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Title:
Synthesis and SAR of a series of mGlu7 NAMs based on an ethyl-8-methoxy-4-(4-phenylpiperazin-1-yl)quinoline carboxylate core

Paper sections:

<p>Recently, metabotropic glutamate receptor subtype 7 (mGlu7) has garnered great interest as a viable target for a broad range of CNS disorders (e.g., PTSD, schizophrenia, depression, epilepsy, ADHD) and neurodevelopmental disorders (e.g., Rett syndrome and autism).1–15 Compared to other mGlu receptors, mGlu7 is one of the least explored and there is limited validation of its therapeutic potential due to a lack of selective, small molecule probes.16,17 To study mGlu7 activation, the field has no selective mGlu7 positive allosteric modulators (PAMs), but must instead rely on mGlu7-preferring PAMs or pan-Group III PAMs in combination with selective NAMs to isolate mGlu7 activity.18 Negative allosteric modulators (NAMs) are more advanced, with essentially four distinct chemotypes (1/2, 3, 4, 5-7) of subtype selective ligands; however, first generation NAMs were electrophilic and suffered from poor disposition.19–24 Recently new CNS-penetrant mGlu7 NAMs, suitable as in vivo tool compounds (e.g. 6 and 7) have become available.25–27</p><p>Based on the pharmacological intricacies of allosteric modulators, we prefer to have multiple chemotypes to probe each mode of pharmacology. Here, we report on the discovery of a fundamentally new mGlu7 NAM chemotype, based on an ethyl8-methoxy-4-(4-phenylpiperazin-1-yl)quinolone carboxylate core, derived from a functional HTS campaign. While SAR was steep, mGlu7 potency could be improved, and key medicinal chemistry insights were gained with respect to non-obvious bioisosteres.</p><p>The structure of VU0226390 (8), based on an ethyl-8-methoxy-4-(4-phenylpiperazin-1-yl)quinolone carboxylate core, and the corresponding multi-dimensional optimization plan is depicted in Figure 2. VU0226390 (8) was an attractive hit for several reasons: 1) it was a novel chemotype with selective sub-micromolar potency (mGlu7 IC50 = 647 nM, 6% L-AP4 min, >30 μM vs. mGlu4 and mGlu8), an acceptable DMPK profile (fu (r,h) = 0.016, 0.018, CLhep = 60 mL/min/kg) and opportunities for a straightforward, modular synthetic plan. However, the ester moiety was a drawback as a potential metabolic liability, as was the inherent DMAP-like core structure of the piperazinyl quinoline. Thus, the optimization plan was to find replacements for these two concerning features, while also exploring alternatives for the distal 2-OMe phenyl ring and the 8-OMe quinolone core.</p><p>The synthetic route shown in Scheme 1, and variations thereof, was employed to access analogs 11, surveying multiple dimensions of the hit 8. Aniline 9 was heated with diethyl ethoxymethylenemalonate, in the absence of solvent to provide enamines, which were suspended in diphenyl ether and heated to 250 oC to promote cyclization to the corresponding quinolone in 31% yield. Standard conversion to the 4-chloroquinoline 10 proceeded under POCl3 conditions in 78% yield. SNAr with various aryl/heteroaryl piperazines afforded analogs 11 in yields ranging from 7–66%. Alternatively, N-Boc-piperazine could be employed in the SNAr step, such that after deprotection, e.g. 12, a wide range of aryl/heteroaryl moieties could be installed via Buchwald-Hartwig couplings to also access analogs 11 in good overall yields. The SAR for analogs 11 proved interesting.</p><p>The ethyl ester moiety of 8 emerged as a necessary pharmacophore for mGlu7 NAM activity, and proved stable under our cell-based assay conditions. Replacement with a methyl ester lost ~6-fold in activity (IC50 = 4 μM), but this was likely due to hydrolysis (the methyl ester partially hydrolyzed in cell buffer) to the corresponding acid, which was inactive (IC50>30 μM). Similarly, the primary carboxamide congener, as well as diverse secondary and tertiary amides all proved devoid of mGlu7 NAM activity. Small alkyl, cycloalkyl and ether replacements for the ethyl ester were uniformly inactive. Thus, we elected to hold the ethyl ester constant, optimize activity elsewhere, and then return to find a more suitable replacement.</p><p>We surveyed a diverse array of substituted aryl and heteroaryl moieties to replace the 2-OMe phenyl moiety in 8 (Table 1) in an attempt to improve mGlu7 NAM potency and/or disposition with analogs 11. SAR was steep, with substitution only tolerated in the 2-position on the distal phenyl ring, e.g., 3-OMe and 4-OMe phenyl congeners were inactive. Ethers in the 2-position larger than methyl (11a-c) lost activity, as did 2-Cl (11d), 2-Me (11e), 2-CN (11f) and 2-F (11h) substitutions. From this survey, only the 2-SMePh derivative (11g) displayed improved potency (IC50 = 510 nM) over 8, with a slight improvement in rat predicted hepatic clearance (CLhep = 51.3 mL/min/kg). However, the corresponding sulfoxide and sulfone of 11g proved devoid of mGlu7 NAM activity. While di-OMe substituted analogs, such as 11i and 11j, retained mGlu7 NAM potency, their disposition (as expected) was inferior to 8. Direct heterocyclic congeners of 8, such as the pyridine 11l or pyrazine 11m displayed modest potency. However, all 5-membered heterocyclic anlaogs (11n-p) proved inactive.</p><p>In parallel, we evaluated replacements for the quinolone core of 8 (Figure 3). Truncation to a monocyclic pyridine, 13a, resulted in a ~6.5-fold loss of potency, as did both regioisomeric thienopyridines 13b and 13c, highlighting the lack of bioisosterism with these substitutions. Replacement with a naphthalene (13d) proved inactive, as did replacement of the 8-OMe moiety with either a hydrogen or fluoride atom (13e). Here, only a 6,8-diOMe congener, 13f, proved equipotent to 8, but once again displayed a poor disposition profile. Thus, moving forward, we would maintain the 8-OMe quinolone core and the 2-OMe phenyl moiety of 8 as we surveyed the piperazine linker (Table 2).</p><p>SAR around the piperazine core of 8 (Table 2), with analogs 13, proved interesting. The addition of a racemic methyl group to either the 3-position (14a) or the 2-position (14b), led to an ~2-fold loss in mGlu7 NAM potency. Cleavage of the piperazine ring, as in 14c, lost ~6-fold, while deletion of the proximal piperazine nitrogen proved inactive. Incorporating unsaturation, as with 14e, regained potency (mGlu7 IC50 = 3.5 μM), while cyclic constraints, e.g, 14f and 14g, proved inactive. Classical piperazine bioisosteres, such as [3.3.0] system (14h) and the spiro-azetidine (14i), were not productive in this context. Surprisingly, a biphenyl monomer we had 'in house' proved intriguing and demonstrated that a simple phenyl ring (14j) could replace the piperazine linker with reasonable potency (IC50 = 2.9 μM). However, the DMPK profile of 14j was poor (fu (r, h) <0.001, CLhep (rat) = 66 mL/min/kg), leading the team to consider replacement of the phenyl ring with polar/basic heterocycles, e.g., analogs 15.</p><p>Analogs 15 were readily prepared according to Scheme 2. Utilizing commercial heteroaryl chloro-boronic acids 16, in a chemoselective Suzuki coupling with intermediate 10, provided derivatives 17 in yields ranging from 45–57%. A second Suzuki coupling with aryl boronic acids delivered final compounds 15 in good overall yields. As shown in Table 3, SAR within this sub-series was more robust than in the preceding saturated systems. Replacement of the phenyl linker with a 6-pyridyl congener, 15a, proved to be equipotent (IC50 = 2.8 μM), but the 5-pyridyl regioisomer, 15b, was a potent and efficacious mGlu7 NAM (IC50 = 0.60 μM, 6.5% L-AP4 min). A slight increase in potency (IC50 = 0.37 μM, 6.3% L-AP4 min and IC50 = 0.41 μM, 5.6% L-AP4 min) was noted with both the 2-OEtPh analog (15c) and the 2-SMePh derivative (15d), respectively. A pyrimidine congener lost (15f) activity (IC50 = 2.4 μM, 12.0% L-AP4 min) while a pyrazine maintained good mGlu7 NAM activity (IC50 = 0.54 μM, 5.1% L-AP4 min). The addition of a pendant methoxy moiety to the pyridine ring of 15b, affording 15h, led to a more potent mGlu7 NAM (IC50 = 0.35 μM, 3.6% L-AP4 min). Even 5-membered heterocycles, such as pyrrole 15j, was a competent surrogate for the phenyl linker. Finally, as with 13f, the incorporation of an additional methoxy group in the 6-position of the quinoline ring system, e.g., 18 and 19 (Figure 4), led to sub-micromolar mGlu7 NAMs.</p><p>While none of these new mGlu7 NAMs possessed in vitro or in vivo DMPK profiles (e.g, 15h, low fraction unbound: fu (rat, mouse) = 0.01, 0.01, BHB fu (rat, mouse) = 0.04, 0.04, high predicted hepatic clearance (CLhep (rat, mouse) = 65.2 and 66.1 mL.mkin/kg) and high clearance in vivo, CLp = 70 mL/min/kg) worthy of advancement as in vivo tools, they are unique in vitro pharmacological tools with selectivity against the other mGlu receptors. Upon closer inspection of analogs 15, these resemble classical terphenyl α-helical mimetic protein-protein inhibitor (PPI) chemotype 20 (Figure 5), with clear overlap of the prototypical i, i+¾ and i+7 moieties in 15h.28,29 As 15h is structurally distinct from previously reported mGlu7 NAMs, and due to the resemblance to α-helical mimetic protein-protein inhibitor (PPI) chemotypes, the exciting prospect exists that this family of NAMs has a unique mode of pharmacological inhibition of mGlu7.</p><p>In summary, we have reported on the multi-dimensional optimization of a fundamentally new mGlu7 NAM chemotype, based on an ethyl-8-methoxy-4-(4-phenylpiperazin-1-yl)quinolone carboxylate core; moreover, we found that aromatic heterocycles, such as pyridines, are an unlikely bioisostere for the central piperazine motif. SAR led to the discovery of a unique terphenyl chemotype, reminiscent of α-helical mimetic PPIs-, a first in the mGlu allosteric ligand world. Studies are underway to evaluate the mechanism of inhibition of mGlu7 by 15h in comparison to chemically distinct mGlu7 NAM 6. These more laborious studies are underway and will be reported in due course.</p>
A High-Throughput Screening (HTS) campaign identified a fundamentally new mGlu7 NAM chemotype, based on an ethyl-8-methoxy-4-(4-phenylpiperazin-1-yl)quinolone carboxylate core. The initial hit, VU0226390, was a potent mGlu7 NAM (IC50 = 647 nM, 6% L-AP4 min) with selectivity versus the other group III mGlu receptors (>30 \xce\xbcM vs. mGlu4 and mGlu8). A multi-dimensional optimization effort surveyed all regions of this new chemotype, and found very steep SAR, reminiscent of allosteric modulators, and unexpected piperazine mimetics (whereas classical bioisosteres failed). While mGlu7 NAM potency could be improved (IC50s ~ 350 nM), the necessity of the ethyl ester moiety and poor physiochemical and DMPK properties precluded optimization towards in vivo tool compounds or clinical candidates. Still, this hit-to-lead campaign afforded key medicinal chemistry insights and new opportunities.