Synthesis of eugenol-derived glucosides and evaluation of their ability in inhibiting the angiotensin converting enzyme

Article abstract of DOI:10.1080/14786419.2020.1827399

We report here a series of glucosides which are active as inhibitors of the angiotensin converting enzyme (ACE). They are structurally related to the natural compound eugenol and exhibited significant inhibition values. Their syntheses were expeditious and we could obtain informative docking plots of them complexed to this enzyme. A glucoside derived from eugenol, carrying a carboxylic group in the aglycone, was the most active of them (with an IC₅₀ of 0.4 mM) and showed good binding energies in docking studies with ACE. Moreover, computational prediction of toxicity risks, physicochemical properties and drug score show that the glucoside derivative of eugenol is a suitable compound for optimisation studies aimed at finding new drug candidates.

Full text of DOI:10.1080/14786419.2020.1827399

Natural Product Research
Formerly Natural Product Letters
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Synthesis of eugenol-derived glucosides and
evaluation of their ability in inhibiting the
angiotensin converting enzyme
Dalila Junqueira Alvarenga , Laira Maria Faria Matias , Cleydson Finotti
Cordeiro , Thiago Belarmino de Souza , Stefânia Neiva Lavorato , Marília
Gabriella Alves Goulart Pereira , Danielle Ferreira Dias & Diogo Teixeira
Carvalho
To cite this article: Dalila Junqueira Alvarenga , Laira Maria Faria Matias , Cleydson Finotti
Cordeiro , Thiago Belarmino de Souza , Stefânia Neiva Lavorato , Marília Gabriella Alves Goulart
Pereira , Danielle Ferreira Dias & Diogo Teixeira Carvalho (2020): Synthesis of eugenol-derived
glucosides and evaluation of their ability in inhibiting the angiotensin converting enzyme, Natural
Product Research, DOI: 10.1080/14786419.2020.1827399
To link to this article: https://doi.org/10.1080/14786419.2020.1827399
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2020.1827399
Synthesis of eugenol-derived glucosides and evaluation
of their ability in inhibiting the angiotensin
converting enzyme
Dalila Junqueira Alvarengaa , Laira Maria Faria Matiasa, Cleydson Finotti
a
b
c
^
ꢀ
Cordeiro , Thiago Belarmino de Souza , Stefania Neiva Lavorato , Marılia
Gabriella Alves Goulart Pereirad, Danielle Ferreira Diase and
Diogo Teixeira Carvalhoa
a
b
^
^
Faculdade de Ciencias Farmaceuticas, Universidade Federal de Alfenas, Alfenas, MG, Brazil; Escola
c
ꢀ
^
de Farmacia, Universidade Federal de Ouro Preto, Ouro Preto, MG, Brazil; Centro das Ciencias
Biologicas e da Saude, Universidade Federal do Oeste da Bahia, Barreiras, BA, Brazil; Instituto de
Ciencias Biomedicas, Universidade Federal de Alfenas, Alfenas, MG, Brazil; Instituto de Quımica,
d
ꢀ
ꢀ
e
^
ꢀ
ꢀ
Universidade Federal de Alfenas, Alfenas, MG, Brazil
ABSTRACT
ARTICLE HISTORY
Received 31 January 2020
Accepted 5 September 2020
We report here a series of glucosides which are active as inhibi-
tors of the angiotensin converting enzyme (ACE). They are struc-
turally related to the natural compound eugenol and exhibited
significant inhibition values. Their syntheses were expeditious and
we could obtain informative docking plots of them complexed to
this enzyme. A glucoside derived from eugenol, carrying a carbox-
ylic group in the aglycone, was the most active of them (with an
IC50 of 0.4 mM) and showed good binding energies in docking
studies with ACE. Moreover, computational prediction of toxicity
risks, physicochemical properties and drug score show that the
glucoside derivative of eugenol is a suitable compound for opti-
misation studies aimed at finding new drug candidates.
KEYWORDS
Glucosides; eugenol;
angiotensin converting
enzyme; enzyme inhibitors
CONTACT Diogo Teixeira Carvalho
diogo.carvalho@unifal-mg.edu.br
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2020.1827399.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

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D. J. ALVARENGA ET AL.
1. Introduction
Angiotensin converting enzyme (ACE) catalyses an important step in the biosynthesis
of mediators that control blood pressure levels. Its hydrolytic action occurs on the
peptide angiotensin I, which in turn leads to the formation of angiotensin II (Zaman
et al. 2002; Atlas 2007). Drugs that act as ACE inhibitors (ACEi) have been used for
more than 30 years and all of them present large numbers of polar groups, which is
essential for affinity to ACE (Lima 1999; Tavares et al. 2015). Although there has been
treated with ACE inhibitors for such a long time (with few side effects) there are some
patient studies to understand the variability of responses to ACE inhibitors, since
about 20% of patients are unable to respond to treatment. This means that in these
patients, the use of ACEi is not able to return the blood pressure to normal levels
(Papademetriou et al. 2004; Schilders et al. 2014; Danilov et al. 2017). These observa-
tions have also raised interest in investigating new inhibitors that have other structural
profiles (Li et al. 2015). In this regard, various natural products are studied as ACE
inhibitors. Some reports have shown that eugenol (1) (Figure 1) can inhibit ACE via
in vitro experiments but its physical properties as the oily state, low palatability, volatil-
ity and propensity to oxidation do not encourage further studies of eugenol (1) as a
candidate for an ACE inhibitor (ACEi) (Jeng et al. 1994). Even so, these limitations may
be overcome by designing optimised derivatives, which would not present these
drawbacks whilst still being active as ACEi. Our rationale for planning eugenol deriva-
tives was based on reports that glycosylation may lead to more stable products, whilst
giving better pharmacokinetic profiles (Li et al. 2015). It has been reported that some
natural glucosides are active as ACEi. Figure 1 shows a number of these glucosides
including (a) calceolarioside B (Li et al. 2015) and (b) junipediol A b-glucoside
(Simaratanamongkol et al. 2014). In addition, Lohith et al. (2006) has shown that

NATURAL PRODUCT RESEARCH
3
Figure 1. Structures of eugenol (1) and some natural glycosides (a-g) with ACE inhibitory activity.
separate mixtures of 1-O-glycosides and 6-O-glycosides of vanillin (c, d and e) and
eugenol (f and g) were active as ACEi in the range of 0.5-1.0 mM. In this study, we
proposed to synthesise and evaluate as ACEi, a pure eugenol b-glucoside and various
analogues resulting from modifications in the aglycone unit.
2. Results and discussion
To prepare the glucosides, we followed the synthetic route depicted in Figure 2
(detailed synthesis information is described in the supplementary file available online).
The purity of all products was confirmed by spectroscopic techniques and the synthes-
ised products demonstrated analytical data in accordance with that previously
described in the literature. These products were subsequently screened as ACEi
according to the methodology described by Friedland and Silverstein (1976). Firstly,
we performed an ACE inhibition assay with all the available compounds using the
Friedland and Silverstein methodology (1976). Compounds (1), (3), (5), (8) and (10) pre-
sented at least 60% ACE inhibition at a concentration of 2 mM. Secondly, these com-
pounds were tested via an IC50 assay, where the value curves were calculated and the
results can be found in Table 1.
IC50 values for eugenol can be found in the literature, with IC50 values ranging from
0.3-0.8 mM (Mnafgui et al. 2013; Chaudhary et al. 2014). However, the results gener-
ated in this study showed an average IC50 value of 1.6 mM. It is noteworthy that the
eugenol glucoside (3) had its potency increased as an ACEi. Whereas, the hydroxyl-
ation of the allyl chain (5) led to an inactive product. The shortening of the methylene
chain in the benzyl derivative (10) reduces the IC50 value slightly when compared to
eugenol inhibition but does not equal it to compound (3). The best result was found
with the glucoside (8), wherein a carboxylic group replaced the allylic chain. In this
case, the glucoside was about four times more potent than eugenol and twice as
potent as the glucoside (3).

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D. J. ALVARENGA ET AL.
Figure 2. Reagents and conditions to the synthesis of compounds: a) 2,3,4,6-tetra-O-acetyl-a-D-glu-
copyranosyl bromide, K2CO3, TBAB, CH2Cl2, H2O, r.t.; b) KOH, MeOH, 25 ꢀC; c) BH3.(CH3)2S, THF, 0 ꢀC
and then 25 ꢀC, followed by 4 M NaOH, 5% H2O2, 0 ꢀC to 25 ꢀC; d) KMnO4, H2O, 80 ꢀC; e) NaBH4,
aqueous NaOH, EtOH, 0 ꢀC to 25 ꢀC, followed by aqueous HCl, 0 ꢀC to 25 ꢀC.
Figure 3. Interaction of compounds 1, 3, 5, 8 and 10 (white carbons) with the active site of ACE
(green carbons). Caption: gray dashed lines - ion-dipole interactions; green dashed lines - hydrogen
bonds; yellow dashed lines - electrostatic interactions; pink dashed lines - pi-pi stacking interaction.
Docking studies were performed to understand how these compounds interact
with ACE active sites (Table 1). In general, the binding energy of more stable com-
plexes between ACE and the evaluated compounds followed the experimental data of
enzyme inhibition. Compound (8), which was the most active via in vitro evaluation,
also had the greatest inhibitory potential in the docking studies which indicated that
they predicted correctly the most potent compound. The main difference in the inter-
action of ACE and the evaluated compounds seems to be the mode of their inter-
action with the zinc atom present in the ACE catalytic site. For compound (8),
electrostatic interaction between its carboxylate group and the ACE zinc is predicted,
while for the others the prevalence of an ion-dipole interaction between zinc and
another oxygen atom of their structure is noted.

NATURAL PRODUCT RESEARCH
5

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D. J. ALVARENGA ET AL.
In the case of eugenol, the phenolic and methoxy oxygen atoms that take part in
this interaction. With glucosides (3) and (10) the saccharide hydroxyls participate in
the interaction with the catalytic zinc. Except in the case of compound (5), glucosyla-
tion appears to contribute to an inhibitory activity improvement via both in silico and
in vitro studies. This may be related to the greater number of hydrogen bonds avail-
able to be made with the ACE active site. This corroborates the study by Li et al.
(2015) that states that these saccharide hydroxyls have a direct influence on the zinc
chelation at the active site of the enzyme. In view of the interesting inhibition results,
a prediction of the toxicity risks was also made (Table 1). In the case of eugenol (1);
mutagenicity, tumorigenicity and irritant effects are expected. The predicted toxic
effects were basically associated with the overall structure of eugenol in the case of
mutagenic potential and the allyl-aryl chain in the case of the two other features. It
can be noted that the mutagenic potential can be reduced with the glucosylation
strategy. However, safety related to tumorigenic and irritant effects is only achieved
when the allyl chain is modified, as predicted for the glucoside (8) (Figure 3).
Despite the improvement in the toxicological profile, the presence of a saccharide
unit and the changes in aglycone contribute to a considerable reduction in the ClogP
values of (5), (8) and (10), which could compromise their oral bioavailability. A good
absorption through gastrointestinal membranes is observed for compounds having a
ClogP ranging from 1 to 3 (Hetal et al. 2010; Gibson 2016). Since these three com-
pounds have negative ClogP values, indicating low lipophilic features, a low ability to
cross lipid membranes can be expected - which compromises its intestinal absorption
and its oral bioavailability. Even with this limitation, the contribution of its toxico-
logical and physicochemical features as a whole provides an intermediate Drug Score
value which is higher than that of eugenol and its glucosylated derivative (3) - indicat-
ing better qualities regarding drug evolution. To improve the lipophilicity of com-
pound (8), one of the alternatives that could be explored would be employing the
synthetic intermediate (7), which is the peracetylated precursor of compound (8). This
could behave as a prodrug to be hydrolysed in vivo to the parent active glucoside.
Peracetylation does not interfere with the toxicological profile of the glucoside (8), as
shown in Table 1, but contributes to enhancing its ClogP value bringing it close to 1 -
which could already improve its intestinal absorption. Moreover, as this value is less
than 2, this would contribute to the lower possibility of the peracetylated derivative
accessing the central nervous system, contributing only to the expected peripheral
action. Finally, the calculated Drug Score for the peracetylated derivative of (8) is
higher (0.66) than that of captopril (0.61), indicating better ADMET properties
(Absorption, Distribution, Metabolism, Excretion and Toxicity). The use of molecular
docking studies, as well as ADMET property prediction findings, would allow for struc-
tural modifications aimed at enhancing both the inhibitory profile of these compounds
and their pharmacokinetic and toxicological behaviour.
3. Conclusions
The glucosides reported herein are promising candidates for additional structural opti-
misation as ACEi, especially glycoside compound (8). The interchange of the allylic

NATURAL PRODUCT RESEARCH
7
chain of the eugenol glycoside by a carboxylic group considerably reduces its IC50
value. The saccharide unit seems to be the main responsible for interaction with the
zinc in the ACE active site, whereas in glycoside (8) the carboxylate unit better does
this role. Moreover, ADMET property prediction shows that glycoside (8) has a good
drug score and that its peracetyl derivative may be an alternative for enhancing its
lipophilicity.
Supporting information
The experimental section and spectroscopic data associated with this article can be
found in the supporting information section.
Disclosure statement
The authors declare that there are no conflicts of interest.
Funding
This work was supported by CAPES (Financial code 001) and Conselho Nacional de
ꢀ
ꢀ
Desenvolvimento Cientıfico e Tecnologico (CNPq, grant 449032/2014-0).
ORCID
Dalila Junqueira Alvarenga
http://orcid.org/0000-0002-0804-648X
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