Synthesis, anticancer activity and cytotoxicity of 7-O-β-d-galactosyl-polyethylene glycol-epothilone B

Article abstract of DOI:10.1111/cbdd.13447

Epothilone, the macrolide compound produced by Sorangium cellulosum, has antitumor activity. Its anti-tumor mechanism is similar to that of paclitaxel, which promotes the polymerization of tubulin and induces apoptosis. Herein, 7-O-β-d-galactosyl-polyethy

Full text of DOI:10.1111/cbdd.13447

DR GANGLIANG HUANG (Orcid ID : 0000-0001-8768-9979)
Article type : Research Article
Running head: Synthesis, anticancer activity and cytotoxicity of 7-O-β-D-galactosyl-PEG-epothilone B
Synthesis, anticancer activity and cytotoxicity of
7-O-β-D-galactosyl-PEG-epothilone B
Shiyu Huang, Gangliang Huang*
Active Carbohydrate Research Institute, Chongqing Key Laboratory of Inorganic Functional Materials,
Chongqing Normal University, Chongqing, 401331, China
E-mail: huangdoctor226@163.com
Abstract: Epothilone, the macrolide compound produced by Sorangium cellulosum, has antitumor
activity. Its anti-tumor mechanism is similar to that of paclitaxel, which promotes the polymerization
of tubulin and induces apoptosis. Herein, 7-O-β-D-galactosyl-PEG-epothilone B 6 was synthesized. It
showed that the toxicity of the synthesized compound was 1/182 of the epothilone B. In addition,
compound 6 also had significant anticancer activity under the action of enzyme.
Keywords: 7-O-β-D-galactosyl-PEG-epothilone B, synthesis, anticancer activity, toxicity
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1. Introduction
Epothilones, the macrolide compounds produced by Sorangium cellulosum, have
antitumor activity. The anti-tumor mechanism of epothilone is similar to that of paclitaxel,
which promotes the polymerization of tubulin and induces apoptosis [1]. Because paclitaxel
has a significant clinical effect, it is recognized by the world as “the most important new
anticancer drug discovered in the past 15 years”, making it one of the three major
achievements in international anticancer drugs since the 1990s [2]. Epothilone is a
non-paclitaxel drug, its efficacy is definite, it is a broad-spectrum and high-efficiency
anticancer drug, which has a good effect on drug-resistant tumors that are resistant to
paclitaxel and other chemotherapeutic drugs. It can be mass-produced by microbial
fermentation, which not only has low cost, but also overcomes the damage to precious
green resources caused by the development of paclitaxel, making it extremely marketable.
Epothilone has good water solubility, simple structure and better chemical modification
potential than paclitaxel, which provides a possibility for obtaining new epothilone
derivatives, and has become a hot spot in the development of new international anti-tumor
drugs. It is expected to replace paclitaxel and become a generation of mainstream
anti-tumor drugs [3]. There are several epothilone analogs for clinical trials, including the
semi-synthetic epothilone B derivatives (BMS-247550, BMS-310705) from Bristol-Myers
Squibb (BMS), chemically synthesized dehydrogenation epothilone D analog (KOS-1584),
Novartis's natural product epothilone B (EPO906) and Bayer's chemically totally synthesized
ZK-EPO. BMS-247550 (Ixabepilone) was officially certified by the Food and Drug
Administration (FDA) as the first epothilone drug IXEMPRA on the market on October 18,
2007, marking a major achievement in the development of new anticancer drugs for
epothilone [3]. However, natural epothilone compounds have been found to have
disadvantages such as high toxicity in vivo, poor selectivity to tumor cells, and susceptibility
to drug resistance [4,5].
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The introduction of glycosyl group into the macrolide compound tends to reduce its
toxicity and enhance its targeting [6]. In addition, the introduction of polyethylene glycol
(PEG) in drugs can also reduce their toxicity [7]. Therefore, the galactose and polyethylene
glycol structural units were introduced into the epothilone B to synthesize
7-O-β-D-galactosyl-PEG-epothilone B 6. So, it would be confirmed whether this could reduce
the toxicity of epothilone B. At the same time, the anticancer activity of compound 6 under
the action of enzyme was also examined.
2. Experimental method
2.1. General methods
Kieselgel 60F254 (E. Merck) was used for TLC. Compounds were visualized using UV light and
charring with 1:4 H2SO4-ethanol. Flash column chromatography was performed on column
of silica gel (Baker, 0.063-0.200nm). The size exclusion chromatography was performed on
Biogel P2. 13C and 1H NMR spectra were recorded using a Bruker DPX-300 spectrometer at
75 and 300MHz, respectively. Mass spectra were recorded with a VG PLATFORM mass
spectrometer using the ESI mode. Some chemical reagents and antibody were purchased
from Sigma company.
2.2. Fermentation synthesis of epothilone B
2.2.1. Culture method
2.2.1.1. Seed culture
The strains deposited in the solid slant medium were connected to the CNST plate on
which the sterilized filter paper was placed. The plate was placed in a constant temperature
incubator and cultured at 30 for 5-7 d, and then transferred to M26 medium. The flask
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with a liquid volume of 50/250mL was shaken at 30 and 170r/min for 72h, and then
obtained as a seed culture for fermentation.
2.2.1.2. Fermentation culture
The resulting seed liquid was inoculated into a fermentation medium at a 5% (V/V)
inoculum for shake flask culture. The fermentation system is a 300 mL triangular flask with a
liquid volume of 50 mL, and shaken at 30 , 170 r/min for 5 d.
2.2.2. Extraction of epothilone B
After the end of the fermentation, the resin was collected. After immersing for 24 h with
10 volumes of methanol, the resin was discarded. The methanol extract was dried in a
vacuum drying oven, reconstituted with 500 μL of methanol, and transferred to a centrifuge
tube. Epothilone B: 1H NMR (CDCl3, 300 MHz) δ 6.94 (s, 1H), 6.56 (s, 1H), 5.39 (dd, 1H), 4.26
(br s, 1H), 4.21 (br s, 1H), 3.82 (t, 1H), 3.32-3.28 (m, 1H), 2.80 (dd, 1H), 2.72-2.63 (m, 1H),
2.69 (s, 3H), 2.56 (dd, 1H), 2.39-2.33 (m, 1H), 2.16-2.11 (m, 1H), 2.05 (s, 3H), 1.95-1.86 (m,
1H), 1.78-1.70 (m, 2H), 1.57-1.36 (m, 5H), 1.33 (s, 3H), 1.29 (s, 3H), 1.20 (d, 3H), 1.10 (s, 3H),
1.05 (d, 3H); 13C NMR (CDCl3, 75 MHz) δ 220.5, 171.0, 165.1, 153.5, 139.6, 120.3, 116.8,
78.0, 73.5, 72.8, 62.7, 60.3, 53.1, 42.8, 40.0, 36.2, 31.9, 31.1, 30.5, 22.6, 22.0, 21.3, 19.7,
18.8, 17.2, 16.0, 14.5; MS (ESI): 530.95 [M+Na]+.
2.2.3. Optimization of fermentation conditions
2.2.3.1. Single factor test design
The effects of initial pH, liquid loading, rotation speed, inoculum size, seed age,
temperature and fermentation time on the yield of epothilone B were investigated by single
factor test.
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2.2.3.2. Orthogonal design
On the basis of the single factor experiment, some factors that had a great influence on the
yield of epothilone B (initial pH, liquid volume, inoculum size, temperature) were selected to
further optimize the fermentation conditions. Three factors were set for each factor, and
the four-factor three-level orthogonal design L9 (34) was used for the test.
2.3. Cell culture
MCF-7 cells were cultured in RPMI-1640 medium. 10% fetal bovine serum and double
antibiotic should be added during use. The concentrations of penicillin and streptomycin
were 100 U/mL and 100 mg/L, respectively. The culture conditions were: 37 , 5% CO2. The
cells were cultured to logarithmic growth phase for experiments.
2.4. Preparation of Herceptin-galactosidase complex
Herceptin-galactosidase complex was prepared by chemical cross-linking of anti HER2
monoclonal antibody Herceptin and galactosidase with N-hydroxysuccinimide
m-(N-maleimide) benzoate (MBS) [8,9]. Study was carried out again on anti-tumor activity
and cytotoxicity in vitro.
2.5. The assay of cell proliferation
The MTT colorimetric method was used. The detection principle of this method is that
succinate dehydrogenase can reduce MTT to blue-violet crystalline formazan, which is
insoluble in water and can be deposited in cells. Succinate dehydrogenase is only present in
living cell mitochondria, and dead cells do not contain this enzyme in mitochondria. The
formazan in the cells was dissolved in dimethyl sulfoxide, and its absorbance at 490 nmol/L
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was detected by a microplate reader. The larger the absorbance value, the more the
number of formazan formed, indicating that the cell viability was stronger.
3. Results and discussion
3.1. Fermentation synthesis of epothilone
Using strong promoter to initiate transcription of subsequent genes can greatly promote
the expression of target genes. In order to further increase the yield of epothilone B, this
study would construct plasmids pR6K-Amp-H.a-f-Ptet-H.a-r and plasmids
pR6K-H.a-f-PBAD-H.a-r by a series of restriction enzyme ligation and Red/ET homologous
recombination genetic engineering techniques. The plasmids used R6K as a replicon,
containing the promoter PTet or PBAD, the upstream and downstream homology arms of
original promoter of epothilone. In order to transfer to the epothilone engineered bacteria,
we replaced its original promoter to promote the fermentation synthesis of epothilone.
In order to improve the yield of epothilone B, on the basis of screening suitable carbon and
nitrogen sources, a multiple high-order regression mathematical model was established by
using uniform design method combined with stepwise regression method to optimize the
fermentation medium of Sorangium cellulosum. The central combinatorial design method
was used to optimize the synthetic precursor medium of epothilone. On this basis, the
central combined response surface analysis method was used to optimize the shake flask
fermentation conditions of Sorangium cellulosum. The optimal fermentation conditions
were determined as: Initial pH 7.4, liquid volume 50/250mL, rotation speed 200r/min, seed
age 60h, inoculum volume 10%, temperature 30 , and fermentation cycle 6 d. Under this
optimal fermentation condition, the yield of epothilone B was 39.76 mg/L, which was
19.75% higher than that before optimization.
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3.2. Chemical synthesis of glycosylated epothilone prodrug
The reactivity of C-3 and C-7 hydroxyl groups of epothilone is not the same (Fig. 1). The C-7
hydroxyl group has a small steric hindrance, and it can be selectively acetylated [10]. The
hydroxyl group at the C-7 position was selectively silanized with the equal mole amount of
TBSCl to obtain compound 2.
The synthetic route of galactose-PEG-linker complex 5 was shown in Fig. 2. In the presence
of NaAc, galactose 3 and acetic anhydride were reacted at 128 to form β-peracetylated
galactose 4 in a yield of 90%. Using BF3•Et2O as the catalyst, compound 4 was reacted with
an excess of 2-[2-(2-chloroethoxy)ethoxy]ethanol to give compound 5 (62%). The
polymerization degree of PEG was 3.
Compound 2 was coupled to compound 5, followed by deacetylation and desilylation in
turn to obtain the target 7-O-β-D-galactosyl-PEG-epothilone B 6 (Fig. 3): 1H NMR (CDCl3, 300
MHz) δ 6.95 (s, 1H), 6.52 (s, 1H), 5.86-5.83 (d, 1H, Hα-1), 5.38 (dd, 1H), 5.33-5.29 (m, 2H, H-2,
H-3), 5.23-5.18 (q, 1H, H-4), 4.40-4.28 (q, 1H, H-6), 4.25 (br s, 1H), 4.19 (br s, 1H), 4.14-4.12
(d, 1H, H-6′), 4.07-3.88 (q, 1H, H-5), 3.79 (t, 1H), 3.77-3.50 (m, 12H, -OCH2CH2O-), 3.31-3.27
(m, 1H), 2.78 (dd, 1H), 2.75-2.64 (m, 1H), 2.68 (s, 3H), 2.57 (dd, 1H), 2.41-2.37 (m, 1H),
2.15-2.09 (m, 1H), 2.06 (s, 3H), 1.97-1.88 (m, 1H), 1.73-1.70 (m, 2H), 1.59-1.40 (m, 5H), 1.37
(s, 3H), 1.33 (s, 3H), 1.25 (d, 3H), 1.16 (s, 3H), 1.09 (d, 3H).
3.3. The cytotoxicity to normal cells and resistance to tumor cells of
7-O-β-D-galactosyl-PEG-epothilone B 6
MTT colorimetry is to detect the survival and growth of cells. It is the reason that succinate
dehydrogenase in mitochondria of living cell can reduce exogenous MTT to form the
water-insoluble blue violet crystal formazan, which deposits in cells. However, dead cells do
not have the function. The formazan can be dissolved with dimethyl sulfoxide in the cells,
the absorbance can be measured at 490 nm, and the number of living cells can be calculated
based on the absorbance value. The amount of MTT crystal formation is linear with the
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number of cells. This method has been widely used for detecting the activity of substances,
screening of large-scale drugs and assaying of cytotoxicity. It is characterized by high
sensitivity and low cost.
Epothilone B was used to treat the cell line MCF-7, and its concentration was in the range
of 0.01-1000 nmol/L. The rate of inhibition to cancer cell was changed from 3.1 to 80.5 %
after 2 d, which exhibited a dose dependent pattern. However, the rate of inhibition to
cancer cells treated with 7-O-β-D-galactosyl-PEG-epothilone B 6 was changed from 6.2 % to
90.7 % under the same conditions in the presence of β-D-galactoside galactohydrolase
(Table 1). So, it proved that the galactosylation and PEGylation of epothilone B could
improve its inhibitory activity against the proliferation of cancer cells under the action of
enzyme.
The IC50 of 7-O-β-D-galactosyl-PEG-epothilone B 6 and free epothilone B was 7016.29 and
38.55 nmol/L, respectively. In the presence of β-D-galactoside galactohydrolase, the
cytotoxicity of 7-O-β-D-galactosyl-PEG-epothilone B 6 was restored to that of the free
epothilone B. The ratio of cytotoxicity between the 7-O-β-D-galactosyl-PEG-epothilone B 6
and the free epothilone B was about 182 (Table 2). The value was compatible with the
antibody-directed enzyme-prodrug therapy (ADEPT)-like strategy where a relatively
non-cytotoxic prodrug could release a cytotoxic compound [11,12].
4. Conclusion
Epothilone is superior to paclitaxel in anti-tumor spectrum, anti-tumor activity, safety,
water solubility and synthetic methods, and is expected to develop into a more effective
anti-tumor drug. However, epothilone is highly toxic in the body and has poor selectivity for
tumor cells. Herein, both galactose and PEG structural units were introduced into
epothilone B. It indicated that 7-O-β-D-galactosyl-PEG-epothilone B 6 was much less toxic
than epothilone B, and compound 6 had good anticancer activity under the action of
enzyme. Therefore, this would provide a new idea for the application of epothilones.
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Acknowledgements
The Project is sponsored by the Scientific Research Foundation for the Returned Overseas
Chinese Scholars, State Education Ministry (No. 2015-1098). The work was also supported
by Chongqing Key Research Project of Basic Science & Frontier Technology (No.
cstc2017jcyjBX0012), Foundation Project of Chongqing Normal University (No. 14XYY020),
Chongqing General Research Program of Basic Research and Frontier Technology (No.
cstc2015jcyjA10054), and Chongqing Normal University Postgraduate's Research and
Innovation Project (No. YKC17004), China.
Conflict of interest statement
The authors confirm that this article content has no conflict of interest.
A list of the figure legends
Fig. 1. Selective protection of C-7 hydroxyl group of epothilone B.
Fig. 2. Synthesis of galactose-PEG-linker complex 5.
Fig. 3. Synthesis of 7-O-β-D-galactosyl-PEG-epothilone B 6.
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References
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1866-1870.
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[7] Zimmerman, R.J., Aukerman, S.L., Katre, N.V., Winkelhake, J.L., Young, J.D. (1989). Schedule
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[8] Syrigos, K. N., Rowlinson-Busza, G., Epenetos, A. A. (1998). In vitro cytotoxicity following specific
activation of amygdalin by β-glucosidase conjugated to a bladder cancer-associated monoclonal
antibody. International Journal of Cancer, 78(6), 712-719.
[9] Huang, G. (2018). Synthesis and biological activities of galactose-aspirin conjugate prodrug
designed for ADEPT and PMT. Medicinal Chemistry Research, 27, 705-708.
[10] Sherrill, M. J., Johnson, R. G. (2006). Therapeutic formulations. US, US 7091193 B2.
[11] Gao, H., Huang, G. (2018). Synthesis, anticancer activity and cytotoxicity of galactosylated
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[12] Springer, C.J., Niculescu-Duvaz, I.I. (1997). Antibody-directed enzyme prodrug therapy (ADEPT):
a review. Advanced Drug Delivery Reviews, 26(2-3), 151-172.
Tables
Table 1. The anticancer activity of epothilone B and 7-O-β-D-galactosyl-PEG-epothilone B 6 (n=5).
Compound
Anticancer activity (0.01-1000 nmol/L)
epothilone B
3.1-80.5 %
6.2-90.7 %
7-O-β-D-galactosyl-PEG-epothilone B 6
Table 2. The cytotoxicity of epothilone B and 7-O-β-D-galactosyl-PEG-epothilone B 6 (n=5).
Compound
IC50 (nmol/L)
38.55 ± 0.11*
7016.29 ± 0.08*
epothilone B
7-O-β-D-galactosyl-PEG-epothilone B 6
*p < 0.05.
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Figures
Fig. 1. Selective protection of C-7 hydroxyl group of epothilone B.
Fig. 2. Synthesis of galactose-PEG-linker complex 5.
Fig. 3. Synthesis of 7-O-β-D-galactosyl-PEG-epothilone B 6.
O
O
O
S
N
S
N
HO
1eq TBSCl
TBSO
7
3
O
O
Epothilone B 1
O
O
2
OH
O
OH
Fig. 1. Selective protection of C-7 hydroxyl group of epothilone B.
OAc
OAc
2-[2-(2-chloroethoxy)ethoxy]ethanol (3 eq)
BF3·Et2O (3 eq)
OH
OH
O
O
Ac2O, NaAc
128
OAc
β
AcO
MS, CH Cl2, 0
rt, 24 h
4A
2
HO
OH
OAc
OH
4
3
galactose
OAc
OAc
O
O
O
O
AcO
Cl
OAc
5
Fig. 2. Synthesis of galactose-PEG-linker complex 5.
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(HF/Py) : Py
3 :1
Et3N
KI
NaOMe (1.5 eq), MeOH, rt, 30 min
2 + 5
O
S
N
HO
O
OH
OH
O
O
O
O
O
O
HO
O
OH
6
Fig. 3. Synthesis of 7-O-β-D-galactosyl-PEG-epothilone B 6.
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