Identification, synthesis, and strategy for the reduction of potential impurities observed in dabigatran etexilate mesylate processes

Article abstract of DOI:10.1021/op500084q

Synthetic impurities that are present in dabigatran etexilate mesylate were studied, and possible pathways by which these impurities are formed during the manufacturing process were examined. The impurities were monitored by high-performance liquid chromatography, and their structures were determined by mass spectrometry and ¹H and ¹³C NMR. Potential causes for the formation of these impurities are discussed, and strategies to minimize their formation are also described.

Full text of DOI:10.1021/op500084q

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Communication
Identification, Synthesis, and Strategy for Reduction of Potential
Impurities Observed in Dabigatran Etexilate Mesylate Processes
Yong-Yong Zheng, chengwu shen, meiyu zhu, yimeng zhou, and jianqi li
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/op500084q • Publication Date (Web): 22 May 2014
Downloaded from http://pubs.acs.org on May 27, 2014
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Identification, Synthesis, and Strategy for Reduction of Potential
Impurities Observed in Dabigatran Etexilate Mesylate Processes
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YongꢀYong Zheng,a,┴ ChengꢀWu Shen,b,┴ MeiꢀYu Zhu,a
YiꢀMeng Zhou,a JianꢀQi Lia*
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aNovel Technology Center of Pharmaceutical Chemistry, Shanghai Institute of
Pharmaceutical Industry, 1111 North Zhongshan No.1 Road, Shanghai 200437, PR
China
bDepartment of Pharmacy, Shandong Provincial Hospital affiliated to Shandong
University, Jinan 250021, PR China
ABSTRACT: Synthetic impurities that are present in dabigatran etexilate mesylate
were studied and possible pathways by which these impurities are formed during the
manufacturing process were examined. The impurities were monitored by HPLC and
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their structures were determined by MS, H and 13C NMR. Potential causes for the
formation of these impurities are discussed and strategies to minimize their formation
are also described.
INTRODUCTION
Dabigatran etexilate mesylate (1) (Figure 1) is as an effective oral prodrug of the
thrombin (Factor IIa) inhibitor dabigatran1. This drug has been approved by FDA and
is widely used to reduce the risk of stroke in patients with nonꢀvalvular atrial
fibrillation (AF). It is also approved by the EMEA for prevention of venous
thromboembolism (VTE) events in adults who have undergone elective total hip or
knee replacement surgery2. It is generally believed that the active pharmaceutical
ingredient (API) can be contaminated by impurities, which may influence the quality
and safety of the drug3. It has been found that the process employed in manufacturing
1 can result in the formation of certain impurities at a level of ≥ 0.10% (HPLC area)4.
One major concern is to maintain the quality of the API while reducing the level of
the impurities. It is therefore significant to isolate and characterize the corresponding
impurities during the manufacturing process.
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Figure 1. Structure of dabigatran etexilate mesylate (1).
Early experimental studies suggested that synthetic method outlined in Scheme 1 is
the most advantageous and convenient route for the synthesis of 15–8.
Cyanoꢀcompound 2 is first converted to the corresponding amidine intermediate 3 by
Pinner reaction using HCl/EtOH. Then, Amidine 3 is reacted with nꢀhexyl
chloroformate 4 to give dabigatran etexilate 5. In the last step, the free base 5 reacts
with methanesulfonic acid to produce its methanesulfonic acid salt 1. A few impurities
are identified. They can be produced by degradation, incomplete reactions, and side
reactions3, 9. Contaminated starting ingredients, however are also major source of the
impurities in 1, such as nꢀhexyl chloroformate, which generally contains 5 impurities
(see Scheme 3 for the impurities investigated in this work).
Scheme 1. Synthesis route of 1
It is extremely challenging to identify impurities that formed in very small
quantities in drug substances. The mechanism is also not straightforward due to the
complexity of the conditions: multiple reactants and multiple impurities can coexist.
This is strongly influenced by the reaction conditions. To solve the puzzle, the
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potential impurities have been explored and synthesized. A mechanism of formation is
here proposed and a practical strategy for lowering the concentration of impurities in
the product is presented.
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RESULTS AND DISCUSSION
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HPLC was used to separate the impurities during the process of synthesizing 1.
Seven impurities were identified and are listed in Table 1. Their structures have been
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determined by MS, H NMR, and 13C NMR. It is essential to understand how these
impurities are introduced in the drug, the mechanism by which the impurities form,
and the strategies for detecting and assessing impurities. These points are elaborated
upon as follows.
Table 1. Impurities detected during the synthetic process
Name
Structure
Relative Retention Time a
8.07 min
ImpurityꢀA
ImpurityꢀB
ImpurityꢀC
ImpurityꢀD
13.93 min
9.39 min
5.34 min
ImpurityꢀE
ImpurityꢀF
13.42 min
23.80 min
ImpurityꢀG
8.67 min
a Ultimate 3000 HPLC (Dionex); column: C18 (5ꢁm, 250mm×4.6mm); flow rate: 1.0 mL/min;
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and detection at 225 nm; injection vol: 10 ꢁL; mobile phase A (0.015mol/L KH2PO4, pH 6.0) and
B (acetonitrile), 48:52 v/v; temp. 35 °C; dabigatran etexilate relative retention time: 10.47 min.
Impurities Identification and Plausible Pathways.
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As shown in Table 1, impurities A and B are the homologous series of dabigatran
etexilate methyl and isopropyl ester, which are similar to 1. The two impurities were
believed to arise from related impurities in the starting material. As shown in Figure 2,
the methyl and isopropyl ester groups in impurities A and B can be traced back to
compounds 6 and 7, which may be the byꢀproducts of the Pinner reaction shown in
Scheme 110. It is generally believed that there are small amounts of methanol and
isopropanol in HClꢀethanol solution, where the methyl and isopropyl ester moieties
can be introduced as the byꢀproducts of intermediate 3 at the first step of synthesis
(Scheme 1).
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Figure 2. Possible pathway to impurity A and B
Impurities C, D, E, F, and G are structural analogues of 1, where the nꢀhexyl group
has been replaced by other alkyl groups. It is reasonable to believe that the
2ꢀethylbutyl moiety in impurity C is derived from the small amount of 2ꢀethylbutyl
chloroformate, which is always present in the nꢀhexyl chloroformate 4. After the
amidation of amidine 3 with nꢀhexyl chloroformate 4, the impurity C was found to be
present at levels greater than 0.15% in the crude product 1. Impurities D, E, F, and G
were present at lower concentrations. They are also caused by the presence of other
impurities (nꢀbutyl chloroformate, heptanꢀ2ꢀyl chloroformate, nꢀoctyl chloroformate,
hexanꢀ3ꢀyl chloroformate) in the nꢀhexyl chloroformate 4. Their concentrations were
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about 0.05–0.12% as indicated by HPLC. Unfortunately, it is virtually impossible to
remove these specific impurities from the final product, and they can affect solubility.
Impurities Synthesis and Control.
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After identification of possible pathways for the formation of the impurities, the
characteristics of the impurities which can influence the quality and safety of the drug
were reevaluated. One path has been presented for the synthesis of all the investigated
impurities. This model explains the quantities found and satisfies the analytical needs
of regulatory requirements. However, the details of the formation of these impurities
are not reported in previous studies, even though impurity A has been identified as
related substance by Miralles and colleagues3.
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Compared to the product 1, replacement of the terminal ethyl propionate moiety
with methyl or isopropyl propionate can produce impurities A and B. It is desirable
that impurities A and B be synthesized from the same starting material
(Cyanoꢀcompound 2) of product 1. As shown in Scheme 2, cyanoꢀcompound 2 was
hydrolyzed by aqueous sodium hydroxide in ethanol, and the carboxylic acid
compound 8 can be furnished by acidification with aqueous hydrochloric acid.
Impurity precursor 6 was synthesized by treating compound 8 with 10 N
hydrochloride methanol. It should be mentioned that precursor 6 has been obtained by
oneꢀpot synthesis, including amidine formation by Pinner reaction and acidꢀcatalyzed
esterification reaction during this synthesis. Finally, the coupling of compound 6 with
nꢀhexyl chloroformate 4 gave the desired impurity A. A similar protocol was utilized
for impurity B, where the difference was that the 10 N hydrochloride methanol was
replaced with 10 N hydrochloride isopropanol for impurity B (Scheme 2). Results
showed that the entire process of the formation of impurities A and B started from 2
with overall yields of about 60% and 51%, respectively without resorting to
separation by column chromatography in any step.
Scheme 2. Synthesis of impurity A and B
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O
O
O
O
H
N
N
H
1) HCl/MeOH,TsOH
2) NH4OH
N
N
OH
N
N
1) aq NaOH, EtOH
2) aq HCl
N
N
O
N
NC
N
NC
HN
8
2
O
O
O
O
H
N
N
H
O
O
N
O
N
N
N
N
O
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4
N
N
N
N
NH2
NH2
TsOH
Impurity A
6
O
O
H
O
O
N
N
N
N
O
4
H
N
N
N
N
OH
1) HCl/IPA,TsOH
HN
N
N
2) NH4OH
NH2
NC
TsOH
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8
O
N
N
O
H
N
N
O
O
N
O
N
NH2
Impurity B
Scheme 3. Synthesis of impurities C–G
O
O
O
O
H
N
H
N
N
N
N
O
O
O
N
N
N
O
R
acetone/H2O
K2CO3
O
O
N
HN
N
N
R
Cl
9 (a-e)
TsOH
NH2
NH2
3
Impurity (C-G)
R for Impurity C-G
O
O
O
O
K2CO3
toluene
R
R
OH
CCl3
Cl
Cl3C
O
10 (a-e)
Impurity C, R=
Impurity D, R=
Impurity E, R=
9 (a-e)
R for9 and 10
d, R=
e, R=
a, R=
b, R=
c, R=
Impurity F, R=
Impurity G, R=
Impurities C–G contain different side chains from product 1. It is favorable to
utilize the available compound 3 and alkyl chloroformate 9a–e as starting material. As
shown in Scheme 3, these impurities can be obtained in good yields and high purity
(yields: 72–80%, purities: 97–99%) through the dabigatran etexilate synthetic route.
Regarding alkyl chloroformate 9a–e, they can be easily synthesized from alkyl
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alcohols 10a–e with triphosgene in toluene11. The structures of all these impurities
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6
1
were determined by mass spectroscopy, H NMR, and 13C NMR, and relative
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retention times were established using HPLC.
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To improve the safety and quality of the drug, the concentrations of these
impurities must be reduced to levels accepted by the International Conference on
Harmonization (ICH)12. Impurities A and B are introduced during the first Pinner
reaction step. They can be removed in the following reaction process. It is conceivable
to use the solvent crystallization as a form of purification. Unfortunately, owing
because of the low solubility of impurities A and B in many organic solvents,
isolation of these impurities from the product 1 proved difficult. Similarly, impurity
precursors 6 and 7 could not be reduced from the amidine intermediate 3. However,
these two impurities can be removed from dabigatran etexilate 5 through
recrystallization.
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In order to identify solvents suitable for purification, several solvents were selected
and the results are shown in Table 2. Entries 1–3 showed that acetone, acetonitrile,
and methyl ethyl ketone suffered a unfavorable effects on recrystallization (impurities
A, B ＞ 0.10%). The ester solvent system (entries 4–8) gave better results and
removed more of impurities A and B. It was here observed that ethyl acetate (12 mL/g)
(enter 6) was the best solvent system for this recrystallization process. It is noticed
that levels of impurities A and B could be reduced to below 0.1%, when the product
was obtained by the conditions reported in entry 6 of Table 2.
Table 2. Solvent screening for purification of dabigatran etexilate (5)
recrystallization results of 5 by HPLCa (%)
enter
solvent
ratio (mL/g)
5
Imp A
0.12
0.14
0.12
0.09
0.09
Imp B
0.13
0.16
0.14
0.10
0.09
1
2
3
4
5
acetone
acetonitrile
8
99.43
99.38
99.45
99.65
99.68
12
10
8
methyl ethyl ketone
ethyl acetate
ethyl acetate
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ethyl acetate
nꢀbutyl acetate
isopropyl acetate
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12
12
99.73
99.56
99.59
0.04
0.08
0.09
0.05
0.12
0.11
a Input quality: purity is 99.27%, Imp A and Imp B content 0.15% and 0.19% respectively
Impurity control by specification of reagents is common in pharmaceutical
manufacturing. It is essential to lower the concentration of reagent impurities to a very
low level and ensure that the derivative impurities that form during API are under
control. It has been found that impurities C–G are derived from the material nꢀhexyl
chloroformate 4 in the amidation reaction process, where other alkyl chloroformate
9a–e may be present. Therefore, the key factor is the supply and purity of the material
4, which can be restricted and monitored by GC.
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To investigate the effect of the degree of purity of 4, different amounts of
impurities in 4 were used to synthesize the final product 1. Results are summarized in
Table 3. It was here noted that product 1 was of high quality (entry 3–5) when the
alkyl chloroformate 9a–e in 4 was below 0.10% by GC. Otherwise, the high
concentration of alkyl chloroformate 9a–e in 4 would cause substantial amounts of
impurities C–G to form in 1 (enter 1–2). These results indicated that it is essential to
obtain highly pure 4.
Table 3. Analytical data for dabigatran etexilate mesylate using different
qualities of 4
input
output
Content of impurities in 4 by GC (%)
Quality of 1 by HPLC (%)
enter
Imp
C
Imp
D
Imp
E
Imp
F
Imp
G
4
9a
9b
9c
9d
9e
1
1
2
3
4
5
98.83 0.19 0.06 0.15 0.11 0.12 99.45 0.15
98.83 0.19 0.06 0.15 0.11 0.12 99.38 0.16
99.25 0.05 ND 0.06 0.03 0.04 99.70 0.05
99.25 0.05 ND 0.06 0.03 0.04 99.74 0.04
99.25 0.05 ND 0.06 0.03 0.04 99.73 0.04
0.07
0.05
ND
ND
ND
0.10
0.12
0.01
ND
ND
0.08
0.09
ND
ND
0.01
0.10
0.11
0.02
0.02
0.01
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CONCLUSION
In summary, seven observed and potential impurities of dabigatran etexilate
mesylate (1) were here synthesized and characterized. The origins of formation
impurities A–G during the preparation of 1 were also mapped out. In addition, a
strategy to lower the concentrations of these impurities to levels accepted by ICH is
here proposed. This information would be immensely useful for process chemists
working in this area.
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EXPERIMENTAL SECTION
Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker NMR
AVANCE 400 (400 MHz) spectrometer with TMS as an internal standard. Chemical
shift (d values) and coupling constants (J values) are given in ppm and Hz,
respectively. ESI mass spectra were performed on an Agilent 6210 TOF spectrometer.
Uncorrected melting points were determined on an electrothermal melting point
apparatus. Solvents and reagents were used without any pretreatment. Reaction
progress and chemical purity were evaluated by HPLC analysis using a Ultimate 3000
(Dionex) C18（5ꢁm, 250mm×4.6mm）with a mobile phase A (0.015mol/L KH2PO4,
pH 6.0) and B (acetonitrile), 48:52 v/v; and detection at 225 nm; flow: 1.0 mL/min;
temp. 35 °C.
Synthesis of Impurity A. A solution of sodium hydroxide (2.6g, 65.0mmol) in
water (40mL) was added dropwise to compound 2 (20.0g, 41.5mmol) in ethanol
(160mL). The mixture was stirred at room temperature for 2h. The mixture was
concentrated in vacuo to remove ethanol (about 100mL), the residue was then diluted
with water (120mL) and adjusted to pH 5ꢀ6 by 10% hydrochloric acid solution. The
precipitate was isolated and washed with water (30mL) to afford compound 8 (18.1g,
1
96.1%) as white crystals. Mp 210ꢀ212 °C; H NMR (400Hz, DMSOꢀd6) δ 8.38ꢀ8.37
(dd, J1 = 1.2Hz, J2 = 4.8Hz, 1H), 7.58ꢀ7.54 (m, 1H), 7.48ꢀ7.47 (m, 2H), 7.45 (s, 1H),
7.40ꢀ7.38 (d, J = 8.4Hz, 1H), 7.21ꢀ7.19 (m, 1H), 7.17ꢀ7.16 (m, 1H), 7.13ꢀ7.10 (dd, J1
= 4.8Hz, J2 = 7.2Hz, 1H), 6.97ꢀ6.95 (d, J = 8.0Hz, 1H), 6.84ꢀ6.81 (d, J = 8.4Hz, 2H),
4.60ꢀ4.59 (d, J = 5.6Hz, 2H), 4.20ꢀ4.16 (t, J = 7.6Hz, 2H), 3.76 (s, 3H), 2.64ꢀ2.60 (t, J
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= 7.6Hz, 2H); 13C NMR (100.6 MHz, DMSOꢀd6) δ 172.8, 170.5, 156.1, 153.3, 151.7,
148.7, 140.9, 137.8, 137.2, 133.3, 129.7, 122.9, 122.0, 121.2, 120.4, 119.6, 112.5,
109.4, 96.9, 43.5, 39.7, 33.1, 29.9; MS m/z 455 [M + H]+ , 477 [M + Na]+.
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4
5
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7
8
9
Compound 8 (9.0g, 19.8mmol) and pꢀtoluenesulfonic acid (4.5g, 23.8mmol) were
dissolved in 10 mol/L HClꢀmethanol solution (20mL), the mixture was stirred for 8h
at room temperature. The mixture cooled to 0℃, and ammonium hydroxide (30mL)
was added dropwise. The reaction mixture was stirred for 5h at room temperature.
Water (30mL) was added to this mixture and stirred for another 5h, the precipitate was
isolated and washed with water (50mL) to afford compound 6 (8.1g, 84.2%) as white
crystals. Mp 188ꢀ190 °C; 1H NMR (400Hz, DMSOꢀd6) δ 8.65 (br, 4H), 8.39ꢀ8.38 (dd,
J1 = 1.2Hz, J2 = 4.8Hz, 1H), 7.64ꢀ7.62 (d, J = 8.8Hz, 2H), 7.57ꢀ7.53 (m, 1H),
7.51ꢀ7.49 (m, 3H), 7.41ꢀ7.39 (m, 1H), 7.31ꢀ7.29 (t, J = 5.6Hz, 1H), 7.18ꢀ7.15 (dd, J1
= 1.2Hz, J2 = 8.4Hz, 1H), 7.12ꢀ7.10 (m, 3H), 6.92ꢀ6.89 (d, J = 8.4Hz, 1H), 6.88ꢀ6.86
(d, J = 8.8Hz, 2H), 4.65ꢀ4.64 (d, J = 5.6Hz, 2H), 4.24ꢀ4.21 (t, J = 7.2Hz, 2H), 3.77 (s,
3H), 3.53 (s, 3H), 2.72ꢀ2.69 (t, J = 7.2Hz, 2H), 2.29 (s, 3H); 13C NMR (100.6 MHz,
DMSOꢀd6) δ 171.4, 170.3, 164.4, 155.9, 153.3, 153.0, 148.6, 144.9, 140.8, 138.1,
137.8, 137.2, 129.6, 129.4, 128.2, 125.4, 122.8, 121.9, 121.2, 119.5, 113.4, 111.8,
109.5, 56.0, 51.3, 44.4, 32.8, 29.9, 20.7; MS m/z 486 [M + H]+.
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Compound 6 (8g, 16.5mmol) and potassium carbonate (6.8g, 49.5mmol) were
dissolved in acetone (100mL) and water (50mL), compound 4 (3.0g, 18.1mmol) was
added at room temperature and stirring was continued for 2h. The mixture was heated
to 50℃ and the organic phase was separated, and concentrated in vacuo. The residue
was finally crystallized in EtOAc (60mL) to give impurity A (7.6g, 75.2%) as a white
solid. Purity by HPLC 97.42%, Mp 88ꢀ90 °C; 1H NMR (400Hz, DMSOꢀd6) δ 8.86 (br,
2H), 8.39ꢀ8.38 (d, J = 4.0 Hz, 1H), 7.81ꢀ7.79 (d, J = 8.4Hz, 2H), 7.56ꢀ7.52 (t, J =
7.2Hz, 1H), 7.48 (s, 1H), 7.40ꢀ7.38 (d, J = 8.4Hz, 1H), 7.18ꢀ7.16 (d, J = 8.0Hz, 1H),
7.13ꢀ7.10 (m, 1H), 6.91ꢀ7.89 (d, J = 8.0Hz, 2H), 6.78ꢀ6.76 (d, J = 8.4Hz, 2H),
4.60ꢀ4.59 (d, J = 5.2Hz, 2H), 4.24ꢀ4.21 (t, J = 6.8Hz, 2H), 4.00ꢀ3.97 (t, J = 6.4Hz,
2H), 3.77 (s, 3H), 3.53 (s, 3H), 2.72ꢀ2.69 (t, J = 6.8Hz, 2H), 1.60ꢀ1.57 (m, 2H), 1.29
(m, 6H), 0.88ꢀ0.85 (t, J = 6.4Hz, 3H); 13C NMR (100.6 MHz, DMSOꢀd6) δ 171.4,
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170.4, 166.4, 164.2, 155.9, 153.7, 151.5, 148.6, 140.8, 137.8, 137.2, 129.4, 129.1,
122.8, 122.0, 121.2, 121.1, 119.5, 111.4, 109.4, 64.1, 51.3, 44.4, 32.8, 30.9, 29.9, 28.5,
25.2, 21.9, 13.8; MS m/z 614 [M + H]+.
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Synthesis of Impurity B. Compound 8 (9.0g, 19.8mmol) and pꢀtoluenesulfonic
acid (4.5g, 23.8mmol) were dissolved in 10 mol/L HClꢀisopropanol solution (20mL),
the mixture was stirred for 8h at room temperature. The mixture cooled to 0℃, and
ammonium hydroxide (30mL) was added dropwise. The reaction mixture was stirred
for 5h at room temperature. Water (30mL) was added to this mixture and stirred for
another 5h, the precipitate was isolated and washed with water (50mL) to afford
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compound 7 (7.5g, 73.7%) as white crystals. Mp 201ꢀ203 °C; H NMR (400Hz,
DMSOꢀd6) δ 8.67 (br, 4H), 8.40ꢀ8.39 (d, J = 4.8Hz, 1H), 7.68ꢀ7.66 (d, J = 8.8Hz, 2H),
7.57ꢀ7.54 (m, 2H), 7.51ꢀ7.49 (m, 2H), 7.41ꢀ7.39 (d, J = 8.4Hz, 1H), 7.30ꢀ7.28 (t, J =
5.6Hz, 1H), 7.19ꢀ7.17 (d, J = 8.4Hz, 1H), 7.12ꢀ7.10 (m, 3H), 6.93ꢀ6.90 (d, J = 8.4Hz,
1H), 6.88ꢀ6.86 (d, J = 8.8Hz, 2H), 4.85ꢀ4.81 (m, 1H), 4.64ꢀ4.63 (d, J = 5.6Hz, 2H),
4.25ꢀ4.22 (t, J = 7.2Hz, 2H), 3.78 (s, 3H), 2.71ꢀ2.68 (t, J = 7.2Hz, 2H), 2.29 (s, 3H),
1.15ꢀ1.13 (d, J = 6.4Hz, 6H); 13C NMR (100.6 MHz, DMSOꢀd6) δ 171.2, 170.5, 166.8,
164.4, 156.1, 154.1, 153.0, 148.9, 144.6, 140.8, 138.2, 137.6, 137.0, 129.6, 129.1,
128.4, 126.0, 123.0, 122.8, 121.9, 121.1, 119.7, 114.0, 111.8, 109.5, 68.0, 56.0, 51.3,
44.6, 32.8, 29.9, 20.7, 19.6; MS m/z 514 [M + H]+.
Compound 7 (7.5g, 14.6mmol) and potassium carbonate (6.1g, 43.8mmol) were
dissolved in acetone (100mL) and water (50mL), compound 4 (2.6g, 16.1mmol) was
added at room temperature and stirring was continued for 2h. The mixture was heated
to 50℃ and the organic phase was separated, and concentrated in vacuo. The residue
was finally crystallized in EtOAc (60mL) to give impurity B (6.7g, 71.6%) as a white
1
solid. Purity by HPLC 97.56%, Mp 103ꢀ105 °C; H NMR (400Hz, DMSOꢀd6) δ 8.85
(br, 2H), 8.41ꢀ8.40 (d, J = 3.6Hz, 1H), 7.85ꢀ7.83 (d, J = 8.4Hz, 2H), 7.56ꢀ7.52 (m,
2H), 7.40ꢀ7.38 (d, J = 8.4Hz, 1H), 7.20ꢀ7.18 (d, J = 8.4Hz, 1H), 7.13ꢀ7.10 (dd, J1 =
5.2Hz, J2 = 6.8Hz, 1H), 6.94ꢀ6.92 (m, 1H), 6.90 (s, 1H), 6.81ꢀ6.79 (d, J = 8.8Hz, 2H),
4.86ꢀ4.81 (m, 1H), 4.62ꢀ4.60 (d, J = 5.2Hz, 2H), 4.27ꢀ4.24 (t, J = 6.8Hz, 2H),
4.02ꢀ3.99 (t, J = 6.8Hz, 2H), 3.78 (s, 3H), 2.70ꢀ2.66 (t, J = 7.2Hz, 2H), 1.61ꢀ1.55 (m,
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2H), 1.35ꢀ1.29 (m, 6H), 1.15ꢀ1.13 (d, J = 6.4Hz, 6H), 0.90ꢀ0.85 (m, 3H); 13C NMR
(100.6 MHz, DMSOꢀd6) δ 170.9, 170.8, 166.9, 164.7, 156.5, 154.2, 152.0, 149.1,
141.4, 138.3, 137.7, 129.9, 129.6, 123.3, 122.6, 121.7, 120.0, 111.9, 109.9, 67.8, 64.6,
44.8, 33.8, 31.5, 30.4, 29.0, 25.7, 22.5, 21.9, 14.3; MS m/z 642 [M + H]+.
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General procedure for the preparation of Impurities CꢀG A mixture of
triphosgene (1.7g, 5.7mmol), potassium carbonate (1.5g, 11mmol) and dimethyl
formamide (0.3g, 4.1mmol) as a catalyst in toluene (10mL) was cooled to 0℃, and
stirred for 1h. A solution of alkyl alcohols 10aꢀe (1.1g, 11mmol) in toluene (10mL)
was added dropwise to this mixture. Then the reaction mixture was stirred for 5h. This
mixture was added to a solution of 3 (5.0g, 10mmol) and potassium carbonate (4.1g,
30mmol) in acetone (60mL) and water (30mL). The reaction was stirred for 2h at
room temperature. The mixture was heated to 50℃ and the organic phase was
separated, and concentrated in vacuo. The residue was finally crystallized in EtOAc
(50mL) to give impurities CꢀG.
Impurity C. Yield 75.4% as a white solid. Purity by HPLC 99.00%, Mp 95ꢀ97 °C;
1H NMR (400Hz, DMSOꢀd6) δ 8.85 (br, 2H), 8.40ꢀ8.38 (dd, J1 = 1.6Hz, J2 = 4.8Hz,
1H), 7.81ꢀ7.79 (d, J = 8.8Hz, 2H), 7.57ꢀ7.52 (m, 1H), 7.50ꢀ7.48 (m, 1H), 7.41ꢀ7.39 (d,
J = 8.4Hz, 1H), 7.18ꢀ7.16 (dd, J1 = 1.2Hz, J2 = 8.4Hz, 1H), 7.14ꢀ7.11 (dd, J1 = 4.8Hz,
J2 = 7.2Hz, 1H), 6.91ꢀ6.89 (d, J = 8.0Hz, 1H), 6.88 (s, 1H), 6.79ꢀ6.77 (d, J = 8.8Hz,
2H), 4.61ꢀ4.59 (d, J = 5.6Hz, 2H), 4.25ꢀ4.22 (t, J = 6.8Hz, 2H), 4.02ꢀ3.96 (q, J =
7.2Hz, 2H), 3.94ꢀ3.93 (d, J = 6.0Hz, 2H), 3.77 (s, 3H), 2.71ꢀ2.67 (t, J = 7.2Hz, 2H),
1.56ꢀ1.46 (m, 1H), 1.40ꢀ1.28 (m, 4H), 1.15ꢀ1.12 (t, J = 6.8Hz, 3H), 0.90ꢀ0.86 (t, J =
7.2Hz, 6H); 13C NMR (100.6 MHz, DMSOꢀd6) δ 170.9, 170.3, 166.4, 164.3, 156.0,
153.6, 151.5, 148.6, 140.8, 137.8, 137.2, 129.3, 129.1, 127.9, 125.5, 122.7, 122.0,
121.2, 121.1, 119.4, 111.4, 109.4, 66.0, 59.9, 44.3, 40.0, 33.0, 29.8, 22.8, 13.9,10.8;
MS m/z 628 [M + H]+.
Impurity D. Yield 77.3% as a white solid. Purity by HPLC 98.53%, Mp
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104ꢀ106 °C; H NMR (400Hz, DMSOꢀd6) δ 8.94ꢀ8.81 (br, 2H), 8.40 (m, 1H),
7.85ꢀ7.83 (d, J = 8.4Hz, 2H), 7.57ꢀ7.52 (m, 2H), 7.40ꢀ7.38 (d, J = 8.4Hz, 1H),
7.19ꢀ7.17 (d, J = 8.4Hz, 1H), 7.13ꢀ7.10 (t, J = 5.6Hz, 1H), 6.99 (s, 1H), 6.92ꢀ6.90 (d,
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J = 7.6Hz, 1H), 6.81ꢀ6.79 (d, J = 8.4Hz, 2H), 4.61ꢀ4.60 (d, J = 4.8Hz, 2H), 4.27ꢀ4.24
(t, J = 6.8Hz, 2H), 4.02ꢀ3.97 (m, 4H), 3.78 (s, 3H), 2.73ꢀ2.69 (t, J = 5.6Hz, 2H),
1.62ꢀ1.55 (m, 2H), 1.40ꢀ1.31 (m, 2H), 1.15ꢀ1.12 (t, J = 7.2Hz, 3H), 0.93ꢀ0.89 (t, J =
7.2Hz, 3H); 13C NMR (100.6 MHz, DMSOꢀd6) δ 171.5, 170.8, 166.9, 164.7, 156.5,
154.2, 152.1, 149.1, 141.4, 138.3, 137.7, 129.8, 129.7, 123.2, 122.5, 121.7, 121.6,
120.9, 120.0, 111.9, 109.9, 64.3, 60.9, 60.5, 44.8, 33.6, 31.1, 30.4, 19.2, 14.4, 14.1;
MS m/z 600 [M + H]+.
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Impurity E. Yield 71.7% as a white solid. Purity by HPLC 99.02%, Mp 86ꢀ88 °C;
1H NMR (400Hz, DMSOꢀd6) δ 8.89 (br, 2H), 8.40ꢀ8.39 (m, 1H), 7.82ꢀ7.80 (d, J =
8.8Hz, 2H), 7.57ꢀ7.52 (m, 1H), 7.49 (s, 1H), 7.41ꢀ7.39 (d, J = 8.4Hz, 1H), 7.18ꢀ7.16
(dd, J1 = 1.2Hz, J2 = 8.8Hz, 1H), 7.14ꢀ7.11 (m, 1H), 6.94ꢀ6.91 (m, 1H), 6.89 (s, 1H),
6.79ꢀ6.77 (d, J = 8.4Hz, 2H), 4.77ꢀ4.69 (m, 1H), 4.61ꢀ4.59 (d, J = 5.2Hz, 2H),
4.26ꢀ4.22 (t, J = 6.8Hz, 2H), 4.02ꢀ3.96 (q, J = 7.2Hz, 2H), 3.78 (s, 3H), 2.71ꢀ2.68 (t, J
= 7.2Hz, 2H), 1.56ꢀ1.51 (m, 1H), 1.49ꢀ1.42 (m, 1H), 1.36ꢀ1.33 (m, 6H), 1.19ꢀ1.17 (d,
J = 6.4Hz, 3H), 1.15ꢀ1.12 (t, J = 7.2Hz, 3H), 0.91ꢀ0.84 (m, 3H); 13C NMR (100.6
MHz, DMSOꢀd6) δ 170.9, 170.3, 166.4, 163.9, 156.0, 153.7, 151.5, 148.6, 140.8,
137.8, 137.2, 129.3, 129.1, 122.7, 122.0, 121.1, 119.5, 111.3, 109.3, 69.9, 59.9, 44.3,
35.6, 33.0, 31.1, 29.9, 24.6, 21.9, 20.0, 13.9, 13.8; MS m/z 642 [M + H]+.
Impurity F. Yield 73.4% as a white solid. Purity by HPLC 98.57%, Mp 85ꢀ87 °C;
1H NMR (400Hz, DMSOꢀd6) δ 8.84 (br, 2H), 8.40ꢀ8.39 (d, J = 4.0Hz, 1H), 7.81ꢀ7.79
(d, J = 8.4Hz, 2H), 7.56ꢀ7.53 (t, J = 7.2Hz, 1H), 7.48 (s, 1H), 7.41ꢀ7.39 (d, J = 8.8Hz,
1H), 7.18ꢀ7.16 (d, J = 8.4Hz, 1H), 7.14ꢀ7.11 (dd, J1 = 5.2Hz, J2 = 6.8Hz, 1H),
6.91ꢀ6.89 (d, J = 7.6Hz, 2H), 6.79ꢀ6.76 (d, J = 8.8Hz, 2H), 4.61ꢀ4.59 (d, J = 5.2Hz,
2H), 4.25ꢀ4.22 (t, J = 7.2Hz, 2H), 4.02ꢀ3.96 (m, 4H), 3.77 (s, 3H), 2.71ꢀ2.67 (t, J =
7.2Hz, 2H), 1.62ꢀ1.56 (m, 2H), 1.27 (m, 10H), 1.15ꢀ1.12 (t, J = 7.2Hz, 3H), 0.88ꢀ0.85
(m, 3H); 13C NMR (100.6 MHz, DMSOꢀd6) δ 170.9, 170.3, 166.4, 164.2, 155.9, 153.6,
151.5, 148.6, 140.8, 137.7, 137.2, 129.3, 129.1, 122.7, 122.0, 121.2, 119.5, 111.4,
109.3, 64.1, 59.9, 44.3, 33.0, 31.2, 29.8, 28.7, 28.6, 25.5, 22.0, 13.9, 13.8; MS m/z
656 [M + H]+.
Impurity G. Yield 79.6% as a white solid. Purity by HPLC 97.28%, Mp
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110ꢀ112 °C; H NMR (400Hz, DMSOꢀd6) δ 8.84 (br, 2H), 8.39ꢀ8.38 (d, J = 3.6Hz,
1H), 7.81ꢀ7.79 (d, J = 8.4Hz, 2H), 7.56ꢀ7.52 (m, 1H), 7.48 (s, 1H), 7.40ꢀ7.38 (d, J =
8.4Hz, 1H), 7.18ꢀ7.16 (m, 1H), 7.13ꢀ7.10 (dd, J1 = 5.2Hz, J2 = 6.8Hz, 1H), 6.91ꢀ6.86
(m, 2H), 6.78ꢀ6.76 (d, J = 8.8Hz, 2H), 4.69ꢀ4.63 (m, 1H), 4.60ꢀ4.59 (d, J = 5.2Hz,
2H), 4.25ꢀ4.21 (t, J = 6.8Hz, 2H), 4.01ꢀ3.96 (q, J = 7.2Hz, 2H), 3.77 (s, 3H),
2.70ꢀ2.67 (t, J = 7.2Hz, 2H), 1.59ꢀ1.46 (m, 4H), 1.38ꢀ1.23 (m, 2H), 1.15ꢀ1.11 (t, J =
7.2Hz, 3H), 0.90ꢀ0.84 (m, 6H); 13C NMR (100.6 MHz, DMSOꢀd6) δ 171.0, 170.4,
166.5, 164.2, 156.0, 153.7, 151.5, 148.6, 140.9, 137.8, 137.2, 129.4, 129.1, 122.8,
122.1, 121.3, 121.2, 119.5, 111.4, 109.4, 74.2, 60.0, 44.3, 35.6, 33.1, 29.9, 26.8, 18.2,
13.9, 13.8, 9.6; MS m/z 628 [M + H]+.
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ASSOCIATED CONTENT
Supporting Information
1H NMR, 13C NMR, MS spectra of impurities AꢀG, and the HPLC results of the
mixture impurities AꢀG and product 1. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
*Phone: +86ꢀ21ꢀ55514600. Fax: +86ꢀ21ꢀ65312830. Eꢀmail: lijianqb@126.com.
Author Contributions
⊥These authors contributed equally to the work described in this paper.
ACKNOWLEDGMENTS
This work is supported by National Natural Science Foundation of China (Grant No.
81302631), Shanghai RisingꢀStar Program (Grant No. 13QB1403800) and the
National Science and Technology Major Project (Grant No. 2012ZX09103101ꢀ038).
REFERENCES
1. Van Ryn, J.; Stangier, J.; Haertter, S.; Liesenfeld, K. H.; Wienen, W.; Feuring, M.; Clemens, A.
Thromb. Haemost. 2010, 103, 1116ꢀ1127.
2. Ma, T. K.; Yan, B. P.; Lam, Y. Y. Pharmacol. Therapeut. 2011, 129, 185ꢀ194.
3. Serra, M. J.; Duran, L. E.; Rao, L. Dabigatran etexilate and related substances, processes and
compositions, and use of the substances as renference standards and markers. World Patent WO
2012152855 A1,2012.
4. Chen, Y.; Liang, J.; Chen, H. S.; Yuan, L.. Heterocycles 2013, 87 (8), 1699ꢀ1710.
5. Hauel, N.; Ries, U.; Priepke, H.; Wienen, W.; Stassen, J. Disubstituted bicyclic heterocycles, their
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production and use as medicaments. World Patent WO 1998037075 A1, 1998.
6. Zerban, G.; Hausherr, A.; Schlarb, K.; Schmitt, H. P.; Weyell, B.; Koch, G.; Hamm, R. Method for
producing 4ꢀ(benzimidazolylmethylamino)ꢀbenzamidines. World Patent WO 2006000353 A1, 2006.
7. Zerban, G.; Hausherr, A.; Hamm, R.; Koch, G.; Schlarb, K.; Schmitt, H. P.; Weyell, B. Improved
process for the preparation of 4ꢀ(benzimidazolylmethylamino)ꢀbenzamidines and the salts thereof.
World Patent WO 2007071742 A1, 2007.
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8. Hauel, N. H.; Nar, H.; Priepke, H.; Ries, U.; Stassen, J. M.; Wienen, W. J. Med. Chem. 2002, 45,
1757ꢀ1766.
9. Jayaraman, V.; Patel, S.; Mistry, S.; Timbadiya, M.; Parmar, B.; Tamboli, P. Novel perference
markers of dabigatran etexilate. World Patent WO 2013024394 A1, 2013.
10. The start cyano compound 2 was obtained commercially, it do not contain methyl and isopropyl
ester.
11. Abdul, R. A. S. D.; Vikas, K. G. Process for preparing alkyl/aryl chloroformates. U.S. Patent
20050065361, 2005.
12. ICH Q3A(R2) Impurities in New Drug Substances, International Conference on Harmonisation of
Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH): Geneva,
Switzerland, October 2006.
O
O
H
R
N
N
O
O
N
N
O
O
O
C6H13
n
H
N
N
N
N
O
O
N
N
O
C6H13
n
NH2
N
N
Impurity (AꢀB)
NH2
MeSO3H
Impuritiy A, R =methyl;
Impuritiy B, R =isopropyl.
O
N
O
F, R=
C, R=
H
N
N
N
O
O
O
R
N
N
D, R=
E, R=
G, R=
NH2
Impurity (C-G)
Table of Contents graphic
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