Discovery of a Selective and CNS Penetrant Negative Allosteric Modulator of Metabotropic Glutamate Receptor Subtype 3 with Antidepressant and Anxiolytic Activity in Rodents

Abstract

Previous preclinical work has demonstrated the therapeutic potential of antagonists of the group II metabotropic glutamate receptors (mGlus). Still, compounds that are selective for the individual group II mGlus (mGlu₂ and mGlu₃) have been scarce. There remains a need for such compounds with the balance of properties suitable for convenient use in a wide array of rodent behavioral studies. We describe here the discovery of a selective mGlu₃ NAM 106 (VU0650786) suitable for in vivo work. Compound 106 is a member of a series of 5-aryl-6,7-dihydropyrazolo[1,5-a]pyrazine-4(5H)-one compounds originally identified as a mGlu₅ positive allosteric modulator (PAM) chemotype. Its suitability for use in rodent behavioral models has been established by extensive in vivo PK studies, and the behavioral experiments presented here with compound 106 represent the first examples in which an mGlu₃ NAM has demonstrated efficacy in models where prior efficacy had previously been noted with nonselective group II antagonists.

INTRODUCTION

Glutamate (l-glutamic acid) is the major excitatory neuro-transmitter in the mammalian central nervous system (CNS) and acts on both ionotropic and metabotropic glutamate receptors (mGlus). While ionotropic glutamate receptors are ligand-gated ion channels, mGlus are a family of eight G-protein coupled receptors (GPCRs). Belonging to family C of the GPCRs, the mGlus possess a seven transmembrane (7TM) α-helical domain connected via a cysteine-rich region to a large bilobed extracellular amino-terminal domain containing the orthosteric binding site. The mGlus have been further categorized into three groups according to their homology, preferred signal transduction mechanisms, and pharmacology: group I (mGlu₁ and mGlu₅), group II (mGlu₂ and mGlu₃), and group III (mGlu₄, mGlu₆, mGlu₇, and mGlu₈).^1–3 Both mGlu₂ and mGlu₃ are primarily located presynaptically in neurons and coupled to G_i/o and the inhibition of adenylyl cyclase activity; in addition, mGlu₃ is also expressed in glial cells. The group II mGlus are widely expressed throughout the CNS, including regions of the brain associated with emotional states such as the amygdala, hippocampus, and prefrontal cortex.^4,5

Researchers have been successful in designing both orthosteric antagonists and negative allosteric modulators (NAMs), also known as noncompetitive antagonists, of the group II mGlus. Although such compounds are generally selective versus the other six members of the mGlu family, selectivity between mGlu₂ and mGlu₃ is negligible.⁶ Still, the use of such compounds in animal models has established a potential role for mGlu_2/3 antagonists in a variety of CNS disorders. Many of these studies have been carried out with two orthosteric antagonists, both of which are highly functionalized glutamate analogues, 1 (LY341495)⁷ and 2 (MGS0039)⁸ (Figure 1). For example, work with these compounds has helped establish mGlu_2/3 inhibition as a potential therapeutic application for obsessive-compulsive disorder (OCD),^9,10 anxiety,¹¹ cognition,¹² and Alzheimer's disease.^13–15 Additionally, antidepressant efficacy has been demonstrated in numerous rodent models of depression with these compounds,^{8,9,11,16–19} including those meant to assess treatment-resistant depression (TRD),²⁰ anhedonia,²¹ and depression associated with withdrawal from addictive substances.^22,23 Compound 1 has also been used as a tool to establish a potential utility for mGlu_2/3 inhibition in the treatment of glioma.^24–27

Figure 1.

mGlu_2/3 orthosteric antagonist tools 1 and 2, mGlu_2/3 NAM tools 3, 4, and 6, and Roche mGlu_2/3 NAM clinical compound 5.

Research with mGlu_2/3 NAMs has been less extensive than with orthosteric antagonists; however, closely related tools 3 (RO4491533)²⁸ and 4 (RO4432717)^29,30 (Figure 1) have been employed in multiple in vivo assays. In particular, these compounds have demonstrated efficacy in multiple models of depression³¹ and cognition.^30,32,33 Furthermore, recent studies in genetically modified mice with 1³⁴ and mGlu_2/3 NAM 6³⁵ (Figure 1) point toward a potential application for group II antagonists in the treatment of certain autism spectrum disorders. Finally, one mGlu_2/3 NAM, 5 (decoglurant, RO4995819)³⁶ (Figure 1), has advanced into human clinical trials, including a phase II trial in patients with major depressive disorder (MDD) and resistant to ongoing treatment with antidepressants (NCT01457677).³⁷ In spite of the wealth of preclinical evidence for the potential utility of mGlu_2/3 antagonists, identification of compounds with ample selectivity between the two group II mGlus and the balance of pharmacology and drug metabolism and pharmacokinetics (DMPK) properties required for use in vivo has been elusive. Such tools are essential for further validation of the precise roles of each receptor in the etiology of disease.

Our first foray into this arena of research began by an observation of occasional weak mGlu₃ NAM activity in a series of 1,2-diphenylethyne mGlu₅ positive allosteric modulators (PAMs).³⁸ An optimization plan for mGlu₃ activity that centered on modification of the functional groups appended to the two phenyl rings within this scaffold delivered first-generation tool 7 (VU0463597, ML289)³⁹ (Figure 2). Importantly, not only was 7 selective versus mGlu₅ but selectivity versus mGlu₂ was also notable (>15-fold). Further optimization within this chemotype identified a second-generation compound 8 (VU0469942, ML337)⁴⁰ (Figure 2) that was devoid of both mGlu₂ and mGlu₅ activity. While 8 has proven quite useful as an in vitro tool compound and can be used in mice at high doses (100 mg/kg),⁴¹ lower CNS penetration and higher protein binding in rats prevent its utility in that species. Furthermore, 1,2-diarylethyne chemotypes similar to this one are prone to bioactivation at the alkyne moiety and subsequent formation of reactive metabolites that can lead to toxicity.^42,43 Thus, the discovery of a superior mGlu₃ NAM from outside the 1,2-diphenylethyne chemotype, with the balance of pharmacology and DMPK properties for convenient use in both rats and mice, remained a worthy goal and is the subject of this manuscript.

Figure 2.

mGlu₃ NAMs from the 1,2-diphenylethyne chemotype.

RESULTS AND DISCUSSION

Lead Identification

The previous success in identification of an mGlu₃ NAM lead with inherently good selectivity versus mGlu₂ from a mGlu₅ PAM chemotype^39,40 prompted the mining of our internal collection of mGlu₅ PAM chemotypes lacking an alkyne moiety. Utilization of this resource provided some examples of compounds with evidence of mGlu₃ NAM activity amid the available associated cross-screening data versus the mGlu family.^44,45 We selected many of these interesting compounds as well as additional closely related analogues to arrive at approximately 160 compounds for full concentration response curve (CRC) measurements in our cell-based functional assay for mGlu₃. This fluorescence-based assay measures calcium mobilization induced by mGlu₃ activation in a cell line stably expressing rat mGlu₃ and the promiscuous G-protein G_α15 and is capable of detecting agonists, PAMs, and NAMs of mGlu₃. We have developed similar assays and cell lines for rat mGlu₂ and rat mGlu₅, and both were used throughout this project for assessing selectivity.

One of the most interesting series to emerge from the full CRC mGlu₃ screen outlined above is exemplified by compounds 9–11 (Figure 3). These compounds are from within a series of 5-aryl-6,7-dihydropyrazolo[1,5-a]pyrazine-4(5H)-ones that includes many potent mGlu₅ PAMs.⁴⁶ An interesting piece of SAR was noted with respect to the presence of a chiral methyl group at the seven position of the pyrazine-4(5H)-one ring in the (R)-configuration (10). Whereas the potency of (R)-methyl analogue 10 at mGlu₅ was only 2-fold less than unsubstituted analogue 9, the efficacy of 10 (Glu Max = 26.5%) was much weaker than that observed with 9 (Glu Max = 93.0%). The (S)-methyl analogue 11 proved highly preferential for mGlu₅ PAM activity and was only weakly active at mGlu₃, inhibiting the glutamate response only at the top concentration tested (30 μM). Gratifyingly, compound 10 was also inactive up to the top concentration tested (30 μM) in our mGlu₂ calcium mobilization assay. Given the potential benefits engendered by this (R)-methyl group, we incorporated this functional group into the design of future compounds.

Figure 3.

Representative compounds from a new mGlu₃ lead series.

Development of SAR in the Western Region

Our initial plan centered on the development of structure–activity relationships (SAR) in the area occupied by the 2-pyridyl ether of 10, termed the western region of the scaffold. Intermediate primary alcohol 20 was envisioned as a valuable intermediate for late stage diversification of this area (Scheme 1). The synthesis of 20 began with commercially available phenoxyacetone 12. The sodium enolate of 12 was prepared and treated in situ with diethyl carbonate to afford 2,4-diketone intermediate 13. Reaction of 13 with hydrazine readily provided 14, which could be N-alkylated via a Mitsunobu reaction⁴⁷ with commercially available chiral alcohol 15. This Mitsunobu reaction was carried out with microwave heating and resulted in inversion of stereochemistry as expected. Treatment of intermediate 16 with acid resulted in cleavage of the tert-butylcarbamate protecting group, and subsequent exposure to aqueous base provided the lactam 17. Chiral HPLC analysis of 17 showed 98.6% ee for this key intermediate. N-Arylation of 17 was accomplished by a copper mediated coupling⁴⁸ with 4-fluorobromobenzene to yield 18. Treatment of 18 with boron tribromide cleaved the phenoxy ether, affording the corresponding primary bromide, which was then reacted with potassium acetate under mild heating to afford acetate ester 19. Hydrolysis of the acetate group was accomplished with aqueous lithium hydroxide to give the desired intermediate alcohol 20.

Scheme 1.

Synthesis of Primary Alcohol Intermediate 20^a

Preliminary mGlu₃ NAM SAR obtained from the testing of analogues of lead 10 that were included in the original set of 160 compounds indicated a preference for phenyl and 2-pyridyl ethers in the western portion of the scaffold. Late stage conversion of intermediate alcohol 20 to new analogues was accomplished through one of three methods (Scheme 2). For the synthesis of substituted phenyl ether analogues 22–39, alcohol 20 was converted to the corresponding mesylate, which was then reacted with the desired phenols 21 and cesium carbonate to afford 22–39. Alternatively, the phenols 21 and alcohol 20 were coupled directly via a Mitsunobu reaction.⁴⁷ For synthesis of 2-pyridyl ether targets 41–51, the sodium alkoxide of 20 was prepared in situ and treated with substituted 2-fluoropyridines 40 to facilitate a S_NAr reaction and provide 41–51.

Scheme 2.

Synthesis of New Western Ether Analogues 22–39 and 41–51^a

The results obtained from preparing and testing unsubstituted phenyl ether intermediate 18 revealed a near 6-fold preference for mGlu₅ vs mGlu₃ activity, which ultimately proved a substantial hurdle with this subset of compounds (Table 1). To determine if substitution of the phenyl group could engender preference for mGlu₃ activity, we systematically installed a variety of functional groups at all positions (22–34). Most substituents at the 2-position (22, 26, 29) only modestly affected mGlu₃ NAM activity relative to 18; however, 2-methoxy analogue 32 was nearly 10-fold less potent than 18. The mGlu₅ PAM activity was improved relative to 18 but remained suboptimal with these 2-substituted analogues. 2,4-Difluoro analogue 25 demonstrated modestly enhanced selectivity versus mGlu₅ relative to its monosubstituted comparators 22 and 24. Although substitution of the 3-position with fluorine (23) provided little impact on activity at either receptor, installation of larger substituents (27, 30, 33) resulted in reductions in potency at both receptors and two instances of pharmacology mode switching at mGlu₅ (27 and 30). Such “molecular switches” have been noted previously in other mGlu₅ chemotypes.^49,50 Substitution of the 4-position with fluorine (24) only minimally impacted potency at either receptor; larger groups (28, 31, 34) reduced potency at both receptors, although the effect on mGlu₅ was more pronounced. Thus, desiring to further examine the effects of substitution at the 4-position, additional analogues were prepared (35–39). The trend toward mGlu₃-preferring compounds continued with the 4-ethoxyphenyl analogue 38 and 4-trifluoromethoxy analogue 39, demonstrating no activity versus mGlu₅ up to the highest concentration tested (30 μM).

Table 1.

mGlu₃ NAM and mGlu₅ SAR of Western Phenyl Ethers 18, 22–39

no.	R	mGlu3 pIC50 (± SEM)a	mGlu3 IC50 (nM)a	% Glu Max (± SEM)a,b	mGlu5 activityc	mGlu5 pEC50 (± SEM)c	mGlu5 EC50 (nM)c	% Glu Max (± SEM)b,c
18	H	6.57 ± 0.15	267	2.21 ± 0.80	PAM	7.34 ± 0.02	46	87.7 ± 1.4
22	2-F	6.28 ± 0.20	530	2.90 ± 1.05	PAM	6.71 ± 0.04	195	89.3 ± 3.0
23	3-F	6.11 ± 0.03	773	1.19 ± 0.75	PAM	7.14 ± 0.02	73	86.1 ± 2.9
24	4-F	6.34 ± 0.04	462	2.09 ± 0.57	PAM	7.03 ± 0.05	92	87.0 ± 1.0
25	2,4-di-F	6.38 ± 0.22	417	1.70 ± 0.35	PAM	6.51 ± 0.05	307	82.3 ± 2.7
26	2-Me	6.14 ± 0.14	721	1.56 ± 0.37	PAM	5.83 ± 0.04	1500	83.7 ± 2.5
27	3-Me	5.84 ± 0.05	1440	2.35 ± 0.18	NAMd	<5.0	>10000	39.7 ± 7.7
28	4-Me	5.92 ± 0.02	1190	1.72 ± 0.55	PAM	5.97 ± 0.04	1080	92.5 ± 2.6
29	2-Cl	6.27 ± 0.18	532	1.63 ± 0.33	PAM	6.38 ± 0.01	419	84.4 ± 2.1
30	3-Cl	5.79 ± 0.02	1620	1.96 ± 0.11	NAMd	<5.0	>10000	32.8 ± 6.1
31	4-Cl	5.93 ± 0.01	1170	1.86 ± 0.52	PAM	6.22 ± 0.04	604	81.8 ± 0.3
32	2-OMe	5.59 ± 0.18	2540	2.32 ± 1.39	PAMd	<5.0	>10000	63.7 ± 3.1
33	3-OMe	5.89 ± 0.02	1290	1.78 ± 0.53	PAM	5.74 ± 0.04	1840	32.4 ± 3.0
34	4-OMe	5.98 ± 0.02	1040	1.76 ± 0.39	PAM	5.87 ± 0.16	1340	40.2 ± 4.8
35	4-CF3	5.73 ± 0.01	1850	4.13 ± 0.09		<4.5	>30000
36	4-Et	6.08 ± 0.05	830	2.29 ± 0.84	PAM	5.49 ± 0.03	3210	50.8 ± 3.7
37	4-CN	6.11 ± 0.12	784	2.53 ± 0.70	PAM	5.71 ± 0.06	1940	38.6 ± 4.8
38	4-OEt	5.94 ± 0.06	1160	3.39 ± 0.87		<4.5	>30000
39	4-OCF3	5.39 ± 0.04	4070	2.66 ± 0.34		<4.5	>30000

^aCalcium mobilization mGlu₃ assay; values are average of n ≥ 3.

^bAmplitude of response in the presence of 30 μM test compound as a percentage of maximal response (100 μM glutamate); average of n ≥ 3.

^cCalcium mobilization mGlu₅ assay; values are average of n ≥ 3.

^dWeak activity; concentration response curve (CRC) does not plateau.

Given the promising profile of initial lead 10, our hope for identifying more attractive compounds within the 2-pyridyl analogues (41–51) remained high; however, this region proved relatively intolerant of substitution (Table 2). Although a 3-fluoro substituent (41) only modestly impacted potency, larger substituents (42, 43) had more deleterious effects. Similarly modest results were observed with 4-position (44–46) and 6-position (49–51) analogues. Still, it was encouraging to identify additional compounds (42, 43, and 50) that displayed no activity versus mGlu₅ up to the highest concentration tested (30 μM) as well as several analogues with only weak activity at mGlu₅. Fortunately, 5-halo analogues 47 and 48 proved an exception to the general trend of modest mGlu₃ potency with these analogues. Furthermore, 5-fluoro analogue 47 demonstrated enhanced selectivity approaching 20-fold versus mGlu₅, while 5-chloro analogue 48 was more modest with regard to selectivity at approximately 5-fold.

Table 2.

mGlu₃ NAM and mGlu₅ SAR of Western 2-Pyridyl Ethers 41–51

no.	R	mGlu3 pIC50 (± SEM)a	mGlu3 IC50 (nM)a	% Glu Max (± SEM)a,b	mGlu5 activityc	mGlu5 pEC50 (± SEM)c	mGlu5 EC50 (nM)c	% Glu Max (± SEM)b,c
41	3-F	5.96 ± 0.02	1100	2.22 ± 0.84	PAM	5.37 ± 0.06	4300	78.5 ± 6.1
42	3-CF3	5.53 ± 0.02	2940	2.11 ± 0.96		<4.5	>30000
43	3-OMe	5.17 ± 0.02	6760	–1.14 ± 2.54		<4.5	>30000
44	4-Me	5.68 ± 0.01	2090	0.86 ± 0.47	NAMd	<5.0	>10000	46.9 ± 5.9
45	4-CF3	5.78 ± 0.01	1670	1.62 ± 0.42	NAMd	<5.0	>10000	12.9 ± 2.9
46	4-OMe	5.43 ± 0.00	3690	–0.18 ± 1.39	NAMd	<5.0	>10000	54.7 ± 5.8
47	5-F	6.27 ± 0.02	539	1.71 ± 0.55	NAMd	<5.0	>10000	46.5 ± 3.4
48	5-Cl	6.22 ± 0.04	605	1.84 ± 0.51	PAM	5.53 ± 0.06	2920	81.4 ± 2.7
49	6-Me	5.62 ± 0.02	2370	1.12 ± 1.08	NAM	5.65 ± 0.07	2250	2.47 ± 0.17
50	6-CF3	5.63 ± 0.02	2360	2.06 ± 0.79		<4.5	>30000
51	6-OMe	5.80 ± 0.02	1590	1.94 ± 0.62	PAMd	<5.0	>10000	31.2 ± 3.1

^aCalcium mobilization mGlu₃ assay; values are average of n ≥ 3.

^bAmplitude of response in the presence of 30 μM test compound as a percentage of maximal response (100 μM glutamate); average of n ≥ 3.

^cCalcium mobilization mGlu₅ assay; values are average of n ≥ 3.

^dWeak activity; CRC does not plateau.

With some initial SAR in hand, we further profiled some of the more promising early analogues (38, 47, 48) to assess other properties important for the development of a useful in vivo tool compound (Table 3). In addition to assessing the degree to which the compounds were bound to rat plasma,⁵¹ the compounds were evaluated in a rat cassette pharmacokinetics (PK) study using intravenous (IV) dosing⁵² to assess their metabolic stability in vivo. In spite of its poor selectivity profile versus mGlu₅, 4-fluorophenyl ether 24 was also included with these compounds as a comparator compound. In fact, analogue 24 exhibited moderate clearance, with a half-life of over 1.5 h. Not surprisingly, replacement of the fluorine atom (24) with the more metabolically labile ethoxy group (38) increased clearance to approximately hepatic blood flow and reduced half-life. The results obtained with the 2-pyridyl analogues 47 and 48 revealed a profound metabolic difference. Whereas 5-fluoro analogue 47 was rapidly cleared, 5-chloro analogue 48 had a moderate clearance and a half-life of approximately 2 h. As indicated by their ligand-lipophilicity efficiency (LLE) values,⁵³ the 2-pyridyl ethers possessed a better balance of potency and lipophilicity than the phenyl ether compounds. Notably, 4-ethoxyphenyl ether 38 also exhibited exceedingly high protein binding to rat plasma. These factors led to the conclusion that optimization should continue in the context of the 2-pyridyl ethers. Superior LLE, selectivity versus mGlu₅, and plasma unbound fraction relative to 48, made 5-fluoro analogue 47 an attractive starting point for further optimization; however, its high plasma clearance remained an issue. To avoid focusing future optimization efforts exclusively in the context of a potential PK liability, the decision was made to prepare analogues of both 47 and 48 in parallel.

Table 3.

DMPK Profiling of Early Analogues

no.	cLogPa	LLEb	mGlu3 IC50 (nM)	fold vs mGlu5	rat plasma Fuc		CLplasma (mL/min/kg)d	VSS (L/kg)d
24	3.96	2.38	462	0.20	0.060	95	40	3.5
38	4.24	1.70	1160	>25	0.005	32	82	2.2
47	3.06	3.21	539	>18	0.135	88	82	6.6
48	3.57	2.65	605	4.8	0.034	141	29	5.2

^aCalculated using Dotmatics Elemental (www.dotmatics.com/products/elemental/).

^bLLE (ligand-lipophilicity efficiency) = pIC₅₀ – cLogP.

^cF_u = fraction unbound.

^dRat IV PK results (n = 2); dose = 0.2 mg/kg; solution in 9% EtOH, 37% PEG 400, 54% DMSO (2 mg/mL).

Development of SAR in the Eastern Region

To enable final step diversification and the rapid synthesis of new analogues, modified syntheses were required for development of SAR in the eastern portion of the chemotype. Synthesis of new 5-fluoropyridin-2-yl ether analogues 55–72 was accomplished via a reordering of the previously outlined reactions (Scheme 3). Intermediate 17 was treated with boron tribromide and subsequently potassium acetate to afford acetate ester 52. Hydrolysis of 52 was accomplished with lithium hydroxide; however, the yield of alcohol 53 suffered due to difficulty in isolation of this polar and water-soluble intermediate. Still, preparation of the 5-fluoropyridin-2-yl ether 54 via S_NAr chemistry was readily accomplished, which enabled a final stage installation of the aryl or heteroaryl eastern ring according to methods described previously to yield the desired products 55–72.

Scheme 3.

Synthesis of 5-Fluoropyridin-2-yl Ether Analogues 55–72^a

Because of the poor yield encountered in the synthesis of intermediate 53, we employed what proved to be a more scalable and shorter synthesis in the preparation of the 5-chloropyridin-2-yl ether analogues (Scheme 4). Synthesis of intermediate 75 from commercially available 73 was accomplished via an analogous two-step Mitsunobu coupling⁴⁷ and deprotection/cyclization sequence as described previously. The lactam nitrogen was protected with a 4-methoxybenzyl group to afford 76. Reduction of the ethyl ester was accomplished with sodium borohydride to yield 77. Formation of the 5-chloropyridin-2-yl ether 78 was carried out via S_NAr chemistry as before. Oxidative removal of the 4-methoxybenzyl protecting group was carried out with ceric ammonium nitrate to provide penultimate intermediate 79. Conversion of 79 into analogues 80–110 utilized the copper mediate N-arylation methods employed previously.

Scheme 4.

Synthesis of 5-Chloropyridin-2-yl Ether Analogues 80–110^a

Testing of new 5-fluoropyridin-2-yl ether analogues 55–72 showed eastern ring modification was a useful strategy for enhancing mGlu₃ NAM activity (Table 4). In the case of monosubstituted phenyl ethers (55–65), several analogues exhibited mGlu₃ IC₅₀ values less than 200 nM. Specifically, at the 2-position, fluorine (55) and chlorine (57) substitution was preferred to methyl (60) and methoxy (63) substituents, while little difference in mGlu₃ activity was observed with variation of the same substituents at the 3-position (56, 58, 61, 64). Substitution at the 4-position (59, 62, 65) was slightly less favorable. Encouragingly, several of these analogues also demonstrated only weak activity at mGlu₅. Some difluorophenyl analogues (66–68) were also prepared and found to exhibit good potency. Simple pyridyl analogues (69–71) were less potent versus mGlu₃ than the majority of the phenyl analogues; however, selectivity versus mGlu₅ was notable in the case of 70 and 71. Finally, simple fluorine substitution of the pyridine ring (72) enhanced mGlu₃ activity relative to unsubstituted comparator 69.

Table 4.

mGlu₃ NAM and mGlu₅ SAR of 5-Fluoropyridin-2-yl Ether Analogues 55–72

no.	R	mGlu3 pIC50 (± SEM)a	mGlu3 IC50 (nM)a	% Glu Max (± SEM)a,b	mGlu5 activityc	mGlu5 pEC50 (± SEM)c	mGlu5 EC50 (nM)c	% Glu Max (± SEM)b,c
55	2-fluorophenyl	6.79 ± 0.17	162	1.53 ± 0.12	NAMf	<5.0	>10000	29.1 ± 7.3
56	3-fluorophenyl	6.72 ± 0.08	192	1.44 ± 0.38	NAM	5.56 ± 0.24	2720	9.71 ± 5.78
57	2-chlorophenyl	6.85 ± 0.16	141	1.37 ± 0.72	NAM	6.00 ± 0.25	1010	8.78 ± 5.29
58	3-chlorophenyl	6.84 ± 0.10	145	2.30 ± 0.17	NAMe	5.73 ± 0.24	1850	19.2 ± 10.0
59	4-chlorophenyl	6.71 ± 0.13	197	2.17 ± 0.40	PAM	6.44 ± 0.03	364	51.6 ± 7.7
60	2-methylphenyl	6.46 ± 0.06	346	1.72 ± 0.16	NAMf	<5.0d	>10000d	35.8d
61	3-methylphenyl	6.67 ± 0.15	216	1.93 ± 0.56	NAMf	<5.0	>10000	36.6 ± 8.7
62	4-methylphenyl	6.57 ± 0.03	269	1.04 ± 0.45	NAMf	<5.0	>10000	54.8 ± 9.4
63	2-methoxyphenyl	6.47 ± 0.10	339	1.49 ± 0.60	NAM	6.52 ± 0.17	301	3.28 ± 0.45
64	3-methoxyphenyl	6.70 ± 0.06	198	1.02 ± 0.12	NAMf	<5.0	>10000	59.7 ± 6.1
65	4-methoxyphenyl	6.49 ± 0.10	320	1.23 ± 0.38	PAM	5.86 ± 0.05	1380	32.3 ± 5.2
66	2,3-difluorophenyl	6.73 ± 0.16	184	2.15 ± 0.33	NAMe	5.68 ± 0.20	2110	21.0 ± 10.6
67	2,5-difluorophenyl	6.91 ± 0.11	123	2.62 ± 0.47	NAMf	<5.0	>10000	18.8 ± 6.1
68	2,6-difluorophenyl	6.74 ± 0.08	181	1.49 ± 0.17	NAMe	5.91d	1230d	50.8d
69	pyridin-2-yl	6.04 ± 0.05	915	0.96 ± 0.63	NAM	5.45 ± 0.04	3520	3.71 ± 0.47
70	pyridin-3-yl	6.36 ± 0.13	436	0.64 ± 0.70	NAMf	<5.0	>10000	46.3 ± 2.9
71	pyridin-4-yl	6.05 ± 0.04	881	0.77 ± 0.44		<4.5	>30000
72	5-fluoropyridin-2-yl	6.46 ± 0.07	349	1.31 ± 0.11	NAM	5.01 ± 0.13	9740	7.0 ± 2.5

^aCalcium mobilization mGlu₃ assay; values are average of n ≥ 3.

^bAmplitude of response in the presence of 30 μM test compound as a percentage of maximal response (100 μM glutamate); average of n ≥ 3.

^cCalcium mobilization mGlu₅ assay; values are average of n ≥ 3.

^dAverage of n = 2.

^ePartial NAM; CRC plateaus above 10% glutamate maximum.

^fWeak activity; CRC does not plateau.

Although several of these new 5-fluoropyridin-2-yl ether analogues demonstrated improved mGlu₃ NAM potency, modest levels of mGlu₅ selectivity, and improved LLE values⁵³ (Table 5), their DMPK profiles remained a critical unanswered question. Thus, selected analogues were profiled in our aforementioned rat protein binding assay,⁵¹ and metabolic stability was also assessed in vitro by measuring the intrinsic clearance of the compound when incubated with rat liver microsomes (RLM).⁵⁴ While the fraction unbound in rat plasma was encouraging for most compounds, metabolic stability was uniformly poor. Unfortunately, on the basis of their intrinsic clearance in RLM, the compounds were predicted to exhibit hepatic clearance near blood flow.⁵⁵

Table 5.

In Vitro DMPK Profiling of Select 5-Fluoropyridin-2-yl Ether Analogues

no.	R	cLogPa	LLEb	mGlu3 IC50 (nM)	fold vs mGlu5	rat plasma Fuc	rat CLhep (mL/min/kg)d
55	2-fluorophenyl	3.06	3.73	162	>61	0.098	64.7
56	3-fluorophenyl	3.06	3.66	192	14	0.094	53.0
57	2-chlorophenyl	3.57	3.28	141	7.2	0.054	66.5
58	3-chlorophenyl	3.57	3.27	145	13	0.034	51.9
61	3-methylphenyl	3.23	3.44	216	17	0.051	58.4
66	2,3-difluorophenyl	3.16	3.57	184	11	0.092	63.6
67	2,5-difluorophenyl	3.16	3.75	123	>81	0.089	49.6
68	2,6-difluorophenyl	3.16	3.58	181	6.8	0.064	64.6

^aCalculated using Dotmatics Elemental (www.dotmatics.com/products/elemental/).

^bLLE (ligand-lipophilicity efficiency) = pIC₅₀ – cLogP.

^cF_u = fraction unbound.

^dPredicted hepatic clearance based on intrinsic clearance in rat liver microsomes.

Fortunately, testing of new 5-chloropyridin-2-yl ether analogues 80–101 ultimately provided another path forward for the design of both potent and highly selective mGlu₃ NAMs (Table 6). Monosubstituted phenyl analogues (80–93) generally exhibited similar activity at mGlu₃ (IC₅₀ = 200–600 nM) regardless of the position of the substituent on the ring. 4-Chlorophenyl analogue 84, 2-methoxyphenyl analogue 88, and 3-cyanophenyl analogue 92 were exceptions to this trend with each exhibiting reduced activity at mGlu₃. Although most of these monosubstituted phenyl analogues (80–93) demonstrated only modest selectivity versus mGlu₅, 4-methylphenyl analogue 87, 2-methoxyphenyl analogue 88, and 3-cyanophenyl analogue 92 exhibited weak activity at that receptor. Preparation of disubstituted phenyl analogues (94–99) yielded additional compounds with improved selectivity versus mGlu₅. 3,5-Difluorophenyl analogue 97 was a weak mGlu₅ PAM, and 2-cyano-5-fluorophenyl analogue 98 was inactive versus mGlu₅ up to the highest concentration tested (30 μM). Finally, encouraging results were observed with unsubstituted pyridyl analogues 100 and 101, where both proved inactive versus mGlu₅ while maintaining good mGlu₃ NAM activity.

Table 6.

mGlu₃ NAM and mGlu₅ SAR of 5-Chloropyridin-2-yl Ether Analogues 80–101

no.	R	mGlu3 pIC50 (± SEM)a	mGlu3 IC50 (nM)a	% Glu Max (± SEM)a,b	mGlu5 activityc	mGlu5 pEC50 (± SEM)c	mGlu5 EC50 (nM)c	% Glu Max (± SEM)b,c
80	2-fluorophenyl	6.65 ± 0.04	226	1.74 ± 0.10	PAM	5.49 ± 0.06	3240	57.3 ± 4.6
81	3-fluorophenyl	6.69 ± 0.04	206	1.38 ± 0.47	PAM	5.22 ± 0.05	5970	63.5 ± 0.8
82	2-chlorophenyl	6.48 ± 0.08	328	1.87 ± 0.29	PAM	5.26 ± 0.03	5450	51.0 ± 3.9
83	3-chlorophenyl	6.25 ± 0.07	558	1.63 ± 0.20	PAM	5.54 ± 0.02	2870	65.8 ± 8.8
84	4-chlorophenyl	5.91 ± 0.25	1230	0.94 ± 1.46	PAM	5.48 ± 0.15	3310	51.2 ± 10.3
85	2-methylphenyl	6.24 ± 0.18	571	1.95 ± 0.70	PAM	5.64 ± 0.03	2280	80.7 ± 0.5
86	3-methylphenyl	6.41 ± 0.05	392	1.71 ± 0.44	PAM	5.44 ± 0.11	3600	69.9 ± 6.5
87	4-methylphenyl	6.39 ± 0.21	410	1.39 ± 0.25	PAMe	<5.0	>10000	70.8 ± 3.1
88	2-methoxyphenyl	6.16 ± 0.15	690	1.29 ± 0.18	NAMe	<5.0	>10000	58.5 ± 1.2
89	3-methoxyphenyl	6.50 ± 0.05	313	2.00 ± 0.32	PAM	5.29 ± 0.08	5070	78.0 ± 1.8
90	4-methoxyphenyl	6.36 ± 0.11	439	2.07 ± 0.68	PAM	5.36 ± 0.06	4330	59.4 ± 7.3
91	2-cyanophenyl	6.37 ± 0.05	429	1.64 ± 0.04	PAM	5.30 ± 0.10	4970	38.1 ± 5.0
92	3-cyanophenyl	6.08 ± 0.26	825	1.75 ± 0.41	PAMe	<5.0	>10000	32.2 ± 3.7
93	4-cyanophenyl	6.27 ± 0.05	539	1.84 ± 0.19	PAM	5.48 ± 0.08	3320	23.1 ± 6.8
94	2,3-difluorophenyl	6.07 ± 0.23	852	0.33 ± 0.71	PAM	5.17 ± 0.29	6680	36.1 ± 8.4
95	2,4-difluorophenyl	6.55 ± 0.08	280	1.34 ± 0.25	PAM	5.59 ± 0.07	2580	70.8 ± 0.8
96	2,6-difluorophenyl	6.65 ± 0.04	225	2.06 ± 0.31	PAM	5.85 ± 0.09	1420	40.3 ± 5.6
97	3,5-difluorophenyl	6.38 ± 0.07	420	2.18 ± 0.19	PAMe	<5.0	>10000	33.1 ± 2.3
98	2-cyano-5-fluorophenyl	6.28 ± 0.06	529	1.55 ± 0.31		<4.5	>30000
99	3-cyano-5-fluorophenyl	6.50 ± 0.07	315	1.92 ± 0.12	PAM	5.98d	1044d	28.0d
100	pyridin-2-yl	6.48 ± 0.07	328	1.07 ± 0.04		<4.5	>30000
101	pyridin-3-yl	6.22 ± 0.10	608	1.73 ± 0.53		<4.5	>30000

^aCalcium mobilization mGlu₃ assay; values are average of n ≥ 3.

^bAmplitude of response in the presence of 30 μM test compound as a percentage of maximal response (100 μM glutamate); average of n ≥ 3.

^cCalcium mobilization mGlu₅ assay; values are average of n ≥ 3.

^dAverage of n = 2.

^eWeak activity; CRC does not plateau.

Encouraged by the selectivity profiles seen with pyridyl analogues 100 and 101 but suspecting that these unsubstituted compounds may be prone to rapid metabolism, we immediately prepared several substituted analogues of each (Table 7). Unfortunately, in the pyridin-2-yl set (102–105), the majority of these modifications enhanced mGlu₅ activity, albeit only slightly. Cyano analogue 103 was the lone exception; however, this compound was approximately 2-fold less potent than unsubstituted analogue 100. Results with the pyridin-3-yl set (106–110) were more encouraging as all new compounds except 6-fluoro analogue 110 maintained the excellent selectivity profile versus mGlu₅ observed with 101 without a loss of activity at mGlu₃. At this point, several compounds with good mGlu₃ NAM activity and devoid of mGlu₅ activity were in hand, which set the stage for further profiling in pursuit of compounds meriting extensive in vivo evaluation.

Table 7.

mGlu₃ NAM and mGlu₅ SAR of Additional 5-Chloropyridin-2-yl Ether Analogues 102–110

no.	series	R	mGlu3 pIC50 (± SEM)a	mGlu3 IC50 (nM)a	% Glu Max (± SEM)a,b	mGlu5 activityc	mGlu5 pEC50 (± SEM)c	mGlu5 EC50 (nM)c	% Glu Max (± SEM)b,c
102	I	3-F	6.44 ± 0.03	362	1.64 ± 0.36	PAMd	<5.0	>10000	32.4 ± 4.4
103	I	3-CN	6.17 ± 0.06	677	1.35 ± 0.55		<4.5	>30000
104	I	5-F	6.59 ± 0.09	260	2.44 ± 0.32	PAMd	<5.0	>10000	47.9 ± 5.1
105	I	6-F	6.53 ± 0.08	294	1.75 ± 0.19	NAMd	<5.0	>10000	24.8 ± 5.5
106	II	2-F	6.41 ± 0.05	392	1.71 ± 0.41		<4.5	>30000
107	II	4-F	6.32 ± 0.03	482	1.45 ± 0.22		<4.5	>30000
108	II	5-F	6.32 ± 0.03	481	1.85 ± 0.32		<4.5	>30000
109	II	5-CN	6.22 ± 0.01	605	1.08 ± 0.50		<4.5	>30000
110	II	6-F	6.39 ± 0.06	408	1.45 ± 0.29	PAMd	<5.0	>10000	30.6 ± 2.4

^aCalcium mobilization mGlu₃ assay; values are average of n ≥ 3.

^bAmplitude of response in the presence of 30 μM test compound as a percentage of maximal response (100 μM glutamate); average of n ≥ 3.

^cCalcium mobilization mGlu₅ assay; values are average of n ≥ 3.

^dWeak activity; CRC does not plateau.

As before, we moved several promising compounds into assays to assess protein binding⁵¹ in rat plasma as well as metabolic stability in RLM⁵⁴ (Table 8). The lone eastern phenyl analogue 99 was slightly more protein bound than its eastern pyridyl comparators, which was not surprising given its higher lipophilicity. A range of predicted hepatic clearance values based on the intrinsic clearance of the compound in RLM were observed including one analogue of less than one-third hepatic blood flow (109) and one analogue near hepatic blood flow (100). The remaining analogues were predicted to have moderate clearance in vivo. Somewhat surprisingly, unsubstituted pyridine-3-yl analogue 101 was more metabolically stable than its regioisomeric comparator 100. Still, substituted versions of 101 (106–109) did exhibit increased stability relative to 101.

Table 8.

In Vitro DMPK Profiling of Select 5-Chloropyridin-2-yl Ether Analogues

no.	R	cLogPa	LLEb	mGlu3 IC50 (nM)	fold vs mGlu5	rat plasma Fuc	rat CLhep (mL/min/kg)d
99	2-cyano-5-fluorophenyl	3.29	2.99	529	>56	0.045	47.7
100	pyridin-2-yl	2.57	3.91	328	>91	0.051	69.5
101	pyridin-3-yl	2.16	4.06	608	>49	0.118	42.0
106	2-fluoropyridin-3-yl	2.67	3.74	392	>76	0.083	36.9
107	4-fluoropyridin-3-yl	2.26	4.06	482	>62	0.085	36.0
108	5-fluoropyridin-3-yl	2.26	4.06	481	>62	0.092	26.6
109	5-cyanopyridin-3-yl	1.88	4.34	605	>49	0.078	22.8

^aCalculated using Dotmatics Elemental (www.dotmatics.com/products/elemental/).

^bLLE (ligand-lipophilicity efficiency) = pIC₅₀ – cLogP.

^cF_u = fraction unbound.

^dPredicted hepatic clearance based on intrinsic clearance in rat liver microsomes.

Several interesting analogues were next advanced into rat cassette PK studies using IV dosing⁵² to assess their metabolic stability in vivo (Table 9). As the most promising analogue with an eastern phenyl group, 99 was selected for these studies. Unsubstituted eastern pyridyl analogue 101 and several substituted comparators (106, 108, and 109) were also chosen based on the totality of data collected to that point. Distinguishing between analogues 108 and 107 was difficult; however, compound 108 was ultimately selected as it had a marginally better in vitro DMPK profile. 5-Cyanopyridin-3-yl analogue 109 was selected as it was predicted to have the lowest clearance, and 2-fluoropyridin-3-yl analogue 106 was chosen as it was the most potent analogue with a moderate predicted clearance. Compound 99 exhibited a lower clearance in vivo than expected and had a long half-life in excess of 3 h. The in vivo clearance for the pyridine-3-yl analogues was generally well predicted by the RLM experiments, with analogue 108 being the lone exception. Analogues 99, 106, and 109 were thus selected for further study in single time point (15 min) tissue distribution studies at a higher dose (10 mg/kg).⁵⁶ Intraperitoneal (IP) dosing was chosen for these studies as this route is convenient for use in our planned behavioral studies. The same three compounds were also examined in protein binding assays with rat brain homogenates.⁵¹ CNS penetration with each compound was excellent, with compound 106 exhibiting the highest plasma and brain levels. Both 99 and 106 exhibited unbound brain to unbound plasma ratios (K_p,uu) of one, indicating distribution equilibrium between the compartments and a low probability of the compounds being substrates for transporters.⁵⁷ Compound 109 had a K_p,uu value of 0.49, indicating possible efflux; however, additional experiments would be required to determine such conclusively. Compound 106 (VU0650786) was deemed the most attractive and targeted for extensive profiling.

Table 9.

In Vivo DMPK Profiling of Select 5-Chloropyridin-2-yl Ether Analogues

			rat IV PK resultsb			rat IP tissue distribution resultsc,d,e
no.	rat plasma Fua	rat brain Fua	t1/2 (min)	CLplasma (mL/min/kg)	VSS (L/kg)	plasma conc (μM)	brain conc (μM)	K p f	K p,uu g
99	0.045	0.032	194	13	2.2	1.68	2.41	1.4	1.0
101	0.118		21	50	1.2
106	0.083	0.052	42	37	1.6	9.49	15.85	1.7	1.0
108	0.092		30	50	1.9
109	0.078	0.033	59	22	1.4	4.52	5.28	1.2	0.49

^aF_u = fraction unbound.

^bn = 2; dose = 0.2 mg/kg; solution in 10% EtOH, 38–40% PEG 400, 50–52% DMSO (2 mg/mL).

^cn = 2; time point = 15 min; dose = 10 mg/kg.

^dFor 99 and 109, fine homogeneous suspension in 0.1% Tween 80 and 0.5% methyl cellulose in H₂O (4 mg/mL).

^eFor 106, fine homogeneous suspension in 10% EtOH and 90% PEG400 (4 mg/mL).

^fK_p = total brain to total plasma ratio.

^gK_p,uu = unbound brain (brain F_u × total brain) to unbound plasma (plasma F_u × total plasma) ratio

Profiling of Compound 106

Profiling of compound 106 began with determination of its full selectivity versus other members of the mGlu family. In addition to mGlu₅, selectivity versus fellow group II receptor subtype, mGlu₂, was critical to assess. We evaluated the selectivity of 106 versus rat mGlu₂ using full CRC analysis, and the compound was inactive up to the highest concentration tested (30 μM). Thus, compound 106 has been established to have no functional activity at either mGlu₂ or mGlu₅ up to a concentration that is more than 75-fold over the functional potency at mGlu₃. The effect of 10 μM 106 on the orthosteric agonist CRC was measured in fold-shift experiments to evaluate selectivity versus the other mGlus.^44,45 No significant effect was found, indicating the compound was inactive at those receptors as well. We further evaluated the nature of the interaction between 106 and mGlu₃ by examining the effects of increasing concentrations of 106 on the glutamate CRC in a progressive fold-shift experiment. If an antagonist acts via a noncompetitive mechanism, we anticipate increasing concentrations of antagonist would shift the glutamate curve to the right and decrease the maximal signal of glutamate. Figure 4 depicts the effects of multiple concentrations of 106 and known mGlu_2/3 NAM 111 (MNI-137)⁵⁸ on the glutamate CRC. As expected, both compounds exhibited the characteristic rightward shift and depressed glutamate maximum typically observed with NAMs, suggesting that 106 does not bind to the orthosteric glutamate binding site but instead acts via an allosteric mechanism.

Figure 4.

Progressive fold-shift of the glutamate CRC by mGlu₃ NAM 106 (A) and mGlu_2/3 NAM 111 (B); compound concentrations shown in M.

To evaluate the ancillary pharmacology of the compound, a commercially available radioligand binding assay panel of 68 clinically relevant GPCRs, ion channels, kinases, and transporters was employed,⁵⁹ and only a single significant response (5-HT_2B, 65% inhibition) was found at 10 μM 106.⁶⁰ Because 5-hydroxytryptamine receptor 2B (5-HT_2B) agonists are associated with cardiotoxicity,⁶¹ we followed this result up with a functional cell-based assay to assess potential agonist or antagonist activity of the compound at 5-HT_2B.⁶² Fortunately, testing up to 10 μM in this assay revealed no functional activity for 106 at 5-HT_2B. Potential for drug–drug interactions was negligible as assessed in a human liver microsomes (HLM) cocktail assay with probe substrates for four common P450s (Table 10).⁶³ Finally, permeability and potential for P-glycoprotein (P-gp) mediated efflux was assessed in Madin–Darby canine kidney (MDCK) cells transfected with the human MDR1 gene.⁶⁴ Not surprisingly, compound 106 was highly permeable with no evidence of efflux in this assay.

Table 10.

P450 Inhibition and Permeability Profile of Compound 106

P450 inhibitiona		permeabilityb
CYP1A2 IC50	>30 μM	A–B Papp	45.0 × 10–6 cm/s
CYP2C9 IC50	25.6 μM	B–A Papp	49.9 × 10–6 cm/s
CYP2D6 IC50	>30 μM	efflux ratio	1.1
CYP3A4 IC50	>30 μM

^aCocktail assay in HLM.

^bMDR1-MDCK cells.

Confident that 106 possessed a favorable profile with respect to its pharmacology, in vitro DMPK properties, and preliminary in vivo DMPK properties, we moved the compound into several definitive in vivo DMPK studies in rats and mice (Table 11).⁵⁶ Time course studies using IP dosing revealed nearly identical and rapidly reached C_max values in both rats and mice. A single time point (30 min) tissue distribution study in mice analogous to the one previously conducted in rats showed excellent CNS penetration in that species as well. Again, the K_p,uu value was near one as would be expected for a highly permeable compound devoid of efflux issues.⁵⁷ A definitive rat IV PK study (1.0 mg/kg) was conducted, and results were essentially identical to those collected with the prior cassette study (0.2 mg/kg). Finally, a rat oral PK study (3.0 mg/kg) was carried out to assess bioavailability, which proved quite good (60%). Taking into account the data summarized herein, it was estimated that the unbound C_max in the CNS following the 10 mg/kg IP studies in both rats and mice was at or beyond the measured functional mGlu₃ NAM activity. With that in mind, evaluation of compound 106 in rodent behavioral models known to be sensitive to mGlu_2/3 antagonists was initiated.

Table 11.

Rodent PK Profile of Compound 106

	Protein Binding (Fu)a		Rat IP PKb
	rat plasma	0.083	dose	10 mg/kg
	rat brain homogenates	0.052	plasma Cmax	4.57 ± 0.67 μM
	mouse plasma	0.163	plasma Tmax	12.3 ± 2.7 minutes
	mouse brain homogenates	0.035	plasma AUC0-∞	11.5 ± 0.4 μM·h

Rat IVc and POd PK		Mouse IP Tissue Distributione				Mouse IP PKh
t1/2	49 minutes	dose		10 mg/kg		dose	10 mg/kg
CLplasma	30 mL/min/kg	plasma concentration		2.59 ± 0.29 μM		plasma Cmax	4.48 ± 0.56 μM
VSS	1.4 L/kg	brain concentration		9.36 ± 0.61 μM		plasma Tmax	30 ± 15 minutes
F	60%	Kpf	Kp,uug	3.6	0.78	plasma AUC0-∞	7.26 ± 0.24 μM·h

^aF_u = fraction unbound.

^bn = 3; fine microsuspension in 0.1% Tween 80 and 0.5% methyl cellulose in H₂O (4 mg/mL).

^cn = 2; dose =1.0 mg/kg; solution in 10% EtOH, 50% PEG 400, 40% saline (1 mg/mL).

^dn = 2; dose = 3.0 mg/kg; fine microsuspension in 0.1% Tween 80 and 0.5% methyl cellulose in H₂O (0.3 mg/mL).

^en = 3; time point = 30 min post dose; fine microsuspension in 0.1% Tween 80 and 0.5% methyl cellulose in H₂O (1 mg/mL).

^fK_p = total brain to total plasma ratio.

^gK_p,uu = unbound brain (brain F_u × total brain) to unbound plasma (plasma F_u × total plasma) ratio.

^hn = 3 per time point; fine microsuspension in 0.1% Tween 80 and 0.5% methyl cellulose in H₂O (1 mg/mL).

Behavioral Pharmacology of Compound 106

It is well-known that naïve mice will bury foreign objects, such as glass marbles, in deep bedding. This behavior can be inhibited by pretreatment with low doses of certain benzodiazepines, such as diazepam,⁶⁵ as well as certain selective serotonin reuptake inhibitors (SSRIs), such as fluvoxamine.⁶⁶ Likewise, this behavior has proven sensitive to a variety of mGlu₅ NAM compounds from diverse chemotypes.^38,67,68 As such, the marble burying assay has often been used as a convenient method for assessing anxiolytic activity. It is worth noting that recent reports have argued that the assay reflects a repetitive and perseverative behavior such as OCD as opposed to novelty-induced anxiety.⁶⁹ For our purposes, the most important fact was that the mGlu_2/3 orthosteric antagonists 1 and 2 have both been previously shown to inhibit marble burying.^9,10 Thus, examination of the selective mGlu₃ NAM 106 in this assay was warranted to determine the contribution of mGlu₂ versus mGlu₃ to this effect (Figure 5). Gratifyingly, dose dependent efficacy was observed in this assay, with statistically significant effects at all three doses and essentially complete inhibition at the highest dose (56.6 mg/kg). The positive control for this assay was the well characterized mGlu₅ NAM 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]-pyridine (112).⁷⁰ On the basis of the available DMPK data, it is estimated that CNS unbound exposure of compound 106 at the 10 mg/kg dose in this study reached a peak of 564 nM or approximately 1.5-fold the functional IC₅₀.

Figure 5.

Inhibition of marble burying in mice by compound 106. n = 8–10 male CD-1 mice per treatment group; vehicle = 10% Tween 80 in H₂O; 15 min pretreatment with compound or vehicle; 30 min burying time; *, p < 0.05 vs vehicle control group. Compound 112 is the mGlu₅ NAM 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]-pyridine.

Having established its efficacy in an anxiolytic/OCD mouse model, profiling of compound 106 in a rat model of depression was considered valuable for illustrating the utility of the compound. The forced swim test (FST) measures immobility time in rats placed in a tank of water from which they cannot escape and is sensitive to many antidepressants including several SSRIs.⁷¹ Importantly, efficacy with the mGlu_2/3 orthosteric antagonists 1 and 2 has been demonstrated in this assay,⁸ making the examination of 106 compelling (Figure 6). In this case, significant effects in decreasing immobility time were observed at the highest dose (56.6 mg/kg). On the basis of the available DMPK data and an assumption of dose linearity, it is estimated that CNS unbound exposure of 106 at the high dose in these studies reached a peak of 2.3 μM or approximately 6-fold over the functional IC₅₀. The positive control in this assay was the N-methyl-d-aspartate (NMDA) receptor antagonist drug ketamine,⁷² which was introduced to the market over 50 years ago.⁷³ Ketamine has recently demonstrated rapid acting antidepressant efficacy in TRD patients.^74–76 Unfortunately, ketamine produces several undesirable side effects including psychotomimetic effects.⁷⁷ Preclinical studies implicate common downstream signaling pathways in the antidepressant effects of ketamine and mGlu_2/3 antagonists, including activation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor and the mammalian target of rapamycin (mTOR) pathway.^{18,19,78–82} Such studies have raised the possibility that mGlu_2/3 antagonists or selective antagonists of each individual group II mGlu might represent novel approaches to rapid acting antidepressants without the side effect profile of ketamine. A selective and CNS penetrant mGlu₃ NAM compound such as 106 will be a valuable tool in shedding light on such questions.

Figure 6.

Decrease in immobility in the FST in rats by compound 106. n = 8–10 male Sprague–Dawley rats per treatment group; 106 vehicle = 10% Tween 80 in H₂O; ketamine vehicle = saline; 30 min pretreatment with compound or vehicle; 6 min testing session; *, p < 0.05 vs vehicle control group.

CONCLUSION

A cross screening hit from a nonalkyne mGlu₅ PAM chemotype served as a successful launching point for the discovery of compound 106, a highly selective mGlu₃ NAM with DMPK properties that enable its convenient use in rodent models of psychiatric disorders. In addition to these features, the compound displays moderate clearance and good bioavailability in rats. Furthermore, the compound is highly permeable, not a substrate for P-gp mediated efflux, and possesses an attractive P450-inhibition profile. The compound has demonstrated efficacy in two rodent models previously shown to be sensitive to mGlu_2/3 inhibition. This highly selective mGlu₃ NAM can thus serve as a useful tool for elucidating the role of selective inhibition of mGlu₃ and its potential utility as a novel therapeutic target. Such studies will constitute the subject of future communications.

EXPERIMENTAL SECTION

Diethyl (R)-1-(1-((tert-Butoxycarbonyl)amino)propan-2-yl)-1H-pyrazole-3,5-dicarboxylate (74)

Diethyl 3,5-pyrazoledicarboxylate 73 (4.24 g, 20 mmol, 1.0 equiv) and tert-butyl (S)-(2-hydroxypropyl) carbamate 15 (7.01 g, 40 mmol, 2.0 equiv) were dissolved in THF (100 mL. 0.2 M), and triphenyl phosphine (9.44 g, 36 mmol, 1.8 equiv) was added. After 5 min, the mixture was cooled to 0 °C and di-tert-butyl azodicarboxylate (8.29 g, 36 mmol, 1.8 equiv) was added. The reaction mixture was then subjected to microwave irradiation for 25 min at 120 °C. The mixture was cooled to room temperature, and the solvent was removed in vacuo. Purification via flash chromatography on silica gel provided the title compound as a semisolid (8.2 g, yield was not determined due to contamination of D^tBAD byproduct, di-tert-butyl hydrazine-1,2-dicarboxylate). ¹H NMR (400 MHz, MeOD) δ 7.29 (s, 1H), 5.69–5.01 (m, 1H), 4.41–4.35 (m, 4H), 3.51–3.39 (m, 2H), 1.51 (d, J = 6.8 Hz, 3H), 1.41–1.38 (m, 15H). ¹³C NMR (100 MHz, CDCl₃) δ 161.7, 159.0, 155.8, 142.6, 134.2, 114.0, 79.4, 61.4, 61.1, 53.4, 45.1, 28.2 (3C), 18.5, 14.3, 14.2. LCMS (method A): R_T = 1.052 min, m/z = 314.2 [M + H]⁺. HRMS, calcd for C₁₇H₂₇N₃O₆ [M], 369.1900; found, 369.1899.

Ethyl (R)-7-Methyl-4-oxo-4,5,6,7-tetrahydropyrazolo[1,5-a]-pyrazine-2-carboxylate (75)

Compound 74 (8.2 g, 22.2 mmol, 1.0 equiv) was treated with a solution of 4 N HCl in 1,4-dioxane (78 mL). Deprotection of the tert-butyl carbamate protecting group was monitored by LCMS. Once deprotection was complete, the reaction mixture was carefully basified with saturated aqueous NaHCO₃ (verified by pH paper) and was allowed to stir at room temperature overnight. The mixture was diluted with dichloromethane, and the aqueous layer was extracted with dichloromethane (3×). The combined organic layers were washed with brine, dried over Na₂SO₄, and concentrated in vacuo to provide the title compound as a white solid (4.4 g, 89% yield over two steps), which was used without further purification. ¹H NMR (400 MHz, CDCl₃) δ 7.35 (s, 1H), 7.13 (bs, 1H), 4.68–4.62 (m, 1H), 4.41 (q, J = 7.1 Hz, 2H), 3.86 (ddd, J = 15.8, 10.2, 1.4 Hz, 1H), 3.51 (ddd, J = 9.4, 6.4, 3.0 Hz, 1H), 1.65 (d, J = 6.6 Hz, 3H), 1.39 (t, J = 7.1 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 161.7, 159.3, 143.8, 134.4, 110.7, 61.3, 53.2, 46.0, 17.4, 14.3. LCMS (method A): R_T = 0.546 min, m/z = 224.2 [M + H]. HRMS, calcd for C₁₀H₁₃N₃O₃ [M], 223.0957; found, 223.0957. [α]²⁵_D = −29.9° (c 0.500, CHCl₃).

Ethyl (R)-5-(4-Methoxybenzyl)-7-methyl-4-oxo-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine-2-carboxylate (76)

Compound 75 (2.23 g, 10 mmol, 1.0 equiv) was dissolved in DMF (50 mL, 0.2 M), cooled to 0 °C, and treated with 60% sodium hydride in mineral oil (480 mg, 12 mmol, 1.2 equiv) in five portions. The reaction mixture was stirred for 15 min, and 4-methoxybenzyl chloride (1.63 mL, 12 mmol, 1.2 equiv) was added. After 16 h, the reaction mixture was diluted with water and extracted with EtOAc (3×). The combined extracts were washed with water and brine, dried over Na₂SO₄, filtered, and concentrated in vacuo. The crude material was purified by flash chromatography on silica gel to provide the title compound (2.51 g, 73% yield) as a pale-yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 7.37 (s, 1H), 7.23 (d, J = 14.1, 2H), 6.87 (d, J = 14.1, 2H), 4.76 (d, J = 14.5, 1H), 4.59–4.50 (m, 2H), 4.44–4.36 (m, 2H), 3.79 (s, 3H), 3.69 (dd, J = 13.1, 4.6 Hz, 1H), 3.35 (dd, J = 13.1, 6.3 Hz, 1H), 1.47 (d, J = 6.6 Hz, 3H), 1.38 (t, J = 7.0 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 161.7, 159.5, 137.1, 143.9, 134.8, 129.9 (2C), 127.9, 114.3, 110.9 (2C), 61.2, 55.3, 52.9, 50.4, 48.8, 17.5, 14.3. LCMS (method A): R_T = 0.955 min, m/z = 344.2 [M + H]⁺. HRMS, calcd for C₁₈H₂₁N₃O₄ [M], 343.1532; found, 343.1533. [α]²⁵_D = −8.1° (c 0.157, CHCl₃).

(R)-2-(Hydroxymethyl)-5-(4-methoxybenzyl)-7-methyl-6,7-dihydropyrazolo[1,5-a]pyrazin-4(5H)-one (77)

Sodium borohydride (1.16 g, 30.6 mmol, 5.0 equiv) was added slowly to a solution of compound 76 (2.1 g, 6.11 mmol, 1.0 equiv) in THF (20 mL) and MeOH (5.0 mL) at 0 °C. The reaction was heated to 60 °C, and after 30 min at that temperature, the reaction mixture was diluted with water and extracted with dichloromethane. The aqueous layer was acidified with a 1 M aqueous HCl solution and extracted with dichloromethane (2×). The combined extracts were dried over Na₂SO₄ and concentrated in vacuo. Purification by flash chromatography on silica gel provided the title compound as a viscous oil (1.55 g, 84% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.23–7.21 (m, 2H), 6.87–6.84 (m, 3H), 4.72 (d, J = 14.5 Hz, 1H), 4.68 (s, 2H), 4.59 (d, J = 14.4 Hz, 1H), 4.43–4.35 (m, 1H), 3.78 (s, 3H), 3.60 (dd, J = 13.0, 4.6 Hz, 1H), 3.31 (dd, J = 13.0, 7.4 Hz, 1H), 1.42 (d, J = 6.5 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 159.4, 157.8, 152.6, 134.5, 129.8 (2C), 128.2, 114.3 (2C), 106.5, 58.7, 55.3, 52.0, 50.8, 48.7, 17.2. LCMS (method A): R_T = 0.680 min, m/z = 302.2 [M + H]⁺. HRMS, calcd for C₁₆H₁₉N₃O₃ [M], 301.1426; found, 301.1428. [α]²⁵_D = −5.3° (c 0.98, CHCl₃).

(R)-2-(((5-Chloropyridin-2-yl)oxy)methyl)-5-(4-methoxybenzyl)-7-methyl-6,7-dihydropyrazolo[1,5-a]pyrazin-4(5H)-one(78)

To a solution of compound 77 (1.5 g, 4.98 mmol, 1.0 equiv) in DMF (25 mL, 0.2 M) at 0 °C was added NaH (300 mg, 12.44 mmol, 2.5 equiv). The resulting mixture was stirred for 15 min, and 5-chloro-2-fluoropyridine (1.25 mL, 12.44 mmol, 2.5 equiv) was added. The mixture was stirred overnight and extracted with EtOAc (3×). The combined extracts were concentrated in vacuo. Purification by flash chromatography on silica gel afforded the title compound (1.72 g, 84% yield) as a viscous oil. ¹H NMR (400 MHz, CDCl₃) δ 8.13 (d, J = 2.6 Hz, 1H), 7.54 (dd, J = 8.8, 2.6 Hz, 1H), 7.25 (d, J = 8.5 Hz, 2H), 6.98 (s, 1H), 6.88 (d, J = 8.5 Hz, 2H), 6.75 (d, J = 8.8 Hz, 1H), 5.39 (s, 2H), 4.75 (d, J = 14.5 Hz, 1H), 4.63 (d, J = 14.5 Hz, 1H), 4.50–4.42 (m, 1H), 3.81 (s, 3H), 3.64 (dd, J = 13.0, 4.6 Hz, 1H), 3.35 (dd, J = 13.0, 7.4 Hz, 1H), 1.48 (d, J = 6.5 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 161.6, 159.4, 157.7, 148.9, 145.1, 138.6, 134.5, 129.8 (2C), 128.2, 124.4, 114.2 (2C), 112.3, 108.2, 61.6, 55.3, 52.1, 50.8, 48.8, 17.2. LCMS (method A): R_T = 1.080 min, m/z = 413.2 [M + H]⁺. HRMS, calcd for C₂₁H₂₁ClN₄O₃ [M], 412.1302; found, 412.1305. [α]²⁵_D = −10.3° (c 1.512, CHCl₃).

(R)-2-(((5-Chloropyridin-2-yl)oxy)methyl)-7-methyl-6,7-dihydropyrazolo[1,5-a]pyrazin-4(5H)-one (79)

Compound 78 (1.65 mg, 4.0 mmol, 1.0 equiv) was dissolved in MeCN (40 mL, 0.1 M). and a solution of ceric ammonium nitrate (6.57 g, 12 mmol, 4.0 equiv) in water (12 mL) was added. After 30 min at room temperature, solvents were removed in vacuo. Purification using flash chromatography on silica gel provided the title compound (764 mg, 65% yield) as a pale-yellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.26 (d, J = 2.7 Hz, 1H), 8.21 (s, 1H), 7.83 (dd, J = 8.8, 2.7 Hz, 1H), 6.92 (dd, J = 8.8, 0.5 Hz, 1H), 6.77 (s, 1H), 5.29 (s, 2H), 4.51–4.46 (m, 1H), 3.66 (ddd, J = 13.0, 8.7, 8.7 Hz, 1H), 3.34 (ddd, J = 13.1, 7.9, 2.2 Hz, 1H), 1.45 (d, J = 6.5 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 161.9, 158.7, 147.7, 145.3, 139.7, 135.1, 124.1, 112.9, 107.4, 61.7, 52.3, 45.6, 17.0. LCMS (method A): R_T = 0.804 min, m/z = 293.2 [M + H]⁺. HRMS, calcd for C₁₃H₁₃ClN₄O₂ [M], 292.0727; found, 292.0727. [α]²⁵_D = −38.3° (c 0.442, CHCl₃).

(R)-2-(((5-Chloropyridin-2-yl)oxy)methyl)-5-(2-fluoropyridin-3-yl)-7-methyl-6,7-dihydropyrazolo[1,5-a]pyrazin-4(5H)-one(106)

Copper(I) iodide (13.7 mg, 0.072 mmol, 2.1 equiv) was added to a suspension of compound 79 (10 mg, 0.035 mmol, 1.0 equiv), 3-bromo-2-fluoropyridine (7.40 μL, 0.072 mmol, 2.1 equiv), potassium carbonate (10 mg, 0.072 mmol, 2.1 equiv), and N,N′-dimethylethylenediamine (20.7 μL, 0.19 mmol, 5.5 equiv) in toluene (0.44 mL) in a sealed reaction vial. The reaction mixture was stirred at 120 °C. After 16 h, the mixture was diluted with EtOAc, filtered through a Celite pad which was rinsed with EtOAc (2×), and concentrated in vacuo. Purification using reserve phase HPLC method 1 with 39–71% CH₃CN in H₂O (0.1% TFA) over 4 min provided the title compound (7.2 mg, 53% yield) as a white powder. ¹H NMR (400 MHz, DMSO-d₆) δ 8.27 (dd, J = 2.7, 0.5 Hz, 1H), 8.23 (ddd, J = 4.8, 2.7, 2.7 Hz, 1H), 8.09 (ddd, J = 9.6, 7.7, 1.8 Hz, 1H), 7.84 (dd, J = 8.8, 2.7 Hz, 1H), 7.50 (ddd, J = 9.0, 4.9, 1.3 Hz, 1H) 6.95 (s, 1H), 6.92 (d, J = 0.5 Hz, 1H), 5.35 (s, 2H), 4.82–4.75 (m, 1H), 4.27 (dd, J = 12.8, 4.3 Hz, 1H), 3.98 (dd, J = 12.8, 7.2 Hz, 1H) 1.55 (d, J = 6.5 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 161.9, 158.2 (d, J_C,F = 239 Hz), 156.6, 148.4, 146.4 (d, J_C,F = 14 Hz), 145.3, 133.9 (d, J_C,F = 13 Hz), 139.8, 134.0, 124.4 (d, J_C,F = 28 Hz), 124.2, 123.2 (d, J_C,F = 4 Hz), 112.9, 108.7, 61.6, 54.0, 52.7, 17.1. LCMS (method A): R_T = 0.944 min, m/z = 388.2 [M + H]⁺. HRMS, calcd for C₁₈H₁₅ClFN₅O₂ [M], 387.0898; found, 387.0899. [α]²⁵_D = −23.6° (c 0.100, DMSO).

ABBREVIATIONS USED

Ac

acetate

AMPA

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

AUC

area under the curve

C

concentration

CL

clearance

CNS

central nervous system

CRC

concentration response curve

DIAD

di-iso-propyl azodicarboxylate

DMF

N,N-dimethylformamide

DMPK

drug metabolism and pharmacokinetics

DMSO

dimethyl sulfoxide

D^tBAD

di-tert-butyl azodicarboxylate

Et

ethyl

FST

forced swim test

F

bioavailability

F_u

fraction unbound

GPCR

G-protein-coupled receptors

HLM

human liver microsomes

IP

intraperitoneal

IV

intravenous

K_p

brain to plasma ratio

K_p,uu

unbound brain to unbound plasma ratio

LLE

ligand-lipophilicity efficiency

max

maximum

MDCK

Madin–Darby canine kidney

MDD

major depressive disorder

Me

methyl

mGlu

metabotropic glutamate receptor

Ms

methanesulfonyl

mTOR

mammalian target of rapamycin

NAM

negative allosteric modulator

NMDA

N-methyl-d-aspartate

OCD

obsessive-compulsive disorder

PAM

positive allosteric modulator

PEG

polyethylene glycol

Ph

phenyl

PK

pharmacokinetics

PO

oral

PS

polystyrene

RLM

rat liver microsomes

SAR

structure–activity relationships

THF

tetrahydrofuran

TRD

treatment-resistant depression

T

time

t_1/2

half-life

V_SS

volume of distribution at steady-state

5-HT_2B

5-hydroxytryptamine receptor 2B

7TM

seven transmembrane