Discovery of 1,3-diaminobenzenes as selective inhibitors of platelet activation at the PAR1 receptor

Article abstract of DOI:10.1021/ml2002696

A high-throughput screen of the NIH-MLSMR compound collection, along with a series of secondary assays to identify potential targets of hit compounds, previously identified a 1,3-diaminobenzene scaffold that targets protease-activated receptor 1 (PAR1). We now report additional structure-activity relationship (SAR) studies that delineate the requirements for activity at PAR1 and identify plasma-stable analogues with nanomolar inhibition of PAR1-mediated platelet activation. Compound 4 was declared as a probe (ML161) with the NIH Molecular Libraries Program. This compound inhibited platelet aggregation induced by a PAR1 peptide agonist or by thrombin but not by several other platelet agonists. Initial studies suggest that ML161 is an allosteric inhibitor of PAR1. These findings may be important for the discovery of antithrombotics with an improved safety profile.

Full text of DOI:10.1021/ml2002696

Letter
pubs.acs.org/acsmedchemlett
Discovery of 1,3-Diaminobenzenes as Selective Inhibitors of Platelet
Activation at the PAR1 Receptor
Chris Dockendorff,*,† Omozuanvbo Aisiku,‡ Lynn VerPlank,† James R. Dilks,‡ Daniel A. Smith,‡
Susanna F. Gunnink,‡ Louisa Dowal,‡ Joseph Negri,† Michelle Palmer,† Lawrence MacPherson,†
Stuart L. Schreiber,†,§ and Robert Flaumenhaft*,‡
†Chemical Biology Platform and Probe Development Center, Broad Institute of Harvard and MIT, 7 Cambridge Center, Cambridge,
Massachusetts 02142, United States
‡Division of Hemostasis and Thrombosis, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School,
Boston, Massachusetts 02215, United States
§Howard Hughes Medical Institute, Broad Institute of Harvard and MIT, 7 Cambridge Center, Cambridge, Massachusetts 02142,
United States
S
* Supporting Information
ABSTRACT: A high-throughput screen of the NIH-MLSMR
compound collection, along with a series of secondary assays to
identify potential targets of hit compounds, previously identified a
1,3-diaminobenzene scaffold that targets protease-activated
receptor 1 (PAR1). We now report additional structure−activity
relationship (SAR) studies that delineate the requirements for
activity at PAR1 and identify plasma-stable analogues with
nanomolar inhibition of PAR1-mediated platelet activation.
Compound 4 was declared as a probe (ML161) with the NIH
Molecular Libraries Program. This compound inhibited platelet aggregation induced by a PAR1 peptide agonist or by thrombin
but not by several other platelet agonists. Initial studies suggest that ML161 is an allosteric inhibitor of PAR1. These findings may
be important for the discovery of antithrombotics with an improved safety profile.
KEYWORDS: platelet activation, PAR1 inhibitor, allosteric inhibitor, 1,3-diaminobenzene, ML161, MLPCN MLSMR
ntiplatelet agents are an important part of regimens for
patients at risk for adverse cardiovascular events,
elevation of liver transaminases and QTc prolongation at higher
doses in phase II trials.6−8 Other small molecule PAR1
inhibitors have been described9−13 but have not been tested
in clinical trials. Safety and efficacy issues with current
antiplatelet therapies, including investigational PAR1 antago-
nists, highlight the need for antiplatelet agents that may act via
alternative mechanisms.
A
decreasing the probability of such events by minimizing
thrombus formation following rupture of atherosclerotic
plaque.1 However, contemporary antiplatelet drugs are only
partially effective as evidenced by the substantial recurrence rate
of arterial thrombosis despite current therapy.2 Furthermore,
the beneficial effect of antiplatelet agents is tempered by an
increased risk of dangerous hemorrhagic complications.3 Many
developmental programs focused on new drug targets on
platelets have been initiated over the past decade. Prominent
among these programs have been those identifying and testing
compounds that block protease-activated receptor 1 (PAR1)-
mediated platelet activation.
Several inhibitors of PAR1 have been developed. The most
advanced in clinical trials is vorapaxar (SCH530348), which
was developed from a lead identified by a radioligand binding
approach using a high affinity Thrombin Receptor Agonist
Peptide.4 Vorapaxar is a potent inhibitor of PAR1 but was
associated with an increased risk of intracranial bleeding when
used in combination with standard therapy in a phase III trial
(TRA-CER).5 Atopaxar (E5555) is a second PAR1 inhibitor in
advanced clinical trials. Atopaxar therapy is associated with anti-
PAR1 activity ex vivo; however, its use was associated with
We recently reported14 a high-throughput screen of the
National Institute of Health Molecular Libraries Small
Molecule Repository (NIH-MLSMR) small-molecule library
(∼300000 compounds), undertaken to identify inhibitors of
granule secretion and/or platelet activation.15,16 The primary
screen measured adenosine triphosphate (ATP) secreted from
dense granules following SFLLRN-induced activation through
PAR19−13 using a luciferin/luciferase detection system. Several
chemically tractable scaffolds were identified that inhibited
dense granule secretion. Target identification studies with hits
from this screen showed that compounds with a 1,3-
diaminobenzene core act at PAR1. This paper describes
structure−activity relationship (SAR) studies of this class of
Received: November 10, 2011
Accepted: January 23, 2012
Published: January 30, 2012
© 2012 American Chemical Society
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Letter
PAR1 inhibitors as well as preliminary studies supporting an
allosteric mode of action.
Table 1. SAR at the East End of the 1,3-Diamidobenzene
Scaffold
Several compounds were identified by high-throughput
screening (HTS) with a 1,3-diamidobenzene core. Of these,
compound 4 was selected as a starting point for SAR studies, as
it showed acceptable potency in the primary assay measuring
inhibition of dense granule release (IC50 = 1−10 μM).17
Importantly, it was inactive in a luciferase counterscreen, and it
showed little or no inhibition of platelet activation stimulated
through other receptors (Figure S1 in the Supporting
Information). Inhibition of PAR1 platelet activation by
compound 4 was readily reversible. In addition, the compound
did not inhibit phosphodiesterase 3A (PDE3A), which is
present within platelets and promotes activation by suppressing
cAMP levels.18 Compound 4 also showed acceptable solubility
in PBS (40 μM) and good inhibition in a standard assay with
human platelets measuring SFLLRN-induced surface expres-
sion of P-selectin (Table 1),19 a transmembrane cell adhesion
molecule that is sequestered in platelet α-granules and
expressed on the platelet surface following activation.20
SARs of the 1,3-diamidobenzene scaffold are described in
Tables 1−4. Compounds were prepared according to
representative Schemes 1 or 2 (see the Supporting Information
for details).
Keeping the west side of compound 4 fixed, the aryl
substituent R1 was investigated comprehensively (Table 1), and
analogues were tested in the P-selectin assay. Maximal activities
were observed with ortho substituents (compounds 2−12);
substituents at the meta and para positions had neutral or
negative effects on potency (compounds 13−26). The best
results were observed with electron-withdrawing or neutral
lipophilic ortho groups, with the bromide substituent of
compound 4 proving optimal, giving an IC50 of 0.26 μM.
Methyl (8) and ethyl (9) also showed good potencies, although
a larger ortho substituent (phenyl, 10) was not tolerated. A
select number of heterocycles (furan, 3-pyridyl, and 3-
quinolinyl, 27−29) were poorly active or inactive. In an
attempt to improve the potency, disubstituted analogues 30−
36 were prepared, but all showed lower potencies than 4.
Concurrent with our explorations at the east end of the
scaffold, the alkyl chain at the west end was investigated (Table
2). Optimal potency was observed with a 3-carbon chain (4).
Some potency was retained with the 2-carbon chain (38), but a
4-carbon chain (39) failed to demonstrate inhibition.
Replacement of the alkyl chain with a phenyl ring (40)
decreased activity at the target. We expected that branched alkyl
chains could provide compounds with improved plasma
stability (PS) over 4, which showed moderate stability in
human plasma (80% remaining after 5 h) but poor stability in
mouse plasma (<2%). Mouse plasma stable compounds are
required for study in a number of our in vivo disease models, so
we attempted to address this liability of 4. We hypothesized
that branched alkyl chains could give compounds more
resistant to proteases and esterases, and in fact, 42 and 43
showed some improvement. Compound 42 had only moderate
potency but acceptable mouse PS (83% after 5 h). Cyclo-
pentane 43 had acceptable potency (IC50 = 0.52 μM) but poor
mouse PS (26%). The lack of activity observed with 1,4-
diamide 44 indicates that the 1,3-substitution pattern about the
central ring is important.
a
% Inhibition of platelet P-selectin expression on human platelets
induced by 5 μM SFLLRN. See the Supporting Information for details.
The IC50 value was only determined for compounds inhibiting
expression by at least 90% at 10 μM. The value is an average of three
b
measurements. Measured at 3 μM.
pounds were prepared, but neither pyridyl, benzimidizole, nor
oxazole analogues (45−50) showed any significant activity in
our assay. Substituents at the 2- and 6-positions of the central
In a search for more potent, plasma-stable compounds, we
continued our investigations by varying the central 1,3-
diamidobenzene ring (Table 3). Several heterocyclic com-
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a
Scheme 1. Synthesis of 4 (ML161)
Table 3. SAR at the Central Ring
Scheme 2. Synthesis of 68
Table 2. SAR at the West End of the 1,3-Diamidobenzene
a
Scaffold
a
See the Table 1 footnotes.
which suggests that both amides act as hydrogen bond donors
with the target. The constrained analogues 61−64 were
similarly inactive. Removal of the eastern carbonyl group of 4
was tolerated somewhat (65), but interestingly, much of the
activity was retained with removal of the western carbonyl
group in aniline 66.
In a final attempt to address the mouse PS liability of 4, we
made additional branched alkyl analogues, building upon the
moderate results of 42 and 43. The more highly branched α,α-
dimethylamide 67 was very plasma stable but poorly active, but
moving the branching point further from the amide carbonyl
(68) gave a compound nearly equipotent to 4 (IC50 = 0.29
μM) with good human PS (90% after 5 h) and mouse PS
sufficient for in vivo work (65% after 5 h), as well as adequate
solubility for a probe compound (20 μM in PBS). The
preparation of 4 is described in Scheme 1,21 and the synthesis
of the mouse plasma-stable analogue 68 is depicted in Scheme
2. Prior to these more recent studies, compound 4 was formally
nominated as a molecular probe (ML161) for the study of
platelet activation.14
a
See the Table 1 footnotes.
ring were not tolerated (51 and 52), although alternative
groups could be worth investigating. Two compounds with
reverse amides were screened (53 and 54), and the compound
with the reverse amide at the western position (54) maintained
much of the activity of the parent compound (3).
To explore SARs further and to search for compounds with
improved potencies and physicochemical and biological
properties (especially PS), additional compounds were
prepared and screened. These results are listed in Table 4,
which includes plasma protein binding (PPB) and PS.
Replacement of the western propylamide of 4 with a carbamate
(55) or sulfonamides (56 and 57) gave compounds with
decreased potencies. N-Methyl amides 58−60 were inactive,
The selectivity at PAR1 is supported by studies involving
various platelet activators in the presence of ML161. Washed
human platelets were separately treated with the peptide
AYPGKF (a PAR4 agonist), PMA (a protein kinase C
activator), U46619 (a thromboxane receptor agonist), or
collagen (an agonist of collagen receptors). ML161 did not
inhibit these activators to any significant degree (Figure S1 in
the Supporting Information). ML161 displayed dose-depend-
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a
which are all agonists at alternative platelet GPCRs (Figure S3
in the Supporting Information).
Table 4. Analogues Designed To Improve PS
Continued work on the 1,3-diaminobenzene scaffold was
inspired by our discovery that its mode of action at PAR1 may
differ from reported PAR1 orthosteric inhibitors.22,23 Activation
of platelets via PAR1 cleaved by thrombin24 leads to multiple
downstream effects and observable phenotypic changes,
including granule release and shape change characterized by
extended pseudopodia. Blockade of PAR1 by orthosteric
antagonists, such as those currently under clinical investiga-
tion,4,25 would be expected to inhibit all phenotypic changes. In
contrast, 4 inhibits granule secretion, but not platelet shape
change, as monitored in a SFLLRN-induced platelet
aggregation assay.26 We hypothesize that it may be acting in
an allosteric manner to inhibit select G-protein-coupled
pathways mediated by PAR1. Our preliminary studies suggest
that it inhibits Gαq, which is required for granule release, but
not Gα12/13 signaling, which affects shape change. Additional
evidence for a nonorthosteric inhibition mechanism was
obtained by evaluating dose−response curves of SFLLRN-
induced P-selectin expression in the presence of varying
concentrations of 4 (Figure 1). Instead of a rightward shift of
Figure 1. Dose−response curves of SFLLRN-induced P-selectin
expression in the presence of varying concentrations of the PAR1
inhibitor 4.
the dose−response curve, as expected of an orthosteric
inhibitor, 4 demonstrated insurmountable antagonism at higher
doses, consistent with a noncompetitive inhibitory mechanism.
A quinolone derivative, termed JF5, was previously discovered
in our laboratories as an inhibitor of platelet activation and has
been recently characterized as a PAR1 inhibitor that requires
helix 8 on the intracellular face of PAR1 for its inhibitory
activity.27 Additional investigations are underway to determine
if compounds such as 4 share a similar binding site.
In summary, the 1,3-diaminobenzene scaffold was identified
as an inhibitor of platelet activation via a high-throughput
screen performed with platelet-rich plasma. Medicinal chem-
istry studies delineated the requirements for optimal potency of
this scaffold at PAR1. These include (1) a secondary benzamide
at the eastern side of the molecule, with a small (ethyl or
smaller) electron-neutral or electron-withdrawing ortho sub-
stituent; (2) a 1,3-substitution pattern about the central
benzene ring; and (3) a secondary amine or amide on the
western side of the molecule, with a linear or branched aliphatic
chain fewer than four carbons long. Potency was maintained
and PS improved with the addition of a methyl group β to the
western amide (68). We have demonstrated that compounds
a
b
c
See the Table 1 footnotes. PPB (% bound). PS (% remaining after
5 h). ND, not determined.
ent inhibition of thrombin-induced platelet activation, as
measured by P-selectin expression (Figure S2 in the Supporting
Information). Inhibition of PAR2, a widely distributed
protease-activated receptor, was not evaluated since this
receptor is not present on platelets. ML161 displayed selective
inhibition of SFLLRN and thrombin-induced platelet aggrega-
tion, both of which operate via PAR1, and had no effect on
AYPGKF, thromboxane, or ADP-induced platelet aggregation,
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such as 4 (ML161) may inhibit PAR1 in an allosteric fashion,
which could enable the selective modulation of platelet
activation pathways. Allosteric inhibition of PAR1 could
provide saturable receptor binding and selective modulation
of downstream G-protein signaling pathways. These pharmaco-
logical attributes may decrease the risk of life-threatening
hemorrhage in the setting of anti-PAR1 therapy.28 Analogues of
the 1,3-diaminobenzene scaffold will be important probes for
evaluating this hypothesis.
ASSOCIATED CONTENT
* Supporting Information
■
S
Additional assay results (Figures S1 to S3), assay protocols, and
synthetic procedures and characterization data for 4, 66, and
68. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*Tel: 1-617-714-7460. E-mail: cjdocken@broadinstitute.org
(C.D.). Tel: 1-617-735-4005. E-mail: rflaumen@bidmc.
harvard.edu. (R.F.).
Funding
This work was funded by the NIH-MLPCN program (1 U54
HG005032-1 awarded to S.L.S.) and P01-HL-87203 (R.F.), an
Established Investigator Award from the American Heart
Association (R.F.), and R03 MH-84076-01 (R.F.).
Notes
The authors declare no competing financial interest.
(12) Nantermet, P. G.; Barrow, J. C.; Lundell, G. F.; Pellicore, J. M.;
Rittle, K. E.; Young, M.; Freidinger, R. M.; Connolly, T. M.; Condra,
C.; Karczewski, J.; Bednar, R. A.; Gaul, S. L.; Gould, R. J.; Prendergast,
K.; Selnick, H. G. Discovery of a nonpeptidic small molecule
antagonist of the human platelet thrombin receptor (PAR-1). Bioorg.
Med. Chem. Lett. 2002, 12, 319−323.
ACKNOWLEDGMENTS
■
We thank Stephen Johnston, Chris Johnson, and Mike
Lewandowski of the Broad Institute for analytical chemistry
support, Benito Munoz for reviewing the manuscript, and
Robert Gould for helpful discussions.
(13) Perez, M.; et al. Discovery of novel protease activated receptors
1 antagonists with potent antithrombotic activity in vivo. J. Med. Chem.
2009, 52, 5826−5836.
ABBREVIATIONS
■
(14) VerPlank, L.; Dockendorff, C.; Negri, J.; Perez, J. R.; Dilks, J.;
MacPherson, L.; Palmer, M.; Flaumenhaft, R.; Schreiber, S. L. Probe
reports from the NIH Molecular Libraries Program. Chemical Genetic
Analysis of Platelet Granule SecretionProbe 3, 2010; https://mli.nih.
gov/mli/?dl_id=1254.
(15) Flaumenhaft, R.; Sim, D. S. The platelet as a model for chemical
genetics. Chem. Biol. 2003, 10, 481−486.
(16) Flaumenhaft, R.; Dilks, J. R. Discovery-based strategies for
studying platelet function. Mini Rev. Med Chem. 2008, 8, 350−357.
(17) Briefly, compounds were incubated in platelet-rich plasma in
384-well plates at a concentration of 7.5 μM (for the primary screen)
or at a dose response (for confirmation assays) for 30 min. Next, a
mixture of the CellTiter-Glo (Promega) reagent (for ATP detection
with a luciferase/luciferin system) and SFLLRN (EC50 for platelet
activation ∼5 μM) was used. Each plate was incubated for 15 min at
22 °C, and then, luminescence resulting from ATP-driven oxyluciferin
production was measured by a plate reader. See ref 13 for full details.
(18) Sim, D. S.; Merrill-Skoloff, G.; Furie, B. C.; Furie, B.;
Flaumenhaft, R. Initial accumulation of platelets during arterial
thrombus formation in vivo is inhibited by elevation of basal cAMP
levels. Blood 2004, 103, 2127−2134.
ATP, adenosine triphosphate; HTS, high-throughput screening;
IC50, concentration of inhibitor giving half-maximal activity;
NIH, National Institute of Health; MLPCN, Molecular
Libraries Probe Center Network; NIH-MLSMR, National
Institute of Health Molecular Libraries Small Molecule
Repository; PAR1, protease-activated receptor 1; PPB, plasma
protein binding; PBS, phosphate-buffered saline; PS, plasma
stability; SAR, structure−activity relationship
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(19) For analysis of P-selectin expression, 20 μL of gel-filtered
platelets (0.5-1 × 108/mL) were incubated with the indicated
antagonists and subsequently stimulated with 5 μM SFLLRN (EC50
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