Platelet activation occurs in response to vascular injury, which triggers an array of cascading signaling events that potentiate the formation of thrombi to control bleeding. One downstream effect of activation is the secretion of granules, which plays a role in the formation of thrombi. Platelet activation inhibitors that target various surface receptors and cytosolic pathways within platelets are well validated for inhibiting thrombosis, clinically preventing adverse cardiovascular events such as intermittent claudication and stroke, and reducing mortality and morbidity in acute myocardial infarction. These inhibitors have provided a greater understanding of the mechanisms of platelet biology. Some inhibitors are currently in use in the clinical setting or in development for the treatment of thrombotic conditions. Unfortunately, many of the characterized platelet inhibitors can cause undesired side effects in patients. None of the characterized inhibitors are known to target the granule secretion machinery. We report the outcome of a high-throughput chemical library screen and a series of secondary conformation assays to identify novel, small-molecule inhibitors of platelet activation, specifically fitting into three possible attribute classes: 1) inhibitors of the pathways required for dense granule secretion, 2) inhibitors of pathways required for granule secretion, and 3) inhibitors of upstream g-coupled protein receptor (GPCR) signaling. A primary assay was developed to measure the release of granules from platelets activated through the thrombin GPCR PAR1. Of 302,457-screened compounds, 28 prioritized compounds were selected for additional characterization in secondary specificity assays, which yielded three probe candidates. These three probe candidates met the criteria as possible modulators of GPCR signaling in platelet activation. Of these, a cyanopyridone scaffold (ML160) showed acceptable potency (av. 3.75 μM ± 4.53) and inhibited P-selectin expression in a concentration-dependent manner (EC₅₀<3 μM), as well as inhibiting SFLLRN-induced thrombus formation. This new GPCR-specific probe will be useful in future cell-based investigations of the complex mechanisms of platelet activation and in vivo studies as an antithrombotic agent in murine models.

Probe Structure and Characteristics

Recommendations for Scientific Use of the Probe

The goal of this project is to identify novel probes that inhibit platelet activation. This project focuses on the pathways involved in the activation of platelets initiated through the thrombin receptor pathway. The SFLLRN peptide activates platelets through the G-protein coupled receptor (GPCR) protease-activated receptor-1 (PAR1). The SFLLRN peptide is derived from the cleavage of the N-terminus of the PAR1 receptor by thrombin (1). Upon activation, the platelets undergo many changes induced by multiple signaling cascades. One downstream effect of activation is the secretion of granules, which then potentiate platelets in controlling bleeding. Granule secretion also contributes to the growth of thrombi, which if not controlled properly can cause blockage of blood vessels and trigger myocardial infarction (MI). Probes from this project are defined as fitting into three possible attribute classes: 1) inhibitors of the pathways required for dense granule secretion, 2) inhibitors of the pathways required for granule secretion, which includes dense granules, alpha-granules, and lysosomes, and 3) inhibitors of upstream GPCR signaling. The first and second classes of inhibitors target distal mechanisms of secretion (i.e., the platelet second messenger systems) (2). The third class of inhibitors targets the proximal cell-surface receptors.

The probes identified in this project will be used to further investigate the complex mechanisms of platelet activation. As these probes have been classified as GPCR-specific, additional assays developed in the Assay Provider’s laboratory (Robert Flaumenhaft, MD) will be performed to determine the specific GPCR target and site of action on the target GPCR signaling pathway. Depending on the results of these additional assays, these probes could be evaluated for their potential as antithrombotic agents in an in vivo murine model.

The GPCR-specific probe presented in this report is a valuable tool compound with unique attributes. Additional experimentation carried out after identification of the probe has demonstrated that this compound acts specifically on the GPCR PAR1. It exhibits several characteristics that distinguishes it from published orthosteric inhibitors of PAR1. This area of investigation is of particular interest to the Assay Provider and others studying clinical aspects of platelet biology as platelets represent a good cellular target for pharmacological manipulation via allosteric modulation (3). The central challenge in developing an antiplatelet agent is how to achieve a potent antiplatelet effect while avoiding hemorrhagic complications. Development of modulators of platelet function with novel modes of action, such as the probe described here, could limit bleeding complications associated with current antiplatelet agents (4).

1. Introduction

Platelets are small, anucleate cells that circulate in the blood of mammals, and are required for maintaining hemostasis. The number of platelets circulating and their relative activity requires strict control. Too few platelets or too little activity can cause uncontrolled bleeding, while too many platelets and too much activity can cause acute thrombosis formation. Thrombi formation can block blood vessels causing myocardial and cerebral infarctions. Platelets undergo dramatic phenotypic changes in response to vascular injury that lead to the formation of thrombosis. These phenotypic changes are the result of an activation process induced by a diverse array of agonists (e.g., thrombin, adenosine diphosphate [ADP], epinephrine, and thromboxane A₂) that act on platelet surface receptors. Phenotypic changes include granule secretion, shape change, and expression of specific cell-surface proteins. Platelet activation inhibitors are well validated for the inhibition of thrombosis and the clinical prevention of adverse cardiovascular events, including MI and stroke (5). These inhibitors target various platelet G-protein coupled receptors (GPCRs), as well as certain cytosolic enzymes, which comprise key parts of the platelet activation pathway. Examples of important drugs within the category of GPCR inhibitors include: clopidogrel (Plavix^®, Bristol-Myers Squibb/Sanofi Aventis), the metabolism of which leads to irreversible inhibition of the ADP receptor P2Y₁₂; and SCH 530348, a potent inhibitor of protease activated receptor (PAR1) currently in Phase 3 clinical trials. Examples of platelet enzyme inhibitors include: aspirin, an irreversible inhibitor of COX1; and cilostazol (Pletal^®, Otsuka), a cyclic nucleotide phosphodiesterase 3 (PDE3) inhibitor in clinical use for intermittent claudication. The structures of these compounds are depicted in Figure 1.

Figure 1

Examples of Platelet Activation Inhibitors.

Unfortunately, many of these drugs and others within the broader category of antithrombotics suffer from significant liabilities. For example, P2Y₁₂ inhibition leads to a significant risk of dangerous hemorrhagic side effects (6). Key platelet enzymes such as COX1 and PDE3 are also present in many other cell types, and their inhibition can lead to undesired complications (7). For this reason, novel inhibitors of platelet activation are of great interest, particularly those that modulate new targets, those that inhibit select features of platelet activation, and those that demonstrate novel pharmacological properties such as allosteric inhibition (3).

Platelets are best studied via chemical genetic analysis due to their anucleate state rendering them unsusceptible to direct genetic manipulation. Platelets in plasma are a readily available reagent in the form of expired donor-collected units of platelet-rich plasma (PRP) from blood centers nationwide. The Assay Provider’s laboratory previously developed and executed a small molecule screen to study platelet activation in PRP using granule secretion as a measurement of platelet activation (2). The assay measures the amount of ATP released into the plasma via secreted dense granules upon activation (8).

The purpose of these studies is to use a cell-based platelet assay to identify novel inhibitors of platelet activation. Inhibitors identified from this project are defined as fitting into three possible attribute classes: 1) inhibitors of the pathways required for dense granule secretion, 2) inhibitors of the pathways required for granule secretion, which includes dense granules, alpha-granules, and lysosomes, and 3) inhibitors of upstream GPCR signaling. The first and second classes of inhibitors target distal mechanisms of secretion (i.e., the platelet second messenger systems) (2). There are no known compounds that selectively inhibit granule secretion in either the first or second class. The third class of inhibitors targets the proximal cell-surface receptors. One important platelet GPCR mentioned above is PAR1, which undergoes a cleavage reaction by the serine protease thrombin that allows its tethered peptide ligand to agonize the receptor (9).

The primary screen was adapted from the cell-based phenotypic screen previously executed by the Assay Provider’s laboratory with an aim to identify inhibitors of platelet activation (2). Platelets were activated with the thrombin receptor activation peptide SFLLRN, which binds the PAR1 GPCR (1). Upon activation, the platelets undergo many changes induced by multiple signaling cascades, which results in the secretion of granules. Platelet activation was quantified by measurement of ATP from the secreted granules, as measured by a luciferin-luciferase assay. Cilostazol, a known platelet aggregation inhibitor that targets PDE3, was used as a positive control in the primary assay (see Figure 1). It was chosen as a positive control for the high level of inhibition seen in the primary assay, although it acts on a target that is not relevant to this project.

The secondary assays in this project served to determine the specificity of the inhibitors identified in the primary assay. Specifically, the first secondary assay distinguishes between inhibitors of granule secretion and those acting nonspecifically on the ATP-detection system. From a list of compounds acting specifically to decrease the levels of secreted ATP-rich granules from platelets, those compounds known to inhibit intracellular platelet targets were filtered out. The final secondary assays were designed to differentiate between inhibitors of the distal granule secretion events and those inhibiting proximal GPCRs.

2. Materials and Methods

2.2. Probe Chemical Characterization

After preparation as described in Section 2.3, the probe (ML160, CID 824820, SID 87556782, MLS002699917) was analyzed by HPLC, both ¹H and ¹³C NMR, and high-resolution mass spectrometry. The data were consistent with the structure of the probe, and HPLC indicated an isolated purity of greater than 99%. The relevant data is provided below. The observed solubility of the probe in PBS buffer (pH 7.4) was determined to be 1.6 μM. Aqueous stability in PBS was monitored over a 48-hour period, and the data is provided in Figure 2, indicating excellent aqueous stability. Plasma protein binding (PPB) was determined to be 96% bound in human plasma and 91% bound in murine plasma. The probe is stable in human and murine plasma with essentially 100% remaining after a 5-hour incubation period. The solubility, PPB, and plasma stability results are summarized in Table 2 (see Section 3.4). The probe and five additional analogs were submitted to the SMR collection [MLS002699917 (probe), MLS002699918, MLS002699919, MLS002699920, MLS002699921, and MLS002699923 (analogs)].

Figure 2

Probe Stability in PBS at 23°C.

2.3. Probe Preparation

Chemistry Experimental Methods

The probe and the analogs of the cyanopyridone class were made via an efficient 2-pot process, without requiring chromatography in any step. 2-cyanoacetic acid was coupled with a series of primary amines, and the resulting amides were subjected to Knoevenagel condensations with several different benzaldehydes. The resulting enamides were condensed with malononitrile; stirring in the air led to the formation of the desired aromatic cyanopyridones (10,11), which readily precipitated and were washed with various solvents to give materials with excellent purity in most cases. The synthesis of the probe is provided in Scheme 1.

Scheme 1

Synthesis of Cyanopyridone Probe.

General Details. All reagents and solvents were purchased from commercial vendors and used as received. NMR spectra were recorded on a Bruker 300 MHz or Varian UNITY INOVA 500 MHz spectrometer as indicated. Proton and carbon chemical shifts are reported in parts per million (ppm;δ) relative to tetramethylsilane, CDCl₃ solvent, or d₆-DMSO (¹H δ 0, ¹³C δ 77.0, or ¹³C δ 39.5, respectively). NMR data are reported as follows: chemical shifts, multiplicity (obs. = obscured, br = broad, s = singlet, d = doublet, t = triplet, m = multiplet); coupling constant(s) in Hz; integration. Unless otherwise indicated, NMR data were collected at 25°C. Flash chromatography was performed using 40–60 μm Silica Gel (60 Å mesh) on a Teledyne Isco Combiflash R_f system. Tandem liquid chromatography/mass spectrometry (LCMS) was performed on a Waters 2795 separations module and Waters 3100 mass detector. Analytical thin layer chromatography (TLC) was performed on EM Reagent 0.25 mm silica gel 60-F plates. Visualization was accomplished with UV light and aqueous potassium permanganate (KMnO₄) stain followed by heating. High-resolution mass spectra were obtained at the MIT Mass Spectrometry Facility with a Bruker Daltonics APEXIV 4.7 Tesla Fourier Transform Ion Cyclotron Resonance mass spectrometer. Compound purity and identity were determined by UPLC-MS (Waters, Milford, MA). Purity was measured by UV absorbance at 210 nm. Identity was determined on a SQ mass spectrometer by positive electrospray ionization. Mobile Phase A consisted of either 0.1% ammonium hydroxide or 0.1% trifluoroacetic acid in water, while mobile Phase B consisted of the same additives in acetonitrile. The gradient ran from 5% to 95% mobile Phase B over 0.8 minutes at 0.45 mL/min. An Acquity BEH C18, 1.7 μm, 1.0 × 50 mm column was used with column temperature maintained at 65°C. Compounds were dissolved in DMSO at a nominal concentration of 1 mg/mL, and 0.25 μL of this solution was injected.

Step 1. Preparation of 2-cyano-N-(p-tolyl)acetamide: p-Toluidine (2.14 g, 20.0 mmol) and 2-cyanoacetic acid (1.79 g, 21.0 mmol) were sealed in a flask with a stir bar and dissolved with tetrahydrofuran (THF, 67 mL) to form a pale yellow solution. Next, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI; 4.41 g, 23.0 mmol) was added, and the reaction was stirred for 22 h. The mixture was then poured onto water (500 mL) in an Erlenmeyer flask, stirred for 1 h, then filtered and washed with water. The white solid was dried under suction for 30 minutes (min), then dried under high vacuum to yield the title compound (3.10 g, 89%) in excellent purity (LCMS). The material was used immediately in the subsequent step without further characterization.

Steps 2 and 3. Preparation of the Probe (6-amino-2-oxo-4-phenyl-1-(p-tolyl)-1,2-dihydropyridine-3,5-dicarbonitrile). 2-cyano-N-p-tolylacetamide (697 mg, 4.00 mmol) and ethanol (13.3 mL) were added to flask with a stir bar, forming a white suspension. Benzaldehyde (0.554 mL, 4.20 mmol) and piperidine (four drops by Pasteur pipette) were then added. A water condenser was attached to the flask, and the reaction was refluxed for 5 h. During this time a yellow solid gradually precipitated. After 5 h, LCMS analysis indicated complete conversion to the intermediate Knoevenagel adduct. Malononitrile (277 mg, 4.20 mmol) and more piperidine (0.40 mL, 4.00 mmol) were added, and the yellow suspension was stirred under air for 17 h. The resulting white solid was filtered and washed with ice-cold ethanol, then dried under high vacuum to yield the title compound (627 mg, 48%). ¹H NMR (300 MHz, d₆-DMSO): δ 7.82 (br s, 1 H), 7.62–7.51 (m, 5 H), 7.40 (d, J = 8.3 Hz, 2 H), 7.26 (d, J = 8.3 Hz, 2 H), 2.41 (s, 3 H). ¹³C NMR (75 MHz, d₆-DMSO): δ 161.2, 159.5, 157.2, 139.4, 134.7, 131.2, 130.9, 130.3, 128.6, 128.2, 127.9, 116.4, 115.6, 88.0, 75.2, 20.87. HRMS (ESI⁺): calculated mass for C₁₇H₁₇BrN₂O₂ [M+H] 361.0546, found 361.0532.

3. Results

3.1. Summary of Screening Results

A high throughput screen (HTS) of 302,457 compounds (AID 1663) was performed in duplicate to measure platelet activation by monitoring granule secretion in PRP. Using expired PRP activated with the thrombin receptor activation peptide SFLLRN, platelet activation was quantified by measurement of ATP from secreted granules, as measured by a luciferin-luciferase assay. A new assay format was developed for this project to aid in scaling up to screen the full MLPCN library with the use of automation. A stable luciferin-luciferase reagent (CellTiter-Glo, Promega) was adapted to measure secreted granules without lysing the platelets. Compounds were screened at a final concentration of 7.5 μM. From the HTS data, 661 compounds were classified as hits, based on inhibiting granule secretion ≥ 50%. A set of 1584 compounds was chosen as cherry picks, including available positive compounds, compounds classified as inconclusive, and additional inactive compounds containing structures similar to active compounds to provide additional SAR information. Cherry-picked compounds were retested in a concentration-dependent manner in the primary assay using PRP activated with SFLLRN (AID 1889). Out of 1584 compounds, 651 compounds were confirmed as active in this assay with IC₅₀ values < 10 μM. The same set of dose-response compound plates were also tested in a biochemical counterscreen to rule out activity seen in the primary assay due to nonspecific inhibition from the luciferin-luciferase detection system (AID 1891). The 446 compounds showing an IC₅₀ > 112.2 μM in the counterscreen were considered inactive in this assay, thus considered specific for inhibiting platelet activation. The results from these secondary assays were taken together to narrow the list to specific active compounds by advancing only compounds that exhibited a ratio of IC₅₀ values of greater than 10-fold (i.e., counter/primary) from both assays. From this list, compounds that had been previously shown to inhibit platelet activation were excluded. Of the remaining compounds, 30 compounds were prioritized for additional characterization in secondary assays. Of these 30 prioritized compounds, 28 were acquired thorough an additional cherry pick and tested in a suite of secondary specificity assays in the Assay Provider’s laboratory (see Table 1, Appendix 1; Figure 3). These assays narrowed the list of compounds to four. The four probe candidates and analogs of each were acquired as dry powders and retested in the confirmatory and secondary assays. Of these, one of the four probe candidates did not retest and was removed from further consideration as a probe. Completion of the series of secondary assays confirmed that the three remaining compounds met the criteria for a Class III probe, possible modulators of GPCR signaling in platelet activation (see Table 1, Appendix 1; Figure 3).

Figure 3

Critical Path for Probe Development.

The second of the three probes to meet the defined probe criteria is a cyanopyridone (ML160, CID 824820). This probe reproducibly retests in the primary assay yielding IC₅₀ values of 0.36 μM and 4.35 μM (see Figure 4) with improved potency over the positive control cilostazol. Variability in IC₅₀ values was observed across experiments and was attributed to differences in platelet response from donor to donor; however, the rank ordering of potency within a probe series was maintained across multiple experiments. The sample (SID 87556782) described in Figure 4 (see Section 3.2) was tested in four different donor samples. The IC₅₀ values from these experiments range from 0.36 μM to 9.93 μM with an average IC₅₀ of 3.75 μM ± 4.53 (data not shown). The cyanopyridone probe also inhibits P-selectin expression in a concentration-dependent manner, with an EC₅₀ less than 3 μM. This result demonstrated that the probe candidate met Class II or Class III probe definitions (see Figure 3). A cyclic AMP (cAMP) assay performed on the cyanopyridone probe demonstrated that the compound did not have activity in modulating cAMP levels, as compared to the potent activity of the positive control compound cilostazol (see Figure 1). Additional testing of the probe candidate in a fluorescent actin polymerization assay determined that it has greater than 50% activity in inhibiting SFLLRN-induced actin filament formation. The results of this assay suggest the probe is acting as a potential Class III probe, at an upstream GPCR target. Counterscreening in two additional assays in which platelets were activated with phorbol myristate acetate (PMA) or Ca2+ ionophore, agents that stimulate activation outside the GPCR signaling pathways, demonstrated that the cyanopyridone meets the criteria as a Class III probe (see Table 2, Section 3.4; Figure 3).

3.2. Dose Response Curves for Probe

Figure 4Cyanopyridone Probe (CID 824820) Concentration-dependent Activity in the Primary Screen Retest (AID 2398, AID 2519)

Primary assay retest measuring extracellular ATP as a measure of granule release: a, Retest 1, EC₅₀ 0.36 μM;b, Retest 2, EC₅₀ 4.35 μM

3.4. SAR Tables

The structures and the activities of the probe and analogs in the primary assay and the suite of secondary assays are summarized in Table 2.

Table 2SAR Analysis and Properties of Cyanopyridone Probe and Analogs

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SAR Analysis							Target Activity (μM)			Anti-target Activity (@ 30 μM)
No	CID/SID	Broad ID	*	R1	R2	R3	EC50 (Dense Granule Release) a	EC50 (P-Selectin expression)	% Inhib. SFLLRN-induced actin polym. @10 μM	% Inhib. PMA-induced P-selectin expression	% Inhib. Ca-ionophore-induced P-selectin expression
1	824820/ 87556782	BRD-K5077615 2-001-07-0	S	4-MePh	Ph	H	0.4	<3	99*	6*	38*
	Solubility (PBS): 1.6 μM; Plasma Protein Binding (Human, Mouse): 96, 91%, Plasma Stability (Human, Mouse): 100, 100%, Purity: >99%
2	1034641 87556784	BRD-K12168891 -001-05-7	S	4-MePh	4-ClPh	H	0.6	<3	NT	NT	NT
	Solubility (PBS): 11.5 μM; Plasma Protein Binding (Human, Mouse): 97, 93%, Plasma Stability (Human, Mouse): 100, 96%, Purity: 96%
3	12425537 87556786	BRD-K12238169 -001-01-2	S	Me	Ph	H	1.7	NT	NT	NT	NT
	Solubility (PBS): 24 μM; Plasma Protein Binding (Human, Mouse): 76, 63%, Plasma Stability (Human, Mouse): 100, 96%, Purity: >99%
4	44629412 87556783	BRD-K12621773 -001-01-7	S	2-MePh	4-MePh	H	2.0	NT	NT	NT	NT
	Solubility (PBS): 6.8 μM; Plasma Protein Binding (Human, Mouse): 97, 91%, Plasma Stability (Human, Mouse): 100, 98%, Purity: 99%
5	1034643 87556785	BRD-K29715937 -001-02-9	S	4-ClPh	4-MePh	H	9.2	<3	NT	NT	NT
	Solubility (PBS): 0.5 μM; Plasma Protein Binding (Human, Mouse): 97, 92%, Plasma Stability (Human, Mouse): 100, 98%, Purity: 99%
6	44629408 87556787	BRD-K84855052 -001-01-6	S	4-MePh	Ph	Ac	>50	NT	NT	NT	NT
	Solubility (PBS): 233 μM; Plasma Protein Binding (Human, Mouse): 97, 88%, Plasma Stability (Human, Mouse): 100, 89%, Purity: >99%

NT = not tested;

*

S = synthesized; P= purchased;

a

Primary HTS assay

The probe (entry 1) demonstrated the highest level of inhibition, but comparable activity was also observed when a para-chloride was added to the “eastern” ring (entry 2). The activity was attenuated by replacement of the N-para-tolyl group of the probe with ortho-tolyl (entry 4). The N-methyl analog (entry 3) retained respectable (but decreased) activity. Acylation of the pendant amino group of the probe greatly decreased the activity (entry 6).

4. Discussion

5. References

1.

Vu TK, Hung DT, Wheaton VI, Coughlin SR. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell. 1991 Mar 22;64(6):1057–68. [PubMed: 1672265]

2.

Flaumenhaft R, Sim DS. The platelet as a model for chemical genetics. Chem Biol. 2003 Jun;10(6):481–6. [PubMed: 12837380]

3.

Dowal L, Flaumenhaft R. Targeting platelet G-protein coupled receptors (GPCRs): looking beyond conventional GPCR antagonism. Curr Vasc Pharmacol. 2010 Mar;8(2):140–54. [PubMed: 19485898]

4.

Shehab N, Sperling LS, Kegler SR, Budnitz DS. National estimates of emergency department visits for hemorrhage-related adverse events from clopidogrel plus aspirin and from warfarin. Arch Intern Med. 2010 Nov 22;170(21):1926–33. [PubMed: 21098354]

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Michelson AD. Antiplatelet therapies for the treatment of cardiovascular disease. Nat Rev Drug Discov. 2010 Feb;9(2):154–69. [PubMed: 20118963]

6.

Wiviott SD, Braunwald E, McCabe CH, Montalescot G, Ruzyllo W, Gottlieb S, Neumann FJ, Ardissino D, De Servi S, Murphy SA, et al. for the TRITON-TIMI 38 Investigators. Prasugrel versus clopidogrel in patients with acute coronary syndromes. N Engl J Med. 2007 Nov 15;357(20):2001–15. Epub 2007 Nov 4. [PubMed: 17982182]

7.

Ding B, Abe J, Wei H, Huang Q, Walsh RA, Molina CA, Zhao A, Sadoshima J, Blaxall BC, Berk BC, et al. Functional role of phosphodiesterase 3 in cardiomyocyte apoptosis: implication in heart failure. Circulation. 2005 May 17;111(19):2469–76. [PMC free article: PMC4108189] [PubMed: 15867171]

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McNicol A, Israels SJ. Platelet dense granules: structure, function, and implications for haemostasis. Thromb Res. 1999 Jul 1;95(1):1–18. [PubMed: 10403682]

9.

Coughlin SR. Thrombin signalling and protease-activated receptors. Nature. 2000 Sep 14;407(6801):258–64. [PubMed: 11001069]

10.

Dyachenko IV, Dyachenko VD, Rusanov EB. N-hetaryl-2-cyanoacetamides in the synthesis of substituted (E)-N-hetaryl-2-cyanoacrylamides, (E)-N-alkyl-N-hetaryl-2-cyanoacrylamides, and 6-amino-2-oxo-4-phenyl-1-(pyridin-2-yl)-1,2-dihydropyridine-3,5-dicarbonitriles. Russ J Org Chem. 2007;43:83–9.

11.

Ismail MMF, Noaman E. Novel pirfenidone analogs as antifibrotic agents: synthesis and antifibrotic evaluation of 2-Pyridones and fused pyridones. Med Chem Res. 2005;14:382–403.

6. Appendices

Appendix 3. Spectroscopic Data for ML160 (probe)

Appendix 4. Spectroscopic Data for SAR Analogs

¹H NMR (300 MHz, d₆-DMSO) (Table 1, entry 2)

LC-MS

¹H NMR (300 MHz, d₆-DMSO) (Table 1, entry 3)

LC-MS

¹H NMR (300 MHz, d₆-DMSO) (Table 1, entry 4)

LC-MS

¹H NMR (300 MHz, d₆-DMSO) (Table 1, entry 5)

LC-MS

¹H NMR (300 MHz, CD₃CN) (Table 1, entry 6)

LC-MS

Acknowledgements

The authors thank Patti Aha, Tyler Aldredge, Frank An, PJ Aspesi, Doug Barker, Jason Burbank, Josh Bittker, Dave DeCaprio, Melanie de Silva, Karen Emmith, Robert Gould, Mary Pat Happ, Kate Hartland, Tom Hasaka, Stephen Johnston, Corrie Lade, Hanh Le, John McGrath, Chris Moore, Evan Mulligan, Myra O’Leary, Gil Walzer, Greg Wendel, and other members of BIPDeC for their assistance with this project. The authors also acknowledge and thank BloodSource (CA), Rhode Island Blood Center, Blood Center of New Jersey, and Blood Center of Wisconsin for supplying platelets for the assays performed at BIPDeC.