Synthesis and biological evaluation of BMS-986120 and its deuterated derivatives as PAR4 antagonists

Article abstract of DOI:10.1016/j.bmc.2018.11.024

BMS-986120 is a PAR4 antagonist that is being investigated as an antiplatelet agent in phase I clinical trial. An improved synthesis of BMS-986120 has been developed. Based on the novel synthetic approach to BMS-986120, a series of deuterated derivatives of BMS-986120 have been synthesized and biologically evaluated to search for more potent antiplatelet agents. The in vitro antiplatelet assay by turbidimetry demonstrated that PC-2 and PC-6 had IC₅₀ values of 6.30 nM and 6.97 nM, respectively, versus BMS-986120 with an IC₅₀ of 7.80 nM. The result of in vitro metabolic stability study showed that all of the deuterated compounds had similar half-life (T_1/2) and intrinsic clearance (Clint) in comparison with BMS-986120. Further probing the metabolic profile of BMS-986120 is worth being conducted.

Full text of DOI:10.1016/j.bmc.2018.11.024

Accepted Manuscript
Synthesis and biological evaluation of BMS-986120 and its deuterated deriva-
tives as PAR4 antagonists
Panpan Chen, Shenhong Ren, Hangyu Song, Cai Chen, Fangjun Chen, Qinglong
Xu, Yi Kong, Hongbin Sun
PII:
S0968-0896(18)31638-9
https://doi.org/10.1016/j.bmc.2018.11.024
BMC 14628
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 September 2018
11 November 2018
15 November 2018
Please cite this article as: Chen, P., Ren, S., Song, H., Chen, C., Chen, F., Xu, Q., Kong, Y., Sun, H., Synthesis and
biological evaluation of BMS-986120 and its deuterated derivatives as PAR4 antagonists, Bioorganic & Medicinal
Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.11.024
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Synthesis and biological evaluation of BMS-
986120 and its deuterated derivatives as
PAR4 antagonists
Leave this area blank for abstract info.
Panpan Chena,1, Shenhong Renb,1,Hangyu Songa, Cai Chena, Fangjun Chena, Qinglong Xua, Yi Kongb* and
Hongbin Suna*
aJiangsu Key Laboratory of Drug Discovery for Metabolic Disease and State Key Laboratory of Natural
Medicines, China Pharmaceutical University, 24 Tongjia Xiang, Nanjing 210009, China .bSchool of Life
Science & Technology, China Pharmaceutical University, 24 Tongjia Xiang, Nanjing 210009, China.

Bioorganic & Medicinal Chemistry
Synthesis and biological evaluation of BMS-986120 and its deuterated derivatives as
PAR4 antagonists
Panpan Chena,1, Shenhong Renb,1,Hangyu Songa, Cai Chena, Fangjun Chena, Qinglong Xua, Yi Kongb,  and
Hongbin Suna, 
aJiangsu Key Laboratory of Drug Discovery for Metabolic Disease and State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24
Tongjia Xiang, Nanjing 210009, China.
bSchool of Life Science & Technology, China Pharmaceutical University, 24 Tongjia Xiang, Nanjing 210009, China.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
BMS-986120 is a PAR4 antagonist that is being investigated as an antiplatelet agent in phase I
clinical trial. An improved synthesis of BMS-986120 has been developed. Based on the novel
synthetic approach to BMS-986120, a series of deuterated derivatives of BMS-986120 have
been synthesized and biologically evaluated to search for more potent antiplatelet agents. The in
vitro antiplatelet assay by turbidimetry demonstrated that PC-2 and PC-6 had IC50 values of
6.30 nM and 6.97 nM, respectively, versus BMS-986120 with an IC50 of 7.80 nM. The result of
in vitro metabolic stability study showed that all of the deuterated compounds had similar half-
life (T1/2) and intrinsic clearance (Clint) in comparison with BMS-986120. Further probing the
metabolic profile of BMS-986120 is worth being conducted.
Available online
Keywords:
PAR4 antagonists
BMS-986120
deuterated derivatives
anti-platelet
2009 Elsevier Ltd. All rights reserved.
metabolic stability
———
 Corresponding author. E-mail: hongbinsun@cpu.edu.cn (H. Sun)
 Corresponding author. E-mail: yikong@cpu.edu.cn (Y. Kong)
1 These authors contributed equally.

receptors (PARs) of platelets.15 As members of the PAR family,
PAR1 and PAR4 are both G protein-coupled receptors inserted in
the cytomembrane of human platelets with a unique self-
activation mechanism following cleavage by thrombin.16 Unlike
PAR1 that has the hirudin like thrombin binding domain, PAR4
is a low-affinity thrombin receptor that can only apperceive
higher concentrations of thrombin while much lower
concentrations of thrombin can be recognized by PAR1.17 With
different kinetics of signal transduction of aggregation in human
platelets, PAR1 and PAR4 are believed to be involved in
different phases of platelet activation. The activation of PAR1
can transmit the signal of hemostasis in the early stage of
bleeding and the activation of PAR4 contributes to stabilize the
1. Introduction
Stroke and myocardial infarction are representative
thromboembolic events that have significant morbidity and
mortality all over the world.1-6 Platelet plays a key role in the
formation of thrombus. At the location of vascular damage,
diverse agonists, such as ADP, collagen, epinephrine and
thrombin can be released. These endogenous substances can
activate platelet rapidly through the corresponding membrane
receptors. Then, the secondary agonists such as ADP and
thromboxane A2 released by the activated platelet can reinforce
the platelet activation again. Finally, platelet activation and
aggregation occur spontaneously through the common central
pathway of the binding of GPIIb/IIIa to fibrinogen resulting in
thrombi.7,8 Thus, antiplatelet therapy prevailed in the clinical
treatment and prevention of atherothrombotic events due to the
role of platelet in the pathological mechanism of thrombus
occurrence and progress.9,10
Figure 1. Structures of BMS-986120 and its deuterated derivatives.
formation of thrombus. This intriguing and valuable mechanism
would make PAR4 to be a promising antiplatelet target with
potent antithrombotic activity with low risk of bleeding.18
The drug combination of asprin and clopidogrel is widely used
as the standard of care in clinical practice.11 Nevertheless,
“aspirin resistance” occurs in the patients with the probability of
5% - 60%, resulting in the increasing recurrence of thrombotic
events in 5 years.12 On the other hand, “clopidogrel resistance”
happens in the CYP2C19 poor metabolizers who also experience
high risk of failure in this fundamental “dual treatment”.13
Furthermore, this drug combination would increase the hazard of
hemorrhage in the elderly patients, especially with
cerebrovascular disease.14 Therefore, agents with strong
antithrombotic activity and low bleeding risk is urgently needed.
Thrombin, a serine protease, is the terminal enzyme of the
coagulation cascade through stimulation of the protease activated
As the first-in-class PAR1 antagonist, Vorapaxar has
undergone two phase III clinical trials. However, the intracranial
bleeding risk hinders it’s clinical usage in patients with a prior
history of stroke or transient ischemic attack.17 On the other hand,
BMS-986120 (Figure 1), an orally active, potent and selective
PAR-4 antagonist has been proven to be safe and well tolerated
by healthy subjects in phase I clinical trials.19 As a part of our
program aiming at the development of novel antiplatelet agents,13
we wished to develop new type of PAR4 antagonists. When we
tried to obtain BMS-986120 as a positive control, we found that
it was extremely difficult to acquire this compound in
satisfactory yields following the literature method.18 Here we
report a practical synthesis of BMS-986120. Moreover, our assay
result of the metabolic stability of BMS-986120 in human liver
microsomes showed that the half-life (T1/2) of this compound was
only about 11.1 min. In fact, the pharmacokinetic studies in a
clinical phase I trial (NCT02439190) demonstrated that the half-
life of BMS-986120 was only 4 hours.20 To overcome the
metabolic instability of BMS-986120, we employed the
deuteration technology21-23 to design and synthesize a series of
deuterated derivatives of BMS-986120 (Figure 1). Biological
evaluation of these deuterated derivatives has also been
conducted.
2. Result and Discussion
2.1. Chemistry
We found that it was not practical to synthesize BMS-986120
following the literature methods.18,24-26 Therefore, we have
developed a new approach to preparation of BMS-986120. As
shown in Scheme 1, formylation of phloroglucinol 1 via
Vilsmeier-Haack reaction provided aldehyde 2 in 80% yield.
Treatment of 2 with MOMCl/Et3N in CH3CN afforded the
desired dually MOM-protected product 3 in 60% yield with high
regioselectivity. Then benzylation of 3 gave compound 4 in 92%
yield. Under reflux condition, the protective group MOM- of 4
could be easily removed by 2 N HCl (aq) to give dihydroxyl
aldehyde 5. Regioselective methylation of 5 was carried out via
Mitsunobu reaction to give aldehyde 6a. Reaction of 6a with 1-
chlorone acetone in acetone at reflux provided 2-acetyl
benzofuran 7a. Treatment of 7a with CuBr2 in ethyl acetate under
refluxing condition provided bromide 8a in 50% yield. Using this
protocol for bromination reaction, low temperature and
sophisticated multistep operations employed by the literature
methods were avoided to obtain the desired product in reasonable
yields. Reaction of 8a with 5-bromo-1,3,4-thiadiazol-2-amine in
isopropanol with heating furnished imidazothiadiazole 9a that

could be used for further reactions without any purification.
Treatment of 9a with sodium methoxide under mild conditions
delivered imidazothiadiazole 10a smoothly. Debenzylation of
10a with BCl3 afforded the key intermediate 11a in quantitative
yield. Alcohol 12a could be synthesized according to the
procedure reported in the patent WO 2013/163279 A1. Initially,
we tried to obtain BMS-986120 through Mitsunobu reaction
between 11a and 12a according to the literature method.18
However, we found that this Mitsunobu reaction was extremely
difficult probably due to the insolubility of 11a in THF. We then
tried to synthesize BMS-986120 through Williamson ether
synthesis. In this regard, bromination of alcohol 12a with PBr3
afforded bromide 13a in 45% yield. A variety of bases (e.g.
K2CO3, NaH, NaOH, t-BuONa) were used for this ether synthesis
with varying yields (6-49%), indicating that a strong base was
necessary for this reaction to occur. Finally, reaction of 11a with
13a in the presence of t-BuONa in DMF afforded BMS-986120
in 49% yield.
reaction of 5 with deuterated methanol gave 6b. Deuterated
imidazothiadiazole 9b could be obtained from 6b following the
procedure as described above (scheme 1). Reaction of 9b with
sodium
methoxide/methanol
or
deuterated
sodium
methoxide/deuterated methanol under ambient temperature
afforded 10b or 10d, respectively. In the similar pattern, reaction
of 9a with deuterated sodium methoxide/deuterated methanol
gave 10c. Debenzylation of 10b-d with BCl3 provided 11b-d in
90-99% yields. Oxidation of alcohol 12a by the Dess-Martin
periodinane afforded aldehyde 14 in 65% yield. Reduction of 14
with sodium borodeuteride in deuterated methanol gave singly -
deuterated alcohol 12b in 74% yield. Bromination of 12b with
PBr3 afforded bromide 13b in 44% yield. Finally, applying
Williamson ether synthesis, reaction of 11a-d with 13a-b in the
presence of t-BuONa in DMF provided all of the deuterated
derivatives PC-1 - PC-7, respectively.
Having established the optimized procedures, an array of
deuterated derivatives of BMS-986120 were synthesized to
examine their
in
vitro
antiplatelet
aggregation
activity and
metabolic
stability
profile.
shown
As
in
Scheme
Mitsunobu
2,
Scheme 1. Reagents and conditions: (a) POCl3, DMF, rt, 80%; (b) MOMCl, Et3N, CH3CN, 0 °C, 60%; (c) BnBr, K2CO3, DMF, rt, 92%; (d) 2 N HCl (aq), MeOH,
reflux, 60%; (e) PPh3, DIAD, MeOH, THF, 0 °C - rt, 60%; (f) 1-chlorone acetone, K2CO3, acetone, reflux, 86%; (g) CuBr2, EtOAc, reflux, 50%; (h) 5-bromo-
1,3,4-thiadiazol-2-amine, isopropanol, sealed tube, 80 °C - 130 °C; (i) MeONa in MeOH (4 M), MeOH/CH2Cl2 (v/v=3:1), rt, 32% (two steps); (j) BCl3,
pentamethylbenzene, dry CH2Cl2, -78 °C, 99%; (k) PBr3, dry CH2Cl2, 0 °C - rt, 45%; (l) t-BuONa, dry DMF, rt, 49%.

2.2. Biological activity assessment
Antiplatelet aggregation assays
on the benzofuran ring of BMS-986120 resulted in improved
potency (PC-2, IC50 = 6.30 nM) in comparison to the parent
compound (IC50 = 7.80 nM). PC-3 with deuteration at both 6-
OCH3 and the C-H bond connected to the phenyl oxygen had a
moderate potency (IC50 = 8.15 nM). Deuteration at 2’-OCH3 on
the imidazothiadiazole ring afforded PC-4 with slightly
decreased potency (IC50 = 8.74 nM). PC-5 with deuteration at the
imidazothiadiazole ring and the C-H bond connected to the
phenyl oxygen was equally potent (IC50 = 7.69 nM) with BMS-
986120 (IC50 = 7.80 nM). Like PC-2, PC-6 possessing both 6-
OCD3 and 2’-OCD3 was more potent (IC50 = 6.97 nM) than
BMS-986120, indicating that deuteration at 6-OCH3 might be
favourable for PAR4 binding and structural modifications at this
position might be considered to improve potency. Further
deuteration of PC-6 at the C-H bond connected to the phenyl
oxygen led to decreased potency (PC-7, IC50 = 9.45 nM), further
implying that structural modifications at this position should be
careful.
Platelet aggregation was measured with a PRECIL LBY-NJ4 4-
channel aggregometer as previously described.27 Briefly, 270 μL
platelets were pipetted into aggregometer cuvettes and were
incubated with 20 μL compounds for 5 min at 37 °C. Saline
served as the vehicle. Platelets aggregation was stimulated with
10 μL PAR4-AP (AYPGKF-NH2) in the presence of magnetic
beads, and the maximum platelet aggregation rate was
determined by a lasting measurement of light transmittance for 5
min. For each compound tested, at least five concentrations were
chosen and the curve of inhibition rate and concentration was
derived, then the IC50 value of this compound was obtained by
using Prism 6.0 (GraphPad, San Diego, CA). The results of
antiplatelet aggregation assays are summarized in Table 1.
Notably, single deuteration at the C-H bond connected to the
Table 1. In vitro antiplatelet activity of the deuterated derivatives of BMS-
986120.
Metabolic stability in human liver microsomes
IC50 (nM)a
7.80±0.16
14.50±1.18
6.30±0.20
8.15±0.36
8.74±0.07
7.69±0.47
6.97±0.24
9.45±0.09
In vitro metabolic study using liver microsomes is the most
common approach to early estimation and prediction of in vivo
metabolic stability of a compound.28 To evaluate the metabolic
stability of the deuterated derivatives of BMS-986120, human
liver microsomes were employed. Half-life (T1/2) and intrinsic
clearance (Clint) of the test compounds are summarized in Table
2. The results show that there is no significant improvement of in
Compound No.
BMS986120
PC-1
PC-2
PC-3
PC-4
PC-5
PC-6
PC-7
vitro metabolic stability for all the deuterated compounds (T1/2
=
11.2 12.5 min) in comparison with BMS-
~
aIn vitro inhibition of PAR4-AP (75 mol)-induced mice platelet aggregation;
values
means from at
least three
are
independent
dose-response
curves.
phenyl
oxygen led to
a significant
decrease in
potency (PC-
1, IC50
=
14.50 nM).
Deuteration
at 6- OCH3

Scheme 2. (a) PPh3, DIAD, MeOH (for 6a) or CD3OD (for 6b), THF, 0 °C - rt, 50% - 60%; (b) 1-chlorone acetone, K2CO3, acetone, reflux, 80% - 86%; (c)
CuBr2, EtOAc, reflux, 43% - 55%; (d) 5-bromo-1,3,4-thiadiazol-2-amine, isopropanol, sealed tube, 80 °C - 130 °C; (e) MeONa in MeOH (4 M), MeOH/CH2Cl2
(v/v=3:1), rt, 32% (two steps) for 10a; or CD3ONa in CD3OD (4 M), CD3OD/CH2Cl2 (v/v=3:1), rt, 32% - 40% (two steps) for 10b-d; (f) BCl3,
pentamethylbenzene, dry CH2Cl2, -78 °C, 90% - 99%; (g) Dess-Martin periodinane, dry CH2Cl2, rt, 65%; (h) NaBD4, CD3OD, 0 °C - rt, 74%; (i) PBr3, dry
CH2Cl2, 0 °C - rt,, 44%; (j) t-BuONa, dry DMF, rt, 22% - 45%.
986120 (T1/2 = 11.1 min). This indicates that deuteration at the
three sites (6-OCH3, 2’- OCH3 and the C-H bond connected to
the phenyl oxygen) might not be suitable for improving
4. Experimental section
All commercially available solvents and reagents were used
without further purification. 1H and 13C NMR spectra were
recorded on an ACF* 300Q Bruker, ACF* 400Q Bruker or
ACF* 500Q Bruker spectrometer in CDCl3, with Me4Si as the
internal reference, or in DMSO-d6. Data for 1H NMR are
recorded as follows: chemical shift (δ, ppm), multiplicity (s =
singlet, d = doublet, t = triplet, m = multiplet or unresolved, br s
= broad singlet, coupling constant (s) in Hz, integration). Low-
and high resolution mass spectra (LRMS and HRMS) were
recorded in electron impact mode. Reactions were monitored by
TLC on silica gel HSGF254 plates (Yantai Jiangyou Company,
China). Column chromatography was carried out on silica gel
(300−400 mesh, Qingdao Ocean Chemical Company, China).
The purity of all final compounds was determined to be ≥95% by
analytical HPLC (equipment: SHIMADZU LC-20AT system
with a SPD-20A UV detector; column, Phecda C18, 4.6 mm ×
250 mm, eluting with 80% MeOH + 20% H2O + 0.1% (for PC-1-
PC-5 and PC-7) or 0.5% (for BMS-986120 and PC-6) TFA,
flow rate 1 mL/min, oven temperature 25 °C, detection UV 254
nm).
Table 2. The metabolic stability of the deuterated derivatives of BMS-986120
in human liver microsomes.
Compound No.
T1/2 (min)a,c
Clint (µL/min/mg
protein)b
BMS-986120
PC-1
11.1±0.33
11.2±0.22
11.6±0.18
11.3±0.18
11.8±0.61
11.2±0.14
11.4±0.18
12.5±0.27
125±3.74
124±2.42
120±1.81
123±1.91
118±6.02
124±1.50
121±1.92
111±2.42
PC-2
PC-3
PC-4
PC-5
PC-6
PC-7
aT1/2 = ln2/k = 0.693/k. bClint (intrinsic clearance) (µL/min/mg protein) =
c
Ln(2)*1000/T1/2/Protein Concentration. The absolute value k of the slope is
measured by the natural logarithm of the percentage of the remaining quantity
of the test compound as a function against time.
metabolic stability.
2,4,6-trihydroxybenzaldehyde (2)29
3. Conclusions
A 100 mL round bottom flask was charged with phloroglucin (3
g, 24 mmol) and DMF (30 mL). After the flask was flushed with
Ar and covered with the rubber plug, POCl3 (2.2 mL, 24 mmol)
was added dropwise under the condition of ice bath. After
completion of adding, the mixture was stirred at room
temperature for 4 h. TLC analysis indicated that the reaction was
completed. After quenching the reaction mixture with ice cold
water (50 mL), the slurry was extracted with CH2Cl2 (20 mL ×
3). The combined organic extracts were washed with 20% LiCl
aqueous solution (10 mL × 2), brine and then dried over
anhydrous sodium sulfate. The solvent was evaporated in vacuo.
The residue was purified by column chromatography on silica gel
In summary, a practical synthesis of PAR4 antagonist BMS-
986120 has been developed, featuring by inexpensive starting
materials, ready scale-up ability, and easy operations. Based on
this novel synthetic approach, a series of deuterated derivatives
of BMS-986120 have been synthesized and biologically
evaluated. The results of antiplatelet aggregation assays showed
that compounds PC-2 and PC-6 with deuteration at 6-OCH3 were
more potent than the parent compound, indicating that structural
modifications at this position might be favourable for PAR4
binding. In vitro metabolic stability studies using human liver
microsomes showed that structural modifications at the three
sites (6-OCH3, 2’- OCH3 and the C-H bond connected to the
phenyl oxygen) might not be suitable for improving metabolic
stability. Further investigation on the metabolic profile and
structural modifications of BMS-986120 are ongoing in our
laboratory.
(petroleum ether/ethyl acetate
trihydroxybenzaldehyde (2.9 g, 80%) as a white solid. H NMR
= 2:1) to provide 2,4,6-
1
(300 MHz, DMSO-d6) δ 11.46 (s, 2H), 10.65 (s, 1H), 9.93 (s,

1H), 5.79 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ 190.9, 167.2,
164.1, 104.6, 94.2. MS (ESI): m/z 153.0 [M-H]-.
mmol) were added to anhydrous THF (10 mL). Diisopropyl
azodicarboxylate (0.40 mL, 2.10 mmol) was added dropwise to
the aforementioned system at 0 oC . The mixture was stirred under
at room temperature for 4 h. TLC analysis indicated that the
reaction was completed. The solvent was evaporated in vacuo.
The residue was purified by column chromatography on silica gel
(petroleum ether/ethyl acetate = 40:1) to provide 2-(benzyloxy)-
6-hydroxy-4-methoxybenzaldehyde (0.31 g, 60%) as a white
solid.1H NMR (300 MHz, CDCl3) δ 12.50 (s, 1H), 10.17 (s, 1H),
7.42-7.35 (m, 5H), 6.03 (d, J = 2.0 Hz, 1H), 5.99 (d, J = 2.0 Hz,
1H), 5.09 (s, 2H), 3.82 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ
191.5, 167.9, 165.1, 162.3, 136.1, 128.5, 128.0, 127.5, 105.6,
93.4, 92.1, 70.0, 55.9. MS (ESI): m/z 281.1 [M+Na]+.
2-hydroxy-4,6-bis(methoxymethoxy)benzaldehyde (3)30
In argon atmosphere, Et3N (0.57 mL, 4.07 mmol) and
MOMCl (4.07 mmol in 5 mL anhydrous acetonitrile) were added
dropwise
successively
to
a
solution
of
2,4,6-
trihydroxybenzaldehyde (0.30 g, 1.95 mmol) in anhydrous
acetonitrile (6 mL) at 0 °C. The mixture was stirred at 0 oC for 80
min. TLC analysis indicated that the reaction was completed.
After quenching the reaction mixture with ice cold water (10
mL), the slurry was extracted with ethyl acetate (5 mL × 3). The
combined organic extracts were washed with brine and then dried
over anhydrous sodium sulfate. The solvent was evaporated in
vacuo. The residue was purified by column chromatography on
silica gel (petroleum ether/ethyl acetate = 10:1) to provide 2-
hydroxy-4,6-bis (methoxymethoxy)benzaldehyde (0.28 g, 60%)
as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 12.17 (s, 1H),
10.10 (s, 1H), 6.29 (d, J = 1.8 Hz, 1H), 6.20 (d, J = 1.8 Hz, 1H),
5.31 (s, 2H), 5.26 (s, 2H), 3.44 (s, 3H), 3.39 (s, 3H). 13C NMR
(75 MHz CDCl3) 192.3, 165.8, 161.4, 119.7, 107.1, 96.8, 94.8,
94.3, 94.3, 56.8, 56.6. MS (ESI): m/z 241.1 [M-H]-.
2-(benzyloxy)-6-hydroxy-4-(methoxy-d3)benzaldehyde (6b)
Following a procedure similar to that described for the
preparation of 6a except that methanol was replaced by
methanol-d4, the title compound was obtained as a white solid in
50% yield, 1H NMR (300 MHz, CDCl3) δ 12.50 (s, 1H), 10.17 (s,
1H), 7.42-7.35 (m, 5H), 6.03 (d, J = 2.0 Hz, 1H), 5.99 (d, J = 2.0
Hz, 1H), 5.09 (s, 2H). 13C NMR (125 MHz, CDCl3) δ 191.8,
168.1, 166.4, 162.6, 135.8, 128.7, 128.4, 127.4, 106.2, 93.3, 91.8,
70.5, 54.9 (hept, J = 21.8 Hz). MS (ESI): m/z 284.1 [M+Na]+.
2-(benzyloxy)-4,6-bis(methoxymethoxy)benzaldehyde (4)31
1-(4-(benzyloxy)-6-methoxybenzofuran-2-yl)ethan-1-one
(7a)18
2-Hydroxy-4,6-bis(methoxymethoxy)benzaldehyde (0.12 g,
0.50 mmol), benzyl bromide (0.07 mL, 0.55 mmol) and
potassium carbonate (0.14 g, 1.0 mmol) were added to DMF (3
mL) and then stirred at room temperature for 4 h. TLC analysis
indicated that the reaction was completed. After quenching the
reaction mixture with ice cold water (5 mL), the slurry was
extracted with ethyl acetate (5 mL × 3). The combined organic
extracts were washed with brine and then dried over anhydrous
sodium sulfate. The solvent was evaporated in vacuo. The
residue was purified by column chromatography on silica gel
(petroleum ether/ethyl acetate = 4:1) to provide 2-(benzyloxy)-
4,6-bis(methoxymethoxy)benzaldehyde (0.15 g, 92%) as a white
solid. 1H NMR (300 MHz, CDCl3) δ 10.46 (s, 1H), 7.48-7.31 (m,
5H), 6.46 (d, J = 2.0 Hz, 1H), 6.38 (d, J = 2.0 Hz, 1H), 5.24 (s,
2H), 5.17 (s, 2H), 5.15 (s, 2H), 3.51 (s, 3H), 3.47 (s, 3H).13C
NMR (75 MHz, CDCl3) δ 187.7, 163.6, 163.0, 161.3, 136.2,
128.7, 128.1, 127.2, 110.9, 96.2, 95.1, 94.9, 94.4, 70.8, 56.7,
56.5. MS (ESI): m/z 355.1 [M+Na]+.
Chlorone acetone (1.88 mL, 23.75 mmol) and anhydrous
potassium carbonate (3.5 g, 25.73 mmol) was added to a solution
of 2-(benzyloxy)-6-hydroxy-4-methoxybenzaldehyde (5.11 g,
19.79 mmol) in acetone (50 mL). The mixture was stirred under
reflux condition for 7 h and then filtered. The filtrate was
concentrated in vacuo. The residue was purified by column
chromatography on silica gel (petroleum ether/ dichloromethane
= 1:2) to provide 1-(4-(benzyloxy)-6-methoxybenzofuran-2-
1
yl)ethan-1-one (5.05 g, 86%) as a light yellow solid. H NMR
(300 MHz, CDCl3) δ 7.56 (d, J = 0.78 Hz, 1H), 7.48-7.36 (m,
5H), 6.65 (dd, J = 1.8, 0.78 Hz, 1H), 6.40 (d, J = 1.8 Hz, 1H),
5.15 (s, 2H), 3.84 (s, 3H), 2.52 (s, 3H). 13C NMR (75 MHz,
CDCl3) δ 186.4, 162.1, 157.3, 153.8, 150.6, 136.4, 128.4, 127.9,
127.7, 112.8, 111.4, 96.6, 88.5, 69.8, 55.9, 25.9. MS (ESI): m/z
295.2 [M-H]-.
1-(4-(benzyloxy)-6-(methoxy-d3)benzofuran-2-yl)ethan-1-
one (7b)
2-(benzyloxy)-4,6-dihydroxybenzaldehyde (5)31
2 N HCl aqueous solution (0.25 mL) was added to a solution of
2-(benzyloxy)-4,6-bis(methoxymethoxy)benzaldehyde (0.18 g,
0.53 mmol) in MeOH (3 mL). The mixture was stirred under
reflux for 3 h. TLC analysis indicated that the reaction was
completed. After quenching the reaction mixture with saturated
sodium bicarbonate solution (5 mL), the slurry was extracted
with ethyl acetate (3 mL × 3). The combined organic extracts
were washed with brine and then dried over anhydrous sodium
sulfate. The solvent was evaporated in vacuo. The residue was
purified by column chromatography on silica gel (petroleum
Following a procedure similar to that described for the
preparation of 7a, the title compound was obtained as a white
1
solid in 80% yield, H NMR (300 MHz, CDCl3) δ 7.57 (s, 1H),
7.48-7.36 (m, 5H), 6.65 (s, 1H), 6.40 (d, J = 1.8 Hz, 1H), 5.15 (s,
2H), 2.53 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 187.1, 162.5,
158.0, 154.3, 151.1, 136.2, 128.7, 128.2, 127.5, 112.3, 112.1,
96.4, 88.2, 70.3, 55.0 (hept, J = 21.8 Hz). 26.0. MS (ESI): m/z
322.1 [M+Na]+.
1-(4-(benzyloxy)-6-methoxybenzofuran-2-yl)-2-
bromoethan-1-one (8a)18
ether/ethyl acetate
= 4:1) to provide 2-(benzyloxy)-4,6-
dihydroxybenzaldehyde (0.077 g, 60%) as a white solid. 1H
NMR (300 MHz, DMSO-d6) δ 12.31 (s, 1H), 10.92 (s, 1H), 10.02
(s, 1H), 7.53-7.30 (m, 6H), 6.08 (d, J = 1.8 Hz, 1H), 5.88 (d, J =
1.6 Hz, 1H), 5.18 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ
190.7, 167.3, 165.2, 162.9, 136.2, 128.5, 128.0, 127.4, 104.9,
95.2, 92.6, 69.8. MS (ESI): m/z 245.0 [M+H]+.
To a suspension of CuBr2 (2.95g,13.24mmol) in ethyl acetate
(50 mL) was added
a solution of 1-(4-(benzyloxy)-6-
methoxybenzofuran-2-yl)ethan-1-one (3.3 g, 11 mmol) in ethyl
acetate (10 mL) dropwise under reflux condition through a
dropping funnel. The mixture was stirred under reflux condition
for 6 h and then filtered. The filtrate was concentrated in vacuo.
The residue was purified by column chromatography on silica gel
(petroleum ether/dichloromethane = 1:1) to provide 1-(4-
(benzyloxy)-6-methoxybenzofuran-2-yl)-2-bromoethan-1-one
(2.1 g, 50%) as a yellow sticky oil. 1H NMR (300 MHz, CDCl3) δ
2-(benzyloxy)-6-hydroxy-4-methoxybenzaldehyde (6a)18
In
argon
atmosphere,
2-(benzyloxy)-4,6-
dihydroxybenzaldehyde (0.50 g, 2.0 mmol), triphenylphosphine
(0.52 g, 2.0 mmol) and absolute methanol (0.073 mL, 1.80

7.72 (s, 1H), 7.47-7.36 (m, 5H), 6.66 (s, 1H), 6.41 (s, 1H), 5.16
Following a procedure similar to that described for the
(s, 2H), 4.35 (s, 2H), 3.86 (s, 3H). MS (ESI): m/z 397.0 [M+Na]+.
preparation of 10a, the title compound was obtained as a white
1
solid in 39% yield. H NMR (300 MHz, CDCl3) δ 7.83 (s, 1H),
1-(4-(benzyloxy)-6-(methoxy-d3)benzofuran-2-yl)-2-
bromoethan-1-one (8b)
7.49-7.33 (m, 5H), 7.09 (s, 1H), 6.69 (s, 1H), 6.40 (s, 1H), 5.19
(s, 2H), 3.83 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 169.4,
159.0, 156.3, 152.5, 149.3, 142.2, 137.0, 135.8, 128.6, 127.9,
127.2, 113.4, 110.3, 98.7, 95.9, 88.8, 70.3, 58.9 (hept, J = 22.5
Hz), 55.8. MS (ESI): m/z 411.1 [M+H]+.
Following a procedure similar to that described for the
preparation of 8a, the title compound was obtained as a white
solid in 43% yield. 1H NMR (300 MHz, CDCl3) δ 7.72 (s, 1H),
7.47-7.34 (m, 5H), 6.65 (s, 1H), 6.40 (s, 1H), 5.16 (s, 2H), 4.35
(s, 2H). MS (ESI): m/z 400.0 [M+Na]+.
6-(4-(benzyloxy)-6-(methoxy-d3)benzofuran-2-yl)-2-
(methoxy-d3)imidazo[2,1-b][1,3,4]thiadiazole (10d)
6-(4-(benzyloxy)-6-methoxybenzofuran-2-yl)-2-
bromoimidazo[2,1-b][1,3,4]thiadiazole (9a)18
Following a procedure similar to that described for the
preparation of 10a, the title compound was obtained as a white
1
solid in 40% yield. H NMR (300 MHz, CDCl3) δ 7.83 (s, 1H),
1-(4-(Benzyloxy)-6-methoxybenzofuran-2-yl)-2-bromoethan-
1-one (1.17 g, 3.12 mmol), 5-bromo-1,3,4-thiadiazol-2-amine
(0.67 g, 3.74 mmol) and 10 mL isopropanol were added to a
pressure flask. After the flask was flushed with Ar and capped,
the mixture was heated under 80 °C for 6 h and then 130 °C for 3
h, TLC analysis indicated that the reaction was completed. The
solvent was evaporated in vacuo. The residue was used for next
step reaction without further purification.
7.49-7.32 (m, 5H), 7.09 (s, 1H), 6.69 (s, 1H), 6.40 (s, 1H), 5.19
(s, 2H). 13C NMR (125 MHz, CDCl3) δ 169.4, 158.9, 156.2,
152.5, 149.3, 142.1, 137.0, 135.7, 128.5, 127.9, 127.2, 113.4,
110.1, 98.6, 95.8, 88.8, 70.2, 58.8 (hept, J = 22.8 Hz), 54.9 (hept,
J = 22.0 Hz). MS (ESI): m/z 436.1 [M+Na]+.
6-methoxy-2-(2-methoxyimidazo[2,1-b][1,3,4]thiadiazol-6-
yl)benzofuran-4-ol (11a)18
6-(4-(benzyloxy)-6-(methoxy-d3)benzofuran-2-yl)-2-
bromoimidazo[2,1-b][1,3,4]thiadiazole (9b)
In argon atmosphere, to a solution of 6-(4-(benzyloxy)-6-
methoxybenzofuran-2-yl)-2-methoxyimidazo[2,1-
b][1,3,4]thiadiazole (0.34 g, 0.83 mmol) and pentamethylbenzene
(0.86 g, 5.81 mmol) in CH2Cl2 (20 mL) was added 1 M BCl3 in
Following a procedure similar to that described for the
preparation of 9a, the crude title compound was obtained.
o
CH2Cl2 (2.18 mL, 2.16 mmol) dropwise at -78 C. The mixture
o
6-(4-(benzyloxy)-6-methoxybenzofuran-2-yl)-2-
methoxyimidazo[2,1-b][1,3,4]thiadiazole (10a)18
was stirred at -78 C for 1 h. TLC analysis indicated that the
reaction was completed. After quenching the reaction mixture
with 5% sodium bicarbonate solution (10 mL), the slurry was
filtered and washed with toluene (5 mL × 3). The filter cake was
dried in vacuum desiccator to provide 6-methoxy-2-(2-
The crude product 6-(4-(benzyloxy)-6-methoxybenzofuran-2-
yl)-2-bromoimidazo [2,1-b][1,3,4]thiadiazole (1.25 g) obtained
from the previous step was added to a mixture solvents of CH2Cl2
(90 mL) and MeOH (30 mL). Sodium methoxide-methanol
solution (2.3 mL, 25 wt.%) was then added to the aforementioned
mixture. The mixture was stirred at room temperature for 1 h.
TLC analysis indicated that the reaction was completed. After
quenching the reaction mixture with saturated ammonium
chloride solution (20 mL), the slurry was extracted with ethyl
acetate (30 mL × 3). The combined organic extracts were washed
with brine and then dried over anhydrous sodium sulfate. The
solvent was evaporated in vacuo. The residue was purified by
column chromatography on silica gel （petroleum ether/ethyl
acetate = 8:1） and recrystallization in petroleum ether/ethyl
methoxyimidazo[2,1-b][1,3,4]thiadiazol-6-yl)benzofuran-
4-ol
(0.25 g, 99%) as a light yellow solid. 1H NMR (300 MHz,
DMSO-d6) δ 9.99 (s, 1H), 8.32 (s, 1H), 6.99 (s, 1H), 6.65 (s, 1H),
6.26 (s, 1H), 4.20 (s, 3H), 3.75 (s, 3H).
4-(4-(bromomethyl)-5-methylthiazol-2-yl)morpholine (13a)
To a solution of (5-methyl-2-morpholinothiazol-4-yl)methanol
(250 mg, 1.16 mmol) in CH2Cl2 (5 mL) was added PBr3 (60 μL,
0.58 mmol in 5 mL CH2Cl2) dropwise at 0 °C. The mixture was
stirred at room temperature for 4 h. After quenching the reaction
mixture with saturated sodium bicarbonate solution (5 mL), the
slurry was extracted with ethyl acetate (5 mL × 3). The combined
organic extracts were washed with brine and then dried over
anhydrous sodium sulfate. The solvent was evaporated in vacuo.
The residue was purified by column chromatography on silica gel
acetate/dichloromethane
to
provide
6-(4-(benzyloxy)-6-
methoxybenzofuran-2-yl)-2-methoxyimidazo[2,1-
b][1,3,4]thiadiazole (0.63 g, two step yield: 32%) as a light
1
yellow solid. H NMR (300 MHz, CDCl3) δ 7.84 (s, 1H), 7.49-
(petroleum ether/ethyl acetate
= 8:1) to provide 4-(4-
7.33 (m, 5H), 7.09 (s, 1H), 6.70 (s, 1H), 6.40 (s, 1H), 5.19 (s,
2H), 4.20 (s, 3H), 3.84 (s, 3H). 13C NMR (75 MHz, CDCl3) δ
169.6, 159.1, 156.4, 152.6, 149.4, 142.3, 137.1, 135.9, 128.7,
128.0, 127.3, 113.6, 110.3, 98.8, 96.0, 88.9, 70.4, 59.8(hept, J =
21.7 Hz), 55.9.
(bromomethyl)-5-methylthiazol-2-yl)morpholine (145 mg, 45%)
1
as a white solid. H NMR (300 MHz, CDCl3) δ 4.39 (s, 2H),
3.80-3.77 (m, 4H), 3.41-3.38 (m, 4H), 2.29 (s, 3H). 13C NMR
(125 MHz, CDCl3) δ 168.3, 143.7, 121.0, 66.3, 48.5, 26.7, 11.3.
MS (ESI): m/z 197.1 [M-Br]+.
6-(4-(benzyloxy)-6-(methoxy-d3)benzofuran-2-yl)-2-
methoxyimidazo[2,1-b][1,3,4]thiadiazole (10b)
5-methyl-2-morpholinothiazole-4-carbaldehyde (14)
Pyridine (45 μL, 0.56 mmol) and Dess-Martin Periodinane
(119 mg, 0.28 mmol) were added successively to a solution of (5-
methyl-2-morpholinothiazol-4-yl) methanol (50 mg, 0.23 mmol)
in CH2Cl2 (5 mL). The mixture was stirred at room temperature
for 1.5 h. TLC analysis indicated that the reaction was
completed. The suspension was filtered. The filter cake was
washed with CH2Cl2 (5 mL × 2). The combined filtrate was
evaporated in vacuo. The residue was purified by column
chromatography on silica gel (petroleum ether/ethyl acetate =
4:1) to provide 5-methyl-2-morpholinothiazole-4-carbaldehyde
Following a procedure similar to that described for the
preparation of 10a, the title compound was obtained as a white
solid in 35% yield. 1H NMR (300 MHz, CDCl3) δ 7.83 (s, 1H),
7.49-7.32 (m, 5H), 7.09 (s, 1H), 6.69 (s, 1H), 6.40 (s, 1H), 5.19
(s, 2H), 4.20 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 169.4,
159.0, 156.2, 152.5, 149.3, 142.2, 137.0, 135.7, 128.6, 127.9,
127.2, 113.4, 110.1, 98.7, 95.9, 88.8, 70.3, 59.7(hept, J = 21.7
Hz), 55.0. MS (ESI): m/z 433.1 [M+Na]+.
6-(4-(benzyloxy)-6-methoxybenzofuran-2-yl)-2-(methoxy-
d3)imidazo[2,1-b][1,3,4]thiadiazole (10c)

1
(32 mg, 65%) as a pale blue solid. H NMR (300 MHz, CDCl3) δ
found: 536.1035. HPLC purity: 98.6%, retention time = 6.71
min.
9.91 (s, 1H), 3.82-3.77 (m, 4H), 3.48-3.44 (m, 4H), 2.65 (s, 3H).
(5-methyl-2-morpholinothiazol-4-yl)methan-d-ol (12b)
4-(4-(((6-methoxy-2-(2-methoxyimidazo[2,1-
b][1,3,4]thiadiazol-6-yl)benzofuran-4-yl)oxy)methyl-d)-5-
methylthiazol-2-yl)morpholine (PC-1)
Sodium borodeuteride (37 mg, 0.88 mmol) was added to a
solution of 5-methyl-2-morpholinothiazole-4-carbaldehyde (187
o
mg, 0.88 mmol) in deuterated methanol (8 mL) at 0 C. Then the
Following a procedure similar to that described for the
preparation of 1 except that 4-(4-(bromomethyl)-5-methylthiazol-
2-yl)morpholine was replaced by 4-(4-(bromomethyl-d)-5-
methylthiazol-2-yl, the title compound was obtained as a white
mixture was stirred at room temperature for 5 h. TLC analysis
indicated that the reaction was completed. After quenching the
reaction mixture with ice cold water (5 mL), the slurry was
extracted with ethyl acetate (5 mL × 3). The combined organic
extracts were washed with brine and then dried over anhydrous
sodium sulfate. The solvent was evaporated in vacuo. The
residue was purified by column chromatography on silica gel
(petroleum ether/ethyl acetate = 1:3) to provide (5-methyl-2-
morpholinothiazol-4-yl)methan-d-ol (140 mg, 74%) as a white
1
solid in 45% yield, H NMR (300 MHz, CDCl3) δ 7.83 (s, 1H),
7.06 (s, 1H), 6.69 (s, 1H), 6.51 (s, 1H), 5.06 (s, 1H), 4.20 (s, 3H),
3.85 (s, 3H), 3.84-3.78 (m, 4H), 3.47-3.38 (m, 4H), 2.37 (s, 3H).
13C NMR (400 MHz, CDCl3) δ 169.5, 168.4, 158.9, 156.2, 152.6,
149.0, 142.9, 142.2, 135.8, 121.2, 113.4, 110.2, 98.9, 96.0, 88.9,
66.3, 64.8 (t, J = 21.7 Hz), 59.8, 55.9, 48.6, 11.2. MS (ESI): m/z
1
+
solid. H NMR (300 MHz, CDCl3) δ 4.45 (s, 1H), 3.81-3.78 (m,
515.1 [M+H]+. ESI-HRMS: calcd. for C23H23DN5O5S2 [M+H]+
4H), 3.41-3.38 (m, 4H), 2.27 (s, 3H).
515.1276, found: 515.1282. HPLC purity: 95.1%, retention time
= 7.51 min.
4-(4-(bromomethyl-d)-5-methylthiazol-2-yl)morpholine
(13b)
4-(4-(((6-(methoxy-d3)-2-(2-methoxyimidazo[2,1-
b][1,3,4]thiadiazol-6-yl)benzofuran-4-yl)oxy)methyl)-5-
methylthiazol-2-yl)morpholine (PC-2)
To a solution of (5-methyl-2-morpholinothiazol-4-yl)methan-
d-ol (140 mg, 0.65 mmol) in CH2Cl2 (5 mL) was added PBr3 (61
μL, 0.65 mmol in 5 mL CH2Cl2) dropwise at 0 °C. The mixture
was stirred at room temperature for 4 h. After quenching the
reaction mixture with saturated sodium bicarbonate solution (5
mL), the slurry was extracted with ethyl acetate (5 mL × 3). The
combined organic extracts were washed with brine and then dried
over anhydrous sodium sulfate. The solvent was evaporated in
vacuo. The residue was purified by column chromatography on
silica gel (petroleum ether/ethyl acetate = 4:1) to provide 4-(4-
(bromomethyl)-5-methylthiazol-2-yl) morpholine (80 mg, 44%)
Following a procedure similar to that described for the
preparation of 1 and 8 except that methanol was replaced by
deuterated methanol, the title compound was obtained as a white
1
solid in 38% yield, H NMR (300 MHz, CDCl3) δ 7.83 (s, 1H),
7.06 (s, 1H), 6.69 (s, 1H), 6.51 (s, 1H), 5.06 (s, 2H), 4.20 (s, 3H),
3.84-3.78 (m, 4H), 3.47-3.38 (m, 4H), 2.37 (s, 3H). MS (ESI):
+
m/z 517.0 [M+H]+. ESI-HRMS: calcd. for C23H21D3N5O5S2
[M+H]+ 517.1402, found: 517.1401. HPLC purity: 95.3%,
retention time = 7.60 min.
1
as a white solid. H NMR (300 MHz, CDCl3) δ 4.38 (s, 1H),
4-(4-(((6-(methoxy-d3)-2-(2-methoxyimidazo[2,1-
b][1,3,4]thiadiazol-6-yl)benzofuran-4-yl)oxy)methyl-d)-5-
methylthiazol-2-yl)morpholine (PC-3)
3.80-3.77 (m, 4H), 3.42-3.38 (m, 4H), 2.29 (s, 3H). 13C NMR
(125 MHz, CDCl3) δ 168.2, 143.4, 120.9, 66.2, 48.5, 26.3 (t, J =
23.3 Hz), 11.2. MS (ESI): m/z 198.1 [M-Br]+.
Following a procedure similar to that described for the
preparation of PC-1 and PC-2, the title compound was obtained
as a white solid in 31% yield, 1H NMR (300 MHz, CDCl3) δ 7.83
(s, 1H), 7.06 (s, 1H), 6.69 (s, 1H), 6.51 (s, 1H), 5.06 (s, 1H), 4.20
(s, 3H), 3.84-3.78 (m, 4H), 3.47-3.38 (m, 4H), 2.37 (s, 3H). MS
(ESI): m/z 518.0 [M+H]+. ESI-HRMS: calcd. for
4-(4-(((6-methoxy-2-(2-methoxyimidazo[2,1-
b][1,3,4]thiadiazol-6-yl)benzofuran-4-yl)oxy)methyl)-5-
methylthiazol-2-yl)morpholine (BMS-986120)18
A 25 mL round bottom flask was charged with 6-methoxy-2-
(2-methoxyimidazo [2,1-b][1,3,4]thiadiazol-6-yl)benzofuran-4-ol
t
+
(50 mg, 0.158 mmol), BuONa (22 mg 0.236 mmol) and DMF (5
C23H20D4N5O5S2 [M+H]+ 518.1464, found: 518.1469. HPLC
mL). After the flask was flushed with Ar and covered with the
rubber plug, a solution of 4-(4-(bromomethyl)-5-methylthiazol-2-
yl)morpholine (65 mg, 0.236 mmol)in DMF (3 mL) was added
dropwise to the above mixture. After completion of adding, the
mixture was stirred at room temperature for 3 h. TLC analysis
indicated that the reaction was completed. After quenching the
reaction mixture with ice cold water (5 mL), the slurry was
diluted with ethyl acetate (15 mL). The separated organic extract
was washed with ice cold water (10 mL), brine and then dried
over anhydrous sodium sulfate. The solvent was evaporated in
vacuo. The residue was purified by column chromatography on
purity: 95.4%, retention time = 7.55 min.
4-(4-(((6-methoxy-2-(2-(methoxy-d3)imidazo[2,1-
b][1,3,4]thiadiazol-6-yl)benzofuran-4-yl)oxy)methyl)-5-
methylthiazol-2-yl)morpholine (PC-4)
Following a procedure similar to that described for the
preparation of 1 and 16 except that methanol was replaced by
deuterated methanol and sodium methoxide was replaced by
deuterated sodium methoxidethe, the title compound was
1
obtained as a white solid in 33% yield, H NMR (300 MHz,
CDCl3) δ 7.83 (s, 1H), 7.06 (s, 1H), 6.69 (s, 1H), 6.51 (s, 1H),
5.06 (s, 2H), 3.85 (s, 3H), 3.84-3.78 (m, 4H), 3.47- 3.38 (m, 4H),
2.37 (s, 3H). MS (ESI): m/z 517.1 [M+H]+. ESI-HRMS: calcd.
for C23H20D3N5O5S2Na+ [M+Na]+ 539.1221, found: 539.1186.
HPLC purity: 98.8%, retention time = 7.58 min.
silica
acetate/triethylamine
methoxy-2-(2-methoxyimidazo[2,1-b]
gel
(petroleum
8:3:0.5:0.2) to provide 4-(4-(((6-
[1,3,4]thiadiazol-6-
ether/dichloromethane/ethyl
=
yl)benzofuran-4-yl)oxy)methyl)-5-methylthiazol-2-
yl)morpholine (38 mg, 47%) as a white solid. 1H NMR (300
MHz, CDCl3) δ 7.82 (s, 1H), 7.05 (s, 1H), 6.69 (d, J = 1.4 Hz,
1H), 6.50 (d, J = 1.4 Hz, 1H), 5.05 (s, 2H), 4.20 (s, 3H), 3.84 (s,
3H), 3.82-3.79 (m, 4H), 3.44-3.40 (m, 4H), 2.36 (s, 3H). 13C
NMR (100 MHz, CDCl3) δ 169.5, 168.4, 158.9, 156.3, 152.7,
149.1, 142.9, 142.2, 135.9, 121.2, 113.5, 110.2, 98.9, 96.0, 89.0,
66.3, 65.1, 59.8, 55.9, 48.6, 11.2.MS (ESI): m/z 514.1 [M+H]+.
ESI-HRMS: calcd. for C23H23N5O5S2Na+ [M+Na]+ 536.1033,
4-(4-(((6-methoxy-2-(2-(methoxy-d3)imidazo[2,1-
b][1,3,4]thiadiazol-6-yl)benzofuran-4-yl)oxy)methyl-d)-5-
methylthiazol-2-yl)morpholine (PC-5)
Following a procedure similar to that described for the
preparation of PC-1 and PC-4, the title compound was obtained
as a white solid in 25% yield, 1H NMR (300 MHz, CDCl3) δ 7.83
(s, 1H), 7.06 (s, 1H), 6.69 (s, 1H), 6.51 (s, 1H), 5.06 (s, 1H), 3.85

(s, 3H), 3.84-3.78 (m, 4H), 3.47-3.38 (m, 4H), 2.37 (s, 3H). MS
(ESI): m/z 518.1 [M+H]+. ESI-HRMS: calcd. for
C23H19D4N5O5S2Na+ [M+Na]+ 540.1284, found: 540.1284. HPLC
purity: 96.3%, retention time = 7.52 min.
The Terfenadine stock solution and Tolbutamide stock solution
were diluted with acetonitrile to 25 ng/mL (Ter) and 50 ng/mL
(Tol) as the stop solution containing mixed internal standard. 3.
Phosphoric acid buffer: 1.83 g K2HPO4·3H2O and 0.28 g
KH2PO4 were dissolved in 200 mL deionized water, diluted to 50
mM, pH = 7.4 and reserved in fridge at 4°C. 4. Preparation of
liver microsomal working solution: The 20 mg/mL liver
microsome solution were freshly diluted to 0.63 mg/mL by
phosphate buffer solution as a working solution just before use.
5. Preparation of NADPH solution: The appropriate amount of
NADPH was freshly dissolved in phosphate buffer to 5 mM just
before use.
4-(4-(((6-(methoxy-d3)-2-(2-(methoxy-d3)imidazo[2,1-
b][1,3,4]thiadiazol-6-yl)benzofuran-4-yl)oxy)methyl)-5-
methylthiazol-2-yl)morpholine (PC-6)
Following a procedure similar to that described for the
preparation of PC-2 and PC-5, the title compound was obtained
as a white solid in 34% yield, 1H NMR (300 MHz, CDCl3) δ 7.83
(s, 1H), 7.06 (s, 1H), 6.69 (s, 1H), 6.51 (s, 1H), 5.06 (s, 2H),
3.84-3.78 (m, 4H), 3.47-3.38 (m, 4H), 2.37 (s, 3H). MS (ESI):
Experimental procedure: Microsomal incubation system was
prepared by mixing liver microsome working fluid (0.63 mg/mL,
197.5 µL) and the test compound or reference compound
working fluid (100 µM, 2.5 µL). The mixture was preincubated
in triplicate at 37 °C in the shaking water bath for 1 h. Then,
NADPH (5 mM, 50 µL) solution was added. The final
concentration of the test compound and liver microsomes in the
mixture was 1 M and 0.5 mg/mL, respectively. At time 0, 5, 15,
30, and 60 min, stop solution was added to terminate the
incubation. After quenching, the mixture was centrifuged and
filtered. The supernatant was then diluted and 5 µL was injected
+
m/z 520.1 [M+H]+. ESI-HRMS: calcd. for C23H18D6N5O5S2
[M+H]+ 520.1590, found: 520.1591. HPLC purity: 97.5%,
retention time = 6.46 min.
4-(4-(((6-(methoxy-d3)-2-(2-(methoxy-d3)imidazo[2,1-
b][1,3,4]thiadiazol-6-yl)benzofuran-4-yl)oxy)methyl-d)-5-
methylthiazol-2-yl)morpholine (PC-7)
Following a procedure similar to that described for the
preparation of PC-1 and PC-6, the title compound was obtained
1
as a gray solid in 22% yield, H NMR (300 MHz, CDCl3) δ 7.83
(s, 1H), 7.06 (s, 1H), 6.69 (s, 1H), 6.51 (s, 1H), 5.06 (s, 1H),
3.84-3.78 (m, 4H), 3.47-3.38 (m, 4H), 2.37 (s, 3H). MS (ESI):
m/z 521.1 [M+H]+. ESI-HRMS: calcd. for C23H16D7N5O5S2Na+
[M+Na]+ 543.1472, found: 543.1470. HPLC purity: 97.1%,
retention time = 7.23 min.
into
liquid
chromatography–mass
spectrometer/mass
spectrometer to detect the remaining amount of the test
compound. Percentage of the test compound remaining was
calculated by dividing the peak area of the test compound
remaining at each time point by the peak area of the test
compound at time 0 min and multiplying by 100%. Natural
logarithm of percentage of the test compound remaining against
time could be plotted. Half-life (T1/2) and intrinsic clearance
(Clint) of the test compound could be calculated based on the
absolute value of slope of the above plot and the following
formula: T1/2 = ln2/k = 0.693/k; Clint (intrinsic clearance)
(µL/min/mg protein) = Ln(2)*1000/T1/2/protein concentration.
Assessment of antiplatelet activity
Animal: Institute of Cancer Research (ICR) mice (male, 18-22
g) were purchased from Nanjing Qinglongshan Animal Center
(Nanjing, Jiangsu, China). The animals were acclimatized for at
least 1 day prior to use.
Preparation of mice Gel-filtered platelets: Gel-filtered
platelets were prepared as described previously.32nticoagulated
blood was taken out from the Abdominal aorta of chloral
hydrate-anesthetized mice by using 2.5 mL syringe, and
centrifugated at 200×g for 10 min at room temperature to get
platelet-rich plasma (PRP). Filtered the PRP by using the column
packed with Sepharose 2B beads and eluted with modified
Tyrode`s buffer(5.56 mM glucose, 137 Mm NaCl, 2.7 mM KCl,
2.56 mM NaH2PO4•2H2O, 20 mM HEPES, 137 mM MgCl2•
6H2O, 0.1% BSA, pH = 7.4）1.5 mL tubes. Collected the high
concentration Gel-filtered platelets, and adjusted to 3 × 108
platelets/mL using modified Tyrode’s buffer.
LC-MS/MS analysis method: Chromatographic column:
Waters HSS C18 column 2.5 μ (2.1 mm * 50 mm); Mobile
phase: A phase: H2O with 0.1% TFA, B phase: ACN with 0.1%
TFA, Flow rate: 0.6 mL/min; Column temperature: 30 °C;
Injector temperature: 4 °C; Injected sample volume: 5 μL;
Running time: 2 min.
Conflicts of interest
There are no conflicts to declare.
Acknowledgments
Metabolic stability of the compounds in human liver
microsomes
Financial support from National Natural Science Foundation
of China (grants 81573299, 81473080 and 81373303) is
gratefully acknowledged. This project is also supported by the
“111 Project” from the Ministry of Education of China, the State
Administration of Foreign Expert Affairs of China (No. B17047).
Materials: Midazolam maleate (Art. No. 171250, Lot No.
200401), as a reference compound, was purchased from National
Institute for the Control of Pharmaceutical and Biological
Products. Human liver microsomes (150-donor with mixed
gender) were from IVT (Lot No. IQF; 20 mg/mL).
References and notes
Solution preparation: 1. Preparation of stock solutions and
working fluids of the test substances and the reference
compound: The test substance was dissolved in DMSO and
prepared into 10 mM stock solution. Appropriate amount of the
reference compound was used to prepare 10 mM stock solution
in DMSO (-20 °C fridge and the validity period was 3 months.).
The stock solution was diluted with MeOH to 100 M as a
working fluid. 2. Preparation of stop solution containing internal
standard: Terfenadine and Tolbutamide were respectively
weighed for about 1 mg to prepare 1 mg/mL stock solution in
DMSO (-20 °C in fridge and the validity period was 3 months.).
1. Ågesen, F. N.; Risgaard, B.; Zachariasardóttir, S.; Jabbari, R.;
Lynge, T. H.; Ingemann-Hansen, O.; Ottesen, G. L.; Thomsen, J.
L.; Haunsø, S.; Krieger, D. W.; Winkel B. G.; Tfelt-Hansen, J. Int.
J. Stroke. 2018, 13, 285-291.
2. Benjamin, E. J.; Blaha, M. J.; Chiuve, S. E.; Cushman, M.; Das,
R. Deo, S. R.; de Ferranti, S. D.; Floyd, J.; Fornage, M.; Gillespie,
C.; Isasi, C. R.; Jiménez, M. C.; Jordan, L. C.; Judd, S. E.;
Lackland, D.; Lichtman, J. H.; Lisabeth, L.; Liu, S.; Longenecker,
C. T.; Mackey, R. H.; Matsushita, K.; Mozaffarian, D.; Mussolino,
M. E.; Nasir, K.; Neumar, R. W.; Palaniappan, L.; Pandey, D. K.;

Thiagarajan, R. R.; Reeves, M. J.; Ritchey, M.; Rodriguez, C. J.;
Roth, G. A.; Rosamond, W. D.; Sasson, C.; Towfighi, A.; Tsao, C.
W.; Turner, M. B.; Virani, S. S.; Voeks, J. H.; Willey, J. Z.;
Wilkins, J. T.; Wu, J. H. Y.; Alger, H. M.; Wong S. S.; Muntner,
P. Circulation, 2017, 135, e146-e603.
19. Wilson, S. J.; Ismat, F. A.; Wang, Z.; Cerra, M.; Narayan, H.;
Raftis, J.; Gray, T. J.; Connell, S.; Garonzik, S.; Ma, X.; Yang, J.;
Newby, D. E.; Arterioscl. Throm. Vas. biol., 2018, 38, 448-456.
20. Han, X.; Nieman, M. T. Arterioscl. Throm. Vas. biol., 2018, 38,
287-289.
3. Feigin, V. L.; Forouzanfar, M. H.; Krishnamurthi, R.; Mensah, G.
A.; Connor, M.; Bennett, D. A.; Moran, A. E.; Sacco, R. L.;
Anderson, L.; Truelsen, T.; O'Donnell, M.; Venketasubramanian,
N.; Barker-Collo, S.; Lawes, C. M. M.; Wang, W.; Shinohara, Y.;
Witt, E.; Ezzati, M.; Naghavi M.; Murray, C. Lancet, 2014, 383,
245-255.
4. Lusis, A. J. Nature, 2000, 407, 233-241.
5. Nakanishi, N.; Tatara, K.; Nishina, M.; Nakajima, K.; Naramura,
H.; Yoneda, H.; J. Epidemiol., 1998, 8, 195-202.
6. Chen, W.; Gao, R.; Zhu, M.; Wang, W.; Wang, Y.; Wu, Z.; Li, H.;
Gu, D.; Yang, Y.; Zheng, Z.; Jiang, L.; Hu, S. Chin. Circ. J., 2018,
33, 1-8.
7. Furie, B.; Furie, B. C. New Engl. J. Med., 2008, 359, 938-949.
8. Ruggeri, Z. M. Nat. Med., 2002, 8, 1227-1234.
9. Wang, Y.; Ouyang, Y.; Liu, B.; Ma, X.; Ding, R. Thromb. Res.,
2018, 166, 28-36.
21. Timmins, G. S. Expert. Opin. Ther. Pat., 2014, 24, 1067-1075.
22. Blake, M. I.; Crespi, H. L.; Katz, J. J. J. Pharm. Sci., 1975, 64,
367-391.
23. Howland, R. H. J. Psychosoc. Nurs. Ment. Health. Serv., 2015, 53,
13-16.
24. Banville, J.; Remillard, R.; Ruediger, EH.; Deon, D.H.; Gagnon,
M.; Dube, L.; Guy, J.; Priestly, E.S.; Posy, S.; Maxwell, B. D.;
Wong, P.C.; WO2013163279A1, 2013.
25. Priestly, E.S.; Posy, S.L.; Tremblay, F.T.; Martel, A.; Marinier, A.
WO2013163241A1, 2013.
26. Lawrence, M.R.; Miller, M.M.; Seiffert, D.A.; Posy, S.L.; Wong,
P.C.; Banville, J.; Ruediger, E.H.; Deon, D.H.; Martel, A.;
Tremblay, F.; Guy, J.; Lavallee J-F.; Gagnon, M.
WO2013163244A1, 2013.
27. Chen, M.; Ye, X.; Ming, X.; Chen, Y.; Wang, Y.; Su, X.; Kong,
Y. Sci. Rep-UK, 2015, 5.
10. Mousa, S. A. In Drug Discovery and Evaluation:
Pharmacological Assays, Hock, F. J., Ed.; Springer International
Publishing, Cham, 2016, pp. 827-831.
28. Baranczewski, P.; Stańczak, A.; Sundberg, K.; Svensson, R.;
Wallin, A.; Jansson, J.; Garberg, P.; Postlind, H. Pharmacol. Rep.,
2006, 58, 453-472.
11. Varenhorst, C.; Jensevik, K.; Jernberg, T.; Sundström, A.;
Hasvold, P.; Held, C.; Lagerqvist, B.; James, S. Eur. Heart. J.,
2014, 35, 969-978.
29. Baldisserotto, A.; Demurtas, M.; Lampronti, I.; Moi, D.; Balboni,
G.; Vertuani, S.; Manfredini, S.; Onnis, V. Eur. J. Med. Chem.,
2018, 156, 118-125.
12. Michos, E. D.; Ardehali, R.; Blumenthal, R. S.; Lange R. A.;
Ardehali, H. Mayo. Clin. Proc., 2006, 81, 518-526.
13. Shan, J. Q.; Zhang, B. Y.; Zhu, Y. Q.; Jiao, B.; Zheng, W. Y.; Qi,
X.W.; Gong, Y. C.; Yuan, F.; Lv, F. S.; Sun. H. B. J. Med. Chem.
2012, 55, 3342–3352.
30. Schultze, C.; Schmidt, B. J. Org. Chem., 2018, 83, 5210-5224.
31. Huang, Y.; Sumida, M.; Kumazoe, M.; Sugihara, K.; Suemasu,
Y.; Yamada, S.; Yamashita, S.; Miyakawa, J.; Takahashi, T.;
Tanaka, H.; Fujimura Y.; Tachibana, H. Chem. Commun., 2017,
53, 1941-1944.
14. Gerasimos, S.; Konstantinos, M.; Evangelos, O.; Manolis, V.;
Georgia, V.; Alexandros, B.; Nikolaos, P.; Theodore, G. P.;
Thodoris, Z.; Angeliki, P.; Athanasios, G. P.; Christodoulos, S.;
Dimitris, T. Curr. Pharm. Design., 2016, 22, 4583-4595.
15. Coughlin, S. R. Nature, 2000, 407, 258-264.
32. Su, X.; Su, W.; He, Z.; Ming, X.; Kong, Y. J. Cardiovasc. Pharm.,
2015, 66, 254-260.
Supplementary Material
16. French Shauna, L.; Hamilton Justin, R. Brit. J. Pharmacol., 2016,
173, 2952-2965.
Supplementary data associated with this article can be found,
in the on line version, at
17. Temple, K. J.; Duvernay, M. T.; Young, S. E.; Wen, W.; Wu, W.;
Maeng, J. G.; Blobaum, A. L.; Stauffer, S. R.; Hamm H. E.;
Lindsley, C. W. J. Med. Chem., 2016, 59, 7690-7695.
18. Wong, P. C.; Seiffert, D.; Bird, J. E.; Watson, C. A.; Bostwick, J.
S.; Giancarli, M.; Allegretto, N.; Hua, J.; Harden, D.; Guay, J.;
Callejo, M.; Miller, M. M.; Lawrence, R. M.; Banville, J.; Guy, J.;
Maxwell, B. D.; Priestley, E. S.; Marinier, A.; Wexler, R. R.;
Bouvier, M.; Gordon, D. A.; Schumacher W. A.; Yang, J. Sci.
Trans. Med., 2017, 9, eaaf5294.
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