2.1. Assays

2.2. Probe Chemical Characterization

Probe compound ML218 (CID: 45115620, SID: 92310249) was prepared according to the above scheme and provided the following characterization data: ¹H NMR (400 MHz, CDCl₃) δ (ppm): 7.78 (d, J = 1.82 Hz, 2H); 7.57 (br, 1H); 7.42 (ap. t, J = 1.76 Hz, 1H); 3.64 (d, J = 10.56 Hz, 2H); 3.32 (t, J = 5.98 Hz, 2H); 2.95–2.83 (m,4H); 1.95 (m, 1H); 1.74 (s, 2H); 1.62 (m, 2H); 0.88 (s, 9H). ¹³C NMR (100 MHz, CD₃OD) δ (ppm): 165.41, 137.46, 135.50, 131.46, 126.36, 55.69, 53.11, 41.94, 39.42, 30.07, 29.44, 21.71; HRMS calc’d for C₁₉H₂₆Cl₂N₂O, 369.1500 [M+H]; found 369.1501.

Solubility. Solubility (kinetic solubility) in PBS was determined to be 542 μM.

GSH Conjugates. No glutathione conjugates detected.

Stability. Stability was determined for ML218 at 23 °C in PBS (no antioxidants or other protectorants and DMSO concentration below 0.1%) and is shown in Table 1. We were surprised by the lack of stability in phosphate buffer, and Merck’s 7²⁴ behaved similarly. Due to the high logP (clogP 5.2, experimentally derived logP 4.9), we proposed that over time, the thermodynamic nature of this assay, the compound precipitates out of solution. In the solubility assay in pH 7.4 phosphate buffer, ML218 shows high solubility, but this is kinetic versus thermodynamic conditions. Upon closer inspection, it does appear that ML218, as well as 7,²⁴ do precipitate out of solution over the time course in the 48 hour PBS assay. Moreover, the ML218 is stable in rat plasma and shows good stability in vivo. Therefore, the physical properties of ML218 appear to be the culprit for the apparent lack of stability in the 48 hour PBS assay, which is actually a lack of solubility and not instability. In vivo, ML218 is stable.

Table 1

Stability in Phosphate Buffer.

Compounds added to the SMR collection (MLS#s): 003431854 (ML 218, CID 45115620, 20 mg), 003431850 (CID 44251479, 5.3 mg), 003431851 (CID 44251485, 5.0 mg), 003431852 (CID 45115609, 9.4 mg), 003431849 (CID 46943243, 5.5 mg), 003431853 (CID 46943258, 8.4 mg).

2.3. Probe Preparation

3,5-dichloro-N-((3-(3,3-dimethylbutyl)-3-azabicyclo[3.1.0]hexan-6-yl)methyl)benzamide (ML218)). tert-butyl 6-((3,5-dichlorobenzamido)methyl)-3 azabicyclo[3.1.0]hexane-3-carboxylate (16). To a solution of exo-3-Boc-6-aminomethyl-3-azabicyclo[3.1.0]hexane (1.0 eq.) and triethylamine (1.0 eq.) in CH₂Cl₂ at 0 °C was added a solution of 3,5-dichlorobenzoyl chloride (1.0 eq.) in CH₂Cl₂. The reaction stirred at 0 °C for approximately 4 hrs. The mixture was then concentrated in vacuo and purified by reverse phase chromatography (MeCN/H₂O/0.1% TFA) to afford the product as a white solid (93% yield). ¹H NMR (400 MHz, CD₃OD) δ (ppm): 7.83 (d, J = 1.92 Hz, 2H); 7.66 (t, J = 1.88 Hz, 1H); 3.57 (m, 2H); 3.35 (m, 4H); 1.60 (m, 2H); 1.46 (s,9H); 0.90 (m, 1H). ¹³C NMR (125 MHz, CD₃OD) δ (ppm): 167.96, 157.61, 139.75, 137.33, 133.02, 127.99, 81.86, 43.65, 29.56, 24.46, 24.02, 23.23. To a vial containing tert-butyl 6-((3,5-dichlorobenzamido)methyl)-3-azabicyclo[3.1.0]hexane-3-carboxylate 16, was added 4.0 M HCl in dioxane. This mixture was allowed to stir at room temp for 2 hrs. The reaction was then concentrated in vacuo and purified by reverse phase chromatography (MeCN/H₂O/0.1% TFA) to afford the product as a white solid (95% yield). ¹H NMR (400 MHz, CD₃OD) δ (ppm): 7.82 (d, J = 1.92 Hz, 2H); 7.68 (m, 1H); 3.45 (s, 4H); 3.34 (m, 2H); 1.90 (m, 2H); 1.21 (m,1H). ¹³C NMR (100 MHz, CD₃OD) δ (ppm): 168.04, 163.95, 139.57, 137.40, 133.17, 127.97, 43.02, 23.35, 22.48. 3,5-dichloro-N-((3-(3,3-dimethylbutyl)-3-azabicyclo[3.1.0]hexan-6-yl)methyl)benzamide (ML218): To a solution of 3,3-dimethylbutyraldehyde (1.0 eq.) in CH₂Cl₂ was added a solution of N-(3-azabicyclo[3.1.0]hexan-6-ylmethyl)-3,5-dichlorobenzamide (1.0 eq.) in CH₂Cl₂ followed by addition of polymer-supported sodium triacetoxyborohydride (1.2 eq.). The reaction was left to stir overnight at room temperature after which the mixture was filtered through a pad of Celite eluting with CH₂Cl₂ and concentrated in vacuo resulting in a crude oil which was then purified by reverse phase chromatography (MeCN/H₂O/0.1% TFA) to afford the product as an off-white solid (88% yield). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 7.78 (d, J = 1.82 Hz, 2H); 7.57 (br, 1H); 7.42 (ap. t, J = 1.76 Hz, 1H); 3.64 (d, J = 10.56 Hz, 2H); 3.32 (t, J = 5.98 Hz, 2H); 2.95–2.83 (m,4H); 1.95 (m, 1H); 1.74 (s, 2H); 1.62 (m, 2H); 0.88 (s, 9H). ¹³C NMR (100 MHz, CD₃OD) δ (ppm): 165.41, 137.46, 135.50, 131.46, 126.36, 55.69, 53.11, 41.94, 39.42, 30.07, 29.44, 21.71; HRMS calc’d for C₁₉H₂₆Cl₂N₂O, 369.1500 [M+H]; found 369.1501.

3.1. Dose Response Curves for Probe

ML218 inhibition of Ca_v3 channels was evaluated in electrophysiological and fluorescent assays (Figure 1). Currents through Ca_v3 channels were recorded using the IonWorks Quattro (patch EP). ML218 is a potent and well-behaved inhibitor of Ca_v3 channels.

Figure 1

Dose-response curves for ML218. A) Structure of ML218; B) Raw fluorescence-based HTS assay traces; C) CRC from fluorescence-based assay (IC₅₀ = 150 nM); D) Inhibition time-course of ML218. Ba²⁺ currents were elicited by pulse depolarized to −20mV (more...)

3.2. Cellular Activity

All assays (fluorescence and EP) for Ca_v3 and other ion channels described in this report are cell-based assays. The activity of ML218 in these assays indicates that the probe compound exhibits adequate cell permeability to support further studies with native cells or tissues. No acute toxicity was observed in the cell based assays. ML218 was also evaluated in our tier 1 DMPK profiling assays as well as in in vivo PK and brain level studies in rats. No acute toxicity was observed in any cell based assay or in vivo. No toxicity was observed in a haloperiodol-induced catalepsy model up to 30 mg/kg p.o.

3.3. Profiling Assays

Beyond the basic CRCs in Figure 1, we assessed ML218 activity on the native T-type currents for selectivity over other ion channels in rat DRG neurons (small diameter 10–20μm) in which the Ca_v3.2 channel is prominently expressed. Using pharmacological tools to isolate the current of interest, ML218 at 1μM produced approximately 70% inhibition of the T-type Ca²⁺ current without affecting the tetrodotoxin (TTX)-sensitive voltage-gated Na⁺ current and K⁺ currents (mainly delay rectifier, Figure 2). Although the potency of ML218 in blocking the native T-type current is less potent compared to its inhibition on the recombinant Ca_v3.2 channel expressed in HEK cells, it shows great selectivity over other major membrane ion channels in native neurons. The effects of ML218 on high-voltage-activated Ca²⁺ channels (e.g., L- and N- type) in the DRG neurons will be investigated in an extended probe characterization project.

Figure 2

ML218 selectively blocks T-type Ca²⁺ currents without affecting voltage-gated Na⁺ and K⁺ currents in DRG neurons. A. ML218 (1μM) reversibly blocks the T-type Ca²⁺ currents elicited by depolarizing steps to −30mV from a Vh of −100mV (more...)

ML218 was evaluated in the parental HEK fluorescent assay which contains native Ca²⁺ channels, presumably both L- and N-Type Ca²⁺ channels. ML218 had no affect on the parental line and was therefore thought to be selective versus L- and N-calcium channels (later confirmed at Ricerca panel, where ML218 showed no significant activity in radioligand binding at L- and N-type Ca²⁺ channels, 49%@10 μM and 17%@10 μM, respectively). As mentioned above, the effects of ML218 on high-voltage-activated Ca²⁺ channels (e.g., L- and N- type) in the DRG neurons via EP will be investigated in a later extended probe characterization effort. To more fully characterize this novel Ca_v3 inhibitor probe molecule, ML218 was tested on Ricerca’s (formerly MDS Pharma’s) Lead Profiling Screen (binding assay panel of 68 GPCRs, ion channels and transporters screened at 10 μM), and was found to only significantly bind two targets (sodium channel site 2 and sigma 1) of the 68 assays conducted (no inhibition of radio ligand binding > 50% at 10 μM). Interestingly, the Merck T-type inhibitors also showed similar activity in binding assays at sodium channel site 2 and sigma 1, but neither Merck T-type inhibitors were active in functional sodium channel EP assays. In radioligand binding assays at Ricerca,43 ML218 had no significant inhibition of L- and N-type calcium channels (17–49% inhibition @10 μM, respectively), K_ATP potassium channel (4% inhibition @10 μM) or hERG (48% inhibition @10 μM). Thus, from the Ricerca selectivity profile and the selectivity versus closely related ion channels in which ML218 affords greater than 60-fold selectivity for block of related ion channels and other distantly related potassium channels.

DMPK Profiling. ML218 was evaluated in our tier 1 in vitro DMPK panel. In plasma protein binding studies (equilibrium dialysis), ML218 possessed good free fraction in both rat (f_u = 9.1%) and human (f_u = 3.3%). The CYP profile across four major CYP450s was also good: 3A4 (IC₅₀ >30 μM), 2C9 (IC₅₀ >30 μM), 1A2 (IC₅₀ = 10.8 μM), 2D6 (IC₅₀ = 1.7 μM). Intrinsic clearance experiments in liver microsomes indicated that ML218 was highly cleared in rat (rat CL_INT = 115 ml/min/kg), but low to moderate clearance in human (human CL_INT = 12.7 ml/min/kg). In vivo rat PK mirrored the in vitro work, with a CL of 56 ml/min/kg; however, the t_1/2 of ML218 was 6.8 hours with a mean residence time of ~7 hours. In a 10 mg/kg po plasma/brain study at a 1 hour time point, the plasma concentration of ML218 was 145 ng/mL and the brain concentration of ML218 was 1018 ng/mL. Importantly, ML218 was found to be highly brain penetrant with a Brain_AUC/Plasma_AUC of ~7.4, making ML218 an exciting tool compound for CNS studies. The profile of ML218 is virtually identical to Merck’s, which showed poor rat PK, but excellent dog, rhesus and predicted human PK. Thus, ML218 (CID 45115620) is highly selective and can be used to dissect the role of Ca T-type inhibition in vitro and potentially in vivo.

In Vivo Efficacy in a Preclinical Model of Parkinson’s disease. Parkinson’s disease (PD) is therapeutic indication for T-Type Ca²⁺ channel inhibitors, and Merck’s displayed robust efficacy in a preclinical cataleptic PD model. As we have this same model, haloperidol-induced catalepsy, running for our other PD programs, we evaluated Merck’s and ML218 in this model, wherein reversal of the cataleptic state is consistent with an anti-Parkinsonian phenotype.^24,42 Here, we dosed i.p. in dose response and observed robust efficacy comparable to an A2A standard.^24,42 Excitingly, ML218 showed similar efficacy upon oral dosing, with a 30 mg/kg dose (1.8 μM plasma (160 nM free) with 17.7 μM brain) essentially equivalent to a 56.6 mg/kg dose of the A2A standard (Figure 3). Thus, ML218 is comparable to Merck’s (both in vitro and in vivo), and a valuable probe to study T-Type Ca²⁺ channel inhibition in vivo without IP restrictions.

Figure 3

ML218 reverses haloperidol-induced catalepsy in a dose-dependent manner upon oral dosing. A) Merck’s 7 dose-dependent reversal of haloperidol-induced catalepsy upon i.p. dosing. B) ML218 dose-dependent reversal of haloperidol-induced catalepsy (more...)

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

A number of structural classes of compounds have been described in the scientific and patent literature that act as Ca²⁺ T-Type channel inhibitors (vide supra). At least 20 structural classes of compounds that inhibit Ca²⁺ T-Type channel have been described in the scientific and/or patent literature. The known compounds either lack the required selectivity for the Ca_v3 channels, or are covered by patents.^11–26,40 This was known prior to running the HTS screen, and NIH desired that we develop a Ca²⁺ T-Type inhibitor free of IP issues so that the biomedical community could possess a potent and selective probe without concern over use restrictions. ML218 is a potent inhibitor (Ca_v3.2, IC₅₀ = 150 nM in Tl⁺ flux; Ca_v3.2 IC₅₀ = 310 nM and Ca_v3.3 IC₅₀ = 270 nM in patch clamp electrophysiology) and selective against the other members of the calcium family of ion channels (L- and N-type calcium channels) as well as multiple sodium and potassium channels. Lastly, ML218 was tested at Ricerca’s (formerly MDS Pharma’s) Lead Profiling Screen (binding assay panel of 68 GPCRs, ion channels and transporters screened at 10 μM) and was found to significantly bind to only 2 of the 68 targets; importantly, ML218 was found to be selective against hERG and many other ion channels. ML218 possess excellent DMPK properties, acceptable in vivo rat PK, excellent brain levels and was efficacious in a haloperidol-induced catalepsy assay, a preclinical Parkinsonian model. Thus, ML218 is a powerful new probe to study Ca²⁺ T-Type function in vitro and in vivo.