Design, Synthesis, And Evaluation of Functionalized Diamino-butylbenzamides As Selective Dopamine D3 Receptor Ligands

: Substance use disorder remains a major, unmet medical need. Cocaine is one of the most commonly abused recreational drugs and in 2018, there were over 5.5 million current cocaine users. There are no approved therapies for the treatment of cocaine use disorder, but the D 3 dopamine receptor has been identified as a potential therapeutic target. We have identified a series of functionalized diamino-butylbenzamides that are potent D3 binders with moderate to high selectivity for D 3 over D 2 . Graphical Abstract:

and avoid situations that will trigger the desire to use cocaine. These methods are, however, far too costly and resources intensive to meet the needs of the patient population in need of treatment. [7] In principle, behavioral interventions and cognitive-behavioral therapies could be paired with a suitable pharmacological intervention, but there are no FDA approved medications for the treatment of cocaine addiction, also known as cocaine use disorder (CUD). Research efforts focused on this issue have led to exploration of novel compounds, as well as effort to repurpose previously approved therapies.
[8] The AMPA/Kainate receptor antagonist Topiramate (2) [9] and the antiepileptic irreversible inhibitor of GABA transaminase Vigabatrin (3), [10] for example have also been examined in clinical trials, but neither have been approved for use as treatments for cocaine addiction. The alcoholism treatment Disulfiram (4) has shown promise in a limited patient population, but its mechanism of action is unknown. [11] Positive results in animal studies with compounds that selectively target the neurokinin-1 receptor, [12] the cysteine glutamate antiporter, [13] and the 5-HT2C serotonin receptor [14] have also been reported.
In an effort to develop novel therapies with the potential to address the needs of patients suffering from CUD, we have focused our efforts on the possible positive impact of modulating dopamine (5) signaling. This neurotransmitter is synthesized in the brain and periphery. In the periphery dopamine (5) can act as a vasodilator, modulate renal sodium excretion, and impact urine output, while in the central nervous system, dopamine (5) has a significant impact on movement, behavioral motivations, and learning. [15] The pharmacological impact of dopamine is mediated by a family of related G-protein coupled receptors (GPCRs) which are designated as designated D1, D2, D3, D4, and D5.. There are two sub-families of dopamine (5) receptors, the D1-like sub-family (D1 and D5) and D2-like subfamily (D2, D3 and D4). These classifications are based on pharmacological properties, amino acid homology, and genetic organization. [16] Multiple lines of evidence suggest that D3 may be a viable therapeutic target for the treatment of CUD. Chronic and acute exposure to cocaine, for example, leads to increased D3 expression in the nucleus accumbens. This area of the brain is responsible for cognitive processing including behaviors associated with CUD. [17] In addition, D3 is highly expressed in key brain regions that have been definitively linked to cocaine addiction. The role of D3 in CUD has been described in multiple review articles.
[18] There is also evidence implicating D2 signaling as a possible therapeutic target for the treatment of CUD, but there are significant risks associated with D2 modulation (e.g. extrapyamidal symptoms, catalepsy). [19] In order to avoid these potential risks, we have focused our efforts on the development of novel D3 ligands that are highly selective for D3 over D2.
We have recently described the identification of a series of N-(4-(4-phenyl piperazin-1yl)butyl)-4-(thiophen-3-yl)benzamides that bind to D3 selectively over D2. As part of these studies, we identified (6) as a highly potent D3 ligand (Ki = 0.5 nM) that demonstrated a significant selectivity for D3 over D2 (Ki = 743 nM). [20] As part of our broader effort to understand the source of D3/D2 selectivity, we determined that while D3 and D2 binding are driven primarily by the aryl piperazine portion of the molecule (via Asp 3.32 ), D3/D2 selectivity is primarily driven by the phenyl thiophene region. [21] Preliminary rat pharmacokinetic studies revealed that (6) has a long terminal T1/2 (10.9 h), but solubility studies revealed that this compound is poorly soluble in aqueous media (Sol < 2 mmol). While there are examples of poorly soluble, FDA approved medication, we viewed this as issue that should be addressed in next generation compounds. We have therefore continued our efforts to identify novel, selective D3 ligands.
Results and Discussion: Our recent efforts have focused on a related series of compounds (7) in which the amide of (6) has been inverted. These compounds are readily prepared using the chemistry described in scheme 1. In this process, 5-bromopentanoyl chloride (8) is reacted with a suitably substituted aniline (9) in the presence of triethyl amine to provide amide (10). Displacement of the primary bromide with a functionalized cyclic diamine (11) in refluxing acetone and in the presence of potassium carbonate provide the final screening targets. Table 1 describes the in vitro screening results for D3 and D2, as well as solubility and rat liver microsomal stability.
In our first series of compounds (7a-7i), we maintained the thiophen-3-y moiety in order minimize the number of changes between our new series and compounds typified by (6). As noted in table 1, electron donating (7a) and election withdrawing groups (7b, 7c) in the 2poistion provide potent D3 binders (Ki = 3.1 nM, 6.2 nM, and 20 nM) with moderate selectivity over D2 (21-40 fold). However, rat liver microsome (RLM) stability of these compounds was low to moderate (19.3 to 5.6 minutes). Potent D3 activity was also observed when electron withdrawing groups were located in the 3-position, but selectivity over D2 was not uniform.

Conclusion:
In summary, we have identified a new series of potent D3 binders that display moderate to high selectivity over D2. In addition, exemplary compounds have high RLM stability and aqueous solubility within an acceptable range. Additional efforts will be focused on further optimizing the physicochemical properties of this series of compounds while maintaining the D3/D2 selectivity observed in better performing compounds.

Preparation of 5-(4-(2-cyanophenyl)piperazin-1-yl)-N-(4-(pyridin-2-yl)phenyl)pentanamide (7j):
The title compound was prepared according to the procedure for 5-(4-(2,3dichlorophenyl)piperazin-1-yl)-N-(4-(pyridin-2-yl)phenyl)pentanamide, except 2-(piperazin-1yl)benzonitrile was substituted for 1- (2,3-dichlorophenyl)     Competitive radioligand studies were performed to determine the concentration of inhibitor that inhibits 50% of the specific binding of the radioligand (IC50 value). The final radioligand concentration was approximately equal to the Kd value for the binding of the radioligand. For each competition curve, triplicates were performed using two concentrations of inhibitor per decade over five orders of magnitude. Binding was terminated by the addition of cold wash buffer (10 mM Tris-HCl/150 mM NaCl, pH = 7.5) and filtration over a glass-fiber filter (Pall A/B filters, #66198). A Packard Cobra Gamma Counter was used to measure the radioactivity of The competition curves were modeled for a single binding site using

Bs = Bo -((Bo +L)/(IC50 + L))
where Bs is the amount of ligand bound to receptor and Bo is the amount of ligand bound to receptor in the absence of competitive inhibitor. L is the concentration of the competitive inhibitor. The IC50 value is the concentration of competitive inhibitor that inhibits 50% of the total specific binding. IC50 values were determined using non-linear regression analysis with