A recently completed functional, high throughput screen of the Molecular Libraries Probe Production Center's Network (MLPCN) screening deck of ∼360,000 compounds conducted by Scripps Research Institute Molecular Screening Center (SRIMSC) against three of the five muscarinic receptor subtypes (M₁, M₄ and M₅) provided a number of interesting hits. As this was the first time a directed effort had been undertaken to identify M₅ selective leads, we were very pleased by the identification of nine M₅ PAMs and nine M₅ antagonists (or negative allosteric modulators, NAMs), despite the complete absence of M₅ agonist hits. The most promising M₅ inhibitor hit (CID 5189681), was quickly developed into a potent, selective and CNS penetrant probe molecule ML375 (hM₅ NAM IC₅₀ = 300 nM, hM_1-4 IC₅₀s >30 μM) displaying enantioselective inhibition. Detailed molecular pharmacology studies demonstrated that ML375 is the first M₅ NAM ever described, and ML375 show good rat and cyno pharmacokinetics to support in vivo studies in rodents and non-human primates.

Probe Structure & Characteristics

(S)-9b-(4-chlorophenyl)-1-(3,4-difluorobenzoyl)-2,3-dihydro-1H-imidazo[2,1-a]isoindol-5(9bH)-one

1. Recommendations for scientific use of the probe

• What limitations in the current state of the art is the probe addressing?

Prior to the discovery of ML375, there was a complete paucity of selective M₅ inhibitors (few antagonists and no NAMs). ML375 is the most potent and selective M₅ NAM, and the only M₅ NAM, currently known and its demonstrated CNS exposure will allow it to find utility in a wide range of in vivo and ex vivo studies. ML375 will find utility in electrophysiology experiments and animal models exploring the function of M₅ receptors (cognition, migraine, ischemia, and addiction/withdrawal), where dose-exposure relationships can now be obtained. Importantly, ML375 is first in class as a selective M₅ NAM.

• How will the probe be used?

Given ML75's sub-micromolar potency (hM₅ NAM IC₅₀ = 300 nM, rM₅ NAM IC₅₀ = 790 nM) and demonstrated CNS penetrance (vide infra), this probe will find utility in various animal models designed to elucidate the specific functions of the M₅ receptor. Importantly, being in vivo quantifiable, exposure-response relationships will be obtainable rather than just dose-response relationships. Additionally, ex vivo electrophysiology experiments on slice preparations from various regions of the brain could help define the neurological functioning of the M₅ receptor. These future experiments will be greatly aided by the very high M₅ receptor selectivity displayed by ML375 over the M₁ – M₄ receptors.

• Who in the research community will use the probe?

Through the use of knock-out (KO) mutant mice, the research community has learned of numerous potential indications for selective M₅ ligands, for example, in models of cognition, addiction and cerebral blood flow. Those researchers interested in confirming and extending those early behavioral studies with the KO mice will find our M₅ NAM probe invaluable in exploring numerous disease states, just as addiction, and identifying new therapeutic possibilities implicated in the genetic models.

• What is the relevant biology to which the probe can be applied?

In particular, M₅ receptor activation has been implicated in modifying cognitive impairment (such as that associated with Alzheimer's disease) and cerebrovascular blood flow (potential use with migraines, ischemia and cognitive enhancement), while M₅ receptor inhibition has been implicated in addiction/withdrawal mechanisms (substance abuse). Validating any of these potential modes of action could help spur increased medical research around selective M₅ ligand development, with the potential to significantly impact human health. It is essential to validate genetic M₅ knock-out data with pharmacological M₅ inhibition.

Introduction. Acetylcholine (ACh) serves as the endogenous agonist for both the nicotinic (nAChR) and muscarinic acetylcholine receptors. There are five mAChR subtypes (M₁ – M₅) widely expressed in both the central nervous system (CNS) and periphery of mammals. These receptors undertake critical roles in regulating a variety of diverse physiological processes. Within the CNS, the M₁, M₄ and M₅ subtypes are believed to be the most important with respect to normal neuronal functioning. Of these three, M₅ is the least studied as a combined result of its lower expression levels(1) (< 2% of total muscarinic receptor population within the brain) and, until recently, a near absence of highly selective M₅ receptor ligands. The majority of our current beliefs surrounding the function of M₅ has come from M₅ receptor localization, phenotypic observations of M₅ knock-out mice(2) and studies conducted with non-selective muscarinic ligands.(3) It is intriguing to note that the M₅ receptor is the only muscarinic receptor observed the substantia nigra pars compacta (based on mRNA detection),4,5 leading to the prediction that M₅ functions in addiction/reward mechanisms. This hypothesis was supported in man through the clinically observed correlation between a specific M₅ gene mutation and an increase in cigarette consumption (+ 27%), as well as, an increased risk for cannabis dependence (+ 290%).(6) M₅ receptors have also been localized on the cerebrovascular system and shown to be critical in the ACh-induced dilation necessary for normal CNS blood perfusion, without an effect extending into the periphery.(7) In a broader sense, M₅ KO mice show decreased prepulse inhibition,(8) CNS neuronal abnormalities and cognitive deficits.(9) In summary, these studies support the potential for M₅ ligands in the treatment of numerous CNS indications including schizophrenia, Alzheimer's disease, addiction, ischemia and migraine. However, in the absence of highly selective M₅ tool compounds, and ultimately pharmaceuticals, this potential will remain unrealized. There is a critical need for subtype selective antagonists, negative allosteric modulators (NAMs) and positive allosteric modulators (PAMs) to truly understand the physiological, and potential therapeutic role, of the M₅ receptor.

Prior Art. To date, only three publications have described the development of M₅ selective ligands. These manuscripts disclosed the successive improvements of a structurally related class of positive allosteric modulators (PAMs) and the development of three MLPCN probe molecules: ML129(10) ML172(11) and ML326(12) (Figure 1). There is little in the way of prior art for M₅ selective inhibitors (either orhtosteric antagonists of NAMs). To date, the majority of mAChR antagonists (Figure 1) are unselective ligands, such as scopolamine (1), atropine (2) or moderately selective ligands such as the ∼10-fold M₁-preferring pirenzepine (3). Recently in 2013, the first M₅-preferring antagonist (based on binding data – not functional) was reported.(13) Compound 4 bound to M₅ with a K_i of 2.2 μM with 11-fold selectivity versus M₁-M₄, but no DMPK or CNS penetration data was disclosed. Moreover, there are no known M₅ NAMs, and no highly selective and CNS penetrant M₅ antagonists/NAMs. ML375 marks a major advancement in the field that will enable detailed in vitro and in vivo studies to be performed.(14)

Figure 1

Prior Art: M5 PAM Probes ML129, ML172 and ML326, and examples of known mAChR antagonists 1-4, which are lacking in terms of mAChR selectivity and/or DMPK profiles.

2. Materials and Methods

Cell culture and transfections.10,14 Chinese hamster ovary (CHO-K1) cells stably expressing rat (r)M₁ were purchased from the American Type Culture Collection and cultured according to their indicated protocol. CHO cells stably expressing human (h)M₂, hM₃, and hM₅ were generously provided by A. Levey (Emory University, Atlanta, GA); hM₁ and hM₄ cDNAs were purchased from Missouri S&T cDNA Resource; rM₄ cDNA was provided by T. I. Bonner (National Institutes of Health, Bethesda, MD). hM₁, hM₄, and rM₄ cDNAs were used to stably transfect CHO-K1 cells purchased from the American Type Culture Collection using Lipofectamine2000. To make stable hM₂, rM₂, hM₄ and rM₄ cell lines for use in calcium mobilization assays, these cells also were stably transfected with a chimeric G-protein (G_qi5) (provided by B.R. Conklin, University of California, San Francisco) using Lipofectamine 2000. rM₁, hM₃, and hM₅ cells were grown in Ham's F-12 medium containing 10% heat-inactivated fetal bovine serum (FBS), 20mM HEPES, and 50µg/mL G418 sulfate. hM₂–G_qi5 and hM₄–G_qi5 cells were grown in the same medium also containing 500 µg/mL Hygromycin B. Stable rM₄–G_qi5 cells were grown in DMEM containing 10% heat-inactivated FBS, 20 mM HEPES, 400 µg/mL G418 sulfate, and 500 µg/mL Hygromycin B. All cell culture reagents were purchased from Invitrogen Corp. (Carlsbad, CA) unless otherwise noted.

Calcium Mobilization Assays - Potency determinations.10,14 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 2x 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. Compounds showing dose-dependency were assigned ‘Outcome’ = ‘Active’, EC₅₀=’Value’, and % ACh min=’Value’.

[³H]-NMS Competition Binding Assay with CRCs.10,14 Membranes were prepared from stable CHO cell lines constitutively expressing human M₅ receptors according to a previously described protocol (Marlo et al., 2009; Brady et al., 2008). Binding reactions were carried out in 2 mL, clear, 96-well, deep well plates (Axygen Scientific) and contained 0.3 nM [³H]-N-methylscopolamine ([³H]-NMS, obtained commercially from PerkinElmer), 10 μg of membrane protein, and an 11 point concentration range of test compound or atropine in a total volume of 500 μL assay buffer (100 mM NaCl, 10 mM MgCl₂, 20 mM HEPES, pH 7.4). Nonspecific binding was determined in the presence of 10 μM atropine. The K_D of [³H]-NMS was determined empirically to be 0.264 nM. Binding reactions were performed at ambient temperature and allowed to incubate for 3 hours on a Lab-Line Titer plate shaker at setting 7 (∼750 rpm). Reactions were terminated by rapid filtration through GF/B filter plates (1 μm pore size) using a 96-well Brandel harvester and washed 3X with ice-cold harvesting buffer (50 mM Tris-HCl, 0.9% NaCl, pH 7.4). Filter plates were dried overnight and counted in a PerkinElmer TopCount scintillation counter (PerkinElmer Life and Analytical Sciences). Actual [³H]-NMS concentration was back-calculated after counting aliquots of 10X [³H]-NMS used in the reaction. Atropine K_i was determined to be ∼2.45 nM. For all assays, radioligand depletion was kept to approximately 15% or less. Data was plotted and atropine K_i was calculated using the curve-fitting software of GraphPad Prism (version 5.01). Data shown represent mean values obtained from three independent determinations performed using three or more replicates (error bars represent +/- SEM).

[³H]-NMS Dissociation Kinetics Assays.10,14 Membranes were prepared from stable CHO cell lines constitutively expressing human M₅ receptors cells according to a previously described protocol (Marlo et al., 2009; Brady et al., 2008). Binding reactions were carried out in 2 mL, clear, 96-well, deep well plates (Axygen Scientific) and initially contained M₅ CHO cell membranes (10 μg ) with 0.3 nM [³H]-NMS. Binding was allowed to equilibrate at ambient temperature for 3 hours on a Lab-Line Titer plate shaker at setting 7 (∼750 rpm). Atropine (10 μM) and either vehicle or test compound (10 μM) were then added at various time points over 3.5 hours to prevent radioligand reassociation in a total volume of 500 μL. Plate agitation was continued between additions. Nonspecific binding was determined in the presence of 10 μM atropine. Reactions were terminated by rapid filtration through GF/B filter plates (1 μm pore size) using a 96-well Brandel harvester and washed 3X with ice-cold harvesting buffer (50 mM Tris-HCl, 0.9% NaCl, pH 7.4). Filter plates were dried overnight and counted in a PerkinElmer TopCount scintillation counter (PerkinElmer Life and Analytical Sciences). Actual [³H]-NMS concentration was back-calculated after counting aliquots of 10X [³H]-NMS used in the reaction. For all assays, radioligand depletion was kept to approximately 15% or less. Data was plotted and half-life was calculated using the curve-fitting software of GraphPad Prism (version 5.01). Data shown represent mean values obtained from three independent determinations performed using three or more replicates (error bars represent +/- SEM).

Crystallographic Data for Compound ML375(14)

Crystal Data

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C23H14ClF2N2O2	F(000) = 868
Mr = 423.81	Dx = 1.473 Mg m−3
Monoclinic, C2	Cu Kα radiation, λ = 1.54178 Å
Hall symbol: C 2y	Cell parameters from 14922 reflections
a = 23.2072 (4) Å	θ = 3.7–1.0°
b = 6.83657 (6) Å	μ = 2.15 mm−1
c = 14.4411 (3) Å	T = 293 K
β = 123.469 (3)°	Rectangular block
V = 1911.26 (11) Å3	× × mm
Z = 4

Data Collection

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Agilent Xcalibur PX II diffractometer	Rint = 0.024
Radiation source: Enhance (Cu) X-ray Source	θmax = 72.0°, θmin = 3.7°
confocal, multilayer	h = −28→27
17619 measured reflections	k = −8→8
3641 independent reflections	l = −17→17
3544 reflections with I > 2σ(I)

Refinement

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Refinement on F2	Secondary atom site location: difference Fourier map
Least-squares matrix: full	Hydrogen site location: inferred from neighboring sites
R[F2 > 2σ(F2)] = 0.027	H atoms treated by a mixture of independent and constrained refinement
wR(F2) = 0.073	w = 1/[σ2(Fo2) + (0.050P)2 + 0.6517P] where P = (Fo2 + 2Fc2)/3
S = 1.02	(Δ/σ)max = 0.001
3641 reflections	Δρmax = 0.15 e Å−3
281 parameters	Δρmin = −0.27 e Å−3
1 restraint	Absolute structure: Flack H D (1983), Acta Cryst. A39, 876-881
Primary atom site location: structure-invariant direct methods	Flack parameter: 0.002 (10)

Special Details

Geometry. All esds (except the esd in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esds is used for estimating esds involving l.s. planes.

Refinement. Refinement of F² against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F², conventional R-factors R are based on F, with F set to zero for negative F². The threshold expression of F² > 2sigma(F²) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F² are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger.

2.2. Probe Chemical Characterization

Probe compound ML375 (CID 71598521, SID 163678873, VU0483253) was prepared according to scheme 1 and provided the following characterization. (S)-9b-(4-chlorophenyl)-1-(3,4-difluorobenzoyl)-2,3-dihydro-1H-imidazo[2,1-a]isoindol-5(9bH)-one: ¹H NMR (400.1 MHz, CDCl₃) δ (ppm): 8.04-7.99 (m, 1H); 7.90-7.85 (m, 1H); 7.65-7.56 (m, 2H); 7.38-7.30 (m, 3H); 7.25-7.19 (m, 2H); 7.18-7.14 (m, 2H); 4.38-4.30 (m, 1H); 4.01-3.93 (m, 1H); 3.82-3.75 (m, 1H); 3.34-3.25 (m, 1H). ¹³C NMR (100.6 MHz, CDCl₃) δ (ppm): 172.07, 166.84, 151.81 (dd, J_CF = 254 Hz, 12.7 Hz), 150.33 (dd, J_C-F = 252 Hz, 13 Hz) 145.77, 136.65, 134.94, 133.55, 132.91 (t, J = 4.8 Hz), 131.88, 130.61, 129.06, 128.97, 127.53, 124.03, 123.62 (dd, J = 6.8 Hz, 4 Hz), 117.94 (d, J = 17 Hz), 116.83 (d, J = 18 Hz), 87.37, 52.24, 39.70. SFC (214 nM) R_T = 3.591 min (>98%). HRMS (TOF, ES+) C₂₃H₁₆N₂O₂F₂Cl [M+H]⁺ calc. mass 425.0868, found 425.0872. Specific rotation [α] $\frac{23}{D}$ = -168.6° (c = 0.75, CHCl₃).

Scheme 1

Preparation of ML375.

Solubility: Solubility for ML375 in PBS (@ pH = 7.4, 23 °C, final DMSO concentration 1%) was determined to be < 4 μM, which is not optimal, but not unexpected in light of its structure. Future efforts will be directed towards improving the solubility for this novel series of M₅ NAMs through the introduction of weakly basic nitrogens or other solubility enhancing groups, and the truncation of lipophilic regions, as allowed by SAR.

Stability: Stability was determined for ML375 at 23 °C in PBS (no antioxidants or other protectorants, initial ML375 concentration = 1.0 µM and final DMSO concentration 10%). After 48 hours, 89% of the initial concentration of ML375 remained, indicating that ML375 was fairly stable to these buffer conditions, or that a small percentage of ML375 precipitated from solution over the course of the experiment despite the presence of additional DMSO relative to the solubility experiment.

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	Percent Remaining (%)
Compound	0 Min	15 Min	30 Min	90 min.	24 Hour	48 Hour
ML375, CID 71598521	100	100	100	97	92	89

Compounds added to the SMR collection (MLS#s): MLS005000729 (ML375, CID 71598521, 26.2 mg); MLS005000730 (CID 71598550, 8 mg); MLS005000731 (CID 71598549, 6.6 mg); MLS005000732 (CID 12204567, 7.1 mg); MLS005000733 (CID 2999410, 5.7 mg); MLS005000734 (CID 71598548, 6.0 mg).

3. Results

Functional, triple-add assay protocols were provided to the screening center detailing a heterogeneous assay conducted in a 384-well format for M₁, M₄ and M₅ human receptor transfected CHO cells and CHO-K1 untransfected cells (assay provider at Vanderbilt). The screening center (SRIMSC) then adapted and modified these procedures into a homogeneous, 1536-well format to facilitate a more rapid and economical screening of the MLSMR (∼360, 000 compounds). From this effort, we identified CID 5189681 (5) as a weak (M₅ IC₅₀ >10 μM, 41% ACh min), but selective (versus M₁ and M₄) antagonist. Figure 2 shows the structure of 5, the CRC window from the confirmation screen at SCRIPPS as well as a CRC generated at Vanderbilt from the HTS DMSO stock. To confirm the structure of the compound, we resynthesized 5 in a two-step sequence (Scheme 2). Commercial acid 6 was condensed with ethylene diamine to produce tricycle 7, followed by acylation with 4-fluorobenzoyl chloride to afford the HTS hit 5 in 74% overall yield. We then assayed fresh powder of 5, and were very pleased to find that new material was significantly more potent (hM₅ IC₅₀ = 3.49 μM, rM₅ IC₅₀ = 5.67 μM, M₁-M₄ IC₅₀s >30 μM), yet retained M₅ selectivity (Figure 3). Thus, we prepared libraries of over 120 analogs, following slight modifications to Scheme 1, surveying SAR in five regions of the HTS hit 5 (Figure 3), and screened in full CRC mode against human M₅.

Figure 2

A) HTS confirmatory data from SCRIPPS for CID 5189681 (5) showing the antagonist window CRCs on M1, M4, M5 and non-transferred parental CHO cell line, highlighting the selectivity of 5 for M5. B) Confirmation CRC at VUMC for on human M5 (a weak M5 inhibitor: (more...)

Scheme 2

Synthetic route to prepare the HTS hit, CID 5189681 (5).

Figure 3

Rat and human M5 potency and mAChR selectivity of fresh powder of CID 5189681 (5), and the chemical optimization plan to establish SAR for the series en route to an MLPCN probe.

SAR was very shallow/flat, and reminiscent of allosteric SAR. Very quickly, one compound stood out form all those prepared, CID 71598549 (8) a potent, sub-micromolar human M₅ antagonist/NAM (hM₅ IC₅₀ = 480 nM, rM₅ IC₅₀ = 1,100 nM), with no activity at hM₁-M₄ (IC₅₀s >30 mM) (Figure 4). As CID 71598549 (8) was a racemic molecule, we then separated the enantiomers by chiral super critical fluid chromatography to >99% ee. Opticcal rotations were obtained, and all of the M5 inhibitory activity was found to reside in the (-)-enantiomer (a ∼-160°). Thus, whereas the racemic 8 had a hM₅ IC₅₀ of 480 nM, the (-)-enantiomer CID71598521 (9) had a hM₅ IC₅₀ of 300 nM, while the (+)-enantiomer's (CID 71598550, 10) hM₅ IC₅₀ was >30 mM (Figure 5). This was an exciting discovery, but we still did not know the absolute stereochemistry at the 9b stereocenter. Thus, we initiated an effort to grow X-ray quality crystals in an attempt to utilize X-ray crystallography to elucidate the absolute stereochemistry at 9b. In short order, crystals were grown that displayed excellent diffraction and enabled our small molecule X-ray facility to definitively assign the absolute stereochemistry of the 9b stereocenter in CID 71598521 (9) as the (S)-enantiomer. Thus, this series displayed enantiospecific inhibition of the M₅ receptor. With the single enantiomer in hand, we assessed mAChR selectivity at both rat and human M₁-M₄ (Figure 7, section 3.1), where CID 71598521 (9) was devoid of activity at M₁-M₄ (IC₅₀s >30 mM) for both species. Subsequent detailed molecular pharmacology studies were performed to establish its mechanism of action. To determine the mechanism of action of (S)-CID 71598521 (9), whether orthosteric or allosteric, we first performed competition binding experiments with the orthosteric mAChR antagonist [³H]-NMS and compared this to atropine. (S)-CID 71598521 (9) displayed no competition [³H]-NMS for binding to hM₅, suggesting an allosteric mode of receptor inhibition. To further investigate an allosteric mechanism, we also performed [³H]-NMS dissociation kinetic experiments, with hM₅ cell membranes, which revealed that (S)-CID 71598521 (9) decreased the dissociation rate of [³H-NMS], further confirming an allosteric effect of (S)-CID 71598521 (9) on the orthosteric site. Thus, (S)-CID 71598521 (9) is the first M₅-selective negative allosteric modulator (NAM), and declared an MLPCN probe, ML375.

Figure 4

Structure and mAChR selectivity of CID 71598549 (8), the first submicrobolar hM5 NAM.

Figure 5

Structures and M5 activity of racemic 8, the (-)-enantiomer 9 and the (+) –enantiomer 10. All of the M5 activity resides in the (-)-enantiomer .

Figure 7

Dose Response Curves ML375. A) Human mAChR selectivity. ML375 is selective for hM₅ (hM5 IC₅₀ = 300 nM, hM₁-M₄ IC_5os >30 μM); B) rat mAChR selectivity ML375 is selective for rM₅ (rM5 IC₅₀ = 790 nM rM₁-M₄ IC_5os >30 μM)

We next assessed the DMPK profile and ancillary pharmacology profile of ML375 (Tables 1-3, section 3.3). ML375 was stable and possessed acceptable solubility. Evaluation of the in vitro and in vivo DMPK profile of ML375 revealed the compound to possess high metabolic stability with low hepatic microsomal intrinsic clearance (CL_int;; human 2.6 mL/min/kg, cynomolgus monkey (cyno), 20 mL/min/kg, rat, 24 mL/min/kg) and a corresponding low predicted hepatic clearance in multiple species (CL_hep; human, 2.3 mL/min/kg, cyno, 14 mL/min/kg rat, 18 mL/min/kg). Correspondingly, ML375 exhibited low clearance (CL_p, 2.5 mL/min/kg) and a long elimination half-life (t_1/2, 80hr) in rodents (male, Sprague-Dawley rat, 1 mg/kg IV, n = 2) and nonhuman primates (male, cynomolgus monkey, 1 mg/kg, CL_p, 3.0 mL/min/kg, t_1/2, 10 hr, n = 3). Consistent with a low clearance compound, ML375 also demonstrated high oral bioavailability (%F, 80) following administration of a suspension-dose to male SD rats (n=2) with a maximal plasma concentration (C_max) of 1.4 µM and a corresponding time to reach C_max (T_max) of 7 hours. The distribution of ML375 was characterized by a low fraction unbound in plasma (f_u,p; human: 0.013, cyno: 0.001, rat: 0.029) and a high nonspecific binding in brain homogenate (f_u,br; rat: 0.003). Following an oral CNS distribution study in rat (male, Sprague-Dawley, n = 2; 10 mg/kg) we observed total and unbound brain-plasma partition coefficients of 1.8 and 0.2 (K_p, K_p,uu, respectively) one hour post-administration. ML375 displayed an acceptable human cytochrome P450 inhibition profile producing acceptable IC₅₀ values for 3A4 (16 µM), 1A2 (25 µM, 2C9 (7.4 µM) and 2D6 (26 µM). Moreover, in a Eurofins radioligand binding panel of 68 GPCRs, ion channels and transporter, ML375 displayed significant binding (>50% inhibition @10 μM) at only 1 target (CB₁, 66%), but no functional activity at this target in a subsequent assay.(14)

Table 1

Pan Labs profiling of ML375.

Table 3

In vitro and in vivo DMPK profile of ML375.

Figure 6X-ray structure of the active (-)-enantiomer, CID 71598521 (9), which proved to have the (S)-configuration

3.3. Profiling Assays

To more fully characterize ML375, and to better inform the scientific community about its potential off target activities, this structurally novel M₅ NAM was tested using Eurofins' Lead Profiling Screen. This battery of radioligand binding assays consists of 68 common GPCRs, ion channels and transporters where the test compound (ML375) was present at 10 µM. Responses were considered significant if > 50% inhibition was observed. However it should be pointed out that these are only single-point values and that functional selectivity may be significantly better than suggested by these “% inhibitions.” Table 1 presents the Pan Labs results for ML375, which showed only a single significant response in just the cannabinoid CB₁ assay. However, in a subsequent functional assay, ML375 exhibited no activity at CB₁.

Additionally, a set of calculated physical properties were determined for ML375 and appear in Table 2. For comparison, this table also shows the averaged values for compounds appearing in the MDDR database (MDL Drug Data Report database, 2010) at two stages of clinical development (Phase I and Launched). ML375 compares reasonably well with the average values for both Phase I and Launched compounds, but obviously has room for improvement with respect to cLogP and solubility. Table 3 summarizes key DMPK parameters for ML375 in both rat and cyno monkey. In rat, ML375 displayed low clearance (2.5 mL/min/kg) with a t_1/2 of 80 hr and with 80% oral bioavailability. In cyno monkey, ML375 possessed similarly low clearance (3.5 mL/min/kg) with a t_1/2 of 10 hours. In a 10 mg/kg oral plasma-brain level (PBL) study, ML375 possessed a brain:plasma ratio of 1.8, indicating excellent CNS exposure.

Table 2

Calculated property comparison between ML375 and MDDR compounds.

4. Discussion

5. References

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