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. 2026 Aug 21;17(9):2085–2094. doi: 10.1021/acsmedchemlett.6c00392

De Novo Design in an M1 Positive Allosteric Modulator Backup Program: The Discovery of VU6069863

Changho Han †,‡,*, Upendra Rathnayake †,‡, Alison R Gregro †,‡, Julie L Engers †,‡, Christopher C Presley †,‡, Nathan D Schley §, Analisa D Thompson Gray †,‡, Michael Bubser †,‡, Hyekyung P Cho †,‡, Alice L Rodriguez †,‡, Valerie M Kramlinger †,‡, Olivier Boutaud †,‡, Colleen M Niswender †,‡,∥,⊥,#, Carrie K Jones †,‡,⊥, Darren W Engers †,‡, Craig W Lindsley †,‡,§,*
PMCID: PMC13573255  PMID: 42741297

Abstract

Recently, we disclosed our M1 PAM that had advanced into Phase I clinical studies, VU0467319, as well as a deuterated back-up compound, VU6045422, to address a major metabolite produced in man and improve tox profile. Ultimately, this series failed to overcome its liabilities and was terminated. However, the team was challenged with developing chemically distinct backup candidates without performing a new HTS campaign. De novo design strategies based upon the other two M1 ago-PAMs that had previously entered clinical development, MK-7622 and TAK-071, led to the discovery of a fundamentally new chemotype based on a 3,4-dihydrobenzo­[f]­[1,4]­oxazepin-5-(2H)-one core, providing VU6069863. VU6069863 proved to be an M1 PAM (EC50 = 248 nM, 87% ACh Max) with minimal M1 agonism, (EC50 > 30 μM, 19% ACh Max) despite being derived from potent M1 ago-PAM chemotypes, with good CNS penetration (rat Kp = 0.59, Kp,uu = 0.13, MDCK-MDR1 ER = 1.8, Papp = 32.8 × 10–6 cm/s) and pro-cognitive efficacy in both contextual fear conditioning (minimally effective dose [MED] = 0.1 mg/kg PO) and novel object recognition (MED = 1 mg/kg PO) in rats.

Keywords: cognition, de novo design, human dose projection, muscarinic acetylcholine receptor subtype 1 (M1), positive allosteric modulator (PAM)

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The selective activation of muscarinic acetylcholine receptors (M1–5), and in particular M1, has been a long sought-after mechanism to address the cognitive deficits in Alzheimer’s disease, schizophrenia and other neuropsychiatric disorders. − Since the discovery that nonselective “M1 agonists” exhibited robust efficacy in improving cognition in Alzheimer’s patients in the 1980s, the focus was on muscarinic subtype selectivity to avoid activation of peripheral M2 and M3 receptors and the undesired SLUDGE reaction (salivation, lacrimation, urination, defecation, gastrointestinal distress and emesis). − Toward this goal, we have been very active in the field of M1 positive allosteric modulators (PAMs), where exquisite subtype selectivity for M1 is achieved by virtue of binding to an allosteric site and potentiating the agonist effects of endogenous acetylcholine. − Both preclinical in vivo tool M1 PAM compounds (with minimal M1 agonism) from our lab and the clinical compound VU0467319 (1) have demonstrated pro-cognitive efficacy and a lack of cholinergic and SLUDGE side effects in preclinical species (mouse, rat, dog, cynomolgus monkey) and in humans (Figure ). However, a major inactive circulating metabolite, VU0481424 (2), accounted for ∼50% of the total drug-related AUC upon increasing the dose of 1 in the Phase I single ascending dose (SAD), coupled with tox findings in 13-week rat and development was stopped. − The incorporation of deuterium into the core of 1, as with VU6045422 (3) to diminish the production of 2, was effective in vitro (e.g., microsome and hepatocyte incubations) as well as in vivo at low doses; however, upon dose escalation in rat and dog toxicology studies, the degree of oxidative metabolism of 3 was comparable to 1, and all efforts around this chemotype were terminated. In need of a chemically distinct M1 PAM backup, we recently disclosed VU6007496 (4), a potent M1 PAM with minimal agonism; yet, disconnect between in vitro and in vivo metabolite identification, and the production of a potent M1 ago-PAM via oxidative metabolism that engendered cholinergic adverse events (AEs), led to the termination of this scaffold. In this paper, we will detail our efforts for the de novo design of a unique M1 PAM chemotype based on a 3,4-dihydrobenzo­[f]­[1,4]­oxazepin-5-(2H)-one core that displayed minimal M1 agonism and robust efficacy in rodent cognition models.

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Structures of the clinical M1 PAM VU0467319 1, and the corresponding oxidative metabolite 2, that accounted for ∼50% of the total plasma AUC in man. A penta-deutero congener 3 (VU6045422) was developed to address the undesired metabolism, which was effective in vitro and at low doses in vivo, but, upon dose escalation in rats and dogs was indistinguishable from 1 in terms of metabolite production. A distinct M1 PAM chemotype VU6007496 (4) was plagued with in vitro:in vivo metabolism disconnects and the production of an M1 ago-PAM metabolite affording cholinergic AEs.

Having exhausted our internal M1 PAM high-throughput screening hits, we turned to scaffold-hopping of the known M1 ago-PAMs that had advanced into the clinic. For this exercise, we employed Merck’s MK-7622 (5) and Takeda’s TAK-071 (6) as a basis for ligand design; however, this required dialing out the potent M1 agonist activity in 5 and 6 to generate an advanceable chemotype (Figure ). It was known that the exocyclic cyclohexyl alcohol moiety of 5 and the exocyclic pyranyl alcohol moiety of 6 were significant contributors to the undesired M1 agonist activity; therefore, we deleted these in our design. − , However, we thought the design should be cautious because previously reported SAR trends in the BQCA scaffold (7) suggested that the exocyclic cyclohexyl alcohol moiety has a significant role in activity upon comparing 8 and 9.

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De novo design of a novel 3,4-dihydrobenzo­[f]­[1,4]­oxazepin-5-(2H)-one based M1 PAM (11) with minimal agonism, derived from the potent M1 ago-PAMs 5 and 6 that had advanced into clinical development, and SAR trend within the BQCA scaffold 6–8. CCDC#: 2477638.

Ago-PAM 5 possesses an additional hydrogen bond acceptor in the pyrimidine ring that is absent in ago-PAM 6; moreover, escaping “flat-land” and increasing sp3 character as with 6 were attractive. Thus, we incorporated an oxygen atom as a hydrogen bond acceptor into 6, and expanded the γ-lactam of 6 into a 7-membered ring to afford a novel 3,4-dihydrobenzo­[f]­[1,4]­oxazepin-5-(2H)-one core 9. Optimization of the southern “tail” group led to the identification of 4-oxazole phenyl heterobiaryl moiety as a preferred group, and the discovery of VU6069863 (11), a low molecular weight M1 PAM with minimal M1 agonism, derived, surprisingly, from potent M1 ago-PAMs. As can be seen in the X-ray crystal structure (Figure ), the carbonyl of the 7-membered ring was distorted and successfully replaced the hydroxy moiety of exocyclic cyclohexanol. To our best knowledge, VU6069863 (11) is the first example showing the importance of the position and angle of the hydrogen bond acceptor, considering that several compounds synthesized for comparison were inactive or very weakly active in which the carbonyl was coplanar with the core.

A convergent synthetic approach was developed for the gram quantity production of M1 PAM 11 (Scheme ) to enable full in vitro and in vivo characterization. Esterification of commercial benzoic acid 12 provides 13 in 93% yield. Bromination proceeded with excellent regio-chemistry to deliver 14 in 98% yield. A Mitsunobu reaction with N-Boc aminoethanol installs the tether for the 7-membered ring 15, followed by removal of the Boc group to give 16, and base-promoted lactam formation provides the 3,4-dihydrobenzo­[f]­[1,4]­oxazepin-5-(2H)-one core 17 in 76% yield for the three steps. A palladium catalyzed borylation of 17 with bis­(pinacolato)­diboron generates Suzuki coupling partner 18 in 88% yield. In parallel, the southern “tail” was prepared from commercial acetyl bromide 19. Standard oxazole formation proceeded smoothly giving 20 in 32% yield, followed by a palladium-catalyzed carbonylation to provide ester 21 in 88% yield. Reduction to the benzyl alcohol with LAH proceeded in high yield, and subsequent chlorination afforded the other Suzuki coupling partner 23. Finally, a Suzuki coupling between 18 and 23 delivered VU6069863 (11) in 82% yield. Overall, the convergent preparation of 11 required 11 steps (7 steps longest linear sequence) in 8.7% overall yield on a 10-g scale.

1. Synthesis of VU6069863 (11)­ .

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a Reagents and conditions: (a) EtOH, H2SO4, 100 °C, 16 h, 93%; (b) Br2, CHCl3, 0 °C, 98%; (c) HOCH2CH2NHBoc, PPh3, DIAD, THF, rt; (d) 4 N HCl in dioxane, rt; (e) K2CO3, EtOH, 80 °C, 18 h, 76% over three steps; (f) bis­(pinacolato)­diboron, KOAc, Pd­(dppf)­Cl2, 1,4-dioxane, 90 °C, 88%; (g) 23, Cs2CO3, Pd­(dppf)­Cl2, 1,4-dioxane/H2O, 100 °C, 80%; (h) HCO2NH2, HCOOH, 115 °C, 2.5 h, 32%; (i) CO (g), Et3N, Pd­(OAc)2, dppp, EtOH/DMSO, 75 °C, 88%; (j) 2 M LiAlH4, THF, 0 °C, 83%; (k) SOCl2, DCM, rt, 97%.

As the basis for the design was known for M1 ago-PAMs, , we hypothesized that our design strategy would mitigate and/or minimize the degree of M1 agonism in 11 to avoid overstimulation of the M1 receptor and resultant cholinergic AEs. Gratifyingly, 11 was found to be a moderately potent M1 PAM (human EC50 = 226 nM, 80% ACh max) with minimal M1 agonism (human EC50 > 30,000 nM, 19% ACh max, an ∼121-fold margin), and thus possessed the desired profile for advancement (Table ). At rat M1, a similar pharmacological profile was noted, with compound activity observed as a potent M1 PAM (rat EC50 = 140 nM, 78% ACh max) with minimal M1 agonism (rat EC50 > 30,000 nM, 19% ACh max, an ∼210-fold margin). Relative to human M1, 11 was an ∼3-fold weaker PAM at both mouse (EC50 = 945 nM, 81% ACh max) and dog (EC50 = 895 nM, 62% ACh max) receptors, but also with minimal M1 agonism (EC50 > 30,000 nM, 20% ACh max). Additionally, PAM 11 was highly selective for M1 (inactive on human and rat M2–5) as well as dog M2 and M4 (dog M3 and M5 not tested). In terms of in vitro DMPK, PAM 11 displayed low predicted hepatic clearance in human (CLhep = 4.8 mL/min/kg), moderate in mouse, dog and cyno (CLhep = 39.4, 22.4, and 28.9 mL/min/kg, respectively) and high in rat (CLhep = 57.2 mL/min/kg) microsomes. A favorable profile was also noted in terms of unbound fraction in plasma (f u (m, r, d, c, h): 0.039, 0.066, 0.032, 0.013 and 0.039) and in rat brain homogenate binding (f u = 0.014), as well as with regards to CYP450 inhibition (IC50 > 30 μM for 3A4, 2D6, 1A2 and 22.2 μM for 2C9). There was weak time-dependent inhibition of CYP3A and CYP2C8 along with weak induction of CYP3A4; however, these weak signals were not showstoppers for PAM 11. Moreover, PAM 11 was predicted to be brain penetrant in human (MDCK-MDR1 ER = 1.8, Papp = 32.8 × 10–6 cm/s). A key issue to improve upon M1 PAMs 1 and 2 was solubility. In our kinetic solubility assay, PAM 11 showed high solubility at both neutral pH (79.7 μM at pH = 6.8) and acidic pH (77.9 μM at pH = 2.2), providing a significant improvement over 1 and 2. Furthermore, PAM 11 displayed good solubility in FaSSIF (12.4 μg/mL) and FaSSGF (16.1 μg/mL) in addition to a sharp, low melting point (180–181.8 °C), suggesting good developability.

1. Pharmacology and in Vitro DMPK Profile of M1 PAM 11 .

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We performed electrophysiology studies in mouse native tissue layer V medial prefrontal cortex (mPFC) to ensure that 11 did not induce long-term depression (LTD) akin to the M1 ago-PAMs 5 and 6. ,, PAM 11 induced no significant changes in field excitatory postsynaptic potentials (fEPSPs) evoked by electrical stimulation in layer II/III at 10 μM concentration (see Supporting Information), ∼11× above the functional mouse EC50, and a projected 40× for human. Thus, in agreement with the minimal agonism in M1 cell lines, PAM 11 maintained activity dependence on PFC regulation.

To confirm a lack of central cholinergic AEs, PAM 11 was dosed by intraperitoneal (I.P.) injection at 100 mg/kg in mice to assess seizure liability using the Racine scale in comparison with a 100 mg/kg I.P. dose of the positive control, the potent M1 ago-PAM BQCA (7) (Figure ). Across the 6-h time course of the study, no seizure activity was induced by PAM 11 (achieving total brain concentrations of 113 μM, or 120-fold the mouse M1 EC50 at 60 min). In contrast BQCA, at the same dose, rapidly induced seizure activity that peaked at Racine scale scores between 3 to 4 for the duration of the study. Combined with the lack of effect on LTD, these data encouraged further progression of PAM 11.

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Seizure liability of M1 PAM 11 compared to the M1 ago-PAM BQCA in male C57BL/6 mice. *p < 0.05; ***p < 0.001 vs. Vehicle (two-way ANOVA followed by Šídák’s multiple comparisons test). Vehicle for M1 PAM 11: aqueous 10% Tween 80; Vehicle for BQCA: aqueous 20% hydroxy-propyl-β-cyclodextrin.

In our standard rat plasma:brain level (PBL) cassette, PAM 11 was CNS penetrant, with a Kp of 0.59 and a Kp,uu of 0.13 (at a 15 min time point), and in agreement with the MDCK-MDR1 efflux data. In vivo IV/PO PK in rat was favorable (CLp = 9.1 mL/min/kg, t 1/2= 0.92 h, Vss = 0.61 L/kg, %F = 100) and a clear IVIVC disconnect with the high rat predicted hepatic clearance. Dog in vivo pharmacokinetics was also better than predicted (CLp = 11.5 mL/min/kg, t 1/2= 1.4 h, Vss = 0.97 L/kg, %F = 100), with high oral bioavailability in both of our presumed toxicology species. In a cyno IV PK cassette study (0.2 mg/kg), a similar favorable profile was obtained (CLp = 13.5 mL/min/kg, t 1/2= 0.67 h, Vss = 0.63 L/kg). Prior to advancing PAM 11 into rat cognition models, we evaluated the broader ancillary pharmacology in a CEREP 44 safety screen radioligand displacement at 10 μM PAM 11). For 42/44 targets, there was no displacement >50%@10 μM; however, in the single point screen, 11 displaced both the 5-HT2A (82.2%) and 5-HT2B (95.7%) agonist radioligands. In follow-up 10-point CRC assays, both proved to be >10 μM, and thus was clear to advance. Transitioning to cardiovascular safety, PAM 11 was clean on hERG (manual patch clamp IC50 > 30 μM) and in the broader CV EP panel (all >10 μM, except KV1.3 IC50 = 6.2 μM). Finally, PAM 11 was negative in a mini-AMES assay (4-strain, ±S9).

Our M1 PAM program had historically been driven by a rat novel object recognition (NOR) assay (Figure ), a preclinical model of prefrontal cortical memory functions. We therefore started our pharmacodynamic characterization of PAM 11 in the NOR; and observed a dose-dependent enhancement in recognition index, consistent with earlier M1 PAMs evaluated in this assay. To assess if PAM 11 also facilitates hippocampal memory processes, we next examined its effects on the acquisition of contextual fear conditioning (CFC) in rats. Compared to NOR, the dose–response in CFC was leftward-shifted with efficacy observed at lower doses of PAM 11 (Figure ). The MED in CFC was 0.1 mg/kg PO, which corresponded to 90 nM total brain levels or 0.62-fold the rat EC50. This PK/PD relationship is consistent with previously observed effects in the history of our M1 PAM program, where total rather than free brain compound exposure drives PD. ,,

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Effects of M1 PAM 11 in the Novel Object Recognition (NOR) test in rats. PAM 11 dose-dependently increased the Recognition Index in male Sprague–Dawley rats. Pretreatment by oral gavage with PAM 11 (0.3, 0.56, 1, and 1.8 mg/kg in aqueous 10% Tween 80 vehicle) 60 min prior to exposure to two identical objects significantly enhanced recognition memory assessed 24 h later. *p ≤ 0.05 vs. Vehicle (one-way ANOVA followed by Dunnett’s posthoc test).

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Effects of M1 PAM 11 on the acquisition of Contextual Fear Conditioning (CFC) in rats. On training day, pretreatment by oral gavage with the PAM 11 (dose range: 0.03, 0.1, 0.3, 1, and 3 mg/kg in aqueous 10% Tween 80 vehicle) 60 min prior to exposure to foot shock stimulus in rats, dose-dependently enhanced the precent of context-dependent freezing behavior 24 h later. N = 9–10/group of male Sprague–Dawley rats. **p < 0.01; ***p < 0.001 vs. Vehicle (one-way ANOVA followed by Dunnett’s posthoc test).

As discussed previously, this is likely due to the fact that we arbitrarily determine the PAM EC50 potency employing an EC20 concentration of ACh. However, in healthy young rats, cortical and hippocampal cholinergic tone may be higher, which would be predicted to increase PAM functional potency. ,, Consistently across dozens of M1 PAM chemotypes, when total brain exposure approaches or meets the M1 PAM EC50, significant pharmacodynamic effects in rodent cognition models were observed. With no observed cholinergic AEs and robust efficacy in both NOR and CFC, PAM 11 was further profiled as a putative backup compound for VU0467319 (1).

To select the second toxicology species and to ensure there were no unique human metabolites, multispecies (rat, dog, monkey, human) hepatocyte incubations were conducted (Figure ). Across all species, PAM 11 was the major species, with more extensive metabolism in rat and monkey. However, there was coverage across species (e.g., all metabolites were detected in all species), no unique human metabolites were detected, and dog was suitable as the second toxicology species. From our work with PAM 4, we knew there was potential to form active metabolites as either M1 PAMs or undesired ago-PAMs. ,, Thus, the team elected to synthesize (see Supporting Information for full synthetic details) the most abundant metabolites identified: the two regioisomeric benzyl alcohols M364a (24) and M364b (25), the ring-opened oxepinone M380a (26), and the α-hydroxy acid M355 (27) resulting from metabolism of the oxazole. In general (Table ), the putative metabolites were weak to inactive M1 PAMs with minimal to no detectable M1 agonism. Interestingly, regioisomeric benzyl alcohol M364a (24) was more potent than M364b (25), and was reasonably active at rat M1 (EC50 = 248 nM, 84%), but weak at human M1 (EC50 = 1,000 nM, 66%). Fortunately, all four metabolites showed poor brain penetration in rat (Kps ∼ 0.05, Kp,uus ∼ 0.06). Unexpectedly, both 24 (CLp = 9.1 mL/min/kg, t 1/2 = 6.1 h, Vss = 2.3 L/kg) and 25 (CLp = 7.9 mL/min/kg, t 1/2 = 7.4 h, Vss = 2.3 L/kg) had longer t 1/2 in vivo than 11, which led to the concern of peripheral metabolite accumulation.

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Metabolism of PAM 11 in rat, dog, monkey and human hepatocytes after a 4 h incubation period.

2. Pharmacology and DMPK Profile of Metabolites 24–27 of PAM 11 .

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With concerns of the potential accumulation of circulating peripheral metabolites notwithstanding, we proceeded with human PK and dose projections to decide whether PAM 11 could advance into IND-enabling studies. Human clearance was estimated using the 3 methods with the highest predictive accuracy as described in the Lombardo assessment (methods C7, C6a, and C3) as well as the rat single species scaling described by Hossea. Human Vss was estimated using the eight methods with the highest predictive accuracy as described in the Lombardo assessment (methods V16, V16a, V7, V9, V10, V2, V3, and V6). For PAM 11, our target C min,tot,hum was 451 nM, the predicted human clearance was 3.12 mL/min/kg, Vss was 0.71 L/kg, and human oral F was estimated at 72%. While this was reasonable, the human dose projection was high, with estimates between 169 mg BID based on rat clearance to >7,000 mg QD based on cyno and dog clearance. With the literature replete with idiosyncratic toxicology findings for drugs dosed at or above 100 mg QD in man coupled with the lengthy synthesis and projected cost of goods, PAM 11 could not advance further.

The necessity for a chemical optimization backup program for VU0467319 led our team to scaffold-hop known M1 ago-PAMs and de novo design a 3,4-dihydrobenzo­[f]­[1,4]­oxazepin-5-(2H)-one-derived M1 PAM (VU6069863, 11) with minimal M1 agonism. PAM 11 exhibited an attractive pharmacological, physiochemical, and DMPK profile that resulted in robust efficacy in rat NOR (MED = 1 mg/kg) and CFC (MED = 0.3 mg/kg) without cholinergic AEs. The potential for accumulating metabolites and high human dose projections prevented PAM 11 from further development. However, PAM 11 represents another exceptional M1 PAM in vivo tool compound, devoid of cholinergic AEs and with a robust PK/PD relationship. Indeed, PAM 11 has become our standard positive control for the CFC assays. Further efforts to find a backup for VU0467319 are underway and will be reported in due course.

Supplementary Material

ml6c00392_si_001.pdf (1.8MB, pdf)

Acknowledgments

The authors thank William K. Warren, Jr. and the William K. Warren Foundation for support of our programs and endowing both the Warren Center for Neuroscience Drug Discovery and the William K. Warren, Jr. Chair in Medicine (C.W.L.).

Glossary

Abbreviations

PAM

positive allosteric modulator

PBL

plasma/brain level

DMPK

drug metabolism and pharmacokinetics

AE

adverse event

M1

muscarinic acetylcholine receptor subtype 1

MED

minimum effective dose

NOR

novel object recognition

CFC

contextual fear conditioning

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.6c00392.

  • Additional experimental details, methods for the synthesis and characterization of all compounds, in vitro and in vivo DMPK protocols, and supplemental tables (PDF)

C.W.L., C.M.N., C.K.J., V.M.K., O.B., A.L.R., D.W.E. and H.P.C. oversaw the medicinal chemistry, target selection and interpreted biological/DMPK data. C.W.L., C.H., C.K.J., and O.B. contributed to the text of the manuscript. C.H., J.L.E., A.R.G., and U.R. performed chemical synthesis. H.P.C., A.L.R, and C.M.N. performed and analyzed in vitro pharmacology assays. C.C.P. performed solubility and physiochemical measurements. A.D.T.G., M.B. and C.K.J. performed in vivo behavior pharmacology assays and in vivo DMPK. O.B. performed in vitro and in vivo DMPK and human dose prediction. All authors have given approval to the final version of the manuscript.

Studies were supported by NIH (NIMH, MH082867, MH073676 and MH108498).

No unexpected or unusually high safety hazards were encountered. All final compounds are >95% pure by HPLC and 1H NMR analysis. Crystallographic data (excluding structure factors) for the structure(s) reported in this paper have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC-2477638. Copies of the data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html or on application to The Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223 336 033; e-mail: deposit@ccdc.cam.ac.uk

The authors declare the following competing financial interest(s): The authors hold IP on M1 PAMs, but this series is open for all to use and we will freely provide.

Published as part of ACS Medicinal Chemistry Letters special issue “Allosteric Modulators in Drug Discovery”.

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