Diacylphloroglucinol derivatives as antioxidant agents: green synthesis, optimisation, in?vitro, and in silico evaluation

Article abstract of DOI:10.1080/14786419.2021.1889542

Several derivatives of diacylphloroglucinol (3a–3c and 5a–5b) as an analogue of natural product compound 2,4-diacetylphloroglucinol 3a, were successfully synthesised in an excellent yield via a greener Friedel–Craft acylation using methanesulfonic acid (MSA) as a catalyst under an ultrasound-assisted condition. Operational simplicity, excellent yield, expedient metal-free synthesis, energy-efficient and mild reaction conditions are the outstanding advantages in this procedure. A scaled-up reaction also revealed the practical suitability of this newly developed procedure. The effects of several process variables on 3a were carefully accomplished using response surface methodology (RSM). Moreover, the green credentials of the present protocol have been assessed using several established green metrics and compared to relevant procedures. Along with the monomers, dimeric diacylphloroglucinols (6a–6e) were also synthesised and their in?vitro antioxidant activity of these species were carried out. Furthermore, drug-likeness, density functional theory (DFT), and molecular docking studies were also established.

Full text of DOI:10.1080/14786419.2021.1889542

Natural Product Research
Formerly Natural Product Letters
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Diacylphloroglucinol derivatives as antioxidant
agents: green synthesis, optimisation, in vitro, and
in silico evaluation
Wahyu E. Prasetyo, Triana Kusumaningsih, Maulidan Firdaus, Soerya D.
Marliana, Venty Suryanti, Anif N. Artanti, Ita Apriana & Septin D. Anggraini
To cite this article: Wahyu E. Prasetyo, Triana Kusumaningsih, Maulidan Firdaus, Soerya
D. Marliana, Venty Suryanti, Anif N. Artanti, Ita Apriana & Septin D. Anggraini (2021):
Diacylphloroglucinol derivatives as antioxidant agents: green synthesis, optimisation, in vitro, and in
silico evaluation, Natural Product Research, DOI: 10.1080/14786419.2021.1889542
To link to this article: https://doi.org/10.1080/14786419.2021.1889542
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2021.1889542
Diacylphloroglucinol derivatives as antioxidant
agents: green synthesis, optimisation, in vitro,
and in silico evaluation
Wahyu E. Prasetyoa, Triana Kusumaningsiha , Maulidan Firdausa,
Soerya D. Marlianaa, Venty Suryantia, Anif N. Artantib, Ita Aprianaa and
Septin D. Anggrainia
aDepartment of Chemistry, Faculty of Mathematics and Natural Sciences, Sebelas Maret University,
b
Surakarta, Indonesia; Department of Pharmacy, Faculty of Mathematics and Natural Sciences,
Sebelas Maret University, Surakarta, Indonesia
ABSTRACT
ARTICLE HISTORY
Received 23 November 2020
Accepted 9 February 2021
Several derivatives of diacylphloroglucinol (3a–3c and 5a–5b) as
an analogue of natural product compound 2,4-diacetylphloroglu-
cinol 3a, were successfully synthesised in an excellent yield via a
greener Friedel–Craft acylation using methanesulfonic acid (MSA)
as a catalyst under an ultrasound-assisted condition. Operational
simplicity, excellent yield, expedient metal-free synthesis, energy-
efficient and mild reaction conditions are the outstanding
advantages in this procedure. A scaled-up reaction also revealed
the practical suitability of this newly developed procedure. The
effects of several process variables on 3a were carefully accom-
plished using response surface methodology (RSM). Moreover, the
green credentials of the present protocol have been assessed
using several established green metrics and compared to relevant
procedures. Along with the monomers, dimeric diacylphlorogluci-
nols (6a–6e) were also synthesised and their in vitro antioxidant
activity of these species were carried out. Furthermore, drug-like-
ness, density functional theory (DFT), and molecular docking
studies were also established.
KEYWORDS
Andraos algorithm;
diacylphloroglucinol;
Friedel-Crafts acylation;
greenness assessment;
molecular docking;
response surface
methodology; ultrasound-
assisted
CONTACT Triana Kusumaningsih
triana_kusumaningsih@staff.uns.ac.id
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2021.1889542.
ß 2021 Informa UK Limited, trading as Taylor & Francis Group

2
W. E. PRASETYO ET AL.
1. Introduction
Over time, natural phenolic compounds have gained increasing attention from
phytochemical research because of their broad applications. Phloroglucinol is belonging
to the natural phenolic class, which exists as an important key intermediate to obtain
better various biologically active compounds (Pal Singh and Bharate 2006; Singh et al.
2010). Approximately, 700 the natural products based phloroglucinol have been reported
to deputize a valuable class of natural products with the enormous arrangement
of biological activities (Kumar et al. 2018; Bourgeais et al. 2019; Wang and Zu 2019).
Among all of the phloroglucinol derivatives, diacylphloroglucinol is one of the promis-
ing derivatives, which belong to privileged medicinal scaffolds in helping the invention
of small-molecule ligands with highly pronounced antioxidant (Kumar et al. 2014), anti-
HIV (Chauthe et al. 2010), antifungal (Gong et al. 2016), antibacterial (Marchand et al.
2000), and anticancer activities (Chauthe et al. 2012). In attempt to improve the level of
biological activity, many researchers carried out chemical structural modifications through
derivatisation processes like a dimeric acylphloroglucinol. Therefore, there has been a
plethora of reports about acylation of phloroglucinol synthetic possibilities via Friedel-
Crafts acylation. However, almost all of the reports record that the diacylphloroglucinol
synthesis approaches less attention to green chemistry aspects involving unfavourable
Lewis acid (AlCl3, ZnCl2 and BF3) or Brønsted acid catalyst (HCl, H2SO4 and HF), employ-
ing hazardous organic solvents, requiring long reaction times and tedious workup proto-
cols (Marchand et al. 2000; Chauthe et al. 2010; Duarte et al. 2014; Mittal et al. 2019).
Thus, there is a need for efforts to be made to find a more convenient and efficient pro-
cedure for implementing green chemistry rules.
As part of our endeavour in searching green and efficient routes for the synthesis
of diacylphloroglucinol derivatives, herein, we carried out an ultrasound-assisted
Friedel-Crafts acylation of phloroglucinol using biodegradable catalyst methanesulfonic
acid (MSA) (Naeimi et al. 2017). Furthermore, this newly developed protocol has opti-
mised according to response surface methodology (RSM). In order to quantify
improvements in this present method by comparing it with previous methods
(Marchand et al. 2000; Chauthe et al. 2012; Duarte et al. 2014; Gong et al. 2016), vari-
ous green metrics have been adapted. Also, the antioxidant activity of the synthesised
diacylphloroglucinol derivatives was investigated using diphenyl-2-picrylhydrazyl
(DPPH) radical scavenging. For further validation of biological results, we also per-
formed in silico studies by drug-likeness, DFT, and molecular docking studies.
2. Result and discussion
2.1. Chemistry
This study started with the Friedel-Crafts C-acylation reaction between 1 mmol of
phloroglucinol (1) and 2 mmol of acetic anhydrides (2) as an acylating agent to obtain
compound 3a under varied conditions, as presented in Table S1. Initially, in term of
green chemistry protocols, an ambient temperature reaction condition with and with-
out catalyst was demonstrated. Inopportunely, there was no product formation
detected for these typical reactions. Similarly, under catalyst-free conditions, this

NATURAL PRODUCT RESEARCH
3
reaction showed an analogous result. Thus, a catalyst that was biodegradable, less
toxic and less corrosive than other mineral acids, MSA, was used in this reaction
(Chauthe et al. 2012). Under the conventional method, the addition of 3 mmol MSA
along with increasing the temperature from room temperature to 60 and 80 ꢀC could
improve the 3a yield until 40–58 and 65–85% in 30–60 min, respectively. Afterwards,
1
the isolated product was further characterised by H NMR and 13C NMR, as presented
in supplementary material. It reveals two singlet signals at d d 5.87 and 2.64 ppm due
to unsubstituted aromatic and CH3 protons, respectively. The 13C NMR spectrum
shows a methyl signal for the acetyl group at d 33.8 ppm and the carbonyl group at d
205.1 ppm, which is in agreement with the structure formed. These interpretations are
in accordance to be 3a with compared to the previous relevant pieces of literature
(Marchand et al. 2000; Chauthe et al. 2012).
Different results might appear from ultrasound-assisted, which could afford better
improvement of yield than the conventional method. Under ultrasound-assisted, the
same typical reactions at 60 and 80 ꢀC were observed to give 77–83 and 86–85% in
20–30 min, respectively. Thus, the increased temperature tends to complete the reac-
tion time within only 15–20 min (the overall results are shown in Table S1).
Additionally, the use of several solvents such as ethanol, chloroform, acetonitrile and
THF did not increase the desired products, or only trace amounts were detected.
Solvent-free conditions are greatly preferable to obtain higher yields. The higher yield
and much faster reactions demonstrated here mean that a reaction can be achieved
with the presence of heating and without any solvent addition. In other words, the
reactants can interact effectively with the other reactants without the presence of a
solvent as a medium (Cave et al. 2001).
In order to achieve a higher yield, CH3COOH (1 mmol) was added, which expected
to provide an H-bond donor solvent for Friedel-Crafts acylation. The result showed
that the presence of acetic acid could enhance the yield of the product 3a to 86–88%
in the conventional method and 88–90% for ultrasound-assisted, respectively. It has
been reported that with the presence of hydrogen bonding (H-bond) from the certain
solvent and/or catalyst could enhance the yield of Friedel-Crafts acylation (Liu and Xu
2018). These results seem likely following the previous study proposed by Chauthe
and co-workers (Chauthe et al. 2012). However, they did not explain further study
between the relationships of acetic acid loading and the yield of the product as well
as the other reaction parameters (i.e. reaction temperature and catalyst loading). Thus,
in this study, we attempted the design of an experiment using RSM to discover the
optimum condition and the detail interaction between the yield and independent vari-
ables. The result analysis of optimisation the synthesis of 3a is given in supplementary
material (see Table S2 and S3, Figure S1 and S2). Overall, the optimum conditions
achieved when a reaction temperature of 80 ꢀC, with the MSA as catalyst loaded at
3 mmol, and with the addition of acetic acid at 0.5 mmol.
Further, we also proposed the plausible reaction mechanism of Friedel-Crafts acyl-
ation of phloroglucinol with the presence of MSA as a catalyst, as presented in
Scheme S1. Initially, through the Brønsted active sites, MSA will lead the protonation
process of acylation agent to form an acylium ion intermediate (I). Because phloroglu-
cinol has adequate nucleophilicity, the electrophilic species represented by the

4
W. E. PRASETYO ET AL.
acylium ion intermediate will be attacked to afford an intermediate (II). After that, this
reaction will be processed either by direct Friedel-Crafts C-acylation or through a Fries
rearrangement reaction from O-acylphloroglucinol or both mechanisms working simul-
taneously to afford a mono-acylphloroglucinol product (III). In the final step, with an
analogous mechanism reaction, the remaining electrophilic species from the acylium
ion will be attacked by another active site from mono-acylphloroglucinol to afford the
3a as the desired product (IV).
According to the standpoint of green chemistry credentials, a ‘greenness’ evaluation
of this newly developed synthetic protocol with respect to existing literature reports
for the synthesis of 3a (Table S4) (Marchand et al. 2000; Chauthe et al. 2012; Duarte
et al. 2014; Gong et al. 2016; Prasetyo et al. 2019) is essential to be performed. To
probe the ‘greenness’ value several important green metrics through the established
working formula like an algorithm advanced by Andraos that permits a calculation
of the footprint of the raw materials including the coefficients of stoichiometric and
the total quantity of material used (Andraos 2009). Two other green metrics called E
and PMI were also implemented. Both of these assessment results are presented
in Figure S3. In general, the presented method clearly exhibited significant
improvement in almost all metrics used. The detail explanation of greenness
assessment is provided in supplementary material.
This remarkably simple and very rapid Friedel-Crafts acylation phloroglucinol
reaction of 3a encouraged us to explore the scalability by using this protocol in a
gram-scale (6 and 10 mmol scales) experiment. To our delight, this experimental out-
come of 3a was very similar to the small-scale (1 mmol scale) experiment. With these
fruitful experimental outcomes, other bulkier acylating agents such as isobutyryl and
isovaleryl chloride also took part in the reaction under the present protocol (Scheme
S2). Remarkably, this protocol can obtain other bulkier diacylphloroglucinol derivatives
in satisfactory yields, as documented in Scheme S2. This approach could clearly enable
the creation of a convergent and versatile protocol, which would present a broad
substrate scope and outstanding functionality tolerance. Also, in order to improve
the level of cytotoxicity for antioxidant activity, we synthesised dimeric diacylphloro-
glucinols via phenol-aldehyde condensation, as illustrated in Scheme S2.
2.2. Antioxidant activity evaluations
The synthesised diacylphloroglucinol derivatives were then subjected to in vitro
antioxidant assessment using DPPH radical scavenging where vitamin C was used as a
standard or positive control. Because of its validity and simplicity, this method is
extensively used to assess the antioxidant radical scavenging activity of various species
both in foods and biological systems (Guerra-Vargas et al. 2018). The results of this
study are shown in Figure S4. Accordingly, as deduced from the IC50 values, all of the
screened diacylphloroglucinol compounds showed strong antioxidant activity level
(IC50 ¼ 10–50 mg/mL). The most active species was dimeric diacylphloroglucinol 6e
(IC50 ¼ 12.704 0.174 mg/mL), with relatively close performance to positive control
ascorbic acid (IC50 ¼ 11.140 0.117 mg/mL). This result can be ascribed to the highest
conjugation of this compound due to the presence of the phenyl substituent group

NATURAL PRODUCT RESEARCH
5
in methylene linkage. It is noteworthy to note that, the dimeric diacylphloroglucinol
possessed higher antioxidant activity compared to their monomers. Furthermore, the
experimental result proved that when the presence of these synthesised compounds
into the radical DPPHꢁ solution, the colour of the solution changed at the certain
concentration, which is indicated to the formation of the radical DPPHꢁ: DPPHꢁ
þ
AH ! DPPH þ Aꢁ. In this case, the redox potentials of the hydroxyl group (-OH) and
potential for electronic delocalisation through their chemical structures will strongly
affect their antioxidant properties.
2.3. In silico pharmacology studies
Moreover, in order to gain insight of the drug-likeness properties, all of the synthes-
ised diacylphloroglucinol derivatives were fully subjected to molecular properties pre-
diction by Osiris Property Explorer software according to the Lipinski’s rule of five
(Ro5) and compared with vitamin C, as listed in Table S5. Through this software, the
lipophilicity and ability of a compound to irritate cell membrane can be represented
by the ClogP value. As listed in Table S5, the ClogP value of all the investigated com-
pounds are in the range from 0.48 to 5.39 whereas vitamin C possessed a ClogP value
of ꢂ2.46. Accordingly, compound 6c showed poor absorption or permeation com-
pared to all the tested compounds. Moving to the drug score values, all of the syn-
thesised diacylphloroglucinol except for compound 6c showed better drug score
values (0.24 to 0.54) rivalled with the vitamin C (0.16). Compounds 3a–3c, 5a, 5b, 6a,
6b, 6d, and 6e revealed good drug score values, thus, these compounds could be
deliberated as potential lead compounds in drug discovery.
2.4. DFT studies
The frontier orbital HOMO and LUMO are a powerful tool in predicting the reactivity
ꢀ
of a molecule, particularly in biological activity result (Flores-Holguın et al. 2019). In
our case, the DFT calculations were performed on the top two compounds with the
highest antioxidant 6d and 6e. The DFT result revealed that both the compound 6d
and 6e have the same stability, which indicated by the same value of energy gap (DE)
of ꢂ4.31, as summarised in Table S6. In both compounds, the HOMOs are mostly
localised over the aromatic ring, hydroxyl, and carbonyl groups whereas the empty
LUMO orbital is delocalised mostly over the hydroxyl and alkyl group, as displayed in
Figure S5. The other quantum chemicals such as g S, v, l and x, which are essential
to describe the reactivity and stability of investigated compounds, were also deter-
mined, as presented in Table S6. Additionally, for the distribution of the electronic
charge over compound 6d and 6e determination, a molecular electrostatic potential
(MEP) map was created based on the DFT optimised geometries, as presented in
Figure S6. For both compounds, the red colour represented the negative electrostatic
potential regions, which are fundamentally indicated for the carbonyl functional
groups, while the blue colour constituted the positive regions, which are mostly
showed over the methyl groups and aromatic rings.

6
W. E. PRASETYO ET AL.
2.5. Molecular docking studies
Finally, along with the experimental data and previous in silico studies, molecular
docking was carried out on compound 6d and 6e with the myeloperoxidase (MPO)
enzyme (PDB ID: 1DNU). This enzyme has been known to play an essential key in the
immune system by producing reactive oxygen species (ROS). From the result, it
observed that all the investigated compounds had a binding affinity for MPO, sur-
passed that of the reference inhibitors (vitamin C), as displayed in Table S7.
Remarkably, these results are in agreement as compared with the previous report
(Ramirez-Duran et al. 2013). Moreover, these compounds occupy in varying ways bind-
ing site of MPO, i.e. Q91, D94, H95, F99, T100, P101, E102, S149, R239, E242, R333,
F366 and F407, R424, and H428, as exhibited in Figure S7.
3. Experimental
Experimental details relating to this paper are in the supplementary material.
4. Conclusion
In summary, we have demonstrated a greener and more efficient one-pot procedure
for the synthesis of diacylphloroglucinol derivatives via an ultrasonic-assisted tech-
nique integrating an environmental catalyst and solvent-free conditions. In a typical
reaction, the optimum conditions of 3a achieved through RSM were as follows: there
was a reaction temperature of 80 ꢀC, the catalyst loaded at 3 mmol, and the amount
of acetic acid was 0.5 mmol. According to the green chemistry point of view, this pre-
sent method presents a significant improvement in all metrics, including AE, Rxn Yield,
SF, RME, MRP, PMI, E and energy efficiency compared to other reported methods. The
in vitro antioxidant assessment revealed that all these synthetic compounds are
showed strong antioxidant activity. Particularly, for compound 6d and 6e displayed a
most potent antioxidant activity with good and positive correlation with drug-likeness,
DFT, and molecular docking studies. Accordingly, it is expected to be useful in design-
ing stronger antioxidant compounds for biomedical purposes in the future.
Disclosure statement
There are no conflicts to declare.
Funding
This work was supported by the Hibah Pascasarjana 2019 under Grant number 516/UN27.21/PP/
2019 and Hibah Pascasarjana 2020 under Grant number 452/UN27.21/PN/2020.
ORCID
Triana Kusumaningsih
http://orcid.org/0000-0002-5795-0125

NATURAL PRODUCT RESEARCH
7
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