Application of Monodisperse PEGs in Pharmaceutics: Monodisperse Polidocanols

Article abstract of DOI:10.1021/acs.molpharmaceut.7b00496

Polydisperse PEGs are ubiquitously used in pharmaceutical industry and biomedical research. However, the monodispersity in PEGs may play a role in the development of safe and effective PEGylated small molecular drugs. Here, to avoid the polydispersity in polidocanol, the active ingredient in a clinically used drug, a macrocyclic sulfate-based strategy for the efficient and scalable synthesis of monodisperse polidocanols, their sulfates, and their methylated derivatives, was developed. TLC and HPLC analysis indicated a complex mixture in regular polidocanol and high purities in monodisperse polidocanols and their derivatives. Assay on HUVEC, L929, and HePG2 cells showed that monodisperse polidocanols have much higher cytotoxicity and safety than that of regular polidocanol. It was found that the monodispersity of PEGs in polidocanols is crucial for achieving the optimal therapeutic results. Therefore, based on this case study, it would be beneficial to optimize PEGylated small molecular drugs with monodisperse PEGs in pharmaceutical research and development.

Full text of DOI:10.1021/acs.molpharmaceut.7b00496

Article
pubs.acs.org/molecularpharmaceutics
Application of Monodisperse PEGs in Pharmaceutics: Monodisperse
Polidocanols
Zeqiong Yu,†,⊥ Shaowei Bo,†,⊥ Huiyuan Wang,‡ Yu Li,† Zhigang Yang,† Yongzhuo Huang,‡
and Zhong-Xing Jiang*,†,§
†Hubei Province Engineering and Technology Research Center for Fluorinated Pharmaceuticals, School of Pharmaceutical Sciences,
Wuhan University, Wuhan 430071, China
‡Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Hai-Ke Road, Shanghai 201203, China
§State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, Dong Hua University, Shanghai 201620, China
S
* Supporting Information
ABSTRACT: Polydisperse PEGs are ubiquitously used in pharmaceutical
industry and biomedical research. However, the monodispersity in PEGs
may play a role in the development of safe and effective PEGylated small
molecular drugs. Here, to avoid the polydispersity in polidocanol, the
active ingredient in a clinically used drug, a macrocyclic sulfate-based
strategy for the efficient and scalable synthesis of monodisperse
polidocanols, their sulfates, and their methylated derivatives, was
developed. TLC and HPLC analysis indicated a complex mixture in
regular polidocanol and high purities in monodisperse polidocanols and
their derivatives. Assay on HUVEC, L929, and HePG2 cells showed that
monodisperse polidocanols have much higher cytotoxicity and safety than
that of regular polidocanol. It was found that the monodispersity of PEGs
in polidocanols is crucial for achieving the optimal therapeutic results.
Therefore, based on this case study, it would be beneficial to optimize
PEGylated small molecular drugs with monodisperse PEGs in pharmaceutical research and development.
KEYWORDS: monodisperse, polydisperse, polyethylene glycols (PEGs), polidocanol, PEGylated drugs
whether the polydispersity of regular PEGs can compromise
the efficacy and safety of PEGylated drugs or not.
INTRODUCTION
■
Polyethylene glycols (PEGs) are the most used polymer in
pharmaceutical industry and biomedical research to improve
solubility and stability, reduce dosing frequency and immuno-
genicity, and prolong blood circulation.1−4 The so-called
“stealth behaviors” of PEGs are the “golden standards” for
biomedical polymers.3 Until 2015, there are 17 PEGylated
drugs approved by US FDA.
Although the importance of enantiomer purity in chiral drugs
has been well recognized by pharmaceutical industry since the
thalidomide tragedy, little attention has been paid to the
monodispersity of PEGs used in either approved drugs or those
in the pipeline. Unfortunately, regular PEGs even with an
excellent polydispersity index (PDI) are still complex mixtures
of oligomers.5 There are two main reasons that led to the
dilemma: (1) Replacing regular PEGs with monodisperse PEGs
will dramatically increase the cost due to the high price and
limited availability of monodisperse PEGs. (2) The polydisper-
sity of PEGs is always supposed to play a minor role in the
efficacy and safety of drugs. To address the first issue, many
novel synthetic strategies have recently been developed for
synthesizing monodisperse PEGs with high efficacy and low
cost.6−14 However, as far as we know, there is no research on
Asclera is an FDA approved drug which has been widely used
to sclerosis small spider veins and reticular veins in the lower
extremities. Its active ingredient, polidocanol, is an ether
mixture of hydrophobic dodecyl alcohol and hydrophilic
polydisperse polyethylene glycols with an average molecular
weight of 400 Da (Scheme 1). As a polymer prepared from
dodecyl alcohol and ethylene oxide, regular polidocanol
contains many analogs with n = 9 as the major component.
The inherent heterogeneity in regular polidocanol may not only
complicate its purification, characterization, quality control, and
clinic application, but also compromise its therapeutic efficacy
and safety. Therefore, it is necessary to identify the most
effective and safe component in regular polidocanol from which
monodisperse polidocanol, the next generation Asclera, may be
developed. Herein, a macrocyclic sulfate-based strategy for
efficient synthesis of monodisperse polidocanols, their sulfates,
and methylated derivatives was developed for a comparative
Received: June 14, 2017
Revised: August 17, 2017
Accepted: August 22, 2017
Published: August 22, 2017
© XXXX American Chemical Society
A
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the macrocyclic sulfate 25 as clear crystal (16.1 g, 42% yield).
1H NMR (400 MHz, CDCl3) δ 3.69 (s, 4H), 3.84−3.95 (m,
4H), 4.43−4.52 (m, 4H).
Scheme 1. Synthetic Strategies for Regular Polidocanol 1,
Monodisperse Polidocanols 9-15, and Their Derivatives 2-8
and 16-22
Macrocyclic Sulfate 26. 26 was prepared from tetraethylene
glycol 24 by following the same procedure for macrocyclic
1
sulfate 25 as white solid (26.6 g, 50% yield). H NMR (400
MHz, CDCl3) δ 3.62−3.78 (m, 8H), 3.80−3.95 (m, 4H),
4.36−4.64 (m, 4H).
Alcohol 27. Under an atmosphere of nitrogen, dodecyl
alcohol (3.7 g, 20.0 mmol) was added to a suspension of NaH
(0.96 g, 60% in mineral oil, 24.0 mmol) in anhydrous THF (50
mL) and the mixture was stirred for additional 30 min at 0 °C.
Then, a solution of macrocyclic sulfate 25 (6.4 g, 30.0 mmol) in
anhydrous THF (50 mL) was added and the reaction was
monitored with TLC until no dodecyl alcohol could be
detected. The reaction was quenched with water (2.0 mL),
acidified with concentrated sulfuric acid to pH 2, and refluxed
for 2 h. After cooled to room temperature, the reaction mixture
was neutralized with saturated NaHCO3 and extracted with
CH2Cl2 (100 mL, 3 times). The combined organic layer was
dried over anhydrous Na2SO4 and concentrated under vacuum.
The residue was purified by flash column chromatography on
silica gel (petroleum ether/ethyl acetate = 1/1 as eluent) to
study on the polydisperse and monodisperse polidocanols
(Scheme 1). This study is intended to address the issue if it is
necessary to replace regular polydisperse PEGs with mono-
disperse PEGs in clinically used small molecular drug
polidocanol.
1
give alcohol 27 as yellowish oil (5.3 g, 83% yield). H NMR
(400 MHz, CDCl3) δ 0.88 (t, J = 8.0 Hz, 3H), 1.23−1.36 (m,
18H), 1.49−1.75 (m, 2H), 3.46 (t, J = 8.0 Hz, 2H), 3.56−3.78
(m, 12H); HRMS (ESI) calcd for C18H38NaO4 [(M+Na)+]
+
MATERIALS AND METHODS
■
341.2662, found 341.2659.
General Information. Unless otherwise indicated, all
reagents were obtained from commercial supplier and used
without prior purification. THF and CH2Cl2 were dried and
freshly distilled prior to use. Regular polidocanol was purchased
from Sigma-Adrich. Flash chromatography was performed on
silica gel (200−300 mesh) with either EtOAc/petroleum ether
Alcohol 28. 28 was prepared from dodecyl alcohol and
macrocyclic sulfate 26 by following the same procedure for
alcohol 27 as clear oil (10.8 g, 86% yield). 1H NMR (400 MHz,
CDCl3) δ 0.85 (t, J = 8.0 Hz, 3H), 1.14−1.42 (m, 18H), 1.46−
1.70 (m, 2H), 3.34 (s, 1H), 3.43 (t, J = 8.0 Hz, 2H), 3.53−3.78
+
(m, 16H); HRMS (ESI) calcd for C20H42NaO5 [(M+Na)+]
(PE, 60−90 °C) or MeOH/CH2Cl2 as eluents. H and 13C
1
385.2924, found 385.2917.
NMR spectra were recorded on a 400 MHz Bruker NMR
spectrometer. Chemical shifts are in ppm and coupling
constants (J) are in Hertz (Hz). 1H NMR spectra were
referenced to tetramethylsilane (d, 0.00 ppm) using CDCl3 as
solvent. 13C NMR spectra were referenced to solvent carbons
Monodisperse Polidocanol 9. 9 was prepared from alcohol
27 and macrocyclic sulfate 25 by following the same procedure
for polidocanol 27 as clear oil (5.4 g, 85% yield). 1H NMR (400
MHz, CDCl3) δ 0.88 (t, J = 8.0 Hz, 3H), 1.18−1.37 (m, 18H),
1.52−1.62 (m, 2H), 3.45 (t, J = 8.0 Hz, 2H), 3.53−3.78 (m,
1
(77.16 ppm for CDCl3). The splitting patterns for H NMR
24H); HRMS (ESI) calcd for C24H54NO7 [(M+NH4)+]
+
spectra are denoted as follows: s (singlet), d (doublet), q
(quartet), m (multiplet). ESI Mass spectra were recorded on a
Thermo Scientific Q Exactive Focus mass spectrometer.
468.3895, found 468.3935.
Monodisperse Polidocanol 10. 10 was prepared from
alcohol 28 and macrocyclic sulfate 25 by following the same
procedure for polidocanol 27 as yellowish oil (6.0 g, 88%
yield). 1H NMR (400 MHz, CDCl3) δ 0.88 (t, J = 8.0 Hz, 3H),
1.22−1.36 (m, 18H), 1.54−1.63 (m, 2H), 3.45 (t, J = 8.0 Hz,
Synthesis of Monodisperse Polidocanols and Their
Derivatives. Macrocyclic Sulfate 25. To a stirring solution of
triethylene glycol 23 (27.1 g, 180.3 mmol), triethylamine (87.6
g, 865.4 mmol), and DMAP (1.1 g, 9.0 mmol) in CH2Cl2 (3.0
L) at 0 °C was slowly added a solution of SOCl2 (53.7 g, 360.6
mmol, in 50 mL CH2Cl2). After the addition, the stirring
mixture was warmed to 25 °C and monitored with TLC until
the complete consumption of triethylene glycol. The reaction
was quenched with 1.5 L water. The organic layer was
collected, filtrated through a pad of silica gel, and concentrated
under vacuum to provide the macrocyclic sulfite intermediate as
brownish oil which was used directly in the next step. To the
macrocyclic sulfite in a mixture of CH2Cl2 (150 mL), CH3CN
(150 mL), and water (225 mL) at 0 °C was added NaIO4 (57.9
g, 270.5 mmol) and RuCl3·3H2O (0.23 g, 0.9 mmol). The
stirring mixture was gradually warmed to 25 °C and monitored
with TLC. Upon complete consumption of the macrocyclic
sulfite, the reaction mixture was filtered through a pad of Celite.
Organic layer was collected, washed with brine, concentrated
under vacuum, and recrystallized in methanol at −15 °C to give
+
2H), 3.56−3.75 (m, 28H); HRMS (ESI) calcd for C26H58NO8
[(M+NH4)+] 512.4157, found 512.4228.
Monodisperse Polidocanol 11. 11 was prepared from
alcohol 28 and macrocyclic sulfate 26 by following the same
procedure for polidocanol 27 as yellowish oil (5.7 g, 87%
yield). 1H NMR (400 MHz, CDCl3) δ 0.88 (t, J = 8.0 Hz, 3H),
1.22−1.35 (m, 18H), 1.54−1.62 (m, 2H), 3.44 (t, J = 8.0 Hz,
+
2H), 3.56−3.74 (m, 32H); HRMS (ESI) calcd for C28H62NO9
[(M+NH4)+] 556.4419, found 556.4503.
Monodisperse Polidocanol 12. 12 was prepared from
alcohol 9 and macrocyclic sulfate 25 by following the same
procedure for polidocanol 27 as yellowish oil (5.4 g, 77%
yield). 1H NMR (400 MHz, CDCl3) δ 0.88 (t, J = 8.0 Hz, 3H),
1.18−1.34 (m, 18H), 1.51−1.64 (m, 2H), 3.45 (t, J = 8.0 Hz,
2H), 3.55−3.76 (m, 36H); HRMS (ESI) calcd for
+
C30H66NO10 [(M+NH4)+] 600.4681, found 600.4776.
B
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Monodisperse Polidocanol 13. 13 was prepared from
alcohol 10 and macrocyclic sulfate 25 by following the same
procedure for polidocanol 27 as yellowish oil (1.7 g, 56%
yield). 1H NMR (400 MHz, CDCl3) δ 0.88 (t, J = 8.0 Hz, 3H),
1.21−1.37 (m, 18H), 1.51−1.64 (m, 2H), 3.44 (t, J = 8.0 Hz,
2H), 3.57−3.75 (m, 40H); HRMS (ESI) calcd for
Monodisperse Polidocanol Sulfate 6. 6 was prepared from
alcohol 10 and macrocyclic sulfate 25 by following the same
procedure for polidocanol sulfate 2 as yellowish wax (9.7 g,
82% yield). 1H NMR (400 MHz, CDCl3) δ 0.81 (t, J = 8.0 Hz,
3H), 1.11−1.32 (m, 18H), 1.43−1.59 (m, 2H), 3.37 (t, J = 8.0
Hz, 2H), 3.45−3.76 (m, 38H), 4.08−4.21 (m, 2H); HRMS
(ESI) calcd for C32H65Na2O14S+ [(M+Na)+] 751.3885, found
751.4060.
Monodisperse Polidocanol Sulfate 7. 7 was prepared from
alcohol 10 and macrocyclic sulfate 26 by following the same
procedure for polidocanol sulfate 2 as yellowish wax (5.6 g,
59% yield). 1H NMR (400 MHz, CDCl3) δ 0.88 (t, J = 8.0 Hz,
3H), 1.20−1.38 (m, 18H), 1.50−1.65 (m, 2H), 3.44 (t, J = 8.0
Hz, 2H), 3.53−3.81 (m, 42H), 4.14−4.29 (m, 2H); HRMS
(ESI) calcd for C34H69Na2O15S+ [(M+Na)+] 795.4147, found
795.4167.
Monodisperse Polidocanol Sulfate 8. 8 was prepared from
alcohol 11 and macrocyclic sulfate 26 by following the same
procedure for polidocanol sulfate 2 as yellowish wax (13.7 g,
70% yield). 1H NMR (400 MHz, CDCl3) δ 0.88 (t, J = 8.0 Hz,
3H), 1.22−1.33 (m, 18H), 1.52−1.63 (m, 2H), 3.44 (t, J = 8.0
Hz, 2H), 3.55−3.79 (m, 46H), 4.18−4.27 (m, 2H); HRMS
(ESI) calcd for C36H73Na2O16S+ [(M+Na)+] 839.4409, found
839.4357.
C32H70NO11 [(M+NH4)+] 644.4943, found 644.4973.
+
Monodisperse Polidocanol 14. 14 was prepared from
alcohol 10 and macrocyclic sulfate 26 by following the same
procedure for polidocanol 27 as yellowish solid (4.0 g, 48%
yield). 1H NMR (400 MHz, CDCl3) δ 0.88 (t, J = 8.0 Hz, 3H),
1.21−1.38 (m, 18H), 1.52−1.64 (m, 2H), 3.45 (t, J = 8.0 Hz,
2H), 3.56−3.75 (m, 44H); HRMS (ESI) calcd for
C34H74NO12 [(M+NH4)+] 688.5206, found 688.5254.
+
Monodisperse Polidocanol 15. 15 was prepared from
alcohol 12 and macrocyclic sulfate 25 by following the same
procedure for polidocanol 27 yellowish solid (1.8 g, 59% yield).
1H NMR (400 MHz, CDCl3) δ 0.88 (t, J = 8.0 Hz, 3H), 1.22−
1.35 (m, 18H), 1.53−1.64 (m, 2H), 3.45 (t, J = 8.0 Hz, 2H),
3.55−3.76 (m, 48H); HRMS (ESI) calcd for C36H78NO13+ [(M
+NH4)+] 732.5468, found 732.5497.
Monodisperse Polidocanol Sulfate 2. Under an atmosphere
of nitrogen, to a suspension of NaH (0.8 g, 60% in mineral oil,
20.0 mmol) in anhydrous THF (50 mL) at 0 °C was slowly
added a solution of alcohol 27 (5.3 g, 16.7 mmol) in anhydrous
THF (50 mL). After stirring for 30 min at 0 °C, a solution of
macrocyclic sulfate 25 (5.3 g, 25.1 mmol) in anhydrous THF
(50 mL) was added and the reaction was monitored until no
alcohol 27 could be detected by TLC. Then, the reaction was
quenched with water (2.0 mL), concentrated under vacuum,
purified with flash column chromatography on silica gel
(dichloromethane/methanol = 10/1 as eluent) to give
monodisperse polidocanol sulfate 2 as yellowish wax (6.0 g,
65% yield). 1H NMR (400 MHz, CDCl3) δ 0.88 (t, J = 8.0 Hz,
3H), 1.20−1.38 (m, 18H), 1.53−1.63 (m, 2H), 3.44 (t, J = 8.0
Hz, 2H), 3.52−3.80(m, 22H), 4.13−4.21 (m, 2H); HRMS
(ESI) calcd for C24H49Na2O10S+ [(M+Na)+] 575.2836, found
575.2841.
Monodisperse Polidocanol Sulfate 3. 3 was prepared from
alcohol 28 and macrocyclic sulfate 25 by following the same
procedure for monodisperse polidocanol sulfate 2 as yellowish
wax (8.9 g, 86% yield). 1H NMR (400 MHz, CDCl3) δ 0.88 (t,
J = 8.0 Hz, 3H), 1.19−1.39 (m, 18H), 1.52−1.64 (m, 2H), 3.44
(t, J = 8.0 Hz, 2H), 3.55−3.82 (m, 26H), 4.16−4.24 (m, 2H);
HRMS (ESI) calcd for C26H53Na2O11S+ [(M+Na)+] 619.3098,
found 619.2982.
Monodisperse Polidocanol Sulfate 4. 4 was prepared from
alcohol 28 and macrocyclic sulfate 26 by following the same
procedure for polidocanol sulfate 2 as yellowish wax (4.1 g,
52% yield). 1H NMR (400 MHz, CDCl3) δ 0.88 (t, J = 8.0 Hz,
3H), 1.19−1.38 (m, 18H), 1.51−1.63 (m, 2H), 3.45 (t, J = 8.0
Hz, 2H), 3.55−3.82 (m, 30H), 4.18−4.28 (m, 2H); HRMS
(ESI) calcd for C28H57Na2O12S+ [(M+Na)+] 663.3361, found
663.3378.
Methylated Monodisperse Polidocanol 16. Under an
atmosphere of nitrogen, to a suspension of NaH (88 mg,
60% in mineral oil, 2.2 mmol) in anhydrous THF (50 mL) at 0
°C was slowly added a solution of alcohol 9 (501 mg, 1.1
mmol) in anhydrous THF (50 mL). After stirring for 30 min at
0 °C, a solution of CH3I (310 mg, 2.2 mmol) in anhydrous
THF (50 mL) was added and the reaction was monitored until
no alcohol 9 could be detected by TLC. Then, the reaction was
quenched with water (2.0 mL), concentrated under vacuum,
purified with flash column chromatography on silica gel
(dichloromethane/methanol = 10/1 as eluents) to give
methylated monodisperse polidocanol 16 as yellowish wax
(450 mg, 87% yield). 1H NMR (400 MHz, CDCl3) δ 0.88 (t, J
= 8.0 Hz, 3H), 1.22−1.43 (m, 18H), 1.51−1.64 (m, 2H), 3.39
(s, 3H), 3.45 (t, J = 8.0 Hz, 2H), 3.53−3.76 (m, 24H); HRMS
(ESI) calcd for C25H52NaO7 [(M+Na)+] 487.3605, found
+
487.3594.
Methylated Monodisperse Polidocanol 17. 17 was
prepared from alcohol 10 and CH3I by following the same
procedure for methylated monodisperse polidocanol 16 as
yellowish wax (1.0 g, 94% yield). 1H NMR (400 MHz, CDCl3)
δ 0.88 (t, J = 8.0 Hz, 3H), 1.19−1.39 (m, 18H), 1.52−1.64 (m,
2H), 3.38 (s, 3H), 3.45 (t, J = 8.0 Hz, 2H), 3.52−3.75 (m,
+
28H); HRMS (ESI) calcd for C27H56NaO8 [(M+Na)+]
531.3867, found 531.3857.
Methylated Monodisperse Polidocanol 18. 18 was
prepared from alcohol 11 and CH3I by following the same
procedure for methylated monodisperse polidocanol 16 as
1
yellowish wax (371 mg, 90% yield). H NMR (400 MHz,
CDCl3) δ 0.89 (t, J = 8.0 Hz, 3H), 1.22−1.39 (m, 18H), 1.53−
1.65 (m, 2H), 3.40 (s, 3H), 3.46 (t, J = 8.0 Hz, 2H), 3.49−3.82
+
(m, 32H); HRMS (ESI) calcd for C29H60NaO9 [(M+Na)+]
Monodisperse Polidocanol Sulfate 5. 5 was prepared from
alcohol 9 and macrocyclic sulfate 25 by following the same
procedure for polidocanol sulfate 2 as yellowish wax (8.7 g,
78% yield). 1H NMR (400 MHz, CDCl3) δ 0.89 (t, J = 8.0 Hz,
3H), 1.13−1.42 (m, 18H), 1.48−1.70 (m, 2H), 3.45 (t, J = 8.0
Hz, 2H), 3.49−3.88 (m, 34H), 4.09−4.23 (m, 2H); HRMS
(ESI) calcd for C30H61Na2O13S+ [(M+Na)+] 707.3623, found
707.3607.
575.4130, found 575.4114.
Methylated Monodisperse Polidocanol 19. 19 was
prepared from alcohol 12 and CH3I by following the same
procedure for methylated monodisperse polidocanol 16 as
yellow wax (891 mg, 94% yield). 1H NMR (400 MHz, CDCl3)
δ 0.84 (t, J = 8.0 Hz, 3H), 1.15−1.34 (m, 18H), 1.47−1.65 (m,
2H), 3.34 (s, 3H), 3.41 (t, J = 8.0 Hz, 2H), 3.47−3.73 (m,
C
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36H); 13C NMR (100 MHz, CDCl3) δ 14.1, 22.6, 26.1, 29.3,
polidocanols or their derivatives concentrations were measured
with the above method. Finally, the pyrene intensity ratios of
29.5, 29.58, 29.63, 31.9, 59.0, 70.0, 70.4, 70.5, 70.6, 71.5, 71.9;
HRMS (ESI) calcd for C31H64NaO10 [(M+Na)+] 619.4392,
I373/I384 were plot against concentrations to calculate the CMC.
+
Procedures for Biological Assay. HUVEC cells were
found 619.4371.
selected as the representative cells. The cells were cultured in a
cell incubator under 5% CO2 at 37 °C with DMEM containing
10% fetal bovine serum and 1% streptomycin double antibody
as the medium. Test compounds were diluted with PBS buffer,
pH 7.4 (NaCl 137 mM, KCl 2.7 mM, Na2HPO4 10 mM,
KH2PO4 2 mM). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) method was employed to
evaluate the cell viability. HUVEC cells were harvested in
logarithmic growth phase and digested with trypsin to prepare
cell suspension with a concentration of about 3 × 104 cells/mL.
Cell suspension (100 μL) was added to each well of 96-well
plates and incubated in a 5% CO2 cell incubator at 37 °C. After
24 h, polidocanol solution of different concentrations was
added and diluted with DMEM medium to the final
concentration. Each concentration is set to six identical wells
and blank control wells were also set. The cells were incubated
for 24 h. Then 20 μL MTT was added to each well and the cells
were incubated for another 4 h. Afterward, the culture medium
was discarded and the cells were dissolved by 200 μL DMSO
and absorbance was read at 570 and 490 nm by a microplate
reader (Thermo, USA). Cell viability (%) was calculated by the
formula:
Methylated Monodisperse Polidocanol 20. 20 was
prepared from alcohol 13 and CH3I by following the same
procedure for methylated monodisperse polidocanol 16 as
yellow wax (900 mg, 70% yield). 1H NMR (400 MHz, CDCl3)
δ 0.89 (t, J = 8.0 Hz, 3H), 1.09−1.47 (m, 18H), 1.52−1.69 (m,
2H), 3.40 (s, 3H), 3.46 (t, J = 8.0 Hz, 2H), 3.54−3.71 (m,
40H); 13C NMR (100 MHz, CDCl3) δ 14.1, 22.7, 26.1, 29.4,
29.5, 29.6, 29.7, 31.9, 59.0, 70.0, 70.5, 70.6, 71.6, 71.9; HRMS
(ESI) calcd for C33H68NaO11 [(M+Na)+] 663.4654, found
663.4636.
+
Methylated Monodisperse Polidocanol 21. 21 was
prepared from alcohol 14 and CH3I by following the same
procedure for methylated monodisperse polidocanol 16 as
yellow solid (1.3 g, 82% yield). 1H NMR (400 MHz, CDCl3) δ
0.89 (t, J = 8.0 Hz, 3H), 1.22−1.35 (m, 18H), 1.53−1.64 (m,
2H), 3.40 (s, 3H), 3.46 (t, J = 8.0 Hz, 2H), 3.54−3.76 (m,
44H); 13C NMR (100 MHz, CDCl3) δ 14.1, 22.6, 26.0, 29.3,
29.6, 29.55, 29.58, 29.6, 31.9, 59.0, 70.0, 70.46, 70.52, 71.5,
71.9; HRMS (ESI) calcd for C35H72NaO12 [(M+Na)+]
707.4916, found 707.4895.
+
Methylated Monodisperse Polidocanol 22. 22 was
prepared from alcohol 15 and CH3I by following the same
procedure for methylated monodisperse polidocanol 16 as
yellow solid (1.5 g, 63% yield). 1H NMR (400 MHz, CDCl3) δ
0.89 (t, J = 8.0 Hz, 3H), 1.19−1.41 (m, 18H), 1.51−1.69 (m,
2H), 3.39 (s, 3H),3.45 (t, J = 8.0 Hz, 2H), 3.53−3.75 (m,
48H); 13C NMR (100 MHz, CDCl3) δ 14.1, 22.6, 26.1, 29.3,
29.4, 29.55, 29.58, 29.6, 31.9, 59.0, 70.0, 70.47, 70.53, 71.5,
Cell Viability(%) = [(ATest − ABlank)/(AControl − ABlank)]
× 100%
ATest, AControl, and ABlank represented the absorbance of cells
with different treatments, untreated cells, and blank culture
media, respectively. The IC50 was calculated by Origin software.
The cytotoxicity assay on L929 cells and HePG2 cells
employed the same procedure as the HUVEC cells. DMEM
and MEM were used as the mediums for HePG2 and L929
cells, respectively.
71.9; HRMS (ESI) calcd for C37H76NaO13 [(M+Na)+]
751.5178, found 751.5154.
+
Purity Analysis by HPLC-ELSD. Polydisperse polidocanol
1, monodisperse polidocanols 9−15, and their derivatives 16−
22 were dissolved in water at a concentration of about 5 mg/
mL. Three mixtures of monodisperse polidocanols and their
derivatives solutions were also prepared, respectively. After
filtration, the solutions were analyzed by HPLC-ELSD using
0.1% (V/V) trifluoroacetic acid in water (mobile phase A) and
0.1% (V/V) trifluoroacetic acid in acetonitrile (mobile phase B)
under the following gradient elution: from 0 to 20 min a linear
increase from 0 to 60% of B, and from 21 to 80 min a linear
increase from 60% to 100% of B (for polydisperse polidocanol
1 and monodisperse polidocanol 9−15); from 0 to 10 min a
linear increase from 0 to 80% of B, from 11 to 35 min a linear
increase from 80% to 100% of B, and from 36 to 40 min an
isocratic step at 100% of B (for methylated monodisperse
polidocanol 16−22). The flow rate was set at 0.5 mL/min and
the gas flow rate was set at 2.9 L/min. The temperature of the
evaporation chamber was 109 °C.
RESULTS
■
The synthesis of each monodisperse component in regular
polidocanol is based on a macrocyclic sulfate strategy
developed in this group (Scheme 2).4 Besides monodisperse
polidocanols, the corresponding sulfates are also highly valuable
because sodium tetradecyl sulfate (STS) has been clinically
used as a substitute for Asclera since 1940s. Interestingly, this
macrocyclic sulfate strategy can conveniently provide these
sulfates by nucleophilic ring opening of the macrocyclic sulfate.
Macrocyclization of triethylene glycol 23 and tetraethylene
glycol 24 with thionyl chloride followed by oxidation with in
situ generated RuO4 gave macrocyclic sulfates 25 and 26,
respectively. Nucleophilic ring opening of macrocyclic sulfates
25 and 26 with dodecyl alcohol followed by hydrolysis in the
presence of sulfuric acid provided alcohols 27 and 28,
respectively. From alcohols 27 and 28, iterative nucleophilic
ring opening reaction on macrocyclic sulfates 25 and 26
followed by acidic hydrolysis of sulfate intermediates provided
all the designed monodisperse polidocanols 9−15 on multi-
gram scale, respectively. The corresponding monodisperse
polidocanol sulfates 2−8 were directly prepared by the
nucleophilic ring opening reaction of macrocyclic sulfates 25
and 26 without hydrolysis. Then their methylated derivatives
16−22 were synthesized by methylation of monodisperse
polidocanols 9−15 with iodomethane (Scheme 2).
CMC Measurement.15,16 To a 10 mL brown bottle was
added a solution of pyrene (0.10 mL, 6.08 mg/L in acetone)
and acetone was allowed to volatize in dark. Then, an aqueous
solution of the polidocanol or its derivative (10 mL with
accurate concentration) was added to the bottle which was
covered with aluminum foil to avoid light. The bottle was first
put in an ultrasound bath for 30 min, then in a water bath at 60
°C for 40 min and 40 °C for 12 h. After it was cooled to room
temperature, pyrene fluorescent intensities at 373 and 384 nm
were measured at 25 °C and the intensity ratio of I373/I384 was
calculated. Then, the intensity ratios of I373/I384 at a series of
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higher critical micelle concentration (CMC) than regular
polidocanol 1 as a result of the sulfates’ higher hydrophilicity
(Figure 1 and Table S1). Monodisperse polidocanols 9−15 and
Scheme 2. Synthesis of Monodisperse Polidocanols 9−15,
Their Sulfates 2−8, and Methylated Derivatives 16−22
It is noteworthy that the synthesis is efficient and convenient.
On one hand, no protecting or activating group was used in this
synthesis which dramatically shorten the synthetic route. On
the other hand, by simple combination of these building blocks,
all the designed monodisperse polidocanol analog can be
conveniently synthesized. Finally, it is also interesting to point
out that these monodisperse polidocanol sulfates are very
difficult to prepare through other means.
Then a purity analysis of monodisperse polidocanols 9−15
(Figure S1) and their methylated derivatives 16−22 (Figure
S2) together with regular polidocanol 1 was carried out on
HPLC. As no UV absorption of these compounds on HPLC,
evaporative light-scattering detector (ELSD) was employed. A
complex mixture on HPLC can be found from regular
polidocanol 1 which clearly show its polydispersity. Over 17
components were observed in regular polidocanol and no
complete separation was obtained on HPLC after many tries. In
contrast, only one component can be find from each
monodisperse polidocanol 9−15 and its methylated derivative
16−22, respectively. In addition, a side-by-side comparison of
regular and monodisperse polidocanols on TLC conveniently
showed the obvious difference in purity (Figure S3).
Figure 1. CMC of monodisperse polidocanols 9−15 (b), their sulfates
2−8 (a), and their methylated derivatives 16−22 (c).
their methylated derivatives 16−22 have lower CMC than
polydisperse polidocanol 1. It was also found that, with the
increasing of ethylene glycol units in monodisperse polidoca-
nols 9−15 and their methylated derivatives 16−22, their CMCs
also gradually increased. The noticeable CMC difference
between monodisperse polidocanols and their methylated
derivatives may result in different biological behaviors.
Then, cytotoxicity of monodisperse polidocanols 9−15, their
sulfates, and methylated derivatives 16−22 together with
regular polidocanol 1 was studied with MTT cytotoxicity
assay on a panel of selected cells, including human umbilical
vein cells (HUVEC cells), fibroblast-like cells (L929 cells), and
liver hepatocellular carcinoma cells (HePG2 cells) (Figures 2,
S4, S5, and S6 and Table S2). HUVEC cells are the target cells
in clinic treatment of sclerose small spider veins and reticular
veins with Asclera. L929 cells and HePG2 cells were chosen as
normal cells for safety assessment and cancer cells for
anticancer efficacy assessment of these compounds, respec-
tively. First, the sulfates of polidocanols has pretty low
cytotoxicity toward the selected cell lines. Second, the
cytotoxicity assay on HUVEC cells indicated that monodisperse
polidocanols 9−15 exhibit higher cytotoxicity than regular
With these polidocanols 9-15, their sulfates 2-8, and
methylated derivatives 16−22 in hand, a physicochemical
investigation was then carried out to reveal the relationship
between their chemical structure and micelle formation
property which may play a role during polidocanol’s clinic
application. Through a pyrene-based fluorescent method,5 it
was found that polidocanol sulfates 2−8 have about 4 times
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cytotoxicity assessment of these compounds on HePG2 cells
indicated that monodisperse polidocanols and their methylated
derivatives have much higher anticancer efficacy than regular
polidocanol 1. Finally, cytotoxicity of two mixtures with equal
amount of monodisperse polidocanols 9-15 (M1) and their
methylated derivatives 16−22 (M2) was investigated. Compar-
ing to the monodisperse polidocanols 9−15 and their
methylated derivatives 16−22, mixture M1 and M2 showed
lower cytotoxicity toward the selected cell lines. Therefore, the
polydispersity in polidocanol indeed plays a role in the
cytotoxicity. It is interesting to point out that mixture M1
and M2 showed even lower cytotoxicity toward the selected cell
lines than regular polidocanol 1 which indicated that some
unknown components in regular polidocanol 1, such as
impurities derived from the polymerization process, interfered
with the cytotoxicity. In all the cases of monodisperse
polidocanols and derivatives, it was also found that the size
of polyethylene glycol moiety has a considerable influence on
their bioactivity, safety, and anticancer efficacy. Based on these
observations, synthesis of monodisperse polidocanols and their
derivatives would be an effective way to improve the drug
efficacy and safety of Asclera.
CONCLUSIONS
■
In this study, we have developed a macrocyclic sulfate-based
strategy for the convenient and scalable synthesis of
monodisperse polidocanols, their sulfates, and methylated
derivatives for a comparative study of monodisperse and
polydisperse polidocanols. Through physicochemical study,
HPLC analysis, and biological assay, it was found that
polydispersity in PEGs can downgrade the purity, bioactivity,
and safety of regular polidocanol. In contrast, monodisperse
polidocanols and their derivatives exhibit a single component,
predictable physicochemical properties, much higher bioactivity
and safety than regular polidocanol. Furthermore, the size of
ethylene glycol in monodisperse polidocanols and their
derivatives also influenced their cytotoxicity and safety.
Although the molecular mechanism behind these phenomena
is still unknown, this study shows, for the first time, that
polydispersity of PEGs indeed can compromise the therapeutic
efficacy and safety of polidocanol. Therefore, in the era of
accurate medicine, it is necessary to pay more attention to
polydispersity of PEGs in small molecular drugs to avoid the
issues in drug purity, efficacy, safety, quality control, regulatory
approval, and beyond.
Figure 2. IC50 of regular polidocanol 1, monodisperse polidocanols 9−
15, their methylated derivatives 16−22, and mixtures with equal
amount of 9−15 (M1) and 16−22 (M2) on HUVEC cells (a), L929
cells (b), and HePG2 cells (c).
polidocanol 1. Although compound 12 with a nonaethylene
glycol moiety is claimed as the major component in Asclera, its
cytotoxicity toward HUVEC cells is actually not the most
potent among monodisperse polidocanols 9−15. It is note-
worthy that methylation of the hydroxyl group in monodisperse
polidocanols 9−15 significantly enhance the cytotoxicity
toward HUVEC cells. Among them, methylated monodisperse
polidocanol 20 with an IC50 of 12.0 μM is 4.2 and 2.8 times
more potent than regular polidocanol 1 and the corresponding
monodisperse polidocanol 13, respectively. Third, the cytotox-
icity assay on L929 cells showed that monodisperse
polidocanols 9−15 have much lower cytotoxicity than regular
polidocanol 1, which is an indication of higher safety for
monodisperse polidocanols. The cytotoxicity of monodisperse
polidocanol 10 with an IC50 of 144.3 μM is 3 times lower than
that of regular polidocanol 1. The same trend was also found
for methylated monodisperse polidocanols 16−22. Forthyl, the
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.molpharma-
ceut.7b00496.
HPLC chromatograms, TLC comparison of purification,
CMC of compounds, MTT cytotoxicity assays, and
copies of 1H/13C NMR, MS/HRMS spectra of
compounds (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: zxjiang@whu.edu.cn.
ORCID
Zhong-Xing Jiang: 0000-0003-2601-4366
F
DOI: 10.1021/acs.molpharmaceut.7b00496
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Author Contributions
⊥Z.Y. and S.B. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are thankful for financial support from the National Key
Research and Development Program of China
(2016YFC1304704), the National Natural Science Foundation
of China (21372181, 21402144 and 21572168), and the State
Key Laboratory for Modification of Chemical Fibers and
Polymer Materials (Donghua University).
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