Continued optimization of the M5 NAM ML375: Discovery of VU6008667, an M5 NAM with high CNS penetration and a desired short half-life in rat for addiction studies

Abstract

This letter describes the continued optimization of M5 NAM ML375 (VU0483253). While a valuable in vivo tool compound, ML375 has an excessively long elimination half-life in rat (t1/2 = 80 hours), which can be problematic in certain rodent addiction paradigms (e.g., reinstatement). Thus, we required an M5 NAM of comparable potency to ML375, but with a rat t1/2 of less than 4 hours. Steep SAR plagued this chemotype, and here we detail aniline replacements that offered some improvements over ML375, but failed to advance. Ultimately, incorporation of a single methyl group to the 9b-phenyl ring acted as a metabolic shunt, providing (S)-11 (VU6008667), an equipotent M5 NAM, with high CNS penetration, excellent selectivity versus M1–4 and the desired short half-life (t1/2 = 2.3 hours) in rat.

Development of VU6019650: A Potent, Highly Selective, and Systemically Active Orthosteric Antagonist of the M5 Muscarinic Acetylcholine Receptor for the Treatment of Opioid Use Disorder

“…High-Throughput Screen and Chemistry. To discover novel scaffolds for compound development, an additional high- 10,20,30,32,33 The IC 50 value was obtained from a 96-well FlexStation II microplate reader. 2a) were selected for follow-up studies.…”

Development of VU6019650: A Potent, Highly Selective, and Systemically Active Orthosteric Antagonist of the M5 Muscarinic Acetylcholine Receptor for the Treatment of Opioid Use Disorder

“…High-Throughput Screen and Chemistry. To discover novel scaffolds for compound development, an additional high- 10,20,30,32,33 The IC 50 value was obtained from a 96-well FlexStation II microplate reader. 2a) were selected for follow-up studies.…”

Discovery and Optimization of Potent and CNS Penetrant M5-Preferring Positive Allosteric Modulators Derived from a Novel, Chiral N-(Indanyl)piperidine Amide Scaffold

“…When the recent optimization campaign initiated, we had developed a rat M 5 cell line and were routinely assessing functional activity at both human and rat M 5 on an industry-standard 384-well-format Hamamatsu FDSS7000 screening system. 22 As a benchmark, we re-evaluated ML380 under these new optimal screening conditions and noted an ~5-fold rightward shift in M 5 PAM potency (hM 5 EC 50 = 1.1 μ M); thus for a robust in vivo tool, we also needed to increase M 5 PAM activity ~10-fold. As all of our M 5 NAMs displayed enantiospecific activity, 18,22,23 we were driven to identify locations within the ML380 scaffold where we could introduce a chiral center and hopefully engender a significant increase in functional potency that might also afford new avenues for productive, tractable SAR.…”

“…22 As a benchmark, we re-evaluated ML380 under these new optimal screening conditions and noted an ~5-fold rightward shift in M 5 PAM potency (hM 5 EC 50 = 1.1 μ M); thus for a robust in vivo tool, we also needed to increase M 5 PAM activity ~10-fold. As all of our M 5 NAMs displayed enantiospecific activity, 18,22,23 we were driven to identify locations within the ML380 scaffold where we could introduce a chiral center and hopefully engender a significant increase in functional potency that might also afford new avenues for productive, tractable SAR. As ML380 possessed a lipophilic CF 3 moiety in the 2 position and in close proximity to the benzylic position, 27 we envisioned the introduction of a cyclic constraint in the form of an indanyl ring system providing racemic 7 (Figure 2), which proved to be an equipotent M 5 PAM (hM 5 EC 50 = 1.06 μ M, pEC 50 = 5.98 ± 0.03, 89% ± 1% ACh Max).…”

“…Initially, optimization of ML380 (hM 5 EC 50 = 190 nM) used a calcium mobilization assay with a 96 well-format FlexStation II microplate reader (Molecular Devices, LLC). When the recent optimization campaign initiated, we had developed a rat M 5 cell line and were routinely assessing functional activity at both human and rat M 5 on an industry-standard 384-well-format Hamamatsu FDSS7000 screening system . As a benchmark, we re-evaluated ML380 under these new optimal screening conditions and noted an ∼5-fold rightward shift in M 5 PAM potency (hM 5 EC 50 = 1.1 μM); thus for a robust in vivo tool, we also needed to increase M 5 PAM activity ∼10-fold.…”

Targeting muscarinic receptors for the treatment of alcohol use disorders: Opportunities and hurdles for clinical development

“…Development of M 5 receptor selective compounds (orthosteric and allosteric) with pharmaceutically suitable ADME (absorption, distribution, metabolism and excretion) profiles has lagged. As discussed previously, several tool compounds VU6019650 (orthosteric) (Capstick et al, 2022; Garrison et al, 2022) and ML375/VU6008667 (allosteric) (Gentry et al, 2013; McGowan et al, 2017; Teal et al, 2023) have led to key insights into the promise of targeting the M 5 receptor to treat addiction in preclinical models (Berizzi et al, 2018; Gould et al, 2019; Gunter et al, 2018). Thus far, none have continued to clinical development.…”

Development of VU6019650: A Potent, Highly Selective, and Systemically Active Orthosteric Antagonist of the M5 Muscarinic Acetylcholine Receptor for the Treatment of Opioid Use Disorder

“…High-Throughput Screen and Chemistry. To discover novel scaffolds for compound development, an additional high- 10,20,30,32,33 The IC 50 value was obtained from a 96-well FlexStation II microplate reader. 2a) were selected for follow-up studies.…”

Discovery and Optimization of Potent and CNS Penetrant M5-Preferring Positive Allosteric Modulators Derived from a Novel, Chiral N-(Indanyl)piperidine Amide Scaffold

“…When the recent optimization campaign initiated, we had developed a rat M 5 cell line and were routinely assessing functional activity at both human and rat M 5 on an industry-standard 384-well-format Hamamatsu FDSS7000 screening system. 22 As a benchmark, we re-evaluated ML380 under these new optimal screening conditions and noted an ~5-fold rightward shift in M 5 PAM potency (hM 5 EC 50 = 1.1 μ M); thus for a robust in vivo tool, we also needed to increase M 5 PAM activity ~10-fold. As all of our M 5 NAMs displayed enantiospecific activity, 18,22,23 we were driven to identify locations within the ML380 scaffold where we could introduce a chiral center and hopefully engender a significant increase in functional potency that might also afford new avenues for productive, tractable SAR.…”

“…22 As a benchmark, we re-evaluated ML380 under these new optimal screening conditions and noted an ~5-fold rightward shift in M 5 PAM potency (hM 5 EC 50 = 1.1 μ M); thus for a robust in vivo tool, we also needed to increase M 5 PAM activity ~10-fold. As all of our M 5 NAMs displayed enantiospecific activity, 18,22,23 we were driven to identify locations within the ML380 scaffold where we could introduce a chiral center and hopefully engender a significant increase in functional potency that might also afford new avenues for productive, tractable SAR. As ML380 possessed a lipophilic CF 3 moiety in the 2 position and in close proximity to the benzylic position, 27 we envisioned the introduction of a cyclic constraint in the form of an indanyl ring system providing racemic 7 (Figure 2), which proved to be an equipotent M 5 PAM (hM 5 EC 50 = 1.06 μ M, pEC 50 = 5.98 ± 0.03, 89% ± 1% ACh Max).…”

“…Initially, optimization of ML380 (hM 5 EC 50 = 190 nM) used a calcium mobilization assay with a 96 well-format FlexStation II microplate reader (Molecular Devices, LLC). When the recent optimization campaign initiated, we had developed a rat M 5 cell line and were routinely assessing functional activity at both human and rat M 5 on an industry-standard 384-well-format Hamamatsu FDSS7000 screening system . As a benchmark, we re-evaluated ML380 under these new optimal screening conditions and noted an ∼5-fold rightward shift in M 5 PAM potency (hM 5 EC 50 = 1.1 μM); thus for a robust in vivo tool, we also needed to increase M 5 PAM activity ∼10-fold.…”

Targeting muscarinic receptors for the treatment of alcohol use disorders: Opportunities and hurdles for clinical development

“…Development of M 5 receptor selective compounds (orthosteric and allosteric) with pharmaceutically suitable ADME (absorption, distribution, metabolism and excretion) profiles has lagged. As discussed previously, several tool compounds VU6019650 (orthosteric) (Capstick et al, 2022; Garrison et al, 2022) and ML375/VU6008667 (allosteric) (Gentry et al, 2013; McGowan et al, 2017; Teal et al, 2023) have led to key insights into the promise of targeting the M 5 receptor to treat addiction in preclinical models (Berizzi et al, 2018; Gould et al, 2019; Gunter et al, 2018). Thus far, none have continued to clinical development.…”

Development of VU6019650: A Potent, Highly Selective, and Systemically Active Orthosteric Antagonist of the M5 Muscarinic Acetylcholine Receptor for the Treatment of Opioid Use Disorder

“…High-Throughput Screen and Chemistry. To discover novel scaffolds for compound development, an additional high- 10,20,30,32,33 The IC 50 value was obtained from a 96-well FlexStation II microplate reader. 2a) were selected for follow-up studies.…”

Discovery and Optimization of Potent and CNS Penetrant M5-Preferring Positive Allosteric Modulators Derived from a Novel, Chiral N-(Indanyl)piperidine Amide Scaffold

“…When the recent optimization campaign initiated, we had developed a rat M 5 cell line and were routinely assessing functional activity at both human and rat M 5 on an industry-standard 384-well-format Hamamatsu FDSS7000 screening system. 22 As a benchmark, we re-evaluated ML380 under these new optimal screening conditions and noted an ~5-fold rightward shift in M 5 PAM potency (hM 5 EC 50 = 1.1 μ M); thus for a robust in vivo tool, we also needed to increase M 5 PAM activity ~10-fold. As all of our M 5 NAMs displayed enantiospecific activity, 18,22,23 we were driven to identify locations within the ML380 scaffold where we could introduce a chiral center and hopefully engender a significant increase in functional potency that might also afford new avenues for productive, tractable SAR.…”

“…22 As a benchmark, we re-evaluated ML380 under these new optimal screening conditions and noted an ~5-fold rightward shift in M 5 PAM potency (hM 5 EC 50 = 1.1 μ M); thus for a robust in vivo tool, we also needed to increase M 5 PAM activity ~10-fold. As all of our M 5 NAMs displayed enantiospecific activity, 18,22,23 we were driven to identify locations within the ML380 scaffold where we could introduce a chiral center and hopefully engender a significant increase in functional potency that might also afford new avenues for productive, tractable SAR. As ML380 possessed a lipophilic CF 3 moiety in the 2 position and in close proximity to the benzylic position, 27 we envisioned the introduction of a cyclic constraint in the form of an indanyl ring system providing racemic 7 (Figure 2), which proved to be an equipotent M 5 PAM (hM 5 EC 50 = 1.06 μ M, pEC 50 = 5.98 ± 0.03, 89% ± 1% ACh Max).…”

“…Initially, optimization of ML380 (hM 5 EC 50 = 190 nM) used a calcium mobilization assay with a 96 well-format FlexStation II microplate reader (Molecular Devices, LLC). When the recent optimization campaign initiated, we had developed a rat M 5 cell line and were routinely assessing functional activity at both human and rat M 5 on an industry-standard 384-well-format Hamamatsu FDSS7000 screening system . As a benchmark, we re-evaluated ML380 under these new optimal screening conditions and noted an ∼5-fold rightward shift in M 5 PAM potency (hM 5 EC 50 = 1.1 μM); thus for a robust in vivo tool, we also needed to increase M 5 PAM activity ∼10-fold.…”

Targeting muscarinic receptors for the treatment of alcohol use disorders: Opportunities and hurdles for clinical development

“…Development of M 5 receptor selective compounds (orthosteric and allosteric) with pharmaceutically suitable ADME (absorption, distribution, metabolism and excretion) profiles has lagged. As discussed previously, several tool compounds VU6019650 (orthosteric) (Capstick et al, 2022; Garrison et al, 2022) and ML375/VU6008667 (allosteric) (Gentry et al, 2013; McGowan et al, 2017; Teal et al, 2023) have led to key insights into the promise of targeting the M 5 receptor to treat addiction in preclinical models (Berizzi et al, 2018; Gould et al, 2019; Gunter et al, 2018). Thus far, none have continued to clinical development.…”