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Development of a Second Generation mGlu3 NAM Probe

, , , , , , and .

Author Information

, , , , , , and *.

Vanderbilt Specialized Chemistry Center, Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center

Received: ; Last Update: March 22, 2013.

Herein we report the discovery and structure activity relationship (SAR) of a novel, second generation metabotropic glutamate receptor 3 (mGlu3) negative allosteric modulator (NAM) probe (ML337) with >50-fold selectivity versus mGlu2 (IC50 >30 μM). The mGlu3 NAM was discovered via an iterative parallel synthesis optimization of the first generation mGlu3 NAM ML289, that was only ~15-fold selective. This mGlu3 NAM (60204017, ML337) displays an IC50 value of 592 nM and is inactive (>30 μM) on mGlu2 (as well as mGlu1,4,5,6,7,8) and clean in a Ricerca ancillary pharmacology panel. ML337 possesses favorable physiochemical properties, a good dystrophia myotonica protein kinase (DMPK) profile and is centrally penetrant. Thus, ML337 is a best-in-class in vitro and in vivo probe for studying non-competitive antagonism of mGlu3.

Assigned Assay Grant #: R01 NS031373

Screening Center Name & PI: A Medicinal Chemistry FastTrack

Chemistry Center Name & PI: Vanderbilt Specialized Chemistry Center for Accelerated Probe Development, Craig W. Lindsley

Assay Submitter & Institution: P. Jeffrey Conn, Vanderbilt University

PubChem Summary Bioassay Identifier (AID): 612451

Probe Structure & Characteristics

ML337.

ML337

CID/ML#Target NameIC50/ (nM) [SID, AID]Anti-target Name(s)IC50 (μM) [SID, AID]Fold SelectiveSecondary Assay(s) Name: IC50/EC50 (nM) [SID, AID]
CID 60204017/ML337mGlu3
(calcium)
592 nM, 0.3% [SID 144223348, AID 602451, AID 651840]mGlu2

mGlu1,4,5,6,7,8
>30 μM

>30 μM

[SID 144223348, AID 623929, AID 588715]
>50-fold
>50-fold
mGlu3 (GIRK)

1.48 μM, −4.4%

[SID 144223348, AID 651830, AID 623885, AID 588715]

1. Recommendations for Scientific Use of the Probe

ML337 (CID 60204017) is the most potent (IC50 = 592 nM, 0.3%) and selective mGlu3 NAM reported to date with >60-fold selectivity vs. mGlu2 as well as mGlu1,4,5,6,7,8 (> 30 μM). ML337 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 70% inhibition @10 μM in the Ricerca Panel) and affords excellent CNS exposure in both mouse (brain:plasma ratio of 0.92) and rat (brain:plasma ratio of 0.3). ML337 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.

2. Materials and Methods

Cell culture. Human Embryonic Kidney (HEK-293) cell lines co-expressing mGlu 2, 3, 4, 6, 7 or 8 and GIRK potassium channels were grown in growth medium containing 45% DMEM, 45% F-12, 10% FBS, 20 mM HEPES, 2 mM L-glutamine, antibiotic/antimycotic non-essential amino acids, 700 μg/ml G418, and 0.6 μg/ml puromycin. Rat mGlu1 and mGlu5 cells were cultured as described in Hempstapat et al., 2007. TREx293 cells stably expressing mGlu3 and the promiscuous G protein Galpha15 were grown in Dulbecco’s Modified Eagle Media (DMEM), 10% Tet-tested fetal bovine serum (Atlanta Biologicals), 100 units/ml penicillin/streptomycin, 20 mM HEPES (pH 7.3), 1 mM sodium pyruvate, 2 mM glutamine, 1× MEM Non-Essential Amino Acids Solution, 500 ug/ml G418 (Mediatech, Inc., Herndon, VA), 100 μg/mL hygromycin, and 5 μg/mL blasticidin S (Growth Media). Cells for experiments were generally maintained for approximately 15–20 passages. All cell culture reagents were purchased from Invitrogen Corp. (Carlsbad, CA) unless otherwise noted. All cells were maintained at 37 °C in the presence of 5% CO2. All cell culture reagents were purchased from Invitrogen Corp. (Carlsbad, CA) unless otherwise noted.

Calcium mobilization assays. Calcium assays were used to assess activity of compounds at mGlus 1, 3, and 5. Briefly, mGlu3 TREx293, mGlu1, and mGlu5 cells were plated into 384 well, black-walled, clear-bottom poly-D-lysine coated plates (Greiner) at a density of 15,000 cells/20 μL/well in DMEM containing 10% dialyzed FBS, 20 mM HEPES, and 100 units/ml penicillin/streptomycin (Assay Media). For the mGlu3 TREx293 cells, this assay medium was also supplemented with 25 ng/mL tetracycline for 20 hours. Plated cells were incubated overnight at 37 °C in the presence of 5% CO2. The following day, plated cells had their medium exchanged to Assay Buffer (Hanks Balanced Salt Solution (Invitrogen) containing 20 mM HEPES and 2.5 mM probenacid, pH 7.3) using an ELX405 microplate washer (BioTek), leaving 20 μL/well, followed by addition of with 20 μL of 4.5 μM Fluo 4 AM (Invitrogen, Carlsbad, CA) prepared as a 2.3 mM stock in DMSO and mixed in a 1:1 ratio with 10 percent (w/v) pluronic acid F-127 and diluted in Assay Buffer for 45 min at 37 °C. The dye was then exchanged to Assay Buffer using an ELX405, leaving 20 μL/well and the plates were incubated at room temperature for 10 min prior to assay. For concentration-response curve experiments, compounds were serially diluted 1:3 into 10 point concentration response curves in DMSO, were transferred to daughter plates using an Echo acoustic plate reformatter (Labcyte, Sunnyvale, CA), and diluted into Assay Buffer to generate a 2× stock. Calcium flux was measured using the Functional Drug Screening System 6000 or 7000 (FDSS6000 or FDSS7000, Hamamatsu, Japan). Baseline readings were taken (10 images at 1 Hz, excitation, 470+/−20 nm, emission, 540+/−30 nm) and then 20 μL/well test compounds were added using the FDSS’s integrated pipettor. Approximately 2.5 minutes later an EC20 concentration of glutamate (10 μL of a 5× final concentration) was added followed approximately 2.0 minutes later by an EC80 concentration of glutamate (12 μL of a 5× final concentration). For fold shift experiments, compounds were added at 2X their final concentration and then increasing concentrations of glutamate were added in the presence of vehicle or the appropriate concentration of test compound. Curves were fitted using a four point logistical equation using Microsoft XLfit (IDBS, Bridgewater, NJ). Subsequent confirmations of concentration response parameters were performed using independent serial dilutions of source compounds and data from multiple days experiments were integrated and fit using a four point logistical equation in GraphPad Prism (GraphPad Software, Inc., La Jolla, CA).

Thallium flux assays. Compound activity at the group II and group III mGlus was assessed using thallium flux through GIRK channels, a method that has been described in detail.1 Briefly, cells were plated into 384 well, black-walled, clear-bottom poly-D-lysine coated plates (Greiner) at a density of 15,000 cells/20 μL/well in DMEM containing 10% dialyzed FBS, 20 mM HEPES, and 100 units/ml penicillin/streptomycin (Assay Media). Plated cells were incubated overnight at 37 °C in the presence of 5% CO2. The following day, plated cells had their medium exchanged to Assay Buffer (Hanks Balanced Salt Solution (Invitrogen) containing 20 mM HEPES pH 7.3) using an ELX405 microplate washer (BioTek), leaving 20 μL/well, followed by addition of with 20 μL of 330 nM FluoZin-2 AM (Invitrogen, Carlsbad, CA) prepared as a 2.85 mM stock in DMSO and mixed in a 1:1 ratio with 10 percent (w/v) pluronic acid F-127 and diluted in Assay Buffer for 1 hour at room temperature. The dye was then exchanged to Assay Buffer using an ELX405, leaving 20 μL/well and the plates were incubated at room temperature for 10 min prior to assay. For concentration-response experiments, compounds were serially diluted 1:3 into 10 point concentration response curves in DMSO, were transferred to daughter plates using an Echo acoustic plate reformatter (Labcyte, Sunnyvale, CA), and diluted into Assay Buffer to generate a 2X stock. Agonists were diluted in Thallium Buffer (125 mM sodium bicarbonate (added fresh the morning of the experiment), 1 mM magnesium sulfate, 1.8 mM calcium sulfate, 5 mM glucose, 12 mM thallium sulfate, 10 mM HEPES, pH 7.3) at 5X the final concentration to be assayed. Thallium flux was measured using the Functional Drug Screening System 6000 or 7000 (FDSS6000 or FDSS7000, Hamamatsu, Japan). Baseline readings were taken (10 images at 1 Hz, excitation, 470+/−20 nm, emission, 540+/−30 nm) and then 20 μL/well test compounds were added using the FDSS’s integrated pipettor. Approximately 2.5 minutes later 10 μL of Thallium Buffer +/− agonist was added. After the addition of agonist, data were collected for an approximately 3 additional min. Data were analyzed as described (Niswender et al., 2008). For fold shift experiments, compounds were added at 2X their final concentration and then increasing concentrations of glutamate were added in the presence of vehicle or the appropriate concentration of test compound. For selectivity experiments, full concentration-response curves of glutamate or L-AP4 (for mGlu7) were performed in the presence of a 10 μM concentration of compound, and compounds that affected the concentration-response by less than 2 fold in terms of potency or efficacy were designated as inactive.

DMPK Methods. In vitro: The metabolism of ML337 was investigated in rat hepatic microsomes (BD Biosciences, Billerica, MA) using substrate depletion methodology (% test article remaining). A potassium phosphate-buffered reaction mixture (0.1 M, pH 7.4) of test article (1 μM) and microsomes (0.5 mg/mL) was pre-incubated (5 min) at 37°C prior to the addition of NADPH (1 mM). The incubations, performed in 96-well plates, were continued at 37 °C under ambient oxygenation and aliquots (80 μL) were removed at selected time intervals (0, 3, 7, 15, 25 and 45 min). Protein was precipitated by the addition of chilled acetonitrile (160 μL), containing glyburide as an internal standard (50 ng/mL), and centrifuged at 3000 rpm (4°C) for 10 min. Resulting supernatants were transferred to new 96-well plates in preparation for LC/MS/MS analysis. The in vitro half-life (t1/2, min, Eq. 1), intrinsic clearance (CLint, mL/min/kg, Eq. 2) and subsequent predicted hepatic clearance (CLhep, mL/min/kg, Eq. 3) were determined employing the following equations:

t1/2 = Ln(2) / k ; where k represents the slope from linear regression analysis (% test article remaining)
1
CLint = (0.693 / t1/2) (rxn volume / mg of microsomes) (45 mg microsomes / gram of liver) (20a gm of liver / kg body weight); ascale-up factors of 20 (human) and 45 (rat)
2
CLhep=Q·CLintQ+CLint
3

Plasma Protein Binding. Protein binding of ML337 was determined in rat plasma via equilibrium dialysis employing Single-Use RED Plates with inserts (ThermoFisher Scientific, Rochester, NY). Briefly plasma (220 μL) was added to the 96 well plate containing test article (5 μL) and mixed thoroughly. Subsequently, 200 μL of the plasma-test article mixture was transferred to the cis chamber (red) of the RED plate, with an accompanying 350 μL of phosphate buffer (25 mM, pH 7.4) in the trans chamber. The RED plate was sealed and incubated 4 h at 37 °C with shaking. At completion, 50 μL aliquots from each chamber were diluted 1:1 (50 μL) with either plasma (cis) or buffer (trans) and transferred to a new 96 well plate, at which time ice-cold acetonitrile (2 volumes) was added to extract the matrices. The plate was centrifuged (3000 rpm, 10 min) and supernatants transferred to a new 96 well plate. The sealed plate was stored at −20 °C until LC/MS/MS analysis.

Liquid Chromatography/Mass Spectrometry Analysis. In vitro experiments. ML337 was analyzed via electrospray ionization (ESI) on an AB Sciex API-4000 (Foster City, CA) triple-quadrupole instrument that was coupled with Shimadzu LC-10AD pumps (Columbia, MD) and a Leap Technologies CTC PAL auto-sampler (Carrboro, NC). Analytes were separated by gradient elution using a Fortis C18 2.1 × 50 mm, 3.5 μm column (Fortis Technologies Ltd, Cheshire, UK) thermostated at 40 °C. HPLC mobile phase A was 0.1% NH4OH (pH unadjusted), mobile phase B was acetonitrile. The gradient started at 30% B after a 0.2 min hold and was linearly increased to 90% B over 0.8 min; held at 90% B for 0.5 min and returned to 30% B in 0.1 min followed by a re-equilibration (0.9 min). The total run time was 2.5 min and the HPLC flow rate was 0.5 mL/min. The source temperature was set at 500°C and mass spectral analyses were performed using multiple reaction monitoring (MRM) utilizing a Turbo-Ionspray® source in positive ionization mode (5.0 kV spray voltage). LC/MS/MS analysis was performed employing a TSQ QuantumULTRA that was coupled to a ThermoSurveyor LC system (Thermoelectron Corp., San Jose, CA) and a Leap Technologies CTC PAL auto-sampler (Carrboro, NC). Chromatographic separation of analytes was achieved with an Acquity BEH C18 2.1 × 50 mm, 1.7 μm column (Waters, Taunton, MA).

2.1. Assays

2.2. Probe Chemical Characterization

Probe compound ML337 (CID 60204017, SID 144223348) was prepared according Scheme 1 and had the following characterization. (R)-(2-fluoro-(4-((4-methoxyphenyl) ethynyl)phenyl)(3-(hydroxypiperidin-1-yl)methanone. [α]D23 = −27.6° (c = 1, MeOH). LC (254 nm) 0.704 min (>99%); MS (ESI) m/z = 354.1. HRMS (TOF, ES+) C21H20FNO3.[M+H]+ calc. mass 354.1505, found 354.1507. 1H NMR (400.1 MHz, d6-DMSO) δ (ppm) [* = Rotamers]: 7.51 (d, J = 8.4 Hz, 2H); 7.46 (d, J = 10.2 Hz, 1H); 7.39 (m, 2H); 6.99 (d, J = 8.9 Hz, 2H); 5.00 (dd, J1 = 76.0 Hz, J2 = 4.2 Hz, 1H); 4.17 (m, 1H*); 3.78 (s, 3H); 3.32 (d, J = 12.5 Hz, 1H*); 3.19 (m, 1H*); 2.99 (m, 1H*); 2.89 (m, 1H); 1.75 (m, 2H); 1.37 (m, 2H). 13C NMR (100.6 MHz, d6-DMSO) δ (ppm): 164.03 (d, J = 19.4 Hz), 160.31, 158.91, 156.47, 133.60, 129.39, 128.15 (d, J =9.7 Hz), 125.79, 124.90*, 124.77*, 118.52 (d, J =22.6 Hz), 114.87, 113.84, 91.91*, 91.88*, 86.73, 65.48*,65.32*, 55.69, 53.76, 48.68*, 47.05*, 41.82, 33.05*, 32.69*, 23.52.

Scheme 1. Probe preparation of ML337.

Scheme 1

Probe preparation of ML337.

Solubility. Solubility for ML337 in PBS was determined to be 7.8 μM (3.2 μg/mL), which is ~13-fold higher than the cellular IC50 for mGlu3 inhibition.

GSH Conjugates. No glutathione conjugates detected.

Stability. Stability was determined for ML337 at 23 °C in PBS (no antioxidants or other protectorants and DMSO concentration below 0.1%). See Table 1. After 48 hours, ~35% of the initial concentration of ML337 remained in solution. The loss is most likely due to limited solubility in buffer. In plasma, ML337 is very stable in vitro as well as in vivo (vide infra).

Table 1. Stability of ML337.

Table 1

Stability of ML337.

MLS004580699 (ML337, CID 60204017, 26.0 mg); MLS004580700 (CID 60204018, 5.7 mg); MLS004580701 (CID 60210783, 5.2 mg); MLS004580702 (CID 60210757, 5.0 mg); MLS004580703 (CID 60204019, 5.1 mg); MLS004580704 (CID 60210795; 4.8 mg)

2.3. Probe Preparation

Probe compound ML337 (CID 60204017, SID 144223348) was prepared according to Scheme 2 and had the following characterization. (R)-(2-fluoro-(4-((4-methoxyphenyl) ethynyl)phenyl)(3-(hydroxypiperidin-1-yl)methanone. 2-fluoro-4-((4-methoxyphenyl)ethynyl)benzoic acid, (6). To a solution of 4-Iodo-2-fluorobenzoic acid (798 mg, 3 mmol) in N,N’-dimethyformamide (5 mL) was added CuI (23 mg, 0.12 mmol), Pd(Ph3P)4 (70 mg, 0.06 mmol), Diethylamine (241 mg, 3.3 mmol), and 1-ethynyl-4-methoxybenzene (475 mg, 3.6 mmol) under argon in a sealed microwave vial. The mixture was allowed to stir and was placed in a microwave reactor and heated to 100 °C for 1 hour. The reaction was allowed to cool to room temperature and was diluted with EtOAc (10 mL), washed with water (10 mL), 5% LiCl (aqueous, 2 × 10 mL), and brine (10 mL). The organic layer was passed through a Celite pad, dried with MgSO4, filtered, and solvent was removed under vacuum. 6 (750 mg, 92.5%) was isolated following purification on HPLC. LC (254nm) 0.752min (>99%); MS (ESI) m/z = 271.1, C16H11FO3

Scheme 2. Probe preparation of ML337.

Scheme 2

Probe preparation of ML337.

(R)-(2-fluoro-4-((4-methoxyphenyl)ethynyl)phenyl)(3-hydroxypiperidin-1-yl)methanone, (7, ML337). To a solution of compound 2 (675 mg, 2.5 mmol) in 20 mL DMF, was added diisopropylethylamine (1.07 g, 8.25 mmol) while stirring. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (560 mg, 3 mmol), hydroxybenzotriazole (337 mg, 2.5 mmol), and (R)-3-hydroxypiperidine hydrochloride (342 mg, 2.5 mmol) were then added. The reaction was allowed to stir for 4 hours at room temperature, then quenched with a solution of saturated NaHCO3 (20 mL), washed with 5% LiCl (aqueous, 2 × 20 mL), and brine (20 mL). The reaction was extracted into dichloromethane (50 mL), and solvent was removed under vacuum. The residue was purified using HPLC, and amine 3 was obtained as an ivory solid (420 mg, 47%). [α]D23 = −27.6° (c = 1, MeOH). LC (254 nm) 0.704 min (>99%); MS (ESI) m/z = 354.1. HRMS (TOF, ES+) C21H20FNO3.[M+H]+ calc. mass 354.1505, found 354.1507. 1H NMR (400.1 MHz, d6-DMSO) δ (ppm) [* = Rotamers]: 7.51 (d, J = 8.4 Hz, 2H); 7.46 (d, J = 10.2 Hz, 1H); 7.39 (m, 2H); 6.99 (d, J = 8.9 Hz, 2H); 5.00 (dd, J1 = 76.0 Hz, J2 = 4.2 Hz, 1H); 4.17 (m, 1H*); 3.78 (s, 3H); 3.32 (d, J = 12.5 Hz, 1H*); 3.19 (m, 1H*); 2.99 (m, 1H*); 2.89 (m, 1H); 1.75 (m, 2H); 1.37 (m, 2H). 13C NMR (100.6 MHz, d6-DMSO) δ (ppm): 164.03 (d, J = 19.4 Hz), 160.31, 158.91, 156.47, 133.60, 129.39, 128.15 (d, J =9.7 Hz), 125.79, 124.90*, 124.77*, 118.52 (d, J =22.6 Hz), 114.87, 113.84, 91.91*, 91.88*, 86.73, 65.48*,65.32*, 55.69, 53.76, 48.68*, 47.05*, 41.82, 33.05*, 32.69*, 23.52.

3. Results

3.1. Dose Response Curves for Probe

Figure 1. In vitro molecular pharmacology characterization of ML337 and three closely related mGlu3 NAMs.

Figure 1In vitro molecular pharmacology characterization of ML337 and three closely related mGlu3 NAMs

Concentration-response curves of mGlu2 and mGlu3 calcium (antagonist mode) for the four lead mGlu3 NAMs, including ML337. All four provide complete inhibition of mGlu3 and are devoid of activity at mGlu2. In addition, all four have no activity (PAM or NAM) at mGlu1,4,5,6,7,8.

3.2. Cellular Activity

The primary screening assays (both GIRK and Calcium) are cell-based assays, indicating that ML337 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 cytotoxicity assays aimed at this parameter indicated ML337 had no cytotoxicity in non-transformed HEK293.

3.3. Profiling Assays

To more fully characterize this potent, selective mGlu3 NAM ML337 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 [KATP]; Potassium channel [hERG]) and class A GPCRs (D1–5, H1–3, etc.). ML337 was found to only inhibit two targets (DAT: 71%@10 μM and 5-HT2B 74%@10 μM) out of the 68 assays (Table 2) conducted (inhibition of radio ligand binding > 50% at 10 μM).2 ML289 hit the same two targets, but at 88% and 77% inhibition@ 10 μM, respectively for DAT and 5-HT2B. Table 3 highlights calculated properties for ML337, which compares favorably with the MDDR.

Table 2. Ricerca Profiling of ML337.

Table 2

Ricerca Profiling of ML337.

Table 3. Calculated Property Comparison with MDDR Compounds.

Table 3

Calculated Property Comparison with MDDR Compounds.

4. Discussion

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

ML337 (CID 60204017) is the most potent (IC50 = 592 nM, 0.3%) and selective mGlu3 NAM reported to date with >50-fold selectivity vs. mGlu2 and inactive on mGlu1,4,5,6,7,8. The existing art, represented by 1–3 (see Figure 2), are inferior to the probe ML337 in terms of both potency and mGlu3 selectivity. ML337 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, ML337 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 in both mouse (brain:plasma ratio of 0.92) and rat (brain:plasma ratio of 0.3). ML337 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, ML337 is free from IP constraints, which will allow the MLPCN to freely provide this probe to the biomedical research community.

Figure 2. Structures of mGlu3 NAMs RO4491533 (1) and LY2399575 (2), both dual mGlu2/mGlu3 NAMs, and the first mGlu3 selective NAM, ML289, with ~15-fold selectivity versus mGlu2.

Figure 2

Structures of mGlu3 NAMs RO4491533 (1) and LY2399575 (2), both dual mGlu2/mGlu3 NAMs, and the first mGlu3 selective NAM, ML289, with ~15-fold selectivity versus mGlu2.

5. References

1.
Niswender CM, Johnson KA, Luo Q, Ayala JE, Kim C, Conn PJ, Weaver CD. Mol. Pharm. 2008;73:1213–1224. [PubMed: 18171729]
2.

For full information on the targets in the Lead Profiling Screen at Ricerca, please see: www​.ricerca.com

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