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Article Cite This: J. Med. Chem. 2018, 61, 9121−9131

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Synthesis and Discovery of Arylpiperidinylquinazolines: New Inhibitors of the Vesicular Monoamine Transporter Brian A. Provencher,*,†,‡ Amy J. Eshleman,§,∥ Robert A. Johnson,§ Xiao Shi,§,∥ Olga Kryatova,† Jared Nelson,† Jianhua Tian,† Mario Gonzalez,† Peter C. Meltzer,† and Aaron Janowsky§,∥,⊥ †

Organix Inc, 240 Salem Street, Woburn, Massachusetts 01801, United States Department of Chemistry and Biochemistry, Merrimack College, North Andover, Massachusetts 01845, United States § Research Service, VA Portland Health Care System, Portland, Oregon 97239, United States ∥ Departments of Psychiatry and Behavioral Neuroscience, Oregon Health and Science University, Portland, Oregon 97239, United States ⊥ The Methamphetamine Abuse Research Center, Oregon Health and Science University, Portland, Oregon 97239, United States

J. Med. Chem. 2018.61:9121-9131. Downloaded from pubs.acs.org by UNIV OF SUNDERLAND on 10/26/18. For personal use only.

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S Supporting Information *

ABSTRACT: Methamphetamine, a human vesicular monoamine transporter 2 (VMAT2) substrate, releases dopamine, serotonin, and norepinephrine from vesicles into the cytosol of presynaptic neurons and induces reverse transport by the monoamine transporters to increase extracellular neurotransmitters. Currently available radioligands for VMAT2 have considerable liabilities: The binding of [3H]dihydrotetrabenazine ([3H]DHTB) to a site on VMAT2 is not dependent on ATP, and [3H]reserpine binds almost irreversibly to VMAT2. Herein we demonstrate that several arylpiperidinylquinazolines (APQs) are potent inhibitors of [3H]reserpine binding at recombinant human VMAT2 expressed in HEK-293 cells. These compounds are biodiastereoselective and bioenantioselective. The lead radiolabeled APQ is unique because it binds reversibly to VMAT2 but does not bind the [3H]DHTB binding site. Furthermore, experimentation shows that several novel APQ ligands have high potency for inhibition of uptake by both HEK-VMAT2 cells and mouse striatal vesicles and may be useful tools for characterizing drug-induced effects on human VMAT2 expression and function.

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INTRODUCTION Methamphetamine is a highly addictive stimulant.1 The abuse of methamphetamine is prevalent and escalates with time for many individuals.2 Stimulant-induced psychosis and long-term neuronal loss are indications of methamphetamine-induced neurotoxicity.3−5 Currently, there are no approved pharmacotherapies for treatment of methamphetamine addiction and behavioral therapy has had limited success.6 Among the primary initial neuronal targets for methamphetamine is the human vesicular monoamine transporter 2 (VMAT2).7−10 Expressed in monoaminergic presynaptic neurons, as well as in peripheral tissues, VMAT2 transports cytosolic dopamine (DA), serotonin (5-HT), or norepinephrine (NE) into storage vesicles.11 By transporting neurotransmitters into vesicles, VMAT2 participates in the regulation of cytosolic levels and vesicular stores of biogenic amines. © 2018 American Chemical Society

Methamphetamine competes with the endogenous neurotransmitters for binding sites and uptake into VMAT2. When VMAT2 binds methamphetamine, vesicular degradation resulting in dopamine efflux into the cytosol of presynaptic neurons is observed.12 This can cause reverse transport by the dopamine transporter to increase dopamine in the extracellular space. Therefore, VMAT2 is a possible target for therapeutics to treat methamphetamine abuse.6,8 The VMAT2, while expressed in vivo on the vesicular membranes of neurons, is also functional when expressed in mammalian cell lines, with the energy source for transport provided by the native V-type H+-ATPase.13 Recent work reveals that in drosophila brain, amphetamine redistributes vesicle contents, diminishes the vesicle pHReceived: April 6, 2018 Published: September 21, 2018 9121

DOI: 10.1021/acs.jmedchem.8b00542 J. Med. Chem. 2018, 61, 9121−9131

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Scheme 1. Synthesis of syn-APQ Ligandsa

a (a) (1) K2CO3, DCM; (2) NaH, PhN(Tf)2, DMF, 0° C; (b) ArB(OH)2, Pd(PPh3)4, LiCl, Na2CO3, toluene/EtOH, 85 °C; (c) Pd/C, H2, rt; (d) 6, toluene or DMF/1,4-dioxane or CH3CN, 90−110 °C.

Scheme 2. Synthesis of anti-APQ Ligandsa

(a) (Boc)2O, Et3N, MeOH, 60 °C; (b) EtONa, EtOH, 70 °C; (c) 4 M HCl; (d) 6, toluene, 100 °C.

a

similar to ATP-dependent VMAT2 function, and [3H]reserpine binds almost irreversibly to VMAT2 and does not lend itself to high-throughput screening.29,30 One model of the binding sites on VMAT2 suggests that the reserpine site has high affinity for substrates and is directed toward the cytosol; in contrast, after the binding of the substrate to VMAT2, a conformational change results in the TBZ-binding conformation.31 Furthermore, it was found that TBZ does not require a proton gradient for binding and that TBZ binding locks the transporter into a confirmation which no longer allows the substrate to bind to VMAT2 by inhibiting the cytoplasmic gate.32 With few available radioligands, the search for novel ligands to serve as chemical probes that bind reversibly and specifically to the VMAT2, and are associated with VMAT2 function, can provide a springboard for the development of pharmacotherapies for symptoms related to methamphetamine addiction. Herein we describe the discovery of new arylpiperidinylquinazoline (APQ) ligands and their biological activity in VMAT2 assays.

gradient needed for biogenic amine uptake, and requires VMAT2 function.14 Current therapy for methamphetamine abuse consists primarily of cognitive behavioral interventions; however, pharmacological interventions could improve the treatment of methamphetamine abuse. Vocci and Appel, when reviewing possible targets for methamphetamine pharmacotherapies, identified the VMAT2 as having an obligatory role in methamphetamine activity.15 Some strategies for pharmacotherapy development have focused on altering psychostimulant interaction with the VMAT2 or with altering VMAT2 function. Lobeline, a lipophilic alkaloid of Indian tobacco, alters VMAT2-mediated DA uptake and release and alters methamphetamine-induced DA release,16,17 and lobeline and its analogs are being investigated as possible therapeutics for methamphetamine abuse.18−20 Reserpine, a high affinity VMAT2 blocker, has been used to treat hypertension but essentially irreversibly binds to VMAT2 which results in depletion of biogenic amines including dopamine and epinephrine. Thus, recovery of biogenic amine storage requires synthesis of new storage vesicles.21 In addition, reserpine binds to both the VMAT2 and the peripheral VMAT1, with side effects including sedation, inability to concentrate or perform complex tasks, and sometimes psychotic depression that appears over many weeks or months.22,23 Tetrabenazine (TBZ) and its analog, dihydrotetrabenazine (DHTB), inhibit VMAT2 function, bind reversibly to the VMAT2 at an allosteric binding site, and have shown effectiveness in treatment of hyperkinesias characterized by abnormal involuntary movements as observed in Huntington’s disease, Tourette’s syndrome, and tardive dyskinesia.22,24 However, TBZ has side effects similar to reserpine including sedation, depression, akathisia, and Parkinsonism.24 Ketanserin binds to VMAT2 with relatively high affinity (∼6−45 nM)25 but is nonselective, displaying high affinity for the 5-HT2A receptor and moderate affinities for the histamine H1 and 5-HT2C receptors.26−28 There are a number of radioligands and drugs that bind to the VMAT2 and affect its function. Currently, available radioligands for labeling VMAT2, such as [3H]DHTB and [3H]reserpine, have considerable liabilities. [3H]DHTB binding is not ATP-dependent and thus mechanistically is less

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CHEMISTRY Novel APQ ligands (±)-5 were synthesized from commercially available ethyl 1-benzyl-4-oxo-3-piperidinecarboxylate hydrochloride 1. Conversion from the ketone to triflate 2 was accomplished using N-phenyl bistrifluorsulfonamide. Suzuki coupling followed by reduction afforded substituted arylpiperidine (4). N-Alkylation with the known quinazoline 6 afforded racemic, syn-APQ ligands (±)-5.33 To ensure water solubility, the APQs were converted to the HCl salt with HCl in ether. The precipitate was filtered, dissolved in water, and freeze-dried to afford the APQ-HCl salt which was confirmed by elemental analysis. Enantiomers could be resolved by crystallizing 4 with tartaric acid. Once purified, single enantiomers were subjected to the same N-alkylation and salt procedure as stated above. anti-APQ isomer 9 was prepared from intermediate 4b. Protection of the free amine with Boc anhydride yielded 7. Heating 7 in the presence of sodium ethoxide converted the cis-diastereomer to the more stable trans-diastereomer. 9122

DOI: 10.1021/acs.jmedchem.8b00542 J. Med. Chem. 2018, 61, 9121−9131

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had micromolar affinity for the reserpine binding site but had 60-fold lower affinity for the DHTB site. Ketanserin displays some affinity for the reserpine binding site albeit 5-fold lower than for the DHTB binding site (Table 1, entry 1). Interestingly, APQs displayed a wide range of binding affinities across both sites and were generally more selective for the reserpine site. syn-APQ (±)-5a bound to the reserpine site with a Ki = 70 nM (Table 1, entry 9). The para-fluoro analog (±)-5b, which contains the same aryl moiety of ketanserin, was found to bind with essentially the same Ki at the reserpine site (74 vs 70 nM). Neither compound showed much affinity for the DHTB site. To explore the diastereoselectivity of the APQ ligand on the receptors, anti-isomer (±)-9 was tested. APQ ligand 9 had less affinity for the reserpine site than the synanalog, (±)-5b. Additionally, (±)-9 was not as selective for the reserpine site and competed at the DHTB site with Ki = 750 nM. With the knowledge that the syn-isomers (5) were more selective for reserpine over DHTB, we explored the structure− activity relationship of the APQ ligands. Compound (±)-5c lost affinity for the reserpine site while 3-trifluorophenyl analog (±)-5d retained some affinity for the reserpine site, albeit much lower than (±)-5b. Other analogs in this series did not bind at the reserpine or DHTB sites (Table 1, entries 15−18). Thiophene analog (±)-5i and dioxane analog (±)-5j were found to bind with moderate affinity to the reserpine site, Ki = 450 and 652 nM respectively. To explore the enantioselectivity of the ligands, (+)-5b and (−)-5b were isolated and subjected to the reserpine and DHTB binding assays. (+)-5b had higher affinity than (±)-5b in the reserpine binding assay with Ki = 62 nM (p < 0.001, one-way ANOVA followed by Tukey’s multiple comparison test) and was essentially inactive in the DHTB binding assay. (−)-5b displayed similar affinity as racemic (±)-5b (p > 0.05) but lower affinity than the enantiomer (+)-5b (p < 0.01) (Ki = 179 nM) for the reserpine site while remaining inactive in the DHTB binding assay. To further study the effect of the APQ ligand on VMAT2, compound (+)-5b, with the highest affinity for the reserpine site, was selected for tritium radiolabeling. The effect of Mg-ATP on radiolabeled [3H](+)-5b binding was determined. The binding of [3H]reserpine and [3H](+)5b to VMAT2 was ATP-dependent. Mg-ATP (2 mM) enhanced specific binding to recombinant VMAT2 approximately 8- and 15-fold for [3H]reserpine and [3H](+)-5b, respectively (Figure 1A and Figure 1B). In contrast, the binding of [3H]DHTB was not changed by the addition of 2 mM ATP (Figure 1C). Thus, ATP increases [3H](+)-5b and [3H]reserpine binding but not [3H]DHTB binding. The ATP enhancement of [3H](+)-5b binding suggests that the binding is energy-dependent and may also be dependent on one or

Deprotection followed by N-alkylation with the known quinazoline afforded (±)-9.

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RESULTS AND DISCUSSION To begin, the binding affinities of 13 APQ derivatives and reference compounds (ketanserin, DHTB, methamphetamine, reserpine, Ro4-1284, serotonin, and lobeline) for VMAT2 were assessed by inhibition of radioligand binding using [3H]reserpine and [3H]DHTB (Table 1). Methamphetamine Table 1. Binding Affinities for the VMAT2 Binding Sites of [3H]Reserpine and [3H]DHTBa

a The core APQ structure is given in Schemes 1 and 2. Experiments were conducted as detailed in the Experimental Section. n ≥ 3 except when the Ki was greater than 10 μM, in which case n = 2. If some experiments yielded IC50 or Ki values less than 10 μM and other experiments yielded IC50 or Ki values greater than 10 μM, the latter experiments were assigned a value of 10 μM and averages calculated. The actual value is greater than that average and no standard error is reported. APQ compounds were tested at concentrations ranging from 1 nM to 10 μM. Data are provided as Ki= nM ± SEM unless otherwise noted.

Figure 1. Effects of Mg-ATP on (A) [3H]reserpine, (B) [3H](+)-5b, and (C) [3H]DHTB. The effects of Mg-ATP on radioligand binding were conducted as described in the Experimental Section. Error bars (SEMs) were derived from multiple experiments conducted with duplicate determinations. 9123

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Figure 2. Kinetics of [3H]reserpine and [3H](+)-5b binding to human VMAT2. (A, B) [3H]Reserpine association and dissociation time curves, respectively. (C, D) [3H](+)-5b association and dissociation time curves, respectively. Note the different X axis time ranges in (B) and (D). Assays were conducted as described in the Experimental Section, with excess concentration of respective nonradiolabeled ligand. n = 3−5 independent experiments conducted in duplicate.

Figure 3. Saturation binding of [3H]reserpine, [3H](+)-5b, and [3H]DHTB binding to HEK-VMAT2 cell membranes: (A) [3H]reserpine, (B) [3H](+)-5b, and (C) [3H]DHTB saturation binding. Assays were conducted as described in the Experimental Section. Saturation curves are the average and SEM of 3−4 independent experiments each conducted in duplicate. A representative Scatchard plot is shown for each radioligand. In the Scatchard analysis, the (−) reciprocal of the slope of the line is an estimate of the Kd value for the radioligand.

ducted, with the caveat that the nearly irreversible [3H]reserpine binding does not obey the reversibility requirement for true equilibrium. Saturation binding of [3H]reserpine, [3H](+)-5b, and [3H]DHTB yielded Kd values of 8.0 ± 1.2 nM, 93 ± 13 nM, and 71 ± 15 nM and Bmax values of 0.942 ± 0.085, 13.5 ± 1.4, and 41.2 ± 2.2 pmol/mg protein, respectively (Figure 3A, Figure 3B, Figure 3C). [3H]Reserpine binding to VMAT2 had a higher affinity but much lower density of binding sites compared to the other two ligands. The fast off rate of [3H](+)-5b compared to the off-rate for 3 [ H]reserpine indicates that binding is reversible, an important characteristic for screening possible therapeutics with high throughput radioligand binding. Likewise, [3H]DHTB binding is reversible,35 with a relatively fast off-rate, but the binding characteristics are not ATP dependent. While APQ ligands did not appear to bind with great affinity for the reserpine or DHTB binding sites, further experimentation with (+)-5b showed evidence that the ligands were binding and affecting VMAT2 function. We lastly screened the APQ ligands in a binding assay competing with [3H](+)-5b (Table 2). Enantiomeric APQ ligand (−)-5b displayed

more proton(s) being countertransported. ATP is required for the proton pump that regulates the vesicle-cytoplasm protongradient that is required for substrate transport by various vesicular transporters.34 Additionally, this finding corroborates data suggesting that reserpine and the APQs, but not DHTB, bind to a site that is coupled to VMAT2 function.32 The rates of association and dissociation for [3H](+)-5b and [3H]reserpine binding were determined in the presence of 2 mM Mg-ATP. Kinetic analysis of [3H]reserpine (14−22 nM) and [3H](+)-5b (24−28 nM) binding to VMAT2 indicates that the observed association rates (kobs) were similar (0.028 ± 0.01, 0.051 ± 0.01, respectively, n = 5, Figure 2A, Figure 2B). By contrast, the dissociation half-lives for [3H]reserpine and [3H](+)-5b are significantly different (p < 0.05), with [3H]reserpine having a dissociation half-life greater than 3 h (data did not fit a one phase exponential decay) and [3H](+)5b having a dissociation half-life of less than 1 min, respectively (Figure 2C, Figure 2D). The kinetic characteristics of [3H](+)5b binding to VMAT2 are quite unlike [3H]reserpine, which binds essentially irreversibly (Figure 2). Saturation binding of [3H]reserpine, [3H](+)-5b, and [3H]DHTB was also con9124

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Table 2. Binding Affinities of APQ Ligands versus [3H](+)-5b at the Human VMAT2, versus [125I]DOI at the 5HT2A Receptor and versus [125I]RTI-55 at the DAT, and Potencies for Inhibition of [3H]5-HT Uptake by Human VMAT2 Expressed in HEK Cells and by Mouse Striatal VMAT2a

Experiments were conducted as detailed in the Experimental Section. n ≥ 3, except when the Ki or IC50 was greater than 10 μM, in which case n = 2. If some experiments yielded IC50 or Ki values less than 10 μM and other experiments yielded IC50 or Ki values greater than 10 μM, the latter experiments were assigned a value of 10 μM and averages calculated. The actual value is greater than that average, and no standard error is reported. APQ compounds were tested at concentrations ranging from 0.01 nM to 10 μM. Data is reported as Ki = nM ± SEM unless otherwise noted. bIC50 = nM ± SEM. cKd= nM ± SEM. ND = not determined. a

moderate affinity in this assay with Ki = 290 nM, whereas the racemate (±)-5b was found to bind with Ki = 161 nM. The other racemic APQ ligands were tested against [3H](+)-5b as well. APQ ligand (±)-5a, displayed moderate binding affinity albeit only two times less potent than (±)-5b. Other APQ ligands with electron withdrawing substituents displayed moderate binding affinity with (±)-5d showing the best results out of the group (entries 13, 14, and 18; Table 2). SAR investigations into electron donating groups also resulted in loss of potency in the [3H](±)-5b binding assay (entries 15−17; Table 2). The potency of the APQ compounds at inhibition of [3H]5HT uptake via human VMAT2 expressed in HEK cells was assessed. APQ ligands (±)-5a and all stereoisomers of 5b had similar potencies, ranging from 16 to 51 nM (Table 2). The next most potent compounds ((±)-5d, (±)-5i, (±)-5j) had potencies ranging from 101 nM to 142 nM. Interestingly, APQ (±)-5d with a meta-trifluormethyl group had greater than 20fold higher potency than (±)-5c with a para-trifluormethyl. APQ ligands (±)-5e, (±)-5f, (±)-5g, and (±)-5h demonstrated little potency in the [3H]5-HT uptake assay. The potencies of a subset of APQ compounds and standards for inhibition of [3H]5-HT uptake via endogenous VMAT2 in mouse striatal vesicular membranes were assessed (Table 2, Figure 4). The potencies of all standard compounds were higher in the mouse vesicular preparations (Table 2). The

Figure 4. Concentration−response curves of inhibition of [3H]5-HT uptake into mouse striatal vesicular membranes. Six APQ compounds and three standards are shown. n = 3−6. All APQ compounds were more potent than ketanserin in this assay.

subset of APQ ligands tested in this preparation had higher potencies compared to potencies in the HEK-VMAT2 uptake assay. APQ ligands (±)-5a and all stereoisomers of 5b were very potent, with IC50 values below 10 nM (Table 2). (±)-5d, (+)-9, and (±)-5i had moderate potencies ranging from 12.8 to 31.5 nM. All APQ compounds tested completely inhibited specific [3H]5-HT uptake, defined with 1 μM reserpine. 9125

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Figure 5. Correlations of binding affinities and uptake potencies for HEK-VMAT2 for select compounds: drug-induced inhibition of (A) log(Ki) for inhibition of [3H](+)-5b binding versus log(IC50) for inhibition of [3H]5-HT uptake, (B) log(Ki) for inhibition of [3H]reserpine binding versus log(IC50) for [3H]5-HT uptake, and (C) log(Ki) for inhibition of [3H]DHTB binding versus log(IC50) for [3H]5-HT uptake. Fourteen APQ compounds and seven standard compounds were included in the analysis. Data were analyzed using Spearman’s nonparametric correlation analysis.

Figure 6. Effects of APQ compounds, standards, and staurosporine (SP) on cell viability in the MTT assay. APQ compounds, RO4-1284, ketanserin, METH, and DHTB were tested at 10 μM, and reserpine was tested at 1 μM. The positive control SP was tested at 0.1, 0.3, 1, and 3 μM. Inset: test for linearity of HEK-hVMAT2 cell number vs OD at 590 nm.

[125I]RTI-55 binding site on the human serotonin transporter and human norepinephrine transporter while cocaine had Ki values of 632 ± 21 and 2,357 ± 39 nM, respectively. No APQs had measurable affinity for 5-HT1A receptor (data not shown). In addition, a subset of APQs was tested in the MTT cell viability assay. (±)-5a, (±)5b, (+)-5b, (−)-5b, (±)-5d, (±)-5i, (±)-9, ketanserin, DHTB, RO4-1284, and methamphetamine at 10 μM and reserpine at 1 μM had no significant effect on HEK-VMAT2 cell viability (one-way ANOVA followed by Tukey’s post hoc test, Figure 6). By contrast, staurosporine, the positive control, decreased cell viability by 45% and 60% at 1 and 3 μM, respectively. Lastly, CNS MPO scores were calculated for the APQ compounds to determine viability of the ligands moving forward.36,37 Ligands scored from 2.2 to 4.4 in the MPO calculation.38 Generally scores greater than 4 are desired, and of note, (±)-5b displayed one of the best MPO scores of 4.1. The moderate MPO values obtained for the APQ ligands lend credence to the viability of APQs as potential CNS therapeutics. The CNS MPO scores will help guide future investigations of the APQ ligands. There are numerous radioligands and drugs that bind to the VMAT2 and affect its function. The results presented here

For the 14 APQ compounds and the seven standard compounds, a good correlation was observed between the log IC50 value for [3H]5-HT uptake and the log Ki value for inhibition of [3H](+)-5b binding (Figure 5A, Spearman’s r = 0.63, p < 0.01). With this same set of compounds, there was an excellent correlation between the log IC50 value for [3H]5-HT uptake and the log Ki value for inhibition of [3H]reserpine binding (Figure 5B, Spearman’s r = 0.90, p < 0.0001). There was also a good correlation between the log IC50 value for [3H]5-HT uptake and the log Ki value for inhibition of [3H]DHTB binding (Figure 5C, Spearman’s r = 0.57, p < 0.01), although the correlation was driven mainly by the control compounds, as the APQs generally had micromolar to no measurable affinity for this site. Affinities for several other neuronal proteins were assessed (Table 2). At the 5-HT2A receptor, (±)-9 had nanomolar affinity similar to ketanserin. All other APQ ligands had little to no measurable affinity for this receptor. Furthermore, (+)-5b and (±)-5a had Ki values of 2440 nM and 1610 nM, respectively, for displacement of [125I]RTI-55 binding to the DAT; all other APQs tested had Ki values greater than 4 μM (Table 2). From assessment of the affinity of the lead compound for other transporters, (+)-5b and the parent compound ketanserin had very low affinities (>10 μM) for the 9126

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General Procedure for the Synthesis of Ethyl 1-Benzyl-4(aryl)-1,2,5,6-tetrahydropyridine-3-carboxylate (3). A 250 mL round-bottom flask was charged with triflate 2 (6.9 mmol), boronic acid (15.1 mmol), PdCl2(PPh3)2 (0.21 mmol), LiCl (20.6 mmol), and K2CO3 (41.3 mmol). The flask was evacuated and back-flushed with argon three times, anhydrous dioxane (100 mL) was added, and the flask was heated to 95 °C overnight. The flask was cooled to room temperature, and CHCl3 (500 mL) was added. The mixture was then washed with water (2 × 150 mL), brine (150 mL), dried (MgSO4), and concentrated. The residue was purified by flash chromatography eluting with 0−30% EtOAc/hexanes to give pure 3. General Procedure for the Synthesis of (±)-syn-Ethyl 4Arylpiperidine-3-carboxylate (4). To a 50 mL round-bottom flask was added 10% Pd/C (225 mg, 0.10 mmol, 50% wet) followed by the addition of a solution of 3 (1 mmol) in EtOH (10 mL). The flask was evacuated and back-flushed with H2 three times, and the reaction mixture was stirred under a static atmosphere of H2 at room temperature (unless otherwise noted) for 24−72 h. The suspension was filtered through a pad of Celite, washing with EtOH. The combined filtrate and washings were concentrated and purified by flash chromatography eluting with 0−10% MeOH/EtOAc (1% Et3N) to give pure 4. Synthesis of (±)-syn-Ethyl 1-(2-(2,4-Dioxo-1,2-dihydroquinazolin-3(4H)-yl)ethyl)-4-arylpiperidine-3-carboxylate (5). A 25 mL high-pressure tube was charged with 4 (0.4 mmol), 633 (0.42 mmol), and toluene or CH3CN (3 mL). The tube was sealed and heated to 110 °C for 2 days. The reaction mixture was concentrated and the residue was purified by flash chromatography, eluting with 0−100% CH2Cl2/EtOAc [Rf ≈ 0.3 (50% CH2Cl2/ EtOAc)] to give 5 as an off-white solid. The ligands were then converted to their hydrochloride salts prior to biological testing. Compounds were dissolved in CHCl3 (1 mL), and 1 N HCl (2 equiv) was then added, and the solution was stirred for 5 min. The solvent was removed under reduced pressure, and the crude solid was dissolved in water. After filtration and removal of the solvent by lyophilization, pure salts could be obtained in good yields. Synthesis of Tritium Labeled [3H](+)-syn-Ethyl 1-(2-(2,4Dioxo-1,2-dihydroquinazolin-3(4H)-yl)ethyl)-4-(4fluorophenyl)piperidine-3-carboxylate ([3H]-5b). The title compound was prepared via direct tritiation of 5b by Morevek Bochemical. Specific activity: 12.8 Ci/mmol. Radiochemical purity: 99.8%. Synthesis of (±)-anti-Ethyl 1-tert-Butyl-4-(4-fluorophenyl)piperidine-1,3-dicarboxylate (7). A 50 mL round-bottom flask was charged with 4b (215 mg, 0.86 mmol), MeOH (5 mL), triethylamine (240 μL, 1.72 mmol), and a magnetic stir bar. Boc2O (375 mg, 1.72 mmol) was added with vigorous stirring, and the mixture was heated to 60 °C for 30 min. Volatiles were removed in vacuo, and the residue was partitioned between CH2Cl2 (15 mL) and brine (5 mL). The layers were separated, and the aqueous layer was further extracted with CH2Cl2 (2 × 10 mL). The combined organic layers were dried (MgSO4), filtered, and concentrated, then purified by flash chromatography, eluting with 0−100% EtOAc/hexanes to give boc protected 4b (214 mg, 71%): Rf = 0.75 (50% EtOAc/ hexanes, 1% Et3N); 1H NMR (300 MHz, CDCl3) δ 7.19 (dd, J = 5.5, 8.8 Hz, 1H), 6.95 (t, J = 8.8 Hz, 2H), 4.09−4.64 (m, 2H), 3.92 (q, J = 7.2 Hz, 2H), 3.01−3.29 (m, 1H), 2.71−3.02 (m, 2H), 2.48−2.71 (m, 1H), 1.54−1.85 (m, 2H), 1.47 (s, 9H), 1.04 (t, J = 7.2 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 171.6, 163.3, 160.0, 154.5, 138.3, 129.1, 129.0, 115.2, 115.0, 79.7, 60.2, 49.1, 45.7, 43.6, 42.5, 28.5, 25.9, 14.1. A 50 mL round-bottom flask was charged with the previous product (194 mg, 0.55 mmol), EtOH (5 mL), and NaOEt (56 mg, 0.83 mmol), then heated to 70 °C for 3 h in an oil bath. The solution was concentrated in vacuo and purified by flash chromatography, eluting with 0−30% EtOAc/hexanes to give pure 7 (137 mg, 71%): Rf = 0.87 (50% EtOAc/hexanes, +1% Et3N); 1H NMR (300 MHz, CDCl3) δ 7.13 (dd, J = 5.2, 8.8 Hz, 2H), 6.96 (t, J = 8.8 Hz, 2H), 4.35 (br s, 1H), 4.24 (br s, 1H), 3.89 (q, J = 7.2 Hz, 2H), 2.72−3.04 (m, 3H), 2.47−2.71 (m, 1H), 1.69−1.87 (m, 1H), 1.56−1.69 (m, H), 1.48 (s, 9H), 0.95 (t, J = 7.2 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ

suggest that the novel APQ compounds bind to a new, heretofore unidentified site on the VMAT2. This site is associated with the region of VMAT2 involved in transport activity, a claim supported by the correlation between binding affinity for this site and inhibition of [3H]5-HT uptake (Figure 4). The modifications of the ketanserin core structure have eliminated affinity for the 5-HT2A receptor and increased selectivity for the [3H]reserpine site for most of the compounds reported. In addition, there is very low affinity for the DAT, an off-target site for some compounds that bind to the VMAT2, such as lobeline.39 The low affinities of the compounds at the two off-target sites indicate that the APQs have high specificity for VMAT2 compared to ketanserin, a ligand for VMAT2, as well as for 5HT receptors. Whether the APQs also lack affinity for other receptors and transporters has not been determined. An important question for additional studies is whether the APQs interact with the vesicular monoamine transporter 1 (VMAT1), expressed in the intestine, stomach, and sympathetic nervous system. 40 Ketanserin has slightly higher potency for inhibition of transport by VMAT2 than VMAT1.41 An interesting finding is that the structural changes made to ketanserin have modified the binding preference for the DHTB binding site; APQ ligands prefer to bind to the reserpine site. Additional detailed structure−activity studies using APQs with varying affinities for VMAT2 may help to characterize the relationships between radioligand binding sites and VMAT2 function, an important precursor to the development of pharmacotherapeutic compounds.

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EXPERIMENTAL SECTION

General Information and Materials. All reactions were magnetically stirred and monitored by analytical thin-layer chromatography (TLC) silica gel 60 F254 plates using UV light to visualize the compounds. Column chromatography was carried out on Teledyne ISCO Combiflash automated chromatography units using standard silica gel cartridges. 1H and 13C NMR spectra were recorded on a JEOL Eclipse 300 MHz spectrometer using tetramethylsilane (TMS) as an internal reference. All target compounds were determined to be >95% pure by HPLC/MS analysis using an Agilent 1200 HPLC apparatus with an ESI mass spectrometer. Melting points were obtained using a Mel-Temp capillary melting-point apparatus and are uncorrected. Reagents and solvents were purchased from commercial suppliers and used without further purification unless otherwise noted. Ethyl 1-Benzyl-4-(trifluoromethylsulfonyloxy)-1,2,5,6tetrahydropyridine-3-carboxylate (2). To a suspension of ethyl 1-benzyl-4-oxo-3-piperidinecarboxylate hydrochloride (3.32 g, 11.1 mmol) in H2O (30 mL) was added potassium carbonate (2.76 g, 20.0 mmol), and the mixture was extracted with CH2Cl2 (3 × 40 mL). The combined organic layers were dried (Na2SO4), filtered, and concentrated to give the free base (100%) as an oil which was used without further purification. This keto ester was dissolved in anhydrous DMF (20 mL), fitted with a septum, nitrogen inlet, and cooled to 0 °C. NaH (60% in mineral oil, 668 mg, 16.7 mmol) was added in one portion, and the solution was stirred at 0 °C for 10 min. N-Phenylbis(trifluoromethanesulfonimide) (4.36 g, 12.2 mmol) was added in one portion, and the solution was stirred at 0 °C for 1.5 h. Water (50 mL) was then added and the resulting mixture was extracted with EtOAc (3 × 50 mL). The combined organic layers were washed with H2O (20 mL), dried (Na2SO4), filtered, and concentrated. The residue was purified by flash chromatography eluting with 0−15% EtOAc/hexanes to give 2 (3.60 g, 83%) as a yellow oil: 1H NMR (300 MHz, CDCl3) δ 7.34−7.25 (comp, 5H), 4.27 (q, J = 7.2 Hz, 2H), 3.67 (s, 2H), 3.42 (t, J = 2.7 Hz, 2H), 2.71 (t, J = 5.4 Hz, 2H), 2.53−2.49 (m, 2H), 1.31 (t, J = 7.2 Hz, 3H). 9127

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[3H]Reserpine Binding Assay. The [3H]reserpine binding assay was modified from the literature procedure.42 [3H]Reserpine binding to the human VMAT2 in HEK-VMAT2 cells was performed as follows. Membranes were prepared as for [3H](+)-5b binding (above). The assays contained approximately 200−300 μg of membrane protein for [3H]reserpine binding (less than 15% of the radioactivity added was bound) and [3H]reserpine (6−8 nM final concentration) in a final volume of 1 mL. Tris assay buffer was used for all assays except as indicated. Specific binding was defined as the difference in binding observed in the absence and presence of 1 μM reserpine. The membranes were incubated for 60 min (equilibrium) at 30 °C. Assays were terminated by addition of 1 mL of ice-cold Tris buffer containing 2 μM nonradiolabeled reserpine and filtration over Whatman 25 mm GF/C filters presoaked in 0.05% polyethylenimine through a 12-channel Millipore filtration chamber. Filters were washed three times with 10 mL of ice-cold Tris buffer. Remaining radioactivity on the filters was counted on a Beckman LS6500 scintillation counter. Saturation binding experiments were conducted with duplicate determinations at each ligand concentration, which included radiolabeled and nonradiolabeled ligand for final concentrations ranging from 1 to 22 nM for reserpine. Protein concentrations were determined using a modified BCA protein assay. [3H]DHTB Binding Assay. [3H]DHTB binding to the human VMAT2 in HEK-VMAT2 cells was performed as described previously, with minor modifications.42 Cells were grown to confluence and washed with calcium−magnesium-free phosphatebuffered saline (cmf-PBS). Cells were scraped from plates in 0.32 M sucrose (4 mL), homogenized with a Polytron homogenizer at setting 6 for 5−10 s, and centrifuged at 30 900g for 20 min at 4 °C. The resulting pellet was resuspended in 2 mL of H2O, homogenized with a Polytron homogenizer, incubated for 10 min on ice, and osmolarity was reestablished by addition of 338 μL of 250 mM Tris, 338 μL of 1 M potassium tartrate, and 10 μL of 0.9 M MgSO4. The assays contained approximately 75−150 μg of membrane protein for [3H]DHTB binding (less than 15% of the radioactivity added was bound) and [3H]DHTB (6−8 nM final concentration) in a final volume of 250 μL. Tris assay buffer without ATP was used for all assays except as indicated. Specific binding was defined as the difference in binding observed in the absence and presence of 2 μM DHTB. The membranes were incubated for 90 min, unless otherwise indicated, at room temperature. Assays were terminated by filtration through Filtermat A filters using a 96-well Tomtec cell harvester, and radioactivity was determined as for [3H](+)-5b using a PerkinElmer 1405 microBeta scintillation counter. Saturation binding experiments were conducted with duplicate determinations at each ligand concentration, which included radiolabeled and nonradiolabeled ligand for final concentrations ranging from 6 to 200 nM for DHTB. Protein concentrations were determined using a modified BCA protein assay. [3H]5HT Uptake Assay, HEK-Human VMAT2. The HEKVMAT2 [3H]5HT uptake assay was performed as described previously, with minor modifications.43 [3H]5HT was used for the uptake assay because it had slightly higher potency than DA or NE in preliminary uptake experiments (data not shown) and has been used as [3H]substrate to characterize VMAT2 activity.44−46 HEK-human VMAT2 cells were grown until confluent. After removal of medium, cells were scraped from plates and homogenized with 12 strokes of a glass/glass homogenizer in sucrose (0.32 M supplemented with protease inhibitors). After centrifugation at 800g for 10 min, the supernatant was removed and centrifuged at 10 000g for 20 min. The pellet was resuspended in sucrose (0.32 M with protease inhibitors). The membranes were osmotically shocked by addition of 3.5 volumes of water (4 °C) and homogenized with a glass/Teflon homogenizer. Osmolarity was reestablished with addition of Tris (25 mM final), potassium tartrate (100 mM), and MgSO4 (0.9 mM), and the membranes were centrifuged at 20 000g for 20 min. After centrifugations, the pellet was resuspended in VMAT2 uptake buffer (100 mM potassium tartrate, 25 mM Tris, 4 mM KCl, 2 mM MgSO4, 1.7 mM ascorbic acid, 100 μM tropolone, and 1 μM pargyline, pH 7.4, at 25 °C with MgATP, 2 mM) using 2 gentle strokes of a Teflon/

172.5, 163.4, 160.2, 154.5, 138.6, 129.0, 128.9, 115.5, 115.2, 80.2, 60.4, 49.1, 46.3, 45.1, 44.1, 32.9, 28.5, 14.0. Synthesis of (±)-anti-Ethyl 4-(4-Fluorophenyl)piperidine-3carboxylate (8). Compound 7 (126 mg, 0.36 mmol) was taken up in HCl (4 M in dioxane, 6.3 mL) under N2. The solution was stirred 45 min and neutralized with solid NaHCO3 (4.60 g). The reaction mixture was diluted with water (20 mL) and extracted with CH2Cl2 (3 × 20 mL). The combined organic layers were dried (MgSO4), concentrated. The residue was purified by flash chromatography, eluting with 0−10% MeOH/CH2Cl2 (1% Et3N) to give 9 (90 mg, 100%); 1H NMR (300 MHz, CDCl3) δ 7.15 (dd, J = 5.2, 8.8 Hz, 2H), 6.94 (t, J = 8.8 Hz, 2H), 3.87 (q, J = 7.2 Hz, 2H), 3.33 (dd, J = 3.6, 11.8 Hz, 1H), 3.10−3.25 (m, 1H), 2.62−2.95 (m, 4H), 2.25 (br s, 1H), 1.74−1.92 (m, 1H), 1.62 (dq, J = 4.1, 12.6 Hz, 1H), 0.95 (t, J = 7.2 Hz, 3H). (±)-anti-Ethyl 1-(2-(2,4-Dioxo-1,2-dihydroquinazolin-3(4H)yl)ethyl)-4-(4-fluorophenyl)piperidine-3-carboxylate (9). The title compound was prepared from 8 and 6 as an off-white solid (68 mg, 47%) using the general procedure described above for the synthesis of 5. Mp = 195.0−197.0 °C; Rf = 0.54 (10% MeOH/ CH2Cl2); 1H NMR (300 MHz, DMSO-d6) δ 11.47 (s, 1H), 7.94 (dd, J = 1.1, 8.0 Hz, 1H), 7.66 (ddd, J = 1.4, 7.4, 8.0 Hz, 1H), 7.14−7.30 (m, 4H), 7.07 (t, J = 8.8 Hz, 2H), 4.04 (t, J = 6.9 Hz, 2H), 3.82 (qd, J = 1.7, 7.2 Hz, 2H), 3.18 (d, J = 10.7 Hz, 1H), 3.02 (d, J = 10.7 Hz, 1H), 2.65−2.78 (m, 2H), 2.60 (t, J = 6.9 Hz, 2H), 2.07−2.27 (m, 2H), 1.51−1.75 (m, 2H), 0.87 (t, J = 7.2 Hz, 3H); 13C NMR (75 MHz, DMSO-d6) δ 172.9, 163.0, 162.5, 159.8, 150.7, 140.2, 140.0, 135.6, 129.7, 129.6, 127.9, 123.1, 115.7, 115.6, 115.3, 114.3, 60.1, 56.2, 55.2, 53.9, 49.1, 44.7, 38.0, 33.3, 14.3. Anal. Calcd for C24H26N3O4F: C, 65.59; H, 5.96; N, 9.56; F, 4.32. Found: C, 65.52; H, 6.09; N, 9.42; F, 4.24. MS (APCI, [M + H]+, m/z) 440.2. Cell Culture. The human VMAT2 cDNA was generously supplied by Dr. Robert Edwards (University of California, San Francisco, CA) and subcloned into pcDNA3.1 with G418 resistance. HEK-293 cells were transfected with the human VMAT2 and characterized as described previously.16,41 Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FetalClone and 0.05 U of penicillin/streptomycin. Stock plates were grown on 150 mm diameter tissue culture dishes in 10% CO2 at 37 °C. [3H](+)-5b Binding Assay. Conditions were optimized for [3H](+)-5b binding, and [3H](+)-5b binding to the human VMAT2 in HEK-VMAT2 cells was performed as follows. Cells were grown to confluence and washed with Tris buffer (25 mM, pH 7.4). Cells were scraped from plates in 5 mL of Tris buffer, resuspended, and centrifuged at 30 900g for 20 min at 4 °C. The resulting pellet was resuspended in 2 mL of H2O and homogenized with a Polytron homogenizer at setting 6 for 5−10 s. After 10 min incubation on ice, osmolarity was reestablished with the addition of 338 μL of 250 mM Tris (pH 7.4), 338 μL of 1 M potassium tartrate, and 10 μL of 0.9 M MgSO4. The binding assays contained approximately 50−75 μg of membrane protein (less than 15% of the radioactivity added was bound) and [3H](+)-5b (50−100 nM final concentration) in a final volume of 250 μL. Tris assay buffer [25 mM Tris, 5 mM KCl, 2.5 mM MgSO4, 0.32 M sucrose, 2 mM MgATP, pH 7.4] was used for all assays except as indicated. Specific binding was defined as the difference in binding observed in the absence and presence of 10 μM O-7443 (an analog of (+)-5b). The membranes were incubated for 60 min (equilibrium) at 4 °C in the dark except where indicated. Assays were terminated by filtration through Filtermat A filters (PerkinElmer) using a 96-well Tomtec cell harvester. Scintillation fluid (50 μL) was added to each filtered spot, and the radioactivity remaining on the filters was determined using a Wallac 1205 Beta plate scintillation counter. Saturation binding experiments were conducted with duplicate determinations at each ligand concentration, which included radiolabeled and nonradiolabeled ligand for final concentrations ranging from 5 to 600 nM for (+)-5b. Protein concentrations were determined using a modified bicinchoninic acid (BCA) protein assay. 9128

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Statistical Analysis. GraphPad Prism 7.02 software (GraphPad Inc., La Jolla, CA) was used to analyze all kinetic, saturation, and competition binding data and was used for analysis of variance (ANOVA) with Bonferroni post-tests. Association experiments with a single concentration of radioligand were analyzed using one phase exponential association nonlinear fit to determine kobs. Dissociation experiments were analyzed using a one phase exponential decay nonlinear fit to determine the dissociation rate constant and half-life. Data shown are the mean ± SEM except as indicated. t tests (twotailed, unpaired) were performed using Microsoft Excel (Microsoft, Redmond, WA). Correlations were assessed with Spearman’s nonparametric correlation.

glass homogenizer. The uptake assay was conducted in duplicate and included membrane preparation, drug, [3H]5-HT (40 nM), and VMAT2 uptake buffer in a final volume of 0.25 mL. [3H]5-HT uptake by HEK-VMAT2 membranes is ATP dependent (data not shown). Membranes were preincubated with drugs for 10 min at 30 °C. After addition of [3H]5HT, uptake was conducted at 30 °C for 6 min and terminated by filtration as described for [3H]DHTB binding, using filters presoaked in 0.05% polyethylenimine. Specific uptake was defined as the difference in uptake observed in the absence and presence of reserpine (1 μM). [3H]5HT Uptake Assay, Vesicular Preparation of Mouse Striata. The method was adapted from the literature.47,48 C57Bl/6J mice (males and females, 44 total, ages 121−152 days) were used. All procedures were performed in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the VA Portland Health Care System. Mice were sacrificed by cervical dislocation, striata were dissected, placed on ice, and homogenized with 12 strokes of a glass/Teflon homogenizer in sucrose (0.32 M supplemented with protease inhibitors). After centrifugation at 800g for 10 min, the supernatant was decanted, the pellet was resuspended in sucrose solution and centrifuged at 800g for 10 min. The supernatants were combined and centrifuged at 10 000g for 20 min. The pellet was resuspended in sucrose (0.32 M with protease inhibitors). The membranes were osmotically shocked by addition of 3.5 volumes of water (4 °C) and homogenized with a glass/Teflon homogenizer. Osmolarity was reestablished with addition of Tris (25 mM final), potassium tartrate (100 mM), and MgSO4 (0.9 mM), and the membranes were centrifuged at 20 000g for 20 min. The supernatant was decanted and centrifuged at 100 000g for 60 min in a Beckman Optima TLX Ultracentrifuge. Uptake was conducted at 30 °C for 8 min and terminated as described above. Specific uptake was defined as the difference in uptake observed in the absence and presence of the VMAT2-selective compound reserpine (1 μM) and was 81−84% of total [3H]5-HT uptake.49 Because reserpine does not bind to the DAT, NET, or SERT, uptake by those transporters in the presence of the VMAT2 blocker, reserpine, is nonspecific uptake.50−52 [125I]RTI-55 Binding to hDAT and [125I]DOI Binding to Human Serotonin Receptor (5HT2A). [125I]RTI-55 binding to the hDAT stably expressed in HEK-293 cells was conducted as previously described using a total particulate membrane preparation.43 The binding assay included test compound or buffer, membranes, [125I]RTI-55 (40−60 pM), and buffer in a final volume of 250 μL. Specific binding was defined as the difference between total binding and binding in the presence of 5 μM mazindol. [125I]RTI-55 binding to the human norepinephrine transporter and the human serotonin transporter was conducted as previously described.43 [125I]DOI binding to the h5-HT2A receptor stably expressed in HEK-293 cells was conducted as previously described.53 The binding assay included test compound or buffer, well-washed cell homogenate, [125I]DOI (50 pM), and buffer in a final volume of 250 μL. Specific binding was defined as the difference between total binding and binding in the presence of 10 μM 5HT. MTT Cell Viability Assay. Compounds were tested for effects on cell viability using a MTT cell proliferation assay kit (Abcam, Cambridge, MA), HEK-VMAT2 cells (5 × 104) were plated in 96well plates in 100 μL of DMEM supplemented with 10% FetalClone, 0.05 U of penicillin/streptomycin, and G418. This density of cells was in the linear response range for the assay (data not shown). On day 2, drugs (100 μL) in unsupplemented DMEM or DMEM were added to triplicate wells. The medium was removed 24 h later, and unsupplemented medium and MTT reagent were added. The plate was incubated for 2 h. at 37 °C, medium was removed, and MTT solvent was added. After 15 min on a shaker, the plate was read at 590 nm. Data were normalized to the response of control cells. Data were analyzed with one-way ANOVA followed with Dunnett’s test. All conditions had 0.1% DMSO. As a positive control, staurosporine, which induces programmed cell death, decreased cell viability at 1 and 3 μM (data not shown).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b00542. Detailed experimental procedures, spectroscopic data (1H NMR) and MPO calculations for the APQ ligands (PDF) Molecular formula strings and some data (CSV)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (978) 8375296. ORCID

Brian A. Provencher: 0000-0003-2607-9530 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. The authors contributed equally. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank John F. Reed for technical assistance. This work was funded by the Department of Veterans Affairs Merit Review Program on Novel Pharmacotherapies for Psychostimulant Addiction (Grant I01 BX000939), Research Career Scientist Program, the National Institute on Drug Abuse (NIDA)-funded Methamphetamine Abuse Research Center (Grant P50 DA018165), and an NIH-VA interagency agreement (Grant ADA15001). The contents do not represent the views of the U.S. Department of Veterans Affairs or the United States Government.

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ABBREVIATIONS USED APQ, arylpiperidinylquinazoline; CCR2, CC chemokine receptor 2; CCL2, CC chemokine ligand 2; CCR5, CC chemokine receptor 5; DAT, dopamine transporter; DHTB, dihydrotetrabenazine; TLC, thin layer chromatography; VMAT1, vesicular monoamine transporter 1; VMAT2, vesicular monoamine transporter 2

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REFERENCES

(1) Abbruscato, T. J.; Tripper, P. C. DARK classics in chemical neuroscience: methamphetamine. ACS Chem. Neurosci. [Online Early Access] DOI: 10.1021/acschemneuro.8b00123. Published Online: March 30, 2018. https://pubs.acs.org/doi/pdf/10.1021/ acschemneuro.8b00123 (accessed July 15, 2018).

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