NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.

Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-.

Cover of Probe Reports from the NIH Molecular Libraries Program

Probe Reports from the NIH Molecular Libraries Program [Internet].

Show details

Extended Probe Characterization: Development of an M4 PAM with Improved Activity and Brain Exposure, while Avoiding Species Bias

, , , , , , , , , , , , , , and *.

Received: ; Last Update: March 7, 2013.

This new M4 positive allosteric modulator (PAM) probe, ML253, is the result of an extended probe characterization project, based upon ML108 and ML173, to improve M4 activity at both human and rat receptors and to increase brain exposure. ML253 achieved these goals by affording balanced, nanomolar PAM activity at both human (M4 EC50 = 37 nM) and rat M4 (M4 EC50 = 170 nM), an unprecedented 139-fold shift of the ACh concentration response curve (CRC) and displaying an oral BrainAUC:PlasmaAUC of 2.5. Moreover, ML253 was selective for M4 (>30 μM versus, M1–3, M5), possessed a clean ancillary pharmacological profile in a Ricerca radioligand binding panel of 68 G-protein-coupled receptors (GPCRs), ion channels and transporters and reversed amphetamine-induced hyperlocomotion in rats.

Assigned Assay Grant #: MH077607-1

Screening Center Name & PI: Vanderbilt Specialized Chemistry Center for Accelerated Probe Development, Craig W. Lindsley

Chemistry Center Name & PI: Vanderbilt Specialized Chemistry Center, Craig W. Lindsley

Assay Submitter & Institution: Colleen M. Niswender, Vanderbilt University Medical Center

PubChem Summary Bioassay Identifier (AID): 588743

Probe Structure & Characteristics



CID/ML#Target NameEC50/ (nM) [SID, AID]Anti-target Name(s)IC50 (μM) [SID, AID]Fold SelectiveSecondary Assay(s) Name: IC50/EC50 (nM) [SID, AID]
CID 53382523/ ML253M4hM4 = 0.037
rM4 = 0.17 [SID 125305152, AID 588743]
M1–3,M5>30 [SID 125305152, AID 588749-AID 588752; AID 588744-AID 588747]>800ACh-Fold Shift (139-fold) [SID 125305152, AID 588751]

1. Recommendations for Scientific Use of the Probe

This extended probe (ML253, CID 53382523) can be used both in vitro and in vivo to study the role of selective M4 receptor activation. This probe possesses excellent selectivity versus M1, M2, M3 and M5, as well as a large panel of GPCRs, ion channels and transporters. Moreover, this probe displays balanced potency at both the human M4 receptor (human M4 EC50 = 37 nM) and the rat M4 receptor (human M4 EC50 = 170 nM) surpassing our initial M4 PAM probes ML108 and ML173, with an unprecedented 139-fold shift of the acetycholine (ACh) CRC. ML253 and all the analogs made to date exhibit moderate to high in vivo clearance. In an oral plasma:brain study, ML253 BrainAUC:PlasmaAUC of 2.5, affording significantly improved CNS exposure versus the earlier probes and efficacy in reversing amphetamine-induced hyperlocomotion in rats. Thus, ML253 is a much improved in vitro and in vivo probe for M4 activation, but novel M4 PAM scaffolds are desperately needed to develop an optimal in vivo tool.

2. Materials and Methods

2.1. Assays

  • AID 588743: hM4_PAM_EC_CRC (Summary)
  • AID 588749: Rat M1 PAM Extended Characterization CounterScreen (CRC)
  • AID 588748: Rat M2 PAM Extended Characterization CounterScreen (CRC)
  • AID 588750: Rat M3 PAM Extended Characterization CounterScreen (CRC)
  • AID 588751: Rat M4 PAM Extended Characterization CounterScreen (Fold-Shift)
  • AID 588752: Rat M5 PAM Extended Characterization CounterScreen (CRC)
  • AID 588744: Human M1 PAM Extended Characterization CounterScreen (CRC)
  • AID 588746: Human M2 PAM Extended Characterization CounterScreen (CRC)
  • AID 588745: Human M3 PAM Extended Characterization CounterScreen (CRC)
  • AID 588747: Human M5 PAM Extended Characterization CounterScreen (CRC)
  • AID 588753: ML253_Ricerca_Binding

2.2. Probe Chemical Characterization

Probe compound ML253 (CID 53382523, SID 125305152) was prepared according to the scheme in Figure 1 and had the following characterization. LCMS: RT = 0.560 min, >99% @ 215 nm, >99% @ 254nm, m/z M(35Cl)+H = 347, M(37Cl)+H = 349. 1H NMR (400 MHz, d6-DMSO, δ (ppm)): 8.5 (d; J = 5.6 Hz; 2H), 8.4 (t; J = 6.0 Hz; 1H), 7.3 (d; J = 5.6 Hz; 2H), 6.9 (bs; 2H), 4.4 (d; J = 6.0 Hz; 2H), 2.8 (s; 3H), 2.6 (s; 3H).

Figure 1. Synthesis of Probe ML253.

Figure 1

Synthesis of Probe ML253.

Solubility: Solubility in PBS was determined to be 0.86 μM, which is ~3-fold higher than the EC50 for M4 activation and better than ML108 (0.7 μM) and ML173 (< 0.10 μM).1

Stability: Stability was determined for ML253 at 23 °C in PBS (no antioxidants or other protectorants and DMSO concentration below 0.1%). See table and Figure 2. After 48 hours, >100% of the initial concentration of ML253 remained. ML253 is very stable and the apparent increase in percent parent remaining is likely a result of variability in the assay.1

Figure 2. Stability of ML253.

Figure 2

Stability of ML253.

Percent Remaining (%)
Compound0 Min15 Min30 Min90 min24 Hour48 Hour
ML253, CID 533825231009098107116115

Compounds added to the SMR collection (MLS#s): MLS003871701 (ML253, CID 53382523, 20.0 mg); MLS003871702 (CID 53382532, 5.5 mg); MLS003871703 (CID 53382486, 5.4 mg); MLS003871704 (CID 53382512, 5.3 mg); MLS003871705 (CID 53382542, 5.3 mg); MLS003871706 (CID 53382531, 5.2 mg)

2.3. Probe Preparation

3-amino-5-chloro-4,6-dimethyl-N-(pyridinyl-4-methyl)thieno[2,3-b]pyridine-2-carboxamide, ML253: To a 20 mL microwave vial fitted with a magnetic stir bar was added 2,5-dichloro-4,6-dimethylnicotinonitrile 1 (1.0 g, 5.0 mmol) and MeOH (10 mL). Methyl thioglycolate (490 μL, 5.5 mmol) was added followed by the addition of a 1 M solution of NaOH (aq, 10 mL, 25 mmol). The microwave vial was sealed and the solution was heated to 125 °C for 30 min. The reaction was cooled to room temperature and the solution was diluted with MeOH (75 mL). The solution was concentrated in vacuo to yield 2 as a powder (870 mg, 68%). The solids were of sufficient purity to use without further purification. To a 20 mL vial fitted with a stir bar and a setum cap, was added sodium 3-amino-5-chloro-4,6-dimethylthieno[2,3-b]pyridine-2-carboxylate 2 (130 mg, 0.50 mmol) and the vial was purged with argon. DMF (3 mL) was added followed by triethylamine (210 μL, 1.50 mmol). 2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate methanaminium (HATU, 190 mg, 0.50 mmol) was added to the solution and stirred at ambient temperature for 10 min. 4-aminomethylpyridine (55 μL, 0.54 mmol) was added and the solution was stirred for an additional hour. The mixture was diluted with deionized water (10 mL) and a precipitate formed. The solids were filtered and dried in a vacuum oven at 50 °C for 24 h. Recrystallization from ethanol provided the target compound (81 mg, 51% yield).

3. Results

3.1. Dose Response Curves for Probe

In calcium mobilization assays, ML253 did not activate either human or rat M4 receptors in the absence of ACh. In the precense of an ~EC20 concentration of ACh, ML253 afforded well-behaved PAM CRCs (Figure 3) with high efficacy (80–100% of ACh max).

Figure 3. Human and rat M4 PAM CRCs of ML253 as assessed using calcium mobilization assays.

Figure 3

Human and rat M4 PAM CRCs of ML253 as assessed using calcium mobilization assays. ML253 exhibited a potency of 37 nM (100% ACh Max) for human M4 and an EC50 of 170 nM (73% ACh Max) for rat M4 (AID 588743).

3.2. Cellular Activity

The primary HTS assay and all secondary assays are cell-based assays, indicating that ML253 can gain access to its molecular target when applied to cells. The compound did not exhibit acute toxicity in cell based assays at concentrations up to 30 μM.

3.3. Profiling Assays

To more fully characterize this novel M4 PAM, ML253 was tested using Ricerca’s (formerly MDS Pharma’s) Lead Profiling Screen (binding assay panel of 68 GPCRs, ion channels and transporters screened at 10 μM).2 Included in the Ricerca screening panel are a number of ion channels (Calcium Channel, L-Type and N-Type; Potassium channel [KATP]; Potassium channel [hERG]) and class A GPCRs (D1–5, H1–3, etc…) ML253 was found to weakly bind in only 2 of the 68 assays conducted (inhibition of radio ligand binding > 50% at 10 μM; Table 1).2

Table 1. Ricerca Profiling of ML253.

Table 1

Ricerca Profiling of ML253.

In order to aid the wider community in the use of these compounds, we further profiled a number of the active analogs in a battery of pharmacokinetic assays including an assessment of intrinsic clearance (CLint) in hepatic microsomes. In addition to intrinsic clearance allowing for the prediction of pertinent rat and human PK parameters (CL and t1/2), intrinsic clearance assessments allow for a rank ordering of compounds with respect to oxidative liability and a subsequent predicted stability in in vivo PK and efficacy models (see Table 3 in full report). The majority of these analogs are predicted to have high clearance in rat, with the exception of CID 53382542, the M1, M2, M4 PAM. ML253 is predicted to have high clearance in both rat and human, but possesses reasonable free fraction (~2% free in human and rat equilibrium dialysis plasma protein binding studies). This data suggests that ML253 would likely not be appropriate for oral dosing. However, several other dosing options are available for compounds that undergo significant first-pass metabolism (e.g., subcutaneous dosing, intravenous dosing). Further evaluation would be warranted in order to establish an in vitro and in vivo correlation.

To ensure that the in vitro experiment was predicting the in vivo clearance, we performed a standard rat IV PK experiment (1 mg/kg, IV). Indeed, there was a good in vitro/in vivo correlation. ML253 displayed a CL of ~100 mL/min/kg, a t1/2 of only 20 minutes and a plasma AUC of ~ 200 (hr·ng/mL). We next evaluated CNS exposure, and based on the IV PK, we chose to examine an IP route of administration for both ML253 and CID 53382542. Here, CID 53382542, at a 1 hour time point, displayed a BrainAUC:PlasmaAUC of 2.5, while ML253 engendered a BrainAUC:PlasmaAUC of 0.88 (BrainAUC = 3410 hr·ng/mL, PlasmaAUC = 3884 hr·ng/mL), better than both ML108 and ML173. This study was followed up with a 10 mg/kg PO plasma/brain level study with ML253. Interestingly, this route of administration afforded an improved BrainAUC:PlasmaAUC of 2.5, though the absolute concentrations in plasma (PlasmaAUC = 214 hr·ng/mL) and brain (PlasmaAUC = 546 hr·ng/mL) were reduced relative to the IP study. Nevertheless, ML253 is CNS penetrant small molecule, superior to ML108 and ML173, and with free brain levels above the EC50 for M4 potentiation.

4. Discussion

4.1. Comparison to Existing Art and How the New Probe is an Improvement

ML253 is superior in most aspects to the limited known art in the M4 field, including LY2033298, ML108 and ML173 (Figure 4).3,4 LY2033298 is known to show species differences in potency, which has recently been attributed to differential cooperativity between rat and human M4, limiting its use in vivo in rodents. ML108 has been a useful tool, validating selective M4 activation in multiple preclinical antipsychotic animal models. However, its potency, solubility, and PK properties are poor. ML173 suffers from the same issues in addition to reduced CNS exposure. ML253 addresses the potency issues regarding the prior art, affording potent, balanced activation of M4 on both human (EC50 = 37 nM) and rat (EC50 = 170 nM) with an impressive 139-fold shift of the ACh CRC. In addition, brain exposure through PO dosing is almost tripled relative to ML108 and ML173, and while still high, rat CL is lower than the other known M4 PAMs.

Figure 4. Comparison of the key profiles of the known M4 PAM art and ML253.

Figure 4

Comparison of the key profiles of the known M4 PAM art and ML253.

5. References

Solubility (PBS at pH = 7.4), Stability and Reactivity experiments were conducted at Absorption Systems. For additional information see: https://www​
For information on the Ricerca Lead Profiling Screen see: https://www​
Brady A, Jones CK, Bridges TM, Kennedy PJ, Thompson AD, Breininger ML, Gentry PR, Yin H, Jadhav SB, Shirey J, Conn PJ, Lindsley CW. J. Pharm. & Exp. Ther. 2008;327:941–953. [PMC free article: PMC2745822] [PubMed: 18772318]
Kennedy JP, Bridges TM, Gentry PR, Brogan JT, Brady AE, Shirey JK, Jones CK, Conn PJ, Lindsley CW. ChemMedChem. 2009;4:1600–1607. [PMC free article: PMC2887613] [PubMed: 19705385]
PubReader format: click here to try


  • PubReader
  • Print View
  • Cite this Page

Related information

Related citations in PubMed

See reviews...See all...

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...