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

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

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Discovery, optimization, and characterization of a novel series of dopamine D2 versus D3 receptor selective antagonists

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Author Information

,a ,b ,a ,b ,b ,a ,a ,a ,a ,a ,a ,a ,b and a,*.

a NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD 20850.
b Molecular Neuropharmacology Section, National Institute of Neurological Disorders & Stroke, National Institutes of Health, 5625 Fishers Lane, Room 4S-04, MSC-9405, Bethesda, MD 20892.
* To whom correspondence should be addressed: Email: vog.hin.liam@jnaguram or 301-217-9198.

Received: ; Last Update: September 3, 2013.

Dopamine receptors (DARs) are members of the G protein-coupled receptors (GPCRs) superfamily which play a critical role in cell signaling processes, especially modulating the transfer of information within the nervous system [1]. Amongst DARs, the D2 receptor is arguably one of the most validated drug targets in neurology and psychiatry [2]. There is a strong correlation between the clinical doses of neuroleptics and their affinity for brain D2 receptors [3]. Despite numerous attempts of producing D2 selective modulators, current drugs display poor selectivity between D2 and D3 DARs. This is because D2 is closest to D3 in terms of sequence homology and signaling transduction pathways hence it pose a challenge to selectively regulate the two receptors [4, 5]. However given the success, a potent and selective D2 DAR antagonist would be of particular interest for the treatment of a variety of related CNS diseases [6, 7]. Here we present the discovery of a novel series of selective small molecule D2 DAR antagonists from a quantitative high-throughput screen (qHTS) campaign. Optimized lead compound in this series exhibits an excellent D2versus D1, D3, D4 and D5 receptor selectivity. In a panel of GPCR binding assays, ML321 shows a cleaner profile compared to the best previously reported selective D2 DAR antagonist. Furthermore, ML321 showed good in vitro ADME data and in vivo pharmacokinetic (PK) properties. We therefore believe that this probe can be a very useful pharmacological tool to perform proof-of-concept studies in animal models and may be an ideal starting point for further development into drug-like molecules for the treatment of a variety of CNS diseases including Tourette’s syndrome, tardive dyskinesia, dystonia, Huntington’s chorea, and especially schizophrenia.

Assigned Assay Grant #: R21-NS064831

Screening Center Name & PI: NIH Chemical Genomics Center, Christopher P. Austin

Chemistry Center Name & PI: NIH Chemical Genomics Center, Christopher P. Austin

Assay Submitter & Institution: David Sibley, Institute of Neurological Disorders & Stroke, National Institute of Health

PubChem Summary Bioassay Identifier (AID): 485359

Probe Structure & Characteristics

ML321.

ML321

CID/ML#Target NameIC50/EC50 (nM) [SID, AID]Anti-target Name(s)IC50/EC50 (μM) [SID, AID]Fold SelectiveSecondary Assay(s) Name: IC50/EC50 (nM) [SID, AID]
CID 57377246/ML321D2 DAR receptorD2 Ca2+ AC50: 70 nM, [SID 136882616, AID 624496]D3 DAR receptorD3 β-arrestin: AC50: 12,893 nM, [SID 136882616, AID 624500]17–22 foldD2 β-arrestin: AC50: 725 nM, [SID 136882616, AID 624495]
D2 binding Ki: 120 nM, [SID 136882616, AID 624494]D3 binding Ki: 2,650 nM, [SID 136882616, AID 624502]

1. Recommendations for Scientific Use of the Probe

Preferential inhibition remains to be a limitation for D2 prior art antagonists. The probe ML321 demonstrates excellent D2versus D3 DAR selectivity with a very clean selectivity profile in a panel of GPCR binding assays. The selective D2 (versus D3) ML321 probe will be of interest to biologist to further elucidate the role of the D2 receptors on CNS diseases. ML321 will be used by researchers as a pharmacological tool for the dissection of D2 DAR signaling pathways in vitro and in vivo in response to a variety of biological conditions. Given its reasonable ADME and pharmacokinetic properties, ML321 will also provide scientists a tool to perform proof-of-concept studies in animal model. Moreover, ML321 can be further development into drug-like molecules subsequently giving clinicians a starting point for the development of novel or improved treatments (with less side effects) for Tourette’s syndrome, tardive dyskinesia, dystonia, Huntington’s chorea, and especially schizophrenia where D2 receptor is observed to play a crucial role.

2. Materials and Methods

General Methods for Chemistry. All air or moisture sensitive reactions were performed under positive pressure of nitrogen with oven-dried glassware. Anhydrous solvents such as dichloromethane, N,N-dimethylformamide (DMF), acetonitrile, methanol and triethylamine were purchased from Sigma-Aldrich (St. Louis, MO). Preparative purification was performed on a Waters semi-preparative HPLC system (Waters Corp., Milford, MA). The column used was a Phenomenex Luna C18 (5 micron, 30 × 75 mm; Phenomenex, Inc., Torrance, CA) at a flow rate of 45.0 mL/min. The mobile phase consisted of acetonitrile and water (each containing 0.1% trifluoroacetic acid). A gradient of 10% to 50% acetonitrile over 8 minutes was used during the purification. Fraction collection was triggered by UV detection at 220 nM. Analytical analysis was performed on an Agilent LC/MS (Agilent Technologies, Santa Clara, CA). Method 1: A 7-minute gradient of 4% to 100% acetonitrile (containing 0.025% trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid) was used with an 8-minute run time at a flow rate of 1.0 mL/min. Method 2: A 3-minute gradient of 4% to 100% acetonitrile (containing 0.025% trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid) was used with a 4.5-minute run time at a flow rate of 1.0 mL/min. A Phenomenex Luna C18 column (3 micron, 3 × 75 mm) was used at a temperature of 50 °C. Purity determination was performed using an Agilent diode array detector for both Method 1 and Method 2. Mass determination was performed using an Agilent 6130 mass spectrometer with electrospray ionization in the positive mode. 1H NMR spectra were recorded on Varian 400 MHz spectrometers (Agilent Technologies, Santa Clara, CA). Chemical shifts are reported in ppm with undeuterated solvent (DMSO at 2.49 ppm) as internal standard for DMSO-d6 solutions. All of the analogs tested in the biological assays have a purity of greater than 95% based on both analytical methods. High resolution mass spectrometry was recorded on Agilent 6210 Time-of-Flight (TOF) LC/MS system. Confirmation of molecular formula was accomplished using electrospray ionization in the positive mode with the Agilent Masshunter software (Version B.02).

2.1. Assays

D2 Ca2+ Primary qHTS of Sytravon Library and Confirmatory Screen. Stable cell line that expresses the D2 DAR regulated by Tetracycline-Regulated Expression (HEK293 T-REx™) and chimeric G-protein (Gqi5) to allow coupling of the D2 DAR to calcium release was developed for the D2 Ca2+ Assay described in detail in Table 1. In this system, D2 DAR gene expression is induced by addition of Tet to the cells prior to the assay. Dopamine stimulation of the D2 DAR activates the chimeric Gqi5 G-protein, which in turn acts on PLC which hydrolyses the membrane phospholipid PIP2 to form Inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 diffuses to the ER, binds to its receptor (IP3 receptor), which is a Ca2+ channel and, releases Ca2+ from the ER to the cytosol. The Screen Quest™ Fluo-8 Calcium Assay Kit (AAT Bioquest, Sunnyvale, CA) was used to measure the cytosolic Ca2+ accumulation. This kit contains a non-polar moleculue, acetoxymethyl (AM) esters bound to Fluo-8™ dye that easily penetrates live cell membranes transporting the dye inside the cell where they are rapidly hydrolyzed by cellular esterases. As Fluo-8™ is freed from AM esters; it binds to Ca2+ and emits a fluorescent signal at 514 nm that escalates with increasing cytosolic Ca2+. A high throughput screening device which allows optical detection of signal transmissions within living cells in a time-resolving fashion, the Functional Drug Screening System (FDSS) (Hamamatsu, Japan) was then used to measure the calcium flux signal.

Table 1. Screen Quest™ Fluor-8 of D2 Ca2+ qHTS assay protocol.

Table 1

Screen Quest™ Fluor-8 of D2 Ca2+ qHTS assay protocol.

DiscoveRx D2 β-arrestin Secondary Assay. For a secondary-screen and selectivity assays, DAR PathHunter® β-arrestin GPCR cell lines from DiscoveRx (Fremont, CA) were used as described in the protocol below (Table 2). In the D2 Receptor PathHunter® β-arrestin GPCR cell line, the D2 GPCR receptor (DAR) is overexpressed and fused with a small 42-amino acid fragment of β-galactosidase called ProLink™ on a CHO cellular background expressing a fusion protein of β-arrestin and a larger N-terminal deletion mutant of β-galactosidase enzyme acceptor. When DAR is activated by dopamine, it stimulates binding of β-arrestin to the ProLink-tagged DAR and the two complementary parts of β-galactosidase form a functional enzyme. When PathHunter® Detection reagent is added, β-galactosidase hydrolyzes it and generates a chemiluminescent signal.

Table 2. Detailed protocol of DiscoveRx D2 β-arrestin secondary assay.

Table 2

Detailed protocol of DiscoveRx D2 β-arrestin secondary assay.

D2 Binding Secondary Assay. Compounds were tested for differences in affinity between three dopamine receptor subtypes using radio-labeled ligand binding assays (Table 3). The first assay was to determine the Ki value of the compounds using the D2 DAR subtype using stable HEK cell lines expressing the D2L human dopamine receptors (Codex Biosciences, Gaithersburg, MD). Cells were cultured in Dulbecco’s modified Eagle’s Medium containing 10% FBS, 1,000 units/mL Penicillin, 1,000 mg/mL Streptomycin, 100 mM Sodium Pyruvate, 1 μg/mL Gentamicin, and 250 mg/mL G418. All cells were maintained at 37 °C in 5% CO2 and 90% humidity. For radioligand binding assays, cells were removed mechanically using calcium and magnesium-free Earle’s Balanced Salt Solution (EBSS(−)). Intact cells were collected by centrifugation and then lysed with 5 mM Tris-HCl and 5 mM MgCl2 at pH 7.4 in a glass homogenizer. Homogenates were centrifuged at 20,000 × g for 30 minutes. The membranes were re-suspended in EBSS (pH 7.4) and protein concentration was determined using a Bradford assay according to the manufacturer’s recommendations (Bio-Rad). Membranes were diluted to 24 mg/mL. It was determined in preliminary experiments that this protein concentration gave optimal binding with minimal ligand depletion. Membrane preparations were incubated for 90 min at room temperature with various concentrations of radioligand in a reaction volume of 250 μL EBSS containing 200 mM sodium metabisulfite. Non-specific binding was determined in the presence of 4 μM (+)-butaclamol. Bound ligand was separated from unbound by filtration through GF/C filters using a PerkinElmer cell harvester with ice cold EBSS (4 washes) and quantified on a Top-count (PerkinElmer) after addition of scintillation solution. Saturation experiments generated a Kd value of 0.2 nM and a Bmax of ~4,200 fmol/mg for [3H]-methylspiperone binding to D2 receptors. In order to determine the affinity of a given compound for a receptor type, competition-binding assays were performed. For these assays the reaction mixture was incubated with a single concentration of radiolabeled ligand (0.2 nM [3H]methylspiperone) and various concentrations of competing compound. Reactions were incubated, terminated, and quantified as indicated above. Ki values of compounds were determined from observed IC50 values using the Cheng-Prussoff equation.

Table 3. Detailed protocol for the D2 binding secondary assay.

Table 3

Detailed protocol for the D2 binding secondary assay.

DiscoveRx D3 β-arrestin Selectivity Assay. To determine the functional selectivity of the compounds for D2versus D3 receptor antagonism, we used a D3 PathHunter® β-arrestin cell line from DiscoveRx (Fremont, CA) using the detailed protocol below (Table 4). A CHO cell line was engineered to overexpress D3 dopamine receptor (DAR) and fused with a small 42-amino acid fragment of β-gal called ProLink™. In addition, these cells stably express a fusion protein of β-arrestin and a larger N-terminal deletion mutant of β-galactosidase (“enzyme acceptor”). When DAR is activated by dopamine, it stimulates binding of β-arrestin to ProLink-tagged DARs, and the two complementary parts of β-galactosidase form a functional enzyme. When substrate (PathHunter® Detection reagent) is added, β-galactosidase hydrolyzes it and generates a chemiluminescent signal.

Table 4. Detailed protocol for the DiscoveRx D3 PathHunter® β-arrestin selectivity assay.

Table 4

Detailed protocol for the DiscoveRx D3 PathHunter® β-arrestin selectivity assay.

D3 Binding Selectivity Assay. Compounds were counter screened for affinity for the D3 dopamine receptor using a radioligand assay (Table 5). This was accomplished by determining the Ki values for the compounds using stable (HEK293 based) cell lines expressing the D3 human dopamine receptors (Codex Biosciences, Gaithersburg, MD). Cells were cultured in Dulbecco’s modified Eagle’s Medium containing 10 % FBS, 1,000 units/mL Penicillin, 1,000 mg/mL Streptomycin, 100 mM Sodium Pyruvate, 1 μg/mL Gentamicin, and 250 mg/mL G418. All cells were maintained at 37 °C in 5% CO2 and 90% humidity. For radioligand binding assays, cells were removed mechanically using calcium and magnesium-free Earle’s Balanced Salt Solution (EBSS(−)). Intact cells were collected by centrifugation and then lysed with 5 mM Tris-HCl and 5 mM MgCl2 at pH 7.4 in a glass homogenizer. Homogenates were centrifuged at 20,000 × g for 30 min. The membranes were re-suspended in EBSS (pH 7.4), and protein concentration was determined using a Bradford assay according to the manufacturer’s recommendations (Bio-Rad). Membranes were diluted to 80 mg/mL, the predetermined optimal protein concentration for binding but minimal ligand depletion. Membrane preparations were incubated for 90 min at room temperature with various concentrations of radioligand in a reaction volume of 250 μL EBSS containing 200 mM sodium metabisulfite. Non-specific binding was determined in the presence of 4 μM (+)-Butaclamol. Bound ligand was separated from unbound by filtration through GF/C filters using a PerkinElmer cell harvester with ice cold EBSS (4 washes) and quantified on a Top-count (PerkinElmer) after addition of scintillation solution. Saturation experiments generated a Kd value of 0.125 nM and a Bmax of ~600 fmol/mg for [3H]-methylspiperone binding to D3 receptors. In order to determine the affinity of a given compound for a receptor type, competition-binding assays were performed. For these assays the reaction mixture was incubated with a single concentration of radiolabeled ligand (0.5 nM [3H]methylspiperone) and various concentrations of competing compound. Reactions were incubated, terminated, and quantified as indicated above. Ki values of compounds were determined from observed IC50 values using the Cheng-Prussoff equation.

Table 5. Detailed protocol for the D3 binding selectivity assay.

Table 5

Detailed protocol for the D3 binding selectivity assay.

2.2. Probe Chemical Characterization

Probe ML321 (CID 57377246).

Probe ML321 (CID 57377246)

10-Methyl-11-oxo-N-(2-(thiophen-2-yl)ethyl)-10,11-dihydrodibenzo[b,f][1,4]thiazepine-8-carboxamide 5-(S)-oxide (ML321). LC-MS Retention Time: t1 (Method 1) = 5.348 min; t2 (Method 2) = 3.188 min; 1H NMR (400 MHz, DMSO-d6) δ ppm 8.76 (t, J=5.7 Hz, 1 H), 7.94 (d, J=2.0 Hz, 1 H), 7.86 (dd, J=8.2, 2.0 Hz, 1 H), 7.69 – 7.77 (m, 2 H), 7.67 (d, J=8.2 Hz, 2 H), 7.51 – 7.60 (m, 1 H), 7.31 (dd, J=5.1, 1.2 Hz, 1 H), 6.92 (dd, J=5.1, 3.1 Hz, 1 H), 6.83 – 6.90 (m, 1 H), 3.55 (s, 3 H), 3.43 – 3.52 (m, 2 H), 3.02 (t, J=7.0 Hz, 2 H); 13C NMR (400 MHz, DMSO-d6) δ ppm 13C NMR (101 MHz, DMSO-d6) δ ppm 165.16, 165.10, 147.66, 145.98, 141.75, 137.87, 137.02, 132.92, 131.69, 131.23, 128.20, 127.39, 126.51, 125.70, 124.57, 124.43, 121.05, 119.40, 41.55, 38.03, 29.49; HRMS (ESI) m/z (M+H)+ calcd. for C21H19N2O3S2 [M+H+] 411.0832, found 411.0831.

Figure 1. Chemical stability evaluation for ML321 for 48 hours at room temperature in (A) D2 Ca2+ assay buffer (Glucose, 10% FBS, 1× NEAA, and Pen/Strep), (B) PathHunter® β-arrestin GPCR assay buffer (DiscoveRx, Catalog # 056R2A) and (C) PBS+ buffer (DPBS, 1 mM CaCl2, 0.

Figure 1

Chemical stability evaluation for ML321 for 48 hours at room temperature in (A) D2 Ca2+ assay buffer (Glucose, 10% FBS, 1× NEAA, and Pen/Strep), (B) PathHunter® β-arrestin GPCR assay buffer (DiscoveRx, Catalog # 056R2A) and (C) PBS+ buffer (DPBS, 1 mM CaCl2, 0.5 mM MgCl2, 0.05% BSA, 0.005% Tween 20).

Table 6List of the D2 DAR inhibitor probe ML321 and related analogs and their corresponding identification numbers

Their corresponding molecular structures are shown above in Figure 2.

Internal IDMLS IDSIDCIDML #TypeSource
NCGC00250206MLS00449724913688261657377246ML321ProbeNCGC
NCGC00241677MLS00449725013688261757377245AnalogNCGC
NCGC00241689MLS00449725113688261857377247AnalogNCGC
NCGC00109352MLS00449725213688261916007752AnalogNCGC
NCGC00248394MLS00449725313688262057377248AnalogNCGC
NCGC00241680MLS00449725413688262157377249AnalogNCGC

2.3. Probe Preparation

Preparation of 10-methyl-11-oxo-N-(2-(thiophen-2-yl)ethyl)-10,11-dihydrodibenzo[b,f][1,4] thiazepine-8-carboxamide 5-(S)-oxide (ML321) is a multi-step process with 8 compound intermediates illustrated above (Scheme 1) and summarized below.

Scheme 1. The synthetic route to ML321 is a multi-step process with 8 compound intermediates (A – H).

Scheme 1

The synthetic route to ML321 is a multi-step process with 8 compound intermediates (A – H).

  1. A solution of methyl 4-fluoro-3-nitrobenzoate and methyl 2-mercaptobenzoate in dimethylformamide (DMF) was treated with caesium carbonate (Cs2CO3) at room temperature. The reaction mixture was stirred at 40 °C for 4 hour and then cooled to room temperature. Ice water was added to induce the precipitation. The precipitate was filtered, washed with water, and dried to give 21.0 g (99%) of methyl 4-(2-(methoxycarbonyl)phenylthio)-3-nitrobenzoate (intermediate A). A yellow solid which was used directly in the next reaction without further purification.
  2. A solution of intermediate A and water was treated at room temperature with lithium hydroxide (LiOH). The reaction mixture was stirred at 60 °C for 2 hr. The organic solvent was removed and the aqueous solution was washed with ethyl acetate (EtOAc) and acidified with 2 N hydrogen chloride (HCl) until pH = ~2. The yellow precipitate was filtered, washed with water, and dried to give 19.1 g (99%) of 4-(2-Carboxyphenylthio)-3-nitrobenzoic acid (intermediate B); a yellow solid which was used directly in the next reaction without further purification.
  3. Intermediate B was treated at room temperature with platinum (IV) oxide (PtO2) and palladium on carbon (Pd/C). A balloon containing hydrogen (H2) was connected to the flask and the reaction flask was repeatedly evacuated and refilled with H2. After 16 hr, additional Pd/C (10%, 600 mg, 5.64 mmol) was added and the reaction mixture was stirred under H2 balloon for an additional 32 hr. The reaction mixture was filtered through a pad of celite and concentrated to give 7.80 g (99%) of 3-amino-4-(2-carboxyphenylthio)benzoic acid (intermediate C); a grey-yellow solid which was used directly in the next reaction without further purification.
  4. A solution of intermediate C in THF was treated at 0 °C with 1,1′-carbonyldiimidazole (CDI) via several portions. The reaction mixture was warmed and stirred at room temperature overnight. The reaction mixture was poured into 140 mL of ice water containing concentrated HCl and stirred for 1 hr. The white precipitate was filtered, washed with water, and dried to give 3.89 g (87%) of 11-Oxo-10,11-dihydrodibenzo[b,f][1,4]thiazepine-8-carboxylic acid (intermediate D), a white solid which was used directly in the next reaction without further purification.
  5. A solution of intermediate D in DMF was treated at 0 °C with sodium hydride (NaH). The reaction mixture was warmed and stirred at room temperature for 1 hr. Then, a solution of methyl iodide (MeI) in DMF was added dropwise to the mixture. The reaction mixture was stirred at room temperature for 1.5 hr. Water was carefully added and the aqueous layer was washed with EtOAc. The aqueous layer was acidified with HCl to induce the precipitation which was filtered, washed, and dried to give 200 mg (91%) of methyl 10-methyl-11-oxo-10,11-dihydrodibenzo[b,f][1,4]thiazepine-8-carboxylate (intermediate E); a yellow solid which was used directly in the next reaction without further purification.
  6. A solution of intermediate E in tetrahydrofuran (THF), methanol (MeOH) and water was treated at room temperature with LiOH. The reaction mixture was stirred at room temperature for 1 hr, diluted with water, and acidified with HCl. The aqueous mixture was extracted with 20% of MeOH in dichloromethane. The organic layer was separated, dried and concentrated to give 140 mg (98%) of 10-Methyl-11-oxo-10,11-dihydrodibenzo[b,f][1,4]thiazepine-8-carboxylic acid (intermediate F); a grey solid which was used directly in the next reaction without further purification.
  7. A suspension of intermediate F in acetic acid was treated at room temperature with hydrogen peroxide (H2O2) for 8 hr. Upon completion, the reaction mixture was poured into a cold saturated solution of sodium thiosulphate (Na2S2O3) in water and stirred at room temperature for 3 hr. The mixture was then extracted with 20% of MeOH in dichloromethane (DCM). The organic layer was separated, dried, and concentrated to give 595 mg (85%) of 10-methyl-11-oxo-10,11-dihydrodibenzo[b,f][1,4]thiazepine-8-carboxylic acid 5-oxide (intermediate G); a white solid containing ~ 5% of dioxide as a by-product.
  8. Intermediate G was enantiomerically purified to > 98% purity to yield 10-methyl-11-oxo-10,11-dihydrodibenzo[b,f][1,4]thiazepine-8-carboxylic acid 5-(S)-oxide (intermediate H) using supercritical fluid chromatography (SFC) preparative systems at Lotus Separations, LLC (Princeton, NJ, USA). For preparative separation, an IC (2 × 15 cm) column was used with an eluent of 40% MeOH (0.1% DEA)/CO2, 100 bar. Flow rate was 60 mL/min and detection wavelength was 220 nm. For analytical separation, an IC (15 × 0.46 cm) column was used with an eluent of 40% MeOH/CO2, 100 bar. Flow rate was 3 mL/min and detection wavelengths were 220 and 280 nm. Retention time was 3.42 min. Retention time for R-configuration enantiomer was 2.40 min. The material was used directly in the next coupling reaction.
  9. A solution intermediate H in DMF (5.00 mL) was treated at room temperature with the peptide coupling reagent HATU and diisopropylethylamine followed by 2-(thiophen-2-yl)ethanamine. The reaction mixture was stirred at room temperature for 3 h and poured into ice water. The white precipitate was filtered and dried to give a white solid, which was purified via silica gel chromatography using a gradient of 10–100% of EtOAc in hexanes to give 118 mg (87%) of 10-methyl-11-oxo-N-(2-(thiophen-2-yl)ethyl)-10,11-dihydrodibenzo[b,f][1,4] thiazepine-8-carboxamide 5-(S)-oxide (ML321).

3. Results

3.1. Dose Response Curves for Probe

Figure 3. Dose response activity of ML321 in (A) D2 Ca2+ assay (green, AC50 = 0.

Figure 3

Dose response activity of ML321 in (A) D2 Ca2+ assay (green, AC50 = 0.070 μM), D2 β-arrestin assay (blue, AC50 = 0.725 μM), and D3 β-arrestin assay (red, AC50 = 12.9 μM). (B) Graphical representation of the dose response curves of ML321 in binding assays for D1 (black, Ki = 67.1 μM), D2 (blue, Ki = 0.1 μM), D3 (red, Ki = 2.9 μM), D4 (green, Ki = 8.48 μM) and D5 (brown) showing preferential inhibition for the D2 receptor.

3.2. Cellular Activity

Table 7Activity profile of the probe ML321 and relative analogs against D2 Ca2+ assay in D2 expressing HK293 T-Rex™ cell line, D2 β-arrestin assay in D2 receptor PathHunter® β-arrestin cells, D3 β-arrestin assay in D3 PathHunter® β-arrestin cells, and D2 or D3 radio-ligand binding assays in HEK cell line expressing either D2L or D3 human dopamine receptor

Results showed probe ML321 is a potent (<1 μM AC50) and selective (>17 fold preference) D2 antagonist.

Internal IDEntryD2 Ca2+ AC50 (μM)D2 β-arrestin AC50 (μM)D3 β-arrestin AC50 (μM)D2 Ki AC50 (μM)D3 Ki AC50 (μM)D3/D2 β-arrestinD3/D2 Ki
NCGC0025020655 (ML321)0.0700.72512.90.12.917.829.0
NCGC00250207R-55InactiveInactiveInactiveInactiveInactiveN/AN/A
NCGC0024841532 Racemic-550.0890.91320.40.236.622.328.7
NCGC0010941410.2812.895.760.080.482.06.0
NCGC00238612380.1411.152.89N/AN/A2.5N/A
NCGC00250200470.0560.5763.240.090.235.62.6
NCGC00250203480.1120.2571.620.0230.136.35.7
NCGC00250211490.2231.299.130.680.887.11.3
NCGC00250215500.3532.8932.40.213.411.216.2
NCGC00248384510.2802.8910.2N/AN/A4.5N/A
NCGC00250205520.0891.024.570.240.374.51.5
NCGC00250213530.4452.8914.50.3121.05.070.0
NCGC00250209540.0700.6364.570.210.357.21.7

3.3. Profiling Assays

Table 8In vitro ADME profile for probe ML321 and a relative analog NCGC00109414 (CID16007814)

Results showed compound ML321 has good stability and permeability.

CompoundAqueous Kinetic SolubilityLiver Microsomal Stability (T1/2 in min)Plasma Stability (% remaining after 2 h)Caco-2 Permeability (10−6, cm/s)
μg/mLμMMouseHumanMouseHumanPapp (A-B)Papp (B-A)
ML32146.5113.324.726.495.298.418.823.8
CID1600781411.526.435.9N/AN/AN/A21.616.2

Table 9Summary of in vivo pharmacokinetic (PK) data for ML321 in C57BL/6 mice (BQL = Below quantifiable limit of 1 ng/mL for ML321 in male C57BL/6 plasma. NA = Not available)

Individual and mean plasma concentration-time data of ML321 after an IP dose of 30 mg/kg in male C57BL/6 mice
Dose (mg/kg)Dose routeSampling Time (hr)Concentration (ng/mL)Mean (ng/mL)μMSDCV (%)
Individual
30IP0BQLBQLBQLBQLNANANA
0.083663967796897677116.4951291.91
0.25801677499061827520.1596948.38
0.5589374736778671516.35779211.8
122572539298625946.31936814.2
22562362572500.60811.64.66
413.09.378.0610.20.0252.5825.4
867.175.794.379.00.19313.917.6
1234.910.713.519.70.04813.267.2
24BQLBQLBQLBQLNANANA
PK parametersUnitEstimate
TmaxHr0.25
Cmaxng/mL8275
Terminal t1/2Hr1.67
AUClasth*ng/mL7796
AUCINFh*ng/mL7844
Figure 3. Mean plasma and brain concentration-time profiles of ML321 after an IP dose of 30 mg/kg in male C57BL/6 mice (N = 3).

Figure 3Mean plasma and brain concentration-time profiles of ML321 after an IP dose of 30 mg/kg in male C57BL/6 mice (N = 3)

The mice appeared less activity at 5 min post dosing, and it lasted for about 2 hr. The IP dosing solution was prepared in 10% NMP + 20% PEG400 + 70% of 25% HP-β-CD in water.

4. Discussion

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

A recent group of publications appeared on a series of N-(1-benzylpiperidin-4-yl)pyridazin-3-amines exemplified by the clinical candidate JNJ-37822681 showing D2 inhibition [8]. However, JNJ-37822681 (Figure 4) is not very selective between D2 (Ki = 158 nM) and D3 (Ki = 1,159 nM) in displacement assays. Another compound L741,626 (Figure 4), a close analog of Haloperidol, had claimed selectivity of ~10–15 fold in D2 and D3 displacement assays [9]. This compound showed a 3-fold selectivity in functional D2 and D3 β-arrestin assays. The binding selectivity was significantly improved through several rounds of medicinal chemistry efforts to obtain analogs 57 and 58 (Figure 4) which was observed to have D2 vs. D3 selectivity greater than 100 fold [10]. However, no further reports related to ADME profiling and in vivo animal data was reported. It is well known that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its derivatives are potential neurotoxins [11] and could be a reason for lack of in vivo data for this chemical series. Hence, the reference compounds 57 and 58 were resynthesized in house and assayed side-by-side with the probe ML321. A stability and GPCR panel inhibition profile for compounds 58 and ML321 were also assessed. Compound 58 was observed to have a 60-fold D2 vs. D3 selectivity in the β-arrestin functional assay and 48.6-fold D2 vs. D3 selectivity in the binding assay (Table 10). However it showed a very short half-life time of 2.4 min in rat liver microsomal test. Results of the GPCR panel inhibition profile also showed that compound 58 at 10 μM is very promiscuous; targeting a large number of receptors with > 50% inhibition (Table 11). Not only did it inhibit dopamine receptors 2–4, but also targeted Sigma 1–2, SERT, MOR and Alpha2 A-B with > 90% inhibition. In contrast, ML321 at 10 μM gave a more selective profile; with > 90% inhibition only against the D2 receptor while the rest (5-HT2C, 5-HT7, D3) were only ~ 60% inhibition. Until this probe, it was very difficult to interpret data using existing compounds in ex vivo preparation because so many of them are “contaminated” by influence from the D3 receptor or the D4 receptor; both of which is antagonized by all existing molecules. Although ML321 (and members of its chemical series) is less potent than the compound 58, ML321 showed a good D2 vs. D3 receptor selectivity and good ADME and PK properties with fewer chemical scaffold liabilities. Therefore, probe ML321 provides sufficient selectivity towards D2 giving scientist a tool to better understand it contributions to dopamine signaling pathways both in vitro and in vivo.

Figure 4. Known chemical series of selective D2versus D3 DAR antagonist.

Figure 4

Known chemical series of selective D2versus D3 DAR antagonist.

Table 10. Comparison of dopamine receptor inhibition assay for ML321 and current D2 antagonists.

Table 10

Comparison of dopamine receptor inhibition assay for ML321 and current D2 antagonists.

Table 11. GPCR panel screening for ML321 and compound 58.

Table 11

GPCR panel screening for ML321 and compound 58. Data represent mean % inhibition (N = 4 determinations) for compound tested at receptor subtypes. > 50% of inhibition (marked red) is considered as significant inhibition. In cases where negative (more...)

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Langlois X, Megens A, Lavreysen H, Atack J, Cik M, te Riele P, Peeters L, Wouters R, Vermeire J, Hendrickx H, Macdonald G, De Bruyn M. Pharmacology of JNJ-37822681, a specific and fast-dissociating D2 antagonist for the treatment of schizophrenia. J Pharmacol Exp Ther. 2012;342(1):91–105. [PubMed: 22490380]
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Grundt P, Husband SL, Luedtke RR, Taylor M, Newman AH. Analogues of the dopamine D2 receptor antagonist L741,626: Binding, function, and SAR. Bioorg Med Chem Lett. 2007;17(3):745–749. [PMC free article: PMC1851912] [PubMed: 17095222]
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Vangveravong S, Taylor M, Xu J, Cui J, Calvin W, Babic S, Luedtke RR, Mach RH. Synthesis and characterization of selective dopamine D2 receptor antagonists. 2. Azaindole, benzofuran, and benzothiophene analogs of L-741,626. Bioorg Med Chem. 2010;18(14):5291–5300. [PMC free article: PMC2946321] [PubMed: 20542439]
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Pitts SMM, SP, Murphy DL, Weisz A, editors. Recommended practices for the safe handling of MPTP MPTP - A Neurotoxin Producing a Parkinsonian Syndrome. Academic Press; New-York: 1995.
12.

Receptor binding profiles was generously provided by the National Institute of Mental Health’s Psychoactive Drug Screening Program, Contract # HHSN-271-2008-025C (NIMH PDSP). The NIMH PDSP is Directed by Bryan L. Roth MD, PhD at the University of North Carolina at Chapel Hill and Project Officer Jamie Driscol at NIMH, Bethesda MD, USA.

Footnotes

*

Purity > 95% as determined by LC/MS and 1H NMR analyses.

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