Synthesis and biological activities of galactose–aspirin conjugate prodrug designed for ADEPT and PMT

Article abstract of DOI:10.1007/s00044-017-2094-4

Aspirin was used as the lead compound, and its structure was modified by galactosylation. Galactose-based aspirin prodrug was synthesized by using the α-d-glactopyranosyl bromide as glycosyl donor and aspirin as acceptor. The experimental method is simple and reproducible, has high yield and great practical value. The galactosylated aspirin prodrug was found to possess reduced cytotoxicity compared to aspirin, and selectively exhibited antiproliferative activity in the presence or in the absence of β-d-galactoside galactohydrolase with the approach of antibody-directed enzyme prodrug therapy (ADEPT) or the prodrug monotherapy (PMT).

Full text of DOI:10.1007/s00044-017-2094-4

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-2094-4
ORIGINAL RESEARCH
Synthesis and biological activities of galactose–aspirin conjugate
prodrug designed for ADEPT and PMT
Gangliang Huang1
Received: 23 May 2017 / Accepted: 3 October 2017
© Springer Science+Business Media, LLC 2017
Abstract Aspirin was used as the lead compound, and its
structure was modified by galactosylation. Galactose-based
aspirin prodrug was synthesized by using the α-D-glacto-
pyranosyl bromide as glycosyl donor and aspirin as
acceptor. The experimental method is simple and repro-
ducible, has high yield and great practical value. The
galactosylated aspirin prodrug was found to possess
reduced cytotoxicity compared to aspirin, and selectively
exhibited antiproliferative activity in the presence or in the
absence of β-D-galactoside galactohydrolase with the
approach of antibody-directed enzyme prodrug therapy
(ADEPT) or the prodrug monotherapy (PMT).
In addition, study has shown that aspirin has anti-cancer
effect (Rothwell et al. 2012). But after taking in aspirin,
there are the gastric mucosal stimulation, rapid metabolism
in vivo, low bioavailability, and indisposition, which greatly
limit the aspirin to be more widely used in clinics (Stéphane
et al. 2005).
In nature, glycosylation of natural products is very
common (Huang and Mei 2014). The sugar moieties of
these natural molecules can change the pharmacological
activities of the parent drugs by increasing the solubility,
mediation of the plasma half-life, and improving the spe-
cificity of binding. For example, enediyne antibiotics
esperamycin and calicheamicin γ1, anthracycline anticancer
drugs doxorubicin, digoxin, vancomycin, and other clinical
therapeutic drugs (Huang et al. 2014). The chemical mod-
ification of glycosylation of molecules provides an impor-
tant field for the development of new drugs. A large number
of studies show that the introduction of sugar-based drugs
can improve the efficacy and reduce the side effects of
drugs. In addition, the use of sugar modification of existing
drugs can also give them some new effects. For example, in
order to make the drug targeting, we can change the drug
absorption, distribution, metabolism, and excretion, and
extend the half-life. At present, few researchers are parti-
cipating in the work about glycosyl derivatives of aspirin.
Because anticancer drugs lack selectivity, tumor che-
motherapy has serious side effects. An ideal solution to the
problem is the antibody-directed enzyme prodrug therapy
(ADEPT) and the prodrug monotherapy (PMT) (Tietze and
Schmuck 2011). Therefore, this galactose–aspirin covalent
complex prodrug was prepared by chemical method in order
to improve its side effects herein. In addition, the inhibitory
activity of aspirin and its galactosylated conjugate prodrug
to the proliferation of cancer cells was also assayed by
ADEPT and PMT.
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●
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Keywords Aspirin Prodrug Synthesis Antiproliferative
●
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activity ADEPT PMT
Introduction
Aspirin is the non-steroidal anti-inflammatory drug as the
first listing. After more than a century of clinical applica-
tion, it proves that aspirin has a truly curative effect, and is
the antipyretic, analgesic, and anti-inflammatory drug. After
in-depth study for many years, people have found that it has
the characteristics for a long-term prevention of myocardial
infarction, thrombosis, stroke, and rheumatism (Jana 2008).
* Gangliang Huang
huangdoctor226@163.com
1
Active Carbohydrate Research Center, College of Chemistry,
Chongqing Normal University, 401331 Chongqing, China

Med Chem Res
Experimental
H-4), 5.21 (dd, 1H, J2,3 10.4 Hz, H-3), 5.00 (dd, 1H, H-2),
4.33 (m, 1H, H-5), 4.15 (dd, 1H, J6a,6b 11.6 Hz, J5,6 6.0 Hz,
H-6a), 3.99 (dd, 1H, J5,6b 6.8 Hz, H-6b); 13C NMR (D2O,
75 MHz): δ = 86.8 (C-1), 70.4 (C-5), 66.3, 66.0 (C-3/C-4),
64.9 (C-2), 59.9 (C-6).
Synthesis of D-galactose pentaacetate 2
Seventy milliters of acetic anhydride was added, and then 5
g of D-galactose was carefully added. While stirring with a
magnetic stirrer, anhydrous pyridine (70 mL) was added.
This mixture was kept at room temperature while stirring
for 20 h. When the reaction had proceeded, the solution was
mixed with ice, which was stirred until all ice was dis-
solved. The filtered product was recrystallized in a solution
of water/methanol (v/v = 1/2). D-Galactose pentaacetate 2:
Synthesis of galactose–aspirin conjugate 5
The anhydrous dimethyl sulfoxide (DMSO) (30 mL) was
added to three-necked flask, and oil bath was heated to
40 ℃. Under violent agitation, α-D-galactopyranosyl bro-
mide 4 (5 mmol), aspirin (5.3 mmol), and triethylamine (5.3
mmol) were sequentially added to the reaction flask. The
reaction went on for 5 h at 40 °C. After the reaction was
completed, a slurry was obtained under the reduced pres-
sure. The slurry was dissolved with absolute alcohol, and
cooled with ice water. The obtained product was recrys-
tallized with anhydrous ethanol to offer galactose–aspirin
conjugate 5 in 80% yield. Galactose–aspirin conjugate 5 1H
NMR (D2O, 300 MHz): δ = 7.09–8.03 (4m, 4H, ArH),
5.89–5.86 (d, 1H, J = 7.73 Hz, Hβ-1), 5.34–5.27 (m, 2H, H-
2, H-3), 5.24–5.16 (q, 1H, H-4), 4.35–4.29 (q, 1H, H-6),
4.13–4.12 (d, 1H, H-6′), 4.08–3.88 (q, 1H, H-5), 3.84 (s,
3H, –OCOCH3); IR (KBr): v (cm−1) = 2939 (C–H), 1755
(C = O), 1605, 1486, 1450 (Ph), 1223 (C–O), 1073
(C–O–C), 918 (β-galactosidic bond), 752 (two adjacent
substituents on benzene ring).
1
yield 92%; H NMR (CDCl3, 300 MHz): δ = 6.34 (d, 1Hα,
J1,2 1.5 Hz, H-1α), 5.63 (d, 1Hβ, J1,2 1.5 Hz, H-1β), 5.50
(dd, 1H, J3,4 < 1.0 Hz, J4,5 1.3 Hz, H-4), 5.34–5.29 (m, 2H,
H-2, H-3), 4.31 (dt, 1H, J5,6 6.5 Hz, H-5), 4.16–4.01 (m,
2H, H-6a, H-6b), 2.14, 2.10, 2.00, 1.98, 1.97, 1.96 (all s,
15H, 10 × C(O)CH3); 13C NMR (CDCl3, 75 MHz): δ =
170.8, 170.5, 170.3, 169.3, 167.5 (5 × C=O), 92.2 (C-1β),
89.9 (C-1α), 68.8 (C-5), 68.0 (C-3 and C-4), 66.6 (C-2),
61.5 (C-6), 21.3, 21.1, 20.9, 20.8, 20.6 (5 × C(O)CH3);
ESI-MS m/z = 413.3397 [M+Na]+.
Synthesis of α-D-galactopyranosyl bromide 4
At 0 °C, a solution of hydrogen bromide in acetic acid
(33%, 50 mL) was added dropwise to 1,2,3,4,6-penta-O-
acetyl-(α,β)-D-galactopyranose 2 (5 g, 12.8 mmol). After
stirring for 2 h at room temperature, CH2Cl2 (150 mL) was
added to the reaction. The solution was carefully washed
with ice-cold water, and the organic layer was dried over
MgSO4. The solvent was evaporated under vacuum to
obtain galactosyl bromide 3 in 97% yield. Compound 3 was
hydrolyzed with dilute hydrobromic acid aqueous solution
(1 mol/L, 20 mL) at room temperature for 2 h to obtain α-D-
galactopyranosyl bromide 4 in 90% yield. α-D-Galactopyr-
Cell culture
The lung adenocarcinoma cell line A549 was cultured in
RPMI-1640 nutrient fluid, and then was cultured with 5%
CO2 at 37 °C in incubator.
MTT colorimetric assay
1
anosyl bromide 4: H NMR (D2O, 300 MHz): δ = 6.79 (d,
1H, J1,2 3.6 Hz, H-1), 5.42 (dd, 1H, J3,4 3.2 Hz, J4,5 1.2 Hz,
The A549 cells in logarithmic growth phase were seeded in
96-well plates for 5 × 103 cells/hole. The culture medium
was replaced after 24 h, the experimental group (with dif-
ferent concentrations of the sample), blank withered group
(not inoculated cells), and control group (with only the same
amount of solvent) were designed. Each group had four
holes. MTT (20 μL) was added into each hole after 48 h.
After the cultivation which lasted for 4 h in the absence or in
the presence of β-D-galactoside galactohydrolase, it was
centrifuged at 1000 r/min for 5 min, and the supernatant was
AcO
HO
HO
OAc
O
OH
OAc
O
Ac2O/pyridine
rt
AcO
OH
OAc
OH
2
1
Fig. 1 Synthesis of galactose pentaacetate 2
Fig. 2 Synthesis of α-D-
galactopyranosyl bromide 4
AcO
AcO
HO
HO
OAc
O
OH
AcO
OAc
HBr/H2O
rt
O
HBr/HAc
rt
O
OAc
AcO
OAc
3
OH
Br
Br
OAc
4
2

Med Chem Res
Fig. 3 Synthesis of
galactose–aspirin conjugate 5
OCOCH3
HO
HO
HO
HO
OCOCH3
Et3N
DMSO
OH
OH
O
O
OOC
HOOC
+
OH
4
OH
Br
5
Table 1 The antiproliferative activity of aspirin and galactose–aspirin
conjugate prodrug (n = 5)
galactose and acetic anhydride was 1:26, the dosage of
catalyst was 13 times of the mass of galactose, reaction time
was 20 h, the reaction temperature was room temperature,
the esterification yield of galactose and acetic anhydride
was up to 92%. The molar ratio of α-galactose pentaacetate
and β-galactose pentaacetate was 3.2:1.
Compound
Antiproliferative activity
(1.25–20 mmol/L) (%)
Aspirin
3.8–63.9
6.3–90.2
Galactose–aspirin conjugate
1,2,3,4,6-Penta-O-acetyl-(α,β)-D-galactopyranose 2 reac-
ted with a solution of hydrogen bromide in acetic acid (33
wt%). After stirring for 2 h at room temperature, 2,3,4,6-
(ADEPT)
Galactose–aspirin conjugate
(PMT)
4.5–71.8
tetra-O-acetyl-α-D-galactopyranosyl bromide
3
was
obtained in the yield of 97%. Compound 3 was hydrolyzed
with dilute hydrobromic acid aqueous solution at room
temperature to obtain α-D-galactopyranosyl bromide 4 in
90% yield (Fig. 2).
Table 2 The cytotoxicity of aspirin and galactose–aspirin conjugate
prodrug (n = 5)
Compound
IC50 (mmol/L)
Aspirin was neutralized with triethylamine to obtain the
corresponding salt. Compound 4 reacted with the salt of
aspirin in anhydrous DMSO at 40 °C for 5 h, and the target
compound, namely galactose–aspirin conjugate 5, was
obtained in 80% yield (Fig. 3).
Aspirin
11.30 0.16*
1256.83 0.10*
Galactose–aspirin conjugate
*p < 0.05
After the human lung adenocarcinoma cell line A549 was
treated with aspirin at a concentration of 1.25–20 mmol/L for
48 h, the inhibition rate of cancer cell proliferation was
3.8–63.9%, which was in a dose-dependent manner. How-
ever, the inhibition rate when treated with galactose–aspirin
conjugate prodrug was 6.3–90.2% under the same conditions
by ADEPT in the presence of β-D-galactoside galactohy-
drolase. Moreover, ADEPT was better than PMT. The related
results are shown in Table 1. Therefore, it was proved that the
galactosylation of aspirin could enhance its inhibitory activity
against the proliferation of cancer cells by ADEPT and PMT.
The IC50 of prodrug 5 and free aspirin were 1256.83 and
11.30 mmol/L, respectively. In the presence of β-D-galac-
toside galactohydrolase, the cytotoxicity of prodrug 5 was
restored to that of the free aspirin. The ratio of cytotoxicity
between the prodrug and the free drug was 111 (Table 2).
This value is compatible with ADEPT or PMT strategy
where a relatively non-cytotoxic prodrug releases a cyto-
toxic compound.
sucked out. DMSO (100 μL) was added into each hole, and
gently shaked for 10 min. The absorbance (A value) in each
hole was determined at 570 nm with the microplate reader.
The inhibition rate of cell proliferation was calculated: the
inhibition rate = (1−the average value of all A values for
experimental group/the average value of all A values for
control group) × 100%.
Results and discussion
In order to prepare α-D-galactose pentaacetate, the catalysts
mainly used had proton acids, Lewis acids, solid acids, and
enzymes. The traditional homogeneous catalysts have small
phase diffusion resistance, are easy to control, and can be
conveniently operated in the catalytic process, but the
selectivity of reactions and the yields of product need to be
further improved. Moreover, the homogeneous catalysts are
not easy to reuse. In the heterogeneous catalysts, solid acid
catalysts have high selectivity and yields, but the prepara-
tion process of catalysts is very complex. So, it was very
important to develop the rapid, simple, and effective cata-
lysts with high catalytic activity and selectivity, improve the
yield of a single configuration, and reduce the production
costs (Huang et al. 2016). Herein, using pyridine as catalyst,
galactose pentaacetate 2 was synthesized by esterification of
galactose 1 and acetic anhydride (Fig. 1). The molar ratio of
Conclusion
The target prodrug 5 was synthesized by four steps, namely,
acetylation, bromination, deacetylation, and coupling. The
method is easy to operate with high yield. In addition, the
galactosylation of aspirin could enhance its inhibitory
activity against the proliferation of cancer cells by ADEPT

Med Chem Res
or PMT in the presence or in the absence of β-D-galactoside
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Acknowledgements The Project Sponsored by the Scientific
Research Foundation for the Returned Overseas Chinese Scholars,
State Education Ministry. The work was also supported by Chongqing
Key Research Project of Basic Science & Frontier Technology (No.
cstc2017jcyjBX0012), Scientific and Technological Research Program
of Chongqing Municipal Education Commission (Grant No.
KJ1400523), Foundation Project of Chongqing Normal University
(No. 14XYY020), Chongqing General Research Project of Basic
Science & Frontier Technology (No. cstc2015jcyjA10054), Chongq-
ing Normal University Postgraduate’s Research and Innovation Project
(No. YKC17004), Open Foundation Project of University Engineering
Research Center for Bioactive Substances in Chongqing (No.
AS201614), and Chongqing University Students’ Training Project of
Innovation & Undertaking (No. 201610637076), China.
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.