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Bioorg Med Chem Lett. Author manuscript; available in PMC 2013 August 1.
Published in final edited form as:
PMCID: PMC3401285
NIHMSID: NIHMS383149

Discovery of N-(4-methoxy-7-methylbenzo[d]thiazol-2-yl)isonicatinamide, ML293, as a novel, selective and brain penetrant positive allosteric modulator of the muscarinic 4 (M4) receptor

Abstract

Herein we describe the discovery and development of a novel class of M4 positive allosteric modulators, culminating in the discovery of ML293. ML293 exhibited modest potency at the human M4 receptor (EC50 = 1.3 µM) and excellent efficacy as noted by the 14.6-fold leftward shift of the agonist concentration-response curve. ML293 was also selective versus the other muscarinic subtypes and displayed excellent in vivo PK properties in rat with low IV clearance (11.6 mL/min/kg) and excellent brain exposure (PO PBL, 10 mg/kg at 1 h, [Brain] = 10.3 µM, B:P = 0.85).

Keywords: Positive allosteric modulator, M4, ML293, CNS penetration, Muscarinic receptor 4

The neurotransmitter acetylcholine (ACh) regulates a diverse set of physiological actions through the activation of two classes of cell-surface receptors. These receptors, the nicotinic ACh receptors (nAChRs)1 are ACh-gated cation channels and the muscarinic ACh receptors (mAChRs) are G protein-coupled receptors (GPCRs).2,3 Both sets of receptors are involved in numerous physiological processes. Molecular cloning has identified five separate subtypes of the mAChRs (M1 – M5) which are subclassified due to their coupling patterns to different G proteins.24 One class couples to Gq, activating phospholipase C (M1,3,5), and the second class couples to Gi/o (M2,4), regulating cAMP levels and various ion channels.24 Two mAChRs are widely distributed in the CNS (M1 and M4) and have been shown in pre-clinical experiments to be potentially viable therapeutic targets for Alzheimer’s disease and schizophrenia.5 In addition to pre-clinical validation, these mechanisms have been shown to be clinically effective utilizing the non-selective M1/M4-preferring agonist xanomeline.6 Finally, significant work utilizing knockout mice has shown the importance of the role of M1 and M4 in cognition and psychosis, respectively.2, 4

Unfortunately, due to high sequence homology and conservation of the ACh binding site among the mAChRs, development of selective orthosteric modulators has been difficult. Due to this, we and others have been investigating the use of allosteric modulators in order to overcome the homology of the orthosteric site and develop selective tools for these receptors. As part of the NIH-sponsored Molecular Libraries Probe Production Center Network, we have recently reported three selective M4 positive allosteric modulator probe molecules (Figure 1). These molecules have been important tool compounds and have shown efficacy in pre-clinical in vivo animal models of schizophrenia; however, these compounds suffer from less than ideal pharmacokinetic properties.7

Figure 1
Previously reported M4 probe molecules ML108, ML173, ML253.

The first SAR library kept the left-hand benzothiazole portion constant and looked at a variety of amide analogs (Table 1). These compounds were synthesized by utilizing the commercially available 4-methoxy-7-methylbenzo[d]thiazol-2-amine and coupling with an appropriate acid chloride under basic conditions. The original molecule, 1, reconfirmed and exhibited micromolar potency (EC50 = 1.8 µM). Addition of a methyl group, 18, led to an inactive compound; in addition, replacement of the dioxine moiety with a saturated pyran was deleterious to activity (16 and 17). Replacement with other heteroaryl groups proved more fruitful with the 2-furyl group (4, 1.3 µM) showing a slight improvement in activity. In addition, 3-pyridyl (20, 2.33 µM), 4-pyridyl (21, 1.3 µM) and 2-pyrazine (23, 1.1 µM) modifications were all well tolerated. However, these analogs showed significant differences in efficacy (%ACh Max) with the 4-pyridyl analog, 20, proving to be the most efficacious (65% vs. <40% for 20 and 23). Replacement of the heterocyclic moiety with alkyl, cycloalkyl or aromatic groups led to inactive compounds.

Table 1
SAR around the eastern amide moiety.8

Due to these shortcomings, we were still interested in the discovery of novel M4 tool compounds that offer improved in vivo PK properties. To this end, we performed a screen at Vanderbilt which revealed a novel structural class of compounds as potential M4 positive allosteric modulators (Figure 2, 1). A small library was generated around this molecule and from this effort there were many compounds that potentiated a submaximal (EC20) concentration of ACh (Figure 2). Based on these promising initial results, a lead optimization campaign was initiated in our laboratories.

Figure 2
Initial ACh EC20 triage screen of M4 analogs. Assay was performed at 10 µM using hM4-Gqi5-CHO cells; activity was monitored using an intracellular calcium mobilization assay. Values represent a single screening experiment performed in triplicate; ...

Next, we turned our attention to amide replacements – an exercise which proved to be less informative and highlights the shallow nature of allosteric modulator SAR (Table 2).9 Moving from the 4-pyridyl amide to the 4-pyridyl urea (29, inactive) led to a complete loss of activity. In fact, all ureas that were analyzed were inactive, with the lone exception being 35, which was weak in terms of both potency (7.1 µM) and efficacy (36%). Other amide replacements (sulfonamide/reverse amides) were not tolerated, resulting in either inactive or weakly active compounds.

Table 2
Amide replacements.8

Lastly, we investigated replacements of the benzothiazole moiety (Table 3). Removal of both the 4-methoxy and 7-methyl groups were not tolerated (50). In addition, replacement of the benzothiazole with thiazolo[5,4-b]pyridine groups also resulted in inactive compounds (5155). However, deletion of the 7-methyl group (while maintaining the 4-methoxy) was tolerated, resulting in compound 56 (2.8 µM).

Table 3
Benzothiazole replacements.8

We determined that compound 21 possessed the appropriate balance between in vitro potency and efficacy, thus, this compound was further evaluated in our muscarinic selectivity panel as well as in a fold-shift assay (an evaluation of a compound’s ability to produce a left-ward shift of the ACh curve). Compound 21 was examined at the other four muscarinic subtypes (M1,2,3,5) and was inactive against each of these receptors at a concentration up to 30 µM. In our fold-shift assay (at a constant concentration, 30 µM), 21 displayed a robust left-ward shift of the ACh curve (14-fold) showing a potentiation of the ACh potency (Figure 3B). Based on the hM4 potency, fold shift and selectivity profile, 21 was declared an MLPCN probe molecule and redesignated ML293.10

Figure 3
A. Concentration-response curve of ML293 in human M4 cells in the presence of an EC20 concentration of ACh. B. ACh fold-shift assay for ML293 at a 30 µM concentration. Data represent three independent determinations performed in triplicate.

ML293 was next profiled in a number of Tier 1 in vitro pharmacokinetic assays (Table 4). ML293 was assessed using an intrinsic clearance assay (CLINT) in hepatic microsomes to evaluate the oxidative metabolism potential. This assay also provides an in vitro prediction for eventual assessment of clearance values in in vivo PK studies (CLHEP). ML293 was predicted to display moderate clearance in both human and rat microsomes (14.9 and 48.5 mL/min/kg). In addition to intrinsic clearance, we determined the plasma protein binding (human and rat equilibrium dialysis studies) of ML293. ML293 showed high plasma protein binding in human plasma (1.0%, unbound); however, in rat plasma, ML293 showed a more favorable free fraction (3.2%, unbound). Lastly, ML293 was evaluated in a rat brain homogenate binding study to predict the fraction of unbound compound in brain. ML293 showed higher brain homogenate binding profile versus the plasma binding (BHB, 0.9% unbound vs. 3.2% unbound, respectively).

Table 4
In vitro and in vivo properties of ML293.12

As a follow-up to the in vitro studies, a standard rat IV PK experiment was performed to determine the in vivo clearance of ML293. In this experiment (1 mg/kg, IV), ML293 exhibited low clearance (CLp = 11.5 mL/min/kg) with a modest half-life (t1/2 = 57 mins) and significant plasma levels (AUC = 1445 hr•ng/mL, 4.8 µM). These values are in contrast to the previously reported M4 probe molecules (Figure 1), as each of those molecules exhibit high clearance in rat when assessed using IV PK experiments (CLp > 100 mL/min/kg).7b,11 Lastly, we evaluated the CNS exposure of ML293 in a PBL (Plasma:Brain level) experiment after a single dose and at a single time point. ML293 was dosed orally at 10 mg/kg and after 1 hour the Brain:Plasma (B/P) ratio was 0.85 with absolute brain levels of 3093 ng/g (~10 µM). In addition, in this experiment we assessed the concentrations in the hepatic portal vein as well as plasma. The levels found in the HPV and plasma can be used as another assessment of the extent of oxidative metabolism, as a ratio at or close to 1:1 indicates a low level of metabolism; ML293 exhibits a roughly 0.8 ratio in these studies. Based on these experiments, ML293 is a potent and selective M4 PAM with favorable pharmokinetics that demonstrates significant CNS penetration following oral administration.

In conclusion, we have discovered an additional M4 tool compound (ML293) with superior in vivo PK properties compared to previously published molecules. ML293 represents a novel chemical scaffold as other reported M4 PAMs have been derived from a common 3-amino-N-(aryl)-4,6-disubstitutedthieno[2,3-b]pyridine-2-carboxamide moiety, and have all suffered from similar poor in vivo PK properties. ML293 is a potent and selective small molecule M4 positive allosteric modulator that significantly leftward shifts the ACh curve (14.9 Fold Shift). In addition, ML293, in contrast to the previously reported compounds, displays low clearance when evaluated in an in vivo rat IV clearance assay and is highly brain penetrant (B:P = 0.85, [brain] = ~10 µM).

Acknowledgments

The authors would like to thank Tammy Santomango, Frank Byers, and Ryan Morrison for technical assistance with the PK experiments and Nathan Kett and Sichen Chang for the purification of compounds. Vanderbilt is a member of the MLPCN and houses the Vanderbilt Specialized Chemistry Center for Accelerated Probe Development (U54MH084659).

Footnotes

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References and notes

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8. Human muscarinic acetylcholine receptor cell line creation and culture. Human M1 (hM1) cDNA in pcDNA3.1(+) was purchased from www.cDNA.org and stably transfected into Chinese hamster ovary (CHO K1) cells purchased from the ATCC ( www.atcc.org). CHO cells stably expressing hM2, hM3, and hM5 were generously provided by A. Levey (Emory University, Atlanta, GA). hM4 cDNA in pcDNA3.1 (+) was purchased from www.cDNA.org and stably transfected into CHOK1 cells. To make stable hM2 and hM4 cell lines for use in calcium mobilization assays, cell lines were cotransfected with the chimeric G protein Gqi5 (Conklin et al., 1993). All transfections utilized Lipofectamine 2000. hM1, hM3, and hM5 CHO cells were cultured in Ham’s F-12; 10% heat-inactivated fetal bovine serum (FBS), 20mM HEPES, and 500 µg/mL G418 (Mediatech, Inc., Herndon, VA). hM2-Gqi5 and hM4-Gqi5 were grown in the same medium supplemented with 500 µg/mL hygromycin B. All cell culture reagents were purchased from Invitrogen Corp. (Carlsbad, CA) unless otherwise noted. Calcium Mobilization Assays - Potency determinations. Assays were performed within the Vanderbilt Center for Neuroscience Drug Discovery’s Screening Center. CHO cell lines expressing muscarinic acetylcholine receptors were plated (15,000 cells/20 µl/well) in black-walled, clear-bottomed, TC treated, 384 well plates (Greiner Bio-One, Monroe, North Carolina) in Ham’s F-12, 10% FBS, 20 mM HEPES. The cells were grown overnight at 37 °C in the presence of 5% CO2. The following day, plated cells had their medium exchanged to Assay Buffer (Hank's balanced salt solution, 20 mM HEPES and 2.5 mM Probenecid (Sigma-Aldrich, St. Louis, MO)) using an ELX405 microplate washer (BioTek), leaving 20 µL/well, followed by addition of with 20 µL of 4.5 µM Fluo-4, AM (Invitrogen, Carlsbad, CA) prepared as a 2.3 mM stock in DMSO and mixed in a 1:1 ratio with 10% (w/v) pluronic acid F-127 and diluted in Assay Buffer for 45 minutes at 37 °C. The dye was then exchanged to Assay Buffer using an ELX405, leaving 20 µL/well and the plates were incubated at room temperature for 10 min prior to assay. Test compounds were transferred to daughter plates using an Echo acoustic plate reformatter (Labcyte, Sunnyvale, CA) and then diluted into Assay Buffer to generate a 2× stock in 0.6% DMSO (0.3% final). Acetylcholine (ACh) EC20 and EC80 were prepared at a 5X stock solution in assay buffer prior to addition to assay plates. Calcium mobilization was measured at 37 °C using a Functional Drug Screening System 6000 or 7000 (FDSS6000 or FDSS7000, Hamamatsu, Japan) kinetic plate reader according to the following protocol. Cells were preincubated with test compound (or vehicle) for 144 seconds prior to the addition of an EC20 concentration of the agonist, ACh. 86 seconds after this addition, an EC80 concentration of ACh was added. Control wells also received a maximal ACh concentration (1 mM) for eventual response normalization. The signal amplitude was first normalized to baseline and then as a percentage of the maximal response to ACh. Microsoft XLfit (IDBS, Bridgewater, NJ) was utilized for curve fitting and EC50 value determination using a four point logistical equation. Calcium Mobilization Assays – Fold-Shift Assays. Assays were performed within the Vanderbilt Center for Neuroscience Drug Discovery’s Screening Center. CHO cell lines expressing muscarinic acetylcholine receptors were plated (15,000 cells/20 µL/well) in black-walled, clear-bottomed, TC treated, 384 well plates (Greiner Bio-One, Monroe, North Carolina) in Ham’s F-12, 10% FBS, 20 mM HEPES. The cells were grown overnight at 37 °C in the presence of 5% CO2. The following day, plated cells had their medium exchanged to Assay Buffer (Hank's balanced salt solution, 20 mM HEPES and 2.5 mM Probenecid (Sigma-Aldrich, St. Louis, MO)) using an ELX405 microplate washer (BioTek), leaving 20 µL/well, followed by addition of with 20 µL of 4.5 µM Fluo-4, AM (Invitrogen, Carlsbad, CA) prepared as a 2.3 mM stock in DMSO and mixed in a 1:1 ratio with 10% (w/v) pluronic acid F-127 and diluted in Assay Buffer for 45 minutes at 37 °C. The dye was then exchanged to Assay Buffer using an ELX405, leaving 20 µL/well and the plates were incubated at room temperature for 10 min prior to assay. Test compounds were prepared in Assay Buffer to generate a 2× stock in 0.6% DMSO (0.3% final). Acetylcholine (ACh) concentration responses were prepared at a 5X stock solution in assay buffer prior to addition to assay plates. Calcium mobilization was measured at 37 °C using a Functional Drug Screening System 6000 or 7000 (FDSS6000 or FDSS7000, Hamamatsu, Japan) kinetic plate reader according to the following protocol. Cells were preincubated with test compound (or vehicle) for 144 seconds prior to the addition of a concentration response of the agonist ACh and the fluorescence was monitored for a total of 5 min. The signal amplitude was first normalized to baseline and then as a percentage of the maximal response to acetylcholine. Microsoft XLfit (IDBS, Bridgewater, NJ) was utilized for curve fitting and EC50 value determination using a four point logistical equation. Fold-Shift values were calculated by dividing the ACh EC50 in the presence of 30 µM compound by the ACh EC50 in the presence of vehicle.
9. (a) Engers DW, Niswender CM, Weaver CD, Jadhav S, Menon UN, Zamorano R, Conn PJ, Lindsley CW, Hopkins CR. J Med Chem. 2009:52. [PMC free article] [PubMed](b) Williams R, Johnson KA, Gentry PR, Niswender CM, Weaver CD, Conn PJ, Lindsley CW, Hopkins CR. Bioorg Med Chem Lett. 2009:19. [PMC free article] [PubMed](c) Zhao Z, Wisnoski DD, O'Brien JA, Lemaire W, Williams DL, Jr, Jacobsen MA, Wittman M, Ha SN, Schaffhauser H, Sur C, Pettibone DJ, Duggan ME, Conn PJ, Hartmann GD, Lindsley CW. Bioorg. Med. Chem. Lett. 2007:17.
10. VU0409524 (ML293) has been declared a probe via the Molecular Libraries Probe Production Centers Network (MLPCN) and is freely available through the network, see: http://mli.nih.gov/mli/.
11. Lewis LM, Bridges TM, Niswender CM, Weaver CD, Lindsley CW. Bookshelf ID: NBK50687. 2010
12. N-(4-methoxy-7-methylbenzo[d]thiazol-2-yl)isonicotinamide, ML293: To a stirred solution of 4-methoxy-7-methylbenzo[d]thiazol-2-amine (0.050 g, 0.26 mmol) and DIEA (0.112 mL, 0.64 mmol) in DCM (1.3 mL) at 0 °C was added isonicotinoyl chloride hydrochloride (0.050 g, 0.28 mmol). The reaction was allowed to warm to room temperature and stir overnight. The reaction was concentrated under vacuum and the residue purified by reverse-phase HPLC to give 55 mg (71%) of the pure product. 1H NMR (400 MHz, DMSO-d6): δ 8.82 (d, J = 6.0 Hz, 2H), 8.02 (d, J = 6.1 Hz, 2H), 7.11 (d, J = 8.1 Hz, 1H), 6.95 (d, J = 8.1 Hz, 1H), 3.90 (s, 3H), 2.45 (s, 3H). LC/MS: RT = 0.60 min., m/z = 300 [M + H]+.