GluN2A-Selective Pyridopyrimidinone Series of NMDAR Positive Allosteric Modulators with an Improved in Vivo Profile

Abstract

The N-methyl-d-aspartate receptor (NMDAR) is an ionotropic glutamate receptor, gated by the endogenous coagonists glutamate and glycine, permeable to Ca²⁺ and Na⁺. NMDAR dysfunction is associated with numerous neurological and psychiatric disorders, including schizophrenia, depression, and Alzheimer’s disease. Recently, we have disclosed GNE-0723 (1), a GluN2A subunit-selective and brain-penetrant positive allosteric modulator (PAM) of NMDARs. This work highlights the discovery of a related pyridopyrimidinone core with distinct structure–activity relationships, despite the structural similarity to GNE-0723. GNE-5729 (13), a pyridopyrimidinone-based NMDAR PAM, was identified with both an improved pharmacokinetic profile and increased selectivity against AMPARs. We also include X-ray structure analysis and modeling to propose hypotheses for the activity and selectivity differences.

Ionotropic glutamate receptors (iGluRs) are transmembrane ion channels primarily expressed in the brain and are key regulators of neurological functions. The iGluR family consists of three congeners sharing structural similarities: the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), the kainate receptors, and the N-methyl-d-aspartate receptors (NMDARs).¹ NMDARs are tetramers, consisting of two GluN1 and two GluN2 subunits. In addition, NMDARs play an important role in synaptic plasticity and memory function. Structurally, NMDAR subunits include an N-terminal domain, a ligand-binding domain (LBD), and a transmembrane pore domain. NMDARs activate when two natural coagonists, glutamate and glycine, bind the LBD concurrently with membrane depolarization, which removes Mg²⁺ blockade. Once the channels are opened, Ca²⁺ and Na⁺ permeate into the cell and contribute to postsynaptic signal transmission.²

Isoforms of the GluN2 subunit of NMDARs, 2A, 2B, 2C, and 2D, are differentially expressed in various brain regions. In addition, it has been demonstrated that a predominant expression of GluN2B in early development shifts to GluN2A in mature synapses.³ Literature precedents have shown that overactivation of GluN2B-containing NMDARs can lead to excitotoxicity.⁴ However, little is known about the selective activation of GluN2C and GluN2D containing NMDARs.^5,6 Selective mutation or microdeletion of NMDARs containing the GluN2A subtype can recapitulate the core symptoms of schizophrenia in animal models.^7,8 NMDAR dysfunction is also implicated in a number of other neurological and psychiatric disorders, such as depression and Alzheimer’s disease,² thus highlighting the importance of this biological target in drug discovery.

Recently, we disclosed the discovery of GNE-0723 (Table 1, compound 1), a potent positive allosteric modulator (PAM) of GluN2A containing NMDARs.⁹ The compound was identified following structure-guided improvements on a high-throughput screening hit. It is well-known that overactivation of AMPARs causes seizures.¹⁰ Therefore, one of the main challenges of the program was to achieve selectivity for NMDARs over AMPARs (GluA2_Flip and GluA2_Flop isoforms),¹ a difficult problem given NMDARs and AMPARs share a PAM binding site. Even though compound 1 demonstrated good AMPAR selectivity (>200×), the pharmacokinetic profile of the molecule was not ideal, with moderate in vivo mouse clearance (Cl = 26 mL min^–1 kg^–1) and low oral bioavailability (F= 24%). During our optimization campaign, we discovered a new pyridopyrimidinone (PP) core, replacing the previous thiazolopyrimidinone (TP) core of compound 1. Herein, we disclose the optimization of the PP core vectors, leading to an improved in vivo pharmacokinetic profile and AMPARs selectivity. We also propose structure-based hypotheses for why the structure–activity relationship and off-target selectivity of the PP core are distinct from the TP core.

Table 1. Comparison of C′ Subsite SAR of Thiazolopyrimidinone and Pyridopyrimidinone Core.

^aNMDAR EC₅₀ values were determined in the presence of EC₃₀ glutamate and saturating glycine. Max potentiation (%) at 125 μM reported if no EC₅₀ could be obtained, where 30% denotes the assay baseline (EC₃₀ glutamate). AMPAR EC₅₀ values were determined in the presence of 100 μM glutamate. Max potentiation (%) at 125 μM reported if no EC₅₀ could be obtained, where 0% denotes the assay baseline due to receptor desensitization. All EC₅₀ values represent geometric means of at least two determinations.

In early studies of a series of GluN2A selective PAMs, we sought to explore various replacements for the thiazolopyrimidinone core. We found that few alternative cores were tolerated, suggesting that several key elements of the original thiazolopyrimidinone core were required for potency (data not shown). One initially tolerated alternative to the TP core (Table 1, Core A) was the pyridopyrimidinone core (Table 1, Core B). The new PP core was first evaluated using a 3D shape search^11,12 of commercially available compounds¹³ using the TP core-containing compound, GNE-3419 as a query (see Supporting Information Figure S-1), as described previously.¹⁴ Moreover, the thiophene-to-phenyl bioisostere is well-precedented in the literature.¹⁵

We first sought to explore the structure–activity relationships (SAR) at R₁, knowing that interactions within the C′-subsite were important for potency based on previous work.⁹ Lower potency was initially observed when an amide (2 vs 3) or a pyrimidine (4 vs 5) was present at R₁ when paired with the PP core. Despite the reduced potency for the PP/TP core matched pairs, very good selectivity against AMPARs was observed for those compounds. To our delight, further investigation revealed that pairing the PP core with cyclopropyl R₁ substituents (6–9) maintained GluN2A potency while improving AMPAR selectivity relative to earlier generation analogues. We found the best balance between GluN2A potency and selectivity against AMPARs with the cyclopropyl nitrile at R₁ (9). Overall, the GluN2A potency for the cyclopropyl analogues at R₁ tracked between the TP- and PP-subseries.

To help explain why the amide-PP subseries is less tolerated relative to cyclopropyl analogues, we first solved the X-ray crystal structure at 2.39 Å resolution of the TP compound 2 bound to the LBD of GluN2A/GluN1 (PDB ID 5TP9, Figure S-2, Table S-1) and then overlaid a model of the PP compound 3 (Figure 1A). We hypothesize that the hydrogen-bond between the ligand amide and GluN2A:Pro129 restricts the position of the amide in both cores. The PP core is slightly larger than the TP core and pushes a core-hydrogen orthogonally into the backbone nitrogen of Glu132 (Glu132:nitrogen to compound 3 phenyl hydrogen distance: 2.1 Å). This compares to the TP X-ray structure, which shows a ligand sulfur to Glu132 nitrogen distance of 3.4 Å. Analysis of similar H–N geometries in small-molecule crystal structures (Figure S-4) suggests 2.1 Å would result in a clash, forcing a shift in the complex and reducing potency of compound 3 compared to compound 2. This rationale may also apply to larger R₁ substituents, which may restrict the core location, such as the pyrimidine in compound 5.

Figure 1.

(A) Overlay of modeled compound 3 (purple) and GluN2A/GluN1 X-ray cocrystal structure with compound 2 (PDB ID 5TP9 gray). The amide in compound 3 may force the larger core to clash slightly with Glu132. Distances are between the Glu132 backbone nitrogen and hydrogen in 3 or sulfur in 2. (B) Overlay of a model of compound 3 (purple) and X-ray crystal structure of compound 9 (PDB ID 5TPA, gray). The cyclopropyl nitrile in 9 may allow the larger core to shift away from Glu132. Distances are between the Glu132 backbone nitrogen and hydrogens in 3 and 9.

The X-ray crystal structure of compound 9 was also solved at 2.48 Å resolution (PDB ID 5TPA, Figure S-3, Table S-1) and a core shift was observed without an amide occupying the C′ subsite (Figure 1B). When we overlaid the X-ray crystal structure of 9 with the model of compound 3, we observed that the cyclopropyl nitrile, in contrast to the amide, does not require a specific interaction with GluN2A:Pro129, allowing the large PP core to shift away from the Glu132 backbone (2.4 Å), thus avoiding a potential steric clash with the protein. The core shift could explain the similar potencies between the PP and TP cyclopropyl nitrile compounds 8 and 9.

We then explored substitutions at R₃ and R₄ on the six-membered pyridine ring of the PP core (Table 2). These positions are distinct from the core vectors of the five-membered thiazole-based core. We started our investigation by removing the methyl group at the R₃ position of compound 9. Compound 10 showed a 2-fold gain in GluN2A potency at the cost of a considerable loss in AMPAR selectivity compared to compound 9. Small R₃ substituents like fluorine (11) demonstrated similar GluN2A potency compared to the unsubstituted phenyl ring (10), but with partially improved AMPAR selectivity. The electron donating methoxy group at R₃ (12) possessed a unique selectivity profile, despite a 3-fold loss in potency compared to compound 10; very low AMPAR potentiation was observed at the highest concentration tested (125 μM). However, increased efflux ratio (ER) in MDCK-MDR1 transfected cells was observed.

Table 2. Representative Examples of Aromatic Ring Substitution SAR.

^aNMDAR EC₅₀ max potentiation (%) and AMPAR EC₅₀ values were determined as described in Table 1.

^bLiver microsome-predicted hepatic clearance.

^cH/R/M = human/rat/mouse.

^dMDCK cells transfected with human MDR1 gene.

^eEfflux ratio.

^fBasolateral-to-apical/apical-to-basolateral.

^gND = not determined

The chloro substituent at the R₃ position (13) showed good GluN2A potency and overall improved GluA2 selectivity compared to compound 9. The chlorine also resulted in a much improved human liver microsome stability compared to all compounds tested (2 mL/min/kg). Furthermore, compound 13 had a lower efflux ratio compared to 12 (B:A/A:B = 1.6 vs 3.2), thus increasing the likelihood of achieving biologically relevant free brain concentrations in vivo.

Finally, we explored the substitution at the newly opened vector at R₄ enabled by the six-membered ring. Unfortunately, no improvement in GluN2A potency or AMPAR selectivity was observed with R₄ as a methyl (14), methoxy (15), or trifluoromethyl (16) substituent (R₃ as hydrogen).

In summary, the substitution at the R₃ position was the most beneficial for AMPAR selectivity, while maintaining good GluN2A potency. We rationalize the importance of the R₃ substituent on the PP core through modeling of compounds 8 and 9 within the previously published X-ray of TP compound GNE-3419 bound to AMPAR/GluA2_Flip (Figure 2). We found that the methyl at R₃ of the PP core (compound 9) is closer to the polar Serine242 in GluA2_Flip compared to the methyl of the TP core of compound 8 (3.2 Å vs 4.0 Å). We hypothesize that compounds presenting either large or hydrophobic substituents near Serine242 may have increased selectivity over AMPAR compared to the TP analogues. Ultimately, compound 13 demonstrated the best balance between GluN2A potency and AMPAR selectivity with good overall metabolic stability across species and no evidence of efflux in vitro. Compound 13 was then scaled up for in vivo PK studies (for synthetic details, see Supporting Information).

Figure 2.

Overlay of modeled ligands in GluA2_Flip (AMPAR) X-ray (PDB ID 5H8S). Compound 8 (gold) and compound 9 (purple). The core methyl group in 9 may clash more with the polar Serine242 in GluA2_Flip.

Compound 13 (GNE-5729) possessed a good overall profile as highlighted in Table 3. Compound 13 showed favorable physicochemical properties as the molecular weight, LogD, and total polar surface area (TPSA) of the molecule were in a range to maximize the odds of success for a central nervous system drug target molecule.¹⁶ Compound 13 had a very selective profile against various off-target ion channels with greater than a 100-fold selectivity over other NMDAR subtypes and greater than a 1000-fold selectivity over AMPAR. This is 5-fold improved selectivity against AMPAR compared to compound 1. Compound 13 also possessed a desirable pharmacokinetic profile, including low to moderate in vitro clearance across species (Table 3), which translated in vivo (CL_Blood = 10 mL min^–1 kg^–1) when dosed IV in mouse at 0.5 mg/kg. We found that compound 13 had a moderate bioavailability (F = 37%) while demonstrating good brain permeability (unbound brain-to-plasma concentration ratio K_p,uu = 0.67 at 1 h) when dosed orally at 5 mg/kg. This result correlated well with the in vitro MDCK-MDR1 ER (B:A/A:B = 1.6) predicting low efflux, thus confirming the viability of compound 13 as potential in vivo tool compound.

Table 3. Receptor Selectivity and DMPK Properties of Compound 13.

^a,bNMDAR EC₅₀ max potentiation (%) and AMAPR EC₅₀ values were determined as described in Table 1.

^cLiver microsome-predicted hepatic clearance.

^dH/R/M = human/rat/mouse.

^eIn vitro stability in cryo preserved hepatocytes.

^fMDCK cells transfected with human MDR1 gene.

^gEfflux ratio.

^hBasolateral-to-apical/apical-to-basolateral.

ⁱPlasma protein binding.

^jH/M = human/mouse.

^kVehicle: 10% DMSO, 10% cremophor EL in saline solution.

^lVehicle: MCT suspension

^mFree plasma concentration at C_max.

ⁿFree brain concentration at 1 h time point.

^oK_p,uu = C_brain,u/C_plasma,u at 1 h time point.

In comparison to our previous lead molecule compound 1 (Table S-2), compound 13 showed similar brain permeability (K_p,uu = 0.62 vs K_p,uu = 0.67) and bioavailability (F = 24% vs F = 37%) in mouse. However, 13 demonstrated a greater free brain concentration at 1 h postdose compared to 1 (C_brain,u = 0.031 μM vs C_brain,u = 0.013 μM). The 3-fold in vivo unbound clearance improvement (Clu= 132 mL min^–1 kg^–1 vs Cl_u= 433 mL min^–1 kg^–1) and the greater brain free fraction (f_u,brain= 0.051 vs f_u,brain= 0.014) of compound 13 resulted in higher free brain concentration. The overall pharmacokinetic profile improvement, in addition to a better kinetic solubility (kinetic solubility = 43.5 μM vs kinetic solubility = 9.3 μM), resulted in compound 13 achieving 1.6-fold free plasma concentrations above the in vitro EC₅₀ at C_max,u. This was an important milestone toward enabling in vivo efficacy studies.

The two compounds share similar deactivation kinetics in whole cell voltage clamp recordings; both compounds are considered moderate deactivators (compound 1 tau = 2102 ms; compound 13 tau = 3164 ms) showing a 4–6-fold slower deactivation than the endogenous coagonists, glycine and glutamate (Figure 3A).⁹ The tau values correlate with the compounds respective maximum potentiation values (compound 1 max potentiation = 152%; compound 13 max potentiation = 136%). Typically, a maximum potentiation greater than 100% is a good indication of a slower deactivator.¹⁷ The improved selectivity of compound 13 over AMPAR, the main off-target liability on the program, was a considerable benefit of the new lead compound that allows for more careful evaluation of the efficacy and safety window of GluN2A PAMs without confounding AMPAR activity. To assess for potentiation of native AMPARs during physiological activation, compounds 1 and 13 were examined by excitatory postsynaptic potential (EPSP) in brain slice field recordings (Figure 3B).¹⁸ The greater selectivity of 13 observed in the cell assay was confirmed when compound 13 showed a significantly lower potentiation of AMPARs compared to compound 1 (greater than 2.6-fold selectivity in EPSP).

Figure 3.

(A) Overlay of whole cell voltage clamp recordings comparing deactivation kinetics for GNE-0723 (1) in red and GNE-5729 (13) in blue compared to the endogenous coagonists (saturating glycine and 100 μM glutamate) in black. Deactivation “tau” is the time constant for the signal decay after glutamate is removed. (B) Dose–response curves of AMPAR excitatory postsynaptic potential (EPSP) area in brain slice field recording for GNE-0723 (1) (EC₅₀ = 5.7 μM) and GNE-5729 (13) (EC₅₀ > 15 μM).

A core-replacement campaign to find alternatives to the thiazolopyrimidinone core of compound 1 led to the identification of the privileged pyridopyrimidinone core, which possessed greater than 2.6-fold improved AMPAR selectivity in the biologically relevant EPSP assay. We found that the cyclopropyl nitrile at R₁ and the chloro at R₃ resulted in a balance of potency, selectivity, and brain penetration. We hypothesize that this increased selectivity originates from a unique R₃ vector situated near the Serine242 in GluA2_Flip. In retrospect, the lower unbound clearance, the improved kinetic solubility, and the increased mouse brain free fraction of compound 13 compared to compound 1 led to increased free brain exposure and EC₅₀ coverage. In summary, the improved pharmacokinetic profile and AMPAR selectivity make GNE-5729 (13) a superior tool compound compare to GNE-0723 (1) for testing the efficacy and safety of GluN2A potentiation.

Glossary

ABBREVIATIONS

NMDA

N-methyl-d-aspartate

NMDAR

N-methyl-d-aspartate receptor

PAM

positive allosteric modulator

iGluR

ionotropic glutamate receptor

AMPAR

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors

LBD

ligand binding domain

PP

pyridopyrimidinone

TP

thiazolopyrimidinone

SAR

structure–activity relationship

ER

efflux ratio

MDCK-MDR1

Madin–Darby canine kidney cells-multidrug resistance protein 1

PK

pharmacokinetic

TPSA

topological polar surface area

DMPK

drug metabolism and pharmacokinetic

LM

liver microsomes

Hep

hepatocytes

PPB

plasma protein binding

AUC

area under the curve

DMSO

dimethyl sulfoxide

MCT

methylcellulose/tween

EPSP

excitatory postsynaptic potential

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmedchemlett.6b00388.

• Three-dimensional shape search identification of pyridopyrimidinone core, X-ray crystal structure of compounds 2 and 9, histograms of Cambridge Structural Database distance statistics, receptor selectivity and DMPK properties of compound 1, experimental procedures and characterization data for compound 13 (PDF)

The authors declare no competing financial interest.