz,
–CH2O–), 7.04–7.24 (m, 9H, Ar-H); Anal. calcd. for C25H25ClN2O5 ꢀ H2O:
C, 61.66; H, 5.59; N, 5.75%; found: C, 61.34; H, 5.78; N, 6.05%.
3-(Nitrooxy)propyl-2-(6-(4-chlorophenyl)-2,2-dimethyl-7-
phenyl-2,3-dihydro-1H-pyrrolizin-5-yl)acetate 6b
Yield 73.9%; MS (m/z): 482 [M]þ; 1H-NMR (CDCl3): d 1.29 (s, 6H,
2 –CH3), 2.03–2.09 (m, 2H, –CH2–), 2.85 (s, 2H, –CH2–), 3.55 (s, 2H,
–CH2COO–), 3.73 (s, 2H, –CH2N–), 4.21 (t, 2H, J ¼ 6.0 Hz, –CH2O–),
4.48 (t, 2H, J ¼ 6.0 Hz, –CH2O–), 7.03–7.24 (m, 9H, Ar-H); Anal.
calcd. for C26H27ClN2O5: C, 64.66; H, 5.63; N, 5.80%; found: C,
64.89; H, 5.25; N, 5.79%.
TꢁC0
T
=
½%ꢃ ¼
ꢄ 100
Ccorr
CꢁC0
where C0: control cells at the time of compound addition; C:
control cells at the time of test end; T: probes/samples at the time
of test end.
4-(Nitrooxy)butyl-2-(6-(4-chlorophenyl)-2,2-dimethyl-7-
phenyl-2,3-dihydro-1H-pyrrolizin-5-yl)acetate 6c
The IC50 value was determined as the concentration causing
50% inhibition of cell proliferation and calculated as mean of at
least two or three independent experiments (OriginPro 8).
1
Yield 82.1%; MS (m/z): 496 [M]þ; H-NMR (CDCl3): d 1.29 (s, 6H, 2
–CH3), 1.74–1.77 (m, 4H, 2 –CH2–), 2.85 (s, 2H, –CH2–), 3.54 (s, 2H,
–CH2COO–), 3.73 (s, 2H, –CH2N–), 4.14 (t, 2H, J ¼ 6.0 Hz, –CH2O–),
4.45 (t, 2H, J ¼ 4.2 Hz, –CH2O–), 7.03–7.24 (m, 9H, Ar-H); Anal.
calcd. for C27H29ClN2O5: C, 65.25; H, 5.88; N, 5.64%; found: C,
65.23; H, 5.79; N, 5.94%.
Inhibition of COX Enzymes
The inhibition of isolated ovine COX-1 and human recombinant
COX-2 was determined with 10 mM of the respective compounds
by ELISA (‘‘COX inhibitor screening assay’’, Cayman Chemicals).
Experiments were performed according to the manufacturer’s
instructions. Absorption was measured at 415 nm (Victor2,
Perkin Elmer). Results were calculated as the means of duplicate
determinations.
8-(Nitrooxy)octyl-2-(6-(4-chlorophenyl)-2,2-dimethyl-7-
phenyl-2,3-dihydro-1H-pyrrolizin-5-yl)acetate 6d
Yield 71.8%; MS (m/z): 552 [M]þ; 1H-NMR (CDCl3): d 1.29 (s, 6H,
2 –CH3), 1.30–1.45 (m, 8H, 4 –CH2–), 1.61–1.71 (m, 4H, 2 –CH2–),
2.84 (s, 2H, –CH2–), 3.51 (s, 2H, –CH2COO–), 3.75 (s, 2H, –CH2N–),
4.11 (t, 2H, J ¼ 6.4 Hz, –CH2O–), 4.41 (t, 2H, J ¼ 6.4 Hz, –CH2O–),
7.03–7.26 (m, 9H, Ar-H); Anal. calcd. for C31H37ClN2O5: C, 67.32;
H, 6.74; N, 5.06%; found: C, 67.01; H, 7.02; N, 5.03%.
In-vitro NO Releasing Assays
In-vitro NO release was assayed according to established pro-
cedures with some modifications [29].
12-(Nitrooxy)dodecyl-2-(6-(4-chlorophenyl)-2,2-dimethyl-
7-phenyl-2,3-dihydro-1H-pyrrolizin-5-yl)acetate 6e
Incubation with 18 mM L-Cysteine in PBS (pH 7.4)
A solution of the test compound (1 mL of 2 mM solution in 0.2 M
PBS, pH 7.4) was mixed thoroughly with a freshly prepared
solution of L-cysteine (1 mL of a 36 mM solution in 0.1 M
PBS, pH 7.4), and the mixture was incubated at 378C for up to
appropriate incubation time in the absence of air. After exposure
to air for 10 min at 258C, an aliquot of the Griess reagent (1 mL)
[freshly prepared by mixing equal volumes of 1.0% sulfanilamide
and 0.1% N-naphthylethylenediamine dihydrochloride in water]
was added to an equal volume (1 mL) of each test compound’s
incubation solution with mixing. After 10 min had elapsed,
absorbance was measured at 540 nm using a Shimadzu UV
2100 UV-VIS scanning spectrophotometer. Solutions of
0–60 mM sodium nitrite were used to prepare a nitrite absor-
bance versus concentration curve under the same experimental
conditions. The percent NO release (quantified as nitrite ion) was
calculated (ꢂ SEM, n ¼ 3) from the standard nitrite versus con-
centration curve.
1
Yield 69.9%; MS (m/z): 608 [M]þ; H-NMR (CDCl3): d 1.26–1.40 (m,
16H, 8 –CH2–), 1.29 (s, 6H, 2 –CH3), 1.61–1.74 (m, 4H, 2 –CH2–),
2.84 (s, 2H, –CH2–), 3.51 (s, 2H, –CH2COO–), 3.75 (s, 2H, –CH2N–),
4.11 (t, 2H, J ¼ 6.4 Hz, –CH2O–), 4.43 (t, 2H, J ¼ 6.4 Hz, -CH2O-),
7.03–7.26 (m, 9H, Ar-H); Anal. calcd. for C35H45ClN2O5: C, 69.00;
H, 7.45; N, 4.60%; found: C, 68.74; H, 7.75; N, 4.80%.
Biological Activity
Cell Culture
The human MCF-7, MDA-MB-231 breast cancer cell lines, and
HT-29 colon cancer cell line were obtained from the American
Type Culture Collection. All cell lines were maintained as a
monolayer culture in L-glutamine containing Dulbecco’s
modified Eagle’s medium (DMEM) with 4.5 g/L glucose (PAA
Laboratories, Austria), supplemented with 5% fetal bovine
serum (FBS; Biochrom, Germany) in a humidified atmosphere
(5% CO2) at 378C.
Incubation with PBS (pH 7.4)
Cytotoxicity
The experiments were performed according to established pro-
cedures with some modifications [26]. In 96 well plates 100 mL of
This assay was performed as described above except that a
solution of the test compound (2 mL of a 1 mM solution in
0.1 M PBS pH 7.4) was used and no L-cysteine was added.
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