Synthesis and cytotoxic activity of 4-O-β-D-galactopyranosyl derivatives of phenolic acids esters

Article abstract of DOI:10.1080/14786419.2017.1375927

The glycosylation of naturally occurring phenolic acids has a significant impact on their solubility, stability and physiochemical properties. D-Galactose residue was found to form a part of glycoconjugates in several tissues and involved in a variety of physiological process. To the best of our knowledge, we have noticed a little information about the glycosylation of the phenolic acids with galactose residue. In this work, we describe the glycosylation of methyl vanillate and methyl ferulate with peracetylated-β-D-galactopyranose in the presence of BF₃·OEt₂. The coupling reaction yielded efficiently and selectively only the acetylated β-D-galactopyranosides 3 and 6. Removal of the acetyl groups using sodium methoxide afforded the corresponding β-D-galactopyranosides 4 and 7 in good yields. Anticancer activity in vitro was evaluated against two human cancer cell lines (MCF-7 breast cancer cell lines and PC-3 prostate cancer cell lines). β-D-galactopyranosides 4 and 7 demonstrated improved cytotoxic activity compared to the parental esters.

Full text of DOI:10.1080/14786419.2017.1375927

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Synthesis and cytotoxic activity of 4-O-β-D-
galactopyranosyl derivatives of phenolic acids
esters
Nader Al Bujuq, Sharif Arar & Raida Khalil
To cite this article: Nader Al Bujuq, Sharif Arar & Raida Khalil (2017): Synthesis and cytotoxic
activity of 4-O-β-D-galactopyranosyl derivatives of phenolic acids esters, Natural Product Research,
DOI: 10.1080/14786419.2017.1375927
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Date: 18 September 2017, At: 04:16

Natural Product research, 2017
https://doi.org/10.1080/14786419.2017.1375927
Synthesis and cytotoxic activity of 4-O-β-D-galactopyranosyl
derivatives of phenolic acids esters
Nader Al Bujuqa , Sharif Ararb and Raida Khalilc
achemistry department, deanship of academic services, taibah university, Medina, Kingdom of saudi arabia;
c
bschool of science, department of chemistry, the university of Jordan, amman, Jordan; department of
Biotechnology and Genetic engineering, Philadelphia university, amman, Jordan
ABSTRACT
ARTICLE HISTORY
received 30 June 2017
accepted 22 august 2017
The glycosylation of naturally occurring phenolic acids has a significant
impact on their solubility, stability and physiochemical properties.
D-Galactose residue was found to form a part of glycoconjugates in
several tissues and involved in a variety of physiological process. To
the best of our knowledge, we have noticed a little information about
the glycosylation of the phenolic acids with galactose residue. In this
work, we describe the glycosylation of methyl vanillate and methyl
ferulate with peracetylated-β-D-galactopyranose in the presence of
BF3·OEt2. The coupling reaction yielded efficiently and selectively
only the acetylated β-D-galactopyranosides 3 and 6. Removal of the
acetyl groups using sodium methoxide afforded the corresponding
β-D-galactopyranosides 4 and 7 in good yields. Anticancer activity
in vitro was evaluated against two human cancer cell lines (MCF-7
breast cancer cell lines and PC-3 prostate cancer cell lines). β-D-
galactopyranosides 4 and 7 demonstrated improved cytotoxic activity
compared to the parental esters.
KEYWORDS
Glycosylation; protection;
methyl ferulate;
methyl vanillate; β-d-
galactopyranosides; Pc-3;
prostate cancer; anticancer
activity; breast cancer; McF-7
Abbreviations: FE Ferulic acid methyl ester (2); VE Vanillic acid
methyl ester (5); Gal-FE β-galactopyranosyl ferulic acid methyl
ester (4); Gal-VE β-galactopyranosyl vanillic acid methyl ester (7)
1. Introduction
Phenolic acids are natural products compounds that are found in many types of plants
(Aruoma et al. 2005) their constituents are considered very important of the human diet due
to their benefit antioxidant activity (Martin and Appel 2010). Recent interest in phenolic
acids comes from their potential protective role, through ingestion of fruits and vegetables,
CONTACT Nader al Bujuq
nbujuq@yahoo.com, nbujuq@taibahu.edu.sa
supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2017.1375927.
© 2017 Informa uK limited, trading as taylor & Francis Group

2
N. AL BUJUQ ET AL.
against oxidative damage diseases (coronary heart disease, stroke) (Bravo 1998), also their
potentially important properties such as anticancer activities (Liu 2004; Harris et al. 2007;
Huang et al. 2010). Wide varieties of phenolic acids exhibit various physiological activities,
including antibacterial, anti-inflammatory and anticarcinogenic (Nohynek et al. 2006; Russell
and Duthie 2011). Researches about biological and pharmacological activities have also
been documented for phenolic compounds, including free radicals scavenging, apoptosis
of cancer cells (Kanai and Okano 1998; Saeki et al. 2000).
The glycosylation of phenolic compounds is considered a vital method due to increase
their solubility and stability in water (Plaza et al. 2014; De Winter et al. 2015). Glycosylation
of phenolic groups protect the hydroxy groups from oxidation (Torres et al. 2011; Zhang
et al. 2013). Also, glycosylation can potentially improve pharmacokinetics, pharmacodynam-
ics, solubility, mechanism and potency.
Variation of the introduced glycosyl residues shows different impact on the properties
of phenolic compounds. The galactosylated hydroquinone is 1.19 times higher antioxidant
than the glucoside counterpart β-arbutin (Kim et al. 2010). More interestingly, there exist
abundant galactose acceptors on the hepatocyte surface that could bind with galactose
derivatives (Mishra et al. 2015). Thus, the glycosylation with galactose could aid targeted
delivery of drugs to liver cancer cells but not to nearby normal cells (Gankhuyag et al. 2015).
Moreover, galactose residue forms part of glycolipids and glycoproteins in several tissues
and considered to be involved in a variety of biological recognition events, the synthesis of
galactosyl phenolic derivatives would be of scientific and practical interest (Ajisaka andYoon
1996). To the best of our knowledge, no information was reported about the glycosylation
of the phenolic acids with certain sugars such as galactose. Most of these procedures were
multisteps, tedious and cumbersome, besides the compounds were not fully characterised
(Ding et al. 1996; Lu et al. 2015; Fei et al. 2016). In this paper, we described a simplified and
straightforward synthetic route of methyl ferulate and methyl vanillate galactopyranosides
4 and 7 under mild conditions. β-D-galactose pentaacetate 1 was utilised as a glycosyl donor,
while the methyl esters 2 and 5 were used as acceptors. As a result of glycosylation, the novel
phenolic β-D-galactopyranosides 4 and 7 were produced selectively in good yield. In this
paper, we also aimed to investigate the cytotoxic activity of the novel phenolic β-D-ga-
lactopyranosides 4 and 7 against two human cancer cell lines (PC-3 prostate cancer cell lines
and MCF-7 breast cancer cell lines). The results of the glycosides 4 and 7 showed superior
cytotoxicity effect on the cell lines compared with its corresponding free ester.
2. Results and discussion
2.1. Synthesis
Currently, phenolic glycosides are available through three different approaches: extraction
from natural sources, chemical synthesis and enzymatic synthesis. Extraction of phenolic
glycosides from natural sources is complex and uneconomical as the target compound in
natural organisms is usually of low content. In contrast, synthetic methods including chemical
synthesis and enzymatic synthesis are efficient and can produce either natural or unnatural
phenolic glycosides with high values (Sears and Wong 2001;Yu et al. 2012). As a consequence,
the synthesis methods have received increasing attention for developing active phenolic
glycosides.

NATURAL PRODUCT RESEARCH
3
The stereoselective formation of anomeric centres is a major problem in the synthesis of
glycosides. Many combinations of leaving group, promoter, and protecting group have been
devised to achieve this. To overcome the formation of mixture of anomers, we started the
synthesis using β anomer of the D-galactose pentaacetate 1. Nevertheless, BF3.OEt2 is a
powerful promoter for the activation of acetyl groups, it was found to be effective for the
coupling between the glycosyl donor 1 and the phenolic acids derivatives acceptors 2 and
5. These couplings were proven to be feasible, and the corresponding glycosides 3 and 6
were produced selectively with only β anomers.
Generally, it is considered that the formation of 3 and 6 follows an SN1-like mechanism.
Practically the glycosylation reaction begins by the interaction taking place between pro-
moter (BF3·OEt2) and the glycosyl donor 1. This results in the formation of an activated species
followed by the dissociation of a leaving group. The reaction may proceed via the formation
of glycosyl cation that can be stabilised via an acyloxonium intermediate which can be
formed as a result of the participation from the carbonyl group at C-2. These intermediates
can be glycosylated with the glycosyl acceptor (ROH). Due to the presence of acetyl group
at C-2 (anchimeric assistance), (ROH) attacks the dioxolenium ion predominantly from
one face to provide a 1,2-trans glycoside which explains the formation of β-glycosides
selectively.
The optimisation condition of the reaction to give high yield and high selectivity of the
β-glycoside was carried out in Dichloromethane at 0 °C for 8 h. The preliminary investigation
1
of H NMR sample of the acetylated intermediate confirmed the formation of β anomer
selectively without any formation of α anomer. The prolonged time above 0 °C led to ano-
merisation. When the reaction was done at higher temperatures, a series of mixture products
were obtained and decomposition appeared. Other lewis acids, for instance, TMSOTf, NIS/
TfOH were employed to improve the yield. However, the selectivity and the efficiency were
not adequate (Table 1).
1
The identification of compound 3 was readily confirmed on the basis of its, H and 13C
NMR spectra. The 1H NMR showed two singlet of methyl signals at 3.80 and 3.73 ppm rep-
resenting (–OMe) and (–COOMe), respectively. The anomeric proton appeared at 5.48 ppm
as doublet with J = 9.0 Hz, the chemical shift and the value of the coupling constant confirm
the formation of only β anomer stereoselectively. The sugar protons appeared at the normal
position range (3.92–4.21 ppm). Three aromatic protons appeared at 7.25–7.64 ppm. The
Table 1. Glycosylation of 1 and 2 in the presence of various acidic catalysts at different reactions condi-
tions.
Glycosyl
donor
Ferulic acid
methyl ester
Reaction
Time
Temperature Acid catalyst
% of starting
material
Entry
(°C)
promoter
β/α
1
2
3
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
15 min
60 min
4 h
8 h
24 h
8 h
0
0
0
0
0
25
0
0
BF3·oet2
BF3·oet2
BF3·oet2
BF3·OEt2
BF3·oet2
BF3·oet2
tMsotf
NIs/tfoh
only β
only β
only β
Only β
82/18
80/20
80/20
75/25
85
34
22
4a
5b
6b
7c
8c
<10
8 h
8 h
Note: Bold values represents the optomized conditions.
aanomeric composition of reaction mixture was monitored by 1h NMr (300 Mhz, cdcl3), based on the integration of peaks.
blonger time reduces the selectivity, increases temperature gives extra side products and less yield.
cthe selectivity is reduced, in addition to side products.

4
N. AL BUJUQ ET AL.
Scheme 1. Glycosylation reaction between 1 and 2. reagents and conditions: (a) BF3·oet2, ch2cl2, Net3
Ms 4Å, 0 °c, 8 h, 70% yield; (b) NaoMe/Meoh, rt, 0.5 h, followed by dowex (h+), 88% yield.
Scheme 2. Glycosylation reaction between 1 and 5. reagents and conditions: (a) BF3·oet2, ch2cl2, Net3
Ms 4Å, 0 °c, 8 h, 80% yield; (b) NaoMe/Meoh, rt, 0.5 h, followed by dowex (h+), 90% yield.
vinyl protons of the Cα and Cβ appeared at 6.30 and 7.60 ppm respectively. Additionally the
value of coupling constant (J = 15.0 Hz) pointed to a trans position of the vinyl protons (cis:
J = 12.0 Hz). Deacetylation of the corresponding 3 was carried out under mild catalytic meth-
oxide/methanol (1 M), followed by neutralisation using dowex acidic (H+) (Scheme 1). This
step was considered very critical, due to labile property of the methyl ester under basic
conditions. The target compound 4 was recrystallised using mixture of methanol and ethyl
acetate solvents. The structure of 4 was confirmed on the basis of its HRFABMS, 1H and 13
C
NMR spectra. Complete proton and carbon chemical shift data for the products are given in
(S1 see supplementary data).
The glycosylation of the methyl vanillate 5 proceeded smoothly under similar conditions
using the same procedure (Scheme 2). The intermediate 6 was used directly. The formation
of only β anomer of compound 7 was deduced from the NMR spectroscopy. Table 1 shows
the values of the chemical shifts for 1H NMR and 13C NMR of the titles compounds.
2.2. Cytotoxic activity
Phenolic acids have been recognised not only for their properties as antioxidant compounds,
but also for their cytotoxic effects towards breast cancer in in vitro and in vivo (Oliveira et al.
2011) and prostate cancer (Weng andYen 2012; Seçme et al. 2015). The present work, aimed
to study the cytotoxicity of ferulic acid methyl ester (FE), β-galactopyranosyl ferulic acid
methyl ester (Gal-FE), vanillic acid methyl ester (VE) and β-galactopyranosyl vanillic acid
methyl ester (Gal-VE) towards human breast cancer and prostate cancer cells. The objective
was to identify whether the molecular alterations by the glycosylation of the test compounds
would display more effective cytotoxic activity.
In the presents study, the anticancer activity of the compounds 2 (FE), 4 (Gal-FE), 5 (VE)
and 7 (Gal-VE) was tested against two cell lines (MCF-7 and PC-3) and all of them showed

NATURAL PRODUCT RESEARCH
5
Table 2. anticancer activity (Ic50, μM)a of compounds 2, 4, 5 and 7 against the cell lines McF-7 and Pc-3.
IC50 values (μM)
Compound
MCF-7
PC-3
(2)
896.9
373.7
529.3
236.7
0.49
324.2
359.9
344.3
729.1
(4)
(5)
(7)
doxorubicinb
2.53
athe values of the Ic50 (concentration of drug yielding a 50% cell viability decrease).
bthe drug doxorubicin was used as positive control.
moderate to good anticancer effects (Figure S29, see the supplementary material). As
expected, the glycosylated compounds 4 and 7 were found to show higher anticancer activ-
ity on MCF-7 cells compared to its free esters 2 and 5 (Table 2). Conversely, compounds 2
and 5 showed noticeable decrease on PC-3 cell viability (IC50 = 324.2 and 344.3 μM respec-
tively) (Table 2). Surprisingly, Compound 7 caused growth inhibition at low dose in MCF-7
cells only with IC50 observed of 236.7 μM with no significant inhibitory effect exerted onto
PC-3. On the other hand, compound 2 showed the most potent cytotoxicity effect with IC50
observed of 324.2 μM on PC-3 cells. Finally, (5 and 7) were less effective than (2 and 4)
whereas most of the effects from (2 and 4) were observed on MCF-7 cells.
3. Conclusion
In summary, our findings, in agreement with data reported by other investigators, clearly
indicate that glycosylated compounds (Gal-FE and Gal-VE) displayed a preferable cytotoxic
activity against MCF-7 cancer cells than the aglycone esters. Therefore, glycosylation proved
to be a promising method in modification of phenolic compounds and could serve as a
novel prodrug. It is necessary to conduct further studies with other cell lines and in vivo
animal models to discover the molecular alteration effect of the glycosylated phenolic com-
pounds on prostate and breast cancer. In addition, the scope of our research will be extended
to cover other active phenolic acids and include other sugars moieties such as glucose and
mannose.
Supplementary material
Supplementary material relating to this article is available online alongside with experimental part
(NMR data and cytotoxicity graphs)
Acknowledgements
We would like to express our deep gratitude for Philadelphia University and the University of Jordan
for their technical and scientific collaborations.
Disclosure statement
No potential conflict of interest was reported by the authors.

6
N. AL BUJUQ ET AL.
ORCID
Nader Al Bujuq
http://orcid.org/0000-0002-3089-7037
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