2.1. Assays

2.2. Probe Chemical Characterization

Synthetic procedure and spectral data for ML273 (CID 53384864, SID 126920341, VU0424465-2, note ML273 is also associated with CID 53382545/SID 125311644 which is used throughout Results and Discussion, also internally associated with VU0424465-1).

Probe compound ML273 (CID 53384864) was prepared according to the scheme in Figure 1 and provided the following characterization data: LC-MS (>98%) m/z = 327.1 [M+H], ¹H NMR (400 MHz, CDCl₃) δ 8.67 (1H, d, J=1.6 Hz), 8.20 (2H, d, J=8.0 Hz), 7.96 (1H, d, J=8.2, 2.2 Hz), 7.36 (2H, m), 7.27 (1H, dd, J=8.2, 1.6 Hz), 7.14 – 7.09 (1H, m), 4.16 (1H, dq, J=9.2, 6.8 Hz), 2.6 (1H, br s), 1.31 (overlapping s and d, 9H, J=10.4 Hz); HRMS (ESI) m/z 327.1508 ([M+H]⁺, 100%) calcd for C₁₉H₂₀FN₂O₂, 327.1509.

Figure 1

(R)-5-((3-fluorophenyl)ethynyl)-N-(3-hydroxy-3-methylbutan-2-yl)picolinamide.

Solubility. Solubility in PBS at pH 7.4 was determined to be >75 μM or >24.5 μg/mL based upon triplicate testing using an assay from Analiza Inc. labs (greater than control concentration). ML273 shows excellent solubility up to 10 mM DMSO. In addition, solubility was determined in-house in Fassif (fasted simulated intestinal fluid) media at 37 °C for 24h. In Fassif ML273 solubility was determined to be 104 μM (34 μg/mL). Collectively, these data demonstrate that ML273 is a moderate to highly soluble tool compound at neutral pH.¹

Stability. Stability was determined for ML273 in PBS buffer at room temperature (Analiza Inc.) with time course evaluation over 48h. After 48 hours, the percent of parent compound remaining was ~100%, indicating excellent stability after prolonged exposure to PBS buffer.¹

Compounds added to the SMR collection (MLS#s): MLS003874879 (ML273, CID 53384864, 21 mg); MLS003874880 (CID 53384876, 7.4 mg); MLS003874881 (CID 53384838, 6.1 mg); MLS003874882 (CID 53384841, 7.6 mg); MLS003874883 (CID 53384852, 6.6 mg); MLS003874884 (CID 53384862, 7.2 mg).

2.3. Probe Preparation

Step 1: Preparation of 5-((3-fluorophenyl)ethynyl)picolinic acid. 5-Bromopicolinic acid (2.0 g, 10.0 mmol), 1-ethynyl-3-fluorobenzene (1.0 mL, 12.0 mmol), Pd(PPh₃)₄ (0.57 g, 0.5 mmol), CuI (0.2 g, 1.0 mmol), and diethylamine (6.2 mL, 60 mmol) were mixed in 15.0 mL of DMF in a sealed 10–20 mL microwave tube. The reaction was subjected to microwave irradiation at 90 °C for 45 min. The crude reaction was diluted with H₂O (60 mL). The product precipitate formed upon the addition of H₂O. The mixture was filtered, affording the title compound as an off-white solid in 94% yield.

Step 2: Preparation of (R)-5-((3-fluorophenyl)ethynyl)-N-(3-hydroxy-3-methylbutan-2-yl)picolinamide. (R)-3-amino-2-methylbutan-2-ol (43 mg, 0.5 mmol, prepared from Boc-D-Ala-OMe according to literature procedures^{17, 18}), 5-((3-fluorophenyl)ethynyl)picolinic acid (100 mg, 0.41 mmol), HATU (155 mg, 0.41 mmol) and N,N-diisopropylethylamine (0.11 mL, 0.8 mmol) were mixed in DMF (1.5 mL) at room temperature. The reaction mixture was stirred at the same temperature for 2 h, diluted with H₂O (10 mL), and extracted with EtOAc (10.0 mL × 3). The organic layers were combined and washed with brine (15.0 mL), dried over MgSO_4, and concentrated under reduced pressure. The crude material was purified by gradient column chromatography over silica (EtOAc:hexanes, 0–45%), affording the title compound as a while solid in 45% yield.

3.1. Dose Response Curves for Probe

Figure 2ML254 (SID 125305189) [3H]methoxyPEPy binding curve

3.2. Cellular Activity

The primary screening assay is a kinetic rat mGlu5 calcium Fluorescence assay using Fluo-4 AM Dye and HEK293A cell background. This biochemical in vitro fluorescence assay screens for potentiators of the glutamate promoted mGlu5-Ga/q-PLC-IPR cascade release of internal calcium stores. Activity from this assay indicates that ML273 can gain access to its molecular target when applied to cells. ML273 did not exhibit acute toxicity in cell based assays at concentrations up to 30 μM.

3.3. Profiling Assays

Off-target ancillary pharmacology for ML273 was assessed using the Ricerca Lead Profiler screen (68 GPCRs, ion channels and transporters screened at 10 μM). No significant activities were observed from this screen (<35% inhibition) indicating ML273 is a highly selective probe.²

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

mGlu5 PAMs with different in vitro profiles are needed in order to fully understand the anticipated activity in vivo. Tool compounds have been limited by poor inherent physicochemical properties and a lack of assessment in systems other than recombinant cell lines. More recently diverse PAM chemotypes^3–5 have appeared and in some cases offer improvements in DMPK properties as well unique pharmacological profiles. In contrast, advances in allosteric agonist development do not exist and to our knowledge, ML273 is the first. Thus, all prior agonists of mGlu₅ are mechanistically orthosteric in nature. The elegant work and discovery of orthosteric mGlu agonists in the 90’s based upon glutamate and phenyl glycine analogs paved the way to a handful of highly selective group I (mGlu_1,5) vs. group II (mGlu_2,3) and group III (mGlu_4,6–8) orthosteric agonist tool compounds (Table 1).^6–7

Table 1

Orthosteric-based mGlu agonists based upon glutamate.

To this end, quisqualate, 3,5-DHPG, and 3-HPG have had the greatest impact for group I mGlu receptor pharmacology based upon their overall potency and selectivity. Although these tool compounds have proven to be tremendously useful in vitro, it’s important to note they are not CNS pentrant and aside from intracerebroventricular (ICV) dosing studies, some previously described,⁸ the impact of orthorsteric agonists in vivo has been limited. Despite the success found with metabotropic group selective orthosteric ligands, mGlu₁ and/or mGlu₅ selective agonists are rare and CHPG (Table 1) represents the first and only mGlu₅ selective agonist to our knowledge, with albeit weak potency for mGlu₅ with a reported EC₅₀ of 750 μM.⁹

Due to the limitations of CHPG and other orthosteric ligands as described in Table 1 and the lack of availability of allosteric agonists of mGlu₅, ML273 should prove to be an interesting and useful probe molecule for the field. ML273 is the first fully characterized, systemically available brain penetrant ago-PAM with agonist activity in both recombinant and native systems. Further studies are warranted with ML273 in order to understand the impact of its profile in our current understandings in basic mGlu₅ signaling.¹⁰ In addition, studies aimed at understanding therapeutic index of ago-PAMs versus pure-PAMs as it relates to therapeutic indications currently being explored for drug discovery using mGlu₅ PAMs are needed.