Polyphenols consumption has been associated to a lower risk of cardiovascular diseases (CVDs) notably through nitric oxide (NO)- and estrogen receptor α (ERα)-dependent pathways. Among polyphenolic compounds, chalcones have been suggested to prevent endothelial dysfunction and hypertension. However, the involvement of both the NO and the ERα pathways for the beneficial vascular effects of chalcones has never been demonstrated. In this study, we aimed to identify chalcones with high vasorelaxation potential and to characterize the signaling pathways in relation with ERα signaling and NO involvement. The evaluation of vasorelaxation potential was performed by myography on wild-type (WT) and ERα knock-out (ERα-KO) mice aorta in presence or in absence of the eNOS inhibitor Nω-nitro-L-arginine methyl ester (L-NAME). Among the set of chalcones that were synthesized, four exhibited a strong vasorelaxant effect (more than 80% vasorelaxation) while five compounds have shown a 60% relief of the pre-contraction and four compounds led to a lower vasorelaxation. We were able to demonstrate that the vasorelaxant effect of two highly active chalcones was either ERα-dependent and NO-independent or ERα-independent and NO-dependent.
Synthetic polyhydroxylated chalcones
,
,
and
were synthesized through a two-step procedure starting from the corresponding methoxymethyl (MOM)-protected acetophenones and benzaldehydes. Although this strategy required an extra deprotection step compared to Claisen-Schmidt condensation of phenolic starting materials, it has led to a better overall yield as observed for the synthesis of chalcone
compared to literature data
. Due to reactivity and efficiency issues in basic medium, chalcones
,
and
bearing a hydroxyl (OH) group in the C-4 position have been prepared through an acid-catalyzed coupling mechanism using thionyl chloride (SOCl
) in ethanol (EtOH). Thus it avoided the need for a synthesis strategy with a MOM protection-deprotection approach
. Nevertheless, in situ generated hydrochloric acid (HCl) catalysis was unsuccessful when applied to the synthesis of diphenolic chalcone
. Thus, it has been prepared under microwave (MW) irradiations in the presence of piperidine
. In this work two other synthetic chalcones
and
, bearing a phenol function on the C-2
position, have been prepared through selective demethylation of the corresponding 2’-methoxychalcones
and
in the presence of aluminum chloride (AlCl
)
. It is worth mentioning that the demethylation of hexamethoxychalcone
led only to
(52% yield) without any trace of
(Scheme 1). Finally, the calculated octanol-water partition coefficient (cLogP) were similar, ranging from 1.80 for synthetic chalcone
to 3.33 for synthetic chalcone
(Table 1).
General scheme for the synthesis of chalcones. Synthetic chalcones were synthesized through the coupling of the corresponding either MOM-protected or phenolic acetophenones and benzaldehydes. Hydrolysis of MOM groups or selective demethylation in the presence of AlCl
were achieved. These procedures led to thirteen synthetic polyoxygenated chalcones whose vasorelaxant effect was evaluated.
Synthesized chalcones and their related cLogP.
Compound |
R2’ |
R4’ |
R6’ |
R3 |
R4 |
R5 |
cLogP |
3 |
OH |
OH |
H |
OCH3 |
OH |
OCH3 |
2.42 |
6 |
,
and
with OH or OCH
group at the C-6’ position or unsubstituted at C-3 and C-5 positions induced an average vasorelaxation between 50% to 80%. Finally, compounds
,
,
and
with both OH and OCH
groups at C-2’, C-4’ and/or C-6’ positions or a bulkier OCH
COOH group at C-3 position exerted a low vasorelaxation effect below 50% (Figure 1 and Table 2).
Concentration-response curves for the vasorelaxation effect on WT mice thoracic aorta of synthetic polyoxygenated chalcones.
: vasorelaxation effect of chalcones with R
= R
= OH and R
= H or OH;
: vasorelaxation effect of chalcones with R
= R
= OCH
and R
= H or OCH
;
: vasorelaxation effect of chalcones with R
= OH, R
= OCH
and R
= H or OCH
;
: vasorelaxation effect of chalcones with R
= R
= OCH
and R
= H. Mice were ovariectomized 7 days before the experiment. Vessels were pre-contracted with U46619, phenylephrine, and serotonin, at 80 % of their maximal response. The presence of functional endothelium was assessed by determining the ability of acetylcholine (10 µM) to induce more than 50% relaxation of pre-contracted rings. Results are expressed in percentage of precontraction (
-axis) following the concentration (
-axis). Compounds
,
,
and
induced higher vasorelaxation, over 80%. Compounds
,
,
,
and
induced an average vasorelaxation between 50% to 80%. Compounds
,
,
and
exerted a low vasorelaxation effect below 50%.
= 4–7, *
< 0.05 (Mann-Whitney).
Summary of the potency and efficacy of chalcones.
Compound |
Potency (EC50, g/L) WT |
Efficacy (Emax, %) |
||||||||||||
WT |
WT with L-NAME |
ERα KO |
ERα KO with L-NAME |
|||||||||||
3 |
OH |
OH |
OH |
OCH3 |
2.44 × 10 |
OH |
OCH3 |
1.80 |
||||||
-3 |
84.28 ± 3.24a,d |
77.62 ± 4.92 |
56.23 ± 4.91£ |
43.22 ± 6.10$ |
8 |
OH |
||||||||
6 |
2.12 × 10-3 |
OH |
57.10 ± 10.40c,d |
H |
33.92 ± 12.66 | OCH3 |
60.69 ± 10.75 |
OCH3 |
38.15 ± 10.91 |
OCH3 |
2.75 |
|||
10 |
OH |
OH |
||||||||||||
8 |
2.99 × 10-3 |
89.96 ± 4.13b,c |
OH |
OCH3 |
OCH3 |
OCH3 |
2.13 |
|||||||
86.69 ± 3.47 | 79.73 ± 3.71 |
69.76 ± 7.99 |
11 |
OCH3 |
||||||||||
10 |
4.08 × 10 |
OCH3 |
-3 |
63.29 ± 12.79a,b |
H |
44.91 ± 15.07 |
H |
52.83 ± 12.88 | OH |
43.33 ± 10.37 |
H |
3.20 |
||
12 |
||||||||||||||
11 |
OH |
OCH3 |
H |
OCH3 |
OH |
OCH3 |
2.99 |
|||||||
1.93 × 10-2 |
67.35 ± 8.94g |
49.06 ± 9.47 |
74.81 ± 2.07 |
36.40 ± 5.81 |
13 |
OCH3 |
OCH3 |
H |
||||||
12 |
NA | OCH | 3 |
30.95 ± 13.23 | OH |
NA |
OCH3 |
NA |
2.84 |
|||||
NA |
14 |
OH |
OCH3 |
H |
OCH | |||||||||
13 | 3 | OCH |
5.06 × 10-2 |
80.86 ± 6.11 | 3 |
19.85 ± 7.81* |
81.96 ± 2.46 |
26.96 ± 6.72 |
OCH3 |
3.33 |
||||
15 |
OCH3 |
|||||||||||||
14 |
NA |
OCH3 |
48.38 ± 7.75 | H |
NA |
OCH3 |
4.64 ± 9.95 |
OCH3 |
NA |
OCH3 |
3.17 |
|||
16 |
||||||||||||||
15 |
OCH3 |
NA |
OCH3 |
90.59 ± 1.56e,f | OCH3 |
76.07 ± 3.72 | OCH3 |
OCH3 |
OCH3 |
3.13 |
||||
79.78 ± 5.51 | 76.74 ± 5.68 |
17 |
OCH3 |
OCH3 |
OCH3 |
OCH3 |
OH |
OCH3 |
2.79 |
|||||
16 |
NA |
75.89 ± 5.99f |
NA |
74.91 ± 10.15 |
NA |
18 |
OH |
OCH3 |
OCH3 |
OCH3 |
OH |
OCH3 |
||
17 |
NA |
72.16 ± 4.59e |
NA | 2.97 | ||||||||||
86.88 ± 4.40 | NA |
20 |
OCH3 |
OCH3 |
H |
OCH3 |
OCH2 | |||||||
18 |
NA | COOH |
24.34 ± 4.60 |
NA |
OCH3 |
2.46 |
Vasorelaxation induced by each synthetic chalcone was first evaluated on WT mice thoracic aorta rings. Compounds
,
,
and
with identical substituents at C-2’ and C-4’ positions, absence of substituent at the C-6’ position and presence of OH and/or OCH
groups at C-3, C-4 and C-5 positions induced a high vasorelaxation reaching more than 80%. Compounds
,
,
NA | |||||
NA | |||||
20 | |||||
NA |
40.77 ± 8.22g |
NA |
NA |
NA |
To evaluate the involvement of the ERα pathway in the chalcones-induced vasorelaxation, experiments have been conducted on ERα KO mice aortas. To focus on the role of OH or OCH
groups at C-6’ position with synthetic chalcones exhibiting a high vasorelaxant effect, compounds
,
,
,
,
,
,
and
have been tested on ERα KO mice thoracic aorta rings. Interestingly, only the synthetic chalcone
induced a significant lower vasorelaxation in ERα KO mice thoracic aorta rings compared to WT mice thoracic aorta rings (56.23 ± 4.91% vs. 84.28 ± 3.24%, respectively) while no significant differences were observed with synthetic chalcones
,
,
,
,
,
and
(Figure 2 and Figure 3 and Table 2).
Concentration-response curves for the vasorelaxation effect on both WT and ERαKO mice thoracic aorta of synthetic chalcones
(
),
(
),
(
) and
(
). Mice were ovariectomized 7 days before the experiment. Vessels were pre-contracted with U46619, phenylephrine, and serotonin, at 80 % of their maximal response. The presence of functional endothelium was assessed by determining the ability of acetylcholine (10 µM) to induce more than 50% relaxation of pre-contracted rings. Results are expressed in percentage of precontraction (
-axis) following the concentration (
-axis). Only the synthetic chalcone
induced a significant lower vasorelaxation in ERα KO mice thoracic aorta rings compared to WT mice thoracic aorta rings (56.23 ± 4.91% vs. 84.28 ± 3.24% respectively) while no significant differences were observed with chalcones
,
and
.
= 4–7, *
< 0.05 (Mann-Whitney).
Concentration-response curves for the vasorelaxation effect on both WT and ERαKO mice thoracic aorta of synthetic chalcones
(
),
(
),
(
) and
(
). Mice were ovariectomized 7 days before the experiment. Vessels were pre-contracted with U46619, phenylephrine, and serotonin, at 80 % of their maximal response. The presence of functional endothelium was assessed by determining the ability of acetylcholine (10 µM) to induce more than 50% relaxation of pre-contracted rings. Results are expressed in percentage of precontraction (
-axis) following the concentration (
-axis). No significant differences were observed with synthetic chalcones
,
,
and
.
= 4–7 (Mann-Whitney).
In order to evaluate the involvement of the NO pathway, experiments have then been performed after incubation for 20 min with NOS inhibitor (10
M) on both WT and ERα KO mice thoracic aorta rings. To focus on the role of groups at C-2’, C-4’ and C-4 positions, synthetic chalcones
,
,
and
have been tested. Interestingly, L-NAME prevented the chalcone-induced vasorelaxation only for chalcone
in WT mice thoracic aorta rings at the highest concentration (10
g/L, Figure 4 and Table 2). It is noteworthy that L-NAME seemed to prevent the chalcone-induced vasorelaxation for compounds
and
at low concentrations and this effect is not recovered at 10
g/L. No difference was observed for the synthetic chalcone
(Figure 4A,B). Similarly, the presence of L-NAME on ERα KO mice thoracic aorta rings prevented the vasorelaxation only with the synthetic chalcone
(81.96 ± 2.46% vs. 26.96 ± 6.72%) while having no effect with synthetic chalcones
,
and
(Figure 5 and Table 2).
Concentration-response curves for the vasorelaxation effect on WT mice thoracic aorta of synthetic chalcones
(
),
(
),
(
) and
(
) in the presence or in absence of L-NAME, a selective eNOS inhibitor. Mice were ovariectomized 7 days before the experiment. Vessels were pre-contracted with U46619, phenylephrine, and serotonin, at 80 % of their maximal response. The presence of functional endothelium was assessed by determining the ability of acetylcholine (10 µM) to induce more than 50% relaxation of pre-contracted rings. Results are expressed in percentage of precontraction (
-axis) following the concentration (
-axis). L-NAME only prevented the chalcone-induced vasorelaxation for the synthetic chalcone
at the highest concentration (10
g/L) and seemed to prevent the chalcone-induced vasorelaxation for compounds
and
at low concentrations. No difference was observed for chalcone
.
= 5–7, **
< 0.01 (Mann-Whitney).
Concentration-response curves for the vasorelaxation effect on ERαKO mice thoracic aorta of synthetic chalcones
(
),
(
),
(
) and
(
). Mice were ovariectomized 7 days before the experiment. Vessels were pre-contracted with U46619, phenylephrine, and serotonin, at 80 % of their maximal response. The presence of functional endothelium was assessed by determining the ability of acetylcholine (10 µM) to induce more than 50% relaxation of pre-contracted rings. Results are expressed in percentage of precontraction (
-axis) following the concentration (
-axis). L-NAME only prevented the chalcone
-induced vasorelaxation (81.96 ± 2.46% vs. 26.96 ± 6.72%) while having no effect with synthetic chalcones
,
and
.
= 4–7, **
< 0.01 (Mann-Whitney).
Structure-activity relationships (SAR) study allowed to identify the 3,5-dimethoxy-4-hydroxybenzene moiety as a pharmacophore for both a ERα- or NO-dependent vasorelaxation (Figure 6). Finally, the current study provides new promising data for the preventive and the therapeutic use of chalcones in the field of CVDs.
Figure 6. Structure-activity relationship of chalcones for vasodilation.