Spiro-oxindole Piperidines and 3-(Azetidin-3-yl)-1H-benzimidazol-2-ones as mGlu₂ Receptor PAMs

Abstract

Starting from two weak mGlu₂ receptor positive allosteric modulator (PAM) HTS hits (4 and 5), a molecular hybridization strategy resulted in the identification of a novel spiro-oxindole piperidine series with improved activity and metabolic stability. Scaffold hopping around the spiro-oxindole core identified the 3-(azetidin-3-yl)-1H-benzimidazol-2-one as bioisoster. Medicinal chemistry optimization of these two novel chemotypes resulted in the identification of potent, selective, orally bioavailable, and brain penetrant mGluR₂ PAMs.

Metabotropic glutamate (mGlu) receptors represent a family of G protein-coupled receptors (GPCRs) that are activated by the excitatory neurotransmitter glutamate.^1,2 To date, eight mGlu receptor subtypes have been identified and classified into three groups based on sequence homology, pharmacologic profile, and preferential signal transduction pathway.³ The mGlu₂ and mGlu₃ receptors are the members of group II that couple negatively to adenylyl cyclase through Gi/Go proteins. Activation of the mGluR₂ results in reduced glutamate release and decreases excitability. mGluR₂ activation can be achieved with orthosteric agonists or can be modulated by positive allosteric modulation of the receptor. A positive allosteric modulator (PAM) can increase the affinity and/or efficacy of the endogenous neurotransmitter glutamate binding to a site topologically distinct from the orthosteric (glutamate) ligand binding sites. PAMs of mGluR₂ have emerged as promising novel therapeutic agents for the treatment of several central nervous system (CNS) disorders.⁴⁻⁶ The mGluR₂ is expressed on presynaptic glutamatergic nerve terminals where it functions as an autoreceptor for glutamate. Thus, a mGlu₂ PAM can normalize excessive glutamatergic neurotransmission, which may be of benefit in disorders such as epilepsy^7,8 and schizophrenia.^9,10

We have previously reported on a series of pyridones originated from a high throughput screening (HTS) campaign of the Addex Pharmaceuticals compound collection.¹¹

As previously described, starting from the HTS hit 1 (Figure 1), potency and druglike properties were significantly improved to deliver the clinical lead JNJ-40411813 (2).¹² Subsequent back up programs focused on novel chemical series identified by pharmacophore overlays and scaffold hopping using the pyridone core as template.^13,14 Additional leads, such as JNJ-42153605 (3),^15,16 with improved potencies and druglike related attributes were found. Later, we confirmed these series bind in the 7-transmembrane domain of the receptor,¹⁷ and the discovery of the first covalent PAM, an analogue of 3, helped confirm our overlay and binding mode hypotheses.¹⁸ Using modeling and mutagenesis, a mechanism was proposed for how PAMs initiate their functional effect.¹⁹

Figure 1.

Previously reported mGlu₂ PAM hit 1 and subsequent lead molecules 2 and 3.

As part of a continuing effort to identify and develop novel mGluR₂ PAMs from structurally different scaffolds, a second HTS campaign, now using the Janssen compound library, was conducted. Nine hit series with structurally distinct chemotypes were found. Hit triaging using mGlu₂ receptor PAM potency and in vitro ADME profiling prioritized two novel series, exemplified by hits 4 and 5 (Figure 2) as the most promising. We hypothesized that the phenylpiperidine motif of hit 4 was embedded into the structure of the spiro-oxindole piperidine hit 5, suggesting that hybrid analogues could be worth exploring. To validate the hypothesis, the hybrid compound 6 was synthesized. Pleasingly, compound 6 showed a 2-fold increase in potency with respect to hit 4 (6, EC₅₀ = 1.02 μM vs 4, EC₅₀ = 2.29 μM) and a significant increase in the E_MAX of 232% indicating a substantial increase in the glutamate response. Herein, we report on the synthesis, SAR exploration, and medicinal chemistry optimization of this novel series as well as on the initial pharmacokinetic evaluation of selected early leads.

Figure 2.

Structure and primary activity of hits 4 and 5 and hybrid molecule 6.

Results and Discussion

The initial exploration of compound 6 focused on two main aspects: study the effect of substituents on the indolone core ring and piridazinyl ring. Functional activity and microsomal stability data for spiro-oxindole piperidine 6 along with new derivatives 7–26 are summarized in Tables 1 and 2.

Table 1. Functional Activity and Metabolic Stability Data for Representative Spiro-oxindole Piperidine mGluR₂ PAMs 6–20.

compd	X	R1	R2	mGlu2 EC50 (nM)a (95% CI)	mGlu2EMAX (%)b ± SD	mGlu2 % effect @ 1 μMb ± SD	HLM (%)c	RLM (%)c
6	5-Br	H	Cl	1020 (822–1257)	232 ± 64	100 ± 17	8	25
7	H	H	Cl	n.d.d	217 ± 6	24 ± 13	45	95
8	7-Br	H	Cl	3090e	65 ± 4	9 ± 2	n.t.e	n.t.
9	6-Br	H	Cl	n.d.	183 ± 2	76 ± 7	n.t.	n.t.
10	4-Br	H	Cl	n.d.	108d	31 ± 5	n.t.	n.t.
11	5-Cl	H	Cl	1000 (904–1132)	187 ± 14	80 ± 8	8	76
12	5-F	H	Cl	n.d.	184 ± 17	31 ± 4	11	57
13	5-Cy	H	Cl	630 (317–1285)	169 ± 5	105 ± 22	45	100
14	5-Br	H	H	n.d.	141d	7 ± 3	n.t.	n.t.
15	5-Br	H	Me	n.d.	228d	36 ± 18	17	69
16	5-Br	H	CF3	400 (242–654)	53 ± 2	46 ± 6	4	20
17	5-Br	H	OEt	>10000	52 ± 11	5 ± 0	7	31
18	5-Br	NHEt	Cl	240 (110–535)	394d	263 ± 18	8	65
19	5-Br	OEt	Cl	210 (150–309)	380d	203 ± 42	11	58
20	5-Br	OEt	CF3	130 (96–164)	216 ± 35	160 ± 23	9	43

^aValues are the mean of at least two experiments and within confidence interval of >95%.

^bValues are the median of at least two experiments with standard deviation.

^cHLM and RLM data refer to % of compound metabolized after incubation with microsomes for 15 min at a 5 μM concentration.

^dn.d.: absolute EC₅₀ could not be calculated since no obvious upper plateau of the concentration–response curve was reached.

^eNot tested.

Table 2. Functional Activity and Metabolic Stability Data for Representative 3-(azetidin-3-yl)-1H-benzimidazol-2-one mGluR₂ PAMs 21–26^a.

d	X	R	Y	Z	mGlu2 EC50a (nM) (95% CI)	mGlu2EMAX (%)b ± SD	HLM (%)c	RLM (%)c
21	6-Br	NHEt	CH	N	230 (200–262)	241 ± 71	9	26
22	H	NHEt	CH	N	280 (229–345)	291 ± 43	0	23
23	H	NHEt	CH	CH	120 (97–160)	381 ± 80	27	88
24	H	OEt	CH	CH	270 (202–368)	259 ± 38	15	46
25	5-CF3	OEt	CH	CH	80 (57–113)	218 ± 32	4	12
26	5-CF3	OEt	N	CH	150 (130–189)	220 ± 16	BQLd	BQLd

^aValues are the mean of at least two experiments and within confidence interval of >95%.

^bValues are the median of at least two experiments with standard deviation.

^cHLM and RLM data refer to % of compound metabolized after incubation with microsomes for 15 min at a 5 μM concentration.

^dBelow quantification limit.

Removing the bromine atom (7) from the indolone core led to decrease in potency and worsened metabolism. Moving the bromine atom from C-5 to other positions of the aromatic ring was also detrimental for activity. Thus, compound 8 (7-Br) was 3-fold less potent showing a low E_MAX, and isomers 9 (6-Br) and 10 (4-Br) weakly active. The role of the bromine atom at C-5 was assessed by exploring alternative substituents. For instance, a chlorine substituent was equipotent (11, EC₅₀ = 1000 nM); however, it displayed higher metabolism in rat liver microsomes (11, RLM 76% vs 6, RLM 25%). The smaller and less lipophilic fluorine (12) was not tolerated leading to an inactive molecule. Nevertheless, the mGluR₂ PAM activity was recovered with the introduction of the more lipophilic cyclopropyl substituent (13) having comparable potency but lacking good stability in both HLM and RLM. Moreover, SAR on the pyridazine ring showed that removing the chlorine atom (14) resulted also in a decrease in activity. Next, several replacements for the chlorine group of the pyridazine such as methyl (15), trifluoromethyl (16), and ethoxy (17) were explored. These modifications turned out to be unfavorable leading to either low E_MAX (16, 53%) or loss of activity (15 and 17, % effect at 1 μM 36% for 15 and 5% for 17, EC₅₀ > 10 μM for 17). Introduction of a second substitution on the pyridazine ring was explored with compounds 18–19. Incorporation of an ethylamino (18) or ethoxy (19) adjacent to the chlorine atom led to a ∼4 and 5-fold increase in potency, respectively, compared to 6 but also with some decrease of the metabolic stability in RLM. In the continued effort to find alternatives for the chlorine group of the pyridazine, we went back to the CF₃ group, which resulted in compound 20 having an additional improvement in potency (EC₅₀ = 130 nM; E_MAX = 216%). Thus, compound 20 is ∼2-fold more potent than 19 and ∼7.5-fold more potent than the initial hit 6, keeping the in vitro metabolic profile in an acceptable range.

We next focused on finding replacements of the central spiro-oxindole core. To that end, a scaffold hopping exercise looking for nonspirocyclic bicyclic cores was conducted. We applied computational techniques based on 3D shape and electrostatic similarity. Such approaches are well suited to scaffold hopping as similarity is assessed using properties important for biological recognition and not the underlying atom connectivity. We have previously assessed these techniques in detail²⁰ and applied them to identify mGlu₂ PAM scaffolds.¹³ Here, we performed a similar approach aimed at replacing the central piperidine with alternative secondary amines, and we also searched databases of prefragmented compounds. The ROCS²¹ and EON software from Openeye Scientific²² were used, which assess shape and electrostatic similarity, respectively. Among the best computational hits was the benzimidazolone core (21) with an azetidine substituent, Figure 3. The shape and electrostatic similarity between the two were high, suggesting this as a strong candidate for follow-up chemistry.

Figure 3.

Comparison of the 3D conformation and electrostatic surfaces for spiro-oxindole piperidine (18) and a new hit containing a 1H-benzimidazol-2-one core (21) with azetidine substitution. The red surface represents the region of negative electrostatic charge and the blue surface is positive charge.

The 3-(azetidin-3-yl)-1H-benzimidazol-2-one core exemplified with compound 21, stood out as an optimal bioisosteric replacement of the spiro-oxindole piperidine. Pleasingly, the new subclass represented by 21 showed comparable mGlu₂ PAM activity and interestingly better metabolic stability when compared to its corresponding match pair 18. This encouraging result triggered a focused exploration in which the 3-(azetidin-3-yl)-1H-benzimidazol-2-one core was kept as template to explore SAR on both the left- and right-hand sides. The most relevant examples prepared along with their primary activity and metabolic stability data are summarized in Table 2. In contrast to the previous subclass, removal of the 6-bromine atom on the benzimidazolone scaffold (22) did not have any effect on the potency or the metabolic stability. Replacement of the pyridazine for pyridine (23) resulted in a ∼2.3-fold potency increase compared to 22, although the compound was found to be metabolically less stable in both HLM and RLM. Substitution of the aminoethyl group on 23 by an ethoxy moiety was unfavorable, and compound 24 showed decreased potency (24, EC₅₀ = 270 nM vs 23, EC₅₀ = 120 nM). Pleasingly, mGlu₂ PAM activity was significantly improved by the introduction of a trifluoromethyl group at the 5 position of the benzimidazolone scaffold (25), resulting in ∼3.4-fold increase in potency compared to the unsubstituted analogue 24 (25, EC₅₀ = 80 nM vs 24, EC₅₀ = 270 nM) and excellent metabolic stability in human and rat liver microsomes (HLM = 4% and RLM = 12%). Interestingly, the pyrimidine analogue 26 displayed comparable activity to 25, and no metabolites could be seen after incubation with HLM and RLM.

Based upon their overall in vitro profile (e.g., good balance between potency and metabolism) and with the aim to maximize the potential to identify suitable in vivo probes, compounds 19, 20, 21, 22, 24, and 25 were evaluated after oral dose in a PK study in rat and for their ability to cross the blood–brain barrier (BBB). Thus, plasma and brain levels measured 2 and 4 h after dosing at 10 or 30 mg/kg are shown in Table 3. With the exception of 19, compounds display relevant plasma exposures after 4 h administration, reflecting low to moderate clearance, in line with the in vitro rat metabolism previously shown. Moreover, the brain penetration was generally low for most of the compounds with K_p values below 0.4, with the exception of compound 20 (K_p = 1.4). To rule out potential interaction with the glycoprotein P (P-gp), the efflux ratio of compounds 24 and 25 was evaluated in LLC-PK1 cell lines transfected with MDR1 showing no indication for P-gp efflux with values of 1.18 and 1.24 × 10^–6 cm/s for both 24 and 25, respectively. Likewise, both compounds possessed moderate permeability (13 and 9 × 10^–6 cm s for 24 and 25, respectively). Interestingly, the K_p values followed a trend with log P, excluding 25, in which its higher lipophilicity may not account for its poor brain penetration. Thus, for example, increasing the lipophilicity by replacing the chlorine atom in the pyridazine ring (19) by a trifluoromethyl group (20) led to an increase in K_p that translated into higher brain exposures. Conversely, the reduction of lipophilicity in compound 21 by removing the bromine atom on the benzimidazolone ring led to compound 22, which displayed lower K_p value. In view of these data, compounds 20, 24, and 25 displayed the best combination of low plasma clearance and relevant brain concentrations maintained at least for 4 h.

Table 3. Brain and Plasma Kinetics in Rat after a Single Oral Dose^a.

	plasma level (ng/mL)		brain level (ng/g)		Kpb
compd	2 h	4 h	2 h	4 h	2 h	ALogP
19c	49	17	18	BQLd	0.37	2.94
20c	260	220	310	290	1.4	3.43
21c	254	416	18	18	0.07	2.64
22	1480	842	48	31	0.03	1.9
24	5770	5780	2050	2374	0.36	2.14
25	2660	5090	371	996	0.14	3.1

^aStudy in male Sprague–Dawley rat (n = 1) dosed at 30 mg/kg p.o. in suspension (20% HP-β-CD at pH 7).

^bK_p is partition coefficient between brain and plasma after 2 h.

^c10 mg/kg dose p.o.

^dBelow quantification limits.

In addition, all compounds displayed a high level of selectivity toward all mGlu receptor subtypes: mGlu₁ and mGlu_3–8 (see compound 20 as example in Supporting Information). Likewise, potential off-target interactions were explored in a limited CEREP panel containing 18 different targets and also in the DiscoverX kinase panel (see Supporting Information) showing overall clean selective profiles with the exception of A2B (IC₅₀ 0.79 μM) for compound 20.

Collectively, our data show that compounds 20, 24, and 25 are suitable tools derived from novel templates to further explore the therapeutic potential of mGlu₂ PAMs in biological models of CNS disorders. In vivo studies to confirm their in vitro activity are in progress and will be reported in due course.

Chemistry

The general synthetic schemes for the preparation of spiro-oxindole piperidines (6–20) and 3-(azetidin-3-yl)-1H-benzimidazol-2-ones (21–26) are outlined in Schemes 1 and 2, respectively. The precursor compounds 27a–e, 28a–d, 28f, and 31a–b were commercially available. For the preparation of compounds 6–20, the general procedure involves a nucleophilic aromatic substitution on a 3,6-dichoropyridazine heterocycle (28a–g) with the corresponding spirocyclic piperidine derivative (27a–g) using DIPEA or K₂CO₃ (Scheme 1) as base. The benzimidazole azetidines 21–26 were prepared following a similar reaction procedure, starting in this case from the corresponding benzimidazole derivative 31a–c and chloro-heterocycles 28e and 32a–c (Scheme 2).

Scheme 1. General Synthesis of Spiro-oxindole Piperidines 6–20.

Reagents and conditions: (a) DIPEA or K₂CO₃, solvent, 85–150 °C. Y: 2–78%, see Supporting Information for detailed protocol. (b) NaOEt, EtOH, 0 to 100 °C, 48 h. Y: 40%. (c) TFA, DCM, rt, 1 h. Y: 42%. (d) 28a, CuI, DMEDA, K₂CO₃, DMF, 120 °C, 12 h. (e) HCl, MeOH, rt, 1 h. Y: 5% (two steps).

Scheme 2. General Synthesis of 3-(Azetidin-3-yl)-1H-benzimidazol-2-ones 21–26.

Reagents and conditions: (a) DIPEA or K₂CO₃, solvent, 85–130 °C. Y: 15–85%, see Supporting Information for detailed protocol. (b) TFA, DCM, rt, 1 h. Y 32–39%; (c) K₂CO₃, MeOH, H₂O, 50 °C, 4 h. Y: 23%.

Conclusions

In summary, we have outlined the medicinal chemistry strategies employed in the optimization of micromolar HTS hits to the identification of two novel mGlu₂ PAM series. A hybrid design approach that combined the two HTS hits gave quick access to a novel series that, after systematic SAR studies including a scaffold hopping approach, delivered compounds with good in vitro potency, selectivity, and appropriate pharmacokinetic attributes to progress to in vivo PD studies.

Glossary

ABBREVIATIONS

mGlu₂

metabotropic glutamate 2

PAM

positive allosteric modulator

GPCR

G protein-coupled receptor

CNS

central nervous system

HTS

high throughput screening

SAR

structure–activity relationship

RLM

rat liver microsomes

HLM

human liver microsomes

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmedchemlett.9b00350.

• Assay protocols, experimental procedures, and analytical data for compounds 6–26, 27f–g, 28e, 28g, 29, 30, 31c, 32a, and 33–35 (PDF)

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.