2.1. Assays

• AID 60245: mGluR3_NAM_Project_Summary
• AID 623885: mGlu3_Thallium_Flux_EC80_CRC
• AID 623888: mGlu3_Thallium_Flux_Schild
• AID 624028: mGluR3_Galpha15_Calcium_CRC
• AID 623929: mGluR2_Thallium_Flux_EC80_CRC
• AID 588715: mGlu5_PAM_SecondaryAssay1
• AID 623931: ML289_Ricerca_Binding

2.2. Probe Chemical Characterization

Probe compound ML289 (CID 56687994, SID 134418959) was prepared according to the scheme in (Figure 1) and had the following characterization. (R)-(3-(hydroxymethyl)piperidin-1-yl)(4-((4-methoxyphenyl)ethynyl)phenyl)methanone.¹H-NMR (400 MHz, CD₃OD) δ 7.58 (d, J = 8.0 Hz, 2H), 7.48 (d, J = 8.0 Hz, 2H), 7.42 (d, J = 8.0 Hz, 2H), 6.96 (d, J = 8.0 Hz, 2H), 4.60-4.48 (m, 1H), 3.84 (s, 3H), 3.86-3.65 (m, 1H), 3.53-3.27 (m, 2H), 3.15-2.74 (m, 2H), 1.87-1.73 (m, 3H), 1.59-1.30 (m, 3H); ¹³C-NMR (100 MHz, CD₃OD) δ 170.37, 160.11, 135.12, 132.71, 131.00, 126.68, 125.18, 114.62, 113.77, 90.50, 86.57, 63.65, 54.37,45.24, 42.76, 38.53, 26.71, 24.07; HRMS (TOF, ES+) C₂₂H₂₄NO₃ [M+H]⁺ calc. mass 350.1756, found 350.1755. Specific rotation [α]23/D = -28.0 (c = 100, MeOH).

Figure 1

Synthesis of ML289.

Solubility. Solubility for ML289 in PBS was determined to be 19.4 μM (6.8 μg/mL), which is ~30-fold higher than the cellular IC₅₀ for mGlu₃ inhibition.¹

GSH Conjugates. No glutathione conjugates detected.¹

Stability. Stability was determined for ML289 at 23 °C in PBS (no antioxidants or other protectorants and DMSO concentration below 0.1%). After 48 hours, ~100% of the initial concentration of ML289 remained in solution. Thus, ML289 is very stable in vitro in assay buffer as well as in vivo (vide infra).¹

Compounds added to the SMR collection (MLS#s): MLS004084522 (ML289, CID 56587994, 27.4 mg); MLS004084523 (CID 53338952, 16.6 mg); MLS004084524 (CID 56587981, 11.5 mg); MLS004084525 (CID 53393871, 7.3 mg); MLS004084526 (CID 56587989, 10.9 mg); MLS004084527 (CID 56642818, 23.1 mg).

2.3. Probe Preparation

Probe compound ML289 (CID 56687994, SID 134418959) was prepared according to the scheme in (Figure 2) and had the following characterization. (R)-(3-(hydroxymethyl)piperidin-1-yl)(4-((4-methoxyphenyl)ethynyl)phenyl)methanone. Ethyl 4-(phenylethynyl)benzoate (5). To a solution of ethyl 4-Iodobenzoate 3 (5.0 g, 18.2 mmol) in DMF (8 mL) was added 4-ethynyl anisole 4 (2.25 g, 22.1 mmol), Pd(Ph3P)4 (502 mg, 0.45 mmol), CuI (172 mg, 0.91 mmol) and diethylamine (2 mL). The reaction vessel was sealed and heated at 60 °C for 1h in a microwave reactor. The reaction was cooled to rt, diluted with EtOAc:hexanes (2:1, 150 mL) and washed with water (2 × 100 mL) and brine (100 mL). The organic phase was dried over MgSO4, filtered, and concentrated under vacuum. The crude product was purified by column chromatography (silica gel) using 0 to 10 % EtOAc/hexanes to afford ester 5 (7.89 g, 86%) as a pale yellow solid: ¹H-NMR (400 MHz, CDCl3) δ 8.05 (d, J = 8.0 Hz, 2H), 7.61 (d, J = 8.0 Hz, 2H), 7.56 (dd, J = 8.0, 2.0 Hz, 2H), 7.41-7.37 (m, 3H), 4.41 (q, J = 7.0 Hz, 2H), 1.44 (t, J = 7.0 Hz, 3H); LC (214 nm) 5.79 min (>98%); MS (ESI) m/z = 250.9. 4-(phenylethynyl)benzoic acid (6). To a solution of ester 5 (7.81 g, 31.2 mmol) in THF (80 mL) was added MeOH (15 mL) and a solution of LiOH (5.24 g, 124 mmol) in water (15 mL). The reaction was stirred at room temperature for 4h. The reaction was acidified with 1 N HCl (50 mL) and isolated benzoic acid 6 (5.78 g, 83%) as a white solid: mp 190.1 °C; ¹H-NMR (400 MHz, CDCl3) δ 8.11 (d, J = 8.0 Hz, 2H), 7.64 (d, J = 8.0 Hz, 2H), 7.62-7.56 (m, 2H), 7.52-7.47 (m, 1H), 7.43-7.36 (m, 3H); ¹³C-NMR (100 MHz, d6- DMSO) δ 167.3, 138.0, 134.5, 131.9, 131.4, 130.9, 130.6, 130.2, 130.0, 129.6, 127.0, 122.15, 101.6, 92.3, 89.0; LC (214 nm) 5.12 min (>98%); MS (ESI) m/z = 222.9. (R)-(3-(hydroxymethyl)piperidin-1-yl)(4-((4-methoxyphenyl)ethynyl)phenyl) methanone (3). To a solution of acid 6 (30 mg, 0.12 mmol) and DIPEA (0.1 mL, 0.6 mmol) in DMF (0.3 mL) was added HATU (45.6 mg, 0.12 mmol). The reaction was stirred for 20 min at rt, then (R)-(3-hydroxymethyl)piperidine 7 (15.0 mg, 0.13 mmol). The reaction was stirred at room temperature for 18 h. The reaction was quenched with water/brine (2 mL) and was extracted with EtOAc (3 × 10 mL). The product was dried over MgSO4, filtered and concentrated under vacuum. The crude product was purified by reverse phase HPLC eluting with MeCN/H₂O/TFA to afford ML289 (31 mg, 73.9%) as a white solid: ¹H-NMR (400 MHz, CD₃OD) δ 7.58 (d, J = 8.0 Hz, 2H), 7.48 (d, J = 8.0 Hz, 2H), 7.42 (d, J = 8.0 Hz, 2H), 6.96 (d, J = 8.0 Hz, 2H), 4.60-4.48 (m, 1H), 3.84 (s, 3H), 3.86-3.65 (m, 1H), 3.53-3.27 (m, 2H), 3.15-2.74 (m, 2H), 1.87-1.73 (m, 3H), 1.59-1.30 (m, 3H); ¹³C-NMR (100 MHz, CD₃OD) δ 170.37, 160.11, 135.12, 132.71, 131.00, 126.68, 125.18, 114.62, 113.77, 90.50, 86.57, 63.65, 54.37,45.24, 42.76, 38.53, 26.71, 24.07; HRMS (TOF, ES+) C₂₂H₂₄NO₃ [M+H]⁺ calc. mass 350.1756, found 350.1755. Specific rotation [α]23/D = -28.0 (c = 100, MeOH).

Figure 2

Complete Synthesis of ML289.

3.1. Dose Response Curves for Probe

We next evaluated the selectivity of ML289 between mGlu₂ and mGlu₅. Utilizing our mGlu₂ GIRK line, the IC₅₀ was much greater than 10 μM, with the CRC not reaching baseline at this highest concentration (Figure 3A, squares). Similarly, ML289 had no effect on potentiating an EC₂₀ concentration of glutamate in our standard mGlu₅ calcium assay (Figure 3C, triangles). As our calcium assays typically drive our mGlu drug discovery programs, we also evaluated ML289 in an mGlu₃ calcium assay in which mGlu₃ is co-expressed with the promiscuous G protein G_α15 (Figure 3B-D). Here, we see improved mGlu₃ NAM potency (IC₅₀ = 649 nM), with high selectivity versus mGlu₂ (~15-fold) and mGlu₅ in both PAM and NAM modes (inactive). Thus, we were able to optimize and develop a potent and selective mGlu₃ NAM starting from a very potent mGlu₅ PAM.

Figure 3

In vitro molecular pharmacology characterization of ML289. A) Concentration-response curves of mGlu₂ and mGlu₃ GIRK (antagonist mode). B) mGlu₃ calcium (antagonist mode) and mGlu₅ calcium (antagonist mode). C) mGlu₃ calcium (antagonist mode) and mGlu (more...)

3.2. Cellular Activity

The primary screening assays (both GIRK and Calcium) are cell-based assays, indicating that ML289 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, and cyctotoxicity assays aimed at this parameter indicated ML289 had no cyctotoxicity in non-transformed HEK293.

3.3. Profiling Assays

To more fully characterize this potent, selective mGlu₃ NAM ML289 was tested using Ricerca’s (formerly MDS Pharma’s) Lead Profiling Screen (binding assay panel of 68 GPCRs, ion channels and transporters screened at 10 μM).² Included in the Ricerca screening panel are a number of ion channels (Calcium Channel, L-Type and N-Type; Potassium channel [K_ATP]; Potassium channel [hERG]) and class A GPCRs (D_1–5, H_1–3, etc.). ML289 was found to only inhibit two targets (DAT: 88%@10 μM and 5-HT_2B 77%@10 μM) out of the 68 assays (Table 1) conducted (inhibition of radio ligand binding > 50% at 10 μM). Table 2 highlights calculated properties for ML289, which compares favorably with the MDDR.

Table 1

Ricerca Profiling of ML289.

Table 2

Calculated Property Comparison with MDDR Compounds.

In order to aid the wider community in the use ML289, we further profiled ML289 in a battery of pharmacokinetic assays (Table 3) including an assessment of intrinsic clearance (CL_INT) in hepatic microsomes, allowing for the prediction of pertinent rat and human PK parameters (CL and t_1/2). ML298 is predicted to have high clearance in rat (CL_INT = 240.8 mL/min/kg, CL_HEP = 54.2 mL/min/kg) and moderate clearance in human (CL_INT = 571.7 mL/min/kg, CL_HEP = 20.3 mL/min/kg), and possesses reasonable free fraction (~1–3% free in human and rat equilibrium dialysis plasma protein binding studies). Moreover, ML289 has an exceptionally clean CYP P450 profile, displaying no inhibition of the four major CYPs (3A4 (>30 μM), 2D6 (>30 μM), 2C9 (>30 μM) and 1A2 (>30 μM)).

Table 3

In vitro DMPK Profile of ML298.

To better understand the rapid clearance and short half-life in rats, we performed metabolite ID studies in S9 fractions. Metabolite ID studies in rat and human liver microsomes (Figure 4) indicated that the principle biotransformation pathway was P450-mediated O-demethylation of ML289 to generate the phenol 7, a metabolite that was subsequently shown to be inactive at mGlu₃.

Figure 4

Oxidative O-dealkylation of ML289 in rat and human liver microsomes.

4.1. Comparison to existing art and how the new probe is an improvement

ML289 (CID 56587994) is the most potent (IC₅₀ = 649 nM) and selective mGlu₃ NAM reported to date with >15-fold selectivity vs. mGlu₂ and inactive on mGlu₅ (the original lead compound was a potent (EC₅₀ = 197 nM) mGlu₅ PAM. The existing art, represented by (Figure 5) 1 and 2,^3,4 are inferior to the probe ML289 in terms of both potency and mGlu₃ selectivity. ML289 can be used both in vitro and in vivo to study the role of selective inhibition of mGlu₃ in the CNS in a manner previously unavailable with the prior art compounds. Moreover, ML289 possess an attractive in vitro and in vivo DMPK profile, ancillary pharmacology (only two activities at 10 μM in the Ricerca Panel) and affords excellent CNS exposure (brain:plasma ratio of 1.67). ML289 can be used for basic biochemical and electrophysiological experiments as well as to help establish potential utility for mGlu₃ inhibition as a target for a wide variety of important CNS therapeutic indications. Finally, ML289 is free from IP constraints, which will allow the MLPCN to freely provide this probe to the biomedical research community.

Figure 5

Structures of mGlu₃ NAMs RO4491533 (1) and LY2399575 (2), both dual mGlu₂/mGlu₃ NAMs.