A novel and efficient synthesis of 3,4-dihydroxyphenylacetic ester and amide derivatives/conjugates and assessment of their antioxidant activity

Article abstract of DOI:10.1080/14786419.2017.1342090

Phenolic acids, a sub-class of polyphenols, are widely studied. By contrary, 3,4-dihydroxyphenylacetic acid is scarcely studied. For this purpose, a series of 3,4-dihydroxyphenylacetic acid ester and amide derivatives/conjugates were synthesised for the first time. A systematic study has been performed to quantitatively identify the functional groups present in these compounds using different techniques such as ¹H NMR, ¹³C NMR and ESI MS. The synthesised compounds were evaluated for their in vitro antioxidant activity by a DPPH radical-scavenging assay. Their physico-chemical profile is also studied using Molinspiration tool. Among all tested compounds, amidoester 36 showed the best scavenging activity possessing an EC₅₀ 17 μΜ and improved physico-chemical properties compared to the parent compound.

Full text of DOI:10.1080/14786419.2017.1342090

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
A novel and efficient synthesis of 3,4-
dihydroxyphenylacetic ester and amide
derivatives/conjugates and assessment of their
antioxidant activity
Aikaterini Pagoni & Stamatia Vassiliou
To cite this article: Aikaterini Pagoni & Stamatia Vassiliou (2017): A novel and efficient synthesis
of 3,4-dihydroxyphenylacetic ester and amide derivatives/conjugates and assessment of their
antioxidant activity, Natural Product Research, DOI: 10.1080/14786419.2017.1342090
To link to this article: http://dx.doi.org/10.1080/14786419.2017.1342090
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Date: 19 June 2017, At: 00:12

Natural Product research, 2017
https://doi.org/10.1080/14786419.2017.1342090
A novel and efficient synthesis of 3,4-dihydroxyphenylacetic
ester and amide derivatives/conjugates and assessment of
their antioxidant activity
Aikaterini Pagoni and Stamatia Vassiliou
laboratory of organic chemistry, department of chemistry, university of athens, athens, Greece
ABSTRACT
ARTICLE HISTORY
received 6 March 2017
accepted 21 May 2017
Phenolic acids, a sub-class of polyphenols, are widely studied. By
contrary, 3,4-dihydroxyphenylacetic acid is scarcely studied. For
this purpose, a series of 3,4-dihydroxyphenylacetic acid ester and
amide derivatives/conjugates were synthesised for the first time.
A systematic study has been performed to quantitatively identify
the functional groups present in these compounds using different
KEYWORDS
Polyphenols; phenolic
acids; antioxidant;
3,4-dihydroxyphenylacetic
acid; molinspiration;
conjugates; dPPh
techniques such as H NMR, 13C NMR and ESI MS. The synthesised
1
compounds were evaluated for their in vitro antioxidant activity by
a DPPH radical-scavenging assay. Their physico-chemical profile is
also studied using Molinspiration tool. Among all tested compounds,
amidoester 36 showed the best scavenging activity possessing an
EC50 17 μΜ and improved physico-chemical properties compared to
the parent compound.
1. Introduction
Polyphenols are secondary metabolites ubiquitously distributed in all higher plants, which
have important roles as defence against plant pathogens and animal herbivore aggression
and as response to various abiotic stress conditions, such as rainfall and ultraviolet radiation.
Over the course of the last 20 years, polyphenols have been studied for their potential
involvement in the prevention of chronic diseases, such as cardiovascular disease, cancer,
osteoporosis, diabetes mellitus and neurodegenerative diseases (Jubaraj Bikash Baruah
2011).
Phenolic acids, a sub-class of polyphenols, seem to be universally distributed in plants.
These compounds are the subject of a great number of studies relating to biological,
CONTACT stamatia Vassiliou
svassiliou@chem.uoa.gr
supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2017.1342090.
© 2017 Informa uK limited, trading as taylor & Francis Group

2
A. PAGONI AND S. VASSILIOU
chemical, agricultural and medical aspects. Among them, caffeic acid, ferulic acid, gallic acid
and protocatechuic acid have been extensively studied (Heleno et al. 2015).
By contrary, 3,4-dihydroxyphenylacetic acid or homoprotocatechuic acid (3,4-DHPAA) is
a member of this family that is scarcely studied. It is part of the so-called Olive Mill Waste
Water (OMWW) (Visioli et al. 2002; Brigante et al. 2006) that derives during the process of
production of olive oil and only recently, the scientific interest has turned to this ortho-di-
hydroxylated compound that seems to have the highest protective effect against oil oxida-
tion (Carrasco-Pancorbo et al. 2005). In the last decade, the first studies concerning DHPAA
started to appear and it seems to exhibit antiproliferative activity in prostate (Gao et al. 2006)
and colon cancer cells (Skrbek et al. 2009), to protect against pancreatic β-cell dysfunction
induced by high cholesterol (Carrasco-Pozo et al. 2015) and to exert neuroprotective action
against oxidative stress-induced cytotoxicity in human neuroblastoma SH-SY5Y cells
(Gonzalez-Sarrias et al. 2017).
One main issue of polyphenols is their poor pharmacokinetic properties and low bio-
availability due to their polar nature. The importance of increasing the lipophilic character
of polyphenols has already been noted. To achieve this goal, chemical reactions e.g. esteri-
fication, etherification or amidation have been proposed which improves their lipophilicity,
while preserving the beneficial properties (Cirillo et al. 2016).
Therefore, the aim of the present study was to synthesise three groups of 3,4-dihydroxy-
phenylacetic acid derivatives/conjugates, esters, amides and mixed amide/ester compounds
to examine their antioxidant properties in comparison with the parent compound and to
perform SAR analysis.
2. Results and discussion
2.1. Chemistry
Single 3,4-dihydroxyphenylacetic acid ester and amide derivatives were synthesised using
3,4-dimethoxyphenylacetic acid 1 as precursor with N-(3-dimethylaminopropyl)-N′-
ethylcarbodiimide hydrochloride (EDC) as coupling agent, in the presence of 1-hydroxyben-
zotriazole (HOBt) in CH2Cl2 in the case of esters 2–5 (Scheme 1), or in the presence of catalytic
amount of 4-(dimethylamino)pyridine (DMAP) in the case of amides 10–11 (Scheme 2).
Protected monoesters 2–5 were obtained after chromatographic purification in 68–91%
yield, while protected monoamides 10–11 were obtained in 85–95% yield. Methyl phenyl
ethers were cleaved using boron trifluoride methyl sulphide complex (BF3·SMe2) in CH2Cl2
at room temperature for 18 h giving monoesters 6–9 in 52-84% yield and monoamides
Scheme 1. synthesis of compounds 6–9.

NATURAL PRODUCT RESEARCH
3
Scheme 2. synthesis of compounds 12–13.
Scheme 3. synthesis of compounds 19–23, 25, 34–37.
12–13 in 75–80% yield after chromatographic purification. Boron trifluoride methyl sulphide
complex is a mild and easy to handle reagent compared to the widely used boron tribromide
(BBr3) which is a rather harsh one and requires special experimental conditions (low tem-
peratures, inert atmosphere).
Using similar reaction conditions and purification methods, dimeric esters and amides
(symmetrical or non) 19–23, 25, 34–37 containing two catechol units were also synthesised
using diols, diamines and aminoalcohols as linkers. Their synthesis is outlined in Scheme 3.
The synthesis of all the 3,4-dihydroxyphenylacetic acid derivatives is reported here for
the first time and the chemical structures of the compounds were confirmed by 1H NMR, 13C
NMR and ESI MS spectral data (see Supplementary material).

4
A. PAGONI AND S. VASSILIOU
2.2. Radical-scavenging studies
In order to evaluate the radical-scavenging properties of these new polyphenolic compounds
we decided to use the DPPH assay, taking also into consideration several studies (Foti 2015;
Tassano et al. 2015) which report that the use of a parameter like EC50 alone, though impor-
tant, is not sufficient to describe the antioxidant or radical power of a catecholic compound.
Indeed, this parameter does not give any information on the kinetics of the process.
When most of the catecholic derivatives herein were added to a methanolic DPPH solu-
tion, we found that a very fast initial reaction was followed by a much slower decrease in
absorbance. To reach the steady state, 60 min or more was often needed. A clear change in
kinetics can be seen for 36, 22, 25 and DHPAA at 250 μΜ and typically happens after 2–3 min
(Figure 1).
This has been also reported and rationalised by two research groups (Alisi et al. 2012; Foti
2015). One possible explanation is a mixed mechanism governing the reaction of DPPH
radical with phenols. According to this rationalisation, DPPH reacts with phenols by a slow
single-step hydrogen transfer mechanism (HAT) and by a fast sequential proton loss transfer
mechanism (SPLET). The predominance of one mechanism over the other depends on var-
ious factors including the medium polarity, the ionisation potentials, phenol
concentration.
Thus, in this study, we preferred to report not only the EC50, but also for each compound
and each concentration the time needed to reach the final steady state (t2), the time repre-
senting the end of the initial fast reaction (t1) and the corresponding radical-scavenging
activity RSA1 and RSA2. These details can be found into Supplementary material (Table S1).
The radical-scavenging activity of the compounds in this study, expressed as EC50 and as
a molar ratio is presented in Table S2 into Supplementary material. Ascorbic acid, a well-
known antioxidant, was used as reference.
DHPAA and monoester 8 showed a similar profile and EC50 was even better for 8 than for
DHPAA which shows that esterification of the carboxylic group increased the radical-scav-
enging efficiency. Amidoester 36, containing two catechol units had the best antioxidant
profile with the lowest EC50 = 17 μΜ of the compounds in this study and also short t1 and t2.
This derivative exhibited higher radical-scavenging activity than the standard ascorbic acid.
Figure 1. radical-scavenging activity (%rsa) of compounds 22, 25, 36 and dhPaa (250 μΜ) vs. time.

NATURAL PRODUCT RESEARCH
5
The second better antioxidant in this study was diamide 25 with EC50 = 39 μΜ and even
shorter t1 and t2 compared to 36.
Comparing monoesters 7, 8 and 9 the cut-off effect (Laguerre et al. 2013) was again
observed, according to which increased lipophilicity is beneficial for the antioxidant activity
until a critical chain length beyond which the activity decreases. Indeed, increasing the
carbon atoms of the ester from four, in 9, to eight in 8, resulted in EC50 decrease from 263 to
239 μΜ, while further increase to 16 in 7 led to an increased EC50 291 μΜ.
A general observed trend is that amidoesters containing two catechol units had better
radical-scavenging activity profile compared to diesters. Diamide 25 had also very good
radical- scavenging profile. This can be attributed to the presence of H-donating group –NH
which can have larger effect on the stability and radical-scavenging activity of the molecule
than the ester bond.
2.3. Physico-chemical properties
The physico-chemical properties of the studied catechols, calculated using Molinspiration tool
(Jarrahpour et al. 2012) are shown in Table S3 into Supplementary material. Molinspiration
methodology for calculation of miLogP implements fragment-based contributions and
correlation factors which makes it robust and widely applicable tool. With the exception of
compounds 25, 34 and 7, all new compounds fit in the range of 1–5 for milogP value and show
improved lipophilicity compared to the parent DHPPA. Their higher lipophilicity is due to their
smaller polar surface area which is another useful descriptor of the oral bioavailability and
drug transport properties. It is being expressed here as topological surface area (TPSA) which
is a sum of the surface areas occupied by oxygen and nitrogen atoms and the hydrogens
attached to them. TPSA represent the hydrogen bonding capacity of the molecules. Molecules
with TPSA less than 140 Å2 are recognised to have good intestinal absorption, and those with
TPSA less than 60 Å2 show good blood-brain barrier penetration. With the exception of 21 and
34, all presented catecholic derivatives are expected to have good intestinal absorption.
On the other hand, with the exception of 19, 21, 22, 7, 34 and 36 the rest of the com-
pounds follow the rule concerning the number of rotatable bonds Nrotb ≤ 10. The number
of rotatable bonds is an important factor for the efficient binding to receptor and channels
as well as for the oral bioavailability. Molecules with more than 10 rotatable bonds tend to
show poor oral bioavailability.
Hydrogen bonding capacity of the drug candidate is also described by the number of
H-bond donors and acceptors. The compounds in the series show 4–9 H-bond acceptors
and 2–6 H-bond donor. Since NON < 10 and NOHNH < 5, with the exception of 25, all com-
pounds fit well to these rules.
Summarising the physico-chemical properties of the studied derivatives, compound 36
presenting the best radical-scavenging activity and improved lipophilicity compared to the
parent compound DHPAA, along with its close in activity analogue 35, obeys also the‘Rule
of five’and meet all criteria for good solubility and permeability.
3. Conclusions
In conclusion, we demonstrated the possibility to convert 3,4-dihydroxyphenylacetic acid,
a natural polyphenol that can be extracted from OMWWs, into a series of derivatives/

6
A. PAGONI AND S. VASSILIOU
conjugates through an ester or and/or amide bond/linker. The chemistry employed is simple.
This series of derivatives has already been proven to maintain, or even increase, some of the
peculiar properties of DHPAA, such as the radical-scavenging activity, though it is definitely
more promising from a pharmacokinetic point of view.
Supplementary material
All experimental sections relating to this article are available online into supplementary material.
Acknowledgements
We thank Dr Charalambos Proestos (Department of Chemistry, University of Athens) for helpful
discussions.
Disclosure statement
No potential conflict of interest was reported by the authors.
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