Concise synthesis of the major metabolite M8 from ticagrelor and simultaneous determination of ticagrelor and M8 by a novel LC/MS method

Article abstract of DOI:10.1177/1747519821991993

Ticagrelor is an oral antiplatelet agent that has been approved for preventing de novo and recurrent acute coronary syndrome. To date, only a few studies have attempted to clarify population differences in the pharmacokinetics and pharmacodynamics of ticagrelor between Caucasians and Asians. Our aim was to develop a simple quantification method for ticagrelor and its pharmacologically active metabolite M8 (AR-C124910XX) in human plasma and urine. First, we concisely synthesized M8 from ticagrelor via a five-step sequence: the hydroxyethyl group of ticagrelor was removed by bromination and subsequent zinc-mediated chemoselective reduction. Then, we developed a simple liquid chromatography–mass spectrometry method using ticlopidine as an internal standard. Ticagrelor, M8, and internal standard were separated with a reverse-phase C₁₈ column, and ticagrelor and M8 were detected at m/z [+] of 523.25 and 479.25, respectively, with good sensitivity and precision in the concentration ranges of 10 to 100 μM and 5 to 50 μM, respectively. This novel liquid chromatography–mass spectrometry system may be attractive to investigators in private laboratories because it is less costly than liquid chromatography/mass spectrometry/mass spectrometry systems. Our method is expected to accelerate further clinical studies on the disposition and pharmacodynamics of ticagrelor.

Full text of DOI:10.1177/1747519821991993

9
research-arti
9cle2021 1993CHL0010.1177/1747519821991993Journal of Chemical ResearchSuzuki et al.
Research Paper
Journal of Chemical Research
–5
1
Concise synthesis of the major
© The Author(s) 2021
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metabolite M8 from ticagrelor and
simultaneous determination of
ticagrelor and M8 by a novel LC/MS method
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1
,2
1
1
Momoko Suzuki , Ryuichi Ogawa , Hirotoshi Echizen ,
Hiroshi Kogen
1
1,2
and Kenichi Kobayashi
Abstract
Ticagrelor is an oral antiplatelet agent that has been approved for preventing de novo and recurrent acute coronary
syndrome. To date, only a few studies have attempted to clarify population differences in the pharmacokinetics and
pharmacodynamics of ticagrelor between Caucasians and Asians. Our aim was to develop a simple quantification
method for ticagrelor and its pharmacologically active metabolite M8 (AR-C124910XX) in human plasma and urine.
First, we concisely synthesized M8 from ticagrelor via a five-step sequence: the hydroxyethyl group of ticagrelor was
removed by bromination and subsequent zinc-mediated chemoselective reduction. Then, we developed a simple liquid
chromatography–mass spectrometry method using ticlopidine as an internal standard. Ticagrelor, M8, and internal
standard were separated with a reverse-phase C column, and ticagrelor and M8 were detected at m/z [+] of 523.25
1
8
and 479.25, respectively, with good sensitivity and precision in the concentration ranges of 10 to 100 µM and 5 to
0 µM, respectively. This novel liquid chromatography–mass spectrometry system may be attractive to investigators
5
in private laboratories because it is less costly than liquid chromatography/mass spectrometry/mass spectrometry
systems. Our method is expected to accelerate further clinical studies on the disposition and pharmacodynamics of
ticagrelor.
Keywords
Ticagrelor, metabolite, antiplatelet agent, AR-C124910XX, liquid chromatography/mass spectrometry
Date received: 18 December 2020; accepted: 14 January 2021
1Graduate School of Pharmaceutical Sciences, Meiji Pharmaceutical
University, Tokyo, Japan
Graduate School of Pharmaceutical Sciences, Health Sciences
Corresponding authors:
Kenichi Kobayashi, Graduate School of Pharmaceutical Sciences, Health
Sciences University of Hokkaido, 1757 Kanazawa, Tobetsu-cho, Ishikari-
gun, Hokkaido 061-0293, Japan.
2
University of Hokkaido, Hokkaido, Japan
Email: kkobayashi@hoku-iryo-u.ac.jp
Ryuichi Ogawa, Graduate School of Pharmaceutical Sciences, Meiji Phar-
maceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan.
Email: smridron@gmail.com
Creative Commons Non Commercial CC BY-NC: This article is distributed under the terms of the Creative Commons
Attribution-NonCommercial 4.0 License (https://creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use,
reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and
Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage).

2
Journal of Chemical Research 00(0)
Introduction
F
F
F
F
HN
N
HN
N
Ticagrelor is a recently approved oral antiplatelet agent for
preventing restenosis after coronary artery revasculariza-
tion in patients with coronary stenosis (e.g. acute coronary
syndrome (ACS)). Ticagrelor suppresses platelet aggrega-
tion by directly inhibiting P2Y12 receptors in the same way
as clopidogrel sulfate and ticlopidine hydrochloride. The
efficacy of ticagrelor for preventing restenosis in ACS
patients treated with coronary intervention was compared
with that of clopidogrel in a large, randomized, double-
blind clinical trial (PLATO study, N = 18,624) in mostly
Caucasian patients (91.7%). Results revealed that the
occurrence of cardiovascular events by 12 months was sig-
nificantly less in the ticagrelor group than in the clopidogrel
group (hazard ratio: 0.84; 95% confidence interval: 0.77 to
N
N
N
N
N
N
N
N
CH3
CH3
S
S
OH
OH
HO
O
OH
Ticagrelor (1)
HO
OH
Ticagrelor metabolite M8 (2)
Figure 1. Structures of ticagrelor (1) and its metabolite M8 (2).
Results and discussion
Preparation of M8
Initially, the three hydroxyl groups in ticagrelor (1)14 were
protected as TBS ethers to give 3 in quantitative yield
Scheme 1). The primary TBS group was removed by treat-
ment with NH F in MeOH at 35°C to afford bis-TBS ether
in 98% yield. Primary alcohol 4 was converted to bromide
in 96% yield by anAppel reaction using CBr and Ph P. The
crucial reduction of bromide 5was accomplished by Muratake
using zinc and NH Cl in 2-pro-
panol/H O (14:1) at 75°C to obtain the desired secondary
alcohol 6 in 90% yield. Finally, the two TBS groups in 6 were
deprotected with TBAF to furnish M8 (2) in 96% yield. Then,
we used the synthesized M8 to develop a simultaneous LC/
MS determination method for ticagrelor and M8.
0
.92). The risk of major bleeding events was similar
1
between the groups. Subsequently, a randomized study
was performed to compare ticagrelor and clopidogrel in
Asian patients (PHILO study, N = 801). In contrast to the
PLATO study, no statistically significant differences were
observed in efficacy and risk of major bleeding events
(
15
4
4
5
4
3
2
between ticagrelor and clopidogrel in the PHILO study.
10–13
and Natsume’s protocol
While the discrepant results may be attributable to the
smaller sample size in the PHILO study than in the PLATO
study, differences in the pharmacokinetics (PK) and/or
pharmacodynamics (PD) of ticagrelor between Caucasians
and Asians may also play a role. In this context, further
studies are needed on the PK and PD of ticagrelor, particu-
larly in Asians.
4
2
The lack of a simple assay method for ticagrelor and its
active metabolite AR-C124910XX (M8) appears to be an
Identification of ticagrelor and M8 by
LC/MS
3
obstacle to clinical studies. While several liquid chroma-
tography/mass spectrometry/mass spectrometry (LC/MS/
MS) methods have been developed mainly for large clinical
The LC/MS traces obtained in selected ion monitoring mode
for a standard solution containing a mixture of ticagrelor, M8,
and ticlopidine (the internal standard) at higher range are
shown in Figure 2. Ticlopidine is a readily available antiplate-
let agent for research and is never used concomitantly with
ticagrelor in clinical settings; therefore, we considered that
ticlopidine would be a good internal standard in this assay.
Clear separation of the peaks corresponding to ticagrelor, M8,
and ticlopidine was obtained without substantial ion suppres-
sion between ticagrelor and M8 using the LC/MS conditions
described in the method section. The peaks were detected at
approximately 4.2, 4.2, and 8.0 min, respectively.
4
–8
trials supported by pharmaceutical companies, to our
knowledge, no assay method is available for LC/MS sys-
tems. In general laboratories at hospitals or academic insti-
tutions, an LC/MS system would be a versatile alternative
to LC/MS/MS systems. Another obstacle is that no pure M8
standard is commercially available. Since M8 may be at
least partly associated with clinical effects and adverse
reactions, simultaneous determination of ticagrelor and M8
is necessary for interpreting clinical outcomes following
administration of ticagrelor. In this context, we decided to
develop a novel synthetic method for M8. Using commer-
cially available ticagrelor and synthetized M8, we devel-
oped a simple LC/MS method that would be suitable for
general laboratories in hospitals and academic institution.
The total synthesis of ticagrelor derivatives, including
Figure 3 shows the calibration curves of ticagrelor and M8
in human plasma and under conditions for a metabolism assay
containing human liver microsomes (approximately 0.28 mg/
mL) deactivated by phosphoric acid, with an ethylenediamine-
tetraacetic acid (EDTA) and nicotinamide adenine dinucleo-
tide phosphate (NADPH)-generating system in potassium
phosphate buffer. Linear calibration curves for ticagrelor and
M8 were obtained up to 100 and 50 µM, respectively. The
intra- and inter-day coefficients of variation were within 10%
over all concentration ranges for both ticagrelor and M8. The
percentage of recovery of ticagrelor and M8 from human
plasma in this protocol was 94.1% ± 11.7% and 103.5% ±
9
M8 (2), has been reported by Sun and co-workers. However,
we envisaged that M8 (2) could be synthetized from ticagre-
lor (1) in fewer steps (Figure 1), which requires only removal
of a hydroxyethyl group. Toward this end, we envisaged that
this could be accomplished by conversion of the hydroxye-
thyl to a bromoethyl group followed by zinc reduction, as
reported by Muratake and Natsume.10–13 Here, we describe
16.1%, respectively. The lowest plasma concentration of tica-
our concise synthesis of M8 (2) from ticagrelor (1) and
simultaneous determination of 1 and synthetic 2 by a novel
LC/MS method for biological research.
grelor in our assay was 0.56µM, whereas the average plasma
concentration range of ticagrelor after 60–90 mg twice daily

Suzuki et al.
3
TBSOTf
2
,6-lutidine
OTBS
OTBS
NH
4
F
OTBS
OTBS
CBr , Ph P
4
3
1
Ticagrelor ( )
CH Cl
MeOH
98%
CH Cl
2
2
2
2
TBSO
HO
96%
quant.
O
O
3
4
Zn, NH Cl
OTBS
OTBS
4
OTBS
OTBS
TBAF
THF
96%
OH
i-PrOH/H O (14:1)
2
0%
Br
9
O
HO
HO
OH
5
6
Ticagrelor metabolite M8 (2)
Scheme 1. Synthesis of ticagrelor metabolite M8 (2).
were measured on a polarimeter (P-2200, JASCO) using a cell
with an optical path length of 100 mm. Column chromatogra-
phy was performed on silica gel (40–100 mesh). Analytical
thin-layer chromatography was conducted using 0.25 mm sil-
ica gel 60 F plates.
A chromatographic system comprising two pumps
(LC-20AD, Shimadzu, Kyoto, Japan), an automatic sample-
injector (SIL-20A, Shimadzu), and a single-quadrupole mass
spectrometer (LCMS-2010, Shimadzu) was used for
our quantitative analysis. The mass spectrometer was
operated in positive ion mode using electrospray ionization.
Figure 2. Typical LC/MS profile of a mixture of ticagrelor, M8,
and ticlopidine (internal standard).
Chromatographic separation was performed using a C col-
would be from 0.2 to 2.5 µM according to its package insert.16
18
umn (Capcell Pak C18 MG II, Shiseido Co., Ltd., Tokyo,
Japan; 3 µm, 50 mm × 4.6 mm ID) with a 10-mm guard col-
umn maintained at 40°C by a temperature-controlled column
oven (CTO-20A, Shimadzu). The mobile phase was a 10/90
Therefore, there is a limitation in detecting the lower plasma
concentration range of ticagrelor in clinical samples. Further
drug–drug interaction studies would be possible within these
concentration ranges using our LC/MS assay.
(v/v) mixture of 10 mM ammonium acetate aqueous solution
(10%; solventA) and 100% acetonitrile (90%; solvent B). The
flow rate of the mobile phase was maintained at 0.2 mL/min,
and the injection sample volume was 2 µL. The analytes sepa-
rated on the column were detected by MS with the following
settings: collision energy of 20 V, capillary temperature of
Conclusion
We accomplished a concise synthesis of the major metabo-
lite M8 (2) from ticagrelor (1) in high yield over five steps.
This synthesis enabled easy access to 2, via zinc-mediated
reduction to remove the hydroxyethyl group of ticagrelor.
In addition, we established a simple LC/MS assay for tica-
grelor and M8. Because LC/MS assays can be used as a
versatile alternative to LC/MS/MS in general laboratories
in hospitals, the novel assay method reported here is
expected to be useful in small clinical studies and in vitro
pharmacological studies or drug–drug interaction studies.
220°C, ionization voltage of 4.5 kV in positive mode, sheath
gas at 60 psi, and nitrogen auxiliary gas at 10 psi. Single-ion
mode was used with m/z values of 523.25, 479.25, and 264.05
for ticagrelor, M8, and ticlopidine (internal standard), respec-
tively. Ticagrelor and ticlopidine were purchased from Sigma-
Aldrich Japan Inc. (Tokyo, Japan). Data acquisition and
analysis were performed using the LC/MS Solution software
(version 3, Shimadzu). To obtain the calibration curves in
human plasma, 100 µL of standard solutions containing tica-
grelor (2.25 to 225 µM), M8 (1.27 to 63.2 µM), and ticlopidine
Experimental
(100 ng/mL) were added to 400 µL of human plasma. Then 5
Materials and methods
mL of diethyl ether were added to each standard sample and 4
1H NMR spectra were recorded at 300 MHz (JNM-AL300, mL of supernatants were collected after shaking and centrifu-
JEOL, Tokyo, Japan) or 400 MHz (JNM-ECS400, JEOL). gation followed by redissolution with 100 µL of mobile phase.
The chemical shifts are expressed in ppm relative to tetra- To obtain the calibration curves under condition for a meta-
methylsilane (δ = 0) as an internal standard (CDCl or CD OD bolic assay, ticagrelor, M8, and ticlopidine (10 µL each) were
3
3
solution). Splitting patterns are indicated as follows: s, singlet; added to a mixture of EDTA and NADPH generating system,
d, doublet; t, triplet; q, quartet; quint, quintet; sext, sextet; sep, potassium phosphate buffer, and human liver microsomes
septet; m, multiplet; br, broad peak. Infrared (IR) spectra were (H0610, Sekisui Medical Co., Ltd., Tokyo, Japan) deactivated
measured on an IR spectrometer (VALOR-III, JASCO, Tokyo, by phosphoric acid (final volume of 470 µL) prior to add sub-
−1
Japan) and are reported in wavenumbers (cm ). Low- strates to give the final concentrations (ticagrelor-M8-ticlopi-
resolution MS and high-resolution mass spectrometry dine: 10-5-50 µM, 50-25-50 µM, and 100-50-50 µM).After 20
(HRMS) were performed on a mass spectrometer (JMS 700, min centrifugation (18,000g, 4°C), ticagrelor, M8, and ticlopi-
JEOL; FAB mode) with a direct inlet system. Optical rotations dine were extracted from 200 µL of the supernatant using 5

4
Journal of Chemical Research 00(0)
Figure 3. Calibration curves of (a) ticagrelor and its (b) active metabolite M8 in (c) human plasma and those in (d) human liver
microsome.
mL of diethyl ether followed by redissolution with 30 µL of Primary alcohol 4. NH F (65.6 mg, 1.77 mmol) was added
4
the mobile phase, and then injected into the LC/MS system.
to a stirred solution of 3 (153 mg, 0.177 mmol) in MeOH
(4.4 mL). The mixture was stirred at 35°C for 24 h, poured
Tris-TBS ether 3. TBSOTf (0.20 mL, 0.87 mmol) was into half-saturated aqueous NH Cl (20 mL), and extracted
4
added to a stirred solution of 1 (92.3 mg, 0.177 mmol) with EtOAc (10 mL, twice). The combined organic layers
and 2,6-lutidine (0.13 mL, 1.1 mmol) in CH Cl (3.5 mL) were dried over Na SO , filtered, and concentrated in
2
2
2
4
at 0 °C. After stirring for 5 min, the reaction mixture was vacuo. The crude residue was purified by flash chromatog-
warmed to room temperature and stirred for 7 h. The raphy on silica gel (n-hexane-EtOAc 4:1→13:7) to afford 4
reaction was quenched by adding H O (10 mL), and the (130 mg, 98%) as a colorless oil.
2
1
mixture was extracted with EtOAc (10 mL, three times).
H NMR (500 MHz, CDCl ) δ: –0.53 (3H, s), –0.16 (3H,
3
The combined organic layers were dried over Na SO , s), 0.135 (3H, s), 0.143 (3H, s), 0.70 (9H, s), 0.94 (9H, s),
2
4
filtered, and concentrated in vacuo. The crude residue 0.99 (3H, t, J = 7.0 Hz), 1.31–1.42 (2H, m), 1.65–1.80 (2H,
was purified by flash chromatography on silica gel m), 2.17 (1H, td, J = 8.0, 3.0 Hz), 2.21 (1H, dd, J = 15.0,
(
n-hexane-EtOAc 9:1) to afford 3 (152 mg, quant.) as a 6.5 Hz), 2.76 (1H, ddd, J = 17.0, 10.5, 6.0 Hz), 2.82 (1H,
colorless oil.
br s), 3.00–3.18 (3H, m), 3.59 (1H, ddd, J = 10.0, 7.5, 3.0
1
H NMR (500 MHz, CDCl ) δ: –0.51 (3H, s), –0.15 (3H, Hz), 3.66 (1H, ddd, J = 10.0, 5.0, 2.5 Hz), 3.72 (1H, m),
3
s), 0.09 (3H, s), 0.10 (3H, s), 0.14 (6H, s), 0.68 (9H, s), 0.91 3.76–3.86 (2H, m), 4.09 (1H, m), 4.83 (1H, dd, J = 7.5, 3.5
(
9H, s), 0.95 (9H, s), 1.01 (3H, t, J = 7.5 Hz), 1.30-1.41 (2H, Hz), 5.27 (1H, ddd, J = 10.5, 8.0, 7.0 Hz), 6.41 (1H, br s),
m), 1.73 (2H, sext, J = 7.5 Hz), 2.02 (1H, m), 2.15 (1H, ddd, 7.00–7.28 (3H, m). IR (CHCl ) 3425, 3269, 2929, 1612,
3
-1
J = 14.0, 7.5, 1.0 Hz), 2.57 (1H, ddd, J = 17.0, 10.0, 7.5 Hz), 1522, 1323, 1104, 838 cm . [α] –78.5 (c = 0.23, CHCl ).
3
+
3
.00–3.19 (3H, m), 3.55 (1H, dt, J = 10.0, 5.5 Hz), 3.60 (1H, HRMS(FAB+)m/z:[M+H] CalcdforC H F N O SSi
3
5
57
2
6
4
2
dt, J = 10.0, 5.0 Hz), 3.72–3.85 (3H, m), 3.93 (1H, d, J = 3.0 751.3669; Found 751.3674.
Hz), 4.61 (1H, dd, J = 8.5, 3.5 Hz), 5.14 (1H, q, J = 9.0 Hz),
6
.20 (1H, br s), 6.99–7.27 (3H, m). IR (CHCl ) 3268, 2929, Bromide 5. Ph P (57.9 mg, 0.221 mmol) and CBr (95.7
3
3
4
-1
1
613, 1521, 1327, 1098, 836 cm . [α] –66.4 (c = 0.09, mg, 0.289 mmol) were added to a stirred solution of 4 (128
+
CHCl ). HRMS (FAB+) m/z: [M + H] Calcd for mg, 0.170 mmol) in CH Cl (3.4 mL), and the mixture was
3
2
2
C H F N O SSi 865.4533; Found 865.4537.
stirred at room temperature for 1 h. After addition of more
41
71
2
6
4
3

Suzuki et al.
5
Ph P (19.3 mg, 0.0736 mmol) and CBr (31.9 mg, 0.0962
mmol), stirring was continued for 30 min. The reaction
mixture was poured into a mixture of saturated aqueous
1
3
4
H NMR (500 MHz, CD OD) δ: 0.91 (3H, t, J = 7.5
3
Hz), 1.39 (1H, m), 1.49 (1H, m), 1.55–1.68 (2H, m), 2.08–
2
.18 (2H, m), 2.79 (1H, ddd, J = 14.0, 9.5, 7.0 Hz), 2.91
NaHCO (10 mL) and brine (5.0 mL) and extracted with
3
(1H, m), 3.07 (1H, m), 3.15 (1H, m), 4.02 (1H, dd, J = 4.5,
EtOAc (10 mL, twice). The combined organic layers were
2
.5 Hz), 4.13 (1H, ddd, J = 7.5, 5.0, 2.5 Hz), 4.85 (1H, m),
dried over Na SO , filtered, and concentrated in vacuo. The
2
4
5.12 (1H, q, J = 8.0 Hz), 7.05 (1H, m), 7.11–7.25 (2H, m).
crude residue was purified by flash chromatography on 13
C NMR (125 MHz, CD OD) δ: 13.6, 15.9, 23.9, 25.6,
3
silica gel (n-hexane-EtOAc 9:1→17:3) to afford 5 (133 mg,
6%) as a colorless oil.
3
4.1, 34.8, 36.8, 63.0, 75.5, 76.5, 78.5, 116.4, (JF-C = 18.0
Hz), 123.9 (JF-C = 3.5, 7.1 Hz), 124.8, 140.2, 150.1 (JF-C
13.1, 245.3 Hz), 150.7, 151.5 (JF-C = 13.1, 246.5 Hz), 155.8,
72.1. IR (CHCl ) 3286, 1615, 1522, 1326 cm . [α] –48.5
c = 1.36, CHCl ). HRMS (FAB+) m/z: [M + H] Calcd
for C H F N O S 479.1677; Found 479.1680.
9
=
1
H NMR (500 MHz, CDCl ) δ: –0.48 (3H, s), –0.14 (3H,
3
s), 0.15 (6H, s), 0.69 (9H, s), 0.95 (9H, s), 1.01 (3H, t, J = 7.0
−1
1
(
3
Hz), 1.31–1.42 (2H, m), 1.74 (2H, sext, J = 7.0 Hz), 2.17
+
3
(1H, td, J = 8.0, 3.0 Hz), 2.30 (1H, ddd, J = 14.5, 8.0, 2.5
2
1
25
2
6
3
Hz), 2.74 (1H, ddd, J = 14.5, 10.5, 7.0 Hz), 3.03–3.18 (3H,
m), 3.50 (2H, t, J = 6.0 Hz), 3.75 (1H, dt, J = 10.5, 6.5 Hz),
Declaration of conflict of interests
3.80 (1H, dt, J = 7.0, 2.0 Hz), 3.89 (1H, dt, J = 11.0, 6.0
The author(s) declared no potential conflicts of interest with respect
to the research, authorship, and/or publication of this article.
Hz), 4.11 (1H, m), 4.75 (1H, dd, J = 8.5, 4.0 Hz), 5.27 (1H,
dt, J = 9.5, 8.0 Hz), 6.41 (1H, br s), 7.02–7.28 (3H, m). IR
-
1
(
[
8
CHCl ) 3268, 2929, 1613, 1520, 1327, 1117, 837, 775 cm .
3
Funding
+
α] –57.4 (c = 0.20, CHCl ). MS (FAB+) m/z: 813 [MH ],
3
The author(s) disclosed receipt of the following financial support for
the research, authorship, and/or publication of this article: This work
was partially supported by a grant from the Dementia Drug Resource
Development Center, Project S1511016, the Ministry of Education,
Culture, Sports, Science and Technology (MEXT), Japan.
+
+
15 [MH + 2]. HRMS (FAB+) m/z: [M + H] Calcd for
C H BrF N O SSi 813.2825; Found 813.2826.
35
56
2
6
3
2
Secondary alcohol 6. Zn (646 mg, 9.88 mmol) and NH Cl
4
(
26.4 mg, 0.494 mmol) were added to a stirred solution of
5
(80.4 mg, 0.0988 mmol) in IPA-H O (14:1, 13.0 mL), and
2
ORCID iDs
the mixture was stirred at 75°C for 3 h. After cooling to
room temperature, saturated aqueous NH Cl (20 mL) and
H O (5.0 mL) were added, and the mixture was extracted
Hiroshi Kogen
https://orcid.org/0000-0002-5885-5138
https://orcid.org/0000-0002-3778-2638
4
Kenichi Kobayashi
2
with EtOAc (20 mL, twice). The combined organic layers
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2
4
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mg, 90%) as a colorless oil.
3
2
2
1
H NMR (500 MHz, CDCl ) δ: –0.53 (3H, s), –0.21 (3H,
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3
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.99 (1H, dd, J = 15.0, 4.0 Hz), 2.20 (1H, m), 2.91 (1H, m),
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5
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878: 2299–2306.
(
1H, m), 3.97–4.08 (2H, m), 4.90 (1H, dd, J = 7.0, 3.0 Hz),
.97–5.07 (1H, m), 5.27 (1H, ddd, J = 11.5, 7.0, 4.0 Hz),
6. Sillén H, Cook M and Davis P. J Chromatography B 2011;
79: 2315–2322.
. Kalyani L and Rao AL. Int J Pharm 2013; 3: 634–642.
8. Crean B, Finnie C and Crosby A. Drugs R D 2013; 13: 153–157.
. Zhang H, Liu J, Zhang L, et al. Bioorg Med Chem Lett 2012;
2: 3598–3602.
0. Muratake H and Natsume M. Tetrahedron 2006; 62: 7056–
070.
M8 (2). Tetra-n-butylammonium fluoride (TBAF; 1 M 11. Muratake H, Natsume M and Nakai H. Tetrahedron 2006;
solution in tetrahydrofuran (THF), 0.31 mL, 0.31 mmol)
62: 7093–7112.
was added to a stirred solution of 6 (54.4 mg, 0.0769 mmol) 12. Muratake H and Natsume M. Tetrahedron Lett 2002; 43:
8
4
6
1
7
.66 (1H, br s), 6.96–7.15 (3H, m). IR (CHCl ) 3274, 2930,
3
-
1
610, 1521, 1328, 1124, 838 cm . [α] –3.4 (c = 0.32,
9
+
CHCl ). HRMS (FAB+) m/z: [M + H] Calcd for
C H F N O SSi 707.3406; Found 707.3406.
3
2
3
3
53
2
6
3
2
1
7
2
913–2917.
3. Muratake H and Natsume M. Angew Chem Int Ed 2004; 43:
646–4649.
4. Ticagrelor was purchased from Sigma-Aldrich Japan Inc
Tokyo, Japan).
5. Zhang W and Robins MJ. Tetrahedron Lett 1992; 33: 1177–
180.
in THF (2.2 mL), and the mixture was stirred at 30°C for 3
h. After addition of more TBAF (0.15 mL, 0.15 mmol),
stirring was continued for 3 h. The mixture was poured into
1
1
1
4
H O (20 mL), extracted with EtOAc (10 mL, three times).
2
(
The combined organic layers were dried over Na SO , fil-
2
4
tered, and concentrated in vacuo. The crude residue was
purified by flash chromatography on silica gel (CHCl3-
MeOH 19:1) to afford 6 (35.2 mg, 96%) as a colorless oil.
The analytical sample was prepared by crystallization from
1
16. BRILINTA® tablets package insert (version 2) June 2020,
https://www.pmda.go.jp/PmdaSearch/iyakuDetail/Result
DataSetPDF/670227_3399011F1027_1_06 (accessed 12
January 2021).
diisopropyl ether and n-hexane.