Discovery of the Potent and Selective M1 PAM-Agonist N-[(3 R, 4 S)-3

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Discovery of the potent and selective M1 PAM-agonist N[(3R,4S)-3-hydroxytetrahydro-2H-pyran-4-yl]-5-methyl-4-[4(1,3-thiazol-4-yl)benzyl]pyridine-2-carboxamide (PF-06767832): Evaluation of efficacy and cholinergic side effects Jennifer Elizabeth Davoren, Che-Wah Lee, Michelle Garnsey, Michael A. Brodney, Jason Cordes, Keith Dlugolenski, Jeremy R. Edgerton, Antony R. Harris, Christopher J. Helal, Stephen Jenkinson, Gregory W. Kauffman, Terry Kenakin, John T. Lazzaro, Susan M. Lotarski, Yuxia Mao, Deane M Nason, Carrie Northcott, Lisa Nottebaum, Steven V. O\'Neil, Betty Pettersen, Michael Popiolek, Veronica Reinhart Bieber, Romelia Salomon-Ferrer, Stefanus J. Steyn, Damien Webb, Lei Zhang, and Sarah Grimwood J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00544 • Publication Date (Web): 08 Jun 2016 Downloaded from http://pubs.acs.org on June 8, 2016

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Journal of Medicinal Chemistry

Pettersen, Betty; Pfizer Global Research and Development Popiolek, Michael; Pfizer Global Research and Development Reinhart Bieber, Veronica; Pfizer Global Research and Development, Neuroscience and Pain Research Unit Salomon-Ferrer, Romelia; Pfizer Global Research and Development, Worldwide Medicinal Chemistry Steyn, Stefanus; Pfizer Global Research and Development, Pharmacokinetics, Dynamics and Metabolism Webb, Damien; Pfizer Global Research and Development, Worldwide Medicinal Chemistry Zhang, Lei; Pfizer Global Research and Development, Worldwide Medicinal Chemistry Grimwood, Sarah; Pfizer Global Research and Development, Neuroscience and Pain Research Unit

ACS Paragon Plus Environment


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Discovery of the potent and selective M1 PAM-agonist N-[(3R,4S)-3hydroxytetrahydro-2H-pyran-4-yl]-5-methyl-4-[4-(1,3-thiazol-4yl)benzyl]pyridine-2-carboxamide (PF-06767832): Evaluation of efficacy and cholinergic side effects Jennifer E. Davoren,†* Che-Wah Lee,‡ Michelle Garnsey,‡ Michael A. Brodney,† Jason Cordes,∥ Keith Dlugolenski,§ Jeremy R. Edgerton,§ Anthony R. Harris,‡ Christopher J. Helal,‡ Stephen Jenkinson,⊥ Gregory W. Kauffman,† Terrence P. Kenakin,∇ John T. Lazzaro,¥ Susan M. Lotarski,§ Yuxia Mao, Deane M. Nason,‡ Carrie Northcott,∥ Lisa Nottebaum,⊥ Steven V. O’Neil,‡ Betty Pettersen,∥ Michael Popiolek,§ Veronica Reinhart,§ Romelia Salomon-Ferrer,† Stefanus J. Steyn,ɸ Damien Webb,† Lei Zhang,† and Sarah Grimwood§

†

Neuroscience and Pain Medicinal Chemistry, §Neuroscience and Pain Research Unit, and ɸPharmacokinetics, Dynamics and

Metabolism, Pfizer Worldwide Research and Development, Cambridge, Massachusetts 02139, United States ‡ 

Neuroscience and Pain Medicinal Chemistry, ∥Drug Safety Research and Development, ¥Primary Pharmacology Group, and Research Formulations, Pfizer Worldwide Research and Development, Groton, Connecticut 06340, United States

⊥Drug

Safety Research and Development, Pfizer Worldwide Research and Development, La Jolla, California 92121, United

States ∇ Department

of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, United

States

KEYWORDS: M1 positive allosteric modulator (M1 PAM), PAM-agonist, allosteric potentiation, M1 activator, GPCR, intramolecular hydrogen bond, cholinergic adverse events

Abstract It is hypothesized that selective muscarinic M1 subtype activation could be a strategy to provide cognitive benefits to schizophrenia and Alzheimer’s disease patients while minimizing the cholinergic side effects observed with non-selective muscarinic orthosteric agonists. Selective activation of M 1 with a positive allosteric modulator


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(PAM) has emerged as a new approach to achieve selective M 1 activation. This manuscript describes the development of a series of M1-selective pyridone and pyridine amides and their key pharmacophores. Compound 38 (PF-06767832) is a high quality M1 selective PAM that has well-aligned physicochemical properties, good brain penetration and pharmacokinetic properties. Extensive safety profiling suggested that despite being devoid of mAChR M2/M3 subtype activity, compound 38 still carries gastrointestinal and cardiovascular side effects. This data provides strong evidence that M1 activation contributes to the cholinergic liabilities that were previously attributed to activation of the M2 and M3 receptors.

Introduction The muscarinic acetylcholine receptors (mAChRs) are a family of five G protein-coupled receptors that serve a wide variety of functions within the central and peripheral nervous systems. Decades of positive preclinical and clinical data on the pharmacology of the mAChRs make them attractive targets for addressing the cognitive and psychiatric symptoms associated with schizophrenia and Alzheimer’s disease (AD). Of the five mAChRs, the M 1 subtype is believed to be important for cognition and attention, with potential benefit for the treatment of cognitive and psychiatric symptoms exhibited by those suffering from AD and schizophrenia. 1 The search for a fully selective M1 agonist has been a pharmaceutical quest for many decades.2 Historically, these efforts have met with little success due to the high sequence homology of the orthosteric binding site across all five mAChRs, 3 although a more recent entry by Heptares appears promising.4 In 2009, Merck disclosed another approach towards M 1 activation with their discovery of the highly M1-selective positive allosteric modulator (PAM) BQCA (1),5 and later PQCA (2)6 (Figure 1). These modulators are believed to bind at an allosteric pocket above the orthosteric site on the



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extracellular side of the M1 receptor (Figure 2). This putative binding site shares 83% homology7 with its nearest mAChR subtype neighbors, M4 and M5, which provides a rationale for the M1 selectivity of these ligands.3, 8, 9 PQCA (2) demonstrated robust efficacy in rodent and non-human primate (NHP) cognition assays,10 and further optimization of 2 resulted in a multitude of unique, potent chemotypes being disclosed in both the primary literature6, 11 and the Merck patent estate.12 Two such examples include lactam 311d and tricyclic pyrimidone 412ae. Furthermore, Merck recently began a phase II clinical study with the M 1 allosteric modulator MK-7622 (structure not disclosed) to assess its efficacy and safety as an adjunct therapy in patients with Alzheimer's disease. 13 These impressive disclosures have garnered considerable attention14 from the medicinal chemistry community and have led to the development of a number of additional structurally unique chemotypes. Examples showcasing this diversity include, but are not limited to, indole 511d, azaindole amide 614z, 4-alkyloxy-pyrrolopyrimidinone 714w, pyrazolepyrimidine 814v and 6-phenylpyrimidinone 914y (Figure 1).

Figure 1: Structural diversity of M1 PAM chemotypes.


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Figure 2: Homology model of the active-state M1 receptor with the orthosteric site (yellow) and the allosteric site (purple). The two sites are separated by a hydrogen bonded typrosine lid3 located superficial to the agonist.

Our own efforts in this area have yielded multiple chemically attractive series.14z, 15 Here we describe the SAR, properties, safety, and pharmacology of an optimized series of M 1 PAMs containing a pyridine core. Multiple design iterations targeting improved CNS penetration and potency furnished lead compound 38 (PF-0676783215), a brain-penetrant M1 PAM with no observed off-target pharmacology across a broad selectivity panel. Compound 38 demonstrates a robust potentiation of acetylcholine (ACh) activity in in vitro cell-based assays and is active in multiple in vivo models. Compound 38 was advanced into safety toxicology studies in both rat and dog. While no significant safety findings were observed in rats, clear signs of GI AEs and convulsion were observed in dogs, bringing into question the strategy of targeting selective M 1 activity to avoid cholinergic liabilities.



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Results and Discussion The program leading to discovery of compound 38 began as an effort to identify the minimum pharmacophore required for M1 modulation. The quinolizinone core of PQCA analogue 1011b was truncated14aa and a methyl group was installed at the 6-position to enforce the optimal 3-dimensional bent geometry of the benzyl group, as depicted below (Figure 3). We believe that the benzyl group orientation and an intramolecular hydrogen bond (IMHB) between the amide N-H and pyridone carbonyl are essential components of the M 1 PAM pharmacophore. To further optimize properties towards a more favorable CNS profile, 16 the piperidine in 10 was replaced with a less basic pyrazole-substituted benzyl group, which we had previously identified in our azaindole amide series.14z These modifications resulted in pyridone 11 as an early lead.

11

Figure 3: Evolution of the pyridone M1 PAM 11 from PQCA analogue 10. The 6-methyl substitution on the pyridone core reinforces the bent 3-dimensional geometry predicted by the docked pose in our homology mode to be optimal for binding.

The initial synthetic approach to analogs in the pyridone series is shown in Scheme 1 (Route A). Conversion of bromide 12 to pinacol boronate 13, followed by Suzuki coupling with a 4-substituted benzyl halide, afforded compounds of type 14. Ester hydrolysis and subsequent amide formation with either (3R,4S)-4aminotetrahydro-2H-pyran-3-ol (THP) or (1S,2S)-2-aminocyclohexan-1-ol (cyclohexanol) furnished analogues of type 16. To effectively explore diversity at R1 using parallel medicinal chemistry (PMC), Route B was developed. This alternative synthetic sequence exploits the recent introduction of N-methyliminodiacetic acid (MIDA) boronates as stable surrogates for boronic acids. 17 MIDA-protected boronic acids are unreactive under anhydrous


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cross-coupling conditions but can be easily deprotected and coupled under basic aqueous conditions, thus permitting iterative cross-couplings and greatly streamlining synthetic sequences. Suzuki-Miyaura coupling of intermediate 19 with 4-MIDA benzyl bromide afforded 20 as a single product in high yield. Compound 20 proved to be a versatile template for R1 SAR expansion using PMC. Hydrolysis of the MIDA boronate in 20 under mild conditions revealed the latent boronic acid, which was successfully coupled with 5- and 6-membered heteroaryl halides to give analogs of type 16. Additionally, we were able to directly couple 5- and 6-membered heterocyclic amines by performing an unprecedented Chan-Lam coupling with the MIDA-functionalized intermediate and stoichiometric copper(II) acetate (step k) to afford N-linked products.

Scheme 1: Synthesis of Pyridone M 1 PAMs

Reagents and conditions: (a) B2pin2, Pd(dppf)Cl2•CH2Cl2, KOAc, toluene, 110 °C, 76%; (b) benzyl halide, Pd(PPh3)4, aq. K2CO3, 110 °C, 85%; (c) NaOH, CH3OH, H2O, 80 °C, 58%; (d) amino alcohol, HATU, Et3N, DMF, rt, 45%; (e) NaOH, CH3OH, H2O, 92%; (f) amino alcohol, HATU, Et3N, DMF, rt, 85%; (g) B2pin2, Pd2(dba)3, PCy3, KOAc, dioxane, rt, 60%; (h) 2-(4-

(bromomethyl)phenyl)-6-methyl-1,3,6,2-dioxazaborocane-4,8-dione, Pd(PPh3)4, KF (dry solid), CH3CN, 80 °C, 90%; (i) aq. NaHCO3, THF, MeOH, rt, 77%; (j) heteroaryl halide, Pd(PPh3)4, aq. K2CO3, dioxane, 80 °C; (k) amine or azole, Cu(OAc)2, aq. NaHCO3, pyridine, 60 °C.



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We were encouraged to find that pyridone 11 was equally potent to quinolizinone 10 (M1 PAM EC50: 31 vs. 29 nM). The proposed binding mode of 11 in our M1 homology model is consistent with our SAR data and with key M1 residues identified through site-directed mutagenesis work.5,

8

The most notable features of this binding

mode are π-stacking of the pyridone core with Trp400 and Tyr179, and the conformation of 11, which is stabilized by the 6-methyl substitution that serves two purposes. First, it extends into a hydrophobic pocket and, second, it orients the aryl-heteroaryl linked portion (in this case phenyl-pyrazole) of the molecule to extend into an aromaticrich pocket between transmembrane (TM) domains 2 and 7 (Figure 4). Lastly, an IMHB locks the amide carbonyl in a conformation where it makes a through water hydrogen bond with GLN177 18.

Trp400 π-stack with core

Gln177 through water H-bond with amide carbonyl

Tyr179 π-stack with core Methyl group in hydrophobic pocket

Tyr82 and Tyr 85 π-stack with tail group

Figure 4: Compound 11 docked in the allosteric site of the M1 homology model with key interactions highlighted

We explored the SAR of the amide in this pyridone series but, with the exception of a THP amide, 19 (Example 21, Table 1), no suitable replacements for the cyclohexanol were identified.20 Although the THP amide provided a considerable improvement in lipophilic efficiency (LipE) 21 relative to the cyclohexanol analog (6.26 vs. 5.43), high MDR efflux ratios (ERs)22 plagued THP amides in this series, preventing their further progression. Exploration at the 4-position of the benzyl group (R1, Table 1) via Suzuki and Chan-Lam libraries (Scheme 1) was slightly more fruitful. A number of alkyl, alkoxy, 5-membered and 6-membered heteroaromatics were well tolerated in this position (EC50 < 500 nM). Saturated heterocyles, such as 25 resulted in a substantial loss in potency


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consistent with our proposed binding mode. However, the majority of these modifications were accompanied by a concomitant increase in both MDR ER and/or human liver microsomal (HLM) clearance (Table 1). No analogues with aligned potency (EC50 < 100 nM), HLM clearance (< 30 mL/min/kg) and MDR ER (< 2) were identified (Figure 5). Compounds 11 and 22, which were close to meeting our criteria except for their MDR ER values, were advanced into a mouse neuro-pharmacokinetic (neuroPK) study to assess their brain penetration. Both compounds exhibited total brain/plasma (B/P) ratios 0.05, indicating a level of brain exposure insufficient to demonstrate the desired pharmacology.23 Table 1: SAR on the benzyl group of the pyridone series

M1 EC50 Compound

R1

X

MDR ER

HLM

LipEc

(nM)a,b

Mouse RRCKf

BA/ABd

(mL/min/kg)e

B/Pg

11

CH2

29

5.42

2.7

20

23

0.05

21

O

20

6.26

> 5.3

3

Given the difficulties encountered in the pyridone series with respect to high MDR ER and consequent low brain penetration, we elected to find an alternative core structure that had a reduced number of hydrogen bond acceptors (HBAs). Towards this end, a concise series of 6-membered ring aromatic cores, both with and without an HBA at the 2-position of the ring, were prepared. These analogs were designed to challenge the need for an IMHB to confer activity. Since these modifications increased the lipophilicity of the target molecules, they were also predicted to increase their oxidative clearance.26 To keep ElogD24 < 3, these analogs were prepared using the THP amide. Our synthetic approach to the new analogues, as represented by a pyridine core, is shown in Scheme 2 (full experimental details for additional analogs and intermediates are provided in the supporting information). The


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synthesis of compounds of type 29 commenced with the palladium-mediated borylation of ethyl 4-chloro-5methylpicolinate 26 to give intermediate 27. Suzuki coupling with p-substituted benzyl halides provided intermediates 28a-c. Ester hydrolysis and amide formation completed the synthesis. Scheme 2: Synthesis of analogues with a pyridine core

Reagents and conditions: (a) B2pin2, Pd(dppf)Cl2•CH2Cl2, KOAc, toluene, 100 °C, 20 h; (b) benzyl halide, Pd(dppf)Cl2•CH2Cl2, K2CO3, dioxane/H2O, 80 °C (50% over two steps) 28a: R1 = N-linked pyrazole, 28b: R1 = 4-oxazole, 28c: R1 = 4-thiazole. (c) NaOH, dioxane, H2O, rt, 85%; (d) amino alcohol, HATU, Et3N, rt, 90%.

Replacing the pyridone core with a pyridine bearing the nitrogen at the 2-position27 (e.g., 30) provided the best analogue in this series. Although pyridine 30 is 5.5-fold less potent than pyridone 21, the MDR ER significantly improved (1.4 vs. > 5.3). Incorporating a fluorine at the 2-position of the corresponding phenyl analogue (31) afforded potency as well as low MDR ER and HLM, but at the cost of increased lipophilicity and lower LipE. In compounds 32 and 33, removal of the amide N-H HBA from the core caused a 2- to 3-fold loss in potency, in addition to higher MDR ER values.28



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To elucidate the conformational preference of these compounds, a torsional energy scan was performed at 15° intervals along the carbonyl-core bond. Profile plots depict the relative energy profiles of analogues 30-34 (Figure 6). The putative bioactive conformation resides at 0° along each contour. As expected, the energy profile of pyridine 30 had an energy minimum at 0° due to the stabilization of that conformation by the IMHB between the pyridine nitrogen and the amide hydrogen. The fluorine in compound 31 is an effective HBA that still favors the bioactive conformation. Removal of the HBA in compounds 32 and 33 resulted in relative destabilization of the desired geometry, by producing a broad minimum around the bioactive conformation and a second minimum at a trajectory opposite to the bioactive conformation at ±180°. Aniline 34, with a HBD in the 2-position, has its energy minimum in a conformation opposite to the bioactive conformation, thus serving as a negative control. Together, this data suggests that an IMHB is beneficial but not critical for potency, as long as the analogue can adopt the bioactive conformation wherein the amide carbonyl forms a predicted through water hydrogen bond with Gln177. Of these analogues, the pyridine core exemplified by compound 30 was optimal for aligning potency with low MDR ER. In a mouse neuroPK study 30 demonstrated modest brain permeability with an unbound brain (Cb,u) to unbound plasma (Cp,u) concentration ratio of 0.2. Table 2: SAR of the core; effect of the IMHB on potency and P-gp liability

Compound

R

M1 EC50 a,b

LipEc

(nM)

MDR ERd

HLM

BA/AB

(mL/min/kg)e

RRCKf

C57BL/6 Mouse B/Pg

Cb,u/Cp,uh

30

109

5.03

1.4

9,000

n.d.

1.8

9.2 µM, PAM EC50= 447 nM) and (C) β-arrestin (Agonist EC50= 3.9 µM, PAM EC50= 365 nM); (D) Functional concentration response curves for compound 38 in the presence (PAM mode) and absence (Agonist mode) of an EC20 Carbachol in hippocampal slice assay. Representative M1 ACh shift data using (E) FLIPR, (F) β-arrestin, and (G) Inhibition of [3H]NMS binding in the presence of fixed concentrations of agonist:  10 µM,  3 µM,  1 µM,  0.3 µM,  0.1 µM,  0 µM. (H) Binding and functional parameters for 38 from the functional allosteric model: a intrinsic efficacy of ACh; b Intrinsic efficacy of 38; c Functional cooperativity of 38 with ACh.

In a recombinant panel of mAChR assays, 38 had no appreciable agonist or PAM activity when tested up to a concentration of 10 µM at M2-M5 receptors (> 180-fold selectivity) (Figure 8A). To confirm these results in a native preparation, we tested 38 in the guinea pig ileum (GPI) tissue bath preparation where M 2 and M3 activation are reported to elicit contractions.36 Although selective in the recombinant mAChR panel, we were surprised that direct treatment with 38 alone elicited what appeared to be a robust partial agonist effect37 (EC50 351 nM, 77% compared to a maximal carbachol response) (Figure 8B). In PAM mode 38, 38 had an EC50 of 82 nM (Figure 8C), a value similar to that determined in the FLIPR PAM assay. We confirmed that these contractions in the GPI were driven by mAChR activation by blocking the response in both the agonist and PAM mode of this assay with the non-selective mAChR antagonist atropine (Figures 8B and 8C). To complement the functional assays we investigated whether 38 modulates ACh binding at either M2 or M3 by running a [3H]NMS radioligand binding assay in the presence of fixed concentrations of compound 38 (Figure 8D and 8E, respectively). Compound 38 did not affect ACh binding confirming that it either does not bind, or is functionally silent at M2 and M3. To see if this effect was compound-specific, we tested > 20 M1 PAMs spanning multiple chemical series, as well as literature standards, in the agonist and PAM mode of this assay. In PAM-mode we saw a correlation (R2= 0.48) between the pEC50 in the FLIPR assay and the pEC50 in the GPI contractility assay (Figure 8F). To rule out off-target effects, 38 was tested against a broad panel of targets that included GPCRs (32 targets, including human M1, M2 and M3), ion channels (12 targets), nuclear receptors (3 targets), amine transporters (5 targets), phosphodiesterases (PDE; 11 targets), and a panel of enzyme and kinase targets (13 targets). Aside from


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human M1, compound 38 had no measurable activity (> 10 µM) at any target with the exception of PDE 2A (IC 50 = 3.09 µM) and PDE 11A4 (IC50 = 4.94 µM). Based on the weight of evidence, we conclude that compound 38 contributes to smooth muscle contraction in the guinea pig ileum through an M 1 mechanism of action.

Figure 8: (A) Representative M1-M5 PAM functional dose response curves for compound 38 in the presence of EC20 of ACh. M1,3,5 used a FLIPR assay while M2,4 used a cAMP readout; Functional concentration response curves for 38 in the absence (B, Agonist mode) and presence (C, PAM mode) of an EC20 donepezil ± the non-selective muscarinic antagonist atropine in the GPI tissue bath assay; Inhibition of [3H]NMS binding to M2- (D) and M3- (E) expressing cell lines by ACh in the presence of fixed concentrations of compound 38:  10 µM,  3 µM,  1 µM,  0.3 µM,  0.1 µM,  0 µM; (F) correlation between pEC50 in the M1 PAM FLIPR assay and pEC50 in the PAM mode of the GPI tissue bath assay (color/shape by chemical series:  Azaindoles14z,  Pyridones,  Pyridines,  Literature standards,  Undisclosed).

Having observed substantial potentiation of ACh affinity in vitro, 38 was advanced into four in vivo studies (Figures 9A-D). The measured Cb,u and Cp,u of 38 from these studies are given in Table 6. Compound 38 robustly increased striatal IP1 levels in a dose-responsive manner in vivo (Figure 9A). Plotting the average fold increase in IP1 accumulation over a 2-hour treatment for each individual animal against its Cb,u provided a partial dose-response curve (Figure 9A). The dynamic range of this assay is large: at a maximum dose of 10 mg/kg (Cb,u = 195 nM) we observed a 42-fold (±3.4) increase in IP1 over baseline, indicating that 38 has excellent cooperativity in vivo under endogenous levels of ACh. A more modest 2-fold (±0.5) IP1 elevation was observed at 1 mg/kg (Cb,u = 14.3 nM). d-



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Amphetamine-stimulated locomotor activity (aLMA) has previously been shown to be sensitive to M 1 PAMs5, 14z and agonists39. As shown in Figure 9B, 38 significantly reduced aLMA in mice administered a 1 mg/kg dose (Cb,u = 18.5 nM, Table 6), when compared to vehicle, aligning with the observation that M 1 PAM activity measured using an in vivo IP1 assay correlates with aLMA activity40. To demonstrate its effects in a cognition assay, 38 was tested in the rat Morris water maze (MWM) assay to assess its ability to reverse spatial learning and memory deficits induced by scopolamine (Figure 9C). Scopolamine was used because anticholinergic treatment has detrimental effects on cognitive functioning in subjects with schizophrenia.41 Significant reversal of scopolamine was observed at doses as low as 0.32 mg/kg (Cb,u = 1.3 nM). Since it had been suggested that M1 activation could potentially provide anti-psychotic effects,1c we next tested 38 in rat amphetamine-disrupted pre-pulse inhibition (PPI) (Figure 9D). PPI deficits have also been observed in patients living with schizophrenia42. At doses that had no effect on startle reflex or PPI alone, 38 significantly attenuated the amphetamine-induced deficit in PPI at 1 mg/kg (Cb,u = 17.4 nM) in the collapsed data set.43 To our knowledge this is the first report of an M1 PAM being tested in this assay.


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A.

B.

A m p h e t a m in e - s t im u la t e d L M A

S t r ia t a l IP 1 R e s p o n s e

V e h ic le /V e h ic le V e h ic le /A m p h H a lo p e rid o l 0 .1 m g /k g /A m p h

40000

F o ld C h a n g e O v e r V e h ic le

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D is t a n c e ( c m )

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C o m p o u n d 3 8 , C b u (n M )

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A m p h e ta m in e -in d u c e d P P I

S c o p o la m in e - in d u c e d M W M a c t iv it y

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D o n e p e z il 1 m g /k g /S c o p

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1500

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ic

ic

le

le

/V

e

/A

h

m

ic

p

le

h

0

V

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 9: Effects of compound 38 on: (A) mouse striatal IP1 levels, (B) mouse amphetamine-stimulated locomotor activity (LMA), (C) rat scopolamine-induced Morris water maze (MWM) activity and (D) rat amphetamine-induced pre-pulse inhibition (PPI); data shown are from 5, 10 and 15 dB (see SI).

Table 6: Summary of exposure data from biology studies on compound 38. Minimally effective dose (MED) from each experiment is is indicated by an asterisk (*)

Assay

0.1 mg/kg

0.32 mg/kg

1 mg/kg

3.2 mg/kg

10 mg/kg

Cp,ua

Cb,ub

Cp,u

Cb,u

Cp,u

Cb,u

Cp,u

Cb,u

Cp,u

Cb,u

(nM)

(nM)

(nM)

(nM)

(nM)

(nM)

(nM)

(nM)

(nM)

(nM)

Species

IP1c

mouse

n.d.

n.d.

n.d.

n.d.

42.1*g

14.3*

176g

52.8

574g

195

aLMAd

mouse

n.d.

n.d.

23.4

6.38

76.1*

18.5*

255

85.2

n.d.

n.d.

MWMe

rat

0.9

0.31h

3.7*

1.3*h

19.6

6.7h

n.d.

n.d.

n.d.

n.d.



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aPPIf a

rat

n.d.

n.d.

11.9

4.3

45.6*

Page 22 of 41

17.4*

123

44.4

n.d.

n.d.

Concentration of unbound compound in plasma; b Concentration of unbound compound in brain; c Compound concentrations

measured in striatal tissue using average exposure from all animals in each dose group (N=5/dose) at T = 2 h. Compound was administered subcutaneously (sc) as a solution in 5/15/80 DMSO/Solutol/water;

d

Compound concentrations measured in

plasma and brain from a satellite study (N=2/dose) at T = 1 h using the same dosing solution (solution in 5/15/80 DMSO/Solutol/water) and route of administration (sc) as the aLMA study; e Compound concentrations measured in plasma at T = 1 h from a satellite study is the average exposure (N=2/dose) using the same dosing solution (suspension in 0.5% methylcellulose in water) and route of administration (oral, po) from the MWM study; f PK measured in plasma and brain is the average of randomly selected (N=2/dose) animals at the end of the PPI study (T = 1.67 h). Animals were dosed sc with a suspension in 0.5% methylcellulose in water; g Plasma Cp,u exposures were estimated from the exposure in striatal brain tissue using the previously measured AUC Cb,u/Cp,u 0.34;

h

Cb,u was estimated from Cp,u using the AUC-derived Cb,u/Cp,u of 0.34;

n.d.: not determined.

The safety profile of 38 was evaluated in a 2-week study in rats (Table 7) and a single-dose study in dogs (Table 8). In the rat study, all doses tested [3, 10, 30 (males)/15 (females) mg/kg] were well tolerated. Treatmentrelated findings were limited to slight increases in food intake and body weight gain. There were no histopathological findings at any tested dose.44 Compared with rat, similar exposures in dogs were poorly tolerated, as evidenced by adverse cholinergic signs. In dogs, dose-dependent cholinergic signs consisted of: soft/watery stools, emesis and salivation at ≥3 mg/kg; and ataxia, side-to-side head movements, and generalized convulsions at 45 mg/kg (Table 9). Although M1 activation is generally not thought to be associated with GI adverse effects, these results are consistent with the in vitro guinea pig ileum data, as well as recent preclinical toxicology data on BQCA (1), PQCA (2) and a related analogue.45 The M1 mechanism is, however, associated with convulsion,46 which was observed in both dogs at 45 mg/kg, corresponding to unbound plasma C max values of 952 and 1099 nM (Cp,u). Using the neuroPK study in dog (Cb,u/Cp,u = 0.3, Table 5), together with the observed plasma C max values, we can estimate the dog Cb,u at the time of convulsion. These estimated brain levels (286 and 330 nM) are at or below this compound’s in vitro agonist EC50. Hence, at these exposure levels it is unlikely that compound 38’s agonist activity is responsible for the observed convulsions. The no-observed-adverse-effect level (NOAEL) of this preclinical study was 5 mg/kg, as the exposures at 15 mg/kg overlapped with those associated with convulsion.


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To put the safety data into context, the Cp,u exposures from the safety studies are compared to the Cp,u exposures from the preclinical animal models to calculate a therapeutic index (TI) range. Marking the lower end of the preclinical rodent efficacy range was the MWM assay (Cp,u at the lowest effective dose = 3.7 nM), while aLMA marked the upper end (Cp,u at the lowest effective dose = 76.1 nM). Compound 38’s TI between efficacy and the onset of cholinergic signs in dog (Cp,u = 568 nM) was between 7.5 and 152-fold, depending on the preclinical efficacy assay. More significantly, due to convulsions, this nonclinical safety data would cap initial clinical studies at 1/10th the Cmax value at the NOAEL (Cp,u = 659 nM).47 The TI between efficacy and the 1/10 th NOAEL (Cp,u = 65.9 nM) was between 0.87 and 17.8-fold, depending on which preclinical assay was considered. To more fully evaluate the effects of 38 on the CV system, a three-dose (0.3, 1 and 3 mg/kg) crossover telemetry study in dogs was performed (detailed CV findings are presented in the supporting information). At 0.3 mg/kg, no change in CV endpoints was observed. At 1 mg/kg, 38 produced increases in maximum systolic left ventricular pressure (+8 mmHg) and at 3 mg/kg statistically significant increases in blood pressure (+7 to +10 mmHg), max systolic left ventricular pressure, left ventricular+dP/dt max (an index of cardiac contractility), activity and heart rate (+25 bpm) were observed, as well as decreases in RR-, QT-, and QTc-intervals up to 9 h post dose. Table 7: Summary of findings from a 2-week rat exploratory toxicology study with 38 Treatment Exposures Dosea Sexb mg/kg

Cmax Cp,u

AUC(0-24h) Cp,u

TI

nM

nM.hr

Range

Treatment Findings

3

M/F

195

1856

3 to 53

None

10

M/F

562

5739

7 to 152

↑ food intake and body weight gain in males.

15

F

708

9768

9 to 191

↑ food intake

30

M

1221

13211

16 to 330

↑ food intake and body weight gain

a

Compound 38 was dosed orally in a 0.5% methylcellulose formulation; b N=4/sex/dose.

Table 8: Summary of clinical signs and exposures following oral administration of 38 from a single-dose escalation study



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Page 24 of 41

Treatment Exposures Dosea Sexb mg/kg

Cmax Cp,u

AUC(0-24h) Cp,u

TI

nM

nM.hr

Range

Treatment Findings

0.3c

M

41d

n.d.

0.5 to 11

Loose stool (1/4 dogs)

1c

M

169d

n.d.

2 to 46

No clinical findings

3c

M

568

5242

7 to 152

Loose/mucoid stool (4/4 dogs), emesis (1/4) & salivation (1/4) 5

M/F

659

5861

9 to 178

Soft/watery feces, emesis, salivation

15

M/F

1050

7765

14 to 284

Mucoid feces, emesis & salivation Soft/mucoid/watery feces, salivation, emesis, ataxia,

45

M/F

n.d.

n.d.

n.d.

side-to-side head movements & convulsions, 45 min post-dose (952 and 1099 nM)e

a

Compound 38 was dosed orally as a spray-dried dispersion (SDD)48 (10% drug loading with hydroxypropyl methylcellulose

acetate succinate, medium grade (HPMCAS-M). The SDD suspension formulation was prepared with mortar and pestle in 0.5 wt% methocellulose/0.5 wt% HPMCAS in 20 mM tris buffer, pH 7.4; b N=1/sex/dose; c From the CV study (N=4); d Extrapolated from 4 hour post-dose in-life plasma samples; e Rising plasma exposures had not yet reached C max.

Conclusion We have identified a series of pyridones and pyridines as a new class of M 1 PAMs. Robust PMC-enabled synthetic routes using MIDA-boronate chemistry enabled extensive study of the SAR in the 4-position of the benzyl ring and identified oxazole and thiazole rings as optimal substituents in this position. Based on our M 1 homology model, we believe that both series bind to the allosteric site of the M 1 receptor in a similar fashion to each other. Although both series have favorable physicochemical properties and potency, the most potent pyridones were not CNS penetrant, whereas exemplified compounds 30 and 38 in the pyridine series exhibited moderate brain exposure in rodent. Based upon results in an NHP brain availability study the more potent pyridine, 38, would be expected to have a human brain availability Cb,u/Cp,u of ≥0.5. Compound 38 showed robust cooperativity with ACh at M1 in both our calcium mobilization and -arrestin assays. More importantly, compound 38 retained its allosteric cooperativity with ACh in-vivo where we saw a robust potentiation response in striatum (> 40-fold) when measuring IP1 accumulation in a wild-type mouse under


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endogenous levels of ACh. In addition to its favorable pharmacology, 38 was shown to have good oral bioavailability, low clearance, and robust in vivo activity in aLMA, PPI and MWM at low free-brain (Cb,u) concentrations. Compound 38 has no significant off-target pharmacology, including M2 and M3 activity, making it an ideal compound for testing the effects of selective M1 activation on the GI and CV systems. In the GPI tissue bath assay we observed a robust response in both the presence (PAM-mode) and absence (agonist-mode) of donepezil which was recapitulated with additional chemically distinct M 1-selective PAMs. This response was blocked by the muscarinic antagonist atropine which is consistent with an M 1-mediated effect. Studies in rat and dog to evaluate safety and cholinergic AE’s in-vivo were performed on 38. While we had no findings in a chronic dose 2-week rat study at exposures as high as 1 µM (Cp,u), at similar exposures dog proved to be a more sensitive species. At exposures > 568 nM (Cp,u) we observed classic cholinergic signs traditionally attributed to M2 and M3, such as increased heart rate, blood pressure, loose stool and emesis. Though these cholinergic signs were present, compound 38 does appear to have a TI relative to our pre-clinical efficacy range. It was, however, convulsion that significantly diminishes 38’s TI. Further, similar GI, CV, and convulsion findings were also observed with structurally distinct M1 PAMs suggesting that these AE’s are not compound or chemotype specific. Further efforts targeting the M1 receptor should acknowledge these risks to ensure that compounds going into the clinic have a sufficient safety window.

Experimental Section All procedures performed on animals in this study were in accordance with established guidelines and regulations, and were reviewed and approved by the Pfizer Institutional Animal Care and Use Committee. Pfizer animal care facilities that supported this work are fully accredited by AAALAC International. Chemistry General Information. All solvents and reagents were obtained from commercial sources and were used as received. All reactions were monitored by TLC (TLC plates F254, Merck) or UPLC-MS analysis (Waters Acquity, ESCI +/-, APCI +/-). Melting points were obtained with a Thomas-Hoover melting point apparatus and are uncorrected. 1H NMR and

13

C NMR spectra were obtained using deuterated solvent on a Varian 400 MHz

instrument. All 1H NMR shifts are reported in δ units (ppm) relative to the signals for chloroform (7.27 ppm) and



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Page 26 of 41

MeOH (3.31 ppm). All 13C shifts are reported in δ units (ppm) relative to the signals for chloroform (77.0 ppm) and MeOH (49.1 ppm) with 1H-decoupled observation. All coupling constants (J values) are reported in hertz (Hz). NMR abbreviations are as follows: br, broadened; s, singlet; d, doublet; t, triplet; q, quartet; p, pentet; m, multiplet; dd, doublet of doublets; ddd, doublet of doublet of doublets. Optical rotations were determined with a Jasco P-2000 polarimeter. Column chromatography was carried out on silica gel 60 (32-60 mesh, 60 Ǻ) or on pre-packed Biotage™ or ISCO columns. HPLC purity analysis of the final test compounds was carried out using one of four methods. Method A: UPLC/UV. WuXi AppTec, Shanghai, China. Column: Agilent Xtimate C18, 5 × 30 mm, 3 µm; UV purity detected at 220 nm; Mobile phase A = 0.2% TFA in H2O; Mobile phase B = 0.1% TFA in CH3CN. Gradient: 1% B to 100% B in 5.0 min. Flow rate: 1.2 mL/min. Method B: UPLC/UV WuXi AppTec, Shanghai, China. Column: Agilent Xtimate C18, 5 × 30 mm, 3 µm; UV purity detected at 220 nm; Mobile phase A = 0.1% TFA in H2O; Mobile phase B = 0.1% TFA in CH3CN. Gradient: 1% B to 5% B in 1.0 min, 5%B to 100% B in 5.0 min, hold at 100% B for 2.0 min. Flow rate: 1.2 mL/min. Method C: Column: UPLC/UV. WuXi AppTec, Shanghai, China. Column: Chiralpak AD-3, 150 x 4.6 mm, 5 µm, UV purity detected at 220 nm; Mobile phase: 60% EtOH (0.05% DEA) in CO2. Flow rate: 1.2 mL/min. Method D: Column: Waters Atlantis C18 4.6 x 50 mm, 5 µm; UV purity detected at 215 nm; Mobile phase A: 0.05% TFA in H 2O (v/v); Mobile phase B: 0.05% TFA in CH3CN (v/v); Gradient: 95.0% H2O/5.0% CH3CN linear to 5.0% H2O/95.0% CH3CN in 4.0 min, hold at 5.0% H2O/95.0% CH3CN to 5.0 min. Flow rate: 2 mL/min. All final compounds were determined to have a purity of > 95% by one of the aforementioned methods unless stated otherwise. Experimental Procedures. Ethyl 4-chloro-5-methylpyridine-2-carboxylate (26): A mixture of 2,4-dichloro-5-methylpyridine (33 g, 0.20 mol), Pd(dppf)Cl2•CH2Cl2 (7.45 g, 10.2 mmol), and triethylamine (61.8 g, 611 mmol) in ethanol (500 mL) was stirred under carbon monoxide (30 psi) at 60 °C for 4h. The reaction mixture was filtered and concentrated in vacuo; silica gel chromatography (Gradient: 10% to 50% EtOAc in petroleum ether) provided 26 as a yellow oil. Yield: 25.0 g, 0.125 mol, 62%. LC-MS m/z 200.0 [M+H]+. 1H NMR (400 MHz, CDCl3)  8.56 (s, 1H), 8.11 (s, 1H), 4.48 (q, J=7.1 Hz, 2H), 2.44 (s, 3H), 1.44 (t, J=7.1 Hz, 3H).

Ethyl 5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine-2-carboxylate (27): A mixture of chloride 26 (16 g, 80 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi-1,3,2-dioxaborolane (30.5 g, 120 mmol),


Page 27 of 41

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Pd(dppf)Cl2•CH2Cl2 (5.86 g, 8.01 mmol), and potassium acetate (28.3 g, 288 mmol) in toluene (1.2 L) was stirred at

130 °C for 20h. The reaction mixture was cooled to rt, then filtered through diatomaceous earth. The filtrate was concentrated in vacuo; silica gel chromatography (Gradient: 10% to 50% EtOAc in petroleum ether) provided a yellow solid (20 g), which was diluted with petroleum ether (50 mL) and stirred at rt for 20 min. The solid was collected via filtration to afford 27 (8.8 g) as a white solid. The corresponding filtrate was concentrated in vacuo and the residue was repurified by silica gel chromatography (Gradient: 0% to 30% EtOAc in petroleum ether); the isolated material (4.5 g) was washed with petroleum ether (5 mL) to yield additional 27 (3.5 g) as a white solid. Combined yield: 12.3 g, 42.2 mmol, 53%. 1H NMR (400 MHz, CDCl3)  8.57 (s, 1H), 8.39 (s, 1H), 4.48 (q, J=7.2 Hz, 2H), 2.57 (s, 3H), 1.45 (t, J=7.2 Hz, 3H), 1.37 (s, 12H).

General Suzuki procedure A: A mixture of 27 (0.310 mmol), benzyl halide (0.258 mmol), Pd(dppf)Cl2•CH2Cl2 (0.026 mmol), and K2CO3 (0.516 mmol) in 1,4-dioxane (2 mL) and water (0.2 mL) was stirred at 100 °C for 18h. The reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated in vacuo.

General hydrolysis/amidation procedure B: A mixture of an ethyl ester of type 28 (1.0 eq) and sodium hydroxide (4.0 eq) in 1:1 MeOH/water (0.1 M) was stirred at 80 °C for 2 h. The reaction mixture was then diluted with water, concentrated in vacuo to remove MeOH, and acidified to a pH of 3 – 4 with conc. HCl. After extraction three times with a mixture of 20:1 DCM/MeOH, the combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo to provide the carboxylic acid as a yellow solid. The residue was combined with DMF, amino alcohol (2.5-3.0 eq), and Et3N (4.0 eq), then treated with HATU (4.1 eq). The reaction mixture was stirred at rt overnight, whereupon it was diluted with saturated aqueous sodium bicarbonate solution and water (1:1), and extracted 2-4 times with EtOAc. The combined organic extracts were washed twice with water, and once with saturated aqueous sodium chloride solution, dried over magnesium sulfate, filtered, and concentrated in vacuo.

4-(4-(1H-Pyrazol-1-yl)benzyl)-N-((3R,4S)-3-hydroxytetrahydro-2H-pyran-4-yl)-5-methylpicolinamide

(30):

Ethyl 4-(4-(1H-pyrazol-1-yl)benzyl)-5-methylpicolinate (28a) was synthesized from 27 according to general Suzuki procedure A using 1-(4-(bromomethyl)phenyl)-1H-pyrazole as the benzyl halide. Silica gel chromatography (Gradient: 10% to 50% EtOAc in petroleum ether) afforded this intermediate as an off-white gum. Yield: 800 mg,



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Page 28 of 41

2.5 mmol, 73%. 1H NMR (400 MHz, CDCl3)  8.53 (s, 1H), 7.89-7.93 (m, 2H), 7.71-7.73 (m, 1H), 7.64 (br d, J=8.5 Hz, 2H), 7.20 (br d, J=8.4 Hz, 2H), 6.47 (dd, J=2.3, 1.9 Hz, 1H), 4.47 (q, J=7.2 Hz, 2H), 4.07 (s, 2H), 2.32 (s, 3H), 1.44 (t, J=7.1 Hz, 3H). Intermediate 28a was subjected to the general hydrolysis/amidation procedure with amino alcohol (3R,4S)-4-aminotetrahydro-2H-pyran-3-ol. Silica gel chromatography (Gradient: 5% to 10% MeOH in DCM), followed by crystallization from a concentrated solution of EtOAc and heptane, provided 30 as a white solid. Yield: 2.00 g, 5.10 mmol, 75%. LC-MS m/z 393.1 [M+H]+. 1H NMR (400 MHz, CD3OD)  8.42 (s, 1H), 8.19 (dd, J=2.5, 0.5 Hz, 1H), 7.86 (s, 1H), 7.70-7.71 (m, 1H), 7.69 (br d, J=8.7 Hz, 2H), 7.30 (br d, J=8.7 Hz, 2H), 6.51 (dd, J=2.4, 1.9 Hz, 1H), 4.15 (s, 2H), 3.87-3.99 (m, 3H), 3.64 (ddd, J=9.7, 9.6, 4.9 Hz, 1H), 3.47 (ddd, J=11.9, 11.9, 2.2 Hz, 1H), 3.19 (dd, J=11.1, 9.8 Hz, 1H), 2.37 (s, 3H), 1.98-2.05 (m, 1H), 1.64-1.76 (m, 1H). 13C NMR (100 MHz, CD3OD)  167.5, 151.4, 151.1, 149.7, 142.4, 140.4, 138.5, 137.5, 131.5, 129.3, 124.1, 121.1, 109.0, 72.9, 70.5, 68.0, 54.8, 39.3, 32.9, 16.9; Elemental analysis: Calcd for C22H24N4O3: C, 67.33; H, 6.16; N, 14.28. Found: C, 67.27; H, 6.12; N, 14.33. []D20 = -7.6o (c 1.25, CHCl3).

4-(4-(1H-pyrazol-1-yl)benzyl)-N-((1R,2R)-2-hydroxycyclohexyl)-5-methylpicolinamide (35): Compound 35 was synthesized according to general hydrolysis/amidation procedure B from ethyl 4-(4-(1H-pyrazol-1-yl)benzyl)-5methylpicolinate (28a) using amino alcohol (1S,2S)-2-aminocyclohexan-1-ol. Silica gel chromatography (Gradient: 0% to 4% MeOH in DCM) yielded 35 as a white solid. Yield: 145 mg, 0.371 mmol, 90%. LC-MS m/z 391.1 [M+H]+. 1H NMR (400 MHz, CD3OD)  8.41 (s, 1H), 8.19 (d, J=2.5 Hz, 1H), 7.86 (s, 1H), 7.70 (d, J=1.6 Hz, 1H), 7.68 (d, J=8.5 Hz, 2H), 7.30 (d, J=8.3 Hz, 2H), 6.51 (dd, J=2.4, 1.9 Hz, 1H), 4.15 (s, 2H), 3.69-3.79 (m, 1H), 3.473.57 (m, 1H), 2.36 (s, 3H), 1.98-2.08 (m, 2H), 1.68-1.81 (m, 2H), 1.31-1.45 (m, 4H).

N-((3R,4S)-3-hydroxytetrahydro-2H-pyran-4-yl)-5-methyl-4-(4-(oxazol-4-yl)benzyl)picolinamide

(37):

Intermediate ethyl 5-methyl-4-(4-(oxazol-4-yl)benzyl)picolinate (28b) was synthesized according to general Suzuki procedure A using 4-(4-(chloromethyl)phenyl)oxazole as the benzyl halide. LC-MS m/z 322.9 [M+H]+. Crude 28b was subjected to the general hydrolysis outlined in procedure B to give 5-methyl-4-(4-(oxazol-4-yl)benzyl)picolinic acid, yield: 62 mg, 0.21 mmol, 81% over 2 steps. LC-MS m/z 294.9 [M+H]+. EDCI (56.5 mg, 0.295 mmol), 1Hbenzotriazol-1-ol (42.7 mg, 0.316 mmol), and Et3N (64.0 mg, 0.632 mmol) were added to a solution of the picolinic acid (62 mg, 0.21 mmol) in a mixture of DCM (5 mL) and DMF (3 mL). The mixture was stirred for 4 h at 25 °C,


Page 29 of 41

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whereupon (3R,4S)-4-aminotetrahydro-2H-pyran-3-ol (29.6 mg, 0.253 mmol) was added and stirring continued for 18 h at 25 °C. Additional EDCI (56.5 mg, 0.295 mmol), 1H-benzotriazol-1-ol (42.7 mg, 0.316 mmol), and triethylamine (64.0 mg, 0.632 mmol) were introduced, and the reaction mixture was stirred for another 30 minutes; additional (3R,4S)-4-aminotetrahydro-2H-pyran-3-ol (29.6 mg, 0.253 mmol) was then added, and stirring was carried out for another 18 h at 25 °C. The reaction mixture was diluted with DCM (20 mL), washed sequentially with saturated aqueous citric acid solution (20 mL) and aqueous sodium hydroxide solution (1 M, 20 mL), and concentrated in vacuo. The residue was subjected to preparative thin layer chromatography on silica gel (Eluent: 33% petroleum ether in EtOAc), followed by reversed phase HPLC purification (Column: Phenomenex Gemini C18, 5 μm; Mobile phase A: water containing 0.225% formic acid; Mobile phase B: acetonitrile containing 0.225% formic acid; Gradient: 23% to 43% B). The product was obtained as a white solid. Yield: 20 mg, 51 μmol, 24%. LCMS m/z 394.1 [M+H]+. 1H NMR (400 MHz, CDCl3)  8.32 (s, 1H), 8.12 (br d, J=6 Hz, 1H), 8.00 (s, 1H), 7.92-7.96 (m, 2H), 7.68 (d, J=8.2 Hz, 2H), 7.17 (d, J=8.2 Hz, 2H), 4.33-4.46 (br m, 1H), 4.09 (dd, J=12, 5 Hz, 1H), 4.06 (s, 2H), 3.90-4.04 (m, 2H), 3.59-3.68 (m, 1H), 3.43-3.52 (m, 1H), 3.23 (dd, J=10.9, 10.2 Hz, 1H), 2.32 (s, 3H), 2.002.08 (m, 1H), 1.74-1.87 (m, 1H).

N-((3R,4S)-3-hydroxytetrahydro-2H-pyran-4-yl)-5-methyl-4-(4-(thiazol-4-yl)benzyl)picolinamide (38): Ethyl 5-methyl-4-(4-(thiazol-4-yl)benzyl)picolinate (28c) was synthesized according to general Suzuki procedure A using 4-(4-(bromomethyl)phenyl)thiazole as the benzyl halide. The residue was purified by silica gel chromatography (Gradient: 20% to 80% EtOAc in heptane) to give the ester intermediate as a white solid. Yield: 872 mg, 2.58 mmol, 52%. LC-MS m/z 339.0 [M+H]+. 1H NMR (400 MHz, CDCl3)  8.86 (d, J=2.0 Hz, 1H), 8.50-8.52 (m, 1H), 7.92 (s, 1H), 7.86 (br d, J=7.8 Hz, 2H), 7.50 (d, J=2.0 Hz, 1H), 7.17 (br d, J=7.8 Hz, 2H), 4.45 (q, J=7.1 Hz, 2H), 4.06 (s, 2H), 2.31 (s, 3H), 1.42 (t, J=7.1 Hz, 3H). The ester intermediate was subjected to the general hydrolysis/amidation procedure B using amino alcohol (3R,4S)-4-aminotetrahydro-2H-pyran-3-ol. The crude product obtained after EtOAc extraction was taken up as a slurry in hot EtOAc (15 mL), which was then allowed to stir and cool to rt over 2 h. The crystals were filtered to afford 38 as a white solid. Yield: 525 mg, 1.28 mmol, 50%. LC-MS m/z 410.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6)  9.18 (br d, J=1 Hz, 1H), 8.51 (d, J=8.4 Hz, 1H), 8.43 (s, 1H), 8.12 (br d, J=1 Hz, 1H), 7.94 (d, J=8.0 Hz, 2H), 7.78 (s, 1H), 7.26 (d, J=8.0 Hz, 2H), 4.93 (d, J=5.7 Hz, 1H), 4.12 (s, 2H), 3.71-3.83 (m, 3H), 3.51-3.61 (m, 1H), 3.27-3.36 (m, 1H, assumed; partially obscured by solvent peak), 3.01 (dd,



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

J=10.5, 10.5 Hz, 1H), 2.33 (s, 3H), 1.77-1.85 (m, 1H), 1.55-1.68 (m, 1H).

Page 30 of 41

13

C NMR (100 MHz, CDCl3)  166.8,

156.0, 152.9, 149.5, 149.4, 147.3, 137.7, 136.0, 132.9, 129.2, 126.9, 123.4, 112.5, 72.2, 71.4, 66.4, 54.1, 38.9, 31.0, 16.7. Elemental analysis: Calcd for add empirical formula C22H23N3O3S: C, 64.53; H, 5.66; N, 10.26. Found: C, 64.52; H, 5.58; N, 10.16. []D20 = -9.4° (c 1.05, CHCl3).

Associated Content Supporting Information: The supporting information is available free of charge on the ACS publications website. Included are full synthetic procedures and characterization for all analogues whose procedures are not included in the main manuscript. Experimental details and additional material including statistics and data analyses for our biology assays. Compound 38 (PF-06767832) is commercially available from Sigma-Aldrich (catalog # PZ0323).

Acknowledgements: We would like to acknowledge Lois Chenard and our Wuxi team for technical assistance in running libraries, Deborah Smith and Nicole Matluck for assistance in running binding assays, Brian Samas for X-ray data, Feng Shao and Bruce Rogers for helpful discussions, Rebecca O’Connor and HD Biosciences for screening assistance, Joy Yang and Xinjun Hou for modeling help, and Neal Sach for reaction optimization assistance.

AUTHOR INFORMATION Corresponding Author * E-mail [email protected] Phone: 1.617.3950655 The authors declare no competing financial interest.

Abbreviations bis(pinacolato)diboron (B2pin2), 1,1′-bis(diphenylphosphino)ferrocene (dppf), O-(7-azabenzotriazol-1-yl)- 1[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium

3-oxid


hexafluorophosphate

(HATU),

Page 31 of 41

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tris(dibenzylideneacetone)dipalladium(0) (Pd2(dba)3), Benzotriazol-1-ol (HOBt), 1-[3-(Dimethylamino)propyl]-3ethylcarbodiimide hydrochloride (EDCI)

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During

the

review

process

of

this

manuscript

the

Merck

clinical

trial

(ClinicalTrials.gov

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Wood, M. R., Discovery and optimization of a novel, selective and brain penetrant M1 positive allosteric modulator (PAM): The development of ML169, an MLPCN probe. Bioorg. Med. Chem. Lett. 2011, 21, 2697-2701; (s) Tarr, J. C.; Turlington, M. L.; Reid, P. R.; Utley, T. J.; Sheffler, D. J.; Cho, H. P.; Klar, R.; Pancani, T.; Klein, M. T.; Bridges, T. M.; Morrison, R. D.; Blobaum, A. L.; Xiang, Z.; Daniels, J. S.; Niswender, C. M.; Conn, P. J.; Wood, M. R.; Lindsley, C. W., Targeting selective activation of M(1) for the treatment of Alzheimer's disease: further chemical optimization and pharmacological characterization of the M(1) positive allosteric modulator ML169. ACS Chem. Neurosci. 2012, 3, 884-895; (t) Mistry, S. N.; Valant, C.; Sexton, P. M.; Capuano, B.; Christopoulos, A.; Scammells, P. J., Synthesis and pharmacological profiling of analogues of benzyl quinolone carboxylic acid (BQCA) as allosteric modulators of the M1 muscarinic receptor. J. Med. Chem. 2013, 56, 5151-5172; (u) Davie, B. J.; Sexton, P. M.; Capuano, B.; Christopoulos, A.; Scammells, P. J., Development of a photoactivatable allosteric ligand for the M1 muscarinic acetylcholine receptor. ACS Chem. Neurosci. 2014, 5, 902-907; (v) Quattropani, A.; Kulkarni, S. S.; Murugesan, K.; Banerjee, J. Preparation of tetraaza-cyclopenta[a]indenyl derivatives as muscarinic M1 mediators. WO 2014198808 Dec 18, 2014; (w) Sugimoto, T.; Shimokawa, K.; Kojima, T.; Sakamoto, H.; Fujimori, I.; Nakamura, M.; Yamada, M.; Murakami, M.; Kamata, M.; Suzuki, S. Nitrogen-containing heterocyclic compounds and their use for pharmaceuticals and cholinergic muscarinic M1 receptor positive allosteric modulation. WO 2015174534 Nov 19, 2015; (x) Davoren, J. E.; O'Neil, S. V.; Anderson, D. P.; Brodney, M. A.; Chenard, L.; Dlugolenski, K.; Edgerton, J. R.; Green, M.; Garnsey, M.; Grimwood, S.; Harris, A. R.; Kauffman, G. W.; LaChapelle, E.; Lazzaro, J. T.; Lee, C.-W.; Lotarski, S. M.; Nason, D. M.; Obach, R. S.; Reinhart, V.; SalomonFerrer, R.; Steyn, S. J.; Webb, D.; Yan, J.; Zhang, L., Design and optimization of selective azaindole amide M1 positive allosteric modulators. Bioorg. Med. Chem. Lett. 2016, 26, 650-655; (y) Mistry, S. N.; Jorg, M.; Lim, H.; Vinh, N. B.; Sexton, P. M.; Capuano, B.; Christopoulos, A.; Lane, J. R.; Scammells, P. J., 4-Phenylpyridin-2-one derivatives: A novel class of positive allosteric modulator of the M1 muscarinic acetylcholine receptor. J. Med. Chem. 2016, 59, 388-409; (z) Davoren, J. E.; O'Neil, S. V.; Anderson, D. P.; Brodney, M. A.; Chenard, L.; Dlugolenski, K.; Edgerton, J. R.; Green, M.; Garnsey, M.; Grimwood, S.; Harris, A. R.; Kauffman, G. W.; LaChapelle, E.; Lazzaro, J. T.; Lee, C. W.; Lotarski, S. M.; Nason, D. M.; Obach, R. S.; Reinhart, V.; SalomonFerrer, R.; Steyn, S. J.; Webb, D.; Yan, J.; Zhang, L., Design and optimization of selective azaindole amide M1 positive allosteric modulators. Bioorg. Med. Chem. Lett. 2016, 26, 650-655; (aa) Mistry, S. N.; Lim, H.; Jorg, M.;



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Capuano, B.; Christopoulos, A.; Lane, J. R.; Scammells, P. J., Novel Fused Arylpyrimidinone Based Allosteric Modulators of the M Muscarinic Acetylcholine Receptor. ACS Chem. Neurosci. 2016. 15. Brodney, M. A.; Davoren, J. E.; Garnsey, M. R.; Zhang, L.; O'Neil, S. V. Pyridine derivatives as muscarinic m1 receptor positive allosteric modulators. WO 2016009297 July 02, 2015. 16. Wager, T. T.; Hou, X.; Verhoest, P. R.; Villalobos, A., Moving beyond rules: the development of a central nervous system multiparameter optimization (CNS MPO) approach to enable alignment of druglike properties. ACS Chem. Neurosci. 2010, 1, 435-449. 17. (a) Li, J.; Grillo, A. S.; Burke, M. D., From synthesis to function via iterative assembly of Nmethyliminodiacetic acid boronate building blocks. Acc. Chem. Res. 2015, 48, 2297-2307; (b) Gillis, E. P.; Burke, M. D., Iterative Cross-Couplng with MIDA Boronates: Towards a General Platform for Small Molecule Synthesis. Aldrichimica acta 2009, 42, 17-27. 18. Water network was modelled by a 20ns molecular dynamics simulation using Desmond in Maestro 2015-3 with an OPLS3 force field. 19. The exemplified THP ring has been highlighted in multiple Merck patents, for a recent relevant example see: WO 2011159554. 20. Though the homology model does not explain the steep SAR observed on the amide bond, it is worth noting that in certain molecular dynamics simulations we did observe a through water hydrogen bond between the THP-OH group and Ser412. 21. Ryckmans, T.; Edwards, M. P.; Horne, V. A.; Correia, A. M.; Owen, D. R.; Thompson, L. R.; Tran, I.; Tutt, M. F.; Young, T., Rapid assessment of a novel series of selective CB(2) agonists using parallel synthesis protocols: A Lipophilic Efficiency (LipE) analysis. Bioorg. Med. Chem. Lett. 2009, 19, 4406-4409. 22. Feng, B.; Mills, J. B.; Davidson, R. E.; Mireles, R. J.; Janiszewski, J. S.; Troutman, M. D.; de Morais, S. M., In vitro P-glycoprotein assays to predict the in vivo interactions of P-glycoprotein with drugs in the central nervous system. Drug. Metab. Dispos. 2008, 36, 268-275. 23. Reichel, A., The role of blood-brain barrier studies in the pharmaceutical industry. Curr. Drug Metab. 2006, 7, 183-203. 24. Lombardo, F.; Shalaeva, M. Y.; Tupper, K. A.; Gao, F., ElogD(oct): a tool for lipophilicity determination in drug discovery. 2. Basic and neutral compounds. J. Med. Chem. 2001, 44, 2490-2497.


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25. Di, L.; Whitney-Pickett, C.; Umland, J. P.; Zhang, H.; Zhang, X.; Gebhard, D. F.; Lai, Y.; Federico, J. J., 3rd; Davidson, R. E.; Smith, R.; Reyner, E. L.; Lee, C.; Feng, B.; Rotter, C.; Varma, M. V.; Kempshall, S.; Fenner, K.; El-Kattan, A. F.; Liston, T. E.; Troutman, M. D., Development of a new permeability assay using low-efflux MDCKII cells. J. Pharm. Sci. 2011, 100, 4974-4985. 26. Johnson, T. W.; Dress, K. R.; Edwards, M., Using the Golden Triangle to optimize clearance and oral absorption. Bioorg. Med. Chem. Lett. 2009, 19, 5560-5564. 27. Independent to our own research efforts Takeda published a patent on a set of compounds containing a similarly substituted pyridine ring, see: WO15190564. 28. Johnson, T. W.; Richardson, P. F.; Bailey, S.; Brooun, A.; Burke, B. J.; Collins, M. R.; Cui, J. J.; Deal, J. G.; Deng, Y. L.; Dinh, D.; Engstrom, L. D.; He, M.; Hoffman, J.; Hoffman, R. L.; Huang, Q.; Kania, R. S.; Kath, J. C.; Lam, H.; Lam, J. L.; Le, P. T.; Lingardo, L.; Liu, W.; McTigue, M.; Palmer, C. L.; Sach, N. W.; Smeal, T.; Smith, G. L.; Stewart, A. E.; Timofeevski, S.; Zhu, H.; Zhu, J.; Zou, H. Y.; Edwards, M. P., Discovery of (10R)-7-amino12-fluoro-2,10,16-trimethyl-15-oxo-10,15,16,17-tetrahydro-2H-8,4-(metheno)pyrazolo[4,3-h][2,5,11]benzoxadiazacyclotetradecine-3-carbonitrile (PF-06463922), a macrocyclic inhibitor of anaplastic lymphoma kinase (ALK) and c-ros oncogene 1 (ROS1) with preclinical brain exposure and broad-spectrum potency against ALKresistant mutations. J. Med. Chem. 2014, 57, 4720-4744. 29. For additional SAR see patent application: Brodney, M. A.; Davoren, J. E.; Garnsey, M. R.; Zhang, L.; O'Neil, S. V. Pyridine derivatives as muscarinic m1 receptor positive allosteric modulators. 2016, WO 2016009297 30. Kenakin, T. P., A Pharmacology Primer: Techniques for More Effective and Strategic Drug Discovery. Fourth ed.; 2014; Vol. 75, p 469-470. 31. FLIPR, IP1 and β-arrestin assays were run using three different cell lines with differing M1 expression levels (Bmax), determined using [3H]NMS binding:

FLIPR, 0.542 pmol/mg; IP1, 20.7 pmol/mg; β-arrestin, 33.2

pmol/mg. 32. (a) Dasari, S.; Gulledge, A. T., M1 and M4 receptors modulate hippocampal pyramidal neurons. J. Neurophysiol. 2011, 105, 779-792; (b) Langmead, C. J.; Austin, N. E.; Branch, C. L.; Brown, J. T.; Buchanan, K. A.; Davies, C. H.; Forbes, I. T.; Fry, V. A.; Hagan, J. J.; Herdon, H. J.; Jones, G. A.; Jeggo, R.; Kew, J. N.; Mazzali, A.; Melarange, R.; Patel, N.; Pardoe, J.; Randall, A. D.; Roberts, C.; Roopun, A.; Starr, K. R.; Teriakidis, A.; Wood,



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M. D.; Whittington, M.; Wu, Z.; Watson, J., Characterization of a CNS penetrant, selective M1 muscarinic receptor agonist, 77-LH-28-1. Br. J. Pharmacol. 2008, 154, 1104-1115. 33. (a) Ehlert, F. J., Analysis of allosterism in functional assays. J. Pharmacol. Exp. Ther. 2005, 315, 740-754; (b) Kenakin, T., New concepts in drug discovery: collateral efficacy and permissive antagonism. Nat. Rev. Drug Discov. 2005, 4, 919-927; (c) Price, M. R.; Baillie, G. L.; Thomas, A.; Stevenson, L. A.; Easson, M.; Goodwin, R.; McLean, A.; McIntosh, L.; Goodwin, G.; Walker, G.; Westwood, P.; Marrs, J.; Thomson, F.; Cowley, P.; Christopoulos, A.; Pertwee, R. G.; Ross, R. A., Allosteric modulation of the cannabinoid CB1 receptor. Mol. Pharmacol. 2005, 68, 1484-1495. 34. Kenakin, T., Gaddum Memorial Lecture 2014: receptors as an evolving concept: from switches to biased microprocessors. Br. J. Pharmacol. 2015, 172, 4238-4253. 35. Agonist EC50 in the M1 FLIPR assay was highly variable. See supporting more detail. 36. (a) Ehlert, F. J.; Thomas, E. A., Functional role of M2 muscarinic receptors in the guinea pig ileum. Life Sci. 1995, 56, 965-971; (b) Ehlert, F. J.; Griffin, M. T.; Sawyer, G. W.; Bailon, R., A simple method for estimation of agonist activity at receptor subtypes: comparison of native and cloned M3 muscarinic receptors in guinea pig ileum and transfected cells. J. Pharmacol. Exp. Ther. 1999, 289, 981-992. 37. A caveat to this data is that we did not determine if this is was a true agonist effect or if compound 38 is modulating an unknown endogenous level of acetylcholine. 38. To measure the effects of compound 38 on GPI in PAM mode we combined compound 38 with 200 nM donepezil, an acetylcholinesterase inhibitor, which produced a contraction that was equivalent to an EC20 of Carbachol's maximal response in this preparation. 39. (a) Vanover, K. E.; Veinbergs, I.; Davis, R. E., Antipsychotic-like behavioral effects and cognitive enhancement by a potent and selective muscarinic M-sub-1 receptor agonist, AC-260584. Behav. Neurosci. 2008, 122, 570-575; (b) Jones, C. K.; Brady, A. E.; Davis, A. A.; Xiang, Z.; Bubser, M.; Tantawy, M. N.; Kane, A. S.; Bridges, T. M.; Kennedy, J. P.; Bradley, S. R.; Peterson, T. E.; Ansari, M. S.; Baldwin, R. M.; Kessler, R. M.; Deutch, A. Y.; Lah, J. J.; Levey, A. I.; Lindsley, C. W.; Conn, P. J., Novel selective allosteric activator of the M1 muscarinic acetylcholine receptor regulates amyloid processing and produces antipsychotic-like activity in rats. J. Neurosci. 2008, 28, 1042210433.


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40. Popiolek, M.; Nguyen, D.; Reinhart, V.; Edgerton, J.; Harms, J.; Lotarski, S. M.; Steyn, S. J.; Davoren, J. E.; Grimwood, S., Quantifying CNS M1 muscarinic aetylcholine receptor modulation using an in-vivo IP1 accumulation assay. Unpublished Results. 41. (a) Tracy, J. I.; Monaco, C.; Giovannetti, T.; Abraham, G.; Josiassen, R. C., Anticholinergicity and cognitive processing in chronic schizophrenia. Biological psychology 2001, 56, 1-22; (b) Vinogradov, S.; Fisher, M.; Warm, H.; Holland, C.; Kirshner, M. A.; Pollock, B. G., The cognitive cost of anticholinergic burden: decreased response to cognitive training in schizophrenia. Am. J. Psychiatry 2009, 166, 1055-1062; (c) Minzenberg, M. J.; Poole, J. H.; Benton, C.; Vinogradov, S., Association of anticholinergic load with impairment of complex attention and memory in schizophrenia. Am. J. Psychiatry 2004, 161, 116-124. 42. Kumari, V.; Soni, W.; Mathew, V. M.; Sharma, T., Prepulse inhibition of the startle response in men with schizophrenia: effects of age of onset of illness, symptoms, and medication. Archives of general psychiatry 2000, 57, 609-614. 43. Data from the individual decibels is in the supporting information. 44. In dog, it was challenging to obtain high plasma exposures due to 38’s the low thermodynamic solubility (2.7 ug/mL at pH 6.5) that limited absorption. To improve exposure the formulation was changed to a spray dried dispersion (SDD). 45. Alt, A.; Pendri, A.; Bertekap, R. L., Jr.; Li, G.; Benitex, Y.; Nophsker, M.; Rockwell, K. L.; Burford, N. T.; Sum, C. S.; Chen, J.; Herbst, J. J.; Ferrante, M.; Hendricson, A.; Cvijic, M. E.; Westphal, R. S.; O'Connell, J.; Banks, M.; Zhang, L.; Gentles, R. G.; Jenkins, S.; Loy, J.; Macor, J. E., Evidence for classical cholinergic toxicity associated with selective activation of M1 muscarinic receptors. J. Pharmacol. Exp. Ther. 2016, 356, 293-304. 46. (a) Cruickshank, J. W.; Brudzynski, S. M.; McLachlan, R. S., Involvement of M1 muscarinic receptors in the initiation of cholinergically induced epileptic seizures in the rat brain. Brain Res. 1994, 643, 125-129; (b) Hamilton, S. E.; Loose, M. D.; Qi, M.; Levey, A. I.; Hille, B.; McKnight, G. S.; Idzerda, R. L.; Nathanson, N. M., Disruption of the m1 receptor gene ablates muscarinic receptor-dependent M current regulation and seizure activity in mice. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 13311-13316. 47.

http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm292340.pdf

(access date February 11, 2016)



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48. Friesen, D. T.; Shanker, R.; Crew, M.; Smithey, D. T.; Curatolo, W. J.; Nightingale, J. A., Hydroxypropyl methylcellulose acetate succinate-based spray-dried dispersions: an overview. Mol. Pharm. 2008, 5, 1003-1019.


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Discovery of the Potent and Selective M1 PAM-Agonist N-[(3 R, 4 S)-3

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