Process development of clinical anti-HBV drug Y101: Identification and synthesis of novel impurities

Article abstract of DOI:10.1007/s11164-015-2169-0

Nine novel process impurities of N-[N-benzoyl-O-(2-dimethylaminoethyl)-l-tyrosyl]-l-phenylalaninol (Y101) observed during the laboratory optimization and later during its bulk synthesis are described in this article. The impurities were monitored by HPLC,

Full text of DOI:10.1007/s11164-015-2169-0

Res Chem Intermed (2016) 42:2577–2595
DOI 10.1007/s11164-015-2169-0
Process development of clinical anti-HBV drug Y101:
identification and synthesis of novel impurities
Zhanxing Hu1 HaiJian Liao2 Qiao An5
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•
•
Weidong Pan1 Peixue Cao1 Changxiao Liu3
•
•
•
Zhengming Huang4 Wen Xia5 Bixue Xu1
Guangyi Liang1
•
•
•
Received: 16 May 2015 / Accepted: 4 July 2015 / Published online: 15 July 2015
Ó Springer Science+Business Media Dordrecht 2015
Abstract Nine novel process impurities of N-[N-benzoyl-O-(2-dimethy-
laminoethyl)-L-tyrosyl]-L-phenylalaninol (Y101) observed during the laboratory
optimization and later during its bulk synthesis are described in this article. The
impurities were monitored by HPLC, and their structures were tentatively assigned
on the basis of fragmentation patterns in LC-MS/MS and NMR spectroscopies. All
of the impurities were synthesized, and their assigned constitutions were confirmed
by co-injection in HPLC. In addition to the formation, synthesis, and characteri-
zation, the strategy for minimizing these impurities to a level accepted by the
International Conference on Harmonisation (ICH) was also described.
Electronic supplementary material The online version of this article (doi:10.1007/s11164-015-2169-0)
contains supplementary material, which is available to authorized users.
&
&
Bixue Xu
bixue_xu@126.com
Guangyi Liang
guangyi_liang@126.com
Zhanxing Hu
huzhxi88@163.com
1
The Key Laboratory of Chemistry for Natural Products of Guizhou Province and Chinese
Academy of Sciences, 202 Shachong South Road, Guiyang 550002, People’s Republic of China
2
3
Gui Yang Medical University, 9 Beijing Road, Guiyang 550004, People’s Republic of China
Tianjin Institute of Pharmaceutical Research, 308 Anshan Xi Dao, Nankai District,
Tianjin 300193, People’s Republic of China
4
5
302 Hospital of PLA, 100 Xisihuan Zhong Road, Fengtai, Beijing 100039,
People’s Republic of China
Guizhou Bailing Enterprise Group Pharmaceutical CO., Ltd, Xihang Road, Anshun 561000,
People’s Republic of China
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Keywords Identification Á Synthesis Á Impurities Á Clinical anti-HBV Á Drug Á
Y101
Introduction
Y101 (Fig. 1) was developed for the treatment of chronic hepatitis B associated with
an increase of alanine aminotransferase (ALT) and evidence of hepatitis B viral
replication as well as active liver inflammation, especially for the treatment of
chronic hepatitis B with the resistance of nucleoside analogues [1–5]. The clinical
trial application of Y101 was approved by the China Food and Drug Administration
(CFDA) at the end of 2013 (No. 2013L02491). Furthermore, a Phase 1 clinical trial
of Y101 is now underway in the First Affiliated Hospital of Soochow University.
The control of impurities observed in the active pharmaceutical ingredient (API)
is critical in delivering an API of high quality. To ensure the desired drug
metabolism, safety, and that clinical studies are not jeopardized by inconsistent
purity or impurities having potential harmful toxicological properties, the detection,
identification, quantification, and control of these impurities originating in the
manufacturing process have become an important element of drug development [6].
When Y101 was synthesized by a practical and scalable synthetic method
(Scheme 1), nine unknown impurities with area percentages ranging from 0.02 to
0.13 % were detected by high performance liquid chromatography (HPLC) [7]. It is
mandatory for the manufacturer to identify and characterize all the unknown
impurities that are present in an API even below a level of 0.05 % [6]. In this
context, a comprehensive study has been undertaken to identify, synthesize, and
characterize all the nine impurities present in the laboratory batches of Y101 using
spectroscopic and spectrometric techniques.
Materials and methods
All materials were purchased from commercial suppliers. Unless specified
otherwise, all reagents and solvents were supplied by their manufacturers. Melting
points were determined with an X-4 melting point apparatus. The 1H nuclear
magnetic resonance (NMR), 13C NMR, and two-dimensional NMR (2D NMR)
spectra were measured in deuterated dimethyl sulfoxide (DMSO-d6 ) on an INOVA
400 MHz Fourier transform (FT) NMR spectrometer. Chemical shifts are reported
Fig. 1 Structure of Y101
H
O
O
N
H
O
HN
O
N
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Process development of clinical anti-HBV drug Y101…
2579
O
O
H
O
H
O
O
H
O
O
NH
NH2
a
O
H
O
N
H
O
c
b
1
HN
O
O
l
C
O
O
3
H
O
5
NH2
2
4
H
O
O
H
O
O
N
H
O
N
H
O
HN
H
O
d
HN
O
N
6
7
Y101
.
N H
l
C
l
C
Scheme 1 Reaction conditions: (a) 0.25-M NaOH, 2 °C, 1 h, 90-% yield; (b) IBCF, NMM, DMF,
CH2Cl2, -10 °C, 1 h, 81-% yield; (c) 2.0-M NaOH, DMF, 30 °C, 4 h, 82-% yield; (d) K2CO3, 1,4-
dioxane, 90 °C, 2 h, 90-% yield
in d (ppm) relative to trimethylsilane (TMS; d 0.0) for 1H NMR spectra, and
DMSO-d6 (d 39.50) for 13C NMR spectra. Mass spectra were determined on an
Hewlett Packard 1100 LC-MSD mass spectrometer.
Synthesis of compound N-benzoyl-O-(2-dimethylaminoethyl)-L-tyrosine
(Impurity G)
Synthesis of intermediate N-benzoyl-L-tyrosine methyl ester (9)
Isobutyl chloroformate (IBCF) (4.1 g, 30 mmol) was added dropwise to the mixture
of benzoic acid (3.05 g, 25 mmol), 8 (6.95 g, 30 mmol), 4-methylmorpholine
(NMM) (5.1 g, 50 mmol), and DMF (100 mL) in CH2Cl2 (250 mL) at -10 °C within
30 min. The mixture was stirred for 1.5 h, and the bulk of CH2Cl2 was removed in
vacuo. The residue was dissolved in ethyl acetate and washed sequentially with water,
5-% HCl, saturated NaHCO3 solution and brine, and dried with MgSO4. Filtration and
solvent evaporation gave a residue which was recrystallized from ethyl acetate to
1
afford target intermediate 9 (5.98 g, 80 %). H-NMR (400 MHz, DMSO-d6) d 9.20
(s, 1H), 8.77 (d, J = 8.0 Hz, 1H), 7.81 (d, J = 7.2 Hz, 2H), 7.53 (t, J = 7.2 Hz, 1H),
7.53 (t, J = 7.2 Hz, 1H), 7.47 (t, J = 7.6 Hz, 2H), 7.09 (d, J = 8.8 Hz, 2H), 6.66 (d,
J = 8.4 Hz, 2H), 4.61-4.55 (m, 1H), 3.62 (s, 3H), 3.08–2.95 (m, 2H).
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Synthesis of impurity G
Intermediate 9 (4.49 g, 15 mmol) was dissolved in 50 mL of 1,4-dioxane under
argon atmosphere. 7 (2.6 g, 18 mmol) and potassium carbonate (12.42 g, 90 mmol)
were added. The reaction mixture was stirred at 90 °C for 2 h. The resulting mixture
was diluted with water (200 mL), extracted with ethyl acetate (300 mL), then the
organic layer was washed with brine (200 mL), dried over MgSO4, filtered and
concentrated. The crude material was dissolved in 100 mL of methanol and reacted
with 20 mL of 2-M NaOH at 30 °C for 3 h. The resulting mixture was cooled to
0 °C and its pH was adjusted to 6 with 10-% acetic acid. The bulk of methanol was
removed in vacuo, the residue was dissolved in n-butyl alcohol and washed with
water. The n-butyl alcohol layer was dried over MgSO4, filtered and concentrated,
and the crude material was recrystallized from CHCl3-EtOAc to give impurity
1
G (3.74 g, 99.22 % purity) in 70-% yield. Melting point (Mp): 154–156 °C; H
NMR (400 MHz, DMSO-d6) d 8.39 (d, J = 8.0 Hz, 1H, 100-CONH), 7.79 (m, 2H,
H-300, 700), 7.51 (ddd, J = 6.4, 3.8, 1.4 Hz, 1H, H-500), 7.47–7.40 (m, 2H, H-600, 400),
7.19 (d, J = 8.7 Hz, 2H, H-5, 9), 6.81 (d, J = 8.7 Hz, 2H, H-6, 8), 4.49 (ddd,
J = 9.6, 8.1, 4.3 Hz, 1H, H-2), 4.05 (t, J = 5.7 Hz, 2H, OCH2CH2N), 3.07 (ddd,
J = 23.4, 13.7, 7.0 Hz, 2H, H-3a, 3b), 2.82 (t, J = 5.6 Hz, 2H, OCH2CH2N), 2.35
13
(s, 6H, N(CH3)2); C NMR (101 MHz, DMSO-d6) d 173.90 (C-1), 165.91 (C-100),
156.66 (C-7), 134.36 (C-200), 131.19 (C-500), 130.80 (C-4), 130.19 (C-5, 9), 128.27
(C-400, 600), 127.21 (C-300, 700), 114.05 (C-6, 8), 64.58 (OCH2CH2N), 56.83
(OCH2CH2N), 55.17 (C-2), 44.54 (N(CH3)2), 35.85 (C-3); MS (ESI): 357.1
[M ? H]?.
Synthesis of compound N-[N-O-(2-dimethylaminoethyl)-L-tyrosyl]-L-
phenylalaniol-N-oxide (Impurity H)
To a stirring solution of Y101 (2.45 g, 5 mmol) in CH2Cl2 (100 mL) was added
3-chloroperbenzoic acid (mCPBA) (1.73 g, 10 mmol) at 30 °C, the reaction mixture
was stirred for 2 h. The bulk of CH2Cl2 was removed in vacuo. The residue was
diluted with ethyl acetate (200 mL), sequentially washed with water (200 mL) and
brine (200 mL). The ethyl acetate layer was dried over MgSO4, filtered,
concentrated, and the crude material was purified by column chromatography
(CHCl3/MeOH, 40/1) to yield impurity H (2.35 g, 98.34-% purity) in 93-% yield.
1
Mp: 218–220 °C; H NMR (400 MHz, DMSO-d6) d 8.65 (d, J = 8.5 Hz, 1H, 100-
CONH), 8.13 (d, J = 8.3 Hz, 1H, 1-CONH), 7.82 (d, J = 8.0 Hz, 2H, H-300, 700),
7.51 (t, J = 7.3 Hz, 1H, H-500), 7.44 (t, J = 7.4 Hz, 2H, H-600, 400), 7.26–7.15 (m,
6H, H-5, 9, 50, 60, 80, 90), 7.12 (t, J = 6.8 Hz, 1H, H-70), 6.83 (d, J = 8.6 Hz, 2H,
H-6, 8), 4.66 (td, J = 9.0, 5.5 Hz, 1H, H-2), 4.46–4.37 (m, 2H, OCH2CH2N),
3.94–3.82 (m, 1H, H-20), 3.55 (d, J = 4.2 Hz, 2H, OCH2CH2N), 3.36–3.21 (m, 2H,
H-10), 3.10 (d, J = 5.8 Hz, 6H, N(CH3)2), 3.02–2.80 (m, 3H, H-3a, 30a, 3b), 2.67
(dd, J = 13.6, 7.8 Hz, 1H, H-30b); 13C NMR (101 MHz, DMSO-d6) d 171.05 (C-1),
166.12 (C-100), 156.33 (C-7), 139.09 (C-40), 134.13 (C-200), 131.28 (C-500), 130.85
(C-4), 130.32 (C-5, 9), 129.24 (C-50, 90), 128.19 (C-400, 600), 128.06 (C-60, 80), 127.47
(C-300, 700), 125.88 (C-70), 114.15 (C-6, 8), 68.26 (OCH2CH2N), 62.10 (C-10,
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OCH2CH2N), 59.14 (N(CH3)2), 55.17 (C-2), 52.54 (C-20), 36.48 (C-3, 30); MS
(ESI): 506.2 [M ? H]?.
Synthesis of compound N-[N-benzoyl-O-(2-aminoethyl)-L-tyrosyl]-L-
phenylalaninol (Impurity I)
Synthesis of intermediate N-[N-benzoyl-O-(2-bromoethyl)-L-tyrosyl]-L-
phenylalaninol (10)
To a solution of 6 (4.2 g, 10 mmol) in anhydrous DMF (100 mL) were added 1,2-
dibromoethane (18.8 g, 0.1 mol) and potassium carbonate (5.52 g, 40 mmol), the
reaction mixture was stirred at 70 °C for 6 h. The reaction mixture was poured into
water and extracted with ethyl acetate. The combined organic layer was washed
with brine, and then dried over MgSO4. Filtration and solvent evaporation gave a
yellow solid. The compound was purified using column chromatography (silica gel,
CHCl3/MeOH, 100/1) to give intermediate 10 (4.46 g) in 85-% yield. Mp:
1
230–232 °C; H NMR (400 MHz, DMSO-d6) d 8.46 (d, J = 8.5 Hz, 1H, 100-
CONH), 7.88 (d, J = 8.3 Hz, 1H, 1-CONH), 7.81–7.77 (m, 2H, H-300, 700),
7.54–7.49 (m, 1H, H-500), 7.44 (t, J = 7.5 Hz, 2H, H-600, 400), 7.20 (ddd, J = 20.4,
11.7, 7.2 Hz, 6H, H-5, 9, 50, 60, 80, 90), 7.14–7.09 (m, 1H, H-70), 6.82 (d, J = 8.7 Hz,
2H, H-6, 8), 4.81 (t, J = 5.4 Hz, 1H, OH), 4.62 (ddd, J = 10.2, 8.6, 4.5 Hz, 1H,
H-2), 4.23 (t, J = 5.1 Hz, 2H, OCH2CH2Br), 3.89 (td, J = 7.9, 2.3 Hz, 1H, H-20),
3.75 (t, J = 5.1 Hz, 2H, OCH2CH2Br), 3.30 (ddd, J = 16.3, 10.6, 4.5 Hz, 2H,
H-10), 2.99–2.83 (m, 3H, H-3a, 30a, 3b), 2.66 (dd, J = 13.6, 8.0 Hz, 1H, H-30b); 13C
NMR (101 MHz, DMSO-d6) d 171.03 (C-1), 166.13 (C-100), 156.41 (C-7), 139.02
(C-40), 134.11 (C-200), 131.33 (C-500), 130.88 (C-4), 130.31 (C-5, 9), 129.23 (C-50,
90), 128.23 (C-400, 600), 128.11 (C-60, 80), 127.44 (C-300, 700), 125.94 (C-70), 114.21
(C-6, 8), 67.64 (OCH2CH2 Br), 62.23 (C-10), 55.06 (C-2), 52.49 (C-20), 36.45 (C-3,
30), 31.59 (OCH2CH2 Br); MS (ESI): 1073.3[2 M ? Na]?.
Synthesis of impurity I
NH3ÁH2O (1.4 g, 40 mmol) was added to the solution of intermediate 10 (1.05 g,
2 mmol) in tetrahydrofuran (THF; 50 mL). After stirring at 50 °C for 4 h, the bulk
of THF was removed in vacuo, and partitioned between ethyl acetate and water. The
organic phase was separated and washed with brine, and dried over MgSO4.
Filtration and solvent evaporation gave a buff oily compound. The compound was
purified using column chromatography (silica gel, CHCl3/MeOH, 25/1) to give
1
impurity I (0.79 g, 99.60-% purity) in 85-% yield. Mp: 213–215 °C; H NMR
(400 MHz, DMSO-d6) d 8.44 (d, J = 8.4 Hz, 1H, 100-CONH), 7.86 (d, J = 8.2 Hz,
1H, 1-CONH), 7.80 (d, J = 7.3 Hz, 2H, H-300, 700), 7.52 (t, J = 7.1 Hz, 1H, H-500),
7.45 (t, J = 7.3 Hz, 2H, H-600, 400), 7.26–7.08 (m, 7H, H-5, 9, 50, 60, 80, 90, 70), 6.80
(d, J = 8.3 Hz, 2H, H-6, 8), 4.63 (td, J = 9.4, 4.8 Hz, 1H, H-2), 3.91 (d,
J = 5.4 Hz, 1H, H-20), 3.84 (t, J = 5.6 Hz, 2H, OCH2CH2N), 3.32 (ddd, J = 24.7,
10.6, 5.5 Hz, 2H, H-10), 2.92 (ddd, J = 23.7, 13.7, 6.9 Hz, 3H, H-3a, 30a, 3b), 2.82
(t, J = 5.8 Hz, 2H, OCH2CH2N), 2.67 (dd, J = 13.5, 8.0 Hz, 1H, H-30b); 13C NMR
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Z. Hu et al.
(101 MHz, DMSO-d6) d 170.96 (C-1), 166.03 (C-100), 157.11 (C-7), 138.96 (C-40),
134.09 (C-200), 131.22 (C-500), 130.15 (C-4), 130.12 (C-5, 9), 129.15 (C-50, 90),
128.14 (C-400, 600), 128.02 (C-60, 80), 127.36 (C-300, 700), 125.84 (C-70), 113.98 (C-6, 8),
69.91 (OCH2CH2N), 62.17 (C-10), 55.04 (C-2), 52.43 (C-20), 40.91 (OCH2CH2N),
36.41 (C-3, 30); MS (ESI): 462.3 [M ? H]?.
Synthesis of compound N-[N-benzoyl-O-(2-methylaminoethyl)-L-tyrosyl]-L-
phenylalaninol (Impurity K)
NH2CH3 (1.24 g, 40 mmol) was added to the solution of intermediate 10 (1.05 g,
2 mmol) in THF (50 mL). After stirring at 50 °C for 4 h, the bulk of THF was
removed in vacuo, and partitioned between ethyl acetate and water. The organic
phase was separated and washed with brine, and dried over MgSO4. Filtration and
solvent evaporation gave a buff oily compound. The compound was purified using
column chromatography (silica gel, CHCl3/MeOH, 25/1) to give impurity
1
K (0.86 g, 99.77-% purity) in 90-% yield. Mp: 223-225 °C; H NMR (400 MHz,
DMSO-d6) d 8.46 (d, J = 8.5 Hz, 1H, 100-CONH), 7.88 (d, J = 8.3 Hz, 1H,
1-CONH), 7.83–7.74 (m, 2H, H-300, 700), 7.56–7.49 (m, 1H, H-500), 7.48–7.40 (m,
2H, H-600, 400), 7.25–7.16 (m, 6H, H-5, 9, 50, 60, 80, 90), 7.14 (dd, J = 5.3, 3.4 Hz,
1H, H-70), 6.80 (d, J = 8.7 Hz, 2H, H-6, 8), 4.82 (s, 1H, OH), 4.62 (td, J = 10.0,
4.6 Hz, 1H, H-2), 3.98–3.83 (m, 3H, H-20, OCH2CH2N), 3.31 (dd, J = 14.4,
4.9 Hz, 2H, H-10), 3.00–2.82 (m, 3H, H-3a, 30a, 3b), 2.77 (t, J = 5.6 Hz, 2H,
OCH2CH2N), 2.66 (dd, J = 13.7, 8.0 Hz, 1H, H-30b), 2.30 (s, 3H, CH3); 13C NMR
(101 MHz, DMSO-d6) d 171.08 (C-1), 166.13 (C-100), 157.08 (C-7), 139.02 (C-40),
134.10 (C-200), 131.34 (C-500), 130.27 (C-4), 130.22 (C-5, 9), 129.24 (C-50, 90),
128.25 (C-400, 600), 128.12 (C-60, 80), 127.45 (C-300, 700), 125.95 (C-70), 114.02 (C-6, 8),
66.89 (OCH2CH2N), 62.25 (C-10), 55.12 (C-2), 52.51 (C-20), 50.26 (OCH2CH2N),
36.47 (C-3, 30), 36.10 (CH3); MS (ESI): 476.2[M ? H]?.
Synthesis of compound N-[N-benzoyl-O-(2-methylaminoethyl)-L-tyrosyl]-O-
benzoyl-L-phenylalaninol (Impurity O)
Intermediate 10 (1.05 g, 2 mmol) was dissolved in 20 mL of anhydrous pyridine
under argon atmosphere, followed by addition of benzoyl chloride (0.35 g,
2.5 mmol) and 4-dimethylaminopyridine (DMAP; 0.1 g, 0.82 mmol). The reaction
mixture was stirred at 50 °C for 4 h. Ethyl acetate and water were charged, and the
mixture was stirred for 30 min. The organic layer was washed with brine, and then
dried over MgSO4, filtered and concentrated. The crude material was dissolved in
100 mL of THF and reacted with NH2CH3 (1.24 g, 40 mmol) at 50 °C for 4 h. The
bulk of THF was removed in vacuo, and partitioned between ethyl acetate and
water. The organic phase was separated and washed with brine, and dried over
MgSO4. Filtration and solvent evaporation gave a buff oily compound. The
compound was purified using column chromatography (silica gel, CHCl3/MeOH,
60/1) to give impurity O (0.81 g, 98.50-% purity) in 70-% yield. Mp: 208–210 °C;
1H NMR (400 MHz, DMSO-d6) d 8.51 (d, J = 8.4 Hz, 1H, 100-CONH), 8.33 (d,
J = 8.3 Hz, 1H, 1-CONH), 7.99 (d, J = 7.3 Hz, 2H, H-3000, 7000), 7.80 (d,
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J = 7.3 Hz, 2H, H-300, 700), 7.66 (t, J = 7.4 Hz, 1H, H-5000), 7.51 (t, J = 7.8 Hz, 3H,
H-4000, 6000, 500), 7.42 (t, J = 7.6 Hz, 2H, H-400, 600), 7.28 (d, J = 7.2 Hz, 2H, H-5, 9),
7.24 (t, J = 7.5 Hz, 2H, H-50, 90), 7.17 (d, J = 8.5 Hz, 3H, H-60, 70, 80), 6.76 (d,
J = 8.5 Hz, 2H, H-6, 8), 4.66 (dd, J = 14.6, 8.7 Hz, 1H, H-2), 4.40– 4.31 (m, 1H,
H-20), 4.26 (dd, J = 11.0, 4.3 Hz, 1H, H-10a), 4.15 (dd, J = 10.9, 6.3 Hz, 1H,
H-10b), 3.95 (t, J = 5.4 Hz, 2H, OCH2CH2N), 2.90 (t, J = 5.4 Hz, 6H, H-3, 30,
OCH2CH2N), 2.38 (s, 3H, CH3); 13C NMR (101 MHz, DMSO-d6) d 171.28 (C-1),
166.02 (C-100), 165.59 (C-1000), 156.77 (C-7), 138.02 (C-40), 134.03 (C-4), 133.32
(C-5000), 131.20 (C-200), 130.30 (C-500), 130.05 (C-5, 9), 129.55 (C-2000), 129.30 (C-
3000, 7000), 129.11 (C-50, 90), 128.61 (C-40000, 6000), 128.23 (C-60, 80), 128.10 (C-400, 600),
127.38 (C-300, 700), 126.22 (C-70), 113.99 (C-6, 8), 65.74 (OCH2CH2N), 65.49 (C-10),
55.11 (C-2), 49.36 (C-20), 49.22 (OCH2CH2N), 36.62 (C-3), 36.47 (C-30), 35.07
(CH3); MS (ESI): 580.3 [M ? H]?.
Synthesis of compound N-[N-benzoyl-O-(2-dimethylaminoethyl)-L-tyrosyl]-
O-acetyl-L-phenylalaninol (Impurity N)
To a solution of Y101 (4.9 g, 10 mmol) and DMAP (100 mg) in CH2Cl2 (150 mL)
was added acetic anhydride (1.23 g, 12 mmol) at 30 °C. The mixture was stirred for
3 h,then adjusted to pH 8 with saturated NaHCO3 solution. The reaction mixture
was extracted with CH2Cl2. The organic layer was washed with brine, and then
dried over MgSO4. Filtration and solvent evaporation gave a buff solid. The
compound was recrystallized from ethyl acetate to afford impurity N (4.52 g, 99.22-%
1
purity) in 85-% yield. Mp: 184–186 °C; H NMR (400 MHz, DMSO-d6) d 8.46
(d, J = 8.5 Hz, 1H, 100-CONH), 8.11 (d, J = 8.5 Hz, 1H, 1-CONH), 7.80 (dd,
J = 8.3, 1.3 Hz, 2H, H-300, 700), 7.52 (ddd, J = 6.5, 3.8, 1.4 Hz, 1H, H-500),
7.48–7.41 (m, 2H, H-600, 400), 7.25–7.18 (m, 6H, H-5, 9, 50, 60, 80, 90), 7.18–7.12 (m,
1H, H-70), 6.81 (d, J = 8.7 Hz, 2H, H-6, 8), 4.61 (td, J = 9.1, 5.4 Hz, 1H, H-2),
4.18 (dd, J = 12.4, 7.3 Hz, 1H, H-20), 4.01 (dd, J = 11.0, 4.8 Hz, 1H, H-10a), 3.96
(t, J = 5.9 Hz, 2H, OCH2CH2N), 3.86 (dd, J = 11.0, 6.7 Hz, 1H, H-10b), 2.95–2.85
(m, 2H, H-3a, 30a), 2.78 (dd, J = 6.9, 4.5 Hz, 2H, H-3b, 30b), 2.56 (t, J = 5.9 Hz,
2H, OCH2CH2N), 2.18 (s, 6H, N(CH3)2), 1.98 (s, 3H, COCH3); 13C NMR
(101 MHz, DMSO-d6) d 171.29 (C-1), 170.30 (COCH3), 166.11 (C-100), 156.98 (C-7),
138.00 (C-40), 134.06 (C-200), 131.29 (C-500), 130.16 (C-4), 130.12 (C-5, 9), 129.14
(C-50, 90), 128.23 (C-400, 600), 128.18 (C-60, 80), 127.43 (C-300, 700), 126.23 (C-70), 114.00
(C-6, 8), 65.60 (OCH2CH2N), 64.63 (C-10), 57.74 (OCH2CH2N), 55.15 (C-2), 49.11
(C-20), 45.55 (N(CH3)2), 36.59 (C-3), 36.45 (C-30), 20.61 (COCH3); MS (ESI): 532.3
[M ? H]?.
Synthesis of compound N-[N-benzoyl-O-(2-dimethylaminoethyl)-L-tyrosyl]-
O-acetyl-L-phenylalaninol-N-oxide (Impurity J)
To a stirring solution of impurity N (2.66 g, 5 mmol) in CH2Cl2 (100 mL) was
added mCPBA (1.73 g, 10 mmol) at 30 °C, the reaction mixture was stirred for 2 h.
The bulk of CH2Cl2 was removed in vacuo. The residue was diluted with ethyl
acetate (200 mL), and sequentially washed with water (200 mL) and brine
123

2584
Z. Hu et al.
(200 mL). The ethyl acetate layer was dried over MgSO4, filtered, concentrated, and
the crude material was purified by column chromatography (CHCl3/MeOH, 40/1) to
yield impurity J (2.46 g, 98.16-% purity) in 90-% yield. Mp: 174–176 °C; 1H NMR
(400 MHz, DMSO-d6) d 8.57 (d, J = 8.4 Hz, 1H, 100-CONH), 8.23 (d, J = 8.3 Hz,
1H, 1-CONH), 7.85–7.78 (m, 2H, H-300, 700), 7.51 (ddd, J = 6.4, 3.8, 1.4 Hz, 1H,
H-500), 7.48–7.40 (m, 2H, H-600, 400)), 7.28–7.20 (m, 6H, H-5, 9, 50, 60, 80, 90),
7.18–7.12 (m, 1H, H-70), 6.85 (d, J = 8.7 Hz, 2H, H-6, 8), 4.68–4.57 (m, 1H, H-2),
4.42 (t, J = 4.8 Hz, 2H, OCH2CH2N), 4.18 (dd, J = 12.6, 7.1 Hz, 1H, H-20), 4.01
(dd, J = 11.0, 4.8 Hz, 1H, H-10a), 3.86 (dd, J = 11.0, 6.7 Hz, 1H, H-10b), 3.52 (t,
J = 4.4 Hz, 2H, OCH2CH2N), 3.08 (s, 6H, N(CH3)2), 2.92 (d, J = 7.9 Hz, 2H,
H-3a, 30a), 2.83–2.71 (m, 2H, H-3b, 30b), 1.98 (s, 3H, COCH3); 13C NMR
(101 MHz, DMSO-d6) d 171.36 (C-1), 170.32 (COCH3), 166.16 (C-100), 156.37 (C-7),
138.03 (C-40), 134.09 (C-200), 131.30 (C-500), 130.76 (C-4), 130.28 (C-5, 9), 129.16
(C-50, 90), 128.24 (C-400, 600), 128.19 (C-60, 80), 127.47 (C-300, 700), 126.24 (C-70), 114.14
(C-6, 8), 68.27 (OCH2CH2N), 64.65 (C-10), 62.04 (OCH2CH2N), 59.19 (N(CH3)2),
55.19 (C-2), 49.12 (C-20), 36.59 (C-3), 36.41 (C-30), 20.64 (COCH3); MS (ESI): 570.2
[M ? Na]?.
Synthesis of compound N-[N-benzoyl-O-acetyl-L-tyrosyl]-O-acetyl-L-
phenylalaninol (impurity L)
Acetic anhydride (2.25 g, 22 mmol) was added to a mixture of compound 6 (4.2 g,
10 mmol) and DMAP (100 mg) in DMF (50 mL). After stirring at 30 °C for 4 h,
the mixture was acidified to pH 8 with saturated NaHCO3 solution, and partitioned
between ethyl acetate and water. The organic phase was separated and sequentially
washed with brine, and dried over MgSO4. Filtration and solvent evaporation gave a
residue which was recrystallized from MeOH-CHCl3 to afford target impurity
1
L (3.92 g, 99.17-% purity) in 78-% yield. Mp: 220–222 °C; H NMR (400 MHz,
DMSO-d6) d 8.54 (d, J = 8.5 Hz, 1H, 100-CONH), 8.16 (d, J = 8.4 Hz, 1H,
1-CONH), 7.80 (dd, J = 5.2, 3.3 Hz, 2H, H-300, 700), 7.56–7.49 (m, 1H, H-500),
7.48–7.42 (m, 2H, H-600, 400), 7.34 (d, J = 8.6 Hz, 2H, H-50, 90), 7.26–7.17 (m, 5H,
H-5, 9, 60, 70, 80), 7.01 (d, J = 8.5 Hz, 2H, H-6, 8), 4.68 (td, J = 9.0, 5.7 Hz, 1H,
H-2), 4.20 (dd, J = 12.5, 7.2 Hz, 1H, H-20), 4.04 (dd, J = 11.0, 4.8 Hz, 1H, H-10a),
3.88 (dd, J = 11.0, 6.8 Hz, 1H, H-10b), 3.05–2.92 (m, 2H, H-3a, 30a), 2.80 (dd,
J = 6.8, 5.0 Hz, 2H, H-3b, 30b), 2.22 (s, 3H, COCH3), 1.99 (s, 3H, COCH3); 13C
NMR (101 MHz, DMSO-d6) d 171.15 (C-1), 170.30 (COCH3), 169.16 (COCH3),
166.22 (C-100), 148.93 (C-7), 137.98 (C-40), 135.81 (C-200), 134.02 (C-500), 131.31
(C-5, 9), 130.06 (C-50, 90), 129.15 (C-4), 128.23 (C-400, 600), 128.18 (C-60, 80), 127.43
(C-300, 700), 126.23 (C-70), 121.33 (C-6, 8), 64.60 (C-10), 54.82 (C-2), 49.17 (C-20),
36.58 (C-3, C-30), 20.84 (COCH3), 20.62 (COCH3); MS (ESI): 525.2 [M ? Na]?.
Synthesis of compound N-[N-benzoyl-O-(2-dimethylaminoethyl)-L-tyrosyl]-
O-benzoyl-L-phenylalaninol-N-oxide (impurity M)
To a stirring solution of impurity F (0.47 g, 0.8 mmol) in CH2Cl2 (40 mL) was
added mCPBA (0.26 g, 1.5 mmol) at 30 °C, the reaction mixture was stirred for
123

Process development of clinical anti-HBV drug Y101…
2585
2 h. The bulk of CH2Cl2 was removed in vacuo. The residue was diluted with ethyl
acetate (100 mL), and sequentially washed with water (100 mL) and brine
(100 mL). The ethyl acetate layer was dried over MgSO4, filtered, concentrated,
and the crude material was purified by column chromatography (CHCl3/MeOH,
40/1) to yield impurity M (0.44 g, 98.73-% purity) in 90-% yield. Mp: 166–168 °C;
1H NMR (400 MHz, DMSO-d6) d 8.54 (d, J = 8.4 Hz, 1H, 100-CONH), 8.37 (d,
J = 8.3 Hz, 1H, 1-CONH), 7.98 (d, J = 7.5 Hz, 2H, H-3000, 7000), 7.79 (d,
J = 7.5 Hz, 2H, H-300, 700), 7.65 (t, J = 7.3 Hz, 1H, H-5000), 7.54–7.47 (m, 3H,
H-4000, 6000, 500), 7.42 (t, J = 7.6 Hz, 2H, H-400, 600), 7.31–7.21 (m, 4H, H-5, 9, 50, 90),
7.17 (dd, J = 12.2, 7.7 Hz, 3H, H-60, 70, 80), 6.78 (d, J = 8.4 Hz, 2H, H-6, 8), 4.67
(dd, J = 14.9, 8.1 Hz, 1H, H-2), 4.38 (t, J = 4.5 Hz, 3H, H-20, OCH2CH2N), 4.26
(dd, J = 10.9, 4.2 Hz, 1H, H-10a), 4.14 (dd, J = 10.9, 6.4 Hz, 1H, H-10b), 3.45 (s,
2H, OCH2CH2N), 3.03 (d, J = 2.9 Hz, 6H, N(CH3)2), 2.89 (d, J = 5.7 Hz, 4H,
H-3a, 30a, 3b, 30b); 13C NMR (101 MHz, DMSO-d6) d 171.53 (C-1), 166.21 (C-100),
165.70 (C-1000), 156.37 (C-7), 138.11 (C-40), 134.08 (C-4), 133.47 (C-5000), 131.33
(C-200), 130.70 (C-500), 130.24 (C-5, 9), 129.61 (C-2000), 129.42 (C-3000, 7000), 129.24
(C-50, 90), 128.74 (C-40000, 6000), 128.36 (C-60, 80), 128.22 (C-400, 600), 127.52 (C-300,
700), 126.35 (C-70), 114.10 (C-6, 8), 68.29 (OCH2CH2N), 65.55 (C-10), 62.02
(OCH2CH2N), 59.31 (N(CH3)2), 55.21 (C-2), 49.26 (C-20), 36.68 (C-3), 36.52
(C-30); MS (ESI):610.2 [M ? H]?.
Results and discussion
To identify the structure of a low-level unknown impurity, LC/MS/high-resolution
MS (HRMS) and tandem MS (MS/MS) were used to determine their molecular
weight (MW), elemental composition, and fragmentation patterns. On the basis of
the MS data and knowledge of the process chemistry, nine possible structures
(Table 1) were assigned for the impurities. MS alone without authentic standards
can never be sufficient for the unequivocal confirmation of the structure of
impurities. Thus, the chemical synthesis of nine impurities was carried out, and their
structures were elucidated on the basis of NMR and MS. The structures of the nine
primary impurities in API were unequivocally confirmed by comparing the HPLC
retention time, the ultraviolet (UV) spectrum profile, and the mass spectrum with
that of the synthetic standards.
Impurity synthesis
The quantity of these impurities required for to confirm their structure, for
validation, and for use as analytical standards was substantial. Thus, a synthetic
route was required for each impurity which could be used to cross-validate the
impurity versus the postulated structure and against the impurity seen in the
analytical method (allowing for confirmation of retention time, relative response
factor, etc.).
The impurity G was prepared from L-tyrosine methyl ester hydrochloride (8) in
three steps, as shown in Scheme 2. The reaction of benzoic acid with 8 gave the
123

2586
Z. Hu et al.
Table 1 Structures of impurities G–O
Entry
1
Name
Structure
Relative retention timea
0.30
O
Impurity-G
5
3
4
2
6
7
1
OH
O
9
1''
HN
O
8
2''
3''
4''
7''
6''
N
5''
2
3
4
5
6
7
Impurity-H
Impurity-I
Impurity-J
Impurity-K
Impurity-L
Impurity-M
0.45
0.57
0.65
0.70
1.20
1.57
8
Impurity-N
1.59
123

Process development of clinical anti-HBV drug Y101…
Table 1 continued
2587
Entry
9
Name
Structure
Relative retention timea
3.16
Impurity-O
a
Agilent 1100 HPLC; column: Phenomenex Gemini-NX C18 (250 mm 9 4.6 mm, 5 lm); column
temperature: 30 °C; mobile phase: MeOH–water (65:35) containing 0.04-% triethylamine, adjusted to pH
9.0 with phosphoric acid; flow rate: 1.0 mL/min; injection volume: 10 lL; wavelength for UV detection:
226 nm
O
OMe
2 .HCl
O
NH
HO
OMe
O
O
IBCF/NMM
8
NH
HO
OH
O
i. K
ii.
2CO3
,
1,4-dioxane
MeOH
HO
O
HN
O
NaOH,
9
N
benzoic acid
N.HCl
Cl
Impurity G
7
Scheme 2 Synthesis of impurity G
compound 9 [8–12]. Alkylation of the hydroxyl group of compound 9 with
dimethylaminoethyl chloride and followed by hydrolysis of the ester bond with
NaOH furnished impurity G.
As shown in Scheme 3, impurity H was obtained by reaction of Y101 with
mCPBA in CH2Cl2 at 30 °C [13, 14].
As outlined in Scheme 4, a straightforward and efficient methodology for the
synthesis of impurities I, K, and O was developed. Compound 10 was the key
OH
OH
O
O
N
H
O
N
H
O
mCPBA
HN
O
HN
O
N
O
N
Y101
Scheme 3 Synthesis of impurity H
Impurity
H
123

2588
Z. Hu et al.
OH
O
N
H
O
HN
O
NH2
NH3
Impurity I
OH
O
Cl
O
OH
O
O
O
O
N
H
O
O
O
Br
N
H
O
N
H
O
Br
O
HN
O
N
H2N
HN
H
O
HN
HO
O
HN
O
Br
NH
H2N
Br
10
6
11
Impurity
O
OH
O
N
H
O
HN
O
NH
Impurity K
Scheme 4 Synthesis of impurities I, K, and O
intermediate for synthesis of the impurities investigated. Treating compound 6 with
excessive amounts of 1,2-dibromoethane at 70 °C for 4 h in anhydrous DMF
catalyzed by potassium carbonate yielded compound 10. Reactions of 10 with
excess ammonia-water and methylamine in DMF at 50 °C for 4 h yielded impurities
I and K. In addition, derivative 11 was prepared by treating compound 10 in situ
with benzoyl chloride at 50 °C for 4 h in pyridine catalyzed by DMAP. Finally,
impurity O was synthesized by treatment of compound 11 with excess methylamine
[15, 16].
The above study indicated that when impurities I and K were synthesized by
treating compound 10 with excess ammonia-water and methylamine in DMF
catalyzed by potassium carbonate, large amounts of by-product (Y101) were
observed. A hypothesis mechanism was shown in Fig. 2. From these results, the
K2CO3 was removed, and the reaction solvent was changed to THF. Using this
newly developed synthetic method, impurities I and K were synthesized in excellent
yield.
O
H
OH
O
K
2CO3
H
N
H
2O
OH
DMF
HN
O
N
H
O
OH
HN
O
O
N
H
O
N
HN
O
Y101
Br
6
Fig. 2 Formation of by-product (Y101)
123

Process development of clinical anti-HBV drug Y101…
2589
O
O
O
OH
O
O
O
O
N
H
O
N
H
O
mCPBA
N
H
O
Acetic anhydride
DMAP
HN
O
HN
O
HN
O
N
O
N
N
Y101
Impurity N
Impurity J
Scheme 5 Synthesis of impurities N and J
Impurities N and J could be synthesized from the same starting material (Y101),
as shown in Scheme 5. Impurity N was obtained by reaction of Y101 with acetic
anhydride in the presence of DMAP in CH2Cl2 at 30 °C [17–19]. Treatment of
impurity N with mCPBA in CH2Cl2 gave impurity J.
Impurity L was synthesized by reaction of compound 6 and acetic anhydride in
the presence of DMAP in DMF at 30 °C (Scheme 6).
A synthetic protocol for impurity M was shown in Scheme 7. Impurity F was
identified as a starting material, and impurity M was synthesized in the presence of
mCPBA in CH2Cl2.
OAc
OH
O
O
N
H
O
N
H
O
Acetic anhydride
HN
HN
AcO
HO
DMAP
L
Impurity
6
Scheme 6 Synthesis of impurity L
O
O
O
O
O
O
N
H
O
N
H
O
mCPBA
HN
O
HN
O
N
N
O
Impurity F
Impurity M
Scheme 7 Synthesis of impurity M
123

2590
Z. Hu et al.
OH
O
N
H
O
OH
O
HN
O
N
H
O
HN
HO
N
OH
O
Y101
N
H
O
NaOH
6
HN
NaOH
Amide disconnection
O
O
O
O
OH
O
OH
O
+
N.HCl
HN
Cl
HO
HN
O
N
5
7
Impurity G
Fig. 3 Possible pathway to impurity G
Plausible pathways to impurities
Impurity G
Impurity G was observed in the range of 0.02–0.05 % in the laboratory
experimental study. This impurity was identified as the hydrolysis product of
Y101, and might be due to the carryover of NaOH to the reaction of 5 to 6 and
workup of the final step (Fig. 3) [7, 20].
O
O
OH
O
O
O
O
O
N
H
O
N
H
O
N
H
O
HN
HN
O
O
HN
O
N
N
N
Impurity F
Y101
Impurity
N
O2 /
O
O
OH
O
O
O
O
O
N
H
O
N
N
H
O
H
O
HN
HN
O
O
HN
O
N
O
N
O
N
O
Impurity
J
Impurity H
Impurity M
Fig. 4 Possible pathway to impurities H, J, and M
123

Process development of clinical anti-HBV drug Y101…
2591
Impurities H, J, and M
The process impurities H, J, and M were observed in the range of 0.03–0.07 %.
Impurity H was the oxidation product of Y101 and might be formed during the
drying process of the drug substance (Fig. 4). Impurities J and M were structural
analogues of impurity H, where the hydroxyl group had been replaced by the
corresponding acetate and benzoate esters. Impurity J might be generated due to the
presence of trace impurity N during the reaction of 6 to Y101 (Fig. 4). The synthetic
pathway for impurity M was shown in Fig. 4; similarly, impurity M might be
formed by oxidation reaction of impurity F [20].
Impurities I, K, and O
Impurity K was found to be present at a level greater than 0.10 % in the crude
product of Y101, and upon being isolated from the filtrate of Y101 recrystallization,
the structure was confirmed by NMR and MS. This impurity was probably formed
due to small amounts of 2-methylaminoethyl chloride hydrochloride presented in
2-dimethylaminoethyl chloride hydrochloride 7 (Fig. 5) [21, 22]. Impurity I pre-
sented at lower concentrations, and was also caused by the presence of an other
impurity (2-aminoethyl chloride hydrochloride) in compound 7. Impurity O was
likely introduced by reaction of impurity B with impurity K (Fig. 5) [20].
Impurities L and N
The process impurities L and N were observed in the range of 0.04–0.06 %.
Impurity L was identified as the acetic ester of compound 6 (the intermediate of
Y101 shown in Scheme 1). Impurity N was a homologous series of Y101 acetic
ester, which was similar to Y101. The two impurities were believed to arise from
related acetic acid in the purification step of the alkylation reaction. As shown in
Fig. 6, the acetic ester groups in impurities L and N can be traced back to ethyl
acetate, which was the extraction solvent of the alkylation reaction (Scheme 1) [7,
OH
O
N
H
O
HN
O
OH
Cl
N
N
HN
N
O
S
Y101
O
O
+
Cl
Cl
N
2CH3OH
NH3
O
OH
Cl
O
H2
N
N
N
H
OH
O
H
O
N
H
O
N
H
O
HN
O
Impurity
OH
Cl
B
NH3
HN
O
H2N
H2N
NH
NH
OH
Impurity
O
O
Impurity
K
N
H
O
OH
O
HN
HO
N
H
O
HN
O
6
NH2
Impurity
I
Fig. 5 Possible pathway to impurities I, K, and O
123

2592
Z. Hu et al.
organic phase
the reaction mixture of
6
OH
O
O
O
K
2CO3
H
3C
O
CH3
H
3C
OH
N
H
O
HN
O
OH
O
OH
N
OH
O
O
N
H
N
H
O
Y101
N
H
O
HN
O
HO
K
2CO3
O/ethyl acetate
H2
OH
HN
O
O
HN
+
HO
O
extraction
N
H
O
N
O
HN
HO
6
7
Y101
6
N.HCl
Cl
6
OAc
O
O
K
2CO3
O
N
H
O
O
N
H
O
HN
N.HCl
AcO
Cl
HN
O
7
N
Impurity L
Impurity N
organic phase
O
O
NaOH
0.1 M HCl
extraction
H
3C
O
CH3
H
3C
OH
the aqueous layer
OH
O
OH
O
N
H
O
N
H
O
HN
O
HN
O
N
1.0 M NaOH
pH 8-9
N
Y101
Y101
ethyl acetate
extraction
O
O
O
O
N
H
O
O
HN
O
O
N
H
O
HN
O
N
N
Impurity
N
Impurity
N
Fig. 6 Possible pathway to impurities L and N
20]. It is generally believed that there are small amounts of acetic acid in the
extraction solution when ethyl acetate was poured into the alkaline aqueous solution
of K2CO3 and NaOH.
Impurity control
Based on the initial input on potential impurities by the process research [20],
impurity tracking from starting materials, through intermediates and finally to the
API manufacturing process was carried out (Scheme 8). The potential impurities of
each stage postulated during the process research were used as the basis for quality
control.
Impurity G was the degradation product, and it could be controlled to a
negligible level during the final step. This was achieved by purification in ethyl
acetate.
123

Process development of clinical anti-HBV drug Y101…
2593
OH
O
O
O
OAc
O
O
O
N
H
O
O
O
N
H
O
N
H
O
N
H
O
HN
O
HN
HN
O
O
O2
HN
AcO
N
O
N
N
O
Impurity-H
Impurity-N
Impurity-L
Impurity-J
O2
process
purification
O
OH
OH
O
HN
O
O
N
OH
H
O
HN
O
HN
O
N
N
Impurity-G
Y101
O
7
OH
4
O
N
H
OH
O
OH
HN
O
HO
HN
O
O
HO
Benzoic acid
Cl
Impurity-A
N
OH
O
O
OH
N
H
O
4
OH
Cl
NH2
O
N
H
HO
HN
HO
OH
HN
O
HO
O
1
N
H
O
Cl
Cl
H2
N
HN
H
3CO
6
CH3Cl
OH
O
O
2
OH
O
Impurity-D
N
H
O
4
O
HN
O
HN
O
O
N
O
O
N
H
O
HN
O
5
3
N
O
OH
Impurity-E
OH
O
N
H
O
Benzoic acid
7
HN
O
O
O
O
O
O
OCH3
NH2
O
O
N
H
O
O
O
N
Y101
MeOH
N
H
O
O2
Impurity-I
HN
O
HN
O
OH
O
N
N
O
Impurity-C
Impurity-B
Impurity-K
N
H
O
HN
O
Impurity-F
Impurity-M
NH
O
O
Impurity-K
O
N
H
O
HN
O
NH
Impurity-O
Scheme 8 Impurity tracking of Y101 process
Impurity H was a thermodynamically controlled impurity, and its rate of
formation increased exponentially along with an increase in temperature. By
conducting the drying temperature below 50 °C, the impurity H can be controlled to
a level of 0.02 % in API.
Following Scheme 8, starting material 7 was known to contain small amounts of
2-methylaminoethyl chloride hydrochloride and a small quantity of 2-aminoethyl
chloride hydrochloride as impurities. These compounds followed the same sequence
as described for the API synthesis (Scheme 1) and leaded to the formation of
123

2594
Z. Hu et al.
impurities K and I. Impurities K and I were analogues of Y101, and hence, the
downstream process was unsuccessful at eliminating these impurities, because of
their similar structural properties. Due to the difficult removal of impurities K and
I from Y101, we controlled the potential impurities of 2-methylaminoethyl chloride
hydrochloride and 2-aminoethyl chloride hydrochloride in 7, and these impurities
are removed by methanol purification. When Y101 was obtained by reaction of 6
with the purified 7, impurities I and K were reduced to lower than 0.06 %.
Impurity O was likely introduced by reaction of impurity B with impurity K, and
was controlled to a non-detectable level in Y101, when the impurities B and K were
reduced to lower than 0.1 %.
Impurities J, L, and N were process impurities and appeared in Y101 due to
carryover of acetic acid in the extraction solution, when ethyl acetate was poured
into the alkaline aqueous solution of K2CO3 and NaOH. The formation of impurities
L and N depended on the basicity of the reaction mixture. Furthermore, the
formation of impurities J, L, and N were controlled to less than 0.04 % when the
extraction solution pH was 8–9, and they were removed to a negligible level during
the purification of Y101 from ethyl acetate.
Impurity M might be formed due to the presence of impurity F in Y101. During
process optimization, we observed that when impurity F was reduced to lower than
0.1 % [20], impurity M was controlled to a non-detectable level in Y101.
Conclusions
The structures of novel potential impurities G–O and their origins of formation
during the preparation of Y101 were identified. The knowledge of the structures led
to the development of a re-work protocol that purged out these impurities to level
accepted by the ICH.
Acknowledgments This work was supported by grants from the Ministry of Science and Technology of
China (No. 2011ZX09102-009-2), the National Natural Science Fund of China (NSFC No. 30760292),
and the Science and Technology Department of Guizhou Province (No. QKHZYZ [2012] 5054).
References
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