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H. Y. Jang et al. / Bioorg. Med. Chem. Lett. 26 (2016) 5438–5443
Table 1
measured the amount of nitric oxide (NO) which is one of the
essential mediators on inflammation induced by lipopolysaccha-
ride (LPS) in macrophage-derived RAW 264.7 cells.22 In fact, it is
difficult to quantify NO intrinsically in view of its short half-life
and existence of other scavenging molecules. Hence, measurement
of its accumulated stable degradation products nitrite (NOꢀ2 ) and
nitrate (NOꢀ3 ) is preferred and the Griess reagent is employed for
this combined (nitrite + nitrate) measurement.
Anti-inflammatory activities of dihydrostilbenes (1–5) and diarylpropanes (6–10)
Compound
NO production (% inhibition)
10
1
lM
lM
Medium(MED)
LPS
1
2
3
4
5
6
7
8
9
10
1.2 0.7 (98.8)⁄⁄⁄
100.0 7.4 (0.0)
92.6 19.6 (7.4)
64.8 2.7 (35.2)
57.3 6.8 (42.7)
75.7 9.7 (24.3)
83.0 22.3 (17.0)
72.8 8.1 (27.2)
61.8 8.4 (38.2)
70.4 20.5 (29.6)
63.1 8.6 (36.9)
59.2 2.1 (40.8)
85.0 4.4 (15.0)
1.2 0.7 (98.8)⁄⁄⁄
100.0 7.4 (0.0)
21.6 5.2 (78.4)⁄⁄⁄
70.5 3.9 (29.5)
53.0 1.4 (47.0)
24.5 5.8 (75.5)⁄⁄⁄
71.7 11.6 (28.3)
78.2 3.4 (21.8)
43.5 1.1 (56.5)
71.4 12.0 (28.6)
0.0 0.0 (100.0)⁄⁄⁄
46.4 8.3 (53.6)
9.9 2.7 (90.1)⁄⁄⁄
Anti-inflammatory activity: Effects of compounds 1–10 on NO
generation by induced macrophages was monitored (Table 1).
Lipopolysaccharide (LPS) treated RAW 264.7 has been used to
stimulate the production of NO through the activation of iNOS
and NG-monomethyl- -arginine acetate (L-NMMA)23 was
L
employed as positive control. Though, NO inhibition activity con-
ducted at 0.1, 1, 5, 10, 50 and 100 M concentrations, significant
activity changes were observed at 1–10 M. At 50 and 100
l
L-NMMA
l
lM
The results are reported as mean value SEM for n = 3. Statistical significance is
based on the difference when compared with LPS-treated groups (⁄⁄⁄P < 0.001).
% Inhibition is based on LPS as shown in parenthesis.
concentrations, all compounds exhibited same level activity (more
than 80% NO inhibition by each compound) (Fig. 2). Hence, we dis-
cussed the activity at 1 and 10 lM concentrations only. At these
concentrations, all the tested compounds decreased NO production
in a concentration-dependent manner except compounds 2, 6 and
8. The percentage of NO production inhibition ranged from 100.0%
and olefinic bond reduction of stilbenes 32–36 were achieved in
one step using excess triethylsilane (12–15 equiv) and Pd/C sys-
tem21 in a very short reaction time (20 min) to furnish the desired
dihydrostilbenes 1–5 in good yields, respectively.
to 21.8% and from 42.7% to 7.4% at the highest (10
lM) and lowest
(1 lM) concentrations, respectively. Of the 10 compounds (1–10)
prepared in the present study, 3 compounds i.e., 9 (hindsiipropane
B) (100.0%), followed by 1 (78.4%) and 4 (75.5%) showed the stron-
Next, we envisioned that the synthesis 1,3-diarylpropanes 6–10
could be achieved from their corresponding chalcone derivatives.
Accordingly, the synthesis began with ethoxymethyl- (EOM-) pro-
tection of phenolic aldehydes 12 and 14 using chloromethyl ethyl
ether (EOM-Cl), K2CO3 and catalytic tetrabutylammonium iodide
(TBAI) in anhydrous DMF (Scheme 2).
gest inhibitory activities at 10
Next, the activated RAW 264.7 cell viability was carried out at
1–100 M concentrations to ensure that cell death was not respon-
lM (Table 1 and Fig. 2).
l
sible for the decreased NO expression in compound (1–10)-treated
group by the MTT cell viability assay (Table 2). Except compound 1
(moscatilin), all the compounds did not had significant cytotoxicity
Selective protection of aldehyde 20 was achieved with N,N-
diisopropylethylamine (DIPEA) to give di-EOM protected aldehyde
40, which upon methylation yielded compound 41, however, pro-
tection of 20 using NaH instead of DIPEA as base produced all-pro-
tected 39 without selectivity. 3-Methoxy-4-hydroxyacetophenone
(42) and 3,5-dihydroxyacetophene (43) were also protected with
EOM-group by treating with EOM-Cl, K2CO3 and TBAI system. Next,
Claisen–Schmidt condensation between acetophenones 44, 48, 45
and aldehydes 37, 38, 39, 41, 25 in the presence of KOH as base
in MeOH/H2O (2/1) at room temperature afforded the EOM-pro-
tected chalcones 46–51, respectively. Finally, deprotection of
EOM- group using 6 N HCl followed by complete enone group
reduction of 46–51 under hydrogen atmosphere furnished the
desired 1,3-diarylpropanes 6–10, respectively. The structures of
the final compounds 1–10 were settled from their spectral (1H &
13CNMR and MS) data.
at 10
production. At 50
whereas at 100
l
M concentration which leading to effective inhibition of NO
M concentration also similar results observed,
M concentration, in addition to compound 1,
l
l
compounds 8–10 also displayed cytotoxicity. IC50 values of com-
pounds 1–10 were evaluated by using GraphPad Prism 4.0 soft-
ware and showed 4.40, 13.77, 6.02, 2.98, 14.53, 16.4, 4.46, 10.72,
1.84 and 1.30
had good inhibition of NO production (78.4%) at 10
tion with an IC50 value of 4.40 M, its inhibitory effect seems to be
more likely related to its cytotoxic effect towards the RAW 264.7
cells (only 52.9% cell viability at 10 M). These results indicate that
9 (hindsiipropane B) possessed the most potent NO inhibition
activity with an IC50 value of 1.84 M which is even better than
lM, respectively (Table 2). Although compound 1
l
M concentra-
l
l
l
the positive control L-NMMA (IC50 2.73). Next, compound 4 had
a little weaker activity than L-NMMA with an IC50 value of
2.98 lM. This findings were also in accordance with the previous
In order to evaluate the anti-inflammatory effects of the pre-
pared dihydrostilbenes (1–5) and 1,3-diarylpropanes (6–10), we
literature reports,24 wherein, compound 1 and analogues of com-
pound 5 were reported as poor NO inhibitors. To understand the
underlying molecular mechanisms by which compounds 1–10
reduces LPS-induced NO production, we further studied the effect
of 1–10 on iNOS protein expression in RAW 264.7 cells using
Western blot analysis. As shown in Figure 3, the results were con-
sistent with the findings related to NO production (Table 1 and
Fig. 2), the protein expression of iNOS induced by LPS in RAW
264.7 cells was dramatically suppressed by compound 9 (hindsi-
ipropane B) treatment. This indicates that the reduced expression
of iNOS due to these compounds exposure was responsible for
the inhibition of NO production.
From the aforementioned pharmacological results, some
structural features that might have influenced the NO inhibitory
activity can be drawn from the comparison of the chemical struc-
tures of the compounds 1–10. (i) Compounds (9 and 4) bearing
300,400-dimethoxyphenyl and 30,40-dihydroxy-20-methoxyphenyl
moieties, were fruitful to show potent NO inhibition with no
Figure 2. Inhibition of iNOS mediated NO production by compounds 1–10.
Jang, Ha Young
