Synthesis and characterization of four process impurities in pazopanib

Article abstract of DOI:10.1691/ph.2018.8538

Pazopanib (trade name Votrient) is a potent and selective multi-targeted tyrosine kinase inhibitor that blocks tumor growth and inhibits angiogenesis. Based on a recently reported procedure, we herein report the first synthesis of four potential process impurities generated in the production of pazopanib. The structure of these impurities were synthesized and characterized by ¹H NMR, ¹³C NMR and HRMS data. The possible formation mechanisms of these impurities were also elucidated. These findings should be useful for the quality control of pazopanib in manufacture.

Full text of DOI:10.1691/ph.2018.8538

ORIGINAL ARTICLES
Department of Medicinal Chemistry1, College of Pharmacy; Chongqing Institute for Food and Drug Control2, Chongqing
Medical University, Chongqing, PR China
Synthesis and characterization of four process impurities in pazopanib
1
2
1
1
1
1
1,*
JIAN-YONG YUAN , DI ZHANG , XIANG-NAN HU , HUA-JUN WANG , DONG-ZHI RAN , SU-QING SHANG , ZONG-JIE GAN
Received May 15, 2018, accepted June 20, 2018
*Corresponding author: Zong-Jie Gan, Department of Medicinal Chemistry1, College of Pharmacy, Chongqing
Medical University, Chongqing, PR China
gzj@cqmu.edu.cn
Pharmazie 73: 494-497 (2018)
doi: 10.1691/ph.2018.8538
Pazopanib (trade name Votrient®) is a potent and selective multi-targeted tyrosine kinase inhibitor that blocks
tumor growth and inhibits angiogenesis. Based on a recently reported procedure, we herein report the first
synthesis of four potential process impurities generated in the production of pazopanib. The structure of these
impurities were synthesized and characterized by 1H NMR, 13C NMR and HRMS data. The possible formation
mechanisms of these impurities were also elucidated. These findings should be useful for the quality control of
pazopanib in manufacture.
determined by HPLC assay during the preparation of pazopanib (Li
et al. 2010). However, to the best of our knowledge, some of these
impurities are not commercially available and no synthetic methods
were reported. As a result, attempts were made to synthesize and
then confirm these new related substances in this work.
1. Introduction
Pazopanib, a novel multi-target inhibitor of vascular endothelial
growth factor receptor (VEGFR)-1,2,3, platelet derived growth
factor receptor (PDGFR) α/β, fibroblast growth factor receptor and
the stem cell receptor/ c-Kit, can increase progression free survival
in renal cell carcinoma and in soft tissue sarcoma compared to
placebo (Goh et al. 2010; Sonpavde et al. 2008; Keisner et al.
2011). It was approved by U.S. Food and Drug Administration
(FDA) for the treatment of metastatic renal cell carcinoma under
the brand name of Votrient® in 2009 (FDA Label 2009).
The detection, identification, quantification control of impurities
especially process impurities originating in the active pharmaceutical
ingredient (API) manufacturing process have become an important
element of drug development (Gorog et al. 1997; ICH 2006). Thus,
it is mandatory to characterize and synthesize the potential process
impurities produced in the manufacture of pazopanib for the quality
control purpose. Some potential process impurities with or without
structures were consistently detected in amounts of 0.1% or above
2. Investigations, results and discussion
One of the most common synthetic method of pazopanib is summa-
rized in Scheme 1 (Okaniwa et al. 2012; Elder et al. 2013). Conden-
sation of the key starting material 2,3-dimethyl-6-amino-2H-indazole
(SM1) with 2,4-dichloropyrimidine (SM2) in the presence of sodium
carbonate afforded intermediate I (IM1), which was then methyl-
ated by CH3I or dimethylcarbonate (DMC) under mild conditions
providing intermediate II, 2,3-dimethyl-N-(2-chloropyrimidin-4-yl)-
N-methyl-2H-indazol-6-amine. (IM2). Nucleophilic substitution of
IM2 with 2-methyl-5-aminobenzene sulfonamide (SM3) resulted
in the target compound pazopanib. Based on the process route and
literature, four possible process impurities were proposed in the Table.
Scheme 1: Manufacturing process of pazopanib hydrochloride
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ORIGINAL ARTICLES
Scheme 2: Synthesis of pazopanib impurity I
Table: Possible structures of impurities I-IV
Name
m/z ([M+H]+)
Retention time (min)
Suggested structure
Impurity I
438.1705
18.877
Impurity II
413.2193
21.641
Impurity III
427.2348
24.414
Impurity IV
(CQA impurtiy)
858.3082
16.119
API
20.487
Pharmazie 73 (2018)
495

ORIGINAL ARTICLES
Impurity I was supposed to be a regioisomer of pazopanib. During
On the other hand, in the manufacturing process of IM1, the pseudo
dimer of IM1 was also detected and mentioned in the literature (Li
et al. 2010). It may be the reaction product of SM1 and IM1. Small
amounts of this impurity leave residue in the second step would
react with CH3I to give N-monomethylation and N-dimethylation
compound, which were proposed to be impurity II and impurity
III, respectively. The synthetic method for impurity II and impu-
rity III is shown in Scheme 3.
Impurity IV, referred to as the dimer of API, was detected in the
final product and defined as a critical quality attribute (CQA)
impurity as previously reported (Li et al. 2010). Plausible forma-
tion of impurity IV can be tracked back to the diamine impurity of
SM3, a by-product formed in the preparation of SM3. However,
the preparation of IM1, the two chlorine atoms of SM2 showed
different reactivities towards SM1 in the presence of base. The
4 position is more prone to be attacked by nucleophile reagents
under basic conditions. However, the 2 position can also respond
to the nucleophile reagents under the same condition producing
the minor product (detected at nearly 4% area level in the reaction
mixture by HPLC). This impurity was thus supposed to be formed
due to the amination competition at 2-Cl position versus 4-Cl
position of SM2. Thus, in order to obtain impurity I, the isomer
of IM1 was firstly enriched and isolated from the mother liquor,
subsequently reacted with CH3I and SM3 afforded impurity I via
the same route as pazopainib (Scheme 2).
Scheme 3: Synthesis of pazopanib impurity II and impurity III
Scheme 4: Synthesis of pazopanib impurity IV
deserved to be mentioned, the literature revealed no more informa-
tion about the dimer impurity, especially the structure. Therefore,
according to the synthetic process of SM3, the possible structure
and synthetic route for impurity IV is firstly proposed by us
(Scheme 4; Yuan et al. 2017). Treatment of 2-methyl-5-nitroben-
zenesulfonamide (IMP1) with 2-methyl-5-nitrobenzenesulfonyl
chloride (IMP2) under mild conditions (NaH/THF) provided
IMP3, H2-Pd/C reduction of IMP3 in CH3OH yielded the diamine
impurity IMP4, which was further condensed with two molecules
of IM2 in isopropanol in the presence of HCl to give impurity IV
(Scheme 4).
Furthermore, a novel HPLC analytical method was established
for the detection of these impurities. The overlay of HPLC chro-
matograms of all impurity samples is given in the Fig. This HPLC
method could separate all impurities and the retention time of each
impurity is included in the Table.
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ORIGINAL ARTICLES
Fig.: Chromatograms of the
HPLC method for all impu-
rity samples
J = 8.8 Hz, 1H), 5.65 (d, J = 5.8 Hz, 1H), 4.04 (s, 3H), 4.02 (s, 3H), 3.49 (s, 3H), 3.28
(s, 3H), 2.60 (s, 3H), 2.58 (s, 3H). 13C NMR (600 MHz, DMSO-d6) (δ, ppm): 162.4,
161.6, 156.0, 147.6, 147.4, 144.1, 142.3, 132.5, 131.8, 122.0, 121.7, 120.1, 119.8,
119.6, 118.8, 114.0, 112.1, 95.8, 39.5, 38.6, 37.8, 37.6, 37.6, 9.87; HRMS m/z calcd
for C24H27N8 [M+H]+ 427.2353, found 427.2348.
3. Experimental
3.1. General
All reagents were obtained from Adamas, tansoole etc and used without further puri-
fication unless otherwise noted. 1H NMR and 13C NMR spectra were obtained from a
solution in DMSO-d6 with TMS as internal standard using a Bruker spectrometer. MS
spectra were obtained with a waters QDA instrument. HRMS spectra were obtained
with a Thermo Fisher Scientific LTQ FTICR-MS.
3.6. Synthesis of impurity IV
Step 1: A solution of IMP1 (0.8 g, 3.7 mmol) in THF (20 mL) was added to a suspen-
sion of NaH (60%, 0.44 g, 11.1 mmol) in THF (20 mL). After stirring for 30 min at
0 °C, IMP2 (0.87 g, 3.7 mmol) was slowly added into the mixture. Then the mixture
was warmed to room temperature and stirred for 2 h. After reaction completion, the
mixture was quenched with H2O 50 mL, extracted with 100 mL CH2Cl2 twice. The
organic layer was washed with H2O (50 mL) and then brine (50 mL), dried over
Na2SO4 (anhyd), filtered, and concentrated under reduced pressure. The residue was
directly used in the next step without purification.
Step 2: The residue above was dissolved in 100 mL MeOH, then 10% Pd–C (0.2 g) and
5mL conc HCl was added, the mixture was stirred for 8 h at room temperature under H2
atmosphere. After the pressure was leaked, the reaction mixture was filtered, concentrated
under reduced pressure to afford IMP4 as a yellow solid. The residue was directly used in
the next step without purification. 1H NMR (600 MHz, DMSO-d6) (δ, ppm): 7.64 (s, 2H),
7.28 (d, J = 7.8 Hz, 2H), 7.20 (d, J = 7.8 Hz, 2H), 2.43 (s, 6H). ESI-MS:356.2[M+H]+.
Step 3: The residue above was dissolved in 50 mL iPrOH, followed by addition
of IM2 (2.13 g, 7.4 mmol) and 0.5 mL conc HCl. The mixture was stirred for 3
h at reflux temperature. After cooling, the reaction mixture was concentrated under
reduced pressure. 0.16 g (overall yield 5.1%) compound was obtained as a yellow
solid after purification by flash column chromatography (CH2Cl2/CH3OH) on silica
gel. 1H NMR (500 MHz, DMSO-d6) (δ, ppm): 10.89 (brs, 2H), 8.12 – 8.22 (m, 2H),
7.86 (d, J = 8.7 Hz, 2H), 7.76 – 7.80 (m, 2H), 7.62 – 7.68 (m, 2H), 7.42 – 7.50 (m,
2H), 7.12-7.15(m, 2H), 6.97 (d, J = 8.7 Hz, 2H), 5.70 – 5.78 (m, 2H), 4.09 (s, 6H),
3.55 (s, 6H), 2.64 (s, 6H), 2.41 (s, 6H). 13C NMR (600 MHz, DMSO-d6) (δ, ppm):
162.4, 151.0, 146.5, 143.9, 142.6, 140.4, 134.5, 133.8, 133.6, 131.9, 123.3, 121.7,
120.6, 120.2, 118.9, 114.6, 96.3, 37.9, 19.8, 9.9; HRMS m/z calcd for C42H44N13O4S2
[M+H]+ 858.3075, found 858.3082.
3.2. HPLC method
A Waters 2695 liquid chromatography apparatus was used. The column was Agilent
RP-C18(ZORBAX Ecipse Plus, 4.6mm×250mm i.d., 5μm particle size). The flow
rate was 1 ml/min. The column temperature was 30 °C. The injection quantity was 20
μl. The mobile phase was employed as the gradient elution method. The conditions
were as follows: Mobile phase A: methanol-deionized water (10:90), B: acetonitrile.
Gradient: 20-56 % B in 20 min.
3.3. Synthesis of impurity I
Step 1: A solution of SM1 (18.0 g, 0.11 mol), SM2 (27.1g, 0.18 mol), NaHCO3
(23.3g, 0.22 mol) in EtOH (360 mL) and THF (90 mL) was placed into a 1L three
necked round-bottom flask. The resulting solution was warmed to 70 °C and stirred at
this temperature for 4 h. The reaction was cooled to 0 °C for another 3 h, then filtered.
The filtrate was concentrated under reduced pressure and the residue was purified by
column chromatography on silica gel using dichloromethane/methanol as eluent to
afford ISO-IM1 (0.5 g, 1.6%) as a yellow solid. 1H NMR (500 MHz, DMSO-d6) (δ,
ppm): 9.98 (s, 1H), 8.45 (d, J = 5.1 Hz, 1H), 8.08 (s, 1H), 7.53 (d, J = 8.9 Hz, 1H),
7.11 (d, J = 8.9 Hz, 1H), 6.94 (d, J = 5.1Hz, 1H), 3.98 (s, 3H), 2.55 (s, 3H).
Step 2: The suspension of ISO-IM1 (1g, 3.6 mmol), CH3I (0.76g, 5.4 mmol) and
Cs2CO3 (2.34g, 7.2 mmol) in DMF (15 mL) was stirred for 2 h at room temperature.
Then to the reaction mixture was added 100 mL H2O, filtered and dried to afford
ISO-IM2 (0.9 g, 85.7%) as a yellow solid. 1H NMR (300 MHz, DMSO-d6) (δ, ppm):
8.28 (d, J = 5.1 Hz, 1H), 7.62 (d, J = 8.8 Hz, 1H), 7.39 (s, 1H), 6.87 (d, J = 8.8 Hz,
1H), 6.80 (d, J = 5.1Hz, 1H), 4.03 (s, 3H), 3.47 (s, 3H), 2.59 (s, 3H).
Step 3: A mixture of ISO-IM2 (0.7g, 2.4 mmol) and SM3 (0.44 g, 2.4 mmol), conc
HCl 0.5ml in isopropanol (50 mL) was stirred at reflux for 2 h. The mixture was
cooled to room temperature and the resultant precipitate was collected by filtration
and washed with isopropanol. The solid was dried to give impurity I (0.52 g, 45.2 %)
as a off-white solid.1H NMR (300 MHz, DMSO-d6) (δ, ppm): 11.84 (s, 1H), 7.84 (d, J
= 7.2 Hz, 1H), 7.73 - 7.75 (m, 2H), 7.67 (s, 1H), 7.40 - 7.42 (m, 2H), 6.98 (d, J = 8.7
Hz, 1H), 6.71 (d, J = 6.8 Hz, 1H), 4.10 (s, 3H), 3.58 (s, 3H), 2.67 (s, 3H), 2.53 (s, 3H).
13C NMR (600 MHz, DMSO-d6) (δ, ppm): 160.8, 153.7, 147.2, 143.1, 142.6, 136.4,
133.0, 131.9, 124.2, 123.3, 120.8, 119.9, 119.1, 115.7, 99.6, 38.0, 19.7, 9.88; HRMS
m/z calcd for C21H24N7O2S [M+H]+ 438.1707, found 438.1705.
Acknowledgements: We appreciate the Fundamental and Advanced Research Projects
of Chongqing City (No. cstc2017jcyjAX0228)
Conflicts of interest: None declared.
References
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3.4. Synthesis of impurity II
To a 100 mL round-bottom flask was charged IM2 (1.1 g, 3.8 mmol), SM1 (0.75 g,
3.8 mmol), 1 ml conc HCl and isopropanol (50 mL). The mixture was stirred at reflux
for 4 h then cooled to room temperature and the resultant precipitate was collected
by filtration and successfully washed with water and ethanol. Recrystallized from
EtOH/H2O to yield impurity II (0.65 g, 41.6 %) as a brown solid. 1H NMR (600 MHz,
DMSO-d6) (δ, ppm): 10.77 (s, 1H), 7.94(s, 1H), 7.84 - 7.88 (m, 2H), 7.63 (d, J = 8.6
Hz, 1H), 7.59 (s, 1H), 7.06 (d, J = 8.7 Hz, 1H), 6.94 – 6.96 (m, 1H), 5.89(s, 1H), 4.08
(s, 3H), 4.02 (s, 3H), 3.58 (s, 3H), 2.65 (s, 3H), 2.58 (s, 3H). 13C NMR (600 MHz,
DMSO-d6) (δ, ppm): 162.6, 150.9, 146.2, 143.4, 142.7, 140.5, 137.6, 137.2, 134.2,
123.4, 122.2, 120.5, 119.0, 117.7, 117.6, 114.1, 103.6, 97.9, 40.1, 37.8, 37.1, 9.86,
9.81; HRMS m/z calcd for C23H25N8 [M+H]+ 427.2353, found 427.2348.
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3.5. Synthesis of impurity III
A solution of impurity II (0.8 g, 1.9 mmol) in DMF (5 mL) was added to a suspension
of NaH (60%, 0.15 g, 3.8 mmol) in DMF (5 mL). After stirring for 30 min at 0 °C,
CH3I (0.32 g, 2.8 mmol) was slowly added to the mixture. Then the mixture was
warmed to room temperature and stirred for another 1 h. After cooling, H2O (50 mL)
was added, filtered, washed with 15 mL MeOH to afford impurity III (0.51 g, 61.7
1
%) as a white solid. H NMR (500 MHz, DMSO-d6) (δ, ppm): 7.69 - 7.73 (m, 2H),
7.54 (d, J = 8.9 Hz, 1H), 7.38 (s, 1H), 7.34 (s, 1H), 6.99 (d, J = 8.8 Hz, 1H), 6.83 (d,
Pharmazie 73 (2018)
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