2.1. Assays

• AID 602453: Chemical Optimization of Advanced mGlu4 Lead Candidates (Summary AID)
• AID 623876: Modulation of the Metabotropic Glutamate Receptor mGluR4: Potency at human mGluR4.
• AID 623875: Modulation of the Metabotropic Glutamate Receptor mGluR4: Rat PAM Potency
• AID 623889: Modulation of Metabotropic Glutamate Receptor mGluR4: Rat PAM Fold-Shift
• AID 623874: Modulation of the Metabotropic Glutamate Receptor mGluR4: Selectivity at mGluR1
• AID 623878: Modulation of the Metabotropic Glutamate Receptor mGluR4: Selectivity at mGluR2
• AID 623879: Modulation of the Metabotropic Glutamate Receptor mGluR4: Selectivity at mGluR3
• AID 623890: Modulation of the Metabotropic Glutamate Receptor mGluR4: Selectivity at mGluR5
• AID 623891: Modulation of the Metabotropic Glutamate Receptor mGluR4: Selectivity at mGluR6
• AID 623893: Modulation of the Metabotropic Glutamate Receptor mGluR4: Selectivity at mGluR7
• AID 623892: Modulation of the Metabotropic Glutamate Receptor mGluR4: Selectivity at mGluR8
• AID 623927: Ricerca Lead Profiling Assay

2.2. Probe Chemical Characterization

Probe compound ML292 (CID 56587900, SID 134213607) was prepared according to the scheme in Figure 1 and provided the following characterization data: R_f = 0.47 (33% EtOAc/hexanes); Mp = 81–83°C; Analytical LCMS: single peak (214 nm), 3.417 min; ¹H NMR (400 MHz, CDCl₃): δ 10.06 (br s, 1H), 8.62 (d, J = 4.4 Hz, 1H), 8.30 (d, J = 8.0 Hz, 1H), 7.95-7.91 (m, 2H), 7.63 (dd, J = 8.0, 1.2 Hz, 1H), 7.51 (dd, J = 7.6, 4.8 Hz, 1H), 7.31 (dd, 8.4, 8.0 Hz, 1H), 7.13 (dd, J = 8.0, 1.2 Hz, 1H); HRMS, calc’d for C₁₂H₁₀N₂OCl (M+H⁺), 233.0482; found 233.0483.

Figure 1

Synthesis of ML 292.

Solubility. Solubility in PBS was determined to be >75 μM, which is ~250-fold higher than the EC₅₀ for rat mGlu₄ activation.¹

Stability. Stability (see Table 1) was determined for ML292 at 23°C in PBS (no antioxidants or other protectorants and DMSO concentration below 0.1%). After 48 hours, 97% of the initial concentration of ML292 remained, indicating ML292 is a very stable compound.¹

Table 1

Stability of ML292.

Compounds added to the SMR collection (MLS#’s): MLS004035809 (ML292, CID 56587900, 22 mg); MLS004035810 (CID 889460, 6.0 mg); MLS004035811 (CID 56587892, 6.5 mg); MLS004035812 (CID 56587906, 6.9 mg); MLS004035813 (CID 56587816, 6.0 mg); MLS004035814 (CID 56587823, 5.0 mg).

2.3. Probe Preparation

N-(3-chlorophenyl)picolinamide (ML292, CID 56587900): To a solution of 3-chloroaniline (29.5 mL, 0.28 mol) in DMF (300 mL) at 0 °C was added DMAP (50 mg) and DIEA (118 mL, 0.84 mol). Picolinoyl chloride hydrochloride (50 g, 0.28 mol) was added portion wise over 30 minutes and after an additional 15 minutes, the ice bath was removed. After 16h at rt, the reaction was added to EtOAc: NaHCO₃ (sat’d) (1:1; 1000 mL). The organic layer was separated and washed with water (5 x 200 mL), brine (200 mL), dried (MgSO₄), filtered and concentrated under reduced pressure. After recrystallization of the crude solid from EtOH, the desired product was obtained as an off-white solid (63.5g, 84% yield).

3.1. Dose Response Curves for Probe

ML292, in the absence of glutamate, did not activate either human or rat mGlu₄ cells. In the presence of a submaximal concentration of glutamate (~EC₂₀), ML292 potentiated the glutamate response in a well-behaved PAM concentration-response curve (CRC) (Figure 2) with excellent potency and efficacy (%GluMax > 100%).

Figure 2

Human (A) and rat (B) mGlu₄ PAM CRC’s for ML292. ML292 exhibited an EC₅₀ at human mGlu₄=1.2 μM (%GluMax = 105%, normalized to PHCCC) and an EC₅₀ at the rat receptor=0.33 μM (%GluMax = 126%, normalized to PHCCC).

3.2. Cellular Activity

The primary HTS assay and all secondary assays are cell-based assays, indicating that ML292 can gain access to its molecular target when applied to cells. The compound did not exhibit acute toxicity in cell based assays at concentrations up to 30 μM.

3.3. Profiling Assays

ML292 (CID 56587900) was further profiled against Ricerca’s Lead Profiling screen² (binding assay of 68 GPCR’s, ion channels and transporters screened at 10 μM) and ML292 (CID 56587900) did not significantly bind to any of the 68 targets (no inhibition of radioligand binding >50% at 10 μM), with the exception of human norepinephrine transporter (80% at 10 μM). From observations that small planar molecules ability can affect monoamine oxidase activity, we obtained IC₅₀ determinations for inhibition of MAO –A and -B. ML292 exhibited K_i values of 8.5 and 0.72 μM for human MAO-A and MAO-B, respectively. Lastly, as the Ricerca panel only measures the displacement of a radioligand binding, we wanted to more fully assess off target functional responses for ML292. Thus, we evaluated ML292 at Millipore in their functional assay of 168 GPCRs which is designed to detect potential agonist, potentiator and antagonist activity. Encouragingly, ML292 displayed a remarkably clean ancillary pharmacology profile in these assays with no activity detected.³

Lastly, ML292 was evaluated for its selectivity against the other seven mGlu subtypes (mGlu_{1–3,5,6–8}). ML292 was selective against the Group II mGlus (mGlu₂,₃) and selective versus mGlu_1,7,8. The compound did exhibit weak antagonist activity against mGlu₅ (IC₅₀ = 17.9 μM) and PAM activity against mGlu₆ (EC₅₀ = 6.8 μM).³ Taking all of these data together, ML292 is a highly selective, potent mGlu₄ PAM with excellent CNS exposure.

4.1. Comparison to Existing Art and How the New Probe is an Improvement

ML292 is a selective and potent positive allosteric modulator of the metabotropic glutamate receptor 4 (mGlu₄). ML292 displays modest potency at the human receptor (hEC₅₀ = 1.2 μM), but shows equivalent potency to the previous probe molecules at the rat receptor (rEC₅₀ = 330 nM) (Table 2). In addition, ML292 produces a robust left-ward shift of the glutamate concentration-reponse curve (fold shift, 30.2 fold). This efficacy is equivalent to ML128 and superior to ML182. Although the in vitro PK properties of ML292 do not allow for oral dosing in vivo, after subcutaneous dosing, ML292 achieves significant brain levels and displays an excellent brain:plasma ratio of 1.4 when evaluated from 0 – 6 h; and had a B:P of ~4 at 1 h. Other reported mGlu₄ PAM molecules display superior potency to ML292 in vitro, but no other published PAMs to date have shown equivalent in vivo efficacy. ML292 is efficacious in a number of pre-clinical anti-Parkinsonian animal models, including HIC (0.75 mpk and 1.5 mpk, halo) and the 6-OHDA forelimb asymmetry model. In fact, ML292 is the only mGlu₄ PAM to show efficacy when administered as a stand-alone treatment in this model. In summary, ML292 is a potent, selective, first-in-class in vivo potency mGlu₄ PAM that will be widely available to the community for further evaluation.

Table 2

Summary of mGlu₄ PAM probe molecules.