The synthesis and Angiotensin Converting Enzyme (ACE) inhibitory activity of chalcones and their pyrazole derivatives

Article abstract of DOI:10.1016/j.bmcl.2010.01.113

A series of chalcones (1-9) and pyrazoles (10-18) was prepared to investigate their potential activity as Angiotensin I-Converting Enzyme (ACE) inhibitors. Their structures were verified by elemental analysis, UV, IR, MS, ¹H NMR, ¹³C

Full text of DOI:10.1016/j.bmcl.2010.01.113

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1990–1993
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The synthesis and Angiotensin Converting Enzyme (ACE) inhibitory activity
of chalcones and their pyrazole derivatives
a
a
b
b
Marco Bonesi a, , Monica R. Loizzo , Giancarlo A. Statti , Sylvie Michel , François Tillequin ,
*
Francesco Menichini a
a Department of Pharmaceutical Sciences, Faculty of Pharmacy, Nutrition and Health Sciences, University of Calabria, I-87030 Rende (CS), Italy
b Laboratoire de Pharmacognosie de l’Université René Descartes, U.M.R./C.N.R.S. No. 8638, Faculté des Sciences Pharmaceutiques et Biologiques, 4, Avenue de l’Observatoire,
F-75006 Paris, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of chalcones (1–9) and pyrazoles (10–18) was prepared to investigate their potential activity as
Angiotensin I-Converting Enzyme (ACE) inhibitors. Their structures were verified by elemental analysis,
UV, IR, MS, 1H NMR, 13C NMR, and 2D NMR experiments. Among tested compounds, chalcone 7 exerted
the highest activity with an IC50 value of 0.219 mM, while the most potent pyrazole was 15 (IC50 value of
0.213 mM).
Received 19 October 2009
Revised 19 January 2010
Accepted 20 January 2010
Available online 25 January 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Chalcones
Pyrazole derivatives
Hypertension
ACE
Hypertension is a common and often progressive disorder that
poses a major risk for cardiovascular and renal disease.1,2 Recent
data revealed that the global burden of hypertension is an impor-
tant and increasing public health problem worldwide and that the
level of awareness, treatment, and control of hypertension varies
considerably among countries. In 2000, 972 million of the world-
wide adult population P18 years of age was affected by hyperten-
sion, and this number is predicted to increase of 1.56 billion
individuals by 2025.3
Angiotensin Converting Enzyme (ACE) is the most important
regulatory site of RAS. ACE is a dipeptidyl carboxypeptidase that
inactivates the vasodepressor compound bradykinin and activates
the potent vasoconstrictor and growth-promoting substance
angiotensin II by the removal of the carboxy-terminal dipeptide
of angiotensin I. The major physiological function of ACE is linked
to the regulation of blood pressure and electrolyte homeostasis by
converting angiotensin I into potent vasoconstrictor angiotensin II
and by inactivating bradykinin.5
From a physiopathological viewpoint, hypertensive disease in-
volves changes in at least one of hemodynamic variables (cardiac
output, arterial stiffness, or peripheral resistance) that determine
the measurable blood pressure. Each of these variables is a poten-
tial therapeutic target, and it is likely that changes in these vari-
ables also contribute to the heterogeneity in the pharmacologic
response of patients with hypertension. Therefore, modern treat-
ment strategies should not only focus on blood pressure reduction
but also in normalizing vascular structure and function.
It is well recognized that the Renin–Angiotensin System (RAS)
has an important role in cardiovascular physiology, water–electro-
lyte balance, and cell function. Excessive activation of this system
has been considered to be a main cause of hypertension.4
The importance of ACE inhibitors in the chronic treatment of
various cardiovascular diseases such as myocardial infarction, con-
gestive heart failure, diabetic nephropathy, or renal dysfunction is
well known.6–10 In fact, ACE inhibitors, namely captopril, enalapril,
fosinopril, and ramepril, are currently available in the market for
the treatment of hypertension. However, some of these ACE inhib-
itors have certain limitations like susceptibility to proteolytic deg-
radation leading to side effects.11
Even today natural products are used for the development of
new drugs. In fact, in the last two decades, about 50% of the drugs
introduced on the market are derived from a natural source.12
Epidemiological evidence suggests an inverse relationship be-
tween dietary intake of flavonoids and isoflavonoids and the risk of
coronary heart disease.13 Chalcones, or 1,3-diaryl-2-propen-1-ones,
are prominent secondary metabolite precursors of flavonoids and
isoflavonoids in plants. Chemically, they consist of open-chain flavo-
noids in which the two aromatic rings are joined by a three-carbon
* Corresponding author. Tel.: +39 984 493169; fax: +39 984 493298.
E-mail address: bonesi@unical.it (M. Bonesi).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.113

M. Bonesi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1990–1993
1991
a
,b-unsaturated carbonyl system. The wide variety of pharmacolog-
The in vitro ACE inhibitory activity was detected through the
cleavage of the chromophore–fluorophore, substrate dansyltrigly-
cine in dansylglycine and unreacted substrate that are separated
and quantified by reversed phase high performance liquid chroma-
tography (HPLC) with UV detection in specific, highly sensitive and
reproducible assay.22,23
On the basis of the role of Angiotensin Converting Enzyme
inhibitors, in the therapeutic approach of hypertension, there have
been renewed efforts in modifying available drugs or in developing
new drugs with less side effects.
Chalcones and their derivatives exerted a lot of biological prop-
erties, but few previous reports referred the ability of these classes
of compounds in lowering blood pressure via the inhibition of
ACE.19 Chalcones and their pyrazole derivatives synthesized by
our procedure inhibited ACE activity, in a concentration-dependent
manner (Figs. 1 and 2). Results are presented and statistically ana-
lyzed in Table 1.24
ical activities reported for these compounds include antimalarial,
anti-inflammatory, cytotoxic, anticancer, and antioxidant proper-
ties.14–18 Recently, the ACE inhibitory activity was also reported.19
Therefore, the aim of the present investigation was to prepare a
number of prototypic molecules (chalcones) and related analogues
(pyrazoles) in order to evaluate the ability to inhibit ACE enzyme.
The antihypertensive potential of these compounds appears to be a
field that is not very explored and needs attention.
Aldolic condensation of 3,4,5-trimethoxy-acetophenone with
appropriately substituted benzaldehydes was carried out in order
to synthesize a set of chalcone derivatives with amino, fluoro, hy-
droxyl, methoxy, and nitro substituents on the B-ring, using com-
mercially available compounds (Scheme 1).20
The reaction of chalcones 1–9 with methyl-hydrazine in tet-
rahydrofuran anhydrous under argon produced the correspond-
ing pyrazole derivatives 10–18 in which the 2-propen-1-one
group was converted into the 4,5-dihydro-1H-pyrazole group
(Scheme 2).21
In the chalcone series the most active compound was 7 (IC50
0.219 mM), which was substituted with an amino group in posi-
O
OCH3
CHO
H3CO
H3CO
H3CO
(i)
+
A
B
CH3
R2
R1
H3CO
R2
OCH3
R1
O
Compds
R1
H
OCH3
H
OH
OCH3
NO2
NH2
OH
F
R2
H
H
OCH3
OCH3
OH
OCH3
OCH3
NO2
Yield (%)
75
1
2
3
4
5
6
7
8
9
66
76
100
75
70
60
100
100
OCH3
Scheme 1. Chemical structure of chalcone derivatives. Reagents and conditions: (i) NaOH 50% (w/v), rt, MeOH, argon.
CH3
O
N
N
H3CO
H3CO
H3CO
H3CO
(ii)
A
B
A
B
R2
R2
R1
OCH3
R1
OCH3
Compds
10
R1
H
OCH3
H
OH
OCH3
NO2
NH2
OH
F
R2
H
H
OCH3
OCH3
OH
OCH3
OCH3
NO2
Yield (%)
41
40
53
46
64
55
58
49
45
11
12
13
14
15
16
17
18
OCH3
Scheme 2. Chemical structure of pyrazole derivatives. Reagents: (ii) CH3NHNH2, THF anhydrous, argon.

1992
M. Bonesi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1990–1993
IC50 value of 0.246 mM, is characterized by a methoxylic group in
R1 and a hydroxylic group in R2. Our data suggests that for the chal-
cones ACE inhibitory activity the presence of hydroxylic groups
could be essential. In particular, the highest activity was found
when this group was positioned in R1 (4 vs 5).
On the basis of the structure-relationship analysis in which R2
was substituted with a methoxylic group, the increase of activity
was observed when we make a reduction of R1 group (NO2/NH2)
as in 6 (IC50 0.516 mM). Maintaining the same group in R1 position
(OCH3 in 2 and 5) an increase of ACE inhibitory activity was ob-
served when the R2 position was substituted with a hydroxylic
group (IC50 values of 0.622 and 0.246 mM for chalcone 2 and 5,
respectively).
The pyrazole derivatives exerted IC50 values ranging from 0.213
to 0.889 mM. The most potent pyrazole is 15, substituted with a ni-
tro group in position R1 and a methoxylic group in R2. Compound
12 presents an activity which verified to be twofold less (IC50 of
0.435 mM). This pyrazole was characterized by the substitution
with hydrogen in R1 and a methoxylic group in R2. When these
substituents were inverted as in 11 an ACE inhibitory activity
reduction was observed (IC50 value of 0.882 mM).
Figure 1. Concentration-dependent inhibition of ACE by the most active com-
pounds (1, 4, 5, and 7) from the chalcone series.
The ACE inhibitory activity decreased when a reduction of R1
group from NO2 (15) to NH2 (16) was made (IC50 values of 0.213
and 0.749 mM for 15 and 16, respectively). Also when the R1 posi-
tion was substituted with a hydroxylic group instead of hydrogen
(12 vs 13) the activity lowered from 0.435 to 0.762 mM.
Perusal of the literature revealed various reports that demon-
strated the ACE flavonoids inhibition.18,19,25–31
Our data (IC50 values ranging from 213 to 889 lM) are in accor-
dance with a previous work that reported the ACE inhibitory activ-
ity of flavonoids with IC50 values ranging from 158.9 to
l
M.25 Moreover, Loizzo et al. (2007) reported the ACE inhibi-
Figure 2. Concentration-dependent inhibition of ACE by the most active com-
pounds (12 and 15) from pyrazole series.
708.8
tion by flavonoids isolated from Ailanthus excelsa [i.e., apigenin
(IC50 of 280 M), luteolin (IC50 of 290 M), kaempferol-3-O-b-
galactopyranoside (IC50 of 260 M), luteolin-7-O-b-glucopyrano-
side (IC50 of 280 M), quercetin glucuronide (IC50 of 200
M)].28
l
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Table 1
l
l
ACE inhibitory activity of chalcones (1–9) and pyrazoles derivatives (10–18)
ACE is a zinc-containing peptidyl dipeptide hydrolase.32 The ac-
tive site of ACE is known to consist of three parts; a carboxylate
binding functionality such as the guanidinium group of arginine,
a pocket that accommodates a hydrophobic side chain of C-termi-
nal amino acid residues and zinc ion. The zinc ion coordinates to
the carbonyl of the penultimate peptide bond of the substrate,
whereby the carbonyl group becomes polarized and is subject to
a nucleophilic attack. Therefore, some flavonoids33 showed an
in vitro activity via the generation of chelate complexes within
the active center of ACE. Kang et al.19 reported that butein exerted
ACE inhibition activity with IC50 of 0.730 mM, supposing that this
action may be due to the ability of this chalcone in creating a che-
late complex with zinc ions within the active center of ACE. Free
hydroxyl groups of phenolic compounds are suggested to be
important structural moieties which chelate the zinc ions, thus
inactivate the ACE activity.30
Our data demonstrated that the electron donator group has an
inhibiting ability on ACE probably due to the same mechanism.
The inhibition of ACE by synthesized molecules may be proba-
bly due to the rigid planar structure of the molecule and the pres-
ence of hydroxylation on the aromatic ring.34 Besides appropriate
hydroxylation, also a planar structure is indispensable for the
metallopeptidases inhibition.
Compds
IC50 (mM)a ( SD)
Chalcones
1
2
3
4
5
6
7
8
9
0.338 ( 0.002)
0.622 ( 0.003)
0.574 ( 0.003)
0.225 ( 0.001)
0.246 ( 0.003)
0.516 ( 0.004)
0.219 ( 0.003)
0.552 ( 0.002)
0.570 ( 0.003)
Pyrazoles
10
11
12
13
14
15
16
17
0.805 ( 0.007)
0.889 ( 0.009)
0.435 ( 0.003)
0.762 ( 0.003)
0.674 ( 0.002)
0.213 ( 0.002)
0.749 ( 0.003)
0.599 ( 0.003)
0.770 ( 0.004)
18
One-way ANOVA Analsyis: ***p <0.0001; Bonferroni’s Multiple Comparison Test:
**p <0.001 (1–18 vs Captopril); °p >0.05 3 versus 9, 4 versus 7, and 13 versus 18;
ꢀp <0.05 13 versus 16.
Values are means of three experiments, standard deviation is given in paren-
theses; captopril was used as positive control (IC50 20 lM).
a
Theuse of ACE inhibitorsis fraught with manyside effects like dry
cough, skin irritation, angioedema, and other problems in particular
with multiple dosing due to less bioavailability.35 Therefore, the
interest in ACE inhibitors research has increased in recent years.
In conclusion, a series of chalcones and pyrazoles was prepared
and tested for their potential ACE inhibitory activity. These com-
pounds offer the advantage of an easy and short synthesis.
tion R1 and a methoxylic group in position R2. This activity was
conserved when the amino group was substituted with a hydrox-
ylic group as in 4 (IC50 0.225 mM). The absence of a hydroxylic
group in position R1 as in 3 caused a reduction of ACE inhibition
activity (IC50 0.574 mM) (3 vs 4). Chalcone 5, which showed an

M. Bonesi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1990–1993
1993
solution of 3,4,5-trimethoxy-acetophenone and substituted benzaldehyde in
alcohol a quantity of an aqueous solution of sodium hydroxide (50% w/v) was
added. The mixture was stirred at room temperature under argon for 16 h,
diluted with dichloromethane (30 mL) and acidified to pH 1 with an aqueous
solution of hydrochloric acid 1 N. The separated aqueous layer was extracted
further with dichloromethane (3 Â 30 mL) and the combined organic fractions
dried over anhydrous magnesium sulfate, filtered and evaporated in vacuo. The
residue was purified by chromatography or crystallization. This procedure
affords the desired chalcones in yields that are always high. Their physico-
chemical properties and the spectra data can be found in Supplementary data.
21. Synthesis of pyrazoles (10–18): A solution of each chalcone 1–9 and methyl-
hydrazine in tetrahydrofuran anhydrous was stirred at room temperature
under argon for 4 h. The resulting mixture was dried over anhydrous
magnesium sulfate, filtered and evaporated in vacuo, and purified by
crystallization from ethyl acetate to yield the desired product. Their physico-
chemical properties and the spectra data can be found in Supplementary data.
The compounds bearing a pyrazole linkage between the two
aromatic rings generally appeared significantly less active than
those bearing a double bound. Therefore, a more flexible linker be-
tween the two phenyl rings appears favorable to increase the ACE
potency in this series. Consequently, compounds derived from 1–9
but with a reduced olefinic linkage should be considered as inter-
esting future prospects.
Acknowledgments
The authors wish to thank Dr. Annamaria Caufin, Faculty of
Pharmacy, Nutrition and Health Sciences, University of Calabria
for revision of the English version of the manuscript.
22. ACE inhibition assay: Dansylglycine and dansyl-
L-glutamine, Angiotensin I-
Converting Enzyme preparation from rabbit lung (EC 3.4.15.1) were purchased
from Sigma–Aldrich, Milan, Italy. Briefly, each compound was dissolved in
Supplementary data
HEPES assay buffer, to obtain the final concentration ranged from 330 to 50
mL. The ACE solution (25 L) was pre-incubated with a test or control solution
(25 L) for 5 min at 37 °C. The enzyme reaction was started by adding a
combined solution (25 L) of the substrate dansyltriglycine (7.86 mM), and the
internal standard, dansyl- -glutamine (0.353 mM) for a time of incubation
chosen by plotting a calibration curve. The reaction was stopped by adding a
solution of 0.1 N Na2EDTA (50 L). The dansylglycine was quantified by a HPLC
reversed phase with UV detection at 250 nm. Instrumentation: HPLC Perkin
Elmer Series 410 LC pump; Injector Perkin Elmer 20 L loop. Detector Perkin
lg/
l
Supplementary data (experimental procedures, NMR, UV, IR,
MS, and elementary analysis) associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.2010.01.113.
l
l
L
l
References and notes
l
Elmer UV/VIS LC290 spectophotometric; solvent system: ALTECH SN 1250-99,
Part. N° 288215 BIN II 43, HYPERSIL ODS 5u Lot. N° 5002.150 mm  4.6 mm
SN:1250-99; mobile phase: isocratic system—10 mM NaH2PO4 buffer (pH 7)/
acetonitrile (88:12); flow rate 2 mL/min, run time 30 min. Linear calibration
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