Isolation, identification and characterization of potential impurities in cabazitaxel and their formation

Full text of DOI:10.1002/mrc.4125

Letter - case report
Received: 2 April 2014
Revised: 16 July 2014
Accepted: 17 July 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/mrc.4125
Isolation, identification and characterization of
potential impurities in cabazitaxel and their
formation
Yongyi Wang,a* Feng Feng,b Lei Chen,a Hongtao Zhaoa and Lianzhong Tiana
1
confirming that impurity I was an isomer of cabazitaxel. The H
Introduction
NMR spectrum (Table 1) showed the appearance of a new methyl
Taxanes, such as paclitaxel, docetaxel and cabazitaxel, were well
known for their remarkable anti-tumor activities. They were pres-
ently applied and evaluated for ovarian, lung, breast and other
gynecological cancers.[1–4] According to International Conference
on Harmonization (ICH) guidelines, the origin of all impurities
(process and degradation) in drug substances should be under-
stood completely and the level of the impurities should be
controlled strictly for quality and safety considerations. The process
impurities of taxanes were different because of the different
manufacturing processes. However, the degradation impurities of
taxanes had their regular patterns as all taxanes were the semisyn-
thetic derivatives of 10-deacetylbaccatin III (10-DAB). Most degra-
dation impurities formed at positon C-7, C-10, C-13, C-3 and C-11
of 10-DAB. β-OH at C-7 of taxanes can be easily epimerized into
α-OH as result of retro-aldol process. β-OH at C-10 of taxanes can
be oxygenated. The side chain at C-13 of taxanes can be lost.[5–8]
Furthermore, C-3 and C-11 of taxanes can be connected under
photolytic condition via intramolecular hydrogen transfer from
C-3 to C-12.[9]
doublet at δH 1.23 ppm instead of a methyl singlet at δH 1.87 ppm,
indicating saturation of C11–C12 double bond. Furthermore, the
signal as a result of H-2, a doublet in cabazitaxel, was now a singlet
at δH 5.90 ppm, while the absorption for H-3 (δH 3.81 ppm in
cabazitaxel) was missing. The 13C and DEPT135 13C NMR spectra
indicated the lack of C11–C12 double bond, the additional one sp3
tertiary carbon at δC 40.7ppm and two sp3 quaternary carbon sig-
nals at δC 57.3 and 58.5 ppm.
In HMBC spectrum, H-2 (δH 5.90 ppm) and 19-CH3 (δH 1.64ppm)
were found to show correlations with δC 58.5ppm (C-3). 18-CH3
(δH 1.23 ppm) had correlations with δC 40.7ppm (C-12) and δC
57.3 ppm (C-11). All of above data confirmed that impurity I was
(1R,2R,4S,5S,7R,10S,11R,12S,13S, 15S,16S)-2,5-dimethoxy,10-acetoxy-
13-hydroxyl-4,16,17,17-tetramethyl-8-oxa-3-oxo-12-phenylcarbonyloxy
pentacyclo[11.3.1.01,11.04,11.07,10]heptadec-15-yl(2R,3S)-3-[[(1,1-
dimethylethoxy)carbonyl] amino]-2-hydroxy-3-phenylpropanoate].
Impurity II
Cabazitaxel (Jevtana@; Sanofi-Aventis) was a novel semisynthetic
taxane drug.[2] In June 2010, it was approved by the US Food and
Drug Administration (FDA) for the treatment of patients with
hormone-refractory metastatic prostate cancer that had been
previously treated with docetaxel. Previous reports have shown
the synthesis routes of cabazitaxel.[10–12] However, no systematic
impurity studies had been reported in the literature.[13] The present
study described the isolation and identification of the impurities of
cabazitaxel. The formation mechanisms of these impurities were
also investigated (Figures 1 and 2).
The molecular formula of impurity II was determined to be
C47H59NO16 based on the pseudo-molecular ion peak at m/z
916.3741 [M + Na]+ (calcd 916.3732) in the HR-TOF-ESI-MS analysis.
1H and 13C NMR spectra (Table 1) of impurity II were similar to those
of cabazitaxel but indicated resonances for the addition of an
O-methylene group, an acetoxy and the absence of an O-methyl
group. The chemical shift value of H-7 proton appeared at δH
4.27 ppm instead of δH 3.87 ppm in cabazitaxel showed the lack
of O-methyl group at C-7. The corresponding carbon of H-7 was
detected at δC 77.2ppm after the analysis of HSQC spectrum.
In HMBC spectrum, the O-methylene protons at δH 5.32 and
5.15 ppm showed correlations with C-7 (δC 77.2 ppm) suggested
that the O-methylene group was determined to be C-7. The
additional acetoxy group (δC 170.4, 20.6 ppm) was located at
O-methylene (δC 86.6ppm) from the correlations of δC 5.32 ppm
and δC 5.15 ppm to carbonyl (δC 170.4 ppm) of the acetyl. Based
Results and Discussion
Cabazitaxel
The ESI mass spectrum of cabazitaxel showed a quasi-molecular ion
peak at m/z 836.3 [M + H]+. The structure of cabazitaxel was con-
1
1
firmed by using H NMR, 13C and DEPT135 13C NMR, H-1HCOSY,
HSQC and HMBC spectra, and all NMR signals were assigned in
Table 1. The NMR data were coincident with those reported.[11]
*
Correspondence to: Yongyi Wang, Jiangsu Yew Pharmaceutical Co., Ltd. Hodo
Group, Wuxi 214199, China. E-mail: wy1107@163.com
Impurity I
a Jiangsu Yew Pharmaceutical Co., Ltd. Hodo Group, Wuxi 214199, China
The molecular formula of impurity I was defined as C45H57NO14 by
HR-TOF-ESI-MS at m/z 858.3687 [M + Na]+ (calcd 858.3677),
b Department of Phytochemistry, China Pharmaceutical University, 24 Tongjiaxiang,
Nanjing 210009, China
Magn. Reson. Chem. (2014)
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Y. Wang et al.
Figure 1. Scheme for the synthesis of cabazitaxel and formation of impurity II and impurity III.
Figure 2. Formation of impurity I, impurity IV and impurity V.
on all of these data, impurity II was established as 7-demethoxy-7-
butyloxycarbonyl group were found to be absent. All of these data
methyleneacetate-cabazitaxel.
indicated impurity IV was de-t-butyloxycarbonyl cabazitaxel.
Impurity III
Impurity V
The ESI mass spectrum of impurity III showed a sodium adduct
The ESI mass spectrum of impurity V showed a sodium adduct
[M+ Na]+ at m/z 595.4, indicating that impurity V had a molecular
1
[M+ Na]+ at m/z 844.4, which together with the H and 13C NMR
1
mass less than that of cabazitaxel by 263 Da. In H and 13C NMR
data (Table 1) indicated a molecular formula C44H55NO14. The
NMR data were similar to those of cabazitaxel except for the
absence of an O-methyl group. The chemical shift value of H-10
proton appeared at δH 5.19 ppm instead of δH 4.79 ppm in
cabazitaxel suggested that the O-methyl group at C-10 was lost.
This was supported by the HMBC correlations of the O-methyl pro-
tons at δH 3.25 ppm with C-7. Hence, impurity III was characterized
as 10-demethoxy-10-hydroxy-cabazitaxel.
spectra (Table 1), all side-chain signals of cabazitaxel were absent.
Thus, impurity
V
was confirmed as 7,10-dimethoxy-10-
deacetylbaccatin III.
Conclusion
The process impurities were isolated during the process
development of cabazitaxel. The major degradation impurities
were obtained in acidic stressed, basic stressed and photolytic
stressed conditions. The name, relative retention time, structure,
molecular weight and nature of impurities in cabazitaxel were
indicated in Table 2. Impurity I appeared in the photolytic
stressed condition because intramolecular hydrogen transferred
from C-3 to C-12 as a result of the favorable geometry.[9]
Impurities II and III were process impurities, which were the
Impurity IV
The ESI mass spectrum of impurity IV showed a proton adduct
[M+ H]+ at m/z 736.2, indicating that impurity IV had a molecular
mass less than that of cabazitaxel (100 Da). The 1H NMR spectrum
(Table 1) showed the loss of methyl signals for the tertiary
butyloxycarbonyl group. The proton data was supported by the
13C NMR spectrum in which the carbon signals of tertiary
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Studies of potential impurities in cabazitaxel
Magn. Reson. Chem. (2014)
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Y. Wang et al.
by-products during the process of methylation. Impurity IV
formed as a result of loss t-butyloxycarbonyl of cabazitaxel un-
der acidic condition. In the presence of base condition, impurity
V formed because the side chain was absent. No β-OH at C-7
epimerize impurities and β-OH at C-10 oxidate impurities ap-
peared as C-7 and C-10 were protected by methoxy. Impurities
I and II were new compounds.
Experimental
Reagents and samples
Cabazitaxel (>99% pure) was synthesized in Jiangsu Yew Pharma-
ceutical Co., Ltd., Hodo Group (Wuxi, China). The scheme for synthe-
sis of cabazitaxel was shown in Figure 1. HPLC grade acetonitrile
was purchased from Tedia (USA). CDCl3 and CD3OD were from
Sigma-Aldrich Co. (St. Louis, MO, USA). Other chemicals were ana-
lytical grade. Purified water was filtrated by 0.22-um membrane.
Analytical liquid chromatographic conditions
The chromatographic separation was performed on Agilent 1200
HPLC system (Santa Clara, CA, USA) with DAD detector. The HPLC
method was developed for the analysis of the process and degrada-
tion impurities of cabazitaxel. Separations were achieved on Agilent
Zorbax Eclipse XDB-C18 column with 250× 4.6mm i.d., 5-um parti-
cle size maintained at 30°C. The mobile phase consisted of water (A)
and acetonitrile (B) with a flow rate of 1 ml/min. The impurities were
detected at 228 nm and eluted according to the step gradient by
changing the percentage of acetonitrile (B), T (min)/%B = 0/25,
10/44, 35/60, 50/85. (Figure 3).
Mass spectroscopy
The high resolution time of flight mass spectra (HR-TOF-ESI-MS)
were recorded on
a Micromass Q-TOF microspectrometer
(Manchester, UK) with an electrospray source. The ESI-MS measure-
ments were performed on an Agilent MSD spectrometer with an
electrospray source. (Agilent Technologies, Palo Alto, CA, USA).
Nuclear magnetic resonance spectroscopy
The 1D and 2D NMR spectra were recorded on a Bruker Avance III
400 NMR spectrometer (equipped with 5-mm probe) at 298 K
using CDCl3 and MeOD as solvent. The sample concentration was
approximately 10 mg/ml. Chemical shifts (δ) in parts per million
1
are referenced to TMS at 0.00 ppm for H and 13C. Coupling con-
stants (J) are given in hertz. The pulse conditions were as follows:
1
for H, spectrometer frequency (SF) = 400.15 MHz, spectral width
(SWH) = 8223.68Hz, pulse 90° width (P1) = 14.94 μs, Fourier trans-
form size (SI) = 65536, line broadening (LB)= 0.30 Hz, acquisition
time (AQ)= 3.98 s, relaxation delay (D1) = 1.00 s and number of
dummy scans (DS)= 2; for 13C, SF= 100.62 MHz, SWH = 28 409.09
Hz, P1= 10.06 μs, SI = 32 768; LB = 1.00 Hz, AQ= 1.15 s, D1= 2.0 s,
DS = 4; for DEPT135, SF = 100.62 MHz, SWH = 22 058.82 Hz, SI = 32 768;
LB=1.00Hz, AQ=1.48s, D1=2.0s, DS=4; for HSQC, SF=400.15 (1H),
100.62 Hz (13C), SWH = 4789.27 Hz, AQ= 0.21 s, D1 = 2.0s, DS = 32;
for HMBC, SF= 400.15 (1H), 100.62 Hz (13C), SWH = 4201.68 Hz,
AQ= 0.49 s, D1 = 1.40 s, DS= 16.
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Studies of potential impurities in cabazitaxel
Table 2. Name, relative retention time (RRT), structure, molecular weight and nature of impurities in cabazitaxel
Name
RRT
Structure
Molecular weight
Nature
Cabazitaxel
1.00
835
Drug substance
Impurity I
1.10
835
Photolytic stressed decstrdation
Impurity II
1.03
893
Process impurity
Impurity III
0.94
821
Process impurity
Impurity IV
0.76
735
Acid stressed decstrdation
Impurity V
0.43
572
Base stressed decstrdation
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Y. Wang et al.
mAU
35
mAU
(A)
(B)
120
100
80
60
40
20
0
30
25
20
15
10
5
cabazitaxel
cabazitaxel
Impurity II
Impurity III
Impurity IV
0
-5
0
10
20
30
40
min
0
10
20
30
40
min
mAU
350
mAU
500
400
300
200
100
0
(C)
(D)
300
250
200
150
100
50
cabazitaxel
cabazitaxel
Impurity I
Impurity V
0
min
0
10
20
30
40
min
0
10
20
30
40
Figure 3. HPLC chromatograms of (A) impurity II and impurity III (B) impurity IV (C) impurity V (D) impurity I.
Forced degradation of cabazitaxel
5 um, Chuangxintongheng, Beijing, China); UV detector (Beijing, China)
was carried for isolation. Acetonitrile-water (45 : 55, v/v) was used as
mobile phase with a flow rate of 300ml/min. The wavelength of
detection was 228 nm, and the injection volume was 50 ml.
Thermal stressed degradation
Ten milligrams of cabazitaxel were subjected to thermal degrada-
tion at 105°C for about 10 days.
Acid stressed degradation
Acknowledgment
Ten milligrams of cabazitaxel were dissolved in 10 ml with 1 : 4 mix-
ture of water and acetonitrile; 1 ml of 1.0 N HCl solution was added
and kept for about 2.0h at room temperature.
Authors thank the accumulation plan of doctor in 2013, Jiangsu
Province, China for providing a research fellowship.
Base stressed degradation
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