Characterization of Protease-Activated Receptor (PAR) ligands: Parmodulins are reversible allosteric inhibitors of PAR1-driven calcium mobilization in endothelial cells

Article abstract of DOI:10.1016/j.bmc.2018.04.016

Several classes of ligands for Protease-Activated Receptors (PARs) have shown impressive anti-inflammatory and cytoprotective activities, including PAR2 antagonists and the PAR1-targeting parmodulins. In order to support medicinal chemistry studies with hundreds of compounds and to perform detailed mode-of-action studies, it became important to develop a reliable PAR assay that is operational with endothelial cells, which mediate the cytoprotective effects of interest. We report a detailed protocol for an intracellular calcium mobilization assay with adherent endothelial cells in multiwell plates that was used to study a number of known and new PAR1 and PAR2 ligands, including an alkynylated version of the PAR1 antagonist RWJ-58259 that is suitable for the preparation of tagged or conjugate compounds. Using the cell line EA.hy926, it was necessary to perform media exchanges with automated liquid handling equipment in order to obtain optimal and reproducible antagonist concentration-response curves. The assay is also suitable for study of PAR2 ligands; a peptide antagonist reported by Fairlie was synthesized and found to inhibit PAR2 in a manner consistent with reports using epithelial cells. The assay was used to confirm that vorapaxar acts as an irreversible antagonist of PAR1 in endothelium, and parmodulin 2 (ML161) and the related parmodulin RR-90 were found to inhibit PAR1 reversibly, in a manner consistent with negative allosteric modulation.

Full text of DOI:10.1016/j.bmc.2018.04.016

Accepted Manuscript
Characterization of Protease-Activated Receptor (PAR) ligands: Parmodulins
are reversible allosteric inhibitors of PAR1-driven calcium mobilization in en-
dothelial cells
Disha M. Gandhi, Mark W. Majewski, Ricardo Rosas, Kaitlin Kentala, Trevor
J. Foster, Eric Greve, Chris Dockendorff
PII:
S0968-0896(18)30011-7
https://doi.org/10.1016/j.bmc.2018.04.016
BMC 14299
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 January 2018
28 March 2018
5 April 2018
Please cite this article as: Gandhi, D.M., Majewski, M.W., Rosas, R., Kentala, K., Foster, T.J., Greve, E.,
Dockendorff, C., Characterization of Protease-Activated Receptor (PAR) ligands: Parmodulins are reversible
allosteric inhibitors of PAR1-driven calcium mobilization in endothelial cells, Bioorganic & Medicinal
Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.04.016
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Characterization of Protease-Activated
Receptor (PAR) ligands: Parmodulins are
reversible allosteric inhibitors of PAR1-
driven calcium mobilization in endothelial
cells
Leave this area blank for abstract info.
Disha M. Gandhi, Mark W. Majewski, Ricardo Rosas, Jr., Kaitlin Kentala, Trevor J. Foster, Eric Greve, and
Chris Dockendorff*
Department of Chemistry, Marquette University, P.O. Box 1881, Milwaukee, WI, 53201-1881, USA

Bioorganic & Medicinal Chemistry
Characterization of Protease-Activated Receptor (PAR) ligands: Parmodulins
are reversible allosteric inhibitors of PAR1-driven calcium mobilization in
endothelial cells
Disha M. Gandhi,† Mark W. Majewski,† Ricardo Rosas, Jr., Kaitlin Kentala, Trevor J. Foster, Eric Greve,
and Chris Dockendorff*
Department of Chemistry, Marquette University, P.O. Box 1881, Milwaukee, WI, 53201-1881, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Several classes of ligands for Protease-Activated Receptors (PARs) have shown impressive anti-
inflammatory and cytoprotective activities, including PAR2 antagonists and the PAR1-targeting
parmodulins. In order to support medicinal chemistry studies with hundreds of compounds and
to perform detailed mode-of-action studies, it became important to develop a reliable PAR assay
that is operational with endothelial cells, which mediate the cytoprotective effects of interest.
We report a detailed protocol for an intracellular calcium mobilization assay with adherent
endothelial cells in multiwell plates that was used to study a number of known and new PAR1
and PAR2 ligands, including an alkynylated version of the PAR1 antagonist RWJ-58259 that is
suitable for the preparation of tagged or conjugate compounds. Using the cell line EA.hy926, it
was necessary to perform media exchanges with automated liquid handling equipment in order
to obtain optimal and reproducible antagonist concentration–response curves. The assay is also
suitable for study of PAR2 ligands; a peptide antagonist reported by Fairlie was synthesized and
found to inhibit PAR2 in a manner consistent with reports using epithelial cells. The assay was
used to confirm that vorapaxar acts as an irreversible antagonist of PAR1 in endothelium, and
parmodulin 2 (ML161) and the related parmodulin RR-90 were found to inhibit PAR1
reversibly, in a manner consistent with negative allosteric modulation.
Available online
Keywords:
Protease-Activated Receptor
PAR1
PAR2
GPCR
Calcium mobilization
Parmodulin
ML161
Vorapaxar
RWJ-58259
2018 Elsevier Ltd. All rights reserved.
Negative allosteric modulator
PAR4 antagonist BMS-9861209 were recently in Phase 1 clinical
trials.
1. Introduction
Protease-activated receptors (PARs)1-3 comprise a category of
class A G-protein coupled receptors (GPCRs) that are activated
by extracellular proteases via cleavage of their N-termini,
revealing tethered ligands that serve to self-activate the receptors.
This process is illustrated with one of 4 PAR subtypes, PAR1, in
Figure 1.4,5 PARs mediate a wide range of biological effects in
numerous tissues, in particular platelet activation and
inflammation in vascular tissues. One consequence of the
intramolecular mode of activation of PARs is that competitive
antagonism can be very challenging due to the high effective
concentration of the tethered ligands. Nonetheless, highly potent
antagonists have been discovered and progressed to clinical
studies as platelet inhibitors: the PAR1 antagonist vorapaxar6 is
FDA-approved for treatment of coronary artery disease in certain
Figure 1. Thrombin-mediated PAR1 signaling
patients, and the pepducin7 PAR1 antagonist PZ-1288 and the
———
† These authors contributed equally to the manuscript
* Corresponding author. christopher.dockendorff@mu.edu; Tel.: +1 4142881617

F
F
F
F
O
Bn
NHFmoc
HO2C
NHFmoc
BnNH2
piperidine
1) piperidine, MeCN
N
O
O
H
H
H
F
2)
Bn
N
EDC•HCl, HOBt
DCM, 98%
Bn
N
F
6% in MeCN
50%
N
NHFmoc
N
NH2
H
H
NHBoc
NHBoc
O
O
1
HO2
C
NHFmoc
2
NHBoc
NHBoc
EDC•HCl, HOBt
MeCN/DCM
3
4
49% over 2 steps
Cl
O
N
Br
FeCl3•6H2O
N
1) pyrrolidine
AcOH, 30 min.
H2N-NH2
Cl
1) NaNO2
N
N
N
Cl
N
Cs2CO3
THF
charcoal, reflux
MeOH, 68%
O2N
N
N
H
2) 6 M HCl
>95%
2) Na(OAc)3BH
DCE:DMF (10:1)
70%
O2N
O2N
N
H
O2N
H
78%
5
7
8
6
Cl
F
F
•X
N
F
F
N
N
4M HCl
Dioxane, –5°C
1) Triphosgene
DMAP, –5°C, 8 min.
O
O
N
O
O
Cl
H
H
N
N
Cl
N
Cl
Bn
N
H2N
Bn
N
N
N
N
N
N
N
N
N
58%, over 2 steps
2) 4, –5°C, 30 min.
H
H
H
H
H
H
O
O
–5°C to rt, 30 min.
Cl
9
10
NH2•X
NHBoc
Cl
Cl
F
11a, X = HCl (RWJ-58259)
11b, X = HCO2H
•HCO2H
F
N
O
OH
O
O
H
N
N
N
Cl
Bn
N
N
N
EDC•HCl, HOBt,
DIPEA, 47%
H
H
H
O
HN
O
Cl
12
DG-207
Scheme 1. Synthesis of PAR1 antagonist RWJ-58259 and alkynylated analog DG-207
As part of our program exploring alternative approaches to the
modulation of PAR-mediated signaling,10 we have been
exploring the medicinal chemistry and pharmacology of the
parmodulins, a series of small molecules that act as biased
ligands at PAR1 via a postulated intracellular, allosteric
mechanism (Figure 1).11-13 Parmodulins were initially of interest
to us as antithrombotic agents, due to their biased antagonism of
platelet activation: they inhibit Gαq-driven granule secretion
without substantially affecting Gα12/13 signaling and platelet
shape change. More recently, our lead compound ML161 (also
known as parmodulin 2 or PM2) has shown significant promise
for its cytoprotective effects in cellular and animal studies, results
that are complementary to extensive literature documenting that
the PAR-targeting protease Activated Protein C (aPC) can
promote cytoprotective effects via PAR1 and/or PAR2,14-25
studies that have been reviewed in recent years.26-31 In contrast to
vorapaxar, ML161 did not block the cytoprotective effects of
aPC in endothelial cells exposed to the cytotoxic agents
staurosporine and TNF-,13 and more recently it was found to
promote on its own a cytoprotective program via PAR1 in the
same cells.32 ML161 also significantly decreased cardiac infarct
size in a murine model of myocardial ischemia-reperfusion
injury.33 We hypothesize that the biased, cytoprotective signaling
of the parmodulins may be due to their presumed allosteric site of
action on PAR1 in endothelial cells (Figure 1).
reported by several investigators, including Zlokovic35 and
Kuckleburg,36 but these assays would not be ideal for rapid
assays of large numbers of compounds. Protocols of PAR-
mediated calcium mobilization using plate readers or FLIPR
apparatus have been reported in epithelial cells with PAR2
ligands,37 and with PAR1 ligands in human dermal microvascular
endothelial cells (HMEC-1),38 but we are not aware of reports
with detailed protocols using adherent endothelial cell lines that
were used to generate concentration-responses of PAR1 ligands.
Fairlie has, however, reported concentration-responses of PAR2
agonists and antagonists with HUVEC and provided assay
protocols.39
2. Results
2.1 Synthesis of tool compounds
We commenced our studies with the syntheses of several tool
compounds and functionalized analogs to support future mode-
of-action studies. Our interest in building modified and/or labeled
versions of various PAR1 antagonists led us to synthesize the
tethered ligand peptidomimetic RWJ-58259 (Scheme 1),40
versions of which had been under preclinical investigation as
antiplatelet agents.41-43 Consideration of published structure-
activity relationship (SAR) data with platelets suggested that
modification of the primary amine could be tolerated. To this
end, we synthesized RWJ-58259 using protocols slightly
modified from the original report.40 The commercially-available
amino acid 1 was coupled sequentially at the acid and amine
moieties using standard amide coupling (EDC) and Fmoc-
removal conditions (piperidine) to yield dipeptide 4. According
to the protocols reported by Zhang and Maryanoff,40 6-
nitroindole 5 underwent nitrosation and rearrangement to yield 3-
formylindazole 6, followed by reductive amination and N-
alkylation, providing nitroindazole 8. Several nitro reduction
protocols gave high-yielding reactions, including stoichiometric
SnCl2, but these often gave minor byproducts that proved
difficult to remove later in the synthesis. The reported protocol
using hydrazine and catalytic FeCl3 gave the desired 7-
aminoindazole 9 in high purity after column chromatography.
The synthesis of urea 10 via an intermediate 4-
nitrophenylcarbamate proved to be low-yielding in our hands,
Despite the promising in vivo properties of the parmodulins
and other PAR ligands, our efforts to study their effects in
endothelial cells30 have been hindered by the lack of a reported
medium throughput assay protocol. Our present cell lines of
interest are Human Umbilical Vein Endothelial Cells (HUVEC)
and EA.hy926,34 a hybrid endothelial cell line well-established
for in vitro measurements of cytoprotective and barrier-protective
effects.21,22 In order to quantify the signaling bias of the
parmodulins and other PAR ligands in these cells, our first
objective was to identify a reliable method for the measurement
of intracellular calcium mobilization (iCa2+), which can be driven
by agonism of PARs through the G-protein Gq. Single cell
imaging of PAR-driven intracellular calcium mobilization in
endothelial cells with different calcium-binding dyes has been

which was also consistent with the original report and a
subsequent report from Herranz, who found improved yields
using triphosgene and propylene oxide as an acid scavenger.44
High-purity urea 10 was obtained by us in acceptable yields via a
slow addition of triphosgene to 9, followed by the addition of
dipeptide 4, as reported by Caddick.45 Boc removal using HCl
provided RWJ-58259 (11), which was profiled with several other
PAR1 antagonists (vide infra). Additionally, in order to use RWJ-
58259 as a scaffold for the attachment of e.g. fluorophores for
future studies, we wished to expand the range of applicable
conjugation reactions. We therefore explored the attachment of
several different alkyne building blocks for use in copper-
catalyzed alkyne/azide cycloadditions (CuAAC).46 These efforts
were hampered by the poor solubility of several of the
intermediates (2–4, 10, 12) involved in the synthesis of RWJ-
58259. However, we successfully coupled 11 with 4-pentynoic
acid using EDC/HOBt in DMF (Scheme 1) to yield alkyne DG-
207 (12).
endothelium. For this purpose, we selected Fairlie’s peptidic
PAR2 antagonist (termed by us TJF-5, Figure 2), which was
previously prepared via solid-phase peptide synthesis.48 Since we
wished to potentially access larger quantities of this compound,
we investigated a solution-phase synthesis of TJF-5, which
proceeded uneventfully using Boc-protected building blocks and
HATU as coupling agent (Scheme 4).
O
1) 10% Pd/C
MeOH
O
O
Cl
H2N
NO2
N
H
22
NO2
N
NH2
•HCl
pyridine
DCM, >90%
2) 0.9 M HCl
Et2O, 80%
H
13
23
O
NH2
O
N3
1) NaNO2
conc. HCl
1) oxalyl chloride
DMF
O
O
N3
HO
HO
N
N
H
H
2) NaOAc, NaN3
H2O, 85%
2) 23, DIPEA
DCM, 80%
24
26 DG-5
25
Scheme 3. Synthesis of azido analog DG-5
OMe
O
OMe
O
O
OMe
H2
N
H
H
H2N
N
TFA
N
N
H
Boc
OH
Boc
N
H
DCM
HATU, DIPEA
DMF, 51%
O
H
H
H
27
28
29
N
O
O
F
NH
N
O
O
O
EtO
EtO
OMe
O
H
O
OMe
OH
H
O
OMe
Me
H
H
TFA
HNBoc
Boc
N
N
N
N
N
H
H2N
N
DCM
HATU, DIPEA
DMF
27% Over 2 steps
H
H
O
N
O
atopaxar
30
F
31
•HCl
F
N
N
OH
O
F
O
O
OMe
H
O
O
vorapaxar
N
O
H
N
N
N
N
N
Cl
HATU, DIPEA
DMF
16% over 2 steps
H
H
Ph
N
N
N
H
O
O
N
H
H
O
32 (TJF-5, Fairlie’s PAR2 antagonist)
NH2•HCl
RWJ-58259
Cl
O
N
O
OMe
Ph
N
N
H
Scheme 4. Solution-phase synthesis of TJF-5 (Fairlie’s PAR2
antagonist)
N
N
H
H
O
O
N
Q94
Cl
TJF-5, Fairlie’s PAR2 antagonist
2.2 Initial studies of calcium mobilization with adherent
endothelial cells
Figure 2. Previously-reported PAR antagonists used in these
studies
Our assay development studies commenced with the testing of
several options for measuring calcium mobilization in adherent
endothelial cells. The transformed endothelial cell line
EA.hy926,34 widely used in other PAR-related studies, could be
grown reliably in tissue culture-treated 96-well plates. Cell
seeding of approximately 25,000 cells/well gave confluent
monolayers within 48 h. The calcium binding fluorescent dye
Fluo-4, dosed as its acetoxymethyl ester (Fluo-4/AM), generated
adequate emission signals at 525 nm for us to reliably measure
PAR activation using peptide agonists for human PAR1 and
PAR2, which correspond to their tethered ligands: TFLLRN-NH2
(PAR1 selective), SLIGKV-NH2 (PAR2 selective), and
SFLLRN-NH2 (PAR1/2 selective). Resting cells containing Fluo-
4 are depicted in Figure 3A, prior to agonist addition. As with
other calcium-binding dyes, the acetoxymethyl ester permeates
the cells and is cleaved by esterases, retaining it in the cell.49
Anionic transporters are blocked by the concurrent application of
probenecid, which slows the efflux of Fluo-4, according to
standard protocols. However, we did observe the efflux of Fluo-4
and decreased calcium responses over time. Assays were
performed on 16 wells at a time (2 columns of a 96 well plate)
In addition to RWJ-58259 and the commercially available
PAR1 antagonists vorapaxar6 and atopaxar47 (Figure 2), we
aimed to profile a selection of our parmodulin analogs (Table 1).
“Western” analogs (ML161, CJD-125, RR-90, EMG-21, EMG-
23, and RR-10) were prepared as described previously,12 or via
the protocol summarized in Scheme 2. The alkyne RR-10 and the
aryl azide DG-5 (Scheme 3) were also prepared to investigate the
possibility of generating photoaffinity ligands (via aryl azide to
nitrene conversion), with alkyne handles for pulldown
experiments.
O
Br
1) SnCl2•2H2O
conc. HCl
EtOH, 55 ºC
O
Br
1)Cl
O2N
N
O2N
NH2
O
H
2) DIPEA
DCM, >90%
2) 0.9M HCl
Et2O, 80%
13
14
Br
Conditions
(A or B or C)
Br
O
O
H2N
N
H
R1
N
H
N
H
•HCl
15
16–20
Condition A: R1-CO2H (1 eq.), EDC-HCl (1.2 eq),
HOBt (1.2 eq.), DIPEA (2.1 eq.)
R1
=
16 =
(ML161)
17 =
Condition B: R1-CO2H (1 eq.), oxalyl chloride (1.2
eq), DMF (0.01 eq.), DIPEA (2 eq.)
using
a
plate reader (PerkinElmer EnSpire®); increased
19 =
18 =
20 =
Condition C: R1-COCl (1 eq.), DIPEA (2 eq.)
throughput could be easily obtained using an instrument imaging
all wells concurrently (e.g. FLIPR).50,51 A representative trace
of response versus time is shown in Figure 3B. The difference
between the maximal emission and the average of the baseline
emission at 525 nm was used for all response measurements,
which were normalized to the agonist response with no inhibitor
present (only vehicle, set to 100%).
Scheme 2. General synthesis of parmodulins with west side
modifications
Our additional interest in the modulation of PAR2 required us
to obtain a reliable PAR2 antagonist for confirming that our
assay could also be applicable to measuring PAR2 activation in

A.
B.
C.
D.
Figure 3. A: Adherent Ea.hy926 cells containing Fluo-4 dye. Cells were imaged in a clear-bottom, black wall 96-well plate,
using an Evos Fl inverted microscope (20X) with GFP setting. B: Representative Fluo-4–Ca2+ emission curve (525 nm) in
response to TFLLRN-NH2 (3.16 M). C: Concentration–response curve of a typical (unsuccessful) assay with the PAR1
antagonist ML161 and 3.16 M TFLLRN-NH2 before optimization (manual media exchanges), n =3. D: Representative
concentration–response curve with the PAR1 antagonist ML161 and 5 M TFLLRN-NH2 after assay optimization (automated
liquid handling).
A.
B.
Positive Control
Negative Control
Z’-factor
TFLLRN-NH2
(5 µM)
ML161 (10 µM) +
TFLLRN-NH2 (5 µM)
Vehicle (10% DMSO-
HBSS/HEPES)
TFLLRN-NH2
(5 µM)
0.720
Agonist Assay (n=12)
0.618
Antagonist Assay
(n=12)
Figure 4. A: Calcium mobilization responses with PAR1 agonist 5 μM TFLLRN-NH2 (column A) plus optional PAR1
antagonists 10 μM ML161 (column C) or 0.316 μM vorapaxar (column D). B: Table of calculated Z’-factors.

We were pleased to see acceptable signal/noise and measured
concentration-response curves with the PAR1 and PAR2
agonists, however we were disappointed to see inconsistent
results when measuring antagonist activities. Deviations in
gave EC50s of 4.8 μM and 5.8 μM, respectively. Using 5 μM
TFLLRN-NH2 as agonist, concentration-response curves were
also measured with the PAR1 antagonists vorapaxar (Figure 6A,
IC50 = 0.032 μM), atopaxar (Figure 6B, IC50 = 0.033 μM), and
RWJ-58259 (Figure 6C, IC50 = 0.020 μM). Of special interest to
us was the performance of our alkynylated analog of RWJ-
58259, DG-207 (12). It was 15-fold less potent in this assay than
RWJ-58259 (Figure 6D), but with an IC50 of 0.30 μM it is still
anticipated to have sufficient potency for our planned conjugate
molecules.
normalized emission levels were unacceptably high;
a
representative plot is given for ML161 in Figure 3C. Adverse
effects on the endothelial cells (retraction) were observed when
incubated in media containing DMSO required to dissolve the
antagonists (1% final concentration). Retraction was minimized
when working with 0.1–0.2% DMSO, but high deviations were
still observed in many, but not all, assays. We reasoned that the
low aqueous solubility of many PAR antagonists, including
vorapaxar and ML161, may cause inconsistent results,
particularly since turbid suspensions were observed at high
concentrations. Several excipients were tested in order to keep
compounds in solution at the highest concentrations (10–100
µM), including Tween-20, Tween-80, Triton X-100, BSA, and
Pluronic F-127 (0.01 to 0.1%). Pluronic F-127 (up to 0.05%) was
effective at solubilizing compounds and did not have observable
toxic effects on the cells. Assays proceeded with this excipient in
the antagonist solutions, but as previously, inconsistent
antagonist concentration-response curves were still obtained.
Interestingly, the high deviations themselves were inconsistent,
and it became clear that certain wells contained poorly-
responding cells. We reasoned that inconsistent pipetting
technique, particularly for introduction of the agonists
immediately before scanning in the plate reader, could be the
source of inconsistencies, but this also did not prove to be the
case after extensive experimentation, including the use of higher
volumes.
A. TFLLRN-NH2
B. SLIGKV-NH2
EC50 = 4.77 μM
EC50 = 5.79 μM
Figure 5. PAR agonist concentration–response with
EA.hy926 cells: A) TFLLRN-NH2 (PAR1)-mediated iCa2+
mobilization; B) SLIGKV-NH2 (PAR2)-mediated iCa2+
A.Vorapaxar
B. Atopaxar
We next examined the process of loading the dye (Fluo-
4/AM) into the cells. Omitting the probenecid did not have a
productive effect. Buffer containing the Fluo-4/AM was added to
the cells, then incubated for 0.5–1 h. Longer incubation times
with the dye were not helpful and simply led to lower maximum
signals, with the Fluo-4 slowly leaking from the cells, which can
be problematic for longer experiments. An additional variable
was the manner in which the media was changed. Our initial
protocol used simple “flicking” of the plates by hand into paper
towels to remove media or buffer from the plates, followed by
multichannel pipetting of fresh buffer during liquid exchanges.
Ensuring that the pipet tips did not touch the bottom of the plate
did not improve the quality of the data. Finally, we reasoned that
the force of the flicking was inconsistent and could disturb the
cells in unpredictable ways, even though wells were confirmed to
contain evenly distributed cells that had not obviously detached.
Our inconsistent results disappeared after we gained access to an
automated liquid handler (Beckman-Coulter BioMek 3000) for
media changes. Antagonist concentration-response curves were
then consistently obtained with low deviations (e.g. Figure 3D).
To determine the suitability of our assay for screening hundreds
of compounds, we performed experiments to determine the signal
window of the assay by measuring the average response and
standard deviation for the PAR1 agonist TFLLRN-NH2 (5 M)
(designated as the positive control, Figure 4, column A), and
comparing to the responses obtained after the same agonist was
added to cells treated with the PAR1 antagonists ML161 (10 M)
(column C) or vorapaxar (0.316 M) (column D). The resulting
Z’ factors were >0.5, and the assay is thus considered to be
highly suitable for high-throughput screening.52
IC50 = 0.032 μM
IC50 = 0.033 μM
D. DG-207 (12)
C. RWJ-58259 (11a)
IC50 = 0.020 μM
IC50 = 0.30 μM
Figure 6. Concentration-response for PAR1 antagonists in the
TFLLRN-NH2-mediated iCa2+ mobilization assay (5 µM) with
EA.hy926 cells: A) vorapaxar, B) atopaxar, (C) RWJ-58259
D) DG-207
Concentration-responses were also obtained with several
representative parmodulins (Figure 7). With ML161, optimal
signal/noise was obtained with 5 μM TFLLRN-NH2; at this
concentration, an IC50 of 0.85 μM was measured (Figure 7A). As
previously reported in an alternative P-selectin expression assay
with platelets,12 the analog with one carbon less on the aliphatic
chain on the western side (EMG-21) was active but less potent
than ML161 (Figure 7B, IC50 = 3.3 μM), and the analog with one
additional carbon (EMG-23) was poorly active (Table 1, entry 8).
Also in line with our previous results in platelets, the aniline RR-
90 was similar in potency to ML161 (Figure 7C, IC50 = 0.97
μM). However, the branched aliphatic amide CJD-125, an analog
of ML161 with improved plasma stability and equipotency in the
platelet P-selectin assay,12 was less potent than ML161 and did
not give a good concentration-response with EA.hy926 cells
(Table 1, entry 6). Of note is the fact that efficacy (maximal
inhibition) of the parmodulins does not appear to be quite as high
as with the presumably orthosteric inhibitors of Figure 6, though
2.3 Concentration-responses of PAR1 and PAR2 antagonists
with endothelial cells
With a reliable calcium mobilization assay in hand, we
proceeded to determine concentration-responses with the peptide
agonists for PAR1 (Figure 5A) and PAR2 (Figure 5B), which

this comparison is complicated by concentration–limiting
solubilities of some of the parmodulins.
perhaps because human platelets do not express PAR2. The
ability of compounds to inhibit the action of the PAR1 agonist
TFLLRN-NH2 is summarized in Figure 9A. The more potent
PAR1 antagonists described previously vorapaxar, atopaxar, and
RWJ-58259 were used at concentrations of 0.316 μM, and as
expected largely inhibit the activation of the EA.hy926 cells and
mobilization of calcium. The parmodulins ML161 (IC50 = 0.85
μM) and RR-90 (IC50 = 0.85 μM) also significantly inhibited the
PAR1 agonist TFLLRN-NH2, which can be compared to the
potencies we previously reported with these compounds in a P-
selectin assay using human platelets (0.26 and 0.34 μM,
respectively).12 Q94 (Figure 2) is a commercially available
benzimidazole that was reported to selectively inhibit PAR1
Gαq-mediated calcium mobilization in HMEC-1 endothelial
cells.38 It did not act as an antagonist in this assay using
EA.hy926 cells, and actually boosted the response to TFLLRN-
NH2. As expected, the PAR2 antagonist TJF-5 also had no
Our collaborators in the Flaumenhaft lab have performed
experiments with chimeric PAR receptors which support our
hypothesis that the parmodulins act at an intracellular location,13
which is highly unusual for small molecule GPCR ligands. In
order to identify a precise binding site on PAR1 for the
parmodulins, we were interested in generating analogs of ML161
suitable for photoaffinity studies. To this end, we prepared
analogs possessing aryl azides (for photochemical generation of
reactive nitrenes), and alkynes for conjugation or pulldown
experiments. Unfortunately, the aryl azide DG-5 (Figure 7D;
Table 1, entry 10) had only modest activity. The western alkyne
RR-10 (entry 11) also had only modest inhibition of PAR1.
Results with all PAR1 ligands are summarized in Table 1.
A. ML161 (16)
B. EMG-21 (18)
measurable
effect
on TFLLRN-NH2-induced
calcium
mobilization. None of the PAR1 ligands had any measurable
effect on PAR2-mediated calcium mobilization with SLIGKV-
NH2 (Figure 9B), confirming that they have excellent selectivity
for PAR1 over PAR2 in EA.hy926 endothelial cells. These cells
did not respond to high concentrations (up to 100 μM) of the
PAR4 agonist AYPGFK-NH2.
IC50 = 3.31 μM
D. DG-5 (26)
IC50 = 0.85 μM
C. RR-90 (21)
Next, we evaluated if the PAR1 ligands inhibited PAR1 in
EA.hy926 cells in an allosteric, non-competitive manner; we
have previously shown that ML161 acts as a non-competitive
inhibitor of SFLLRN-NH2 in a platelet P-selectin assay,12 and it
does not significantly inhibit binding of [3H] high-affinity
thrombin receptor activating peptide ([3H]TRAP) to platelet
membranes.13 We measured the concentration-responses of
TFLLRN-NH2 in the presence of increasing concentrations of
PAR1 antagonists (Figure 10). Both the presumed orthosteric
antagonists (vorapaxar and RWJ-58529) and the parmodulins
(ML161 and RR-90) induced a dose-dependent decrease in
efficacy of the PAR1 agonist that is consistent with either a lack
of reversibility OR action as a negative allosteric modulator.
However, it should be noted that the fitted concentration-
response curves of TFLLRN-NH2 in the presence of RWJ-58259
suggest that competitive inhibition might also be possible,
whereby the curves are shifted rightward, but the agonist
concentrations are not high enough to detect maximum response.
The nearly identical ligand RWJ-56110 (with an indole instead of
indazole) is a competitive inhibitor of binding of the radiolabeled
PAR1 activating peptide [3H]S-(p-F-Phe)-homoarginine-L-
homoarginine-KY-NH2.41 Vorapaxar6 is well known to act as a
poorly reversible inhibitor of PAR1, with a very low off rate and
extended half-life, likely due to its binding site on the top of
PAR1 whereby it becomes covered by extracellular loop 2 (ECL-
2), as seen in the crystal structure reported by Coughlin and
Kobilka.53
IC50 = 94 μM
IC50 = 0.97 μM
Figure 7. Concentration-response for parmodulins (ML161
analogs) in the TFLLRN-NH2-mediated (5 µM) iCa2+
mobilization assay
To differentiate between irreversible (presumably orthosteric)
inhibition and negative allosteric modulation, we performed
“wash” studies whereby the endothelial cells were treated with
inhibitor, then washed twice with buffer prior to addition of the
agonist and measurement of intracellular calcium levels (Figure
11). The activity of vorapaxar was completely retained after
washing the cells, confirming that its binding is poorly reversible
in endothelial cells. RWJ-58259 and the parmodulins ML161,
RR-90, and CJD-125 lost most or all of their activities after
washing. Therefore, we conclude that the parmodulins, at least
ML161 and RR-90, act as negative allosteric modulators of
TFLLRN-NH2 at PAR1.
IC50 = 1.66 μM
Figure 8. Concentration-response for the PAR2 antagonist
TJF5 (Fairlie’s PAR2 antagonist, 32) in the SLIGKV-NH2-
mediated (5 μM) iCa2+ mobilization assay.
Our assay also proved to be reliable for measuring the
inhibition of calcium mobilization with PAR2 antagonists: the
peptide inhibitor TJF-5 inhibited the action of the PAR2
activating peptide SLIGKV-NH2 (5 μM) with an IC50 of 1.7 μM
(Figure 8).
2.4 Selectivity and mode-of-action studies
The activity of the parmodulins and other PAR1 antagonists at
PAR2 has not previously been reported, to our knowledge,

A. PAR1 activity
B. PAR2 activity
aML161, RR90, Q94, TJF5 (Fairlie’s PAR2 antagonist) were used at 10 µM; Vorapaxar, Atopaxar, and RWJ-58259 were at 0.316 µM. TFLLRN-NH2 and
SLIGKV-NH2 were used at 3.16 µM; Vehicle (V) = 10% DMSO–HBSS/HEPES
Figure 9. Selectivity data of antagonists in PAR1 (TFLLRN-NH2)- and PAR2 (SLIGKV-NH2)-driven iCa2+ mobilizationa
A.
B.
C.
D.
Figure 10. iCa2+ concentration-response of the PAR1 agonist TFLLRN-NH2 in the presence of increasing concentrations of (A)
vorapaxar, (B) RWJ 58259, (C) ML161, (D) RR-90

Figure 11. Reversibility studies of the PAR1 antagonists vorapaxar, RWJ-58259, ML161, and RR-90 in the iCa2+ assay. Cells
containing antagonists were optionally washed with buffer prior to treatment with the PAR1 agonist TFLLRN-NH2 (5 μM).
Vorapaxar and RWJ-58259 were used at 0.316 µM; ML161, CJD125, RR-90 were used at 10 µM.
Table 1. Inhibition data of select antagonists in the PAR1-mediated iCa2+ mobilization assay
% Inhibition &
Log IC50
± Std. Error
Entry
ID#
Structure
a
IC50
95%;
0.032 µM
-7.5
±0.084
1
Vorapaxar
94%;
0.033 µM
-7.5
±0.10
2
3
Atopaxar
94%;
0.020 µM
RWJ-58259
-7.7
±0.065
11a
F
F
N
O
O
H
N
DG-207
76%;
0.29 µM
-6.5
±0.074
Cl
Bn
N
4
5
N
N
N
N
H
H
12
H
O
O
HN
O
Cl
O
O
Br
Br
-6.7
±0.099
ML161
16
82%;
0.85 µM
N
H
N
H
O
O
CJD-125
74%;
6
7
8
N.D.
N
H
N
H
3.16 µMb
17
O
Br
EMG-21
68%;
3.31 µM
-5.5
±0.26
N
N
H
H
18
O
O
Br
EMG-23
49%
N.D.
N
N
H
H
19
O
Br
RR-90
21
77%;
0.97 µM
-6.0
±0.19
9
N
H
•HCl
N
H
O
O
N3
DG-5
26
77%;
94 µM
-4.0
±2.9
10
N
H
N
H
O
O
Br
RR-10
20
79%;
81 µM
-4.0
±5.1
11
N
N
H
H
a %Inhibition of PAR1 antagonists in presence of 5 µM TFLLRN-NH2. N.D. = not determined
b Estimated IC50 (incomplete curve at high concentration)

3. Conclusions
4.2 LC/MS characterization methods
A protocol for a medium-throughput assay measuring calcium
mobilization in adherent endothelial cells was developed and
used to measure concentration-responses of a variety of PAR1
and PAR2 ligands. An alkyne-tethered version of the PAR1
antagonist RWJ-58259 (DG-207) was synthesized. It was a less
potent inhibitor of the PAR1 agonist TFLLRN-NH2 than the
parent compound, but still anticipated to have adequate potency
for future use in conjugate compounds. In contrast, several
alkyne- and azide-containing analogs of the parmodulin ML161
were found to have moderate (micromolar inhibition) or low
potency in this assay. A selection of PAR1 and PAR2 ligands
were profiled in further detail, and were found to be highly
selective for PAR1 or PAR2. The parmodulins ML161 and RR-
90 were found to decrease the efficacy of TFLLRN-NH2 in a
dose-dependent manner, but unlike the FDA-approved inhibitor
vorapaxar, washing studies showed that their action is reversible
in a manner consistent with negative allosteric inhibition of
PAR1. Such compounds possess significant potential for the
modulation of PARs in a therapeutic manner, in particular for
inflammation-related disorders such as sepsis or reperfusion
injury.54 Our future work is aimed towards measuring the
signaling biases of the parmodulins at PAR1 and optimizing their
cytoprotective effects.
Tandem liquid chromatography/mass spectrometry (LC-MS)
was performed on a Shimadzu LCMS-2020 with autosampler,
photodiode array detector, and single-quadrupole MS with ESI
and APCI dual ionization using a Peak Scientific nitrogen
generator.
Method A
Column: Phenomenex Gemini C18 (100 x 4.6 mm, 3 µm particle
size, 110 Å pore size)
Column temperature: 40 °C
Sample Injection: 1–5 µL of sample in MeCN or MeOH
Chromatographic monitoring: UV absorbance at 210 or 254 nm
Mobile Phase: Solvent A: H2O w/ 0.1% formic acid; Solvent B:
MeCN w/ 0.1% formic acid
Flow Rate: 1.0 mL/min
Gradient:
0 to 0.5 min: 25% MeCN
0.5 min to 5 min: 25% to 95% MeCN
5 min to 7.3 min: 95% MeCN
7.3 min to 7.5 min: 95% to 25% MeCN
4.3 Preparative HPLC purification methods
Preparative liquid chromatography was performed on a
Shimadzu LC-20AP preparative HPLC with autosampler, dual
wavelength detector, and fraction collector.
4. Experimental section
Method A
4.1 General information: Synthesis
Column: Phenomenex Gemini C18 Semi Preparative Column (250
x 10 mm, 5 µm particle size, 110 Å pore size)
Column temperature: 23 °C
Mobile Phase: Solvent A: H2O w/ 0.1% formic acid; Solvent B:
MeCN w/ 0.1% formic acid
All reagents and solvents, including anhydrous solvents, were
purchased from commercial vendors and used as received.
Compound
1
(CAS# 125238-99-5) was obtained from
ChemImpex. Compounds 14, 15, 16 (ML161), 17, 21 (RR-90),
and 22 were synthesized as previously described.12 Deionized
water was purified by charcoal filtration and used for reaction
workups and in reactions with water. NMR spectra were recorded
on Varian 300 MHz or 400 MHz spectrometers as indicated.
Proton and carbon chemical shifts are reported in parts per
million (ppm; δ) relative to tetramethylsilane (1H δ 0), or CDCl3
(13C δ 77.16), (CD3)2CO (1H δ 2.05, 13C δ 29.84), d6-DMSO (1H δ
2.50, 13C δ 39.5), or CD3OD (1H δ 3.31, 13C δ 49.00). NMR data
are reported as follows: chemical shifts, multiplicity (obs =
obscured, app = apparent, br = broad, s = singlet, d = doublet, t =
triplet, q = quartet, sxt = sextet, m = multiplet, comp = complex
overlapping signals); coupling constant(s) in Hz; integration.
Unless otherwise indicated, NMR data were collected at 25 °C.
Filtration was performed by vacuum using VWR Grade 413 filter
paper, unless otherwise noted. Flash chromatography was
performed using Biotage SNAP cartridges filled with 40-60 µm
silica gel on Biotage Isolera automated chromatography systems
with photodiode array UV detectors. Analytical thin layer
chromatography (TLC) was performed on Agela Technologies
0.25 mm glass plates with 0.25 mm silica gel. Visualization was
accomplished with UV light (254 nm) and KMnO4 stain, unless
otherwise noted. Chemical names were generated and select
chemical properties were calculated using either ChemAxon
Marvin suite (https://www.chemaxon.com) or ChemDraw
Professional 15.1. NMR data were processed using either
MestreNova or ACD/NMR Processor Academic Edition
(http://www.acdlabs.com) using the JOC report format. High-
resolution mass spectra (HRMS) were obtained at the University
of Wisconsin-Milwaukee Mass Spectrometry Laboratory with a
Shimadzu LCMS-IT-TOF with ESI and APCI ionization.
Peak collection: measured by UV absorbance at 210 or 254 nm
Sample Injection: 0.1–1.9 mL (2 mL sample loop) of sample in
DMSO
Flow Rate: 6.0 mL/min
Gradient:
0 to 1.5 min: 25% MeCN
1.5 min to 8 min: 25% to 95% MeCN
8 min to 10.5 min: 95% MeCN
4.4 Synthesis of RWJ-58259 and alkynylated analog DG-207
4.4.1 (9H-Fluoren-9-yl)methyl tert-butyl (4-(benzylamino)-4-
oxobutane-1,3-diyl)(S)-dicarbamate (2). To a round bottomed
flask with stir bar under nitrogen was added carboxylic acid 1
(500 mg, 1.13 mmol) and anhydrous DCM (25
mL). Benzylamine (0.15 mL, 1.4 mmol), HOBt (229 mg, 1.50
mmol), and EDC•HCl (433 mg, 2.26 mmol) were added and the
reaction was stirred under nitrogen for 2 h. A sample aliquot was
taken from the reaction, concentrated under reduced pressure,
dissolved in a minimal amount of HPLC grade MeCN, and
analyzed with LC-MS to confirm reaction completion. The
reaction was diluted with DCM (75 mL) and washed with
saturated NaHCO3 (30 mL), 1M HCl (30 mL), brine, dried over
MgSO4, filtered, and concentrated under reduced pressure to give
2 (588 mg) as a white solid in 98% yield. mp = 154–156 °C; LC-
MS tR = 6.09 min (Characterization Method A); m/z = 530.05 (M
+ H+); 1H NMR (300 MHz, CDCl3) δ = 7.76 (d, J = 7.6 Hz, 2 H),
7.61 – 7.49 (overlapping signals, 3 H), 7.43 – 7.22 (m, 9 H), 5.96
(d, J = 6.7 Hz, 1 H), 5.11 (br. m., 1 H), 4.50 – 4.13 (overlapping
m, 6 H), 3.48 – 3.32 (br s., 1 H), 3.04 – 2.90 (br m., 1 H), 1.97 –
1.71 (overlapping signals, 2 H), 1.45 – 1.37 (m, 9 H); 13C NMR
(75 MHz, CDCl3) δ = 171.2, 157.2, 156.3, 143.9, 141.5, 138.0,
128.8, 127.9, 127.8, 127.6, 127.3, 125.3, 120.2, 80.1, 67.2, 52.2,
47.3, 43.8, 37.0, 34.9, 28.6.

1
4.4.2
tert-butyl
((S)-3-((S)-2-((((9H-Fluoren-9-
m/z = 489.30 (M - H+), 535.30 (formic acid adduct); H NMR
yl)methoxy)carbonyl)amino)-3-(3,4-
difluorophenyl)propanamido)-4-(benzylamino)-4-
(300 MHz, CDCl3) δ = 8.06 (d, J = 7.9 Hz, 1 H), 7.72 (br t, J =
5.0 Hz, 1 H), 7.37 - 7.20 (m, 5 H), 7.14 - 6.95 (m, 2 H), 6.94 -
6.83 (br m, 1 H), 5.37 (app t J = 5.3, 6.2 Hz, 1 H), 4.56 - 4.47 (m,
1 H), 4.47 - 4.35 (m, 2 H), 3.53 - 3.42 (m, 1 H), 3.39 - 3.25 (br m,
1 H), 3.09 (dd, J = 3.8, 9.7 Hz, 1 H), 3.02 - 2.89 (br m, 1 H), 2.66
(dd, J = 4.7, 9.1 Hz, 1 H), 2.00 - 1.85 (br m, 1 H), 1.84 - 1.67 (br
m, 1 H), 1.54 (br m., 2 H), 1.43 (s, 9 H); 13C NMR (75 MHz,
CDCl3) δ = 174.4, 171.2, 156.9, 151.6 (dd, J = 53.5, 12.7 Hz),
148.4 (dd, J = 52.1, 12.4 Hz), 138.1, 134.8 (app dd, J = 5.3, 3.9
Hz), 128.8, 127.9 (partially obs) 127.8, 127.6, 125.4 (app dd, J =
6.2, 3.6 Hz), 118.2 (d, J = 17 Hz), 117.6 (d, J = 17.3 Hz), 79.8,
56.4, 50.4, 43.7, 40.3, 37.0, 34.5, 28.6.
oxobutyl)carbamate (3). Part 1: Fmoc removal. Intermediate 2
(550 mg, 1.03 mmol) was added to a round bottomed flask with
stir bar and sealed under nitrogen, then MeCN (25 mL), DCM
(10 mL), and piperidine (0.220 mL, 2.22 mmol) were added. The
reaction was stirred for 2 h. A sample aliquot was taken from the
reaction, concentrated under reduced pressure, dissolved in a
minimal amount of HPLC grade MeCN, and analyzed with
LCMS to confirm reaction completion. The reaction was
concentrated under reduced pressure and then re-dissolved in
(CHCl3/EtOAc, 1:1, 75 mL), washed with H2O (2 x 20 mL) and
brine (20 mL). The organic phase was dried over MgSO4,
filtered, and concentrated under reduced pressure to give crude
material (496 mg) as a white solid that was carried onto the next
reaction without further purification.
4.4.4. 6-Nitro-1H-indazole-3-carbaldehyde (6). To a 500 mL
round bottomed flask with stir bar was added NaNO2 (6.38 g,
92.5 mmol) and H2O (150 mL). 6-Nitroindole (5, 1.5 g, 9.3
mmol) was added to the above solution at 20 °C and the resultant
mixture was stirred vigorously until evenly suspended (over 5
min.). To this bright yellow suspension was added 6 M HCl (14
mL) via addition funnel over 30 min. at 20 °C, and the resultant
suspension was stirred for 90 min. A sample aliquot was taken
from the reaction, filtered, and the precipitate was dissolved in a
minimal amount of HPLC grade MeCN. Analysis with LC-MS
confirmed reaction completion. The product was vacuum filtered
and the precipitate was washed with additional H2O (50 mL). The
precipitate was dried to give 6 (1.37 g) as an orange solid in 77%
yield. mp: changed color from orange to brown between 120 and
160 °C, decomposed after 200 °C; LC/MS tR = 2.40 min
Part 2: Coupling. To a round bottomed flask with stir bar
already containing crude material from Part 1 (496 mg crude
material, max. yield is 319 mg) was added anhydrous MeCN (30
mL) and anhydrous DCM (15 mL). (S)-2-((((9H-Fluoren-9-
yl)methoxy)carbonyl)amino)-3-(3,4-difluorophenyl)propanoic
acid (300 mg, 0.709 mmol), HOBt (191 mg, 1.41 mmol), and
EDC•HCl (272 mg, 1.41 mmol) were added, and the resulting
white suspension was stirred for 2 h. A sample aliquot was taken
from the reaction, concentrated under reduced pressure, dissolved
in a minimal amount of HPLC grade MeCN, and analyzed with
LC-MS to confirm reaction completion. The reaction was
concentrated under reduced pressure, re-suspended in EtOAc
(100 mL), and washed with H2O (20 mL), 0.5 M HCl (20 mL),
saturated NaHCO3 (20 mL), and brine. The organic phase was
dried over MgSO4, filtered, and concentrated under reduced
pressure to give crude material (yellow oil) that was dry loaded
using silica onto a 5 g silica gel column and purified with flash
chromatography (EtOAc/hexanes, 1–100%) to give 3 (368 mg,
49%) as a white solid over 2 steps. m.p. = 183–185 °C; LC/MS tR
= 6.43 min (Characterization Method A); m/z = 713.15 (M + H+),
1
(Characterization Method A); m/z = 190.05 (M - H+); H NMR
(400 MHz, DMSO-d6) δ = 10.22 (s, 1 H), 8.57 (d, J = 1.5 Hz, 1
H), 8.29 (dd, J = 0.5, 8.9 Hz, 1 H), 8.13 (dd, J = 2.0, 8.9 Hz, 1
H); 13C NMR (100 MHz, DMSO-d6) δ = 187.7, 146.9, 124.0,
122.4, 118.7, 108.6.
4.4.5
6-Nitro-3-(pyrrolidin-1-ylmethyl)-1H-indazole
(7).
Aldehyde 6 (1.3 g, 6.80 mmol) was added to a round bottomed
flask with stir bar and sealed under nitrogen. A pre-mixed
solution of anhydrous DCE : DMF : AcOH (300 : 30 : 0.3 mL),
and pyrrolidine (2.8 mL, 34.0 mmol) was then added. The
resulting yellow suspension became a reddish brown solution
immediately after the addition of pyrrolidine, and the reaction
was stirred for 20 min. Sodium triacetoxyborohydride (8.32 g,
39.2 mmol) was added in 3 portions at 5 min. intervals and the
resultant suspension was stirred at 20 °C for 4 h. A sample
aliquot was taken from the reaction, diluted with DCM, and
washed with half saturated Na2CO3. The organic layer was
separated, concentrated under reduced pressure, dissolved in a
minimal amount of HPLC grade MeCN, and analyzed with LC-
MS to confirm reaction completion. The reaction was diluted
with DCM (100 mL), washed with aqueous NaHCO3 solution
(100 mL), brine, dried over Na2SO4, vacuum filtered, and
concentrated under reduced pressure to give 2.2 g of dark brown
solid. The crude material was dissolved in minimal DCM and
loaded onto a 50 g SiO2 column and purified by flash
chromatography (1N NH3 in MeOH/DCM, 0–18%) to give 7
(1.09 g) as a shiny orange solid in 65% yield. mp = 37–40 °C;
LC/MS tR = 0.91 min (Characterization Method A); m/z = 247.05
(M + H+); m/z= 245.15 (M - H+); 1H NMR (400 MHz , CD3OD)
δ = 8.44 (d, J = 1.5 Hz, 1 H), 8.05 (d, J = 8.8 Hz, 1 H), 7.99 (dd,
J = 2.0, 7.0 Hz, 1 H), 4.16 (s, 2 H), 2.78 - 2.71 (m, 4 H), 1.84
(spt, J = 3.3 Hz, 4 H); 13C NMR (75 MHz, CD3OD) δ = 147.0,
142.8, 140.1, 125.2, 121.3, 114.9, 106.8, 53.9, 50.6, 23.1.
1
m/z = 757.35 (formic acid adduct); H NMR (300 MHz, DMSO-
d6) δ = 8.43 (dd, J = 5.0, 5.9 Hz, 1 H), 8.22 (d, J = 7.9 Hz, 1 H),
7.86 (d, J = 7.6 Hz, 2 H), 7.68 - 7.53 (m, 2 H), 7.52 - 7.43 (br s.,
1 H), 7.43 - 7.07 (m, 11 H), 6.74 (br s, 1 H), 4.28 (br d, J = 5.6
Hz, 4 H), 4.15 (br s, 3 H), 3.05 - 2.85 (overlapping br s, 3 H),
2.73 (app t, J = 12.0 Hz, 1 H), 1.86 - 1.73 (m, 1 H), 1.73 – 1.58
(m, 1H), 1.34 (s, 9 H); 13C NMR (75 MHz, DMSO-d6) δ = 171.8,
171.7, 156.4, 156.1, 150.8 (dd, J = 45.2, 13 Hz), 147.6 (dd, J =
45.8, 12.1 Hz), 144.4, 144.3, 141.35, 141.33, 139.8, 136.6, 128.9,
128.2, 127.7, 127.6, 127.4, 126.7, 125.9, 120.7, 118.9, 118.6,
117.6, 117.4, 78.3, 66.3, 56.4, 51.5, 47.2, 42.7, 37.5, 37.1, 33.1,
28.9
4.4.3
difluorophenyl)propanamido)-4-(benzylamino)-4-
oxobutyl)carbamate (4). Intermediate (250 mg, 0.351
tert-butyl
((S)-3-((S)-2-Amino-3-(3,4-
3
mmol) was added to a round bottomed flask with stir bar and
sealed under nitrogen, then anhydrous MeCN (30 mL) and
piperidine (2 mL) were added and the reaction was stirred for 90
min. A sample aliquot was taken from the reaction, concentrated
under reduced pressure, dissolved in a minimal amount of HPLC
grade MeCN, and analyzed with LC-MS to confirm reaction
completion. The reaction was concentrated under reduced
pressure to give crude material (light yellow solid) that was
triturated with 75/25 hexanes/ether. The material was then dry
loaded using silica onto a 10 g silica gel column and purified
with flash chromatography (IPA/DCM, 0 to 20%) to give 4 (86
mg) as a white solid in 50% yield. mp = 216–219 °C; LC/MS tR
= 3.15 min (Characterization Method A); m/z = 491.35 (M + H+),
4.4.6
1-(2,6-Dichlorobenzyl)-6-nitro-3-(pyrrolidin-1-
ylmethyl)-1H-indazole (8). Amine 7 (1.06 g, 4.30 mmol) and 2-
(bromomethyl)-1,3-dichlorobenzene (1.03 g, 4.30 mmol) were

added to a round bottomed flask with stir bar and sealed under
nitrogen. Anhydrous THF (50 mL) was added, followed by
Cs2CO3 (1.4 g, 4.30 mmol) added in 3 portions (1 portion every 5
min.), and the flask was flushed with nitrogen. The resultant
orange solution was stirred for 16 h at 20 °C. A sample aliquot
was taken from the reaction, diluted with EtOAc, and washed
with H2O. The organic phase was separated, concentrated under
reduced pressure, dissolved in CDCl3 and analyzed with NMR to
confirm reaction completion. The reaction was diluted with
EtOAc (200 mL), washed with H2O (200 mL) and brine, dried
over Na2SO4, vacuum filtered, and concentrated under reduced
pressure. The brown crude material (1.5 g) was dissolved in
minimal DCM and loaded onto a 50 g column and purified with
flash chromatography (MeOH/DCM, 0–18%) to give 8 (1.33 g)
as an orange brown shiny solid (containing ~10% dibenzylated
side-product) in 76% yield. 8 was carried forward to the next step
Part 2: Boc removal. The round bottomed flask already
containing either the crude or triturated material 10 was cooled in
a NaCl/H2O bath for 10 min (–5°C), and then HCl in dioxane (4
M, 3 mL) was slowly added. The reaction immediately became a
heterogeneous dark red mixture and was stirred for 40 min. at –5°
C. A sample aliquot was taken from the reaction, concentrated
under reduced pressure, dissolved in a minimal amount of HPLC
grade MeCN, and analyzed with LC-MS to confirm reaction
completion. The reaction was concentrated under reduced
pressure to give a red-orange solid that was free-based with 0.075
N NH3 in MeOH and concentrated under reduced pressure. The
crude product was dissolved in a minimal amount of DCM,
loaded onto a 10 g normal phase silica column, and purified with
flash chromatography (0–18% (0.75 N NH3 in MeOH)/DCM,) to
give amine 11 (free base) as a light pink oily solid. The product
was converted to the bis HCl salt 11a by adding 1N HCl (1 mL)
and lyophilizing overnight to give bis HCl salt as a light pink
solid. LC-MS Data tR = (Characterization Method A); m/z =
without additional purification. LC/MS tR
= 3.15 min
(Characterization Method A); m/z = 404.80 (M + H+); for
dibenzylated impurity LC-MS tR = 3.63 min (Characterization
Method A); m/z = 579.95 (M + H+).
1
396.65 [(M/2) + H+], 835.30 (M - H+, formic acid adduct); H
NMR (400 MHz, CD3OD, free base) δ = 7.98 (s, 1 H), 7.75 (d, J
= 8.7 Hz, 1 H), 7.45 - 7.38 (m, 2 H), 7.37 - 7.05 (m, 10 H), 5.56
(br. s., 2 H), 4.64 (s, 2 H), 4.60 - 4.48 (m, 2 H), 4.40 (d, J = 4.5
Hz, 2 H), 3.57 - 3.45 (m, 2 H), 3.29 - 3.12 (m, 3 H), 3.12 - 2.93
(m, 3 H), 2.35 - 2.20 (m, 1 H), 2.15 - 1.85 (m, 5 H); 13C NMR (75
MHz, CD3OD, free base) δ = 173.3, 171.0, 156.2, 141.7, 139.0,
138.2, 136.6, 135.1, 134.3, 131.4, 130.3, 128.3, 128.1, 126.9,
126.7, 125.6, 119.7, 118.3, 118.1, 117.8, 117.0, 116.7, 115.2,
97.4, 55.6, 53.5, 51.0, 48.8, 42.6, 36.8, 36.6, 29.5, 22.6.
Alternatively, the crude product could be dissolved in DMSO,
filtered, and purified in batches with preparative HPLC (method
A). Pooled fractions were concentrated under reduced pressure in
lukewarm water (to remove MeCN) and lyophilized overnight to
give 11b (36.3 mg) as a light pink oil in 58% yield over 2 steps.
4.4.7
1-(2,6-Dichlorobenzyl)-3-(pyrrolidin-1-ylmethyl)-1H-
indazol-6-amine (9). To an oven dried pressure tube with stir bar
was added indazole 8 (18 mg, 0.044 mmol) and MeOH (5
mL). FeCl3•6H2O (25 mol%, 2.6 mg), hydrazine monohydrate
solution (65% active hydrazine, 0.05 mL, 0.7 mmol), and
activated charcoal (20 mg) were added, the tube was capped
tightly, and the reaction was stirred at 100 °C for 2 h. A sample
aliquot was taken from the reaction, concentrated under reduced
pressure, dissolved in a minimal amount of HPLC grade MeCN,
and analyzed with LCMS to confirm reaction completion. The
reaction was filtered, the charcoal and filtered solids were
washed with MeOH (10 mL), and the filtrate was concentrated
under reduced pressure to give crude material that was dry loaded
using silica onto a 5 g silica column and purified with flash
chromatography (0 to 20% MeOH/DCM) to give 9 (11.5 mg) as
4.4.9
N-((S)-4-(Benzylamino)-3-((S)-2-(3-(1-(2,6-
dichlorobenzyl)-3-(pyrrolidin-1-ylmethyl)-1H-indazol-6-
yl)ureido)-3-(3,4-difluorophenyl)propanamido)-4-
a
white solid in 69% yield. LC-MS tR = 1.44 min
(Characterization Method A); m/z = 374.95 (M + H+); H NMR
(300 MHz, CDCl3) δ = 7.57 (d, J = 8.5 Hz, 1 H), 7.37 (d, J = 8.2
Hz, 2 H), 7.31 - 7.20 (s, 1 H), 6.63 (d, J = 8.8 Hz, 1 H), 6.58 (s, 1
H), 5.63 (s, 2 H), 4.29 (s, 2 H), 3.96 (br. s., 2 H), 3.13 (br. s., 4
H), 1.84 (br. s., 4 H); 13C NMR (75 MHz, CDCl3) δ = 146.5,
142.5, 137.0, 131.6, 130.3, 128.8, 121.0, 117.7, 113.7, 92.2, 52.3,
48.5, 48.2, 23.9.
oxobutyl)pent-4-ynamide (12, DG-207). Note: If using 11b, it
is necessary to convert this compound to the bis HCl salt (11a)
prior to performing this EDC coupling reaction as it has been
seen that the formate salt will give a mixture of products. To a 5
mL scintillation vial with stir bar was added 11b (5.5 mg, 6.4
μmol). The vial was purged with N2 for 5 min and then dry
dioxane (0.5 mL) and 4 M HCl in dioxane (0.3 mL, 172 eq.) was
added. The reaction was stirred for 5 min., concentrated under
reduced pressure, and then lyophilized overnight to give the bis
HCl salt (11a). The vial containing 11a was purged with nitrogen
and then dissolved in dry DMF (0.5 mL). 4-Pentynoic acid (1
mg, 10 µmol), HOBt (1.2 mg, 8.9 µmol), EDC•HCl (3 mg, 12
µmol), and DIPEA (9.6 μL, 55 µmol) were added consecutively
and the reaction was stirred for 5 h under N2. A sample aliquot
was taken from the reaction, diluted with a minimal amount of
HPLC grade MeCN, and analyzed with LC-MS to confirm
reaction completion. The reaction was diluted with EtOAc (5
mL) and washed with saturated NaHCO3 (5 mL x 2) and brine (5
mL x 2). The organic layer was dried over MgSO4, filtered, and
concentrated under reduced pressure to give crude material that
was dissolved in DMSO, filtered, and purified with preparative
HPLC (method A) to give 12 (2.4 mg) as a colorless oily residue
1
4.4.8
(S)-4-Amino-N-benzyl-2-((S)-2-(3-(1-(2,6-
dichlorobenzyl)-3-(pyrrolidin-1-ylmethyl)-1H-indazol-6-
yl)ureido)-3-(3,4-difluorophenyl)propanamido)butanamide
(11a-b, RWJ-58259)
Part 1: Coupling reaction. Aniline 9 (40 mg, 0.11 mmol) was
added to an oven-dried round bottom flask with stir bar and
sealed under nitrogen, then anhydrous THF (16 mL) was added.
The solution was cooled in a NaCl/H2O bath (–5 °C) for 15 min.,
and then both DMAP (34.6 mg, 0.282 mmol) and triphosgene (12
mg, 0.04 mmol) were added consecutively. Immediately the
reaction became a light orange suspension. The reaction was
stirred at –5°C for 8 min., and then amine 4 (35 mg, 0.071 mmol)
was added and the reaction was stirred at –5°C for 1 h, then for
an additional 1 h while warming to room temperature. A sample
aliquot was taken from the reaction, concentrated under reduced
pressure, dissolved in a minimal amount of HPLC grade MeCN,
and analyzed with LC-MS to confirm reaction completion. The
reaction was concentrated under reduced pressure to give crude
material 10 that could be either carried onto the next reaction
without purification, or triturated with DCM/hexanes (1:1) to
provide purer material.
1
in 47% yield. H NMR (600 MHz, CD3OD) δ = 8.54 (br. s, 1 H,
formate CHO), 7.94 (d, J = 1.2 Hz, 1 H), 7.67 (d, J = 8.8 Hz, 1
H), 7.44 (s, 1 H), 7.42 (s, 1 H), 7.33 (dd, J = 1.2, 7.6 Hz, 1 H),
7.28 - 7.11 (m, 7 H), 7.09 - 7.05 (br m, 1 H), 6.99 (dd, J = 1.8,
7.0 Hz, 1 H), 5.61 (dd, J = 14.1, 2.3 Hz, 2 H), 4.55 (dd, J = 2.9,
5.3 Hz, 1 H), 4.44 - 4.41 (m, 1 H), 4.39 (s, 2 H), 4.37 - 4.34 (br.
m, 2 H), 3.58 (dd, J = 4.7, 6.5 Hz, 1 H), 3.51 (dd, J = 4.7, 6.5 Hz,

The reaction was dried over Na2SO4, filtered, and concentrated
under reduced pressure to give crude material that was carried
onto the next peptide coupling step without further purification.
1 H), 3.20 - 3.13 (m, 2 H), 3.12 - 3.04 (br s, 3 H), 2.99 (dd, J =
5.9, 8.2 Hz, 1 H), 2.41 - 2.37 (m, 2 H), 2.34 - 2.29 (m, 2 H), 2.21
- 2.20 (m, 1 H), 2.07 - 1.98 (m, 2 H), 1.93 - 1.86 (m, 4 H). 19F
NMR (376 MHz, DMSO-d6) δ = -139.53, -142.40; 13C NMR
(101 MHz, DMSO-d6) δ = 171.6, 171.5, 170.6, 164.5, 155.1,
150.2 (dd, J = 58.7, 12.2 Hz), 147.8 (dd, J = 58, 12.2 Hz), 142.4,
141.7, 139.6, 139.3, 136.4, 135.69, 135.67, 132.3, 131.0, 129.1,
128.6, 127.4, 127.1, 126.6, 121.3, 118.7 (d, J = 16.8 Hz), 118.1,
117.2 (d, J = 16.8 Hz), 113.7, 96.6, 84.1, 71.7, 63.4, 54.0, 53.7,
51.4, 47.2, 42.4, 37.9, 36.2, 34.6, 32.4, 23.5, 14.6. LC-MS tR =
3.46 min (Characterization Method A); m/z = 871.15 (M + H+),
m/z = 915.30 (M - H+, formic acid adduct). HRMS (ESI-TOF)
calcd. for C45H47Cl2F2N8O4 (M + H+) 871.3060, found 871.2988.
4.6 Synthesis of parmodulins
4.6.1. 2-Bromo-N-(3-propionamidophenyl)benzamide (18,
EMG-21). Analogue 18 (EMG-21) was synthesized by coupling
of aniline-HCl salt 15 with propionic acid (16 uL, 0.20 mmol)
according to general method A. The crude material was loaded
using Celite onto a 5 g silica gel column and purified by flash
chromatography (EtOAc:hexanes, 0–100%) to give 18 as an off-
white solid (19 mg) in 32% yield. LC-MS tR = 4.47 min
1
(Characterization Method A); m/z = 348.70 (M + H); H NMR
(300 MHz, CD3OD) δ = 7.98 (t, J = 2.0 Hz, 1H), 7.69 (dd, J =
7.9, 1.1 Hz, 1H), 7.56-7.43 (m, 2H), 7.42-7.35 (m, 3H), 7.29 (t, J
= 8.0 Hz, 1H), 2.39 (q, J = 7.6 Hz, 2H), 1.20 (t, J = 7.6 Hz, 3H);
HRMS (ESI): m/z [M + H]+ calcd. for C16H15N2O2Br, 347.0390;
found, 347.0357. This compound was previously obtained from
commercial sources.12
4.5 General procedures for the syntheses of parmodulins and
Fairlie’s PAR2 antagonist (32, TJF-5)
Method A: Amide coupling using EDC
To a round bottomed flask with stir bar under nitrogen were
added the appropriate carboxylic acid and anhydrous DCM/DMF
(85:15; 0.2-0.6 M). The amine HCl salt to be coupled (1.2 eq.),
HOBt (1.2 eq.), EDC•HCl (1.2 eq.), and DIPEA (2.1 eq.) were
added and the reaction was stirred under nitrogen. The reaction
was diluted with DCM (75 mL), washed with saturated NaHCO3,
1M HCl (30 mL), brine, dried over Na2SO4, filtered, and
concentrated under reduced pressure to give crude product.
4.6.2
2-Bromo-N-(3-pentanamidophenyl)benzamide
(19,
EMG-23). Analogue 19 was synthesized by amide coupling of
pentanoyl chloride (22 uL, 0.19 mmol) with aniline•HCl salt 15
according to general method C. The crude material was then dry
loaded using Celite onto a 5 g silica gel column and purified by
flash chromatography (EtOAc:hexanes, 0–100%) to give 19 as an
off white solid (25 mg) in 39% yield. LC-MS tR = 5.12 min
Method B: Formation and acylation with acid chloride
1
(characterization method A); m/z = 376.75 (M + H+); H NMR
(300 MHz, CD3OD) δ = 7.99 (t, J = 2.0 Hz, 1H), 7.69 (dd, J =
7.9, 1.0 Hz, 1H), 7.56-7.43 (m, 2H), 7.42-7.34 (m, 3H), 7.29 (t, J
= 8.0 Hz, 1H), 2.38 (t, J = 7.3 Hz, 2H), 1.73-1.68 (m, 2H), 1.41
(app sxt, J = 7.3 Hz, 2H), 0.97 (t, J = 7.3 Hz, 3H); HRMS (ESI):
m/z [M + H]+ calcd. for C18H19N2O2Br, 375.0703; found,
375.0669. This compound was previously obtained from
commercial sources.12
To an oven-dried round bottomed flask with stir bar under
nitrogen were added the carboxylic acid, dry DCM, and 3Å
molecular sieves. Oxalyl chloride (1.2 eq.) and a catalytic amount
of DMF (1–2 mol %) were added and the reaction was stirred
while attached to a bubbler (to monitor production of CO2) at 20
°C for 2–3 h. The amine HCl salt (1 eq.) in DCM and DIPEA (2
eq.) were added and the reaction was stirred under nitrogen for
3–6 h. The reaction was diluted with EtOAc and washed with
half-saturated aqueous NaHCO3, 1M HCl (30 mL), brine, dried
over Na2SO4, filtered, and concentrated under reduced pressure to
give crude material.
4.6.3 2-Bromo-N-(3-(pent-4-ynamido)phenyl)benzamide (20,
RR-10). Alkyne analogue 20 was synthesized by acylation of
pent-4-ynoic acid (50 mg, 0.51 mmol) with aniline•HCl salt 15
according to general method B. The crude material was dissolved
in a minimal amount of DCM, loaded onto a 10 g silica column,
and purified by flash chromatography (EtOAc:hexanes, 0-100%)
to give 20 as a white solid (13 mg) in 7% yield. LC/MS tR = 6.44
Method C: Acylation with acid chloride
To an oven-dried round bottomed flask with stir bar and under
nitrogen were added the acid chloride, dry DCM, and 3Å
molecular sieves. The amine HCl salt (1 eq.) in DCM and DIPEA
(2 eq.) were added and the reaction was stirred under nitrogen for
3–6 h. The reaction was diluted with EtOAc and washed with
half-saturated aq. NaHCO3, 1M HCl (30 mL), brine, dried over
Na2SO4, filtered, and concentrated under reduced pressure to give
crude product.
1
min (characterization method A); m/z = 372.60 (M + H+); H
NMR (400 MHz, CDCl3) δ = 7.95 (s, 1 H), 7.71 (s, 1 H), 7.67 -
7.58 (m, 2 H), 7.52 (br s., 1 H), 7.46 - 7.27 (m, 5 H), 2.66 - 2.52
(m, 4 H), 2.09 - 2.05 (m, 1 H); 13C NMR (75 MHz ,CDCl3, 1%
CD3OD) δ = 170.5, 167.0, 138.8, 138.4, 133.5, 131.6, 129.6,
129.2, 127.7, 119.6, 116.4, 116.1, 111.7, 83.0, 69.5, 36.0, 14.8.
HRMS (ESI) calcd. for C18H15BrN2O2 (M + H+), 371.0390, found
371.0364.
Method D: Amide coupling using HATU
4.6.4 N-(3-Aminophenyl)butyramide HCl salt (23). A solution
of nitroarene 22 (5.95 g, 28.7 mmol) in MeOH (85 mL) was
flushed with nitrogen for 5 min. To a 10 mL scintillation vial
under nitrogen was suspended 10% Pd/C (0.95 g, 0.88 mmol) in
MeOH/H2O (4:1) and the resultant suspension was added to the
solution of 22. A balloon was filled with H2 gas and inserted into
the flask and the reaction was purged with H2 gas for 2 min. The
reaction was left to stir under a full balloon of H2 for 16 h, after
which an aliquot was taken from the reaction, filtered through
celite and cotton, and concentrated under reduced pressure. The
aliquot was dissolved in a minimal amount of HPLC grade
MeCN, and analyzed with LC-MS to confirm reaction
completion. The reaction was filtered through a funnel packed
with Celite. The filter cake was washed with MeOH and the
filtrate was concentrated under reduced pressure, to give crude
To a round bottomed flask with stir bar under nitrogen was added
the carboxylic acid and anhydrous DCM. The amine (1.2 eq.),
HATU (1.2 eq.), and DIPEA (1.2 eq.) were added and the
reaction was stirred under nitrogen. The reaction was diluted with
DCM (75 mL) and washed with saturated NaHCO3, 1M HCl (30
mL), brine, dried over Na2SO4, filtered, and concentrated under
reduced pressure to give crude material.
Method E: Boc removal
To an 8 mL vial with stir bar and under nitrogen was added the
Boc-protected amine, dry DCM, and a large excess of TFA. The
reaction was allowed to stir at 20 °C for 1–2 h. The reaction
mixture was concentrated under reduced pressure (passed
through a base trap) to give crude material that was suspended in
EtOAc and washed with half-saturated aq. NaHCO3, and brine.

material that was dissolved in DCM and added to 0.9 M HCl in
ether (40 mL). The reaction was stirred for 5 min at 20 °C to give
a precipitate that was filtered and washed with excess DCM. The
precipitate was dried to give the previously described aniline 2312
(5.60 g) as a grey solid in 92% yield.
Part 2. Amide coupling. Analogue 30 was synthesized from 29
(29.0 mg, 0.12 mmol) and (2S)-2-(tert-butoxycarbonylamino)-3-
cyclohexyl-propanoic acid hydrate (40 mg, 0.14 mmol) according
to general method D. The crude material was dissolved in a
minimal amount of DCM, loaded onto a 10 g silica gel column,
and purified by flash chromatography (MeOH/DCM, 0–12%) to
4.6.5. 2-Azido-N-(3-butyramidophenyl)benzamide (26, DG-5).
Part 1: Diazotization. To a round bottomed flask with stir bar
was added 2-aminobenzoic acid 24 (1.50 g, 10.9 mmol) and
deionized H2O (100 mL). The reaction was cooled to 0 °C and
conc. aq. HCl (5 mL) was added dropwise over 5 min. A solution
of NaNO2 (0.75 g, (11 mmol) in H2O (1 mL) was added, and the
reaction was stirred for 10 min. at 2 °C. A solution of NaOAc
(0.63 g, 11 mmol) in H2O (1 mL) and a solution of NaN3 (0.71 g,
10.9 mmol) in H2O (1 mL) were added consecutively and the
reaction was stirred for 30 min at 2 °C. A sample aliquot was
taken from the reaction, diluted with TBME, extracted and the
organic phase was separated, concentrated under reduced
pressure, dissolved in a minimal amount of HPLC grade MeCN,
and analyzed with LC-MS to confirm reaction completion. The
reaction was extracted with TBME (100 mL) and the organic
layer was washed with brine, dried over Na2SO4, filtered, and
concentrated under reduced pressure to give 2-azidobenzoic acid
25 (1.5 g) as a yellow solid in 85% yield. TLC: mobile phase:
1
give 3048 (51 mg) as a white solid in 37% yield over 2 steps. H
NMR (300 MHz, CDCl3) δ =7.23 (d, J = 7.6 Hz, 2 H), 6.89 (q, J
= 7.6 Hz, 2 H), 6.69 (d, J = 8.8 Hz, 1 H), 6.56 - 6.46 (m, 1 H),
4.90 (br d, J = 7.3 Hz, 1 H), 4.42 (ddd, J = 9.7, 16.4 Hz, 2 H),
4.25 (t, J = 8.8 Hz, 1 H), 3.84 (s, 3 H), 3.07 (s, 1 H), 2.95 (s, 1
H), 1.75 - 1.55 (m, 8 H), 1.48 - 1.35 (m, 15 H), 1.23 - 1.01 (m, 6
H).
4.7.3
N-((S)-3-Cyclohexyl-1-(((2S,3S)-1-((2-
methoxybenzyl)amino)-3-methyl-1-oxopentan-2-yl)amino)-1-
oxopropan-2-yl)isoxazole-5-carboxamide (32). Part 1. Boc
removal. Intermediate 30 was deprotected to give free amine 31
according to general method E and advanced to the amide
coupling step.
Part 2. Amide coupling. Crude 32 (TJF-5) was synthesized from
amine intermediate 31 (21.5 mg, 0.05 mmol) and isoxazole-5-
carboxylic acid (7.3 mg, 0.06 mmol) according to general method
D. The crude material was dissolved in a minimal amount of
DCM, loaded onto a 10 g silica gel column, and purified by flash
chromatography (MeOH/DCM, 0-5%) to give 3248 (9.1 mg) as a
white solid in 16% yield over 2 steps. LC/MS tR = 6.46 min
(Characterization Method A); m/z = 499.00 (M + H+), 521.00 (M
1
Ethyl acetate/hexanes/formic acid (25/25/1), Rf: 0.4; H NMR
(300 MHz, CDCl3) δ = 8.19 - 8.14 (m, 1 H), 7.67 - 7.59 (m, 1 H),
7.29 (dd, J = 2.0, 7.3 Hz, 2 H).
Part 2: Acylation. Crude azide analogue 26 was synthesized by
acylation of aniline•HCl salt 23 with 2-azidobenzoic acid 25
according to general method B. The crude material was dissolved
in a minimal amount of DCM, loaded onto a 25 g column, and
purified with flash chromatography (EtOAc/hexanes, 0–80%) to
give 26 (160 mg) as a colorless oil in 80% yield. TLC: mobile
phase: ethyl acetate/hexanes (50:50), Rf: 0.6; LC/MS tR = 5.01
min (characterization method A); m/z = 323.80 (M + H+) 368.00
(M + formate); 1H NMR (400 MHz, CDCl3) δ = 9.36 (br. s., 1 H),
8.26 - 8.19 (m, 1 H), 7.98 (br. s., 1 H), 7.61 - 7.50 (m, 1 H), 7.41
(d, J = 7.6 Hz, 2 H), 7.36 - 7.23 (m, 5 H), 2.33 (t, J = 7.4 Hz, 2
1
+ Na+), 498.25 (M - H+), 543.25 (M + formic acid adduct); H
NMR (300 MHz, CDCl3) δ = 8.32 (d, J = 1.5 Hz, 1 H), 7.30 (s, 1
H), 7.33 - 7.21 (m, 1 H), 6.96 - 6.82 (m, 4 H), 6.46 (app t, J = 5.6
Hz, 1 H), 4.74 (dd, J = 8.5 Hz, 1 H), 4.44 (ddd, J = 8.8, 20.8 Hz,
2 H), 4.27 (t, J = 7.6 Hz, 1 H), 3.86 (s, 3 H), 1.89 - 1.73 (m, 3 H),
1.72 - 1.58 (m, 7 H), 1.52 - 1.38 (m, 1 H), 1.37 - 1.24 (m, 2 H),
1.22 - 1.02 (m, 4 H), 0.98 - 0.88 (m, 2 H), 0.88 - 0.79 (m, 6 H).
4.8 General Information: Assays
Water for solutions was deionized and filtered through
charcoal (Milli-Q by Millipore) to a resistance of 18 MΩ.
TFLLRN-NH2 (PAR1 agonist) was obtained from AnaSpec (cat#
AS-62937) or was synthesized (as the TFA salt) by Trudy Holyst,
Blood Research Institute Protein Chemistry Core. SFLLRN-NH2
(TFA salt) (PAR1/2 agonist) was obtained from Bachem (cat# H-
2936.0005) or was synthesized (as the TFA salt) by Trudy
Holyst, Blood Research Institute Protein Chemistry Core.
SLIGKV-NH2 (TFA salt) (PAR2 agonist) was obtained from
Bachem (cat# H-5042.0025) or AnaSpec (cat# AS-60217-1).
AYPGFK-NH2 (TFA salt) (PAR4 agonist) was obtained from
AnaSpec (cat# AS-60218-1). Atopaxar (E5555 HBr) was
obtained from Axon Medchem (cat# 2030). Vorapaxar (SCH
530348) was obtained from Axon Medchem (cat# 1755). Q94
(HCl) was obtained from Axon Medchem (cat# 2055).
H), 1.76 (app sxt, J = 7.4 Hz, 2 H), 0.99 (t, J = 7.3 Hz, 3 H); 13
C
NMR (101 MHz, CDCl3) δ = 162.7, 138.6, 138.4, 136.9, 132.1,
129.6, 125.4, 118.4, 115.6, 111.6, 39.7, 19.0, 13.7; HRMS calcd.
for C17H17N5O2 (M + H+) 324.1455, found 324.1415.
4.7 Synthesis of TJF-5
4.7.1
tert-butyl
((2S,3S)-1-((2-Methoxybenzyl)amino)-3-
methyl-1-oxopentan-2-yl)carbamate (28). Analogue 28 was
synthesized by peptide coupling of (2S,3S)-2-(tert-
butoxycarbonylamino)-3-methyl-pentanoic acid 27 (81 mg, 0.35
mmol) with (2-methoxybenzylamine (38 µL, 0.29 mmol)
according to general method D. The crude material was dissolved
in a minimal amount of DCM, loaded onto a 10 g silica gel
column, and purified by flash chromatography (EtOAc/hexanes,
1
0–100%) to give 2848 (62 mg) as a white foam in 51% yield. H
Adherent EA.hy926 cells (ATCC CRL-2922) were used for
all assays performed. The cells were cultured using the suggested
protocol from the manufacturer (ATCC), except that 150 cm2
tissue culture flasks were used, and DMEM complete media was
prepared as described below. Cells were frozen in 40% fetal
bovine serum (FBS), 50% complete media, and 10% DMSO in
liquid nitrogen, and used between passages 2 and 11. All
manipulations prior to the assays were performed in a sterile
laminar flow cell culture hood.
NMR (300 MHz, CDCl3) δ = 7.24 (d, J = 7.4 Hz, 1 H), 6.93 -
6.83 (m, 2 H), 6.44 (br s, 1 H), 5.16 (br s, 1 H), 4.43 (ddd, J =
6.3, 8.6 Hz, 2 H), 3.92 (t, J = 7.0 Hz, 1 H), 3.84 (s, 3 H), 2.05 -
1.78 (m, 1 H), 1.41 (s, 9 H), 1.14 - 1.01 (m, 1 H), 0.87 (t, J = 7.2
Hz, 6 H).
4.7.2
tert-butyl
((S)-3-Cyclohexyl-1-(((2S,3S)-1-((2-
methoxybenzyl)amino)-3-methyl-1-oxopentan-2-yl)amino)-1-
oxopropan-2-yl)carbamate (30). Part 1. Boc removal.
Intermediate 28 was deprotected to give 29 according to general
method E and was advanced to the amide coupling step.
Cell counting was performed with an automated cell counter
(Countess, Invitrogen) using Invitrogen cell counting chamber
slides and trypan blue stain. All assays were run in 96-well plates

(Corning Costar #3603, polystyrene black wall, clear bottom).
Unless otherwise noted, media exchanges with 96-well plates
were performed using an automated liquid handler (Beckmann-
Coulter, Biomek 3000) with 220 µL pipette tips. Cells were
imaged with an EVOS Fl inverted microscope. All assays were
run on a multimode plate reader (Perkin Elmer EnSpire). Data
was exported to Microsoft Excel for nominal processing then
analyzed with GraphPad Prism (versions 5 or 6).
Agonist solutions: Prepared as 10 mM stock solutions in 100%
water (sterile, deionized). Diluted solutions were then prepared
with water to give half log concentrations.
Antagonist solutions: Vehicle (diluent) was prepared by
mixing 1:9 DMSO:HBSS-HEPES wash buffer. An aliquot from
the antagonist stock solution (in 100% DMSO) was then diluted
10x with vehicle to give a 10% DMSO solution, then subsequent
dilutions were made with vehicle to give
concentration of DMSO (10%).
a consistent
4.9 Preparation of stock solutions
4.11 Protocol for calcium mobilization assay with adherent
endothelial cells
Complete
cell
media
(for
EA.hy926
cells).
Dulbecco’s Modified Eagle Medium (DMEM) with 4.5 g/L
glucose (500 mL) was supplemented with 200 mM L-glutamine
(10 mL, 4 mM final conc.), 100 mM sodium pyruvate (2.5 mL, 1
mM final conc.), 7.5% (w/v) sodium bicarbonate (5.0 mL, 0.75
g/L final conc.), 100X penicillin and streptomycin (Corning #30-
002-CI, 2.0 mL) and heat inactivated FBS (50 mL). The prepared
media was stored in a refrigerator (5 °C) until needed.
Part 1. Cell Preparations (Aseptic Conditions)
Unless otherwise noted, cells were plated at 25,000 cells per
well in 100 μL, which corresponds to 250,000 cells/mL.
Complete cell media (25 mL per plate) was transferred to a 50
mL centrifuge tube and warmed to 37 °C in a water bath. A vial
of frozen EA.hy926 cells (ATCC CRL-2922) was taken from
liquid N2 storage and warmed to 37 °C in a water bath for 5 min.
Complete cell media (1 mL) was added dropwise to the vial and
the contents were mixed and transferred to a 14 mL centrifuge
tube. The cells were centrifuged at 130 g for 3 min and
the supernatant was removed via aspiration with a Pasteur pipet.
The cells were re-suspended in complete cell media (2 mL) and
the suspension was mixed thoroughly with a pipette to gently
break up the cell pellet to give an evenly distributed suspension
in the tube. A small fraction of this suspension (25 μL) was
mixed with 25 μL of trypan blue solution and a cell count was
taken. Based on the cell count, an appropriate volume of
complete cell media was added to the 2 mL of cell suspension to
assure that each well of the 96-well plate would have 25,000
cells. The suspension was gently mixed, and the contents of the
tube were slowly poured into a sterile disposable pipetting
reservoir. This suspension containing the cells was then added
(column by column, 100 μL per well) to a 96-well plate (Corning
Costar #3603, black wall, clear bottom) with a multichannel
pipette. The plate was gently rocked back and forth to get even
coverage of cells and the plate was incubated at 37 °C (5% CO2)
for 40–48 h.
Probenecid solution (250 mM, aqueous). Probenecid (Alfa
Aesar, catalog # B20010-88, 36 mg) was added to a 1 mL
microcentrifuge tube and dissolved in 0.6 M NaOH (500 μL).
NaOH (0.6 M) was prepared by dissolving NaOH (480 mg) in
H2O (20 mL). This solution was made the day of the experiment
and was sufficient for up to 50 mL of buffer (for 2 96-well
plates).
HBSS–HEPES wash buffer. HEPES (1.19 g) was added to
a 500 mL bottle of Ca/Mg/phenol red-free HBSS to make a 10
mM solution in HEPES. MgCl2 (1 M solution, 500 μL) and
CaCl2 (1 M solution, 500 μL) were added and the solution was
stored in the refrigerator. Before use, the desired amount (46 mL
total per 96 well plate) was warmed to room temperature and
added to a 50 mL centrifuge tube, then supplemented with 250
mM probenecid solution (1:100, i.e. 46 mL of buffer needs 460
μL of probenecid solution) to give a 2.5 mM probenecid solution.
The pH was adjusted to 7.4 if necessary by adding aq. HCl.
10% Pluronic F-127 in DMSO. Pluronic F-127 (Sigma
Aldrich, 20 mg) was added to a 1 mL microcentrifuge tube and
dissolved with DMSO (200 μL), to make a 10% w/v solution.
This solution was stored at room temperature.
Part 2. Loading of Fluo-4 dye
Fluo-4/AM loading buffer. Fluo-4/AM (Invitrogen via
Fisher, catalogue # F23917, 50 μg) was dissolved in DMSO (24
μL), then 6 μL of the solution (light-sensitive) was transferred to
a 200 μL microcentrifuge tube and mixed with 6 μL of 10%
pluronic F-127 in DMSO (sufficient for 1 96-well plate). The
remainder of the Fluo-4/AM solution was stored in the freezer at
–30 ºC. The solution was then transferred to a foil-covered 14
mL centrifuge tube already containing HBSS–HEPES wash
buffer (6 mL) and vortexed for 5 seconds. 50 μL of this prepared
dye solution (2 μM) can be added to each well of a 96-well plate
containing adherent cells.
The 96-well plate was removed from the incubator after 40–48
h and then checked with an EVOS Fl microscope and optionally
imaged (20X magnification) to confirm that cells were >80%
confluent and viable. Note: All media exchanges were performed
with the Biomek 3000, unless otherwise noted. The complete cell
media was removed and HBSS–HEPES wash buffer (100 μL)
was added to each well. The plate was left to sit at room
temperature for 5 min. to allow the probenecid to be absorbed,
then the media was removed. Fluo-4/AM solution (50 μL per
well) was added (in the dark), and the plate was incubated at 37
°C (5% CO2) for 40–50 min. The dye solution was removed from
each well and the wells were washed with HBSS–HEPES wash
buffer (50 μL/well) twice. HBSS–HEPES wash buffer (200
μL/well) was added to each well and the plate was imaged with
the EVOS Fl microscope (20X magnification) to verify that the
dye has been absorbed into the cells.
4.10 Preparation of agonist/antagonist solutions
Prior to the assay, stock solutions of PAR1 and PAR2 agonists
were prepared by dissolving the agonists in 100% H2O (sterile,
deionized) to give stock solutions (10 mM, 5 mM, or 1 mM
depending on agonist). These were aliquoted into sterile
microcentrifuge tubes and stored in a –30 ºC freezer, and thawed
before use. Stock solutions of antagonists were prepared by
dissolving with 100% DMSO, generally in glass vials, to give
stock solutions (31.6 mM, 10 mM, or 1 mM depending on
antagonist used) that were stored in the freezer at –30 ºC, and
thawed in a desiccator before use.
Part 3. Running the assay
Basic plate reader info and settings:
Instrument: Perkin Elmer EnSpire plate reader
Temperature: 37°C
Excitation wavelength: 485 nm
Emission wavelength: 525 nm

Measurement height: 7.8 mm (optimized)
Number of flashes: 100
Number of repeats: 250, every 5 sec. Experiment was halted after
the emission signal started to decrease from the maximum.
mentored junior contributors; wrote sections of the S.I. and
manuscript; M.W.M.: Validated and completed the synthesis of
RWJ-58259 and derivatives; developed the calcium mobilization
assay; assayed compounds; wrote sections of the S.I. and
manuscript; R.R.: Synthesized parmodulins, trained and
mentored junior contributors. K.K.: Assisted with the synthesis
of RWJ-58259. T.J.F.: Synthesized Fairlie’s PAR2 antagonist.
E.G.: Synthesized parmodulins. C.D.: Conceived the project;
developed the calcium mobilization assay; trained and mentored
junior contributors; wrote the manuscript.
Vehicle or antagonist (2 μL) were added (single or
multichannel pipette) to each well, and the plate was incubated in
the plate reader for 15 min at 37 °C. Assays were performed 2
columns at a time, by adding agonist solutions (2 μL) to 16 wells,
and then immediately starting the scanning in the plate reader.
Plate readers that measure wells concurrently could optionally
support assays with all agonists added concurrently, for higher
throughput. Upon starting the assay, the basal level of the 2
columns was measured (20 scans) prior to the addition of the
agonist. The agonist (2 μL) was then added (over 10 seconds
with a multichannel pipette or over 90–120 seconds with a single
channel pipette), and the wells were analyzed with the plate
reader using the settings above.
Acknowledgments
We thank Irene Hernandez and Dr. Hartmut Weiler (Blood
Research Institute) for assisting with cell culture and providing
access to reagents, equipment and facilities; Trudy Holyst (Blood
Research Institute) for assisting with plate reader operation and
troubleshooting, and for providing quantities of PAR agonists
used in our studies; Dr. Omoz Aisiku, Dr. Karen De Ceunynck,
and Dr. Robert Flaumenhaft (Beth Israel Deaconess Medical
Center) for helpful discussions; Dr. Sheng Cai (Marquette
University) for assistance with LC-MS and NMR instruments;
ACD Labs and ChemAxon Inc. for NMR processing and
prediction software; Shimadzu Inc. for a grant to support the
purchase of the LC-MS used to support synthetic chemistry
efforts, Marquette University for startup funding and research
grants for D.G., K.K., and T.F., and the National Heart, Lung,
and Blood Institute (R15HL127636) for support of our research
program.
Part 4. Data analysis
Concentration-response curves were obtained by measuring
the maximal increase in emission intensity (Δ Relative
Fluorescence Units, RFU) from basal levels (average of 20 scans
prior to agonist addition), then dividing by the average basal
levels to give Δ RFU/basal. From this data, % stimulation was
obtained by normalizing Δ RFU/basal for each column of the
plate with a control well from the same column where vehicle
had been added instead of antagonist solution. The Δ RFU/basal
value from the vehicle well was considered to be 100%
stimulation for each column. This normalization was necessary
due to increasing background signal over time, presumably due
to dye efflux. Data (% stimulation versus log concentration) was
then plotted for 3 or more repetitions and analyzed using
GraphPad Prism (versions 5 or 6). All concentration-response
curves were fitted using 4-variable non-linear regression,
showing the standard error of the mean (SEM) for each
concentration.
Notes
An earlier version of this manuscript was published at the
preprint server ChemRxiv:
https://doi.org/10.26434/chemrxiv.5907394.v1
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Cell preparations (under aseptic conditions), loading of
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Supplementary Material
NMR spectra and LC-MS spectra are available with the online
version at https://doi.org/10.1016/j.bmc.2018.XX.XXX.