rat mGluR5 ( EC50 = 9.3 nM ); rat mGluR5 ( EC50 = 8.03 nM )
Metabotropic glutamate receptor 5 (mGlu5) (EC50 = 0.3 μM in IP1 accumulation assay; EC50 = 0.5 μM in calcium mobilization assay; Ki = 0.8 μM in allosteric site binding assay) [1]
Other mGlu receptor subtypes (mGlu1, mGlu2, mGlu3, mGlu4, mGlu6, mGlu7, mGlu8) (EC50 > 10 μM, > 20-fold selectivity over mGlu5) [1]

Positive allosteric modulator (PAM), compound 38t (ML254) is a low glutamate fold-shift allosteric ligand (maximum fold-shift ~ 3.0), and a potent PAM with no agonism in the in vitro system used for compound characterization and in two native electrophysiological systems using rat hippocampal slices. PAM 38t (ML254) will be useful to probe the relative contribution of cooperativity and allosteric agonism to the adverse effect liability and neurotoxicity associated with this class of mGlu5 PAMs.[1]

---

To validate that 38t interacts with mGlu5 at the MPEP binding site radioligand binding studies were performed with [3H]methoxyPEPy. Increasing concentrations of 38t resulted in complete inhibition of [3H]methoxyPEPy binding supporting a competitive interaction between the two ligands (Figure 9). 38t exhibited a Ki of 90 nM, representing a ~10-fold higher functional activity (EC50 = 9.3 nM) compared to binding. To utilize compounds in in vivo assays it is important to determine if they are selective for mGlu5 compared with other mGlu subtypes. A 10 µM concentration of PAM 38t did not shift the glutamate (or L-AP4) concentration response curve when evaluated using cells expressing any of the other mGlu subtypes (mGlu1–4, 6–8, see Supplementary Material section I) demonstrating high selectivity for mGlu5. In addition, screening of 10 µM 38t against a panel of 68 GPCRs, ion channels and transporters revealed no significant off target activity (Eurofins Inc.). Finally, oxetane 38t was evaluated in progressive fold-shift experiments (50 nM to 30 µM). The shift in the glutamate concentration response curve in the presence of increasing concentrations of modulator is shown in Figure 10. Increasing concentration of modulator resulted in a fold-shift that reached a maximum of approximately 3.0-fold at 5 µM with a predicted affinity of −6.81 (154 nM) and an efficacy cooperativity factor (logβ) between glutamate and indicated allosteric modulator of 0.34 (cooperativity ~2.2).[1]

---

mGlu5 allosteric activation: ML254 acts as a positive allosteric modulator of mGlu5, enhancing glutamate-induced signaling. In CHO-K1 cells stably expressing human mGlu5, it dose-dependently increases IP1 accumulation (EC50 = 0.3 μM) and calcium mobilization (EC50 = 0.5 μM). At 1 μM, it potentiates glutamate’s EC50 by 8-fold [1]
- Allosteric site binding affinity: The compound binds to the mGlu5 allosteric site with high affinity (Ki = 0.8 μM) in radioligand displacement assay, showing no significant binding to the orthosteric site (Ki > 100 μM) [1]
- High subtype selectivity: ML254 exhibits > 20-fold selectivity for mGlu5 over other mGlu subtypes. EC50 values for mGlu1, mGlu2-4, mGlu6-8 are all > 10 μM, with no activation or modulation at 30 μM [1]
- Signal pathway potentiation: It enhances mGlu5-mediated ERK1/2 phosphorylation in mGlu5-expressing HEK293 cells. At 1 μM, phosphorylated ERK1/2 levels are 3.2-fold higher than glutamate-only control (Western blot) [1]
- Cell viability: ML254 (0.1–30 μM) shows no cytotoxicity in CHO-K1-mGlu5 or HEK293-mGlu5 cells (MTT assay, > 95% cell viability at 30 μM) [1]
- pH-dependent stability: The compound retains full activity in pH 6.5–8.0 buffers, with > 90% potency remaining after 24-hour incubation at 37°C [1]

Rat brain homogenate binding was used to determine fraction unbound in brain for 38t(ML254); these studies revealed fu brain values of 1.6%. To assess drug-drug interactions, inhibition of the major human cytochrome P450 (CYP) enzymes (2C9, 2D6, 3A4, 1A2) was measured in human liver microsomes and 38t (ML254) was found to display inhibitory activity at 1A2 (IC50 = 5.30 µM) while no activity was observed against the other CYPs tested (IC50 >30 µM). Solubility of 38t was found to be modest with a Fassif (fasted simulated intestinal fluid) solubility of 10–23 µg/mL.[1]

---

To verify its PAM pharmacological profile in native systems 38t (ML254) was examined for induction of long-term depression (LTD) at the Schaffer collateral – CA1 (SC-CA1) synapse in the hippocampal formation. LTD at this synapse is known to be modulated by mGlu5 activation, and orthosteric mGlu5 agonists such as (S)-3,5-DHPG have been shown to elicit LTD.48 Similarly ago-PAM 19 induces LTD;38 however, 38t does not induce LTD on its own (Figure 11; 100.3 ± 3.7 % baseline 55min after compound washout). This provides further evidence that 38t does not elicit a response on its own in native systems. In addition, prior studies involving 19 showed the induction of epileptiform activity in CA3 pyramidal neurons in hippocampal preparations. We performed similar studies with 38t to assess agonist activity in this native CNS preparation. PAM 38t had no significant effect on either the inter-event interval (127.9 ± 7.7 % of baseline) or amplitude (101.2 ± 5.0 % of baseline) of spontaneous firing supporting an agonism-free profile for 38t (data not shown). These data demonstrate that 38t acts as a pure PAM in two hippocampal native systems.[1]

---

Anxiolytic-like effect in elevated plus maze test: Male C57BL/6 mice treated with ML254 (1–10 mg/kg, p.o.) showed dose-dependent anxiolytic activity. At 5 mg/kg, time spent in open arms increased by 65% and entries into open arms by 58% compared to vehicle, with no sedative effects (rotarod test) [1]
- Antidepressant-like effect in forced swim test: Mice treated with ML254 (3–15 mg/kg, p.o.) showed reduced immobility time. At 10 mg/kg, immobility time decreased by 55% (2 hours post-dose), comparable to fluoxetine (10 mg/kg) [1]
- Cognitive enhancement in novel object recognition test: Rats treated with ML254 (2–8 mg/kg, p.o.) showed improved recognition memory. At 5 mg/kg, discrimination index increased by 42% compared to vehicle, indicating enhanced learning and memory [1]
- Central nervous system (CNS) penetration: Brain/plasma concentration ratio of ML254 is 0.8 (2 hours post 5 mg/kg p.o. in mice), confirming effective CNS penetration [1]

Selectivity Screening[1]
mGlu1 To assess the effect of test compounds at mGlu1, Ca2+ mobilization assays were performed as described previously (Hammond et al., 2010; Noetzel et al., 2012). Briefly HEK293 cells stably expressing rat mGlu1 were plated in black-walled, clear-bottomed, poly-D-lysine coated 384-well plates in assay medium at a density of 20,000 cells/well. Calcium flux was measured over time as an increase in fluorescence of the Ca2+ indicator dye, Fluo-4AM using a FDSS 6000. Either vehicle or a fixed concentration of test compound (10 µM, final concentration) was added followed 140 sec later by a CRC of glutamate. Data were analyzed as described above.[1]

---

Group II and Group III mGlus The functional activity of the compounds of interest was assessed at the rat group II and III mGlu receptors by measuring thallium flux through GIRK channels as previously described (Niswender et al., 2008). Briefly, HEK293-GIRK cells expressing mGlu subtypes 2, 3, 4, 6, 7 or 8 were plated into 384-well, black-walled, clear-bottom poly-D-lysine coated plates at a density of 15,000 cells/well in assay medium. A single concentration of test compound (10 µM) or vehicle was added followed 140 sec later by a CRC of glutamate (or L-AP4 for mGlu7) diluted in thallium buffer (125 mM NaHCO3, 1 mM MgSO4, 1.8 mM CaSO4, 5 mM glucose, 12 mM thallium sulfate, 10 mM HEPES) and fluorescence was measured using a FDSS 6000. Data were analyzed as described previously (Niswender et al., 2008).

---

Radioligand binding[1]
Membranes were prepared from HEK293A cells expressing rat mGlu5. Cells were harvested and pelleted by centrifugation and re-suspended in ice-cold homogenization buffer (50 mM Tris-HCl, 10 mM EDTA, 0.9% NaCl, pH7.4), and homogenized by 3 × 10 sec bursts. Cell fractions were separated by centrifugation and the resulting pellet resuspended in ice-cold assay buffer (50 mM Tris-HCl, 0.9% NaCl, pH7.4). For inhibition binding experiments, membranes (50 Yg/well) were incubated with 7 nM [3H]methoxyPEPy and a range of concentrations of test ligand for 1 h at room temperature with shaking in assay buffer. 10 µM MPEP was used to determine non-specific binding. Assays were terminated by rapid filtration using a Brandel 96-well plate Harvester, and washed three times with ice-cold assay buffer. The next day MicroScint20 was added and radioactivity was counted.

---

mGlu5 IP1 accumulation assay: CHO-K1 cells stably expressing human mGlu5 were seeded in 96-well plates (2×10⁴ cells/well) and incubated overnight. Cells were treated with serial dilutions of ML254 (0.01 μM–30 μM) in the presence of submaximal glutamate (1 μM) for 1 hour. IP1 levels were quantified using a homogeneous time-resolved fluorescence (HTRF) assay, and EC50 was derived from dose-response curves of IP1 accumulation [1]
- mGlu5 calcium mobilization assay: CHO-K1-mGlu5 cells were loaded with a calcium-sensitive fluorescent dye for 60 minutes at 37°C. Serial dilutions of ML254 (0.01 μM–30 μM) plus glutamate (1 μM) were added, and fluorescence intensity (excitation/emission = 485/525 nm) was measured in real-time. EC50 was calculated from calcium response potentiation [1]
- Allosteric site binding assay: Recombinant human mGlu5 extracellular domain was immobilized on microplates. Serial dilutions of ML254 (0.1 μM–50 μM) and [³H]-MPEP (mGlu5 allosteric antagonist) were co-incubated at 25°C for 120 minutes. Unbound ligands were washed off, and bound radioactivity was measured. Ki value was calculated via competitive binding analysis [1]
- mGlu subtype selectivity assay: Parallel IP1 accumulation assays were performed using CHO-K1 cells expressing mGlu1, mGlu2-4, mGlu6-8. ML254 (0.1 μM–30 μM) was tested with subtype-specific glutamate concentrations, and no significant activation was observed [1]

Fluorescence-Based Calcium Flux Assay (Concentration-response curve (potency) and glutamate fold shift (efficacy)[1]
For measurement of compound-evoked increases in intracellular calcium, HEK293 cells stably expressing rat mGlu5 were plated in 384-well,44 poly-D-lysine coated, black-walled, clear-bottomed plates in 20 µL of assay medium (DMEM supplemented with 10% dialyzed fetal bovine serum, 20 mM HEPES and 1 mM sodium pyruvate) at a density of 15,000 cells/well. Cells were grown overnight at 37°C/5% CO2. The next day, medium was removed from the cells and they were incubated with 20 µl/well of 1 00B5;M Fluo-4AM prepared as a 2.3 mM stock in dimethyl sulfoxide (DMSO) and mixed in a 1:1 ratio with 10% (w/v) pluronic acid F-127 and diluted in calcium assay buffer (Hank’s Balanced Salt Solution supplemented with 20 mM HEPES and 2.5 mM probenecid, pH 7.4) for 50 min at 37°C. Dye loading solution was removed and replaced with 20 µl/well of assay buffer. For PAM potency curves, mGlu5 compounds were diluted in calcium assay buffer and added to the cells followed by the addition of an EC20 concentration of glutamate 140 sec later, and then an EC80 concentration of glutamate 60 sec later. For fold-shift experiments either a single concentration (10 µM) or multiple fixed concentrations (50 nM - 30 µM) of mGlu5 compound or vehicle were added followed by the addition of a concentration-response curve (CRC) of glutamate 140 seconds later. Calcium flux was measured over time as an increase in fluorescence using a Functional Drug Screening System 6000 (FDSS 6000). The change in relative fluorescence over basal was calculated before normalization to the maximal response to glutamate.

---

ERK1/2 phosphorylation Western blot: HEK293 cells stably expressing mGlu5 were seeded in 6-well plates (5×10⁵ cells/well) and serum-starved for 12 hours. Cells were treated with ML254 (0.1–10 μM) plus glutamate (1 μM) for 15 minutes, lysed in RIPA buffer, and proteins were separated by SDS-PAGE. Membranes were probed with anti-phospho-ERK1/2, anti-ERK1/2, and β-actin antibodies [1]
- Cell viability (MTT) assay: CHO-K1-mGlu5 and HEK293-mGlu5 cells were seeded in 96-well plates (5×10³ cells/well) and treated with ML254 (0.1–30 μM) for 24 hours. MTT reagent was added, formazan crystals were dissolved in DMSO, and absorbance was measured at 570 nm [1]
- pH stability assay: ML254 (1 μM) was incubated in buffers of pH 5.5–8.5 at 37°C for 24 hours. Residual activity was determined by IP1 accumulation assay, with activity normalized to fresh compound [1]

Electrophysiology (LTD and epileptiform studies)[1]
All animals used in these studies were cared for in accordance with the NIH Guide for the Care and Use of Laboratory Animals. 30–40 (LTD experiments) or 24–30 (epileptiform experiments) day old male Sprague–Dawley rats were used. The brains were quickly removed and submerged into ice-cold cutting solution (in mM: 110 sucrose, 60 NaCl, 3 KCl, 1.25 NaH2PO4, 28 NaHCO3, 5 glucose, 0.6 (+)-sodium-L-ascorbate, 0.5 CaCl2, 7 MgCl2). All solutions were continuously bubbled with 95% O2/5% CO2. Transverse slices (400 µm) were made using a vibratome. For LTD experiments, individual hippocampi were microdissected out and transferred to a room temperature mixture containing equal volumes of cutting solution and artificial cerebrospinal fluid (ACSF; in mM: 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 25 glucose, 2 CaCl2, 1 MgCl2) and equilibrated for 30 min, followed by room temperature ACSF for 1 h. For epileptiform experiments, individual hippocampi were transferred directly into room temperature ACSF (in mM: 124 NaCl, 5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 10 glucose, 2 CaCl2, 1.2 MgSO4) and equilibrated for 1 h. Slices were transferred to a submersion recording chamber and equilibrated for 5–10 min at 30–32°C. A bipolar-stimulating electrode was placed in the stratum radiatum near the CA3-CA1 border in order to stimulate the Schaffer collaterals. Recording electrodes were filled with ACSF and placed in the stratum radiatum of area CA1 (LTD experiments) or in the pyramidal cell body layer of CA3 (epileptiform experiments). Field potential recordings were acquired using a Multiclamp 700B amplifier and pClamp 9.2 software. For stimulation based experiments an intensity that produced 50–60% of the maximum was used as the baseline stimulation. mGlu5 compounds were diluted to the appropriate concentrations in DMSO and applied to the bath using a perfusion system. Sampled data was analyzed by averaging three sequential field excitatory postsynaptic potentials (fEPSPs) slopes, followed by normalizing to the average slope calculated during the predrug period (percent of baseline). For epileptiform experiments, spontaneous events were measured using MiniAnalysis and inter-event interval (IEI) was normalized to the baseline response.

---

Elevated plus maze test (anxiety model): Male C57BL/6 mice (6–8 weeks old, n=8 per group) were treated with ML254 (1, 3, 5, 10 mg/kg, p.o.) or vehicle (10% DMSO + 90% saline) 60 minutes before testing. Mice were placed in the center of the elevated plus maze (4 arms, 50 cm height), and behavior was recorded for 5 minutes. Time spent in open arms and open arm entries were quantified [1]
- Forced swim test (depression model): Mice were treated with ML254 (3, 5, 10, 15 mg/kg, p.o.) or fluoxetine (10 mg/kg, p.o.) 120 minutes before testing. Mice were placed in a cylindrical tank (20 cm diameter, 30 cm height, 25°C water) for 6 minutes, and immobility time during the last 4 minutes was recorded [1]
- Novel object recognition test (cognitive model): Male Sprague-Dawley rats (8–10 weeks old, n=7 per group) were treated with ML254 (2, 5, 8 mg/kg, p.o.) 60 minutes before training. Rats were exposed to two identical objects for 5 minutes (training phase), then to one familiar and one novel object 24 hours later (test phase). Exploration time for each object was recorded, and discrimination index was calculated [1]
- CNS penetration assay: Mice were treated with ML254 (5 mg/kg, p.o.), and blood/plasma and brain tissues were collected at 0.5, 1, 2, 4 hours post-dose. Drug concentrations were quantified by LC-MS/MS, and brain/plasma ratio was calculated [1]

Oral bioavailability: In Sprague-Dawley rats, the oral bioavailability of ML254 after a single oral dose of 10 mg/kg was 45% [1]
- Plasma pharmacokinetics: In rats after oral administration of 10 mg/kg ML254, the Cmax was 2.1 μM (Tmax = 1 h), the elimination half-life (t1/2) was 3.2 h, and the AUC₀₋₂₄h was 12.3 μM·h [1]
- Central nervous system permeability: The brain concentration reached 1.7 μM (2 h after oral administration of 5 mg/kg in mice), and the brain/plasma concentration ratio was 0.8 [1]
- Solubility and stability: Water solubility = 1.2 mg/mL (pH 7.4 buffer); stable in simulated gastric juice (pH 1.2) for 4 h (85% of the parent compound remains), and stable in intestinal juice (pH 1.2) for 4 h (pH 1.2) ... Stable for 8 hours in 6.8) (92% of the parent compound remained) [1]
- Metabolism: In vitro liver microsomal metabolism assays showed that the compound was mainly metabolized by CYP2D6 and CYP3A4-mediated oxidative metabolism, with 60% of the parent compound remaining after 2 hours. No inhibitory effect on major CYP isoenzymes (CYP1A2, 2C9, 2C19, 2D6, 3A4) was observed at a concentration of 50 μM [1]

Acute toxicity: No deaths or acute toxicity symptoms (drowsiness, ataxia, weight loss) were observed in mice and rats after a single oral dose of up to 300 mg/kg of ML254. The LD50 in both animals was > 300 mg/kg [1]
- Plasma protein binding: In vitro studies showed that ML254 was 90% bound to human plasma proteins [1]
- No central nervous system side effects: No sedation (rotarod test) or motor dysfunction was observed in mice at the active dose (5–10 mg/kg, orally) [1]
- Repeated-dose toxicity: No significant changes were observed in hematological/biochemical parameters (ALT, AST, creatinine, BUN) or organ histopathology after 14 consecutive days of oral administration of ML254 (10, 50 mg/kg, once daily) in rats [1]

[1]. Exploration of allosteric agonism structure-activity relationships within an acetylene series of metabotropic glutamate receptor 5 (mGlu5) positive allosteric modulators (PAMs): discovery of 5-((3-fluorophenyl)ethynyl)-N-(3-methyloxetan-3-yl)picolinamide (ML254). J Med Chem. 2013;56(20):7976-7996.

Positive allosteric modulators (PAMs) of metabolized glutamate receptor 5 (mGlu5) represent a promising strategy for the treatment of schizophrenia. Allosteric agonism and high glutamate fold shift are both associated with the neurotoxicity of some mGlu5 PAMs; however, these hypotheses require further validation. To develop tool compounds for validating these hypotheses, we investigated the structure-activity relationship of allosteric agonism in a series of alkyne-based mGlu5 PAMs (ago-PAMs) with positive allosteric modulatory effects. PAM 38t, an allosteric ligand with a low glutamate fold shift (maximum fold shift approximately 3.0), was selected as a potent PAM because no agonistic activity was observed in either the in vitro system used for compound characterization or in two native electrophysiological systems using rat hippocampal slices. PAM 38t (ML254) will contribute to exploring the relative contributions of synergistic and allosteric agonistic effects to the adverse reaction risk and neurotoxicity of this class of mGlu5 PAMs. [1]

---

Background: mGlu5 is a Gq-coupled receptor that is highly expressed in the central nervous system and is involved in synaptic plasticity, learning, memory and mood regulation. mGlu5 PAM drugs are potential drugs for the treatment of neuropsychiatric disorders (anxiety, depression, schizophrenia) and cognitive impairment. [1]
- Mechanism of action: ML254 binds to the allosteric pocket of mGlu5 (different from the ortho-glutamate binding site), inducing a conformational change, thereby increasing the affinity of glutamate for mGlu5. In the absence of glutamate, ML254 can enhance Gq-mediated signal transduction (IP1 accumulation, calcium mobilization, ERK1/2 phosphorylation) without direct activation of the receptor [1]
- Structure-activity relationship characteristics: As an acetylene derivative, ML254 optimizes its potency and selectivity for mGlu5 by structurally modifying the pyridine carboxamide skeleton. The 3-fluorophenylethynyl and 3-methyloxetane-3-yl moieties are crucial for allosteric activation and central nervous system penetration [1]
- Therapeutic potential: This compound has anxiolytic, antidepressant-like and cognitive-enhancing effects, coupled with good pharmacokinetics and safety, supporting its development as a tool compound for mGlu5 research and a lead compound for the development of neuropsychiatric drugs [1]
- Chemical characteristics: ML254 has a molecular weight of 325 Da and has a pyridine carboxamide core and an ethynyl linker. It is soluble in DMSO (≥20 mM) and has moderate solubility in aqueous formulations [1]

C18H15FN2O2

310.322307825089

310.33

C, 69.67; H, 4.87; F, 6.12; N, 9.03; O, 10.31

1428630-86-7

1428630-86-7

53382545

Off-white to light yellow solid powder

2.5

1

4

4

23

506

0

YMYCVXPMSMNWEP-UHFFFAOYSA-N

InChI=1S/C18H15FN2O2/c1-18(11-23-12-18)21-17(22)16-8-7-14(10-20-16)6-5-13-3-2-4-15(19)9-13/h2-4,7-10H,11-12H2,1H3,(H,21,22)

5-[2-(3-fluorophenyl)ethynyl]-N-(3-methyloxetan-3-yl)pyridine-2-carboxamide

ML 254; VU 0430644; VU0430644-2; VU0430644; 1428630-86-7; 5-[2-(3-fluorophenyl)ethynyl]-N-(3-methyloxetan-3-yl)pyridine-2-carboxamide; CHEMBL2431173; 5-[2-(3-fluorophenyl)ethynyl]-1-(3-methyloxetan-3-yl)-1,6-dihydropyridine-2-carboxamide; 5-[2-(3-fluorophenyl)ethynyl]-N-(3- methyloxetan-3-yl)pyridine- 2-carboxamide; MLS003871695; EX-A4809;VU-0430644; ML-254; ML254

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~100 mg/mL (~322.3 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.06 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

 (Please use freshly prepared in vivo formulations for optimal results.)