A novel class of ion displacement ligands as antagonists of the αiIbβ3 receptor that limit conformational reorganization of the receptor

Article abstract of DOI:10.1016/j.bmcl.2013.12.122

A collection of αIIbβ3 integrin receptor antagonists possessing a unique MIDAS metal ion displacement mechanism of action is presented. Insight into these agents' structure-activity relationships, binding modality, and pharmacokinetic and pharmacodynamic

Full text of DOI:10.1016/j.bmcl.2013.12.122

Accepted Manuscript
A novel class of ion displacement ligands as antagonists of the αIIbβ3 receptor
that limit conformational reorganization of the receptor
Jian-kang Jiang, Joshua G. McCoy, Min Shen, Christopher A. LeClair, Wenwei
Huang, Ana Negri, Jihong Li, Robert Blue, Amanda Weil Harrington, Sarasija
Naini, George David III, Won-Seok Choi, Elisabetta Volpi, Joseph Fernandez,
Mariana Babayeva, Mark A. Nedelman, Marta Filizola, Barry S. Coller, Craig
J. Thomas
PII:
S0960-894X(14)00003-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2013.12.122
BMCL 21230
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Accepted Date:
24 November 2013
30 December 2013
Please cite this article as: Jiang, J-k., McCoy, J.G., Shen, M., LeClair, C.A., Huang, W., Negri, A., Li, J., Blue, R.,
Harrington, A.W., Naini, S., III, G.D., Choi, W-S., Volpi, E., Fernandez, J., Babayeva, M., Nedelman, M.A., Filizola,
M., Coller, B.S., Thomas, C.J., A novel class of ion displacement ligands as antagonists of the αIIbβ3 receptor that
limit conformational reorganization of the receptor, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://
dx.doi.org/10.1016/j.bmcl.2013.12.122
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A novel class of ion displacement ligands as antagonists of the αIIbβ3 receptor
that limit conformational reorganization of the receptor.
Jian-kang Jiang1, Joshua G. McCoy1, Min Shen1, Christopher A. LeClair1, Wenwei Huang1, Ana
Negri2, Jihong Li3, Robert Blue3, Amanda Weil Harrington3, Sarasija Naini3, George David III3,
Won-Seok Choi3, Elisabetta Volpi3, Joseph Fernandez4, Mariana Babayeva5, Mark A.
Nedelman6, Marta Filizola2, Barry S. Coller3 and Craig J. Thomas*1
1.
2.
3.
NIH Chemical Genomics Center, Division of Preclinical Innovation, National Center for
Advancing Translational Sciences, National Institutes of Health, Bethesda, MD
Department of Structural and Chemical Biology, Icahn School of Medicine at Mount
Sinai, New York, NY
Allen and Frances Adler Laboratory of Blood and Vascular Biology, Rockefeller
University, New York, NY
4.
5.
6.
Proteomics Resource Center, Rockefeller University, New York, NY
Touro College of Pharmacy, New York, NY
Ekam Imaging, Boston, MA
* Corresponding author:
Craig J. Thomas
NIH Chemical Genomics Center
National Center for Advancing Translational Sciences
National Institutes of Health
Bethesda, MD 20892, USA
craigt@mail.nih.gov
Tel: + 1-301-217-4079
Fax: +1-301-217-5736

ABSTRACT: A collection of αIIbβ3 integrin receptor antagonists possessing a unique MIDAS
metal ion displacement mechanism of action is presented. Insight into these agents‟ structure-
activity relationships, binding modality, and pharmacokinetic and pharmacodynamic profiles
highlight the potential of these small molecule ion displacement ligands as attractive candidates
for clinical development.
2

The αIIbβ3 integrin receptor is an important mediator of platelet aggregation and to date
three αIIbβ3 antagonists have been approved for human use by the U.S. Food and Drug
Administration, including the small molecules eptifibatide and tirofiban (Figure 1).1-3 Integrin
receptors are heterodimeric complexes that rely upon coordinated conformational changes to the
 and  subunits to initiate ligand binding, and upon ligand binding to convey signals to within
cells that ultimately alter a wide array of phenotypes ranging from cytoskeletal reorganization, to
cellular differentiation, to specified immune responses.4 The use of αIIbβ3 antagonists to alter
platelet aggregation results in clinical utility for these agents as antithrombotic therapies. The
aforementioned small molecule αIIbβ3 antagonists are modeled after the Arg-Gly-Asp (RGD)
motif found in some of the naturally occurring ligands.5 The binding of both eptifibatide and
tirofiban are particularly reliant upon interactions with a conserved Asp (224) residue found in
the αIIb subunit and span a defined binding pocket to also engage a Mg2+ ion found in the 3
subunit‟s metal ion-dependent adhesion site (MIDAS) domain.5,6 Crystallographic analysis of
these drugs (and other RGD mimetics) demonstrates that after these antagonists (and a related
peptide from the ligand fibrinogen) bind, the 3 unit undergoes a „swing-out‟ motion resulting in
a major change in conformation.5-8 This conformational change has been theorized to contribute
to the thrombocytopenia caused by the RGD mimetic agents by exposing neoepitopes for which
some individuals have pre-formed antibodies.9 Attempts to develop oral RGD mimetic agents to
inhibit αIIbβ3 failed because the agents produced thrombocytopenia and some were associated
with a paradoxical increase in mortality.10,11 This latter effect has been theorized to be due to
these agents‟ ability to “prime” the receptor to bind fibrinogen as the conformational change
induced by the agents leaves the receptor in a high affinity ligand binding state.3,11-15 Thus, while
αIIbβ3 represents a validated drug target, there remains a need to identify small molecule αIIbβ3
antagonists that do not alter the β3 subunit conformation since these may have better safety
profiles.
In an attempt to identify a novel small molecule capable of retaining the advantageous
physiological effects associated with αIIbβ3 receptor antagonism without the negative
consequences of receptor „priming‟ we screened and identified
a
novel 5H-
[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one based small molecule antagonist.16 This agent, named
3

RUC-1 (1, Figure 1), was found to inhibit adhesion of platelets to fibrinogen and block ADP-
induced platelet aggregation at modest potencies. It was also noted that 1 was selective for the
αIIbβ3 receptor over related integrins αVβ3 and α2β1 and for human αIIbβ3 over murine and rat
αIIbβ3.16,17 The specificity for human αIIb/β3 resided in the αIIb subunit and thus its
antithrombotic properties could be determined in a transgenic murine model in which mice
express human αIIb in complex with murine β3 (hαIIb/mβ3).16,17 Intriguingly, the 3 domain of
the receptor was shown to undergo little or no swing-out and the receptor did not undergo
priming upon compound binding when compared with eptifibatide and tirofiban binding.16-18 An
X-ray crystallographic analysis of this agent bound to the αIIbβ3 receptor confirmed molecular
dynamic (MD) simulations suggesting that 1 bound exclusively to the αIIb domain of the
receptor (PDB codes: 3NID, 3NIG, 3NIF).18 Pharmacokinetic studies conducted in dogs
demonstrated rapid oral absorption (Tmax ≈ 0.5 hr), high oral bioavailability (~92%), and rapid
elimination (t½ ≈ 2hr)(Table S1).
To optimize this agent a series of analogues were synthesized and examined for improved
potency in the aforementioned platelet/fibrinogen adhesion and platelet aggregation assays.
From these efforts, an analogue of 1 named RUC-2 (2, ML165, Figure 1) was identified that
possessed increased affinity for the receptor and maintained the favorable lack of effect on
receptor conformation as judged by several analyses, including electron microscopy of αIIbβ3
nanodiscs, Stokes radii measurements by gel filtration, exposure of ligand-induced binding site
epitopes for monoclonal antibodies, and dynamic light scattering.19 Importantly, 2 also did not
prime the receptor to fibrinogen binding nor in the majority of cases, induce recruitment to
platelets of αIIbβ3-dependent antibodies from the serum of patients who developed
thrombocytopenia when treated with eptifibatide or tirofiban.19 X-ray crystallographic studies
revealed that 2 possessed a unique binding modality whereby the primary amine of the chemical
structure of the ligand replaced the Mg2+ found within the MIDAS domain by binding to Glu220,
one of the major Mg2+ coordinating residues (PDB code: 3T3M).19 This agent represents the first
in a novel pharmacological class, which we term “ion displacement ligands” with remarkable
selectivity and a lack of detrimental conformational events for the ligand-bound receptor. Here,
we detail the structure-activity relationships (SAR) revealed during the optimization effort,
highlight additional MD simulations that underscore the unique binding of this class of
molecules and provide pharmacokinetic (PK) parameters for advanced analogues.
4

Figure 1. Chemical structures of tirofiban, eptifibatide, RUC1 (1) and RUC2 (ML165, 2).
The optimization of 1 focused on structural alteration to the piperazine domain and the 2-
and 6-positions of the core 5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one heterocycle. Numerous
analogues were synthesized and tested and a collection of representative SAR is presented in
table 1. It rapidly became apparent that the secondary amine of the piperazine domain would not
tolerate alteration as judged by conversions to piperidine (analogue 3), morpholine (analogue 4)
and N-methyl-piperazine (analogue 5). These results were consistent with the crystallographic
analysis that demonstrated the key interaction of this moiety with αIIbAsp224.18 All additional
attempts to alter the piperazine ring system led to a loss in activity, including steric additions to
the 2- and 6-positions (for instance 2,6-dimethylpiperazine), bicyclic analogues (for instance 2,5-
diazabicyclo[2.2.2]octane), alternate diamines (for instance N-phenylazetidin-3-amine), and
selected spirocyclic compounds (for instance 2,7-diazaspiro[4.4]nonane)(data not shown).
Changes to the 2-position of the 5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one were limited to
fluorine and selected alkyl groups (R2 in Table 1). A methyl group at this domain remained
active (slight loss in activity) while any larger substitution resulted in an inactive analogue (data
not shown). The fluorine substitution (analogue 6) retained equivalent activity to that of 1.
Changes to the 6-position of the heterocycles included modified alkyl and aryl groups. Changing
5

from the ethyl in 1 to a tert-butyl (analogue 7) produced an agent with equivalent activity as did
the phenyl analogue 8. Multiple ring substitutions on the phenyl ring were explored including
carboxylic acids (analogues 9 and 10). In general, potencies for these agents were equivalent to
that displayed by 1. Incorporation of a 2-aminoacetamide at the meta position of the phenyl ring
(analogue 2), however, produced a striking increase in potency in both the adhesion assay (83%
at 1 M) and the aggregation assay (0.096 M). The aforementioned crystallographic analysis
identified the novel interaction between 2 and the MIDAS domain19 and we endeavored to fully
explore the SAR of this new binding modality. Interestingly, a single methyl group on the alkyl
chain [(S)-2-aminopropanamide](analogue 12) results in a significant loss in activity. This result
conforms to the crystallographic analysis that demonstrates a limited amount of free space as the
amide NH forms a key hydrogen bond with the backbone Asn215 carbonyl, resulting in proximal
binding to Asn215 limiting the required space for the incorporated methyl group. Furthermore,
additional bulk placed upon the primary amine (data not shown) eliminated activity. Again,
crystallographic analysis of 2 highly suggests that the replacement of the Mg2+ ion by the
primary amine allows for a nearly identical conformation for the amino acid side-chains that
define the MIDAS domain and mitigates any alteration to this key pharmacophore. Modification
of the phenyl ring (substitutions X2 and X3 in Table 1) maintained similar activity and selected
analogues incorporating ring nitrogens [13 and 14] provided slight increases in potency and
improved key physicochemical properties (see below). Based upon the modest potency
enhancement demonstrated by 13 and on selected pharmacokinetic and pharmacodynamic
studies with this agent, we further confirmed that 13 neither primes the receptor for fibrinogen
binding nor changes the β3 conformation as judged by the binding of a conformation-specific
monoclonal antibody; it also does not possess any activity versus the V3 receptor suggesting a
high degree of selectivity for αIIbβ3 akin to our studies with 2 (manuscript in preparation).19
6

We previously reported the synthesis of 2 from an 5-(3-nitrophenyl)-1,3,4-thiadiazol-2-
amine (see reference 19). Analogues reported here-in, including 13 were synthesized utilizing
the same protocol from commercially available materials [for instance, 5-nitronicotinic acid for
the production of 13 (Scheme S1)]. To facilitate the multiple follow-up studies undertaken with
13 we optimized this route utilizing 5-bromonicotinic acid (Scheme 1). Conversion to the acid
chloride followed by addition of hydrazinecarbothioamide yielded the required acyl-
hydrazinecarbothioamide (17) in greater than 50% yield over two steps. Addition of 17 to PPA
at 100 °C cleanly formed the 1,3,4-thiadiazol-2-amine 18 which was converted to the 7-chloro-
7

5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one 19 by condensing with methyl-3-chloro-3-
oxopropanoate followed by chlorination via POCl3 treatment. Nucleophilic displacement of the
3-chloro moiety with 1-Boc-piperazine was followed by a Buchwald-Hartwig amination using
Boc-protected 2-aminoacetamide which proceeded with an 80% yield. Treatment with TFA
afforded deprotection of both Boc protecting groups.
To better understand the subtle improvement in potency found through incorporation of
the ring nitrogens into the core of 2 we carried out molecular dynamic simulations of the integrin
αIIbβ3 headpiece bound to 2, 13, or analogue 14. Starting from the crystallographic pose of 2,
each agent‟s initial geometry was optimized ab initio using a restricted Hartree-Fock (RHF)
calculation with a 6-31G (d) basis set, as implemented in the Gaussian 09 program,20 and the
8

resulting geometries used to calculate electrostatic potential-derived (ESP) point charges
employing the RESP methodology, as implemented in the AMBER suite of programs,21 and
reported previously.19 Ligands were then docked in an equivalent geometric orientation within
the binding pocket to that seen in the crystal structure of the 2-αIIbβ3 headpiece. All three
resulting IIb3 headpiece-drug systems, including the SyMBS and ADMIDAS Ca2+ metal ions
and the crystallographic water molecules found in all metal binding sites, were immersed in
truncated octahedral boxes of ~22,000 TIP3P water molecules and neutralized by the addition of
7 Na+ counter-ions at random locations. Each system was simulated for 20 ns by all-atom,
standard MD simulations, using the general AMBER force field (GAFF) for the ligands and the
ff12SB force field for the protein, within the AMBER12.0 suite of programs.21
Detailed analysis of representative snapshots from the 20 ns simulation trajectories of 2-
αIIbβ3 (Figure 2, panel A), 14-αIIbβ3 (Figure 2, panel B), and 13-αIIbβ3 (Figure 2, panel C)
shows that both 14 and 13 maintain a similar geometry to the pose adopted by 2 during
dynamics. Stable interactions monitored during simulations included the piperazine-Asp224
hydrogen bond, the primary amine interactions with Glu220 and the backbone carbonyl oxygen
of Asp218, and a hydrogen bond between the phenylacetamide and the backbone carbonyl
oxygen of 3 Asn215. We additionally noted a stable π-π stacking interaction between each of
the compounds‟ fused ring and the αIIb Tyr190 aromatic ring and a water-mediated hydrogen
bond between the carbonyl group in each of the compounds‟ fused ring and the side chain
carboxyl group of αIIb Asp232. The average distances between the aforementioned atoms and
protein residues monitored during simulation are reported in Supplemental Table 2. There are no
direct interactions between residues of the αIIbβ3 headpiece and 13 and 14 that are not also
present in 2, making understanding of the slight potency enhancement for these agents not
obvious. There are additional water-mediated interactions between the inserted ring nitrogen in
13 and Tyr166 that may differentiate the binding of this congener from that of 2 (Figure
2). Similarly, the inserted nitrogen in 14 appears to support additional water-mediated
interactions with Tyr166 and Arg214 (Figure 2B). These additional water-mediated interactions
with the 3 subunit may account for the slightly higher affinity of 13 and 14 relative to 2.
9

Figure 2. Visualization of MD simulations following 20 ns simulation trajectories of 2- αIIbβ3
(panel A), 14- αIIbβ3 (panel B), and 13- αIIbβ3 (panel C).
10

The novel binding modality demonstrated by these agents make them ideal probe
molecules for further study of ion-displacement antagonists as a novel means of pharmacological
intervention. We were also interested in examining 2 and its structural congeners for potential
translation into clinical reagents. Given the limitations associated with the current approved
drugs there is a need to develop novel αIIbβ3 antagonists that possess the appropriate
combination of attributes to establish them as new drugs. As such, we examined key analogues
for selected physicochemical and preclinical ADME properties. From the outset, we prioritized
2, 13 and 14 for advanced study. Prior to ADME analysis we noted that 14 possessed poor
chemical stability as a solution (both DMSO-based and aqueous) and consequently this analogue
was deprioritized. Initial assessments included an analysis of the aqueous solubility and
biological stability of 2 and 13 (Table 2). Both analogues were highly stable in mouse plasma
and each agent possessed a positive solubility profile across a range of pH values. Neither
eptifibatide nor tirofiban possess oral bioavailability, which may be attributed to their having
formal positively and negatively charged elements in their chemical structure. Likewise, 2 and
13 are charged drugs at physiological pH (pKa values for 2 were experimentally determined at
6.41 and 8.08) and thus, as expected, PAMPA and Caco2 permeability analyses indicated that it
was unlikely that either agent possesses high oral bioavailability (data not shown). However, the
intended clinical application for this agent is in the early, pre-hospital treatment of patients with
myocardial infarction in hopes of ameliorating the mortality and morbidity by decreasing the
resulting tissue death and long-term cardiac dysfunction. Several studies have explored the use of
αIIbβ3 antagonists in a pre-hospital setting and present evidence that such therapeutic
intervention translates into both acute and long-term benefit.22-24 To apply novel αIIbβ3
antagonists in such a setting requires a facile administration route and method of delivery. The
anticipated use of autoinjector technology to provide a bolus dose of drug via IM injection would
provide a means to deliver an anticipated dose (~ 1 mg/kg) of drug. Autoinjectors are limited in
their volume of delivery and therefore require the API to possess significant solubility in the
delivery vehicle (a primarily aqueous formulation). Both 2 and 13 are soluble small organic
molecules as judged by both kinetic and thermodynamic solubility assessments, but 13 is much
more soluble as judged by chemiluminescent nitrogen detection (CLND)(Table 2) and, in fact,
many of the measured values exceeded the detection limit of these methods. An LC method was
also performed and correlated well with the data shown in Table 2. Additional studies have
11

demonstrated that 13 can be solubilized long-term up-to and exceeding levels of 60 mg/mL,
which far exceeds the level that can be achieved for 2. As a result, for the ideal delivery
mechanism, 13 represents a strong candidate agent.
Effort was also put forth to examine several predictive in vitro toxicology markers. Both
2 and 13 were further profiled for hERG inhibition with neither agent presenting activity versus
this common target at all concentrations tested. As these agents represent cationic, amphiphilic
drugs we also examined the capacity of 13 to produce a drug-induced phospholipidosis event.25
No appreciable induction of phospholipidosis was noted nor did 13 alter nuclear size or induce
stenosis (data not shown). The fraction unbound (fu) for 13 in both whole blood and plasma
protein was high (>85% and >30%, respectively; data not shown).
An analysis of the microsomal stability for 2 and 13 suggested that 2 was highly resistant
to metabolism in three species (mouse, rat and human microsomes) while 13 possessed a more
modest stability profile. Hepatocyte stability (mouse) was also assessed for 13 and found to be
negligible (>95% remaining after 120 minutes). The exposure (PK) profile for each agent (both
IV and IP administration route) suggested that each agent has a rapid onset and a limited time of
exposure following a dose of 0.03 mgs. These data are generally contradictory to the
microsomal stability results, suggesting an alternate route of metabolism/clearance. The limited
exposure times are beneficial for use in the proposed pre-hospital infarction indication where a
drug-induced antiplatelet effect is desired for a limited time interval, thus allowing first
responders to administer drug without preventing the physicians in the receiving hospital from
instituting whichever drugs and interventions they think best.
12

In summary, we report the discovery and optimization of a novel class of αIIbβ3
antagonists that demonstrate a novel ion displacement mechanism of action. This binding
modality allows our lead agents 2 and 13 to inhibit receptor function without significant β3
conformational reorganization, thus avoiding receptor priming and perhaps decreasing the
likelihood of developing thrombocytopenia. Our results suggest that advanced analogues 13 and
14 bind to the protein in a similar fashion as 2 with additional water-mediated interactions that
potentially account for modest affinity gains. Neither 2 nor 13 possess any predictive toxicology
liabilities and 13 possesses both the required aqueous solubility and chemical properties for the
specified clinical applications. Pharmacodynamic analysis of both 2 and 13 in an engineered
mouse model and 13 in a non-human primate model (manuscript in preparation), support the
advancement of this class of αIIbβ3 antagonists into advanced preclinical studies.
13

Acknowledgments: The authors thank Jim Bougie, Thomas Daniel and William Leister for
compound purification, as well as Paul Shinn, Danielle VanLeer for assistance with compound
management. This research was supported by the Molecular Libraries Initiative of the National
Institutes of Health Roadmap for Medical Research grant U54HG005033 and the Intramural
Research Program of the National Human Genome Research Institute at the National Institutes
of Health. This work was further supported, in part, by grants HL19278, HL13629, and
HL48675, and from the National Heart, Lung, and Blood Institute, and CTSA grant
ULRR024143 from the National Center for Research Resources, NIH. Computations were run,
in part, on resources available through the Scientific Computing Facility at Icahn School of
Medicine at Mount Sinai and in part on TeraGrid advanced computing resources provided by
Texas Advanced Computing Center through TG- MCB080077.
Supporting Information
Supplementary data associated with this article can be found, in the online version, at…
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