The replacement of a problematic indazole core present in a known class of PAR4 antagonists was successfully implemented through the introduction of an indole core structure. Subsequent libraries of compounds exploring the structure activity relationship (SAR) for this indole series provided PAR4 antagonists with potencies only in the micro-molar range. As a result, a similarity search was conducted which identified 160 candidates from our in-house sample collection for testing against PAR4. From this redirected effort arose ML354, a selective PAR4 antagonist with good potency (PAR4 IC₅₀ = 140 nM), and reasonable selectivity versus PAR1. A lead profiling screen (Pan Labs) identified only 3 potential off-target binding activities, which were all quite weak and unrelated to relevant PAR4 biology. This compound/series also displays improved physical properties and is very amenable to future medicinal chemistry development. Although this molecule contains a nitro group, it still represents the best-in-class PAR4 antagonist for use in vitro, and holds great promise for the development of future in vivo tools.

Probe Structure & Characteristics

ML354

1. Recommendations for Scientific Use of the Probe

2. Materials and Methods

Platelet Isolation. Platelets were prepared via the standard washed platelet protocol as described below. Briefly, written informed consent was obtained from healthy volunteers in accordance with the Vanderbilt University Internal Review Board approved protocols. Blood drawn into syringes containing 3.2% sodium citrate Platelet rich plasma was prepared by centrifugation at 170g for 15 minutes. 10× acid citrate dextrose was added to platelet rich plasma and centrifuged at 800g for 10 minutes at room temperature. The supernatant was aspirated and the platelet pellet was suspended in Tyrode's buffer (15 mM HEPES, 0.33 mM NaH2PO4 (pH 7.4), 138 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 5.5 mM dextrose) containing 0.1% Bovine Serum Albumin fraction V (BSA) and counted on a Beckman Z1 Coulter particle counter (Brea, CA).

Calcium Mobilization Assays – Single Point Screening. Washed human platelets were prepared via the standard procedure (vide supra) and suspended in Tyrode's buffer containing 0.1% BSA. Platelets were dye loaded for 1 hour with Fluo4-AM (Invitrogen, Eugene, OR) in calcium assay buffer (1× HBSS, 20 mM HEPES, 2.5 mM probenecid without calcium or magnesium). The calcium assay buffer containing dye was mixed with platelets to yield a final concentration of 2.5 μg/mL Fluo4-AM and 1.0×10⁸ platelets/mL. 60 μL of dye loaded platelets were added to each well of a NUNC 384 well plate black optical bottom plate (Thermo, Rochester, NY). Fluorescence measurements were recorded in the Vanderbilt Center for Neuroscience Drug Discovery's Screening Center using a Functional Drug Screening System 6000, Hamamatsu (Hamamatsu, Japan). 10 μM of each compound was added in triplicate 6 minutes prior to the addition of 80 μM Protease activated receptor 4-activating peptide (PAR4-AP) (GL Biochem, Shanghai, China). 480:540 (ex:em) was measured each second for a total of 12 minutes at 37 °C. The final concentration of DMSO in the assay was 0.5%. The difference between basal and maximal relative fluorescence unit values (RFUs) for ex:em 480:540 was determined for each well. The change in RFU for DMSO treated control stimulated with 80 μM PAR4-AP was set to 100% for each donor. IC₅₀ values were determined using GraphPad PRISM v.5.0 inhibitory sigmoidal dose response ‘variable slope’ parameter.

PAC-1 Binding – Potency and Selectivity. Briefly, 60 μL of washed platelets (Tyrodes buffer containing 0.1% BSA) at a concentration of 0.15 ×10⁸ platelets/mL were added to 5 mL round bottom polystyrene tubes (BD, Franklin Lakes, NJ). FITC conjugated PAC-1 (BD Biosciences, San Jose, CA) antibody was diluted (to the manufacturers recommended concentration) in Tyrode's buffer containing 0.1% BSA. 40 μL of diluted antibody was added to the platelets and allowed to bind for 5 minutes. Platelets were pre-treated with indicated concentrations of antagonist or DMSO control for 5 minutes followed by addition of PAR1-AP (GL Biochem, Shanghai, China) or PAR4-AP for 10 minutes. Platelet activity was quenched by the addition ice cold 1.5% paraformaldehyde followed by dilution in 1× phosphate buffered saline. The final DMSO concentration was 0.5%. Platelets were stored up to 18 hours at 4 °C before flow cytometric analysis. Analysis was carried out on a BD FACS Canto II (Franklin Lakes, NJ). Fluorescent intensity was determined for 100,000 events within the platelet gate. Data was collected and analyzed via FACS DiVa software. Flow cytometric data analysis was conducted by the following method. 100% response for PAR4-AP was determined for each individual as the DMSO treated control stimulated with either 200 μM PAR4-AP, or 20 μM PAR1-AP. Data was plotted in GraphPad PRISM v.5.0. Dose response curves and subsequent IC₅₀ values were generated using the inhibitory sigmoidal dose response ‘variable slope’ parameter. PAR4 results were plotted is mean ± SEM. Compounds displaying IC₅₀'s less than 1 μM were assigned ‘Outcome’ = Active, and subject to PAR1 PAC1 selectivity screens.

2.2. Probe Chemical Characterization

Probe compound ML354 (CID 752812, SID 161003819, VU0099704) could be prepared according to the above route, but was most efficiently obtained from commercial sources and provided the following characterization. (1-methyl-5-nitro-3-phenyl-1H-indol-2-yl)methanol: ¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.58 (d, J = 2.1 Hz, 1H); 8.17 (dd, J₁ = 9.1 Hz, J₂ = 2.2 Hz, 1H); 7.55-7.49 (m, 2H); 7.48-7.36 (m, 4H); 4.90 (s, 2H); 3.97 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 142.11; 140.07; 137.73; 133.03; 130.00; 129.14; 127.52; 126.06; 119.46; 118.42; 117.61; 109.37; 55.08; 30.80. HRMS (TOF, ES⁺) for C₁₆H₁₄N₂O₃ [M+Na⁺] calc. mass: 283.1083; found: 283.1081.

Solubility: Solubility for ML354 in PBS (@ pH = 7.4, final DMSO concentration 1%) was determined to be 3.3 ± 0.5 μM, which is 26-fold higher than its PAR4 IC₅₀.

Stability. Stability was determined for ML354 at 23 °C in PBS (no antioxidants or other protectorants, initial ML354 concentration = 10 μM and final DMSO concentration 10%). After 48 hours, 94.5% of the initial concentration of ML354 remained. There was no significant change in concentration between the 24 and 48 hour time points indicating that ML354 is very stable in solution under these conditions and may even be stable for longer periods of time in solution.

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	Percent Remaining (%)
Compound	0 min	19 min	37 min	90 min	24 hr	48 hr
ML354, CID 752812	100	97.6	98.8	96.9	94.2	94.5

Compounds added to the SMR collection (MLS#s): MLS004820369 (ML354, CID 752812, 25.0 mg); MLS004820370 (CID 71148622, 17.0 mg); MLS004820371 (CID 71148620, 15.8 mg); MLS004820372 (CID 71148614, 6.2 mg); MLS004820373 (CID 71148612, 17.7 mg); MLS004820374 (CID 71148613, 5.0 mg)

3. Results

Published preparations for the known PAR4 antagonists, YD-31 and related analogs,2 entail 9-step routes with complex mixtures of N-1 and N-2 indazole benzylation products. Using YD-3 as a starting point, we first sought to prepare analogs which possessed a simplified core and a higher-yielding synthesis route. Figure 2 shows YD-3 and two early attempts at replacing the indazole core. It should be pointed out that the differences in potency for YD-3 appearing in Figures 1 and 2 are a result of the different assays used to assess PAR4 inhibition. Although introduction of the hydroxyl indolinone core, in MLS-E-6, resulted in an inactive compound, the indole substitution provided a compound with nanomolar potency (PAR4 IC₅₀ = 66 nM). Observing only a 2.5-fold decrease in potency for EBIB relative to YD-3 clearly validated the indole core as an attractive replacement for the indazole. Furthermore, these new analogues could be prepared via the highly efficient, 3-step route generally depicted in Scheme 1. Briefly, substituted indoles can be N-benzylated with benzyl halides and sodium hydride, followed by NBS bromination at the 3-position of the indole. These indole bromides can then undergo efficient palladium(0) catalyzed coupling with a large variety of boronic acids/esters to provide a diverse range of substituted indole PAR4 antagonist.

Figure 2

Replacements for the indazole core of YD-3.

Figure 1

Known PAR4 antagonists.

Scheme 1

Generalized preparation for the compounds appearing in Tables 1 and 2.

These successful initial results and the simplified preparation of this novel class of PAR4 antagonists enabled the submission, and acceptance, of an MLPCN Fast Track application. Looking to improve the physical properties and metabolic stability of EBIB our next goal was to replace the ethyl ester, which represented a potential metabolic liability, and introduce polarity or weakly basic amines. Towards this goal, libraries were produced which explored alternative Ar² groups, as depicted in Scheme 1. Table 1 lists some of the more interesting structures which were initially tested in a single point screen, at 10 μM, to quickly obtain an idea of their ability to inhibit PAR4. The data appearing in the right-most column represents PAR4 activity remaining, relative to a DMSO control. Replacing the ethyl ester with a methyl ether, SID 152145949, provided a compound which inhibited PAR4 activity down to 27%, a very encouraging result among our initial libraries. Ethers with larger alkyl groups (SIDs 160870441 and 160870439), more electron withdrawing ethers (SID 160870442), or ethers in the meta positions (SID 152145950) did not show improved inhibition. Moving away from substituted phenyls, a pyrazole (SID 152145945) and pyrimidine analogues (SID 160870438, etc.) did not show improved activity over the 4-OMe phenyl compound. However, the pyrimidines suggested that 3-pyridyl should be explored. With the exception of the 4-pyridyl analog SID 152145951, the mono- and di-substituted 3-pyridyl compounds all displayed some level of PAR4 antagonism. The most encouraging compounds were SID 152145947 and SID 152145946, with just 28% and 17% activity remaining when tested at 10 μM. Those PAR4 antagonists showing less than 30% activity remaining (SID 152145949, SID 152145947 and SID 152145946) were further characterized in a related concentration response experiment to determine their IC₅₀s for PAR4. Quite disappointingly, the first two failed to return a measurable IC₅₀ (> 10 μM), while SID 152145946 yielded a PAR4 IC₅₀ of only 5.4 μM. Although this did represent an approximately 80-fold decrease in potency relative to EBIB, we felt that replacing the ethyl benzoate with a 6-MeO-pyridin-3-yl heterocycle was a reasonable trade off.

Table 1

Indole PAR4 antagonists.

We next held the top aryl group constant, as the 6-MeO-pyridin-3-yl, while we attempted to improve potency through modifications of the N-benzyl group. Table 2 highlights only the most successful analogues from this endeavor, along with their single point screening values and the subsequently obtained PAR4 IC₅₀s. Consistent with previous SAR developed around the indazole series of PAR4 antagonists,2 meta-substituents provided the best results in these indole analogues. Of the halogenated analogues, SID 160870425 displayed the best IC₅₀ of 3.1 μM. More polar substituents which showed promising results in the single-point screen (SID 160870449 and SID 160870443) both possessed IC₅₀ values > 10 μM, despite showing encouraging levels of antagonism in the single point screen (14% and 24% respectively).

Table 2

Indole PAR4 antagonists with m-Substituents.

Beginning to realize that we might not be able to obtain reasonable potencies for this indole class of compounds without including the ethyl benzoate present in YD-3 and EBIB (Figure 2), we conducted a rudimentary structural similarity search around the lead indole. We selected compounds which were somewhat similar to EBIB and which were readily available within Vanderbilt's sample repository. Approximately 160 compounds were selected for single point testing, and Figure 3 illustrates some of the structural diversity which was explored. Four ‘actives’ were obtained from this similarity screen (SID 161004578, SID 161004613, SID 161004645 and SID 161004647). Among these four, SID 161004645 (CID 752812) proved to be the most attractive hit despite the presence of an aryl nitro group. We consulted with the assay provider and decided that since the use of this compound would most likely be limited to in vitro experiments with isolated platelets, the presence of a nitro group was of limited concern. That being said, we believe it would still be advantageous to explore molecules which do not contain such a potentially offensive moiety. One distinct advantage of this new lead was its commercial availability, both with an eye towards activity confirmation and subsequent derivatization. As such, we promptly ordered a gram of CID 752812 (Chembridge# 5280909) and set about characterizing its structure and confirming its activity. The spectral data appearing in Section 2.2 reliably confirmed its structure and we were very pleased to determine its PAR4 antagonist IC₅₀ = 140 nM! Simultaneously we explored a small subset of the readily accessible analogues shown in Figure 4. Direct alkylation of the hydroxyl provided ether analogues of varing activity, the best of which was a sub-nanomolar compound against PAR4, SID 161780288. Oxidation of the alcohol to an aldehyde could be followed by reductive aminations or Grignard additions under standard conditions to provide amines and secondary alcohols, respectively. However, neither of these latter two approaches provided any improvement over CID 752812. In fact, of the three classes of derivatives prepared around CID 752812, none has yet to provide an improvement in potency relative to CID 752812 (ML354). These results thus far would suggest that SAR around this indole series of PAR4 antagonists is fairly steep. However, given the low molecular weight for ML354 (MW = 282), its commercial availability and its easy-to-derivatize structure, we are confident that future work will result in further improvements in PAR4 potency and are optimistic with regard to replacing, or removing, the nitro functionality.

Figure 3

Sampling of the diversity explored around the lead compound EBIB, as obtained from a structural similarity search.

Figure 4

Derivatives of CID 75812 (ML354).

Since only PAR1 and PAR4 are expressed on platelets, we determined ML354's IC₅₀ against PAR1 and were encouraged to measure a PAR1 IC₅₀ ∼ 10 μM, indicating a roughly 70-fold selectivity for PAR4 over PAR1 (Figure 5). In summary, ML354 represents a very attractive probe compound for the in vitro study of platelet activation and will likely represent a critical starting point for the future development of a highly selective in vivo tool compound. An additional advantage of ML354 is its commercial availability, rendering it readily and immediately available to the broader scientific community.

Figure 5

PAR4 and PAR1 CRC for ML354 (VU0099704).

3.3. Profiling Assays

To more fully characterize ML354, and to better inform the scientific community about its potential off target activities, this 1^st generation PAR4 antagonist was tested using Eurofins' (formerly Ricerca's, formerly MDS Pharma's) Pan Labs Lead Profiling Screen (AID 686926). This battery of radioligand binding assays consists of 68 common GPCRs, ion channels and transporters where the test compound (ML354) was present at 10 μM. Responses were considered significant if > 50% inhibition was observed. However it should be pointed out that these are only single-point values and that functional selectivity may be significantly better than suggested by these “% inhibitions.” Table 3 presents the Pan Labs results for ML354, which showed a significant response in only three assays. Importantly, none of these alleged activities would be expected to participate in the coagulation cascade, or be present on human platelets, indicating that studies performed with isolated human platelets and ML354 are highly focused towards PAR4 effects.

Table 3

Pan Labs profiling of ML354.

Additionally, a set of calculated physical properties were determined for ML354 and compared to the averaged values for compounds appearing in the MDDR database (MDL Drug Data Report database, 2010) at two stages of clinical development (Phase I and Launched, Table 4). ML354 compares favorably with the average values for both Phase I and Launched compounds. The most notable departure from this favorable comparison occurs in the comparison of cLogP values, and although ML354 is on the high side of the MDDR ranges, it is still a substantial improvement over the cLogP value for the lead compound YD-3 (cLogP = 5.4).

Table 4

Calculated property comparison between the PAR4 probe and MDDR compounds.

4. Discussion

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