[
M.-D. Lu et al. / Chinese Chemical Letters 24 (2013) 415–418
417
8
100
80
60
40
20
0
B
A
LO2
LO2
7
6
5
4
3
2
1
HepG2
HepG2
0
6f
6h
6n
6p
0
2
4
8
12
16
Concentrations of 12 µmol/L
Concentrations of 6f (µmol/L)
Fig. 1. In vitro biological assessment of compound 6f. (A) Cytotoxicity of 6f against HepG2 and nontumor LO2 cells. HepG2 and LO2 cells were cultured in media with indicated
concentrations of 6f for 24 h. The cytotoxicity of 6f was determined by MTT assay. Data are means Æ SEM of cytotoxicity (%) from three independent experiments. (B) The levels
of nitrite/nitrate. HepG2 and LO2 cells were incubated in triplicate with each compound (12 mmol/L) for 5 h. The levels of nitrite/nitrate produced in the lysates of these cells were
determined using a calorimetric Griess assay. Data shown are the mean values Æ SEMs of individual compounds at 5 h post-treatment in individual types of cells from three
experiments.
6a–p. The structures of the target compounds were shown in
Table 1, and the data of MS, IR, 1H NMR spectra and elemental
analysis (EA) of selected compounds were shown as follow: 6f:
Yield 68%, mp 117–120 8C; MS (ESI) m/z 492 [M+H]+; 1H NMR
proliferation in vitro, indicative of a selective cytotoxicity against
tumor cells (Fig. 1A). Furthermore, the levels of nitrite/nitrate
produced by individual compounds with strong cytotoxicity in
HepG2 cells were much higher than those in LO2 cells (Fig. 1B).
Therefore, these new compounds could produce high levels of NO
selectively in HCC cells, which was associated with their strong
inhibition against human cancer cell proliferation in vitro.
Anti-tumor activities of target compounds were influenced by
the molecular structure of the chemicals. Analysis of structure
activity relationship (SAR) revealed that the inhibitory effects of
6a–h linked with CA were slightly better than those of 6i–p linked
with FA. In addition, target compounds 6a–p with different linkers
displayed variable inhibitory effects on the proliferation of cancer
cells. Compounds 6g–h and 6o–p linked with the cyclic amines,
such as piperidine, piperazine, exhibited stronger inhibitory effects
than compounds 6a–e and 6i–n linked with alkyl chains.
Furthermore, compounds 6a and 6e with two-carbon alkylol
amines displayed stronger anticancer activity than compounds
(300 MHz, CDCl3):
d 8.06 (d, 2H, J = 7.5 Hz), 7.78 (d, 1H), 7.66 (m,
2H), 7.59 (d, 1H, J = 16.2 Hz), 6.78–7.12 (m, 3H), 6.41 (d, 1H,
J = 16.2 Hz), 4.39 (t, 2H, J = 4.5 Hz), 4.29 (m, 2H), 3.90 (t, 2H,
J = 4.5 Hz), 3.81 (m, 2H); Anal. Calcd. for C21H21N3O9S: C, 51.32; H,
4.31; N, 8.55. Found: C, 51.25; H, 4.56; N, 8.43. 6h: Yield 73%, mp
130–132 8C; MS (ESI) m/z 517 [M+H]+; 1H NMR (300 MHz, CDCl3):
d
8.09 (d, 2H, J = 7.5 Hz), 7.79 (m, 1H), 7.68 (m, 2H), 7.62 (d, 1H,
J = 16.2 Hz), 6.77–7.16 (m, 3H), 6.43 (d, 1H, J = 16.2 Hz), 4.53 (t, 2H,
J = 4.5 Hz), 3.84 (d, 2H, J = 4.5 Hz), 2.93 (m, 4H), 2.64 (m, 4H); Anal.
Calcd. for C23H24N4O8S: C, 53.48; H, 4.68; N, 10.85. Found: C, 53.65;
H, 4.86; N, 10.69. 6n: Yield 70%, mp 121–123 8C; MS (ESI) m/z 506
[M+H]+; 1H NMR (300 MHz, CDCl3):
d 8.06 (m, 2H), 7.75 (m, 1H),
7.64 (m, 2H), 7.57 (d, 1H, J = 16.2 Hz), 6.75–7.13 (m, 3H), 6.38 (d,
1H, J = 16.2 Hz), 4.40 (m, 2H, J = 4.5 Hz), 4.31 (m, 2H), 3.93 (m, 2H),
3.82 (t, 2H, J = 4.5 Hz); Anal. Calcd. for C22H23N3O9S: C, 52.27; H,
4.59; N, 8.31. Found: C, 52.03; H, 4.78; N, 8.13. 6p: Yield 67%, mp
137–139 8C; MS (ESI) m/z 531 [M+H]+; 1H NMR (300 MHz, CDCl3):
6b–d with
a three-carbon or four-carbon linker. However,
compounds 6f with diglycolamine linker displayed a significant
anti-tumor activity. The precise mechanisms of these active
compounds require further investigation.
d
8.05 (d, 2H, J = 7.5 Hz), 7.76 (m, 1H), 7.66 (m, 2H), 7.60 (d, 1H,
J = 16.2 Hz), 6.73–7.14 (m, 3 H), 6.41 (d, 1H, J = 16.2 Hz), 4.55 (t, 2H,
J = 4.5 Hz), 3.84 (d, 2H, J = 4.5 Hz), 2.95 (m, 4H), 2.66 (m, 4H); Anal.
Calcd. for C24H26N4O8S: C, 54.33; H, 4.94; N, 10.56. Found: C, 54.15;
H, 5.20; N, 10.38.
4. Conclusion
In summary, a series of novel furoxan-based nitric oxide-
releasing derivatives 6a–m of hydroxylcinnamic acids were
synthesized by coupling the carboxyl group of hydroxylcinnamic
acids with furoxan through various alkylol amines. Compounds 6a,
6e–i and 6m–p displayed more potent anti-tumor activities
superior to control 5-fluorouracil (5-FU) in most cancer cells
tested. Furthermore, 6f could selectively inhibit tumor cells, but
not non-tumor cell proliferation. This inhibition was attributed to
high levels of NO released in cancer cells and the potentially
synergistic effect of NO donor moieties and the bioactivity of
hydroxylcinnamic acids.
3. Results and discussion
The cytotoxicities of target compounds 6a–p against human
hepatocellular carcinoma cells (SMMC-7721 and HepG2) and
human breast cancer cells (MCF-7) were evaluated in vitro by MTT
method, using 5-fluorouracil (5-FU), 2h (NO donor moiety) and
adriamycin as control. The results (shown in Table 1) illustrated
the IC50 values of the target compounds against each tumor cell
line. Compounds 6a, e–i and m–p showed significant cytotoxic
activities which were stronger than that of 5-FU. Especially, the
IC50 of 6f (3.8–7.3
less than those of 5-FU (16.7–43.3
those of 2h (11.2–20.3 mol/L), and slightly weaker than those of
adriamycin (0.9–2.1 mol/L). More importantly, the hybrids 6h
and 6p had stronger anticancer activity than their corresponding
NO donor moiety 2h, suggesting a synergistic effect of furoxan and
hydroxylcinnamic acid in inhibition of cancer cell proliferation.
m
mol/L) against three tumor cells was 4-6-fold
References
m
mol/L), nearly 3-fold less than
m
m
Next, it was found that compound 6f (2–16
mmol/L) inhibited
HepG2 cell proliferation in a concentration-dependent manner,
but 6f had little inhibitory effect on non-tumor liver LO2 cell
Lu, Ming-Dong
