An effective method to produce 7-epitaxol from taxol in HCO3–

Article abstract of DOI:10.1016/j.bmcl.2020.127285

It is known that 7-epitaxol has much stronger cytotoxicity than taxol does. However, the content of 7-epitaxol in yew is much less than taxol, which makes it more costly to obtain. We describe here a method to effectively convert taxol to 7-epitaxol. The

Full text of DOI:10.1016/j.bmcl.2020.127285

Journal Pre-proofs
-
An effective method to produce 7-epitaxol from taxol in HCO3
Yanjie Liu, Xiaoyu Ma, Mengkai Zhou, Xiaoran Hao, Xudong Zhu
PII:
S0960-894X(20)30395-4
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127285
BMCL 127285
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
7 March 2020
16 May 2020
23 May 2020
Please cite this article as: Liu, Y., Ma, X., Zhou, M., Hao, X., Zhu, X., An effective method to produce 7-epitaxol
-
from taxol in HCO3 , Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.
2020.127285
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version
will undergo additional copyediting, typesetting and review before it is published in its final form, but we are
providing this version to give early visibility of the article. Please note that, during the production process, errors
may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier Ltd.

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
-
An effective method to produce 7-epitaxol from taxol in HCO3
Yanjie Liu, Xiaoyu Ma, Mengkai Zhou, Xiaoran Hao, Xudong Zhu
Beijing Key Laboratory of Genetic Engineering Drug and Biotechnology, Institute of Biochemistry and Biotechnology, College of Life Science, Beijing Normal
University, Beijing, 100875, P.R. China
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
It is known that 7-epitaxol has much stronger cytotoxicity than taxol does. However, the content
of 7-epitaxol in yew is much less than taxol, which makes it more costly to obtain. We describe
here a method to effectively convert taxol to 7-epitaxol. The key condition for reaction needs
NaHCO3 in solvent acetonitrile (ACN). The conversion rate can be over 82%.
Accepted
Available online
2009 Elsevier Ltd. All rights reserved.
Keywords:
7-epitaxol
epimerization
taxol
-
HCO3
acetonitrile
 Corresponding author. Tel.: +86-010-58804266; e-mail: zhu11187@bnu.edu.cn

7-epitaxol1 was one of the natural-existing epimers of the antitumor drug taxol which has been used widely in clinic for decades.2,3 It
was isolated and structurally characterized by Huang et al. in 1986 from the bark of yew Taxus brevifolia.1 Also, in the study, they
demonstrated that taxol could be transformed to 7-epitaxol via the catalysis of azobis (isobutyronitrile) in toluene at 80℃.1 It is well
established that 7-epitaxol exhibits the same antitumor mechanism, mainly by stabilizing of microtubule polymers,4 yet shows a higher
cytotoxicity than taxol, to a number of tumor types, e.g. 12.7 times to murine leukemia L1210 cells and 5.9 times to human epidermoid
carcinoma KB cells.5,6 Still, observations on in vivo biotransformation of taxol revealed the fact that 7-epitaxol is more stable than taxol
in patients.1 When patients were only given with taxol in treatment, 7-epitaxol was formed and detected in both plasma and urine
samples,7,8 suggesting a contribution of 7-epitaxol to treatment. Thus, 7-epitaxol is presumably recognized to own advantages of cure
over taxol. Unfortunately, industrial production of 7-epitaxol mainly depend on the raw material of yew tree, and content of 7-epitaxol
in the plant is even less than taxol in Taxus sp.1,9 and Corylus avellana which are by far the major material supplier for taxol
-
production,10 resulting in high cost in the production of 7-epitaxol. Here we report a finding that HCO3 and some other basic salt could
effectively turn taxol into 7-epitaxol in the solvent acetonitrile (ACN) which itself facilitates subsequent purification steps of the
product, for instance, via extraction hereby by ethyl acetate or dichloromethane to desalinate.
The reaction was set as the following: 20 μL 0.02 mg/mL of freshly prepared taxol in ACN solution, mixed with an equal volume of
NaHCO3 solution at 0.02 mg/mL, was incubated at 60℃ for 30 min, then to centrifuge. An aliquot of 20 μL supernatant was taken for
HPLC analysis. A new signal peak marking for 7-epitaxol was detected at 16.135 min on HPLC spectrum (Fig 1A).7,8 The newly
formed compound was purified via HPLC (ACN:H2O, 70:30, 2 mL/min), and subject to HRMS analysis, which gave rise to ions at m/z
854.3382 [M+H]+ and 876.3227 [M+Na]+, consistent with the mass pattern of 7-epitaxol (Figs 1B and 1C).1,11 1H NMR and 13C NMR
spectrum in CD3CN of purified compound were further performed to confirm the structure by comparing with that of taxol. The major
difference from purified compound to taxol in NMR spectrum occurred in the protons signals of C-7 (δH=3.57 ppm to 4.33 ppm), C-10
(δH=6.72 ppm to 6.33 ppm), C-20 (a pair of doublets at δH=4.27 ppm and 4.38 ppm to a two-proton singlet peak δH=2.00 ppm), and
carbon signals of C-7 (δC=76.10 ppm to 71.57 ppm) and C-19 (δC=15.94 ppm to 9.31 ppm) (Table S1 and Figs S1-S4), similar to the
description of Huang et al.1
To determine the exact role of cation from anion in the reaction, we further tested seven salts, i.e. NaHCO3, CH3COONa, NaCl,
Na2CO3, Na2HPO4, NaOH and NaH2PO4, which all had the same cation Na+, but anion groups are distinct. As shown clearly, NaHCO3
and Na2HPO4 displayed high efficiency in the reaction, with a conversion rate of 23.6% and 10.3%, respectively, under such condition,
while the other salts hardly drove the conversion (Fig 2A). Extend assays showed that KHCO3 had a similar conversion rate of taxol in
-
reactions with NaHCO3 (Fig S5). These results suggest that the active catalytic ingredient was HCO3 rather Na+, consistent with former
conclusion that base promotes the epimerization as well as degradation in aqueous solution though.12 Considering the conversion rate,
NaHCO3 was chosen over Na2HPO4 as the reactant in further assay to optimize reaction parameters, including the reaction time,
Fig. 1 Characterization of 7-epitaxol from taxol. (A) HPLC
analysis of standard Taxol, reaction products, purified 7-
epitaxol and 7-epitaxol. The retention time of taxol and 7-
epitaxol was at 11.831 min and 16.135 min, respectively.
(B) HRMS of standard and purified 7-epitaxol. (C)
Molecular structure of taxol and 7-epitaxol.
Fig. 2 Effects of reactants (A), reaction time (B), reactant
ratio (C) and reaction temperature (D) on the epimerization
of taxol to 7-epitaxol. Yield of taxol and 7-epitaxol after
reaction was measured by HPLC. Each data was measured
in triplicate.

reactant rates and temperatures (Tables S2-S5). As shown in Fig 2B, the conversion efficiency increased with the incubation time, and
nearly half of taxol was turned to 7-epitaxol in 50 min. The conversion rate naturally depended on initial concentration of NaHCO3 and
taxol and their ratio. When the ratio of NaHCO3 to taxol in mass was 2, the highest reaction efficiency (51.4%) was achieved (Fig 2C).
The temperature was also a key factor for the reaction. The epimerization required a temperature over 40℃ (Fig 2D). Notably,
approximately 82% of transformation rate could be achieved when 10 mg taxol, plus 10 mg NaHCO3 in 1 mL ACN was incubated at
60℃, for 1.5 h, i.e. approximately 8.2 mg 7-epitaxol was obtained, and 1.5 mg taxol was recovered, in experiments. Using a taxol
sensitive yeast strain Saccharomyces cerevisiae AD1-8,13 our product 7-epitaxol after purified had a stronger cytotoxicity than taxol
(Table 1. Strain S288C is the wild type).
Table 1 Cytotoxicity of taxol and our 7-epitaxol against Saccharomyces cerevisiae S288C and AD1-8
S288C
IC50 (μM)
>100
AD1-8
IC50(μM)
>100
Compound
Taxol
7-epitaxol
>100
71.5
There are two possible epimerization routes from taxol to 7-epitaxol (Fig 3). The first one is a retroaldol/aldol reaction under the
Fig. 3 Deduced reaction routes to epimerization of taxol to
7-epitaxol.
-
-
catalysis of HCO3 reported by Tian et al.12 (Fig 3). The 7-OH was first deprotonated by Lewis base, HCO3 , and turned into [O]-. The
deprotonated intermediate made the bond of C-7 and C-8 easier to cleave, resulting in a high possibility to produce epimer, 7-epitaxol.
The second route seems plausible to be an intramolecular reaction described by Mclaughlin et al.14 (Fig 3). In this mechanism, the
proton of 7-OH was first transferred to [O] at C-9, concomitant the formation of aldehyde group at C-7 and the cleavage of C-7 and C-8.
However, as analyzed by Tian et al.12, the secondary mechanism was occurred in a ‘closed system’, which may depend on a natural or
acid environment, rather alkaline environment. Together with that fact that 7-epitaxol was rarely produced when acidic NaH2PO4 or
neutral NaCl were added in reaction system (Fig 2), the epimerization occurred through more likely via the first mechanism (Fig 3).
-
In summary, we reported taxol could be converted to the only product 7-epitaxol epimerized in the presence of HCO3 and ACN,
instead of the aqueous Windows Userphase which may causes simultaneous severe degradation of taxol.12 Comparatively, our method
is straightforward with high efficiency to obtain 7-epitaxol which has a higher bioactivity in respect of stability and a stronger antitumor
efficacy.
Acknowledgments
This study was formerly initiated and supported by the National High Technology Research and Development (“863”) Program (no.
2012AA022105) and currently is under the support by the National Science Foundation of China (#81871629).
References and notes
1. C. H. Oliver Huang, D.G.I.K., Neal F. Magri, G. Samaranayake, Journal of Natural Products, 1986, 49, 665-669.
2. Yang, H., et al., Clin Cancer Res, 2019, 25, 5702-5716.
3. Egorin, M.J., Clin Cancer Res, 2008, 14, 2517; author reply 2517-8.
4. Peter B. Schiff, J.F., Susan B. Horwitz, Nature, 1979, 227, 665-667.
5. Hideyuki Shigemori, J.i.K., Journal of Natural Products, 2004, 67, 245-256.
6. Kobayashi, J. and H. Shigemori, Med Res Rev, 2002, 22, 305-328.
7. I. Royer, P.A., J. P. Armand, L. K. Ho, M. Wright, B. Monsarrat, Rapid Commun Mass Spectrom, 1995, 9, 495-502.
8. D. Fraier, V.C., E. Frigerio, Journal of Chromatography A, 1998, 797, 295-303.
9. M. C. Wani, H.L.T., Monroe E. Wall, Journal of the American Chemical Society, 1971, 93, 2325-2327.
10. Ottaggio, L., et al., Journal of Natural Products, 2008, 71, 58-60.
11. Ge, G.B., et al., Rapid Commun Mass Spectrom, 2009, 23, 425-32.
12. Tian, J. and V.J. Stella, J Pharm Sci, 2008, 97, 1224-35.
13. Entwistle, R.A., et al., FEBS Letters, 2008, 582, 2467-2470.
14. JL Mclaughlin, RW Miller, et al., Journal of Natural Products, 1981, 44, 312-319
Declaration of interests

☐ The authors declare that they have no known competing financial interests or personal relationships that
could have appeared to influence the work reported in this paper.
☒The authors declare the following financial interests/personal relationships which may be considered as potential
competing interests:
Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
An effective method to produce 7-epitaxol
-
from taxol in HCO3
Yanjie Liu, Xiaoyu Ma, Mengkai Zhou, Xiaoran Hao, Xudong Zhu*