Summary /

ML321 is potent (< 1 uM AC50) and found to have > 17 – 22 fold selective against the D2 dopamine receptor (DAR) vs. other DAR.

Figure 1. Dose response activity of ML321 in (A) D2 Ca++ assay (green, AC = 0.070 μM), D2 β-arrestin assay (blue, AC = 0.725 μM), and D3 β-arrestin assay (red, AC = 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 D receptor.

Table 1. Activity profile of the probe ML321 and relative analogs against D2 Ca++ 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.

Summary /

ADME profile for probe ML321 and a relative analog showed that the probe has good stability and permeability.

Table 2. In vitro ADME profile for probe ML321 and a relative analog NCGC00109414 (CID16007814).

Table 3. Summary of in vivo 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).

Summary /

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.

Figure 2. Mean plasma and brain concentration-time profiles of ML321 after an IP dose of 30 mg/kg in male C57BL/6 mice (N = 3).

Summary /

A recent group of publications appeared on a series of N-(1-benzylpiperidin-4-yl)pyridazine 3-amines exemplified by the clinical candidate JNJ-37822681 showing D inhibition (Langlois 2012). However, JNJ-37822681 is not very selective between D2 (Ki = 158 nM) and D3 (Ki = 1,159 nM) in displacement assays. Another compound L741,626, a close analog of Haloperidol, had claimed selectivity of ~10–15 fold in D2 and D3 displacement assays (Grundt 2007). 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 which was observed to have D2 vs. D3 selectivity greater than 100 fold (Vangveravong, 2010). 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 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 4). 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 5). 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 (Xiao 2010).

Table 4. Comparison of dopamine receptor inhibition assay for ML321 and current D antagonists.

Table 5. GPCR panel screening for ML321 and compound 58. Data represent mean % inhibition (N = 4 determinations) for compound tested at receptor sub-types. > 50% of inhibition (marked red) is considered as significant inhibition. In cases where negative inhibition (−) is seen, this represents a stimulation of binding. Occasionally, compounds at high concentrations will non-specifically increase binding. The default concentration for primary binding experiments is 10 μM. 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.