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Article Evaluation of Substituted N-Phenylpiperazine Analogs as D3 vs. D2 Dopamine Receptor Subtype Selective Ligands

Boeun Lee 1 , Michelle Taylor 2, Suzy A. Grifﬁn 2, Tamara McInnis 2, Nathalie Sumien 2 , Robert H. Mach 1 and Robert R. Luedtke 2,*

1 Department of Radiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; [email protected] (B.L.); [email protected] (R.H.M.) 2 Department of Pharmacology and Neuroscience, University of North Texas Health Science Center-Fort Worth, Fort Worth, TX 76107, USA; [email protected] (M.T.); suzy.grifﬁ[email protected] (S.A.G.); [email protected] (T.M.); [email protected] (N.S.) * Correspondence: [email protected]

Abstract: N-phenylpiperazine analogs can bind selectively to the D3 versus the D2 dopamine receptor subtype despite the fact that these two D2-like dopamine receptor subtypes exhibit substantial amino acid sequence homology. The binding for a number of these receptor subtype selective compounds was found to be consistent with their ability to bind at the D3 dopamine receptor subtype in a bitopic manner. In this study, a series of the 3-thiophenephenyl and 4-thiazolylphenyl fluoride substituted N-phenylpiperazine analogs were evaluated. Compound 6a was found to bind at the human D3 receptor with nanomolar affinity with substantial D3 vs. D2 binding selectivity (approximately 500-fold). Compound 6a was also tested for activity in two in-vivo assays: (1) a hallucinogenic-dependent head twitch response inhibition assay using DBA/2J mice and (2) an Citation: Lee, B.; Taylor, M.; Griffin, L-dopa-dependent abnormal involuntary movement (AIM) inhibition assay using unilateral 6- S.A.; McInnis, T.; Sumien, N.; Mach, 6a R.H.; Luedtke, R.R. Evaluation of hydroxydopamine lesioned (hemiparkinsonian) rats. Compound was found to be active in Substituted N-Phenylpiperazine both assays. This compound could lead to a better understanding of how a bitopic D3 dopamine Analogs as D3 vs. D2 Dopamine receptor selective ligand might lead to the development of pharmacotherapeutics for the treatment Receptor Subtype Selective Ligands. of levodopa-induced dyskinesia (LID) in patients with Parkinson’s disease. Molecules 2021, 26, 3182. https:// doi.org/10.3390/molecules26113182 Keywords: D2-like dopamine receptors; D3 dopamine receptor subtype; G-protein coupled receptor (GPCR); dopamine receptor subtype selective ligands; bitopic ligands Academic Editor: Ingebrigt Sylte

Received: 27 April 2021 Accepted: 21 May 2021 1. Introduction Published: 26 May 2021 Dopamine is a neurotransmitter that controls a variety of physiological functions in both the periphery and in the central nervous system (CNS). In the periphery, dopamine Publisher’s Note: MDPI stays neutral receptors have been implicated in the modulation of renal sodium excretion, urinary with regard to jurisdictional claims in output, inhibition of norepinephrine release, vasodilatory activity of blood vessels and the published maps and institutional affil- β iations. regulation of insulin secretion in pancreatic -cells [1,2]. Dopamine receptor expression has also been reported to be expressed on regulatory T cells and antigen-presenting dendritic cells associated with the immune system [3,4]. Dopaminergic pathways in the brain have been implicated in the reward system, psychostimulant seeking behaviors, movement, memory and learning, sleep regulation, feeding, attention, olfaction, vision, hormonal Copyright: © 2021 by the authors. regulation, and emotional tone [5]. Licensee MDPI, Basel, Switzerland. There are five human dopamine receptor subtypes classified into two major categories: This article is an open access article D1-like (D1 and D5) and D2-like (D2, D3 and D4) dopamine receptor subtypes. The D1-like distributed under the terms and conditions of the Creative Commons and D2-like dopamine receptor classes have been categorized based upon similarities in Attribution (CC BY) license (https:// genomic organization, amino acid sequence homology and pharmacological properties [6]. creativecommons.org/licenses/by/ All five of the dopamine receptor subtypes are members of the G protein-coupled receptor 4.0/). (GPCR) protein superfamily. The two genes which encode for the D1 and D5 are both

Molecules 2021, 26, 3182. https://doi.org/10.3390/molecules26113182 https://www.mdpi.com/journal/molecules Molecules 2021, 26, 3182 2 of 16

intronless genes, whereas the D2, D3 and D4 dopamine receptor genes contain exons and introns. The length, organization, and amino acid homology of the two D1-like dopamine receptors are similar, with the D1-like receptors having a shorter third intracellular loop and a longer carboxy terminus compared to D2-like dopamine receptor subtypes. The three-dimensional structures of all three D2-like dopamine receptors has been determined by x-ray diffraction [7–9]. The structure of the D2 dopamine receptor in complex with a Gi-protein has recently been reported using cryo-electron microscopy [10]. The three- dimensional structure of the D1-like receptors has not been reported. Agonist stimulation of the D1-like dopamine receptors has been shown to increase the production of cAMP, whereas D2-like receptor agonists inhibit forskolin-dependent stimulation of adenylyl cyclase [11,12]. Agonist binding at both the D1-like and D2-like dopamine receptors leads to β-arrestin recruitment [13,14]. Dopamine receptor agonists are used therapeutically to treat Parkinson’s disease and restless legs syndrome, but adverse side effects can include nausea, orthostatic hypotension, hallucinations and impulse control disorders. Chronic administration of dopaminergic agonists that are used of L-dopa for the treatment of Parkinson’s disease in humans can lead to L-dopa-induced dyskinesia (LIDs). In preclinical studies, selective blockade of the D3 dopamine receptor has been reported to reduce L-dopa dependent dyskinesia-like movements, without affecting the antiparkinsonian efficacy of L-dopa [15,16]. Despite having similar amino acid sequences and pharmacologic profiles, the D2 and D3 dopamine receptor subtypes exhibit differences in how they couple to signal transduction pathways. For example, agonist stimulation of the D2 receptor activates ERK via a Giα-dependent pathway, whereas D3 receptors activate ERK by a mechanism that is Gβγ dependent [17]. In addition, sigma-1 receptors have been reported to selectively interact with D2 dopamine receptors, but not with the D3 or D4 receptor subtypes [18]. Further, the S100B calcium binding protein has been reported to interact with the D2, but not the D3, dopamine receptor [19]. Since the orthosteric binding site of D2-like dopamine receptors is constructed by conserved amino acid residues in the transmembrane helical spanning regions, it is not surprising that many of the original D2 vs. D1 selective ligands bind nonselectively at the D2 and D3 dopamine receptor subtypes. Ligands that can simultaneously interact with two different regions (an orthosteric and secondary binding site (SBS) on a single GPCR have been described as bitopic ligands [20–24]. The ability of some N-phenylpiperazine benzamides to bind selectively at the D3 dopamine receptor subtype has been attributed to the ability of the N-phenylpiperazine moiety to occupy either the D2 or D3 dopamine orthosteric binding site, while the benzamide moiety interacts with an SBS that is unique to the D3 dopamine receptor subtype [21,24]. Consequently, a number of research groups have been able to develop D3 vs. D2 dopamine receptor selective ligands using a substituted N-phenylpiperazine benzamide template to identify ligands that bind selectively and with high affinity at the D3 dopamine receptor subtype [25–28]. Previously we reported how incorporating a 4-(thiophen-3-yl)benzamide could lead to compounds that bind to the D3 receptor subtype with high affinity and with D3 vs. D2 receptor binding selectivity. Maintaining the thiophene moiety while varying the composi- tion or position of substituents on the phenyl ring of the N-phenylpiperazine moiety leads to a panel of compounds with varying D2/D3 dopamine receptor subtype affinity and binding selectivity [22,23]. In this communication we explored the effect of varying the benzamide portion of our compounds while maintaining the fluorinated N-phenylpiperazine portion. The result of this study provides further insight into how structural variation in the dopaminergic ligands might impact ligand binding affinity and binding selectivity of N-phenylpiperazine benzamide at human D2 and D3 dopamine receptors. Molecules 2021, 26, x FOR PEER REVIEW 3 of 17 Molecules 2021, 26, 3182 3 of 16

2. Results 2. Results 2.1.2.1. Chemistry Chemistry The structure and binding affinities (Ki values) for several previously published N- The structure and binding affinities (Ki values) for several previously published N- phenylpiperazinesphenylpiperazines at at the the human human D2 D2 and D3 do dopaminepamine receptor areare shownshown inin FigureFigure1 .1. TheseThese compounds compounds exhibit exhibit varying varying binding binding affini affinitiesties at atthe the D3D3 dopamine dopamine receptor receptor and and var- yingvarying D3 D3vs. vs.D2 D2binding binding selectivity selectivity [29–33]. [29–33 ].Compound Compound LS-3-134LS-3-134 exhibits highhigh affinityaffinity bindingbinding at at the the D3 D3 receptor (Ki value of approximatelyapproximately 0.20.2 nM)nM)with with >150-fold >150-fold D3 D3 vs. vs. D2 D2 dopaminedopamine receptor receptor binding selectivity,selectivity, whereas whereasWW-III-55 WW-III-55exhibits exhibits a higher a higher level level of D3 of vs.D3 vs.D2 D2 dopamine dopamine receptor receptor binding binding selectivity selectivity (>800-fold) (>800-fold) but but with with lower lower binding binding affinity affinity at atthe the D3 D3 dopamine dopamine receptor receptor (K i(Kvaluesi values approximately approximately 20 nM).20 nM). It is It noteworthy is noteworthy that thatLS-3-134 LS-3- 134and andWW-III-55 WW-III-55both both contain contain an N -phenylpiperazinean N-phenylpiperazine and a 4-(thiophen-3-yl)benzamideand a 4-(thiophen-3-yl)ben- zamidemoiety, moiety, while KX-2-67 while KX-2-67binds with binds low with affinity low predominantlyaffinity predom toinantly the SBS toof the the SBS D2 of and the D3 D2 anddopamine D3 dopamine receptor receptor [21,22]. [21,22].

FigureFigure 1. 1.ChemicalChemical structures structures and and binding binding data dataof a variety of a variety of arylamide of arylamide phenylpiperazine phenylpiperazine D3 dopamine D3 dopamine receptor receptor selective compoundsselective compounds are shown. are KX-2-67 shown. bindsKX-2-67 withbinds low withaffinity low to afﬁnity the secondary to the secondary binding site binding of the site D3 of dopamine the D3 dopamine receptor [19,20,26,27,30].receptor [19,20, 26,27,30].

TheThe synthesis synthesis strategystrategy forfor the the compounds compounds described described in thisin this communication communication is outlined is out- linedin Scheme in Scheme1. Brieﬂy, 1. Briefly, the orthosteric the orthostericN-phenylpiperazine N-phenylpiperazine moieties moieties2a–f were 2a–f affordedwere afforded by a byone-pot a one-pot Pd-catalyzed Pd-catalyzed Buchwald-Hartwig Buchwald-Hartwig amination amination reaction reaction of six of different six different aryl aryl halides hal- ideswith with piperazine piperazine [34]. [34]. The TheN-alkylation N-alkylation of aryl of aryl piperazines piperazines2a– f2awith–f with 1-bromobutane 1-bromobutane in inacetone acetone using using potassium potassium carbonate carbonate as a as base a base provided provided fragments fragments of D3 of dopamine D3 dopamine receptor re- ceptorselective selective compounds, compounds,3a–f. Butyl 3a–f. amine Butyl compoundsamine compounds5a–f, which 5a–f were, which used were for used conjugation for con- jugationwith substituted with substituted benzoic acids,benzoic were acids, synthesized were synthesized using a Gabriel using a amine Gabriel synthesis amine synthesis reaction. reaction.N-arylpiperazine N-arylpiperazine compounds compounds2a–f were reacted2a–f withwereN -(4-bromobutyl)phthalimidereacted with N-(4-bromobu- in tyl)phthalimideacetonitrile to give in acetonitrile butyl phthalimide to give butyl compounds phthalimide4a–f andcompounds the hydrazinolysis 4a–f and the of hydra- these zinolysiscompounds of these gave thecompounds corresponding gave aminesthe corresponding5a–f in high yieldsamines (70–95%). 5a–f in Thehigh free yields amines (70– 95%).5a–f were The free coupled amines with 5a 4-(thien-3-yl)benzoic–f were coupled with acid 4-(thien-3-yl)benzoic or 4-(1,3-thiazol-4-yl)benzoic acid or 4-(1,3-thiazol- acid using 1- 4-yl)benzoicethyl-3-(3-dimethylaminopropyl)carbodiimide acid using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-hydroxybenzotriazole (EDC) and (HOBt) 1-hy- hydrate in dichloromethane to give the targeted benzamide compounds 6a–f (52–88% droxybenzotriazole (HOBt) hydrate in dichloromethane to give the targeted benzamide yields) and 7a–f (64–84% yields). The ﬁnal products were converted to the corresponding compounds 6a–f (52–88% yields) and 7a–f (64–84% yields). The final products were con- hydrochloride salts. Detailed synthesis procedure and chemical analysis of synthesized verted to the corresponding hydrochloride salts. Detailed synthesis procedure and chem- compounds (1H, 13C NMR, and Mass) were reported in Supplementary Material. ical analysis of synthesized compounds (1H, 13C NMR, and Mass) were reported in Sup- plementary Material.

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Scheme 1. SchemeSynthesis 1. ofSynthesis Thiophenephenyl of Thiophenephenyl and Thiazolylphenyl and Thiazoly Substitutedlphenyl Substituted Phenylpiperazine Phenylpiperazine Analogs. Ana- Reagents and logs. Reagents and conditions: (a) Aryl halide (1a–f), piperazine, NaOtBu, Pd◦ 2(dba)3, RuPhos, diox- conditions: (a) Aryl halide (1a–f), piperazine, NaOtBu, Pd2(dba)3, RuPhos, dioxane, 100 C, 1 h; (b) 1-bromobutane, K2CO3, ane, 100 °C, 1 h; (b) 1-bromobutane, K2CO3, acetone, rt, overnight; (c) N-(4-bromobutyl)phthalimide, acetone, rt, overnight; (c) N-(4-bromobutyl)phthalimide, Et3N, acetonitrile, rt, overnight; (d) hydrazine hydrate, ethanol, ◦ Et3N, acetonitrile, rt, overnight; (d) hydrazine hydrate, ethanol, 80 °C, 2 h; (e) 4-(thien-3-yl)benzoic 80 C, 2 h; (e) 4-(thien-3-yl)benzoic acid, EDC, HOBt hydrate, CH2Cl2, rt, 1 h; (f) 4-(1,3-Thiazol-4-yl)benzoic acid, EDC, acid, EDC, HOBt hydrate, CH2Cl2, rt, 1 h; (f) 4-(1,3-Thiazol-4-yl)benzoic acid, EDC, HOBt hydrate, HOBt hydrate, CH Cl , rt, 1 h. CH2Cl2,2 rt,2 1 h.

2.2. Pharmacological2.2. Pharmacological Studies Studies 2.2.1. D2-Like Dopamine Receptors 2.2.1. D2-Like Dopamine Receptors Competitive radioligand binding techniques were used to determine the Ki values of Competitive radioligand binding techniques were used to determine the Ki values of ligands at human D2 and D3 dopamine receptors expressed in stably transfected HEK cells ligands at human D2 and D3 dopamine receptors expressed in stably transfected HEK (Table1). As anticipated, compounds 3a–f exhibited the lowest binding afﬁnities for the cells (Table 1). As anticipated, compounds 3a–f exhibited the lowest binding affinities for human D2 (Ki values = 349–7522 nM) and D3 (Ki values = 96–1413 nM) dopamine receptor the human D2 subtypes.(Ki values These = 349–7522 compounds nM) and bound D3 (K essentiallyi values =nonselectively 96–1413 nM) dopamine at the D2and re- D3 dopamine ceptor subtypes.receptor These subtypescompounds (binding bound selectivity essentially ratio nonselectively = 1.0–7.5-fold). at the The D2 4-thiophene-3-yl-benzamide and D3 dopamine receptorN-phenylpiperazines subtypes (binding selectivity (6a–f) and ratio corresponding = 1.0–7.5-fold). 4-thiazolyl-4-ylbenzamide The 4-thiophene-3-yl- N-piperazine benzamide N-phenylpiperazinesanalogs (7a–f) exhibited (6a–f) a and similar corresponding range of binding 4-thiazolyl-4-ylbenzamide afﬁnities at the D3 dopamine N- receptor piperazine analogs (7a–f) exhibited a similar range of binding affinities at the D3 dopa- (Ki = 1.4–43 nM and 2.5–31 nM, respectively) and a similar range of D3 vs. D2 receptor mine receptor binding(Ki = 1.4–43 selectivity nM and (67–1831-fold 2.5–31 nM, respectively) vs. 73–1390-fold, and a respectively). similar range Representative of D3 vs. examples D2 receptor bindingof competitive selectivity radioligand (67–1831-fold binding vs. 73–1390-fold, curves of compound respectively).6a–c andRepresenta-7a–c at human D2 and tive examples D3of competitive dopamine receptors radioligand are shownbinding in curves Supplementary of compound Material. 6a–c and 7a–c at human D2 and D3 dopamine receptors are shown in Supplementary Material. A comparison of the binding affinity and binding selectivity of the N-phenylpiperazine orthosteric ligands (compounds 3a–f) with the longer thiophene-containing (compounds 6a–f) and thiazole-containing (compounds 7a–f) analogs further suggest that the benzamide N-phenylpiperazine class of compound likely engage the D3 dopamine receptor, but not the D2 dopamine receptor, in a bitopic binding mode, leading to enhanced affinity at the D3 receptor subtype and consequently increased D3 vs. D2 binding selectivity. In general, the thiophene analogs (compounds 6a–f) bound with slightly higher affinity at D3 dopamine receptors and/or with slightly greater D3 vs D2 binding selectivity than the corresponding thiazole analogs (compounds 7a–f).

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Table 1. Binding Affinity and cLogP Values for Compounds.

R2 Molecules 2021, 26, x FOR PEER REVIEW Table 1. BindingN Affinity and cLogP Values for Compounds.H 5 of 17 Molecules 2021, 26, 3182 N 5 of 16 N N N O R2 R 1 N H R N 1 N Table 1. BindingN 3a-f Affinity and cLogP Values for Compounds.6a-f, 7a-f Table 1. Binding Affinity and cLogP ValuesN for Compounds.O R1 R Structure KRi1 Values (nM) 1 D2:D32 3a-fN 6a-f, 7a-fH 2 # N cLogP R1 N R2 D2 Receptor ND3 Receptor Selectivity Structure Ki ValuesN (nM) 1 O 3a 2-F R1 384 ± 39.6 96.2 ± 16.9 D2:D34.0 4.06 # cLogP 2 R1 3b R3-F1 R2 D22516 Receptor ± 252 D31003 Receptor ± 118 Selectivity2.5 4.06 3a-f 6a-f, 7a-f 3a3c 2-F4-F 3843091 ± ±39.6 269 96.211761 ± ±16.9 240 4.02.6 4.064.06 Structure Ki Values (nM) # Structure − Ki Values (nM) 1 D2:D3D2:D3 Selectivity cLogP 2 3b 3-F 2516 ± 252 1003 ± 118 2.5 4.062 # 3d 2-F, 5-CNR1 R2 D25431 Receptor ± 1059 D3 Receptor726 ± 101 7.5 cLogP3.78 R1 R2 D2 Receptor D3 Receptor Selectivity 3c3e 2-F,4-F 4-CF 3 30917522 ± ± 269 466 11761413 ±± 24059.1 2.65.3 4.065.13 3a 2-F − 384 ± 39.6 96.2 ± 16.9 4.0 4.06 3a 3b2-F 3-F384 2516 ± 39.6± 252 100396.2 ± 16.9118 4.0 2.5 4.06 4.06 3d3f 2-OC2-F, 25-CNH4F, 4-F 5431349 ±± 105921.1 726351 ±± 10142.1 7.51.0 3.784.31 ± ± 3b 3c3-F 4-F− 2516 3091 ± 252269 11761003 ± 240118 2.5 2.6 4.06 4.06 3e6a 3d2-F, 2-F, 2-F4-CF 5-CN 3 7522 5431648 ±± 4661059100 72614131.4± 101 ±± 59.10.2 7.55.3467 5.135.15 3.78 3c 4-F 3091 ± 269± 1176± ± 240 2.6 4.06 3f6b 3e2-OC 2-F,2H3-F4F, 4-CF 4-F3 − 34975222334 ± ±21.1 466485 14133517.9 ±59.1 ± 42.1 1.4 5.31.0296 4.315.15 5.13 3d 3f2-F, 2-OC 5-CN2H 4F, 4-F5431 349 ± 1059± 21.1 351726± ± 42.1101 7.5 1.0 3.78 4.31 6a6c 2-F4-F 6487200 ± ±100 215 1.412.4 ± ±0.2 1.6 467579 5.155.15 S 3e 6a2-F, 4-CF 2-F3 7522648 ± ±466100 1413 1.4 ± ± 0.259.1 5.3 467 5.13 5.15 6b 3-F 2334 ± 485 7.9 ± 1.4 296 5.15 6d 6b2-F, 5-CN 3-F 23344502± ± 485565 7.915.5± 1.4 ± 1.3 296290 4.87 5.15 3f 6c2-OC2H4F, 4-F4-F 349 7200 ± 21.1± 215 351 12.4 ±± 42.11.6 1.0 579 4.31 5.15 6c6e 2-F,4-F 4-CF 3 7200>75,000 ± 215 12.443.3 ± ± 1.6 9.2 >1800579 5.156.22 6d 2-F, 5-CNS 4502 ± 565 15.5 ± 1.3 290 4.87 6a6d 2-F,2-F 5-CN 6484502 ± 100 ± 565 1.415.5 ± 0.2 ± 1.3 467290 5.154.87 6f 6e2-OC 2-F,2H4 4-CFF, 4-F3 411>75,000 ± 27.3 43.36.1± 9.2 ± 0.2 >180067.4 5.40 6.22 6b 3-F 2334 ± ±485 7.9 ±± 1.4 296 5.15 6e7a 6f2-F, 2-OC 2-F4-CF2H 43F, 4-F 411478>75,000 ± 27.371.0 6.143.32.50.2 ±± 0.229.2 >1800 67.4190 6.224.16 5.40 6c 4-F 7200 ± ±215 12.4± ± 1.6 579 5.15 6f7b 7a2-OC2H3-F4 2-FF, 4-F S 4113946478 ± ±27.371.0 146 2.5 6.124.00.22 ± ±0.2 5.5 19067.4165 5.404.16 4.16 6d 7b2-F, 5-CN 3-F 4502 3946 ± 565± 146 24.015.5 ±± 5.51.3 290 165 4.87 4.16 7a7c 2-F4-F N 4784012 ±± ±71.0 694 2.528.6± ± 0.22± 2.1 190140 4.164.17 7c 4-FS 4012 694 28.6 2.1 140 4.17 6e 2-F, 4-CF3 >75,000 43.3 ± 9.2 >1800 6.22 7b 7d 2-F,3-F 5-CN3946 7717 ±± 1461497 28.124.0± 0.8± 5.5 274165 4.16 3.89 7d 2-F, 5-CN 7717 ± 1497 28.1 ± 0.8 274 3.89 6f 7e2-OC2H 2-F,4F, 4-CF4-F 3 411 >40,000± 27.3 30.96.1 ± 0.22.6 67.4 1390 5.40 5.24 7c 4-F N 4012 ± 694 28.6 ± 2.1 140 4.17 7e 7f2-F, 2-OC 4-CF2H4F,3 4-FS 349>40,000± 50.3 4.8 30.9± 0.28 ± 2.6 72.91390 5.24 4.42 7a7d 2-F,2-F 5-CN 4787717 ± 71.0 ± 1497 2.528.1 ± 0.22 ± 0.8 190274 4.163.89 7fThe Ki values2-OC for2 theH4F, binding 4-F of test compound at human349 D2 and ± 50.3 D3 dopamine receptors4.8 ± 0.28 are shown. The D372.9 vs. D2 dopamine receptor4.42 1 7b binding selectivity3-F and cLogP values of the test compound3946 are ± also146 shown. Mean24.0± S.E.M.;± 5.5 Ki values were165 determined by at4.16 least three 7eThe Ki values2-F, for 4-CF the 3binding of test compound at human>40,000 D2 and D3 dopamine30.9 ± 2.6 receptors are1390 shown. The D3 5.24vs. D2 experiments. 2 cLogP values were calculated using ChemDraw Professional 15.1. N 1 7c dopamine receptor4-F binding selectivity and cLogP 4012values ± 694of the test compound28.6 ± 2.1are also shown.140 Mean ± S.E.M.; 4.17Ki values 7f 2-OC2H4F, 4-F S 349 ± 50.3 4.8 ± 0.28 72.9 4.42 were determined by at least three experiments. 2 cLogP values were calculated using ChemDraw Professional 15.1. 7d The Ki values2-F, for5-CN the binding of testA comparison compound 7717at of human the ± binding1497 D2 and affinity D3 28.1dopamine and ± binding0.8 receptors selectivity are274 shown. of the TheN-phenylpiperazine D33.89 vs. D2 dopamine receptor binding selectivityorthosteric and ligands cLogP values (compounds of the test3a compound–f) with the are longer also shown. thiophene-containing 1 Mean ± S.E.M.; Ki (compoundsvalues 7e 2-F, 4-CF3 2.2.2. 5-HT1A Receptor>40,000 Binding 30.9 ± 2.6 1390 5.24 were determined by at least three6a–f experiments.) and thiazole-containing 2 cLogP values (compounds were calculated7a –usf)ing analogs ChemDraw further Professional suggest that 15.1. the benzamide 7f 2-OC2H4F, 4-F 349 ± 50.3 4.8 ± 0.28 72.9 4.42 N-phenylpiperazineBecause the 5-HT1A class receptor of compound is a commonly likely engage observed the D3 off-site dopamine binding receptor, site for but ben- not The Ki values for the binding2.2.2. zamideofthe test D25-HT1A compound N dopamine-phenylpiperazine Receptor at receptor, human Binding D2 in ligands, aand bitopic D3 compounds dopamine binding mode, receptors were leading simultaneously are shown. to enhanced The screened D3 affinity vs. D2 forat thebind- D3 dopamine receptor binding selectivitying of our and compounds cLogP values at of thethe humantest compound 5-HT1A are re alsoceptor shown. expressed 1 Mean ± in S.E.M.; CHO-K1 Ki values cells, using receptorBecause subtype the2 5-HT1A and consequently receptor is increaseda commonly D3 vs.observed D2 binding off-site selectivity. binding Insite general, for ben- the were determined by at least three experiments. cLogP3 values were calculated using ChemDraw Professional 15.1. zamidethethiophene radioligand N-phenylpiperazine analogs [ H]-8-hydroxy-DPAT (compounds ligands,6a– compoundsf) bound(8-OH-DPAT) with we slightlyre (Tablesimultaneously higher 2). Based affinity screened upon at D3the for dopamine analysis bind- ofreceptors the displacement and/or with assay slightly shown greater in Table D3 vs2, the D2 bindingorthosteric selectivity compounds than the3c–f corresponding appeared to 2.2.2.ing 5-HT1Aof our compounds Receptor Binding at the human 5-HT1A receptor expressed in CHO-K1 cells, using thebindthiazole radioligand with analogslow affinity [3H]-8-hydroxy-DPAT (compounds at human7a 5-HT1A–f). (8-OH-DPAT) receptors. However, (Table 2). at Based a concentration upon the ofanalysis 91 nM Because the 5-HT1A receptor is a commonly observed off-site binding site for ben- ofcompounds the displacement 3a and 3bassay appeared shown toin beTable able 2, to the partially orthosteric displace compounds the 5-HT1A 3c– fselective appeared radi- to zamide2.2.2. N-phenylpiperazine 5-HT1A Receptor Binding ligands, compounds were simultaneously screened for bind- bindoligand. with Compounds low affinity at6b, human 7a, 7b 5-HT1Aand 7f displaced receptors. tritiated However, 8-OH-DPAT at a concentration binding inof 91a man- nM ing of our compounds at the human 5-HT1A receptor expressed in CHO-K1 cells, using compoundsner thatBecause suggested 3a and the 5-HT1A3bsubstantial appeared receptor af tofinity be is able afor commonly theto partially 5-HT1A observed displace receptor. off-site the 5-HT1A binding selective site for radi- benza- the radioligand [3H]-8-hydroxy-DPAT (8-OH-DPAT) (Table 2). Based upon the analysis oligand.mide N Compounds-phenylpiperazine 6b, 7a, ligands,7b and 7f compounds displaced weretritiated simultaneously 8-OH-DPAT screened binding forin a binding man- of the displacement assay shown in Table 2, the orthosteric compounds 3c–f appeared to nerof that our suggested compounds substantial at the human affinity 5-HT1A for the receptor 5-HT1A expressed receptor. in CHO-K1 cells, using the bindradioligand with low affinity [3H]-8-hydroxy-DPAT at human 5-HT1A (8-OH-DPAT) receptors. However, (Table2 ).at a Based concentration upon the of analysis 91 nM of

compoundsthe displacement 3a and 3b assayappeared shown to be in able Table to2 partially, the orthosteric displace compounds the 5-HT1A selective3c–f appeared radi- to oligand.bind Compounds with low afﬁnity 6b, 7a, at human7b and 5-HT1A7f displaced receptors. tritiated However, 8-OH-DPAT at a concentration binding in a man- of 91 nM ner thatcompounds suggested3a substantialand 3b appeared affinity to for be the able 5-HT1A to partially receptor. displace the 5-HT1A selective radioligand. Compounds 6b, 7a, 7b and 7f displaced tritiated 8-OH-DPAT binding in a manner that suggested substantial afﬁnity for the 5-HT1A receptor.

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Table 2. Screen for Binding Affinity at the 5-HT1A Receptor.

R Molecules 2021, 26, x FOR PEER REVIEWTable 2. Screen for Binding Affinity at the 5-HT1A Receptor. 2 6 of 17 N H Molecules 2021, 26, 3182 N 6 of 16 N N R2 N N H O R1 N N TableN 2. Screen for Binding AffinityR1 at the 5-HT1A Receptor. Table 2. Screen3a-f for Binding Affinity at theN 5-HT1A6a-f, Receptor. 7a-f O R1 R1 R2 3a-f N Structure 6a-f, 7a-f H5-HT1A Receptor Inhibition # N N N R1 R2 (%) Structure N 5-HT1AO Receptor Inhibition 3aR 1 # 2-F 20.3 ± 16.2 R1 R1 R2 (%) 3b 3a-f 3-F 6a-f, 7a-f 36.1 ± 2.1 3a 2-F 20.3 ± 16.2 3c 4-F Structure 5.1 ± 11.2 # Structure 5-HT1A5-HT1A Receptor Receptor Inhibition Inhibition (%) 3b # 3-F − 36.1 ± 2.1 3d 2-F,R1 5-CN R2 −18.0 ± 11.1 3c 4-F R1 R2 5.1 ± 11.2(%) 3e 3a2-F, 2-F 4-CF3 − −11.620.3 ± ±15.416.2 3d 3a 3b2-F, 3-F 5-CN2-F −18.020.3 36.1 ± 11.1 ± 16.22.1 3f 3c2-OC 4-F2H4F, 4-F 1.7 5.1 ± 21.7± 11.2 3e 3b 2-F, 4-CF3-F3 − −11.636.1 ± 15.4 ± 2.1 6a 3d 2-F, 5-CN2-F −3.3−18.0 ± 15.1± 11.1 3f 3c 2-OC2H4F,4-F 4-F 1.7 ±5.1 21.7 ± 11.2 3e 2-F, 4-CF3 − −11.6 ± 15.4 6b 3-F 74.7 ±± 1.9 6a 3d 3f 2-OC2H2-F2-F,4F, 4-F5-CN −3.3− 1.718.0± 15.1 ±21.7 11.1 6c 4-F −7.7− ± 23.5± 6b 3e 6a 2-F3-F2-F, 4-CF3 74.7−11.6 3.3± 1.9 ± 15.115.4 6d 6b2-F, 3-F 5-CN 2.5 74.7 ± 3.5± 1.9 6c 3f 6c2-OC 4-F4-F 2H4F, 4-F −7.7− 1.7±7.7 23.5 ±± 21.723.5 6e 2-F, 4-CF3 6.1 ± 6.8± 6d 6a 6d 2-F,2-F, 5-CN 5-CN2-F 2.5− 2.5±3.3 3.5 ± 3.515.1 6e 2-F, 4-CF 6.1 ± 6.8 6f 6b 2-OC2H43-FF,3 4-F 16.974.7 ± 24.4 ± 1.9 6e 6f 2-OC2-F,2H 4-CF4F, 4-F3 6.1 16.9 ± 6.8± 24.4 7a 2-F 65.8 ± 5.0 6f 6c 7a2-OC 2-F2H4F,4-F 4-F 16.9−65.8 ±7.7 24.4 ± 23.55.0 7b 7b 3-F3-F 74.1 74.1 ± 5.3± 5.3 7a 6d 2-F2-F, 5-CN 65.8 2.5± 5.0 ± 3.5 7c 7c 4-F4-F 12.2 12.2 ± 3.1± 3.1 7b 6e 7d 2-F,3-F 5-CN2-F, 4-CF3 74.1− 6.15.6± 5.3 ±± 6.818.3 7d 2-F, 5-CN −5.6 ± 18.3± 7c 6f 7e 2-F,2-OC 4-CF4-F 2H3 4F, 4-F 12.216.97.6 ± 3.1 ± 6.5 24.4 7f 2-OC H F, 4-F 37.1 ± 24.4 7e 7a 2-F,2 44-CF2-F3 7.665.8 ± 6.5 ± 5.0 7dPhenylpiperazine ligands2-F,3a –5-CNf, 6a–f , and 7a–f were screened for the binding activity−5.6 of ± [318.3H]-8-hydroxy-DPAT at 7fhuman 5-HT1A receptors2-OC expressed2H4F, 4-F in CHO-K1 cells using a single point radioligand37.1 ± displacement 24.4 assay. The 7e 7b 2-F, 4-CF3-F3 7.6 74.1± 6.5 ± 5.3 Phenylpiperazineradioligand displacement ligands percentages 3a–f, 6a–f were, and determined 7a–f were by screened at least three for the experiments binding usingactivity 91 nM of concentration[3H]-8-hy- 7c 4-F 12.2 ± 3.1 droxy-DPAT7fof test compound. at human Nonspecific2-OC 5-HT1A2H4 bindingF, receptors4-F was defined expressed using in 10 CHO-K1µM metergoline. cells using Compounds37.1 a single± 24.43a point and 3bradiolig-exhibited moderate displacement of the radioligand at the 5-HT1A receptor. Thiophenyl compound 6b and thiazolyl and 7ddisplacement assay. The2-F, radi 5-CNoligand displacement percentages were determined−5.6 ± 18.3 by3 at least Phenylpiperazinecompounds 7a, 7b ligands, and 7f 3aexhibited–f, 6a–f, a and greater 7a– percentagef were screened of radioligand for the displacement.binding activity Data ofis [ presentedH]-8-hy- as droxy-DPATthree experiments at human using 5-HT1A 91 nM receptorsconcentration expressed of test in compound. CHO-K1 cells Nonspecific using a single binding point was radiolig- defined percent7e displacement of radioligand2-F, 4-CF (n 3≥ 3) ± S.E.M. 7.6 ± 6.5 andusing displacement 10 μM metergoline. assay. The Compounds radioligand 3a displacementand 3b exhibited percentages moderate were displacement determined of the by radiolig-at least 7f 2-OC2H4F, 4-F 37.1 ± 24.4 threeand atexperiments theA comparison5-HT1A using receptor. 91 of nM the Thiophenyl concentration effect of compound compounds of test 6bcompound. and3a, thiazolyl 6a and Nonspecific 7acompoundson [ 3bindingH]-8-hydroxy-DPAT 7a, 7bwas, and defined 7f ex- 3 usinghibitedbindingPhenylpiperazine 10 a μ greaterM was metergoline. conducted.percentage ligands Compounds of Based3a radioligand–f, 6a upon– f3a, and and displace the 7a 3b radioligand– fexhibited werement. screened Data moderate displacement is presentedfor thedisplacement binding as assessment percent activity of displacementthe theofradiolig- [ orthos-H]-8-hy- andof tericradioligand droxy-DPATat the ligand 5-HT1A (n (compound at ≥ receptor. 3)human ± S.E.M. 5-HT1A Thiophenyl3a) exhibited receptors compound moderate expressed 6b and affinity in CHO-K1thiazolyl at 5-HT1A cellscompounds using receptors. a single7a, 7b , Thepoint and thiazole7f radiolig- ex- hibitedand a displacement greater percentage assay. of The radioligand radioligand displace displacementment. Data percentages is presented were as percent determined displacement by at least analog of compound 3a (compound 7a) exhibited increased affinity at 5-HT1A, while the of radioligandthreeA comparison experiments (n ≥ 3) ± using ofS.E.M. the 91 effect nM concentration of compounds of test 3a, compound. 6a and 7a Nonspecific on [3H]-8-hydroxy-DPAT binding was defined thiophene analog (compound 6a) appeared to exhibit decreased binding affinity at the bindingusing 10was μM conducted. metergoline. Based Compounds upon 3athe and radioligand 3b exhibited displacement moderate displacement assessment of the the radiolig- or- 5-HT1A receptor compared to compound 3a. thostericandA comparisonat theligand 5-HT1A (compound of receptor. the effect 3a)Thiophenyl exhibitedof compounds compound moderate 3a, 6b 6aaffinity and and thiazolyl 7aat 5-HT1Aon compounds [3H]-8-hydroxy-DPAT receptors. 7a, 7b The, and thi- 7f ex- Compound 6a bound at the D3 dopamine receptor with high affinity (Ki value = 1.4 ± bindingazolehibited analog was a greater conducted.of compound percentage Based of3a radioligand (compoundupon the displaceradioligand 7a) exhibitedment. Datadisplacement increasedis presented assessmentaffinity as percent at displacement5-HT1A, the or- 0.21 nM) and exhibited >400-fold D3 vs. D2 receptor binding selectivity, while exhibiting thostericof radioligand ligand (compound (n ≥ 3) ± S.E.M. 3a) exhibited moderate affinity at 5-HT1A receptors. The thi- whilea low the level thiophene of radioligand analog (compound displacement 6a) appeared at 5-HT1A to receptors,exhibit decreased suggesting binding that affinity it bound azoleat the analog 5-HT1A of receptor compound compared 3a (compound to compound 7a) exhibited3a. increased affinity at 5-HT1A, with lowA comparison affinity at the of 5-HT1Athe effect receptor of compounds binding site.3a, 6a To verifyand 7a the on low [3H]-8-hydroxy-DPAT affinity binding of whileCompound the thiophene 6a analogbound (compoundat the D3 dopamine 6a) appeared receptor to exhibit with high decreased affinity binding (Ki value affinity = 1.4 compoundbinding was6a atconducted. the 5-HT1A Based receptor, upon a the dose radioligand dependent competitivedisplacement radioligand assessment binding the or- at± 0.21the 5-HT1A nM) and receptor exhibited compared >400-fold to D3 compound vs. D2 receptor 3a. binding selectivity, while exhibiting curvethosteric was ligand performed. (compound Compound 3a) exhibited6a was found moderate to bind affinity to the at 5-HT1A5-HT1A receptorreceptors. with The a thi- Ki a lowCompound level of radioligand 6a bound at displacement the D3 dopamine at 5-HT1A receptor receptors, with high suggesting affinity (K thati value it bound = 1.4 valueazole ofanalog 199 nM of (Figurecompound2), which 3a (compound is approximately 7a) exhibited 140-fold increased lower than affinity its affinity at 5-HT1A, at the ±with 0.21 lownM) affinityand exhibited at the 5-HT1A>400-fold receptor D3 vs. D2 binding receptor site. binding To verify selectivity, the low while affinity exhibiting binding D3while dopamine the thiophene receptor. analog The rank(compound order of 6a affinity) appeared of these to exhibit analogs decreased at the 5-HT1A binding receptor affinity aof low compound level of 6aradioligand at the 5-HT1A displacement receptor, aat dose 5-HT1A dependent receptors, competitive suggesting radioligand that it bound bind- wasat the found 5-HT1A to be receptor7a > 3a >compared6a (Figure to2). compound 3a. withing curve low affinity was performed. at the 5-HT1A Compound receptor 6a bindingwas found site. to To bind verify to the the 5-HT1A low affinity receptor binding with Compound 6a bound at the D3 dopamine receptor with high affinity (Ki value = 1.4 of compound 6a at the 5-HT1A receptor, a dose dependent competitive radioligand bind- ± 0.21 nM) and exhibited >400-fold D3 vs. D2 receptor binding selectivity, while exhibiting ing curve was performed. Compound 6a was found to bind to the 5-HT1A receptor with a low level of radioligand displacement at 5-HT1A receptors, suggesting that it bound with low affinity at the 5-HT1A receptor binding site. To verify the low affinity binding

of compound 6a at the 5-HT1A receptor, a dose dependent competitive radioligand binding curve was performed. Compound 6a was found to bind to the 5-HT1A receptor with

Molecules 2021, 26, x FOR PEER REVIEW 7 of 17

a Ki value of 199 nM (Figure 2), which is approximately 140-fold lower than its affinity at Molecules 2021, 26, 3182 7 of 16 the D3 dopamine receptor. The rank order of affinity of these analogs at the 5-HT1A receptor was found to be 7a > 3a > 6a (Figure 2).

FigureFigure 2.2. CompetitiveCompetitive RadioligandRadioligand Binding Binding Assay Assay for for the the Inhibition Inhibition of of [3 H]-8-hydroxy-DPAT[3H]-8-hydroxy-DPAT with with 2- fluorophenyl2-fluorophenyl Compounds Compounds at 5-HT1Aat 5-HT1A Receptors Receptors. 2-Fluorophenyl. 2-Fluorophenyl piperazine piperazine compounds compounds3a (–3a• –)(–,●6a–), 6a (–■–), and 7a (–▲–) were evaluated for their affinity at 5-HT1A receptor using competitive radi- (––), and 7a (–N–) were evaluated for their affinity at 5-HT1A receptor using competitive radioligand oligand binding techniques. Ki values were determined by at least three experiments. Compound binding techniques. Ki values were determined by at least three experiments. Compound 7a exhibited 7a exhibited the highest affinity for the 5-HT1A receptor (Ki = 14.3 ± 7.1 nM) while compound 6a the highest affinity for the 5-HT1A receptor (Ki = 14.3 ± 7.1 nM) while compound 6a had the lowest had the lowest affinity for the receptor (Ki = 199 ± 34.3 nM). The orthosteric fragment, compound 3a, affinity for the receptor (K = 199 ± 34.3 nM). The orthosteric fragment, compound 3a, exhibited exhibited an intermediate affinityi for 5-HT1A receptors (Ki = 67.8 ± 4.6 nM). This rank-order of af- anfinity intermediate is consistent affinity with the for re 5-HT1Asults obtained receptors in affinity (Ki = 67.8 displacement± 4.6 nM). screen This rank-order shown in Table of affinity 2. is consistent with the results obtained in affinity displacement screen shown in Table2. 2.2.3. Off-Target Binding Assessment 2.2.3. Off-Target Binding Assessment Further evaluation of the off-target binding of compound 6a was provided by the Further evaluation of the off-target binding of compound 6a was provided by the Psychoactive Drug Screening Program (PDSP) for a range of receptors including serotonin Psychoactive Drug Screening Program (PDSP) for a range of receptors including serotonin receptor subtypes, α-adrenergic receptor subtypes, β-adrenergic receptor subtypes, hista- receptor subtypes, α-adrenergic receptor subtypes, β-adrenergic receptor subtypes, his- taminergicminergic receptor receptor subtypes, subtypes, muscarinic muscarinic recept receptoror subtypes, subtypes, opioid receptor subtypessubtypes andand thethe twotwo sigmasigma receptor receptor subtypes. subtypes. The The PDSP PDSP evaluation evaluation confirmed confirmed the the high high affinity affinity binding bind- ofing compound of compound6a at 6a the at the D3 D3 dopamine dopamine receptor receptor (0.2 (0.2 nM) nM) and and the the low low affinity affinity binding binding atat thethe D1,D1, D2,D2, D4D4 (>1500(>1500 nM)nM) andand D5D5 dopaminedopamine receptorreceptor subtypes,subtypes, asas wellwell asas thethe dopaminedopamine transportertransporter (DAT)(DAT) (>190(>190 nM),nM), thethe norepinephrinenorepinephrine transportertransporter (NET)(NET) (>650(>650 nM)nM) andand thethe serotoninserotonin transportertransporter (SERT)(SERT) (>750(>750 nM).nM). TheThe highesthighest off-siteoff-site bindingbinding affinityaffinity waswas observedobserved for the several of the alpha-adrenergic receptor subtypes (α1a, 9.8 nM; α2a, 15.2 nM; α1d, for the several of the alpha-adrenergic receptor subtypes (α1a, 9.8 nM; α2a, 15.2 nM; α1d, 16.6 nM; α2c, 27.1 nM), with lower affinity at the α2b (>100 nM) and α1b(>100 nM) receptor 16.6 nM; α2c, 27.1 nM), with lower affinity at the α2b (>100 nM) and α1b(>100 nM) recep- torsubtypes. subtypes. Off-site Off-site binding binding was was observed observed for forthree three of the of the serotonin serotonin receptor receptor subtypes subtypes (5- (5-HT1A,HT1A, 27 27nM; nM; 5-HT2B, 5-HT2B, 36.3 36.3 nM; nM; 5-HT7A, 5-HT7A, 73.6 73.6 nM). nM). Off-site Off-site binding binding affinity affinity was was higher higher at atthe the sigma-2 sigma-2 receptor receptor (33.1 (33.1 nM) nM) compared compared to tothe the sigma-1 sigma-1 receptor receptor (>175 (>175 nM). nM).

2.2.4.2.2.4. LigandLigand EfficacyEfficacy AnalysisAnalysis CompoundsCompounds exhibitingexhibiting high high affinity affinity binding binding at at the the D3 D3 dopamine dopamine receptor receptor were were subse- sub- quentlysequently evaluated evaluated for for efficacy efficacy at the at D3the dopamineD3 dopamine receptor receptor subtype subtype using using both aboth forskolin- a for- dependentskolin-dependent inhibition inhibition of adenylyl of adenylyl cyclase cyclase assay andassay a βand-arrestin a β-arrestin binding binding assay assay (Figure (Fig-3). Quinpiroleure 3). Quinpirole was used was as used a reference as a reference prototypic prototypic full agonist full andagonist haloperidol and haloperidol was used was as aused reference as a reference prototypic protot antagonist.ypic antagonist. Compounds Compounds7e and 7e6d andwere 6d found were found to be antagoniststo be antag- inonists both in assays. both assays. Compound Compound6c was 6c foundwas found to be to functionally be functionally selective selective because because it wasit was a weak partial agonist for the adenylyl cyclase assay while being a very weak partial agonist/antagonist in the β-arrestin binding assay. Compounds 6a and 6b were found to β be weak partial agonists in both the adenylyl cyclase inhibition assay and the -arrestin binding assay. Molecules 2021, 26, x FOR PEER REVIEW 8 of 17

a weak partial agonist for the adenylyl cyclase assay while being a very weak partial ago- Molecules 2021, 26, 3182 nist/antagonist in the β-arrestin binding assay. Compounds 6a and 6b were found8 to of 16be weak partial agonists in both the adenylyl cyclase inhibition assay and the β-arrestin binding assay.

FigureFigure 3.3.Comparison Comparison ofof thethe EfﬁcacyEfficacy ofof Phenylthiophene Phenylthiophene AnalogsAnalogsfor for Adenylyl Adenylyl Cyclase Cyclase Inhibition Inhibition andand ββ-Arrestin-Arrestin BindingBinding atat HumanHuman D3D3 DopamineDopamine ReceptorsReceptors.. AA comparisoncomparison ofof thethe efﬁcacyefficacy ofof com-compounds 6a–d and 7e for the inhibition of adenylyl cyclase and the binding of β-arrestin is shown. pounds 6a–d and 7e for the inhibition of adenylyl cyclase and the binding of β-arrestin is shown. Values were normalized to reference full agonist quinpirole (10,000 nM). The concentration of the Values were normalized to reference full agonist quinpirole (10,000 nM). The concentration of the test compound was equal to 10× the Ki value as determined from competitive radioligand binding × teststudies, compound therefore was 90% equal of the to 10 bindingthe K sitesi value were as occupied. determined from competitive radioligand binding studies, therefore 90% of the binding sites were occupied.

2.3.2.3. In-VivoIn-Vivo AnalysisAnalysis ofof CompoundCompound6a 6a BasedBased uponupon thethe datadata indicatingindicating thatthat compoundcompound 6a6a boundbound withwith highhigh afﬁnityaffinity toto thethe humanhuman D3D3 dopamine receptor receptor and and with with low low affinity afﬁnity at at both both the the human human D2 D2 dopamine dopamine re- receptorceptor subtype subtype (Table (Table 1)1 )and and the the 5-HT1A 5-HT1A receptor receptor (Table (Table 22 and and Figure Figure 2),2), two two in-vivo in-vivo as- assayssays were were performed performed to to determine determine if ifcompound compound 6a6a waswas capable capable of of in-vivo in-vivo engagement engagement of ofthe the D3 D3 receptor receptor in inthe the brain. brain. While While we wedo a doppreciate appreciate the thepotential potential that thatpossible possible in-vivo in- vivodifferences differences in pharmacological in pharmacological responses responses for the for same the same drug drug in experimental in experimental animals animals and andhumans humans of sex of differences, sex differences, these these initial initial in-vivo in-vivo studies studies were wereprimarily primarily conduced conduced to assess to assessthe ability the abilityof compound of compound 6a to cross6a to the cross blood the brain blood barrier brain (BBB) barrier and (BBB) engage and the engage pharma- the pharmacologicallycologically relevant relevant target. target.Further Further sex differences sex differences in the inpharmacological the pharmacological response response of an- ofimals animals to compound to compound 6a will 6a willbe conduction be conduction as further as further in-vivo in-vivo studies studies are necessitated. are necessitated. First,First, wewe have have previously previously published published that that D3 D3 receptor receptor selective selective compounds compounds could could atten- at- uatetenuate a hallucinogen-dependent a hallucinogen-dependent head head twitch twitch response response (HTR) (HTR) in male in DBA/2J male DBA/2J mice [20 mice,33]. Figure[20,33].4 indicatesFigure 4 indicates that at a dosethat at of a 10 dose mg/kg of 10 compound mg/kg compound6a attenuated 6a attenuated the number the ofnumber head twitchesof head inducedtwitches byinduced 5 mg/kg by of5 mg/kg the hallucinogen of the hallucinogen 2,5-dimethoxy-4-iodoamphetamine 2,5-dimethoxy-4-iodoampheta- (DOI) overmine a (DOI) 30 min over time a period.30 min Atime two-way period. ANOVA A two-way with ANOVA repeated with measures repeated revealed measures a main re- effectvealed of a Group main effect (F2,18 of= 6.49;Groupp = (F 0.008),2,18 = 6.49; a main p = 0.008), effect of a Timemain (Feffect5,90 =of 348.16; Time (Fp 5,90<0.001 = 348.16;) and p an< 0.001) interaction and an between interaction Time between and Group Time (F10,90 and= Group 14.92; p(F<10,90 0.001). = 14.92; Second, p < 0.001). we previously Second, re-we portedpreviously that D3reported vs. D2 that dopamine D3 vs. receptor D2 dopamine subtype receptor selective subtype ligands couldselective attenuate ligands L-dopa could dependentattenuate L-dopa abnormal dependent involuntary abnormal movement involuntary (AIM) scoresmovement in unilaterally (AIM) scores lesioned in unilater- (hemiparkinsonian)ally lesioned (hemiparkinsonian) male rats, which is male a model rats, for which L-dopa-Induced is a model for Dyskinesia L-dopa-Induced (LID) [ 35Dyski-,36]. Figurenesia 5(LID) indicates [35,36]. that Figure at a dose 5 indicates of 10 mg/kg that at compound a dose of 6a10 couldmg/kg attenuate compound the 6a AIM could scores at- inducedtenuate the by AIM 8 mg/kg scores of induced L-dopa by and 8 benserazidemg/kg of L-dopa (each) and in benserazide unilaterally (each) lesioned in unilater- rats. A two-wayally lesioned ANOVA rats. withA two-way repeated ANOVA measures with revealed repeated a mainmeasures effect revealed of Group a (Fmain1,10 =effect 26.98; of p < 0.001), a main effect of Time (F7,70 = 15.61; p < 0.001) and an interaction between Time and Group (F7,70 = 7.44; p < 0.001).

Molecules 2021, 26, x FOR PEER REVIEW 9 of 17

Molecules 2021, 26, 3182 Group (F1,10 = 26.98; p < 0.001), a main effect of Time (F7,70 = 15.61; p < 0.001) and an interac- 9 of 16 tion between Time and Group (F7,70 = 7.44; p < 0.001). Group (F1,10 = 26.98; p < 0.001), a main effect of Time (F7,70 = 15.61; p < 0.001) and an interaction between Time and Group (F7,70 = 7.44; p < 0.001).

FigureFigure 4. 4.InhibitionInhibition of the of theMurine Murine DOI-Dependen DOI-Dependentt Head Twitch Head Response Twitch Response by Compound by Compound 6a. The 6a. The inhibition of the DOI-dependent Head Twitch Response (HTR) by compound 6a as a function of Figureinhibition 4. Inhibition of the DOI-dependentof the Murine DOI-Dependen Head Twitcht Head Response Twitch (HTR)Response by by compound Compound6a 6a.as The afunction of time (minutes) is shown. Male DBA/2J mice were dosed with 5 mg/kg of DOI in the presence of 10 inhibition of the DOI-dependent Head Twitch Response (HTR) by compound 6a as a function of mg/kgtime (minutes)of compound is shown.6a. The vehicle Male DBA/2Jcontrol studies mice were pe dosedrformed with either 5 mg/kg one week of DOIbefore in (– the□–), presence of time (minutes) is shown. Male DBA/2J mice were dosed with 5 mg/kg of DOI in the presence of 10 or10 one mg/kg week after of compound (–○–) the compound6a. The vehicle6a (–▲–) control was evaluated. studies The were data performed shown is the either mean onenum- week before mg/kg of compound 6a. The vehicle control studies were performed either one week before (–□–), ber(– of–), head or onetwitches week ± afterS.E.M. (– averaged–) the compoundby two observers6a (– cumulatively–) was evaluated. at 5 min.The Intervals data for shown n = 7 is the mean or one week after (–○–) the compound 6a (–▲–) was evaluated.N The data shown is the mean num- mice. * A p < 0.05 was observed compared to both controls at times 15–30 min (one-way ANOVAs bernumber of head of twitches head twitches ± S.E.M. averaged#± S.E.M. by averaged two observers by two cumulatively observers at cumulatively5 min. Intervals at for 5 n min. = 7 Intervals revealed main effects of Groups (F2,18 ranging from 5.1 to 10.4 with p < 0.019) and were followed by mice.for n * = A 7 p mice < 0.05.*A wasp observed< 0.05 was compared observed to both compared controls at to times both 15–30 controls min at(one-way times15–30 ANOVAs min (one-way post-hoc analyses). revealedANOVAs main revealed effects of main Groups effects (F2,18of ranging Groups from (F 2,185.1 toranging 10.4 with from p < 0.019) 5.1 to and 10.4 were with followedp < 0.019) by and were post-hocfollowed analyses). by post-hoc analyses).

Figure 5. The Effect of Compound 6a on Abnormal Involuntary Movement (AIM) Scores in Hemi- parkinsonian Rats. Male hemiparkinsonian rats were purchased from Charles Rivers Laboratories. Figure 5. The Effect of Compound 6a on Abnormal Involuntary Movement (AIM) Scores in Hemi- AfterFigure a 21 5. dailyThe intraperitoneal Effect of Compound injection 6aregimen on Abnormal of 8 mg/kg Involuntary of L-dopa and Movement benserazide (AIM) (each) Scores the in Hemi- parkinsonianparkinsonian Rats Rats. Male. Male hemiparkinsonian hemiparkinsonian rats were rats purcha weresed purchased from Charles from Rivers Charles Laboratories. Rivers Laboratories. After a 21 daily intraperitoneal injection regimen of 8 mg/kg of L-dopa and benserazide (each) the After a 21 daily intraperitoneal injection regimen of 8 mg/kg of L-dopa and benserazide (each) the animals were administered L-dopa/benserazide (8 mg/kg each) either with vehicle (10% DMSO

in sterile water) or compound 6a at a dose of 10 mg/kg. Animals were scored on a scale from 0 to 4 for a) dystonic torsion of the trunk and neck contralateral to the lesion, b) dystonic movements of the forelimb contralateral to the lesion, c) orolingual movements with protrusion of the tongue and d) increased locomotion activity. Average AIM scores ± S.E.M. is shown for n = 6 animals in the presence (–•–) or absence (––) of compound 6a.*A p < 0.05 value compared to vehicle control was observed for time points 15–120 min (one-way ANOVAs revealed main effects of Groups (F1,10 ranging from 11 to 38.4 with p < 0.009). Molecules 2021, 26, 3182 10 of 16

3. Discussion This communication describes part of our ongoing studies on the development of dopamine receptor selective ligands in vivo to dissect the role of the D2 and D3 dopamine receptor subtypes in the rewarding and motivational aspects of psychostimulant abuse [35,37–39], the treatment of neurological and neurodegenerative disorders [36,40] and for the development of selective imaging agents [41]. We previously reported that structural variations in the aryl portion of 4-(thiophen- 3-yl)benzamides play an important role in D3 dopamine receptor affinity, D3 vs. D2 receptor binding selectivity and D3 dopamine receptor functional selectivity (biased ago- nism) [20–22,30,35,42]. The structural templates for these studies included LS-3-134 and WW-III-55 (Figure1). Both compounds possess a common 4-(thiophen-3-yl)benzamide with a saturated four-carbon chain adjacent to a substituted phenylpiperazine moiety. LS-3- 134 was found to bind at the human D3 dopamine receptor with high affinity (Ki = 0.17 nM) and demonstrated >150-fold D3 vs. D2 dopamine receptor binding selectivity [30]. LS- 3-134 was found to be a partial agonist at the D3 dopamine receptor (35% of maximum efficacy) using an adenylyl cyclase inhibition assay. While WW-III-55 exhibits only moderate binding affinity (approximate Ki of 20 nM) at D3 dopamine receptors, this compound binds with high (>800-fold) D3 vs. D2 dopamine receptor binding selectivity. Using an adenylyl cyclase assay, WW-III-55 was found to be a strong partial agonist (67.6 ± 12.5% of maximum efficacy) while LS-3-134 was found to be a weak partial agonist (34.4 ± 1.7%). Therefore, we projected that structurally related analogs of WW-III-55 and LS-3-134 might be identified that could exhibit high affinity binding at the D3 dopamine receptor, substantial D3 vs. D2 receptor binding selectivity and varying efficacy for G-protein and β-arrestin mediated signaling. This expectation was verified when we examine the binding properties of a panel of structural analogs of WW-III-55 and LS-3-134, where the 4-(thiophen-3-yl)benzamide structure, which we project interacts with the SBS, was invariant while substituents on the phenyl moiety of the phenylpiperazine (the structure binding to the orthosteric binding site) were varied. This synthetic strategy led to the identification of several compounds that exhibited subnanomolar affinity (Ki values < 1.0 nM) at the human D3 dopamine receptor subtype with high D3 vs. D2 dopamine receptor binding selectivity (≥1000-fold) [22]. In the context of a dynamic ensemble theory of GPCR structure and activation [43–45], this observation suggests that each of the 4-(thiophen-3-yl)benzamide analogs used in that study interacts with the D3 dopamine receptor protein in a slightly different manner (depending on the position and chemical properties of the substituent), stabilizing a unique ensemble of receptor structures resulting in a collection of ligand-dependent structural variations in the D3 dopamine receptor energy landscape [42–45]. In this communication our initial goal was first to characterize a number of synthetic analogs in which the 4-(thiophen-3-yl)benzamide group and the adjacent saturated four carbon chain was invariant, while the substitution pattern on the phenyl ring of the phenylpiperazine moiety substituted at the 2nd, 3rd and 4th position with a fluorine. Once we established that the 2nd position was optimal for a fluoride substitution (compounds 3a and 6a), our second goal was to attempt to further optimize ligand binding affinity by exploring additional substitutions on the phenyl ring of the N-phenylpiperazine moiety (compounds 6d–f). Compound 6a remained our best candidate, with the highest affinity at the D3 receptor subtype (Ki value = 1.4 nM), while it exhibited substantial D3 vs. D2 receptor binding selectivity (>450-fold). Our third synthetic strategy was to substitute the thiophene ring (6a–f) with a thiazole ring (7a–f). In general, this structural alteration led to a decrease in D3 vs. D2 receptor binding selectivity. Using the aforementioned strategy, we were able to identify a number of ligands which exhibited high affinity binding at the D3 dopamine receptor or substantial D3 vs. D2 dopamine receptor binding selectivity. However, compound 6a remained of particular interest because it bound at the D3 dopamine receptor with high affinity (Ki value = 1.4 nM) and exhibited substantial D3 vs. D2 binding selectivity (>400-fold). Molecules 2021, 26, 3182 11 of 16

A direct comparison of the binding affinities for compounds 3a–f and 6a–f suggests that compound 6a–f likely bound at the D3 dopamine receptor subtype, but not the D2 dopamine receptor, in a bitopic mode. This observation was similar to what we previously reported [23]. While the identification of a ligand that can interact with a dualistic ligand binding mode may not be applicable for the development of receptor subtype selective ligands for every GPCR subfamily, it appears not to be unique to the dopamine receptor subtypes. Bitopic ligands have also been reported for other GPCRs, including the muscarinic and angiotensin II receptors [46–48]. Furthermore, binding data provided by NIMH Psychoactive Drug Screening Program (PSDP) indicated that compound 6a had reduced affinity at the 5-HT1A receptor, which is a common off-site binding component that has limited the development of in-vivo D3 dopamine receptor selective imaging agents. How- ever, this binding data indicated that several alpha-adrenergic receptor subtypes bound compound 6a. The studies in this communication also demonstrated that after intraperitoneal injection compound 6a was capable of engaging CNS D3 dopamine receptors in vivo leading to a significant decrease in the DOI-dependent head twitch response in mice and AIM scores in dyskinetic hemiparkinsonian rats. These observations suggest that compound 6a was capable of traversing the blood-brain barrier in a manner such that a therapeutic concentration could be achieved. We anticipate that studies such as those reported in this communication could lead to a better understanding of how the development of bitopic ligands might be designed and used to selectively target structurally similar GPCR receptor subtypes in vivo.

4. Materials and Methods 4.1. Chemistry All reagents and solvents for synthesis were purchased and used without further purification. Structures of synthesized chemicals were identified using 1H and 13C nu- clear magnetic resonance (NMR) spectra, and mass spectroscopy. 1H and 13C NMR were recorded in δ units relative to deuterated solvent (CDCl3) as an internal reference by Bruker DMX 500 MHz NMR instrument (Bruker, Billerica, MA, USA). 1H chemical shifts are reported in parts per million (ppm) and measured relative to tetramethylsilane (TMS). Mass spectra were acquired using a 2695 Alliance LC/MS (Milford, MA, USA). Purifi- cation of synthesized chemicals was conducted on Biotage Isolera One (Biotage, Salem, NH, USA) with a dual-wavelength UV-VIS detector. A detailed description of the synthesis and characterization of the compounds described in this paper can be found in the Supporting Information.

4.2. D2 and D3 Dopamine Receptor Competitive Radioligand Binding Assays For these competitive binding studies, transfected HEK293 cell homogenates were suspended in homogenization buffer and incubated with radioligand [125I]IABN, in the presence or absence of inhibitor at 37 ◦C for 60 min (total volume = 150 µL), as previously described [49]. The final radioligand concentration was approximately equal to the Kd value for the binding of the radioligand. Nonspecific binding was defined as the binding of the radioligand in the presence of 25 µM (+)-butaclamol. 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 (PerkinElmer, Waltham, MA, USA) was used to measure the radioactivity of [125I]IABN. 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 Molecules 2021, 26, 3182 12 of 16

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 TableCurve 2D v 5.01 (Jandel, SYSTAT, Systat Software, Inc., San Jose, CA, USA). The values for Bs and Bo were constrained using experimentally derived values. The IC50 values were converted to equilibrium dissociation constants (Ki)[50,51]. Mean Ki values ± S.E.M. are reported for at least three independent experiments.

4.3. 5-HT1A Receptor Binding Assays 10 µg of membranes from CHO-K1 cells expressing the human 5-HT1A receptor were suspended in 50 mM Tris-HCl, 10 mM MgSO4, 0.5 mM EDTA, and 0.1% (w/v) ascorbic acid, pH 7.4 and test ligands (91 nM for single point radioligand displacement assay and 1 nM to 1 µM for full competition assay) were incubated with 0.25 nM [3H]8- hydroxy-DPAT (PerkinElmer, Boston, MA, USA) for 60 min at room temperature. The nonspecific binding was determined using 10 µM metergoline (Sigma, St. Louis, MO, USA). After incubation, the bound ligands were filtered using an M-24 Brandel filtration system (Brandel, Gaithersburg, MD, USA), collected on glass fiber papers (Whatman grade 934-AH, GE Healthcare Bio-Sciences, Pittsburgh, PA, USA), and the radioactivity was quantitated using a MicroBeta2 Microplate scintillation counter 2450 (PerkinElmer, Boston, MA, USA).

4.4. Ligand Efﬁcacy Analysis 4.4.1. Cyclase Inhibition Assay The forskolin-dependent adenylyl cyclase inhibition assay was performed using a cAMP-Glo Assay kit (Promega). For this assay cAMP stimulates protein kinase A (PKA) activity, which leads to a decrease in ATP levels and a decrease in the luciferase-coupled production of light. This is a 96 well plate assay using HEK cells stably transfected to express the human D3 dopamine receptor (10,000 cells/well/0.1 mL complete media). After removing media and rinsing with phosphate buffered saline, cells were incubated with 20 µL of forskolin (6000 nM) and test compound or vehicle. The dose of test compound was equal to 10x the Ki value obtained from our competitive radioligand binding studies, such that 90% of the binding sites are occupied. After cells, test compound (or vehicle) and forskolin (or vehicle) were incubated for 15 min, cells were lysed using 20 µL lysis buffer (provided with the kit) and mixed for 15 min at room temperature. A cAMP Detection Solution (40 µL) was added to each well, gently mixed for 60 s and incubated at room temperature for 20 min. The Kinase-Glo Solution was added (40 µL) to terminate the PKA reaction. The reaction mixture was gently mixed for 60 s and then incubated at room temperature for 10 min. The remaining ATP was detected via a luciferase reaction. The plates were read using an Enspire Alpha 2390 Multilabel Reader/Luminometer. Haloperidol (200 nM) and quinpirole (10,000 nM) was used as a prototypic reference antagonist and full agonist, respectively.

4.4.2. β-Arrestin Binding Assay The β-arrestin binding assay was performed using a DiscoverX Pathhunter® kit according to the manufacturer’s instructions. PathHunter® eXpress β-Arrestin GPCR cells are engineered to co-express the ProLink™ (PK) tagged GPCR and the Enzyme Acceptor (EA) tagged β-Arrestin. Activation of the GPCR-PK induces β-Arrestin-EA recruitment, forcing complementation of the two β-galactosidase enzyme fragments (EA and PK). The resulting functional enzyme hydrolyzes substrate to generate a chemiluminescent signal. DRD3-U2OS cells were seeded according to kit instructions into white plastic 96-well plates (provided with the kit) 48 h prior to the assay in a volume of 100 µL/well using the Cell Plating Reagent (CPR) provided. Vehicle or test drug, diluted in CPR, was added to the designated wells and incubated for 90 min at 37 ◦C. Working detection solution (a component provided with the kit) was added to the wells and incubated for 1 h in the dark at room temperature. As discussed for the cyclase assay, an Enspire Alpha 2390 Multilabel Molecules 2021, 26, 3182 13 of 16

Reader/Luminometer was used to detect the luminescent signal with haloperidol and quinpirole used as reference antagonist and full agonist, respectively.

4.5. In-Vivo Studies The treatment of the animals and the experimental procedures were approved by the UNTHSC Institutional Animal Care and Use Committee (IACUC). Animal care and housing were in adherence with the conditions set forth in the “Guide for the Care and Use of Laboratory Animals” (Institute of Laboratory Animal Resources on Life Sciences, National Research Council, 1996).

4.5.1. Head Twitch Response Upon arrival DBA/2J mice (Jackson Labs) were allowed to acclimate for at least one week prior to testing. Mice were housed at ≤4 animals per cage with unlimited access to food and fresh water. The protocol for the HTR assessment has been previously reported [32]. Briefly, on the day of testing mice were weighed and placed individually in an open-ended Plexiglas cylinder (21 × 34 cm) with a clean paper towel on the floor in a dimly lighted room. Animals were allowed to habituate to the cylinder for 10 min prior to the intraperitoneal (i.p.) injection of vehicle (90% sterile deionized water and 10% dimethylsulfoxide (DMSO: Sigma) in sterile deionized water) or test drug (10 mg/kg). Five minutes later DOI (5 mg/kg) was administered, and the mouse was immediately returned to the cylinder. The mice exhibited various responses to DOI administration including a vertical jerking movement. However, a head twitch was defined as a rapid movement of the head, without the involvement of the front paws during grooming. We estimate that the HTR lasted approximately 0.6 s. At least two observers counted the number of head twitches by visual examination and the number of head twitches was recorded in 5-min cumulative intervals. The data points are presented as the mean values obtained by two observers. The effect of Group was assessed using two-way analysis of variance (ANOVA) with Time as the repeated measure. The data was further analyzed at each time point with a one-way ANOVA with Group as the factor followed by planned individual comparisons between different groups using a single degree-of-freedom F test involving the error term from the overall ANOVA when the Time and Group interaction was significant. The alpha level was set at 0.05 and Systat 13 statistical package was used.

4.5.2. AIMs Studies The Abnormal Involuntary Movement (AIMs) studies were performed using male Sprague-Dawley rats that were unilaterally lesioned, using injections of 6-hydroxydopamine (6-OHDA) in the medial forebrain bundle (MFB) by the commercial vendor (Charles River Laboratories). Upon arrival to our vivarium, the lesioned rats were housed under a 12-h light: 12-h dark cycle. The animals were housed singly with unlimited access to food and water. 8 mg/kg L-dopa with 8 mg/kg benserazide was administered to each rat as a daily i.p. injection for 21 consecutive days to induce the development of dyskinesia-like movements. Benserazide was included because it is a peripherally-acting aromatic L-amino acid decarboxylase inhibitor which prevents the peripheral metabolism of L-dopa. The L-dopa/benserazide solutions were dissolved in water. Test drug (compound 6a) was prepared using 90% sterile water with 10% DMSO. 8 mg/kg of L-dopa combined with 8 mg/kg benserazide was administered via intraperitoneal injection followed by either the vehicle control or test drug (compound 6a, 10 mg/kg). Experiments were designed such that animals received test drug only once per week. Throughout the course of these studies each animal received a minimum of one dose of L-dopa/benserazide (8 mg/kg each) per week to maintain the involuntary movements. AIMs ratings were performed as previously described [31,38,52]. The severity of the AIMs was quantiﬁed using lesioned rats that were observed individually in their home cages at 15 min intervals, starting 15 min after the injection of L-dopa for approximately 2 h. Molecules 2021, 26, 3182 14 of 16

The AIMs were scored in four categories: (1) axial AIMs, including dystonic or choreiform torsion of the trunk and neck towards the side contralateral to the lesion; (2) limb AIMs, including jerky and/or dystonic movements of the forelimb contralateral to the lesion; (3) orolingual AIMs, including twitching of orofacial muscles with empty masticatory movements and protrusion of the tongue towards the side contralateral to the lesion and (4) locomotive AIMs, including increased locomotion with contralateral side bias. Each of the four categories were scored on a severity scale from 0 to 4, where 0 = absent, 1 = present for less than half of the observation time, 2 = present for more than half of the observation time, 3 = present all the time but suppressible by external stimuli and 4 = present all the time and not suppressible by external stimuli. For these experiments the external stimuli that was used was gentle tapping on the cage with a pencil. The change in crossovers from controls was calculated for each test compound dosage group by subtracting the average number of crossovers of the respective vehicle group from the number of crossovers of individual subjects in the test compound dosage groups. The latter measure is graphed to illustrate the effects of the compounds on locomotion. The effect of Group was assessed using two-way analysis of variance (ANOVA) with time as the repeated measure. The data was further analyzed at each time point with a one-way ANOVA with Group as the factor. The alpha level was set at 0.05 and Systat 13 statistical package was used.

Supplementary Materials: The following are available online, procedure of synthesis; chemical analysis of synthesized compounds (1H, 13 C NMR, and Mass); Figure S1: representative examples of competitive radioligand binding studies for human D2 and D3 dopamine receptors (compound 6a–c and 7a–c). Author Contributions: Conceptualization, R.H.M. and R.R.L.; methodology, B.L., M.T., S.A.G., N.S. and T.M.; software, B.L., M.T. and R.R.L.; validation, R.H.M. and R.R.L.; formal analysis, B.L., R.H.M. and R.R.L.; investigation, R.H.M. and R.R.L.; resources, R.H.M. and R.R.L.; data curation, R.H.M. and R.R.L.; writing—original draft preparation, B.L., R.H.M., M.T. and R.R.L.; writing—review and editing, R.H.M., M.T. and R.R.L.; visualization, R.H.M.; supervision, R.H.M. and R.R.L.; project administration, R.H.M. and R.R.L.; funding acquisition, R.H.M. and R.R.L. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by National Institute on Drug Abuse, grant number, DA029840 (R.H.M.) and DA023957 (R.R.L.). Institutional Review Board Statement: This study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of the University of North Texas Health Science, protocol codes 2017-0006 and 2019-0008 (approved 3 July 2017 and 6 June 2019, respectively). Informed Consent Statement: Not applicable. Data Availability Statement: The article contains complete data used to support the findings of this study. Conflicts of Interest: The authors declare no conflict of interest. Sample Availability: Samples of the compounds described in this paper are available from the authors.

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