Design and Synthesis of New Quinazolin-4-one Derivatives with Negative mGlu7 Receptor Modulation Activity and Antipsychotic-Like Properties

Following the glutamatergic theory of schizophrenia and based on our previous study regarding the antipsychotic-like activity of mGlu7 NAMs, we synthesized a new compound library containing 103 members, which were examined for NAM mGlu7 activity in the T-REx 293 cell line expressing a recombinant human mGlu7 receptor. Out of the twenty-two scaffolds examined, active compounds were found only within the quinazolinone chemotype. 2-(2-Chlorophenyl)-6-(2,3-dimethoxyphenyl)-3-methylquinazolin-4(3H)-one (A9-7, ALX-171, mGlu7 IC50 = 6.14 µM) was selective over other group III mGlu receptors (mGlu4 and mGlu8), exhibited satisfactory drug-like properties in preliminary DMPK profiling, and was further tested in animal models of antipsychotic-like activity, assessing the positive, negative, and cognitive symptoms. ALX-171 reversed DOI-induced head twitches and MK-801-induced disruptions of social interactions or cognition in the novel object recognition test and spatial delayed alternation test. On the other hand, the efficacy of the compound was not observed in the MK-801-induced hyperactivity test or prepulse inhibition. In summary, the observed antipsychotic activity profile of ALX-171 justifies the further development of the group of quinazolin-4-one derivatives in the search for a new drug candidate for schizophrenia treatment.

Out of the group III mGlu receptors, the mGlu7 receptor has a low affinity for endogenous agonists [27,28], which is several orders of magnitude lower than that for mGlu4 and mGlu8 receptors, requiring a high glutamate concentration (almost 10 mM) for a full receptor activation.
Regarding the importance of mGlu 7 receptor modulation for potential therapeutic applications, our previous study explored the role of negative mGlu 7 receptor modulation in animal models of schizophrenia [7]. The promising results of this study showed that the new mGlu 7 ligands could accelerate the development of a unique drug candidate in this class of molecular targets.  [37,40] (NAMnegative allosteric modulator; PAM-positive allosteric modulator). EC50-effective concentration 50; IC50-inhibitory concentration 50; assay type indicated in parenthesis; rat CL hep-predicted hepatic clearance in rat; Kp(rat)-rat brain/plasma ratio.
Regarding the importance of mGlu7 receptor modulation for potential therapeutic applications, our previous study explored the role of negative mGlu7 receptor modulation in animal models of schizophrenia [7]. The promising results of this study showed that the new mGlu7 ligands could accelerate the development of a unique drug candidate in this class of molecular targets.
With this in mind, we now focus on designing and discovering new structural scaffolds and potential mGlu7 NAMs, and assessing their therapeutic potential based on behavioral tests commonly used in antipsychotic drug development.

Scaffold Design
Starting the search for new scaffolds, the structures of the primary tool compounds available at that time (MMPIP and ADX71743) were applied. In addition to a bioisosteric replacement, which was the primary strategy to design the A1-A13 and A18-A22 chemotypes, the Cresset field methodology was used to develop the A14-A17 scaffolds, as shown in Figure 3.  [37,40] (NAMnegative allosteric modulator; PAM-positive allosteric modulator). EC 50 -effective concentration 50; IC 50 -inhibitory concentration 50; assay type indicated in parenthesis; rat CL hep-predicted hepatic clearance in rat; Kp(rat)-rat brain/plasma ratio.
With this in mind, we now focus on designing and discovering new structural scaffolds and potential mGlu 7 NAMs, and assessing their therapeutic potential based on behavioral tests commonly used in antipsychotic drug development.

Scaffold Design
Starting the search for new scaffolds, the structures of the primary tool compounds available at that time (MMPIP and ADX71743) were applied. In addition to a bioisosteric replacement, which was the primary strategy to design the A1-A13 and A18-A22 chemotypes, the Cresset field methodology was used to develop the A14-A17 scaffolds, as shown in Figure 3.  Figure 2. Chemical structures of the last reported mGlu7 receptor tool compounds [37,40] (NAMnegative allosteric modulator; PAM-positive allosteric modulator). EC50-effective concentration 50; IC50-inhibitory concentration 50; assay type indicated in parenthesis; rat CL hep-predicted hepatic clearance in rat; Kp(rat)-rat brain/plasma ratio.
Regarding the importance of mGlu7 receptor modulation for potential therapeutic applications, our previous study explored the role of negative mGlu7 receptor modulation in animal models of schizophrenia [7]. The promising results of this study showed that the new mGlu7 ligands could accelerate the development of a unique drug candidate in this class of molecular targets.
With this in mind, we now focus on designing and discovering new structural scaffolds and potential mGlu7 NAMs, and assessing their therapeutic potential based on behavioral tests commonly used in antipsychotic drug development.

Scaffold Design
Starting the search for new scaffolds, the structures of the primary tool compounds available at that time (MMPIP and ADX71743) were applied. In addition to a bioisosteric replacement, which was the primary strategy to design the A1-A13 and A18-A22 chemotypes, the Cresset field methodology was used to develop the A14-A17 scaffolds, as shown in Figure 3.  Initially, the terminal groups present in the reference structures were used to substitute the designed cores ( Figure 3), i.e., 4-methoxyphenyl and 4-pyridyl in MMPIP and 2,4-dimethylphenyl and ethyl in ADX71743. Additionally, selected scaffolds were decorated with other R1 and R2 substituents (Figure 4), which were previously used in SAR studies of ADX compounds [35].  Tables S1 and S2) via analogy to reference tool compounds MMPIP and ADX71743.

Crystal Structure Analysis
The molecular structures of both most active compounds, ALX-065 and ALX-171, were confirmed with the use of a single-crystal X-ray diffraction experiment. The asymmetric unit of the crystal ALX-065 consisted of two independent molecules, which represented two alternative conformations. This solid-state observation indicated the significant rotational freedom of the benzene substituent in the solution. This was not observed for the structure of ALX-171, where, for both aromatic fragments at C-2 and C-

A9-12 Inactive
a All derivatives of the A9 chemotype depicted in Table S2, Supplementary Materials; b inhibition of the forskolin-stimulated (5 µM) cAMP response using the EC80 concentration of LSP4-2022, according to methodology described in previous papers [7,41]; c T-REx 293 cells expressing the human mGlu7 receptor (depicted in Section 2.1 of the Supplementary Materials); data are the mean of three independent experiments. The substances were incubated with 5 µM LSP4-2022 (EC80).
In the next step, selectivity tests of both ALX-065 and ALX-171 at other group III mGlu receptors (mGlu4 and mGlu8) were conducted, revealing only mGlu4 NAM activity of ALX-065 (19.6% at 10 µM) (Supplementary Materials, Section 2.3, Figures S5 and S6). Finally, the crystal structures were determined for these two modulators, and initial DMPK profiling and in vivo behavioral tests were performed.

Crystal Structure Analysis
The molecular structures of both most active compounds, ALX-065 and ALX-171, were confirmed with the use of a single-crystal X-ray diffraction experiment. The asymmetric unit of the crystal ALX-065 consisted of two independent molecules, which represented two alternative conformations. This solid-state observation indicated the significant rotational freedom of the benzene substituent in the solution. This was not observed for the structure of ALX-171, where, for both aromatic fragments at C-2 and C-

A9-12 Inactive
a All derivatives of the A9 chemotype depicted in Table S2, Supplementary Materials; b inhibition of the forskolin-stimulated (5 µM) cAMP response using the EC80 concentration of LSP4-2022, according to methodology described in previous papers [7,41]; c T-REx 293 cells expressing the human mGlu7 receptor (depicted in Section 2.1 of the Supplementary Materials); data are the mean of three independent experiments. The substances were incubated with 5 µM LSP4-2022 (EC80).
In the next step, selectivity tests of both ALX-065 and ALX-171 at other group III mGlu receptors (mGlu4 and mGlu8) were conducted, revealing only mGlu4 NAM activity of ALX-065 (19.6% at 10 µM) (Supplementary Materials, Section 2.3, Figures S5 and S6). Finally, the crystal structures were determined for these two modulators, and initial DMPK profiling and in vivo behavioral tests were performed.

Crystal Structure Analysis
The molecular structures of both most active compounds, ALX-065 and ALX-171, were confirmed with the use of a single-crystal X-ray diffraction experiment. The asymmetric unit of the crystal ALX-065 consisted of two independent molecules, which represented two alternative conformations. This solid-state observation indicated the significant rotational freedom of the benzene substituent in the solution. This was not observed for the structure of ALX-171, where, for both aromatic fragments at C-2 and C-6, there were spatial substituents in the ortho positions, leading to a stabilization and

A9-12 Inactive
a All derivatives of the A9 chemotype depicted in Table S2, Supplementary Materials; b inhibition of the forskolin-stimulated (5 µM) cAMP response using the EC80 concentration of LSP4-2022, according to methodology described in previous papers [7,41]; c T-REx 293 cells expressing the human mGlu7 receptor (depicted in Section 2.1 of the Supplementary Materials); data are the mean of three independent experiments. The substances were incubated with 5 µM LSP4-2022 (EC80).
In the next step, selectivity tests of both ALX-065 and ALX-171 at other group III mGlu receptors (mGlu4 and mGlu8) were conducted, revealing only mGlu4 NAM activity of ALX-065 (19.6% at 10 µM) (Supplementary Materials, Section 2.3, Figures S5 and S6). Finally, the crystal structures were determined for these two modulators, and initial DMPK profiling and in vivo behavioral tests were performed.

Crystal Structure Analysis
The molecular structures of both most active compounds, ALX-065 and ALX-171, were confirmed with the use of a single-crystal X-ray diffraction experiment. The asymmetric unit of the crystal ALX-065 consisted of two independent molecules, which represented two alternative conformations. This solid-state observation indicated the significant rotational freedom of the benzene substituent in the solution. This was not observed for the structure of ALX-171, where, for both aromatic fragments at C-2 and C-6, there were spatial substituents in the ortho positions, leading to a stabilization and In the next step, selectivity tests of both ALX-065 and ALX-171 at other group III mGlu receptors (mGlu4 and mGlu8) were conducted, revealing only mGlu4 NAM activity of ALX-065 (19.6% at 10 µM) (Supplementary Materials, Section 2.3, Figures S5 and S6). Finally, the crystal structures were determined for these two modulators, and initial DMPK profiling and in vivo behavioral tests were performed.

Crystal Structure Analysis
The molecular structures of both most active compounds, ALX-065 and ALX-171, were confirmed with the use of a single-crystal X-ray diffraction experiment. The asymmetric unit of the crystal ALX-065 consisted of two independent molecules, which represented two alternative conformations. This solid-state observation indicated the significant rotational freedom of the benzene substituent in the solution. This was not observed for the structure of ALX-171, where, for both aromatic fragments at C-2 and C-6, there were spatial substituents in the ortho positions, leading to a stabilization and Inactive a All derivatives of the A9 chemotype depicted in Table S2, Supplementary Materials; b inhibition of the forskolinstimulated (5 µM) cAMP response using the EC 80 concentration of LSP4-2022, according to methodology described in previous papers [7,41]; c T-REx 293 cells expressing the human mGlu 7 receptor (depicted in Section 2.1 of the Supplementary Materials); data are the mean of three independent experiments. The substances were incubated with 5 µM LSP4-2022 (EC 80 ).

Crystal Structure Analysis
The molecular structures of both most active compounds, ALX-065 and ALX-171, were confirmed with the use of a single-crystal X-ray diffraction experiment. The asymmetric unit of the crystal ALX-065 consisted of two independent molecules, which represented two alternative conformations. This solid-state observation indicated the significant rotational freedom of the benzene substituent in the solution. This was not observed for the structure of ALX-171, where, for both aromatic fragments at C-2 and C-6, there were spatial substituents in the ortho positions, leading to a stabilization and reduction in rotation due to steric hindrance.
The orientation of both aromatic fragments at C-2 and C-6 to the 3-methyl-quinazolin-4-one moiety was a result of two effects. First, it was strictly dependent on the ortho substituent. Second, both mentioned rings influenced their mutual arrangement, which was observed in structure ALX-065. The angular position of o-Cl-benzene with respect to the 3-methyl-quinazolin-4-one ring system (approx. 65 • ) favored the angular orientation of benzene at C-6 (approx. 25 • ), whereas the perpendicular orientation of o-Cl-benzene observed for conformer two led to a nearly flat arrangement of the remaining molecular fragment (see Figure 7; conformers one and two of ALX-065). Despite o-Cl-benzene being in proximity to the perpendicular orientation (approx. 85 • ) in structure ALX-171, the angular orientation of the o,m-dimethoxy benzene was preferential.   Figure S1, Table S3, Supplementary Materials.

Pharmacokinetic
The pharmacokinetic properties and brain uptake of the new 2,6-disubstituted (3H)quinazolin-4-one derivatives (ALX-065 and ALX-171) and tool compounds (ADX71743 and MMPIP) were determined in mice after an i.p. administration of a dose of 10 mg/kg in 3% DMSO + 20% Captisol in water. The results shown in Table 2 confirmed that all analyzed compounds entered the circulation system and reached the brain quickly (Tmax (plasma and brain): 15 min for ADX71743, ALX-065, and ALX-171, and 30 min for MMPIP). Of these compounds, ALX-171 exhibited the longest half-life (T1/2 = 5.56 h) and the highest brain exposure (AUC0-t = 13.85 µM × h/L). As shown in Table 3, ALX-171 also exhibited a good oral bioavailability (F = 58%) after the administration of a 5 mg/kg dose.  The pharmacokinetic properties and brain uptake of the new 2,6-disubstituted (3H)quinazolin-4-one derivatives (ALX-065 and ALX-171) and tool compounds (ADX71743 and MMPIP) were determined in mice after an i.p. administration of a dose of 10 mg/kg in 3% DMSO + 20% Captisol in water. The results shown in Table 2 confirmed that all analyzed compounds entered the circulation system and reached the brain quickly (T max (plasma and brain): 15 min for ADX71743, ALX-065, and ALX-171, and 30 min for MMPIP). Of these compounds, ALX-171 exhibited the longest half-life (T 1/2 = 5.56 h) and the highest brain exposure (AUC 0-t = 13.85 µM × h/L).  Table 3, ALX-171 also exhibited a good oral bioavailability (F = 58%) after the administration of a 5 mg/kg dose. Table 3. Pharmacokinetic parameters for ALX-171. Plasma (A) and brain (B) concentrations of ALX-171 in female BALB/c mice following intravenous (i.v., 3 mg/kg) and peroral (p.o., 5 mg/kg) administration.

Administration
T max

In Vitro Metabolic Stability and CYP450 Inhibition
The data presented in Table 4 show that ALX-171 exhibited a significantly higher microsomal stability with the lowest intrinsic clearance compared to the other compounds tested. Moreover, this compound was also characterized by relatively good kinetic solubility. Regarding the cytochrome P450 inhibition profile, all compounds weakly inhibited (IC 50 > 10 µM) isoforms 2B6 and 2D6, while strong inhibition (IC 50 < 1.1) was observed only for MMPIP for isoforms 3A4 and 2C19.

In Vivo Behavioral Tests
Based on our previous study regarding the putative antipsychotic-like activity of mGlu 7 NAMs [7], we aimed to assess the efficacy of newly synthesized compounds to reverse schizophrenia-like symptoms in several behavioral tests. Since ALX-171 had better drug-like properties (kinetic solubility and metabolic stability) than ALX-065, and relatively good bioavailability, this compound was selected for the in vivo profiling. The antipsychotic-like activity was evaluated, assessing each group of symptoms (positive, negative, and cognitive) that may manifest in schizophrenic patients.
Negative symptoms are associated with anhedonia, a blunted affect, or social withdrawal. In animals, social disturbances can be measured with social interaction tests. Cognitive symptoms (memory and attention deficits) can be assessed with the novel object recognition test, spatial delayed alternation test, and prepulse inhibition test.

Tests Predictive of Positive Symptoms DOI-Induced HTR
The administration of DOI induced characteristic head shakes in mice. ALX-171 significantly attenuated the number of head shakes at all tested doses (2.5, 5, and 10 mg/kg), compared with the control group ( Figure 8A). Additionally, mGlu 7 knockout (KO) animals were used to confirm that the observed effects are mGlu 7 -receptor-dependent. The deletion of the mGlu 7 receptor resulted in a marked reduction in DOI-induced head twitches, and ALX-171 had no further effect on head twitches in KO mice, which suggested that the effect of ALX-171 was mGlu 7 receptor-specific ( Figure 8B). The administration of DOI induced characteristic head shakes in mice. ALX-171 significantly attenuated the number of head shakes at all tested doses (2.5, 5, and 10 mg/kg), compared with the control group ( Figure 8A). Additionally, mGlu7 knockout (KO) animals were used to confirm that the observed effects are mGlu7-receptor-dependent. The deletion of the mGlu7 receptor resulted in a marked reduction in DOI-induced head twitches, and ALX-171 had no further effect on head twitches in KO mice, which suggested that the effect of ALX-171 was mGlu7 receptor-specific ( Figure 8B).

MK-801-Induced Hyperactivity
The administration of MK-801 resulted in an increase in locomotor activity in mice. ALX-171 was not able to reverse the MK-801-induced increase in locomotor activity at any of the administered doses (2.5, 5, or 10 mg/kg) ( Figure 8C). The administration of ALX-171 had no effect on spontaneous locomotor activity in naïve mice ( Figure 8D).

MK-801-Induced Hyperactivity
The administration of MK-801 resulted in an increase in locomotor activity in mice. ALX-171 was not able to reverse the MK-801-induced increase in locomotor activity at any of the administered doses (2.5, 5, or 10 mg/kg) ( Figure 8C). The administration of ALX-171 had no effect on spontaneous locomotor activity in naïve mice ( Figure 8D).

Test Predictive of Negative Symptoms Social Interaction Test
The administration of MK-801 resulted in significant disruptions in both the number of social episodes and the total time of interactions between mice. ALX-171 reversed the MK-801-induced disruptions in the interaction time and number of episodes at all tested doses ( Figure 9A,B).

Social Interaction Test
The administration of MK-801 resulted in significant disruptions in both the number of social episodes and the total time of interactions between mice. ALX-171 reversed the MK-801-induced disruptions in the interaction time and number of episodes at all tested doses ( Figure 9A,B).

Novel Object Recognition Test
The administration of MK-801 induced a profound decrease in the recognition index in the NOR test in mice. ALX-171 ameliorated the effects of the MK-801 administration at all tested doses (1, 2.5, and 5 mg/kg) ( Figure 10A). It was observed that the compound had no significant impact on the recognition index after the administration of the compound alone ( Figure 10B).

Tests Predictive of Cognitive Symptoms Novel Object Recognition Test
The administration of MK-801 induced a profound decrease in the recognition index in the NOR test in mice. ALX-171 ameliorated the effects of the MK-801 administration at all tested doses (1, 2.5, and 5 mg/kg) ( Figure 10A). It was observed that the compound had no significant impact on the recognition index after the administration of the compound alone ( Figure 10B).
The administration of MK-801 resulted in significant disruptions in both the number of social episodes and the total time of interactions between mice. ALX-171 reversed the MK-801-induced disruptions in the interaction time and number of episodes at all tested doses ( Figure 9A,B).  Figure 10A). It was observed that the compound had no significant impact on the recognition index after the administration of the compound alone ( Figure 10B).

Spatial Delayed Alternation Test
The administration of MK-801 decreased the choice accuracy in the spatial delayed alternation test in rats. AXL-171 at the highest dose tested (5 mg/kg) reversed the MK-801-induced disruptions in the number of alternations ( Figure 8C). The compound, when administered alone, had no negative effect on choice accuracy in this test ( Figure 8C).

Prepulse Inhibition Test
The administration of MK-801 decreased the prepulse inhibition in rats. ALX-171 had no effect on the MK-801-induced effects at any of the investigated doses ( Figure 8D). The compound disturbed the PPI response (~50%) when given alone at the highest investigated dose of 10 mg/kg.

Discussion
Although the efficacy of currently available antipsychotics toward positive symptoms of schizophrenia is satisfactory, the reversal and treatment of negative and cognitive symptoms are debatable. The latter two groups of symptoms persist chronically, have the most significant negative impact on daily functioning, and remain treatment-resistant [42,43]. Therefore, the search for new antipsychotic drugs focuses on providing relief to these groups of symptoms, and the determination to find new targets for the drug is wellfounded. Metabotropic glutamate receptors have been widely studied in this field [42,44], and activators of mGlu 2 or mGlu 4 receptors have been indicated as promising targets. The mGlu 7 subtype has been the least investigated thus far. According to the glutamatergic theory of schizophrenia, an increased glutamate efflux is responsible for schizophrenia arousal [42,[45][46][47]. Therefore, the inhibition of this increased activity of glutamatergic neurons presumably constitutes the most appropriate way to reverse schizophrenia symptoms. The first choice is to use activators of receptors mGlu 2 or mGlu 4 , which serve as autoreceptors.
In contrast to receptors mGlu 2 and mGlu 4 , which inhibit the glutamate release when activated, the mGlu 7 receptor acts rather as a heteroreceptor expressed on GABAergic terminals that regulate the GABA release. NAMs of mGlu 7 , including ALX-171, inhibit receptors expressed on the terminals of GABAergic neurons, thus, facilitating the GABA release. This neurotransmitter is a principal inhibitory amino acid in the brain that controls and inhibits excitation, including the glutamatergic network. Consequently, increasing its activity with mGlu 7 inhibition may counteract an abnormal glutamatergic excitation.
Although new subtype-selective negative allosteric mGlu 7 receptor modulators have recently been identified, only a few have been shown to have acceptable DMPK profiles allowing for a robust validation of their therapeutic potential. Here, we reported on an earlylead selection in the search for new mGlu 7 NAMs and their preliminary in vitro and in vivo preclinical characterization. The efforts to identify ALX-171 were not straightforward, and confirmed complexities in designing allosteric modulators of mGlu receptors. Indeed, steep and flat SAR and subtle structural changes that affect CNS penetration and DMPK properties are frequently reported problems in numerous studies devoted to developing ligands with an allosteric mechanism of action [36,46,48,49].
The synthesized compound library, containing 103 chemicals representing 22 chemotypes (A1-A17 and M1-M5), included only three compounds (quinazoline analogs, chemotype A9) that exhibited NAM mGlu 7 activity in the T-REx 293 cell line. Furthermore, the structural modification around the active 4(3H)-quinazolinone basic skeleton at the C-2 and C-6 positions showed that the incorporation of a wide range of substituents was not tolerated; the phenyl moieties were found to be important for interactions with the mGlu 7 receptor on both sides of the basic nucleus. The incorporation of a 2,3-dimethoxyphenyl substituent at the C-6 position of the basic molecule did not significantly change the NAM activity of the mGlu 7 receptor; however, ALX-171 showed an improved selectivity toward the mGlu 4 -receptor-enhanced stability and increased solubility, compared to the unsubstituted derivative ALX-065. Interestingly, despite the comparable activity and slight differences in chemical structure between ALX-065 and ALX-171, the crystal structure analysis showed some diversity in the examined crystals, e.g., the presence of two conformers in ALX-065 crystals, and various angular orientations at the C6 position for both molecules. The differences observed in the analyzed crystals suggested that the mutual orientation of terminal aromatic substituents may be important for interactions inside the allosteric binding site. This hypothesis is currently under investigation.
An in vivo pharmacokinetic characterization of ALX-171 revealed that this compound possessed good exposure in the brain and oral bioavailability. As reported in our previous studies, a positive allosteric modulator of the mGlu 7 receptor, AMN082, exerted propsychotic activity in animal models [50], while the modulation of the mGlu 7 receptor through NAMs, e.g., ADX71743 or MMPIP, exerted antipsychotic activity in animal models of schizophrenia [7]. ADX71743 was active in all the tests used, including the DOI-induced head twitches, MK-801-induced disruptions in social interaction, cognitive impairment in novel object recognition, and prepulse inhibition, while the activity of MMPIP was not observed in MK-801-induced disruptions of social interaction or in PPI.
In the present study, ALX-171 was screened for its potential antipsychotic-like activity. The compound reversed DOI-induced head twitches and MK-801-induced disruptions of social interactions or cognition in the novel object recognition test and spatial delayed alternation test. The efficacy of the compound was not observed in the MK-801-induced hyperactivity test or prepulse inhibition at any dose tested.
Despite some differences observed in vivo between ALX-171 and the reference compounds ADX71743 and MMPIP, the antipsychotic activity profile of ALX-171 justified the further development of the group of quinazolin-4-one derivatives in the search for a new drug candidate. At the same time, our results confirmed, once again, the involvement of the inhibition of the mGlu 7 receptor in the therapeutic effects on schizophrenia.

Materials
All the chemicals employed in the syntheses were purchased from commercial vendors such as Merck (Darmstadt, Germany), Fluorochem (Hadfield, UK), Apollo (Bredbury, UK), and Combi-Blocks (San Diego, USA) and were used without purification. Solvents and inorganic reagents were acquired from Chempur (PiekaryŚląskie, Poland). Reaction progress was monitored with TLC on Merck Silica Gel 60 F 254 on aluminum plates. Column chromatography was performed on Merck Silica Gel 60 (0.063-0.200 mm; 70-230 mesh ASTM).

Analytical Methods
A UPLC/MS analysis was performed on a Waters TQD spectrometer combined with a UPLC Acquity H-Class with PDA eLambda detector. A Waters Acquity UPLC BEH C18 1.7 µm 2.1 × 50 mm chromatographic column was used at 40 • C, a 0.3 mL/min flow rate, and 1.0 µL injection volume (the samples were dissolved in LC-MS-grade acetonitrile, typically used at a concentration of 0.1-1 mg/mL prior to injection). All mass spectra were recorded under electrospray ionization in the positive mode (ESI+) and chromatograms were recorded with UV detection in the range of 190-300 nm. The gradient conditions used were: 80% phase A (water + 0.1% formic acid) and 20% phase B (acetonitrile + 0.1% formic acid) to 100% phase B (acetonitrile + 0.1% formic acid) at 3.0 min, kept for 3.5 min, and then to initial conditions for 4.0 min and kept for an additional 2.0 min. Total time of analysis-6.0 min.

Purity Analysis
The 1 H NMR spectra were measured at 300 MHz or 500 MHz, and the 13 C NMR spectra at 75 MHz or 126 MHz on a Varian Mercury-VX (300 MHz/500 MHz) spectrometer in CDCl 3 or d 6 -DMSO solutions with TMS as an internal standard. The spectral data of new compounds refer to their free bases. Chemical shifts are expressed in (ppm). Splitting patterns describe apparent multiplicities and were designated as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and br s (broad singlet). Coupling constants are given in units of hertz (Hz). In cases where it was possible, multiplets in 13 C NMR were identified and denoted in the experimental part. Unless otherwise noted, presented compounds were of at least 95% purity as determined with LC-MS. Syntheses and characterization details for Method A. A mixture of 6-bromo(3H)-quinazolin-4-ones (4, Scheme 1) (0.48 mmol), appropriate phenylboronic acid (0.72 mmol, 1.5 eq), potassium carbonate (1.44 mmol, 3 eq), and 1 mL of 2N Na 2 CO 3 aq solution in 4 mL of toluene and 1 mL of 1,4-dioxane was degassed with argon, and a Pd(dppf)Cl 2 complex in DCM (0.024 mmol, 0.05 eq) was added. The reaction was run under microwave irradiation at 120 • C for 20 min, cooled to room temperature (RT), and diluted with chloroform (50 mL) and water (30 mL). The layers were separated and the aqueous layer was extracted with chloroform (3×). Combined organic layers were washed with brine, dried over MgSO 4 , and evaporated to give a dark oily product. The crude product was purified with silica gel column chromatography followed by slurring with 2-PrOH/hexane (1:3) to yield the desired products.
Method C. We started with 6-bromo(3H)-quinazolin-4-ones (4, Scheme 1) (0.54 mmol), corresponding phenylboronic acid (0.70 mmol, 1.3 eq), potassium phosphate tribasic (1.36 mmol, 2.5 eq), a Pd(dppf)Cl 2 complex in DCM (0.05 mmol) in 4 mL of DMF, and 3 mL of water. The reaction was run for 1 h in a sealed tube at 80 • C, cooled to RT, and diluted with ethyl acetate (60 mL) and water (20 mL). The layers were separated and the aqueous layer was extracted with ethyl acetate (3×). The combined organic layers were washed with brine, dried over MgSO 4 , and evaporated, and the crude product was purified with silica gel column chromatography using hexane/EtOAc, followed by slurring with 2-PrOH/hexane (1:3) to yield A-9 derivatives. source (λ = 1.5418 Å). Additionally, the diffractometer was equipped with the CryoStream cryostat system, allowing for low-temperature experiments performed at 100(2) K. The obtained datasets were processed with CrysAlisPro software [51]. The phase problem was solved with direct methods using SIR2014 [43]. Parameters of the obtained models were refined with full-matrix least-squares on F 2 using SHELXL-2014/6 [52]. Calculations were performed using the WinGX integrated system (v2014.1) [53]. Figures were prepared with Mercury 4.0 software [54]. All nonhydrogen atoms were refined anisotropically. All hydrogen atoms attached were positioned with the idealized geometry and refined using the riding model with the isotropic displacement parameter U iso [H] = 1.2 U eq [C] for all but the methyl groups, for which U iso [H] = 1.5 U eq [C] was applied. Crystal data and refinement results are shown in Table S6. Asymmetric units, presenting an atom numbering scheme, are shown in Figure S1. The crystallographic data were deposited with the Cambridge Crystallographic Data Centre as supplementary publication nos.: CCDC2130726(ALX-065), CCDC2130727(ALX-171). Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK (e-mail: deposit@ccdc.cam.ac.uk).

cDNA Constructs and Cell Lines
The cell lines with the tetracycline-inducible expression of human metabotropic receptors 4, 7, and 8 were described in detail by Chruścicka et al. [41] (mGlu 4 receptor NM_000841, mGlu 7 receptor NM_000844.2, and mGlu 8 receptor NM_000845). Cells were grown under standard cell culture conditions (37 • C, 5% CO 2 ) in DMEM supplemented with 10% tetracycline-free FBS. The expression was induced through the addition of tetracycline to the culture medium at 0.75 µg/mL.

Forskolin-Induced cAMP Production Assay
The determination of the intracellular cAMP through homogeneous time-resolved fluorescence (HTRF) was described in detail previously [41]. Briefly, 48 h before the experiments, the expression of the receptor was induced through the addition of tetracycline to the culture medium; the next day, FBS and L-Glu were removed from the medium. Just before the cAMP measurement, the cells were detached and centrifuged. Then, the cell suspension in Hanks-HEPES (130 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl 2 , 0.8 mM MgSO 4 , 0.9 mM NaH 2 PO 4 , 20 mM HEPES, and 3.25 mM glucose; pH 7.4) was incubated in the presence of 10 µM forskolin (mGlu 7 receptor, 3 µM forskolin), the agonist LSP4-2022 or L-Glu (for mGlu 4 and mGlu 8 ), and a compound for 5 min. The intracellular concentration of cyclic AMP was measured by means of a cAMP Gi HTRF kit from Cisbio, according to the manufacturer's instructions. After 1 h of incubation at room temperature, the fluorescence at 620 nm and 665 nm was read (Tecan Infinite M1000). The results were calculated as the 665 nm/620 nm ratio multiplied by 10 4 . Each sample was prepared in triplicate. Data were analyzed using GraphPad Prism version 5.04 for Windows (GraphPad Software, San Diego, USA).

In Vitro Activity
The activity of the compounds was examined in the T-REx 293-cell-line-expressing recombinant human mGlu 7 receptor by detecting the level of cyclic AMP in the presence of 5 µM forskolin [7,41]. Firstly, all compounds were screened at 10 µM using three cell lines expressing the mGlu 4/7/8 receptor to evaluate selectivity. For the mGlu 4 and mGlu 8 receptors, glutamic acid was used as an agonist and had good potency. For the mGlu 7 receptor, we used LSP4-2022 as an agonist due to its homogeneity and better potency, compared to L-Glu (~1 mM). VU0155041 and AZ12216052 were used as reference compounds for the mGlu 4 and mGlu 8 receptors, respectively. The parameter described as "% of inhibition" was introduced to compare the bioactivity of the new compounds to the reference NAM at a 10 µM concentration in the presence of 5 µM LSP4-2022 (0%) and 3 µM forskolin (100%). For ADX71743, the percent inhibition was 42.61% (±7.56; n = 10). For compounds that passed the screening procedure, the dose-response curve in the presence of LSP4-2022 in 5 µM was evaluated compared to reference NAM ADX71743. Only compounds ALX-063, ALX-065, and ALX-171 ( Figure S3, Supplementary Materials) met our conditions regarding bioactivity. All of them were further investigated to determine the EC 50 and receptor selectivity.

Pharmacokinetic Studies
The method described below was successfully applied to a pharmacokinetic study of all tested compounds (ALX-065, ALX-171, ADX71743, and MMPIP) in mice (Albino Swiss) after an i.p. injection. Compounds were administered to mice at a dose of 10 mg/kg i.p. Plasma and tissue samples were collected at 0.25, 0.50, 1.0, 2.0, 4.0, 6.0, and 24 h. Plasma and tissue samples from all drug-treated animals were thawed at room temperature prior to use. The standard protocol for sample preparation was used: 200 µL of acetonitrile was added to the Eppendorf tubes with 50 µL of studied plasma samples or tissue homogenate. Samples were mixed for 5 min on a mixer at 25 • C and 1400 rpm. The tubes were then centrifuged at 2000× g for 15 min at 4 • C. A total of 180 µL of each supernatant was transferred to a plate well. Finally, each sample was injected into the LC-MS column. Calibration curve serial dilution method: Plasma was spiked with a standard at different concentrations. Acetonitrile was added. After mixing and centrifugation, the supernatant was collected.

LC-MS Analysis
Chromatographic conditions. Plasma and tissue samples from all drug-treated animals at selected time points were analyzed using a previously developed nonvalidated LC-MS/MS method. A sensitive and highly selective liquid chromatography-tandem mass spectrometry (LC-MS) method was used to determine the drug concentration in mouse plasma samples or tissue homogenates. The LC-MS analysis was carried out on a Bruker amaZon SL mass spectrometer using the positive/negative ion ESI mode. Chromatographic separation was achieved on an Ascentis Express C18 column (5 cm × 2.1 mm, 2.7 µM, Supelco Technologies) at room temperature with a thermostatted column oven. A gradient elution of eluents A (acetonitrile (LiChrosolv, Reag. Ph Eur) + 0.1% formic acid (Sigma Aldrich, 98-100%)) and B (water + 0.1% formic acid) were used for separation. The flow rate was set at 1 mL/min. The injection volume was 20 µL, and the time of injection was 4 min.
Mass spectrometric conditions. An ion trap mass spectrometer (Bruker amaZon SL, Bruker, Bremen, Germany) was equipped with an electrospray source operating in the positive/negative ion mode. Data were collected and processed using Bruker Quant Analysis (Bruker, Bremen, Germany) software. A quantification of the analytes was performed in the SIM mode.

CYP Inhibition Assay
CYP inhibition was performed with the recombinant enzymes CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 obtained from BD Biosciences. Compounds for CYP inhibition testing were prepared as 10 mM stock solutions in DMSO. Isoform-specific substrates were incubated at 37 • C individually with P450 enzymes (Table 5) and a range of test compound concentrations (1.1, 3.3 and 10 µM) in duplicate. Each isoform was tested separately with one reference compound, a known positive control inhibitor. During preincubation, the 96-well black plates were scanned with a fluorescence plate reader to eliminate false results originating from the autofluorescence of the test compounds. At the end of the incubation, product formation was monitored with fluorescence detection. A decrease in the formation of the metabolite compared to the "no inhibition" control samples was used to calculate the IC 50 value.

Kinetic Solubility Assay
The kinetic solubility assay was performed to determine the solubility of the compounds in the in vitro assay conditions. The assay investigated the solubility based on the precipitation process in the HBSS buffer. For the preparation of the assay buffer, the following compounds were dissolved in 800 mL purified water: − 130 mM sodium chloride, NaCl (7.600 g); − 5.4 mM potassium chloride, KCl (0.403 g); − 0.8 mM magnesium sulfate, MgSO 4 × 7H 2 O (0.197 g); − 0.9 mM sodium phosphate monobasic, NaH 2 PO 4 × 2H 2 O (0.140 g); − 25 mM D-glucose (4.500 g); − 20 mM HEPES sodium salt (5.210 g).
The buffer was adjusted to pH 7.4, and the following compound was added to the mixture: − 1.8 mM calcium chloride, CaCl 2 × 2H 2 O (0.265 g).
The total volume was adjusted to 1000 mL. Compounds for the kinetic solubility testing were prepared as 10 mM stock solutions in DMSO. The investigated compounds were diluted (in triplicate) with the HBSS buffer at pH 7.4 to a final concentration of 500 µM.
The mixture was shaken on a filter plate at 500 rpm for 1.5 h at RT. After incubation, the filter plate was placed inside a vacuum manifold and filtrated. Samples were collected and the concentration of the filtrate was determined spectrophotometrically by measuring the UV-VIS absorption spectrum. Concentrations were calculated based on equations resulting from the calibration curves (in duplicates, 8 calibration points, dilution Factor ×2, 500 µM → 3.91 µM, including blank sample).

Metabolic Stability
A metabolic stability assay was performed using a mouse microsomal fraction (phase I of metabolism) obtained from XenoTech (Kansas City, MO, USA). Compounds for metabolic stability testing were prepared as 10 mM stock solutions in DMSO. Compounds were incubated in triplicate at a 1 µM initial concentration with the microsomal fraction in the presence of NRS (NADPH-regenerating system, 1.3 mM). The final microsomal incubation (0.3 mg/mL) contained the components mentioned below in a pH 7.4 phosphate buffer. Control incubations were conducted without cofactors. The reference compound verapamil was used as a positive control. Incubations were carried out for 1 h on 96-well plates using a heated (+37 • C) orbital shaker (350 rpm). After incubation, the reaction was quenched through the addition of cold acetonitrile. The plate containing the samples was centrifuged at 2000 rpm for 15 min at 4 • C. The supernatants were analyzed using LC-MS.

Conditions of the Liquid Chromatography-Mass Spectrometry
The HPLC-MS consisted of a Dionex, Ultimate 3000 HPLC, and a Bruker Daltonics amaZon SL mass spectrometer (ESI-IT). The conditions were as follows: Column HPLC Ascentis Express C18 (5 cm × 2.1 mm, 2.7 µm) and an injection volume of 20 µL. An acetonitrile gradient was used with 0.1% formic acid in water at a constant flow rate of 1 mL/min. The programming was as follows: 0-0.5 min 5% ACN, 0.5-3.0 increase of 5-95%, 3.0-3.3 remaining at 95% ACN, 3.3-3.4 min decrease of 95-5% ACN, and 3.4-4.0 min 5% ACN. Samples were analyzed in a single-ion monitoring scan mode in positive mode. The conditions of the main parameters were as follows: a trap drive of 48.2, capillary exit voltage of 140.0 V, dry temperature (set) of 300 • C, nebulizer pressure (set) of 40.00 psi, dry gas flow (set) of 9.00 L/min HV capillary voltage of −4500 V, HV end-plate offset voltage of −500 V.
The percent loss of compounds was calculated through the normalization of the peak area against time 0, where the peak area at T 0 was 100%. The elimination rate constant (h) was determined from the plot of ln (percent loss of compound) as a time function and equal to the minor value of the linear curve slope. The half-life time was calculated from the following equation: Male CD1 mice (Charles River, Sulzfeld, Germany) weighing 20-25 g at the time of arrival were used in the behavioral experiments. Animals were kept under standard laboratory conditions (12:12 light:dark cycle, 22 ± 2 • C) with free access to food and water. Animal welfare was regularly controlled by a veterinarian and animal welfare committee. After 2 weeks of acclimatization and handling, the experiments began. Experimental groups consisted of 4 to 10 animals, depending on the procedure. Drugs were administered intraperitoneally (i.p.) at a volume of 10 mL/kg. Experimental assessments were performed by an observer who was blinded to the treatment conditions. All procedures were conducted in accordance with the European Communities Council Directive of 22 September 2010 (2010/63/EU) and Polish legislation acts concerning animal experimentation, and were approved by the II Local Ethics Committee of the Maj Institute of Pharmacology, Polish Academy of Sciences, in Krakow (272/2019).

Drugs
MK-801 was purchased from Tocris Bioscience, Bristol, UK. MK-801 was dissolved in 0.9% NaCl. Compounds 15 and 18 were dissolved in 0.9% NaCl. All compounds were administered intraperitoneally (i.p.) in a volume of 10 mL/kg. Vehicle-treated animals received appropriate solvents. The vehicle was administered to animals in any case when drug administration was omitted (e.g., control or MK-801-treated groups).

DOI-Induced Head Twitch Test
The experiments were performed according to the procedure described in our previous studies [7,53,54]. Briefly, to habituate mice to the experimental environment, each animal was transferred to a 12 cm (diameter) × 20 cm (height) glass cage lined with sawdust 30 min before treatment. Test compounds were administered intraperitoneally (i.p.) at doses of 2.5, 5, and 10 mg/kg body weight 60 min before the test was performed. The head twitch response (HTR) in mice was induced using DOI (2.5 mg/kg, i.p.). Immediately after treatment, the number of head twitches was counted during a 20 min session.

Novel Object Recognition Test
This procedure was adapted from [55] and performed as described in a previous paper [7]. Habituation, training and test trials were performed in a black plastic rectangular arena (40 × 30 × 35 cm) illuminated with a light intensity of 335 lux. During the habituation trial (2 consecutive days), each animal was allowed to explore the arena for 10 min. The next day, during the training trial (T1), mice were placed in the arena and were presented with two identical objects (red glass cylinders; 6.5 cm in diameter and 4.5 cm high) for 5 min. After 1 h, animals were placed back into the arena for a 5 min test trial, during which one of the previously presented familiar objects was replaced with a novel object (a transparent glass elongated sphere-like object with an orange cap; 5.5 cm in diameter and 8.5 cm high). Time spent exploring (i.e., sniffing or touching) the familiar (Tfamiliar) and novel (Tnovel) objects was measured by a trained observer, and the recognition index (%) was calculated for each mouse [(Tnovel − Tfamiliar)/(Tfamiliar + Tnovel)] "×" 100.

MK-801-Induced Hyperactivity
The locomotor activity was recorded individually for each animal in locomotor activity cages [55,56] with modifications [50]. The mice were placed individually into activity cages (13 × 23 × 15 cm; Opto-M3; Columbus Instruments) for an acclimatization period of 30 min; then, they were injected i.p. with compound 15 (0.5, 1, 3 mg/kg) or compound 18 (0.05, 0.1, 0.5, 1, 3 mg/kg) and placed again in the same cages. After 30 min, all of the mice were injected i.p. with MK-801 at 0.35 mg/kg and once again placed in the same cage. From then on, the ambulation scores were counted for 60 min. All of the groups were compared with the MK-801 control group. The experiment also included a control group treated with NaCl only.

Social Interaction Test
The method was adapted from de Moura Linck et al., 2008 [57], and Woźniak et al., 2016 [58]. After the 2-day habituation trial (10 min/day), a pair of mice was placed in the open field for 5 min. The social interactions between the two mice were determined based on the total time spent participating in social behavior, such as genital investigation, sniffing, chasing, and fighting each other. The total number of social episodes was also measured. The test was video-recorded and viewed by a trained observer. MMPIP (5, 10, and 20 mg/kg, i.p.) or ADX71743 (1, 5, and 15 mg/kg, i.p.) were administered 30 min before MK-801 (0.3 mg/kg, i.p.), which was administered 30 min before the test.

Statistics
The statistical analysis was performed using GraphPad Prism v.9.1.0. The results of behavioral studies were analyzed using Student's t-test (comparison vs control group), oneway ANOVA followed by Dunnett's post hoc comparison or two-way ANOVA followed by Tukey's post hoc comparison. Data are presented as mean ± SEM.