Development of a series of (1-benzyl-3-(6-methoxypyrimidin-3-yl)-5-(trifluoromethoxy)-1h-indol-2-yl)methanols as selective protease activated receptor 4 (PAR4) antagonists with in vivo utility and activity against γ-thrombin

Article abstract of DOI:10.1021/acs.jmedchem.6b00928

Here, we describe the development of a series of highly selective PAR4 antagonists with nanomolar potency and selectivity versus PARI, derived from the indole-based 3. Of these, 9j (PAR4 IC₅₀ = 445 nM, PARI response IC₅₀ > 30 μM) and lOh (PAR4 IC₅₀ = 179 nM, PARI response IC₅₀ > 30 μM) maintained an overall favorable in vitro DMPK profile, encouraging rat/mouse in vivo pharmacokinetics (PK) and activity against γ-thrombin.

Full text of DOI:10.1021/acs.jmedchem.6b00928

Brief Article
pubs.acs.org/jmc
Development of a Series of (1-Benzyl-3-(6-methoxypyrimidin-3-yl)-5-
(trifluoromethoxy)‑1H‑indol-2-yl)methanols as Selective Protease
Activated Receptor 4 (PAR4) Antagonists with in Vivo Utility and
Activity Against γ‑Thrombin
Kayla J. Temple,†,‡ Matthew T. Duvernay,† Summer E. Young,† Wandong Wen,∥ Wenjun Wu,∥
Jae G. Maeng,† Anna L. Blobaum,†,‡ Shaun R. Stauffer,†,§ Heidi E. Hamm,*,† and Craig W. Lindsley*,†,‡,§
†Department of Pharmacology, ‡Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University School of Medicine, 9281
Wardley Park Lane, Nashville, Tennessee 37232, United States,
§Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37232, United States
∥College of Science, Northwest Agriculture & Forestry University, Yangling, Shaanxi 712100, China
S
* Supporting Information
ABSTRACT: Here, we describe the development of a series of highly selective PAR4 antagonists with nanomolar potency and
selectivity versus PAR1, derived from the indole-based 3. Of these, 9j (PAR4 IC50 = 445 nM, PAR1 response IC50 > 30 μM) and
10h (PAR4 IC50 = 179 nM, PAR1 response IC50 > 30 μM) maintained an overall favorable in vitro DMPK profile, encouraging
rat/mouse in vivo pharmacokinetics (PK) and activity against γ-thrombin.
Thrombin receptor antagonists (TRAs) as a therapeutic
alternative have been eagerly anticipated in cardiovascular
medicine; vorapaxar is a PAR1 TRA that underwent two phase
III clinical trials, TRA•CER,10 and TRA 2°P.11,12 After safety
review, the TRA•CER trial was halted early and the TRA 2°P
secondary prevention trial was partially discontinued due to an
alarming increase in intracranial hemorrhage.12 As a result,
attention has turned to PAR4 as a potential target on the
platelet that could strike the desired balance between safety and
efficacy.
In addition to delayed engagement, PAR4 displays a more
robust and sustained responses in the platelet,13 suggesting
distinct roles for the receptors; contrary to prior convention,
recent findings suggest PAR4 is more important for the
thrombin-induced procoagulant effect on platelets.13 Unfortu-
nately, tool compounds to probe the role of PAR4 in
hemostasis and thrombosis have been lacking and the field
has relied almost exclusively on PAR4 antibodies.14 Addition-
ally, thrombin activates PARs through cleavage of the
INTRODUCTION
■
Heart attack is the leading cause of death and morbidity in the
western world,1 with up to one-third of patients experiencing a
second event within six months.2 The role of platelet activation
is reflected by the efficacy of antiplatelet reagents (aspirin +
P2Y12 antagonist) in preventing the recurrent thrombosis that
causes heart attack and stroke.3,4 Despite this progress, 10−12%
of patients on currently available antiplatelet therapeutics will
face a second heart attack one year after being treated and a
significant risk of bleeding.5−7 Thus, there is an urgent need for
additional antiplatelet therapeutics to prevent thrombosis
without causing bleeding.
Thrombin is the terminal enzyme of the coagulation cascade
responsible for platelet activation and the generation of fibrin,
essential processes for both thrombosis and hemostasis. Human
platelets respond to thrombin through two G protein-coupled
receptors, PAR1 and PAR4.8 PAR1 contains a hirudin like
thrombin binding domain and therefore displays higher affinity
for thrombin.9 EC50s for PAR1 (∼0.2 nM) and PAR4 (∼5 nM)
differ by more than an order of magnitude, suggesting PAR4 is
engaged after PAR1 as the concentration of thrombin increases
at the local site of injury.
Received: June 21, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jmedchem.6b00928
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Brief Article
a
extracellular domain of the receptor, revealing an encrypted
tethered ligand (TL) that binds intramolecularly to activate the
receptor.15 PARs can also be activated artificially with a
synthetic soluble “activating peptide” (AP) corresponding in
sequence to the naturally derived TL.15,16 Becaue of its covalent
nature, the TL is much more difficult to inhibit than AP, but the
ability to target TL would be a critical attribute of an in vivo
probe molecule.
Scheme 1. Synthesis of PAR4 Antagonist Analogues 8
PAR4 antagonist described to date in the literature (Figure
1), including YD-3 (1),17 SEY-3 (2),18 and ML354 (3),19
a
Reagents and conditions: (a) THF, NBS (1.0 equiv), 0 °C, 1 h, 90−
99%; (b) aryl/heteroarylboronic acid (1.5 equiv), Pd(PPh3)4 (0.1
equiv), Na2CO3 (2.0 equiv), DMF−H2O (4:1), μw, 120 °C, 15 min,
76−95%; (c) K2CO3 (2 equiv), benzyl or alkyl bromide (2 equiv,
DMF, 45 °C, 16 h, 60−90%; (d) LAH, THF, 0 °C, 1 h, 50−85%.
antagonists 8 in yields ranging from 50 to 85%. Initially, we
held the 3-positon constant with a 2-methoxypyridine moiety,
providing analogues 9, as this 3-position substituent showed
modest activity (∼10 μM) in analogues lacking the 5-NO2
group. If a competent NO2 replacement could be identified in
the context of the 2-methoxypyridine moiety, we hoped to see
an increase in PAR4 activity.
Our screening strategy relied on an initial single-point screen
at 10 μM and assessing the maximum % of PAC-1 binding,
followed by full concentration−response curves (CRCs) to
determine PAR4 IC50s. As shown in Table 1, initial SAR for
Figure 1. Structures and properties of reported PAR-4 antagonists.
proved to be selective versus PAR1 but suffer from both poor
DMPK profiles as well as a lack of activity upon γ-thrombin
activation. As we previously reported, the 2-methyl carbinol
moiety within 3 plays an important role in maintaining potency
while reducing lipophilicity, positively impacting free fraction
and plasma stability.19 A unique challenge remaining within 3
was the identification of an appropriate electron-withdrawing
functionality to replace the aromatic nitro group, as limited
success was found in our initial campaign (all analogues ∼10
μM).19 To further address this challenge, we now report the
results from ongoing iterative lead optimization efforts within
the indole chemotype, leading to compounds with comparable
potency and selectivity while improving DMPK profiles and
achieving activity against the TL.
Table 1. Initial SAR of Analogues 9
RESULTS AND DISCUSSION
Chemistry. Our multidimensional optimization approach is
summarized in Figure 2, and PAR4 antagonist activity was
■
PAR4-AP %
a
PAR4-AP PAC-1 IC50
b
compd
R1
R2
max PAC-1
(μM), pIC50 SEM
9a
9b
9c
9d
9e
9f
CF3
H
106.5
53.4
89.0
108.5
80.6
19.7
19.7
95.5
27.1
4.95
>10
>10
OCF3
OCF3
CF3
Ph
2-FPh
>10
3-OMePh
3-FPh
>10
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
>10
2,4-diFPh
3-CF3Ph
3-ClPh
3-BrPh
2-ClPh
5.11, 5.29 0.33
4.82, 5.32 0.04
>10
9g
9h
9i
2.19, 5.66 0.33
0.45, 6.31 0.05
9j
a
Initial single point screens were conducted using 10 μM analogue,
Figure 2. Library optimization strategy for 3 to improve PAR4
antagonist activity, selectivity, and the DMPK profile.
and values indicate the percentage of max PAC-1 binding after PAR4-
AP stimulation of human platelets. Average of three independent
b
determinations.
evaluated through αIIbβ3 activation upon PAR4-AP stimulation
using a PAC-1 binding assay to drive SAR.20 Scheme 1
highlights the four-step route to access analogues 8, surveying
diversity at three positions.20 Here, bromination of commercial
5- or 6-substituted indoles 4 occurred smoothly, affording 3-
bromo congeners 5 in 90−99% yield. Suzuki couplings with
aryl or heteroaryl boronic acids afforded analogues 6 in good
yields, followed by alkylation to deliver derivatives 7. Finally,
LAH reduction of the esters provided putative PAR4
series 9 was steep, with NO2 replacements such as CF3 (9a and
9d) and, in many cases, OCF3 (9b, 9c, 9e, and 9h) being
devoid of PAR4 activity. Moreover, the nature of the benzyl
moiety was critically important for PAR4 inhibitory activity. For
example, 9f, with a 2,4-diF benzyl group, displayed
submicromolar activity (IC50 = 820 nM), highlighting that
the undesirable 5-NO2 moiety could be replaced while
maintaining PAR4 activity. However, the most potent analogue
B
DOI: 10.1021/acs.jmedchem.6b00928
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Journal of Medicinal Chemistry
Brief Article
in this initial series 9 was 9j, a 2-Cl benzyl derivative, with an
IC50 of 445 nM.
methoxy-6-methylimidazo[2,1-b][1,3,4]thiadiazole bicyclic ring
system.21
Having identified optimal benzyl moieties in analogues 9, our
next SAR exploration focused on the identification of alternate
heterocyclces for the 2-methoxypyridine in analogues 10 (Table
2). Here, a more basic, unsubstituted 4-pyridyl analogue, 10a,
Table 2. Initial SAR of Analogues 10
Figure 3. Structures of 10h and 12, PAR4 anatgonists containing the
−methoxy-6-methylimidazo[2,1-b][1,3,4]thiadiazole bicyclic ring sys-
tem.
Finally, we elected to explore the impact of 6- versus 5-
substiuents on the indole core in analogues 11 (Table 3), as
Table 3. Initial SAR of Analogues 11
a
Initial single point screens were conducted using 10 μM analogue and
values indicate the percentage of Max PAC-1 binding after PAR4-AP
b
stimulation of human platelets. Average of three independent
determinations.
a
Initial single point screens were conducted using 10 μM analogue,
and values indicate the percentage of max PAC-1 binding after PAR4-
AP stimulation of human platelets. Average of three independent
determinations.
was inactive as was an unsubstituted pyrimidine 10b.
Interestingly, introduction of either a alkoxy group or a
thiomethyl moiety to the 2-position of the pyrimidine core, as
in 10c−g, restored PAR4 inhibition; however, PAR4 IC50s
varied from 490 nM (10d) to >10 μM (10f). Introduction of
either the 2-methoxy or a 2-thiomethoxy group had significant
b
this had never been assessed. Interestingly, simple alkoxy
moieties (11a−c) did afford modest PAR4 inhibition, but once
again, the 6-OCF3 group, in the context of 2-OMe and 2-SMe
pyrimidines, 11d (IC50 = 930 nM) and 11e (IC50 = 210 nM),
respectively, showed submicromlar potency. Thus, multiple
new PAR4 antagonists resulted that proved worthy of further
profiling.
Molecular Pharmacology. Of the six submicromolar
PAR4 antagonists identified (9f, 9j, 10d, 10h, 11d, and 11e),
all were selective versus the key antitarget PAR1 (IC50s > 30
μM). On the basis of physiochemical and DMPK data (vide
infra), only 9j and 10h were subjected to more detailed
molecular pharmacology profiling. As shown in Figure 4A, 9j
was an equipotent PAR4 antagonist at both assay readouts
(both PAC-1, IC50 = 445 nM, and P-Selectin, IC50 = 435 nM).
impact. Comparison of 10b (IC50 > 10 μM) to 10c (IC50
=
1.49 μM) and 10b (IC50 > 10 μM) to 10e (IC50 = 1.50 μM)
highlights how this small polar moiety enhances PAR4
inhibition. As we were concerned that 2-substituted pyrimidines
with OMe or SMe functionalities may be electrophilic in vivo
and be displaced by biological nucleophiles, we also surveyed a
variety of pyrimidine replacements and found that only a 2-
methoxy-6-methylimidazo[2,1-b][1,3,4]thiadiazole bicyclic ring
system, as in 10h, was active. Moreover, it was the most potent
analogue made in the series (PAR4 IC50 = 179 nM) and on par
with 3. Recently, Bristol-Myers Squibb disclosed a novel
benzofuran-based series of PAR4 antagonists (Figure 3),
exemplified by 12 (BMS986120), that also possesses the 2-
C
DOI: 10.1021/acs.jmedchem.6b00928
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Brief Article
affording comparable inhibition (IC50s of 4.35 μM and 39.4%
maximum inhibition). Thus, while weak partial antagonists
against the TL, 9j and 10h represent the first reported small
molecule PAR4 antagonists to have activity.
DMPK Disposition Attributes. Of the six submicromolar
PAR4 antagonists identified (9f, 9j, 10d, 10h, 11d, and 11e),
9f, 11d, and 11e quickly fell out of contention as in vivo probes
due to either high plasma protein binding (rat and human fu <
0.001) or high predicted in vitro clearance in microsomes that
was equal to or greater than hepatic blood flow (rat CLhep > 60
mL/min/kg and human CLhep > 20 mL/min/kg).20 Table 4
Table 4. DMPK Characterization of Select PAR4
Antagonists
9j
462.8
10d
479.9
10h
Figure 4. Molecular pharmacology profile of 9j and 10h. (A) PAR4-
AP concentration−response curves (n = 3) with equivalent inhibition
of both PAC-1 (IC50 = 445 nM) and P-Selectin (IC50 = 435 nM). (B)
Progressive fold-shift inhibition assay with 9j, showing complex mode
of PAR4 inhibition. (C) PAR4-AP concentration−response curves (n
= 3) with comparable inhibition of both PAC-1 (IC50 = 179 nM) and
P-Selectin (IC50 = 132 nM). (D) Progressive fold-shift inhibition assay
with 10h, also showing a complex mode of PAR4 inhibition. Note:
compound solubility was not an issue at the concentrations evaluated,
i.e., no precipitate was observed.
MW
508.9
69.8
5.1
TPSA
cLogP
54.2
5.2
57.3
5.3
CYP (1A2, 2C9, 2D6, 3A4) 4.2,6.8,
2.9, 6.1, <0.1, >30, 11.2, 1.2,
17.6 >30
IC50 (μM)
0.12, 14
In Vitro PK
rat CLHEP (mL/min/kg)
45.1
58
36.1
human CLHEP (mL/min/kg) 5.3
18.5
1.1
rat PPB (fu)
0.04
0.10
0.001
0.002
0.003
0.004
human PPB (fu)
in Vivo Rat PK (IV Cassette, 0.2 mg/kg, 0−6 h)
A progressive fold-shift inhibition assay/Schild analysis with 9j
(Figure 4B) displayed an unexpected mixed competitive/
noncompetitive mode of PAR4 inhibition with neither a clean,
parallel rightward shift in the concentration−response curve
(competitive) nor a defined decrease in AP-max (non-
competitive). Similarly, 10h displayed comparable PAR4
antagonism (Figure 4C) at both assay readouts (both PAC-1,
IC50 = 179 nM, and P-Selectin, IC50 = 132 nM). However, a
progressive fold-shift inhibition assay/Schild analysis with 10h
(Figure 4D) displayed a similar, complex mode of inhibition yet
clearly more potent. The mixed mechanism of PAR4 inhibition
is intriguing and suggests with further SAR exploration that it
might be possible to develop both competitive and non-
competitive inhibitors of PAR4 and then validate each in vivo.
In addition, both 9j and 10h were devoid of activity at PAR1
(IC50s > 30 μM).
As mentioned earlier, no small molecule PAR4 antagonist
reported to date has demonstrated PAR4 inhibition against the
tethered ligand (TL). As an ideal tool compound that would
possess activity against the TL, we evaluated the ability of both
9j and 10h to inhibit PAR4 when activated by 100 nM γ-
thrombin (Figure 5). While the results with 9j were variable
and weak (IC50 > 10 μM with P-Selectin and IC50 = 9.4 μM,
48.7% max inhibition with PAC-1), efficacy was noted against
the TL. However, 10h proved far more effective against the TL,
t1/2 (h)
2.83
118
21.4
0.25
380
5.82
6.1
45
CLp (mL/min/kg)
Vss (L/kg)
21.1
highlights representative physiochemical and DMPK profiles of
9j, 10d, and 10h. We were concerned about the lability of the
2-SMe pyrimidine moiety in 10d, and this concern was born
out, as 10d showed little fraction unbound in rat or human
plasma and predicted hepatic clearance near blood flow in each
species and superhepatic clearance in vivo. In contrast, the
pyrimidine bioisostere 10h displayed low predicted hepatic
clearance in human microsomes (CLhep = 1.1 mL/min/kg) and
low to moderate in rat (CLhep = 36.1 mL/min/kg). In vivo rat
PK showed a 6.1 h half-life with moderate clearance (CLp = 45
mL/min/kg); however, 10h was highly protein bound in both
species. PAR4 antagonist 9j represented middle ground
between the 10d and 10h. Analogue 9j displayed low predicted
hepatic clearance in human microsomes (CLhep = 5.3 mL/min/
kg) and moderate in rat (CLhep = 45.1 mL/min/kg), and the
best free drug levels to date (human fu = 0.10, rat fu = 0.04). In
vivo rat PK showed a 2.8 h half-life with high total plasma
clearance (CLp = 118 mL/min/kg) and a high volume of
distribution at steady state (Vss = 21.4 L/kg). As mouse is the
species in which the thrombosis models are performed, and in
which PAR4 is expressed on platelets, we performed an IV PK
study (0.2 mg/kg, PEG:saline (1:1) for 9j in five male c57bl/6
mice to mimic the route of administration in these models.
Plasma levels of 9j were collected at a single 40 min time point
and were incredibly consistent (plasma 450 20 ng/mL, 1000
50 nM) and above the in vitro IC50 of 9j. When corrected for
free drug levels, the unbound plasma concentration of 9j was
reduced to ∼38 nM; however, a question to be addressed in
later studies and with more refined PAR4 inhibitors is if efficacy
will be driven by total or free drug levels in plasma.
Figure 5. Molecular pharmacology profile of (A) 9j and (B) 10h
against activation of PAR4 with the tethered ligand, 100 nM γ-
thrombin.
D
DOI: 10.1021/acs.jmedchem.6b00928
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Journal of Medicinal Chemistry
Brief Article
by UV215 nm, UV254 nm, and ELSD analysis on LCMS. LCMS: RT =
1.229 min. MS (ESI+) m/z = 463.0 [M + H]+. 1H NMR (400.1 MHz,
DMSO-d6): 3.84 (s, 1H), 3.93 (s, 3H), 4.51 (s, 2H), 5.71 (s, 2H),
6.33−6.35 (m, 1H), 6.98−7.01 (dd, J = 0.5, 8.5, 1H), 7.16−7.20 (m,
2H), 7.28−7.31 (m, 1H), 7.45−7.48 (m, 2H), 7.54−7.56 (m, 1H),
7.91−7.94 (dd, J = 2.5, 8.5, 1H), 8.36 (d, J = 1.8, 1H). 13C NMR
(DMSO-d6): 162.4, 146.7, 142.7, 140.1, 138.6, 135.0, 134.7, 131.0,
129.3, 128.8, 127.6, 127.5 (q, J = 259.6), 127.3, 126.7, 122.7, 116.2,
112.1, 111.5, 111.2, 110.6, 53.2, 53.0, 48.7. HRMS (TOF, ES+)
C23H19ClF3N2O3 [M + H]+ calcd mass 463.1031, found 463.1035.
CONCLUSION
■
In summary, we report significant advances in the field of small
molecule PAR4 antagonists relative to the known art 1−3, for
which we have addressed the plasma instability of 1 and 2 and
found suitable replacements for the aromatic nitro group of 3.
New PAR4 antagonists described here have also lowered
plasma protein binding (increased fraction unbound, e.g., free
drug principle) as well as both improved in vitro and in vivo
DMPK such that 9j may serve as a first-generation PAR4 in
vivo tool. Importantly, both 9j and 10h are the first reported
small molecules to show PAR4 inhibitory activity against the
native ligand, γ-thrombin, and were inactive against PAR1
(IC50s > 30 μM). Finally, the mode of inhibition for 9j and 10h
is complex, showing a mixed competitive/noncompetitive
mechanism of action, suggesting it might be possible to
develop both competitive and noncompetitive PAR4 antago-
nists. Additional refinements and in vivo studies are in progress
and will be reported in due course.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.6b00928.
Experimental procedures and spectroscopic data for
selected compounds, detailed pharmacology and DMPK
methods (PDF)
Molecular formula strings (CSV)
EXPERIMENTAL SECTION
■
Chemistry. The general chemistry, experimental information, and
syntheses of all other compounds, including 10h, are supplied in the
Supporting Information, as well as pharmacology and DMPK
methods.
AUTHOR INFORMATION
Corresponding Authors
*For H.E.H.: phone, 615-343-9536; fax, 615-343-1084; E-mail,
heidi.hamm@vanderbilt.edu.
*For C.W.L.: phone, 615-322-8700; fax, 615-343-3088; E-mail,
craig.lindsley@vanderbilt.edu.
■
(1-(2-Chlorobenzyl)-3-(6-methoxypyridin-3-yl)-5-(trifluoro-
methoxy)-1H-indol-2-yl)methanol (9j): Ethyl 3-bromo-5-(tri-
fluoromethoxy)-1H-indole-2-carboxylate. To a THF solution (3
mL) of commercially available ethyl 5-(trifluoromethoxy)-1H-indole-
2-carboxylate (300 mg, 1.098 mmol, 1 equiv) was added dropwise the
THF (5 mL) solution of NBS (214.7 mg, 1.206 mmol, 1.1 equiv). The
mixture was stirred at room temperature for 2 h. Then THF was
removed under reduced pressure and the crude product was purified
via ISCO flash chromatography (0−10% ethyl acetate in hexane) (382
mg, 99%). LCMS: RT = 1.276 min, MS (ESI+) m/z = 351.8 [M + H]+.
1H NMR (400.1 MHz, CDCl3): 1.46 (t, J = 7.1, 3H), 4.44−4.50 (q, J =
Author Contributions
K.J.T. and M.T.D. contributed equally
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Dr. Lindsley thanks William K. Warren, Jr. and the William K.
Warren Foundation who funded the William K. Warren, Jr.
Chair in Medicine, and for support of our research. This work
was generously supported by the NIH (NS081669 and
NS082198)
7.1, 2H), 7.23 (d, J = 1.4, 1H), 7.38−7.42 (dd, J = 4.7, 8.9, 1H), 7.53
(d, J = 16.1, 1H), 9.12 (s, 1H).
Ethyl 3-(6-Methoxypyridin-3-yl)-5-(trifluoromethoxy)-1H-in-
dole-2-carboxylate. To a microwave vial was added ethyl 3-bromo-
5-(trifluoromethoxy)-1H-indole-2-carboxylate (300 mg, 0.852 mmol, 1
equiv), 6-methoxy-3-pyridinyl-boronic acid (169.5 mg, 1.107 mmol,
1.3 equiv), Pd(PPh3)4 (98.5 mg, 0.085 mmol, 0.1 equiv), Na2CO3
(180.6 mg, 1.704 mmol, 2 equiv), DMF (4 mL), and water (1 mL).
The vial was sealed and put in the microwave reactor and then heated
for 15 min at 120 °C. On completion, the solvents were passed
through a Celite pad. The solvents were removed under reduced
pressure and pure 3-(6-methoxypyridin-3-yl)-5-(trifluoromethoxyl)-
1H-indole-2-carboxylate was afforded; ISCO flash chromatography
(0−20% ethyl acetate in hexane) (253.2 mg, 78%). LCMS: RT = 1.233
min. MS (ESI+) m/z = 380.8 [M + H]+. 1H NMR (400.1 MHz,
CDCl3): 1.27 (t, J = 7.1, 3H), 4.05 (s, 3H), 4.29−4.34 (q, J = 7.1, 2H),
6.90 (d, J = 8.5, 1H), 7.26−7.27 (m, 1H), 7.43 (s, 1H), 7.45 (d, J = 9.0,
1H), 7.81−7.84 (dd, J = 2.4, 8.5, 1H), 8.37 (d, J = 2.1, 1H), 9.1(s, 1H).
(1-(2-Chlorobenzyl)-3-(6-methoxypyridin-3-yl)-5-(trifluoro-
methoxy)-1H-indol-2-yl)methanol (9j). To a dry vial was added 3-
(6-methoxypyridin-3-yl)-5-(trifluoromethoxyl)-1H-indole-2-carboxy-
late (10 mg, 0.039 mmol, 1 equiv), a series of 2-chlorobenzyl bromide
(1.5 equiv), K2CO3 (5.5 mg, 0.078 mmol, 2 equiv), and DMF. The
mixture was stirred at room temperature for 18 h. The desired indole
esters were purified by the Gilson reverse-phase preparative LC
(CH3CN/H2O/TFA). To the solution of the indole esters in THF
was added LAH (1 M in THF) (2 equiv) at 0 °C, and the solution was
allowed to stir for 1 h. Then, 2 N HCl aqueous (10 μL) was added to
consume the remaining LAH, followed by addition of 10 μL of
saturated NaHCO3 aqueous. The solvents of reaction were removed
on a heated air-blowing block, and then the title compound was
purified by the Gilson reverse-phase preparative LC, with >95% purity
ABBREVIATIONS USED
■
PAR4, selective protease activated receptor 4; CRC, concen-
tration−response-curve; PPB, plasma protein binding
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