Structure-Activity Relationships for a Recently Controlled Synthetic Cathinone Dopamine Transporter Reuptake Inhibitor: α-Pyrrolidinohexiophenone (α-PHP)

Abstract

α-Pyrrolidinohexiophenone (α-PHP) is the one-carbon unit α-extended homolog of the better-known and widely abused synthetic cathinone central stimulant α-PVP (“flakka”); both are now U.S. Schedule I controlled substances. Structurally, α-PVP and α-PHP possess a common terminal N-pyrrolidine moiety and differ only with respect to the length of their α-alkyl chain. Using a synaptosomal assay, we previously reported that α-PHP is at least as potent as α-PVP as a dopamine transporter (DAT) reuptake inhibitor. A systematic structure-activity study of synthetic cathinones (e.g. α-PHP) as DAT reuptake inhibitors (i.e., transport blockers), a mechanism thought responsible for their abuse liability, has yet to be conducted. Here, we examined a series of 4-substituted α-PHP analogs and found that, with one exception, all behaved as relatively (28- to >300-fold) selective DAT versus serotonin transporter (SERT) reuptake inhibitors with DAT inhibition potencies of most falling within a very narrow (i.e., <3-fold) range. The 4-CF₃ analog of α-PHP was a confirmed “outlier” in that it was at least 80-fold less potent than the other analogs and displayed reduced (i.e., no) DAT vs SERT selectivity. Consideration of various physicochemical properties of the CF₃ group, relative to that of the other substituents involved here, provided relatively little insight. Unlike with DAT releasing agents, as previously reported by us, a QSAR study was precluded because of the limited range of empirical results (with the exception of the 4-CF₃ analog) for DAT reuptake inhibition.

Introduction

α-Pyrrolidinohexiophenone (also referred to as α-pyrrolidinohexanophenone, α-PHP; 1) (Figure 1) is the one-carbon unit α-extended side-chain homolog of the better known and controlled (i.e., U.S. Schedule I) synthetic cathinone stimulant α-pyrrolidinovalerophenone (α-PVP). α-PVP (2, Figure 1), commonly known as “flakka” and by other “street” names, is the aryl ring-unsubstituted parent of another earlier controlled (i.e., U.S. Schedule I) substance, 3,4-methylenedioxypyrolovalerone (MDPV; 3) (Figure 1).^1–3 Certain synthetic cathinones have been generically referred to as “bath salts”, of which MDPV (3) was, early on, sometimes a component; but, the term “bath salts” no longer refers to a specific or combination of agents. The term “bath salts” has radically evolved to include any of one or more synthetic cathinone analogs and even analogs of amphetamine/methamphetamine.^{1, 4}

Figure 1.

Chemical structures of α-PHP (1), α-PVP (2), and MDPV (3).

α-PHP (1) has been found on the clandestine market and now is also a controlled U.S. Schedule I substance.⁵ Various synthetic cathinones produce their central stimulant actions either by acting as substrates (i.e., as selective or non-selective monoamine neurotransmitter releasing agents), or as reuptake inhibitors (i.e., transport blockers), primarily at dopamine transporters (DAT) in the brain.^{1–4, 6–8} Synthetic cathinones can also act, depending upon their specific chemical structures (i.e., appended substituent groups), on norepinephrine and/or serotonin transporters (NET and SERT, respectively) (reviewed^{1, 4}). That is, synthetic cathinones can influence synaptic monoamine neurotransmitter levels via one or more of several mechanisms (i.e., as substrates/releasing agents or as reuptake inhibitors/blockers of one or more monoamine transporters).^{4, 7, 8} Although many of these agents have yet to be examined in a comprehensive or systematic fashion, we have suggested on the basis of preliminary structure-activity evidence that synthetic cathinones seem to belong to two broad groups of agents: i) those possessing an extended α-alkyl side chain (i.e., longer/larger than an α-methyl group) and/or a tertiary amine or bulky secondary terminal amine (i.e., complex synthetic cathinones), that typically behave as DAT reuptake inhibitors with reduced efficacy/potency at SERT, and ii) those with a primary amine or an N-methyl amine and either no α-, or an α-methyl, substituent (i.e., simple synthetic cathinones) that usually act as releasing agents at DAT but display little selectivity for DAT relative to SERT, depending upon pendant aryl substituents.^{3, 4, 9–11} These generalities are based on studies with a wide range of (often structurally-unrelated) agents or with relatively little direct systematic comparison. Furthermore, we have suggested that molecular determinants (i.e., various cathinone amine- and/or aryl-substituents) might differ for synthetic cathinone-related agents acting as DAT reuptake inhibitors versus those acting as DAT releasing agents.¹¹

Prior to the recent control of α-PHP (1) as a Scheduled substance, we examined 1 at DAT and found it to be at least as potent an inhibitor of DAT reuptake as its better known cousin α-PVP (2)¹², consistent with more recent observations reported by others^{7, 8}; α-PHP (1) was, coincidently, confiscated from the clandestine market at nearly the same time we reported the structure-activity relationships (SAR) of side-chain modified α-PVP analogs (that is, while our manuscript¹² was in press). We showed that α-PHP possesses hallmarks of a new drug of abuse;¹² and, this eventually was found to be the case.^13–15 α-PHP has since become an established abused substance (e.g. see: Odoardi et al.,¹⁶ and Beck et al.,¹⁷). A few aryl-substituted analogs of α-PHP have also appeared on the illicit market including its 4-methyl (i.e., MPHP; 4)^18–20 and 4-fluoro analogs.^{17, 21} In addition, previous work suggested that aryl substitutions in the 4- position of pyrrolidine cathinones might not affect their potency at inhibiting DAT or NET transport activity.^{3, 7, 8} With the exception of an early study by Meltzer et al.,²² in pursuit of agents for the potential treatment of cocaine abuse (but prior to the current widespread abuse of synthetic cathinones of the type described here), the SAR for the inhibition of dopamine reuptake at DAT by abused (or potentially abusable) aryl-substituted synthetic cathinones possessing a terminal pyrrolidine moiety has yet to be examined in a systematic manner.

Because we have examined the SAR of simple synthetic cathinones as releasing agents at DAT in the past, ^{e.g. 11, 23–25} we now turned our attention to the SAR of pyrrolidine-containing complex synthetic cathinones. α-PHP (1) served as our model because i) α-PHP (1), by definition, falls into the class of agents we have termed complex synthetic cathinones; that is, α-PHP possesses a bulky (i.e., a pyrrolidine) tertiary amine and an extended α-alkyl chain, ii) α-PHP has been shown to be a reuptake inhibitor at DAT with comparable potency of α-PVP (2),^{7, 8, 12} and iii) α-PHP is an abused and recently controlled (U.S. Schedule I) substance. Here, we investigated a limited series of 4-substituted α-PHP analogs for their ability to act at DAT with the intent of developing quantitative structure-activity relationships (QSARs) in the same manner we reported for simple synthetic cathinone releasing agents.^{1, 13, 24} Although complex synthetic cathinones are typically less potent at SERT than DAT, we also examined these agents at SERT for comparative purposes. A series of 4-position aryl-substituted α-PHP analogs (i.e., 4-9), that included α-PHP (1) itself, was synthesized and examined for their ability to act as DAT reuptake inhibitors. Analogs included electron donating and electron withdrawing substituents selected for their different physicochemical properties (e.g. size, length, volume) to gain insight on what might be important for their actions. This study, an initial foray into the SAR/QSAR of pyrrolidine-containing complex synthetic cathinones as DAT reuptake inhibitors, was limited to 4-substituted analogs because 2- and 3-substituted analogs might bind to transporters in a rotameric fashion that could confound possible SAR and QSAR findings. Another intent of the present investigation was to provide information on some of the structural requirements of the DAT to interact with these new agents.

Results

Synthesis.

Synthesis of the racemic α-PHP analogs was accomplished by one of two general methods (Scheme 1 for 5, 6, and 8, and Scheme 2 for 4, 7, and 9). The first route was based on an approach previously used by us¹² to prepare α-PHP (1). Friedel-Craft’s acylation of the appropriately-substituted benzene analog using hexanoyl chloride with a catalytic amount of freshly sublimed AlCl₃ afforded hexanophenones 10-12, followed by halogenation with bromine to give intermediates 13-15, respectively (Scheme 1). Subsequent treatment of the α-bromohexanophenones with pyrrolidine provided compounds 5, 6, and 8, respectively. In some instances, the reaction scheme resulted in very poor yields (e.g. 4) or failed to provide the desired product (e.g. 7, 9).

Scheme 1.

^a Synthesis of compounds 5, 6, and 8.

^aReagents and conditions: (a) i. Br₂, AlCl₃ (cat.), 0 °C, 30 min; ii. rt, 1 h; (b) i. pyrrolidine, 0 °C, 10 min; ii. rt, 30 min.

Scheme 2.

^a Synthesis of compounds 4, 7, and 9.

^aReagents and Conditions: (a) n-bromopentane, magnesium, 1,2-dibromoethane, THF, N₂, rt, 5–24 h; (b) i. NBS (1.3 equiv); ii. 1,4-dioxane, rt; iii. pyrrolidine (24–48 h).

Consequently, a different approach was employed for the synthesis of 4, 7, and 9. Here (Scheme 2), the necessary 4-hexanophenones were obtained via a Grignard reaction using benzaldehydes 16-18 to afford their corresponding alcohols (i.e., 19-21, respectively). The Grignard procedure was adapted from a patent from Sebti and coworkers²⁶ whereby the resultant alcohols were oxidized to their corresponding keto analogs by Jones’ reagent and subsequently reacted with bromine to afford the corresponding α-bromohexanophenones. Here, a one-pot procedure utilizing the milder N-bromosuccinamide (NBS) in place of both Jones’ Reagent and bromine, and which also simplified several steps into a one-pot reaction (oxidation, bromination, and amination) was utilized. This route was inspired by Guha et al.²⁷ who had previously used this method for the synthesis of several closely related pyrovalerone derivatives. In this procedure, NBS oxidizes the alcohol, then generates bromine in situ, which can be observed in a burst of a brown gas that evolves from the reaction mixture, to afford the α-brominated phenone. Reaction of the brominated carbonyl compound with pyrrolidine afforded the desired target compounds.

APP⁺ Uptake.

The α-PHP analogs were evaluated for their ability to inhibit transport of 4-(4-(dimethylamino)phenyl)-1-methylpyridinium (APP⁺), a fluorescent DAT and SERT substrate.^{28, 29} Uptake studies were performed in cell cultures expressing human DAT or SERT.^{30, 31} APP⁺ is not fluorescent in solution but, once transported, interacts with intracellular components locking its structure in a fluorescent conformation.³² The uptake of APP⁺ was measured using fluorescence microscopy in live cells exposed to APP⁺ for 30 s under constant perfusion. The maximal fluorescence at the end of the experiment was assigned to 100% in the control wells (APP⁺ alone), and the inhibitory effect of each compound was tested at different concentrations to construct dose-response curves. Figure 2 shows the dose-response curves obtained for cells expressing DAT. Calculated potency values (pIC₅₀ ± SEM) and confidence intervals (95% CI) for each compound are shown in Table 1 for data obtained at DAT and SERT. The unsubstituted parent compound 1 and the methoxy analog 5 were the most potent at DAT (IC₅₀ = 97 and 75 nM, respectively), and the trifluoromethyl analog 9 was the least potent (IC₅₀ = 7,502 nM). The remainder of the series differed only very slightly (i.e., <3-fold) in potency from 1 as DAT reuptake inhibitors. All compounds were very weak at SERT; the weakest was the parent compound 1, with an IC₅₀ value of >30,000 nM. All para-substituted analogues were more potent than 1 at SERT; the most potent in the series were 8 and 9 with approximate IC₅₀ values of 3,600 and 5,800 nM, respectively (Table 1). Compound 9 displayed similar potency in inhibiting APP⁺ uptake at both transporters.

Figure 2.

Effects of α-PHP analogues on APP⁺ uptake in DAT-expressing HEK cells. APP⁺ is a fluorescent substrate of monoamine transporters. Maximal fluorescence intensity after 30s of APP⁺ exposure in the absence (control) or presence of a test compound was determined in 30–60 cells per well and averaged to determine the maximal intensity of that well. Each well was exposed to a single concentration of the tested molecule. The values were normalized assigning 100% to the control wells. Each point represents the mean ± SEM of at least four wells analyzed per concentration.

Table 1.

Inhibition of APP⁺ transport by α-PHP analogues in DAT- or SERT-expressing HEK293 cells. The potency of each compound inhibiting APP⁺ uptake was estimated by fitting the results to a dose-response inhibition curve: pIC₅₀ ± SEM in molar (the total number of wells analyzed per curve are shown in parenthesis), 95% confidence interval (CI) of the pIC₅₀, and the IC₅₀ values for each compound in nmolar are shown.

a) hDAT
Compound	R	pIC50 ± SEM	95%CI	IC50, nM	DAT Selectivitya
α-PHP (1)	-H	7.02 ± 0.04 (45)	7.10; 6.93	97	>300
4-CH3 α-PHP (4)	-CH3	6.91 ± 0.06 (63)	7.03; 6.78	124	83
4-OCH3 α-PHP (5)	-OCH3	7.12 ± 0.04 (57)	7.20; 7.05	75	200
4-CH2CH3 α-PHP (6)	-Et	6.61 ± 0.06 (33)	6.72; 6.49	247	40
4-Cl α-PHP (7)	-Cl	6.74 ± 0.11 (28)	6.95; 6.52	184	55
4-Br α-PHP (8)	-Br	6.90 ± 0.07 (27)	7.04; 6.75	127	28
4-CF3 α-PHP (9)	-CF3	5.13 ± 0.39 (48)	5.91; 4.30	7,502	0.8

b) hSERT
Compound	R	pIC50 ± SEM	95%CI	IC50, nM
α-PHP (1)	-H	<4.5 (43)	---	>30,000
4-CH3 α-PHP (4)	-CH3	4.99 ± 0.37 (62)	5.73; 4.25	10,230
4-OCH3 α-PHP (5)	-OCH3	4.82 ± 0.43 (65)	5.68; 3.96	15,150
4-CH2CH3 α-PHP (6)	-Et	5.01 ± 0.33 (72)	5.67; 4.35	9,834
4-Cl α-PHP (7)	-Cl	5.00 ± 0.11 (67)	5.22; 4.77	10,110
4-Br α-PHP (8)	-Br	5.44 ± 0.10 (72)	5.64; 5.24	3,613
4-CF3 α-PHP (9)	-CF3	5.24 ± 0.07 (81)	5.37; 5.10	5,794

^aDAT selectivity = SERT IC₅₀ value (from Table 1b) ÷ DAT IC₅₀ value.

Discussion

Synthetic cathinones bearing a tertiary amine and/or an extended α-alkyl chain convert methcathinone (MCAT, 10) analogues from relatively non-selective releasing agents to (typically, more DAT-selective) reuptake inhibitors; aryl substituents further modulate potency and selectivity.^{1, 4, 11, 25} The present investigation examined a series of 4- (i.e., para-) substituted cathinones with a tertiary amine and an extended α-alkyl side chain (i.e., α-PHP analogues). Consistent with the above SAR generalizations, most α-PHP analogues were found to be relatively selective DAT versus SERT reuptake inhibitors to varying degrees. Although enantiomers were not studied in this report, it was previously shown that the S enatiomer of 2 and 3 were at least one log-unit more potent than the R enantiomer, both at DAT and NET.^{33, 34} It is likely that this stereselectivity would be preserved in the 4-substituted α-PHP series studied here. The aryl-unsubstituted parent compound (i.e., α-PHP; 1) behaved as a potent DAT reuptake inhibitor confirming what we earlier demonstrated using a different (i.e., a synaptosome-based) assay¹² and by others in mammalian expression systems in vitro.^{7, 8} Also α-PVP, α-PHP, and several related analogs have similar potency inhibiting uptake at DAT and NET, but are much weaker at SERT.^{8, 34} The blocade of DAT activity in vivo produces an elevation of mesolimbic dopamine neurotransmission that is related to abuse and addiciton, whereas the inhibition of NET in peripheral organs is linked to cardiovascular effects such as elevated blood pressure and incresed heart rate.³⁴

Here we show that with the exception of the 4-CF₃ analogue 9 being >80-fold less potent than 1, there was little (i.e., not much more than a 2- or 3-fold) difference in DAT potency among the examined analogues, and 24- to >300-fold DAT versus SERT selectivity. It would seem that the DAT tolerates substituents of various physicochemical character (except for CF₃) at the 4-position of α-PHP as reuptake inhibitors, but this remains to be examined in greater detail (see below).

The IC₅₀s here reported for the 4-substituted α-PHP series using the APP⁺ uptake inhibition assay are not an absolute measure. We have found, for purpose of comparison, that threo-methylphenidate a known potent reuptake inhibitor at DAT showed an IC₅₀ of ~70 nM when assessed using the APP⁺ procedure.^{28, 35} Cocaine, another relevant psychostimulant, showed an IC₅₀ of 340 nM inhibiting APP⁺ uptake in DAT-expressing cells.²⁹ Here, α-PHP (1) and 4-OCH₃ α-PHP (5) the most potent agents of the series showed IC₅₀s of 97 nM and 75 nM, respectively, indicating that these compounds have higher potency than cocaine and similar potency to threo-methylphenidate at inhibiting DAT-mediated uptake.

We have previously published on the quantitative SAR (or QSAR) for DAT action, and DAT versus SERT selectivity, of a series of simple synthetic cathinones, specifically, aryl-substituted methcathinone (10) analogues, as DAT/SERT releasing agents.^23–25 However, due to the narrowness of the present DAT reuptake inhibition data, with the exception of the 4-CF₃ analogue 9, a QSAR study for DAT inhibition was impractical. The overall <10-fold difference in SERT potency (Table 1) also precluded a SERT QSAR investigation.

The 4-CF₃ analogue 9 was only weakly active at DAT and much less potent than its unsubstituted parent, α-PHP (1). Cozzi et al., reported that the simple 4-substituted methcathinone series, 4-CF₃ methcathinone behaved as a very weak DAT substrate (releasing agent) compared to its parent, methcathinone (10),³⁶ a phenomenon that we also have observed several times in the past.^23–25 Niello et al.,³⁷ upon examination of several 4-substituted analogues of MCAT (10), also reported that the 4-CF₃ analogue was much less potent at DAT than 10 or the other analogues investigated. In a complex cathinone-related 2-benzoylpiperidine series the corresponding CF₃ analogue was only a weak DAT reuptake inhibitor.²⁸ Furthermore, the 4-CF₃ analogue of 2 has been shown to bind at DAT with >300-fold lower affinity than 2.²² To the extent that it might be instructive for future SAR/QSAR studies involving synthetic cathinones, we sought a possible explanation for why trifluoromethyl compound 9 is a seeming “outlier” (i.e., lacks DAT potency and selectivity) compared to the other compounds in Table 1. That is, what is it about the CF₃ group, or what makes this substituent unique, so that compounds bearing this substituent result in reduced potency at DAT?

Comparing the Van der Waals radius of a CF₃ group as found in 9 with that of the 4-C₂H₅ substituent found in the more potent 4 (both being approximately 39–40 ų),³⁸ it would seem unlikely that the radius (i.e., size) of the CF₃ substituent is solely responsible for the reduced DAT potency of 9. Furthermore, although the size of the CF₃ substituent has been estimated to be greater than that of C₂H₅ when legacy Taft steric E_s values are considered (E_s = −2.40 vs −1.31, respectively, as reviewed by Karelson³⁹), more recent Verloop Sterimol calculations of substituent length (L) and maximal width (B5) suggest the contrary. That is, the maximal substituent width as reported by Bethome et al.⁴⁰ (i.e., Verloop B5 = 3.16 Å) and substituent length (Verloop L = 4.59 Å) of C₂H₅ exceeds that of CF₃ (B5 = 2.60 Å, L = 3.75 Å) (see Table S1, SI). Santiago et al.⁴¹ previously reported similar findings a few years earlier regarding the width and length of these two substituents when positioned at the 4-position of a phenyl ring. Furthermore, the calculated⁴² whole-molecule volume of 1 and 4-9 evinced that the ethyl analogue 6 (ca 289.4 À³) is comparable in volume to the trifluoromethyl analogue 9 (ca 287.3 À³), and it might be noted that the more potent compound, 1 (ca 256.0 À³), possesses the lowest calculated volume (see Table S1, SI). Hence, the size (volume, steric bulk, length, maximal width), alone, of the CF₃ group is unlikely to play a defining role for the reduced potency of 9. Lipophilicity also doesn’t seem to be a major factor here because the lipophilicity of the CF₃ group (π = 0.88), although less than that of C₂H₅ (π = 1.02), is comparable to that of the higher-potency 4-Br-substituted analogue 8 (π = 0.86) (see Table S1, SI).⁴³ Electron withdrawing and electron donating groups both seem to be accommodated by DAT (although neither is essential as evidenced by the high potency of 1 where Hσ_p = 0); for example, the Hammett σ_p values of CF₃ and Br are 0.54 and 0.23, respectively, whereas those of C₂H₅ and OCH₃ are −0.15 and −0.27, respectively.⁴³ It might be noted, however, that the CF₃ group found in the least potent DAT compound is the most electron withdrawing in character, whereas the most potent compound in the series, 4-methoxy analogue 5, possesses a substituent with the greatest electron donating character (see Table S1; SI). At this time, no simple explanation can be advanced for the low potency of 9 at DAT and, in fact, potency might be related to more than a single physicochemical property of the 4-position substituent, or to a property yet to have been considered. Although a QSAR study was not conducted, Meltzer et al.,²² investigating a series α-PVP (2) analogues as DAT reuptake inhibitors, commented that their inhibitory activities were not readily explained by the electronic or lipophilic nature their various aryl substituents. To examine DAT action in greater detail and in a comprehensive QSAR manner, it will be necessary to synthesize and evaluate additional compounds with a broader potency range and with varying/diverse physicochemical properties to achieve statistical validity.

In conclusion, α-PHP and its 4-substituted analogues examined here, consistent with their amine bulk and extended α side chain, generally acted (with the exception of 9) as potent and relatively selective DAT versus SERT reuptake inhibitors, and their DAT potencies (again with the exception of the 4-CF₃ analogue 9) seemed relatively impervious to the physicochemical nature of the substituents examined. However, various aryl substituents modulated DAT versus SERT selectivity. Some interesting SAR similarities and differences were noted between the present DAT reuptake inhibitors and their like-substituted MCAT (10)-related releasing agents. In both cases, the parent aryl-unsubstituted molecule was among the most potent in the series (1, DAT IC₅₀ = 97 nM; 10, EC₅₀ = 13.5 nM²³) and the 4-CF₃ analogue was the least potent (9, IC₅₀ = 7,502 nM; 4-CF₃ 10, EC₅₀ = 2,710 nM²³); in both series, the unsubstituted parent displayed >300-fold DAT versus SERT selectivity, whereas the 4-CF₃ analogues displayed the least (i.e., no) selectivity. In contrast, the 4-OCH₃ analogue 5 displayed the highest DAT potency as a DAT reuptake inhibitor and 200-fold selectivity, whereas the corresponding methcathinone (i.e., 4-OCH₃ 10)²³ displayed much lower potency as a releasing agent (40-fold lower than its parent, 10) and 4-fold SERT versus DAT selectivity.

At least two of these analogues, α-PHP (1) itself, and its 4-methyl analogue (i.e., MPHP; 4), already are on the clandestine market; additional analogues might appear. Further monoamine transporter studies on complex synthetic cathinone analogues are required to better understand their abuse liability and DAT versus SERT selectivity.

Experimental

Synthesis.

Melting points were measured using a Thomas-Hoover melting point apparatus with glass capillary tubes and are uncorrected. Compounds were characterized by ¹H NMR spectrometry and IR spectroscopy, and some by mass spectrometry (MS). ¹H NMR spectra were recorded using a Bruker AXR 400 MHz spectrometer with tetramethylsilane (TMS) as internal standard, IR spectra were recorded using a Thermo Nicolet iS10 FT-IR, and MS were recorded, where applicable, using a Waters Acquity tandem quadrupole (TQD) instrument with electrospray ionization. Reactions were monitored by thin-layer chromatography (TLC) using silica gel GHLF plates (250 mm, 2.5 × 10 cm; Analtech Inc. Newark, DE). Flash chromatography, where applicable, was performed on a CombiFlash Companion/TS (Teledyne Isco Inc.; Lincoln, NE). All novel compounds were prepared as their water soluble hydrochloride salts, and their purity was determined by TLC homogeneity, and microanalysis for C, H and N (Atlantic Microlab Inc.; Norcross, GA), and all results were within 0.4% of theoretical values.

Racemic 1-phenyl-2-(pyrrolidin-1-yl)hexanone (α-PHP; 1) was prepared as previously reported by us as its oxalate salt¹² using the general procedure shown in Scheme 1.

1-(4-Methylphenyl)-2-pyrrolidin-1-yl)hexanone Hydrochloride (MPHP; 4)

The compound was reported in a patent, but with no physical characterization.⁴⁴ Under an open atmosphere and constant stirring, N-bromosuccinamide (1.21 g, 6.8 mmol) was added directly (i.e., solventless) to the oil 19 (1.01 g, 5.26 mmol) at room temperature. The mixture turned yellow, then orange, and released a burst of a brown gas over the course of 2 min, at which time 1,4-dioxane (4 mL) was added. After 10 min of additional stirring, pyrrolidine (1.3 mL, 1.12 g, 16 mmol) was added in a dropwise manner to the mixture, which became cloudy and colorless, then returned to a clear yellow solution. The mixture was allowed to stir at room temperature for 36 h, quenched with saturated aqueous NaHCO₃ (20 mL), then acidified with HCl (1N, 50 mL, to pH 1). The aqueous portion was separated, basified with NaOH (3M, 50 mL, to pH 13), and extracted with EtOAc (3 × 20 mL). The combined organic portion was dried (Na₂SO₄), filtered, and the filtrate was evaporated under reduced pressure resulting in a yellow-colored oil. The oil was dissolved in anhydrous Et₂O (5 mL) at room temperature and, under constant stirring, a saturated solution of gaseous HCl in anhydrous Et₂O (10 mL) was added; stirring was allowed to continue at room temperature overnight. The solvent was evaporated to yield a brown solid that was recrystallized from acetonitrile to yield 0.23 g (15%) of 4 as a beige-colored solid: mp 179–182 °C; ¹H NMR (DMSO-d₆) δ 0.77 (t, J = 7.1 Hz, 3H, CH₃), 0.99–1.29 (m, 4H, 2 x CH₂), 1.94–2.09 (m, 6H, 3 x CH₂), 2.45 (s, 3H, CH₃), 3.05–3.07 (m, 1H, CH₂), 3.20–3.29 (m, 1H, CH₂), 3.49–3.51 (m, 1H, CH₂), 3.63–3.64 (m, 1H, CH₂), 5.47–5.50 (m, 1H, CH), 7.47 (d, J = 8.1 Hz, 2H, ArH), 8.02 (d, J = 8.3 Hz, 2H, ArH), 10.47 (br s, 1H, NH⁺); Anal. Calcd for (C₁₇H₂₅NO•1.0 HCl•0.6 H₂O) C, 66.58; H, 8.94; N, 4.57. Found: C, 66.56; H, 8.83; N, 4.47; HRMS-ESI⁺ (m/z) calcd for C₁₇H₂₆NO⁺ (M+H⁺) 260.2014, found 260.2017, calcd for C₁₇H₂₅NONa⁺ (M+Na⁺) 282.1834, found 282.1831.

1-(4-Methoxyphenyl)-2-(pyrrolidin-1-yl)hexanone Hydrochloride (5)

Compound 13 (460 mg, 1.8 mmol) was stirred in pyrrolidine (2.0 mL, 24.0 mmol) for 1 h at room temperature. The stirred reaction mixture was quenched by the careful addition of ice-cold H₂O (10 mL), acidified with HCl (6 N, 3 × 5 mL, to pH 1), and extracted with EtOAc (3 × 10 mL). The combined acidic portion was basified with NaOH (3 M, 3 × 5 mL, to pH 13), and re-extracted with Et₂O (3 × 15 mL). The combined organic portion was washed with brine (15 mL), dried (Na₂SO₄), filtered, and the filtrate was evaporated under reduced pressure. The crude free base was dissolved in anhydrous Et₂O (2 mL) to which a saturated ethereal HCl solution was added in a dropwise manner at −78 °C (dry ice/acetone) under constant stirring. The mixture was allowed to stir at room temperature for 24 h, after which the solvent was evaporated to yield a white solid that was recrystallized from acetonitrile to yield 97 mg (19%) of 5 as white needles: mp 195–198 °C; ¹H NMR (DMSO-d₆) δ 0.76 (t, J = 7.1 Hz, 3H, CH₃), 1.01–1.26 (m, 4H, 2 x CH₂), 1.93–2.09 (m, 6H, 3 x CH₂), 3.00–3.01 (m, 1H, CH₂), 3.17–3.25 (m, 1H, CH₂), 3.36–3.48 (m, 1H, CH₂), 3.60–3.61 (m, 1H, CH₂), 3.90 (s, 3H, CH₃), 5.43–5.51 (m, 1H, CH), 7.16 (d, J = 8.8 Hz, 2H, ArH), 8.08 (d, J = 8.9 Hz, 2H, ArH), 10.28 (br s, 1H, NH⁺); Anal. Calcd for (C₁₇H₂₅NO₂·HCl) C, 65.48; H, 8.40; N, 4.49. Found: C, 65.76; H, 8.54; N, 4.46.

1-(4-Ethylphenyl)-2-(pyrrolidin-1-yl)hexanone Hydrochloride (6)

Compound 14 (520 mg, 1.8 mmol) was allowed to stir in pyrrolidine (2.0 mL, 24.0 mmol) for 1 h at room temperature. The stirred reaction mixture was quenched by the careful addition of ice-cold H₂O (10 mL), acidified with HCl (1 N, 3 × 15 mL, to pH 1), and extracted with Et₂O (3 × 5 mL). The combined acidic portion was basified with NaOH (3 M, to pH 13), and re-extracted with Et₂O (3 × 15 mL). The combined organic portion was washed with brine (15 mL), dried (Na₂SO₄), filtered, and the filtrate was evaporated under reduced pressure. The crude free base was dissolved in anhydrous Et₂O (2 mL) to which a saturated ethereal HCl solution was added in a dropwise manner at −78 °C (dry ice/acetone) under constant stirring. The mixture was allowed to stir at room temperature for 24 h; the solid material was collected by filtration and recrystallized from acetonitrile to yield 152 mg (27%) of product as yellow crystals: mp 182–184 °C; ¹H NMR (DMSO-d₆) δ 0.75 (t, J = 7.0 Hz, 3H, CH₃), 0.92–1.27 (m, 7H, 2 x CH₂, CH₃), 1.87–2.09 (m, 6H, 3 x CH₂), 2.73 (q, J = 7.6 Hz, 2H, CH₂), 2.97–3.10 (m, 1H, CH₂), 3.21–3.28 (m, 1H, CH₂), 3.45–3.49 (m, 1H, CH₂), 3.60–3.61 (m, 1H, CH₂), 5.42–5.47 (m, 1H, CH), 7.49 (d, J = 8.2 Hz, 2H, ArH), 8.01 (d, J = 8.3 Hz, 2H, ArH), 10.24 (br s, 1H, NH⁺); Anal. Calcd for (C₁₈H₂₇NO·HCl) C, 69.77; H, 9.11; N, 4.52. Found: C, 69.81; H, 9.21; N, 4.57.

1-(4-Chlorophenyl)-2-(pyrrolidin-1-yl)hexanone Hydrochloride (7)

Under an open atmosphere and with constant stirring, N-bromosuccinimide (5.49 g, 31 mmol) was added directly to 20 (5.08 g, 24 mmol) at room temperature. The temperature was increased to 30 °C, and the mixture was allowed to stir for 1 h. Upon the release of a brown gas, 1,4-dioxane (10 mL) was added. The mixture turned clear, then yellow after 10 min of stirring at room temperature; pyrrolidine (5.90 mL, 5.09 g, 72 mmol) was added in a dropwise manner and stirring was allowed to continue for 24 h. The mixture was washed with saturated aqueous NaHCO₃ (20 mL) and extracted with EtOAc (3 × 20 mL). HCl (3N, 15 mL, to pH 1) was added to the organic portion; the aqueous portion was separated, basified with NaHCO₃ (3M, 50 mL, to pH 13), and extracted with EtOAc (3 × 20 mL). The combined organic portion was dried (Na₂SO₄), filtered, and the filtrate was evaporated under reduced pressure to afford 3.5 g of a crude brown oil. The oil was dissolved in anhydrous Et₂O (5 mL) at room temperature and, under constant stirring, a saturated solution of gaseous HCl in Et₂O (10 mL) was added and the reaction mixture was allowed to stir overnight. The precipitate was collected by filtration to yield a brown solid that was recrystallized from i-PrOH/Et₂O to provide 0.15 g (2%) of 7 as an off-white powder: mp 198–200 °C; ¹H NMR (DMSO-d₆) δ 0.74 (t, J = 7.2 Hz, 3H, CH₃), 0.89–1.27 (m, 4H, 2 x CH₂), 1.93–2.05 (m, 6H, 3 x CH₂), 3.07–3.29 (m, 2H, CH₂), 3.41–3.61 (m, 2H, CH₂), 5.54–5.55 (m, 1H, CH), 7.72 (d, J = 8.6 Hz, 2H, ArH), 8.10 (d, J = 8.6 Hz, 2H, ArH), 10.59 (br s, 1H, NH⁺); Anal. Calcd for (C₁₆H₂₂ClNO•HCl) C, 60.76; H, 7.33; N, 4.43. Found: C, 60.66; H, 7.21; N, 4.53.

1-(4-Bromophenyl)-2-(pyrrolidin-1-yl)hexanone Hydrochloride (8)

Compound 15 (860 mg, 2.6 mmol) was allowed to stir in pyrrolidine (3.0 mL, 36.0 mmol) for 30 min at room temperature. The stirred reaction mixture was quenched by the careful addition of ice-cold H₂O (10 mL), acidified with HCl (2 N, to pH 1), and extracted with Et₂O (3 × 10 mL). The combined acidic portion was basified with aqueous NaOH (15%, 20 mL, to pH 13), and re-extracted with Et₂O (3 × 20 mL). The combined organic portion was washed with brine (15 mL), dried (Na₂SO₄), filtered, and evaporated under reduced pressure. The residual oil was purified by flash chromatography (silica gel; hexanes/EtOAc; 95/5 to 80/20) to afford the free base of the product as a yellow oil. The free base was dissolved in anhydrous Et₂O (2 mL) under an N₂ atmosphere and HCl gas was bubbled in. The precipitate was recrystallized from acetonitrile to yield 152 mg (27%) of 8 as an off-white powder: mp 199–201 °C; ¹H NMR (DMSO-d₆) δ 0.75 (t, J = 6.7 Hz, 3H, CH₃), 1.19 (m, 1H, CH₂), 1.78–1.94 (m, 3H, 2 x CH₂), 1.78–2.05 (m, 6H, 3 x CH₂), 2.99–3.07 (m, 1H, CH₂), 3.21 (m, 1H, CH₂), 3.49 (m, 1H, CH₂), 3.61 (m, 1H, CH₂), 5.39–5.58 (m, 1H, CH), 7.88 (d, J = 7.6 Hz, 2H, ArH), 8.01 (d, J = 8.5 Hz, 2H, ArH), 10.38 (br s, 1H, NH⁺); Anal. Calcd for (C₁₆H₂₂BrNO·HCl·0.4 H₂O) C, 52.23; H, 6.52; N, 3.81. Found: C, 52.01; H, 6.30; N, 3.96.

1-(4-Trifluoromethylphenyl)-2-pyrrolidin-1-yl)hexanone Hydrochloride (9)

Under an open atmosphere and with constant stirring, N-bromosuccinimide (2.08 g, 11.7 mmol) was added directly to 21 (2.21g, 9.0 mmol) at room temperature. The temperature was increased to 30 °C, and the mixture was allowed to stir for 40 min. Following the release of a brown gas, 1,4-dioxane (10 mL) was added and, subsequent to 10 min of additional stirring, pyrrolidine (2.21 mL, 1.92 g, 26.9 mmol) was added in a dropwise manner. The mixture was allowed to stir, loosely covered, at room temperature overnight, carefully quenched with saturated aqueous NaHCO₃ (20 mL) at 0 °C, and extracted with Et₂O (3 × 20 mL). HCl (3N, 15 mL, to pH 1) was added to the organic portion, and the aqueous portion was separated, basified with NaHCO₃ (3M, 50 mL, to pH 13), and extracted with Et₂O (3 × 20 mL). The combined organic portion was dried (Na₂SO₄), filtered, and the filtrate was evaporated under reduced pressure. The resulting crude oil was dissolved in anhydrous Et₂O (5 mL) at room temperature. Under constant stirring, gaseous HCl was bubbled in and the reaction mixture was allowed to stir at room temperature overnight. The precipitate was collected by filtration to yield a brown solid that was recrystallized from EtOH/Et₂O resulting in 0.17 g (5%) of 9 as a white powder: mp 219–222 °C; ¹H NMR (DMSO-d₆) δ 0.74 (t, J = 7.2 Hz, 3H, CH₃), 0.99–1.25 (m, 4H, 2 x CH₂), 1.96–2.07 (m, 6H, 3 x CH₂), 3.13–3.26 (m, 2H, CH₂), 3.52–3.62 (m, 2H, CH₂), 5.61 (m, 1H, CH), 8.03 (d, J = 8.1 Hz, 2H, ArH), 8.28 (d, J = 8.2 Hz, 2H, ArH), 10.59 (br s, 1H, NH⁺); Anal. Calcd for (C₁₇H₂₂F₃NO•HCl) C, 58.37; H, 6.63; N, 4.00. Found: C, 58.18; H, 6.45; N, 3.99.

4-Bromohexanophenone (12)

Using a 2-neck flask, hexanoyl chloride (1.9 g, 14.0 mmol) was added to a solution of freshly sublimed AlCl₃ (1.9 g, 14.0 mmol) in anhydrous DCM (10 mL) under an N₂ atmosphere and cooled to −10 °C (salt/ice-bath). The contents were allowed to stir at −10 to −5 °C for 10 min. A solution of 4-bromobenzene (2.0 g, 12.7 mmol) in anhydrous DCM (10 mL) was added in a dropwise manner over 30 min at −10 to −3 °C. The ice bath was removed and the contents were allowed to stir at room temperature for an additional 1 h, quenched by the careful addition of ice-cold HCl (1N, 50 mL), and extracted with DCM (3 × 20 mL). The combined organic portion was washed with brine (20 mL), dried (Na₂SO₄), filtered, and the filtrate was evaporated under reduced pressure. The residual semi-solid was crystallized by trituration with hexanes and then recrystallized from hexanes, yielding 0.9 g (27%) of 12 as a yellow-colored solid: mp 61–65 °C (lit.⁴⁵ mp 59–60 °C); ¹H NMR (CDCl₃) δ 0.91 (t, J = 4.1 Hz, 3H, CH₃), 1.25–1.41 (m, 4H, 2 x CH₂), 1.68–1.76 (m, 2H, CH₂), 2.91 (t, J = 7.4, 2H, CH₂), 7.59 (d, J = 7.9, 2H, ArH), 7.81 (d, J = 7.9, 2H, ArH).

4-Methoxy-α-bromohexanophenone (13)

Freshly sublimed AlCl₃ (5 mg) was added at room temperature to a stirred solution of 10⁴⁶ (1.7 g, 8.3 mmol) in anhydrous Et₂O (10 mL). Upon cooling the reaction mixture to 0 °C (ice-bath), a solution of bromine (1.3 g, 8.3 mmol) in anhydrous Et₂O (10 mL) was added in a dropwise manner over 20 min, and the reaction mixture was allowed to stir at room temperature for 1 h. The reaction was quenched by the careful addition of H₂O (30 mL), washed with brine (10 mL), dried (Na₂SO₄), filtered, and the filtrate was evaporated under reduced pressure resulting in 1.45 g of a yellow-colored oil, which upon crystallization with hexane and recrystallization from hexane afforded 1.2 g (50%) of product as a yellow-colored solid: mp 51–53 °C (lit.⁴⁷ mp 51–52 °C); IR (solid, cm⁻¹) 1667 (s, C=O); ¹H NMR (CDCl₃) δ 0.92 (t, J = 6.9 Hz, 3H, CH₃), 1.52–1.59 (m, 4H, 2 x CH₂), 2.05–2.25 (m, 2H, CH₂), 3.89 (s, 3H, CH₃), 5.10 (t, J = 7.2 Hz, 1H, CH), 6.96 (d, J = 8.9 Hz, 2H, ArH), 8.00 (d, J = 8.9, 2H, ArH).

4-Ethyl-α-bromohexanophenone (14)

Freshly sublimed AlCl₃ (5 mg) was added at room temperature to a stirred solution of 4-ethylhexanophenone (11)⁴⁶ (1.05 g, 5.14 mmol) in anhydrous Et₂O (10 mL). After cooling the reaction mixture to 0 °C (ice-bath), a solution of bromine (0.81 g, 5.14 mmol) in anhydrous Et₂O (10 mL) was added in a dropwise manner over 20 min and the reaction mixture was allowed to stir at room temperature for 1 h. The reaction was quenched by the careful addition of H₂O (30 mL), washed with brine (10 mL), dried (Na₂SO₄), filtered, and evaporated under reduced pressure. The residual oil was purified by flash chromatography (silica gel; hexanes/EtOAc; 80/20) to afford 0.52 g (36%) of 14 as a clear yellow oil: ¹H NMR (CDCl₃) δ 0.85 (t, J = 7.1 Hz, 3H, CH₃), 1.18–1.21 (m, 3H, CH₃), 1.25–1.47 (m, 4H, 2 x CH₂), 1.97–2.19 (m, 2H, CH₂), 2.65 (q, J = 7.6 Hz, 2H, CH₂), 5.05 (t, J = 6.9, 1H, CH), 7.25 (d, J = 8.3, 2H, ArH), 7.87 (d, J = 8.3, 2H, ArH).

4-Bromo-α-bromohexanophenone (15)

Freshly sublimed AlCl₃ (5 mg) was added at room temperature to a stirred solution of 12 (890 mg, 3.5 mmol) in anhydrous Et₂O (10 mL). After cooling the reaction mixture to 0 °C (ice-bath), a solution of bromine (550 mg, 3.5 mmol) in anhydrous Et₂O (10 mL) was added in a dropwise manner over 30 min, and the reaction mixture was allowed to stir at room temperature for 6 h. Additional bromine (270 mg, 1.7 mmol) was added at 0 °C (ice-bath), and the reaction was allowed to stir for an additional 30 min. The reaction was quenched by the careful addition of saturated aqueous NaHCO₃ (60 mL), extracted with Et₂O, washed with brine (10 mL), dried (Na₂SO₄), filtered, and the filtrate was evaporated under reduced pressure. The resulting 860 mg (74%) of the white solid, 15, was used without further purification: mp 37–38 °C; ¹H NMR (CDCl₃) δ 0.92 (t, J = 7.1 Hz, 3H, CH₃), 1.32–1.57 (m, 6H, 3 x CH₂), 2.06–2.26 (m, 2H, CH₂), 5.04 (dd, J = 7.7, 6.6 Hz, 1H, CH), 7.63 (d, 8.7 Hz, 2H, ArH), 7.87 (d, 8.7 Hz, 2H, ArH).

1-(4-Methylphenyl)hexan-1-ol (19)

In a three-neck flask under N₂ at room temperature, 1-bromopentane (2.5 mL, 20 mmol) and 1,2-dibromoethane (several drops as catalyst) diluted by THF (12 mL) were added dropwise to a stirred solution of magnesium (0.48 g, 20 mmol) in THF (8.0 mL). After stirring at room temperature for two hours, the mixture was cooled to 0 °C and p-tolualdehyde (16) (2.4 mL, 20 mmol) was added in a dropwise manner over the course of 20 min. The mixture was allowed to come to room temperature and stirred overnight (24 h). The reaction was quenched carefully with saturated aqueous NH₄Cl (50 mL) and extracted with EtOAc (3 × 20 mL). The combined organic portion was washed with brine (30 mL), dried (Na₂SO₄), filtered, and the filtrate was evaporated under reduced pressure, resulting in an orange liquid that was purified by flash chromatography (silica gel; hexanes/EtOAc; 80/20) to afford 19 as a clear yellow oil: (1.01 g, 26%) ¹H NMR (CDCl₃) δ 0.89 (t, J = 6.2 Hz, 3H, CH₃), 1.11–1.31 (m, 6H, 3 x CH₂), 1.42–1.53 (m, 1H, OH), 1.55–1.77 (m, 2H, CH₂), 2.37 (s, 3H, CH₃), 4.61–4.65 (m, 1H, CH), 7.16 (d, J = 7.9 Hz, 2H, ArH), 7.24 (d, J = 8.0 Hz, 2H, ArH). The compound was used without further characterization in the preparation of 4.

1-(4-Chlorophenyl)hexan-1-ol (20)

Magnesium turnings (2.4 g, 100 mmol), ground with a mortar and pestle, were added to a three-neck flask, and brought under an N₂ atmosphere at room temperature. THF (12 mL) was added and the suspension was allowed to stir for 5 min, after which 1,2-dibromoethane (several drops as catalyst) was added. The mixture was allowed to stir for another 10 min, whereupon it turned opaque gray, and 1-bromopentane (11.0 mL, 89 mmol) was added, followed by additional THF (6 mL). The mixture was allowed to stir at room temperature for 2 h, and then heated at reflux for 1 h. Upon cooling the reaction mixture to 0 °C (ice-bath), 4-chlorobenzaldehyde (17) (10.0, 71 mmol) was added in a dropwise manner over the course of 20 min. The mixture was allowed to equilibrate to room temperature while stirring for 1 h. The reaction mixture was carefully quenched with H₂O, followed by the addition of saturated aqueous NH₄Cl (50 mL). The organic portion was washed with brine (30 mL), dried (Na₂SO₄), filtered, and the filtrate was evaporated under reduced pressure to afford 20 as a clear yellow oil: (5.45 g, 36%). ¹H NMR (CDCl₃) δ 0.89 (t, J = 5.8 Hz, 3H, CH₃), 1.22–1.45 (m, 6H, 3 x CH₂), 1.64–1.83 (m, 2H, CH₂), 1.88 (d, J = 3.0, 1H, OH), 4.65–4.70 (m, 1H, CH), 7.28–7.35 (m, 4H, ArH). The compound was used without further characterization in the synthesis of 7.

1-(4-(Trifluoromethyl)phenyl)hexan-1-ol (21)

The compound was prepared in 33% yield from 18 according to a slightly modified literature procedure⁴⁸ in that the reaction was terminated when all starting materials had been consumed as determined by TLC. The product was obtained as a clear yellow oil: ¹H NMR (CDCl₃) δ 0.88 (t, J = 6.7 Hz, 3H, CH₃), 1.25–1.46 (m, 6H, 3 x CH₂), 1.64–1.82 (m, 2H, CH₂), 1.95 (d, J = 2.9, 1H, OH), 4.72–4.76 (m, 1H, CH), 7.46 (d, J = 8.1 Hz, 2H, ArH), 7.60 (d, J = 8.1 Hz, 2H, ArH). Although a somewhat shorter reaction time was required here, the product was identical to the literature product. Compound 21 was used without further characterization in the synthesis of 9.

APP^± uptake studies using fluorescence microscopy.

Stable cell lines expressing the human dopamine transporter (hDAT) or the human serotonin transporter (hSERT) were previously generated using the Flp-In T-REx system.^{30, 31} The APP⁺ uptake studies were performed as previously described;²⁸ briefly, cells were plated in 96-well plates and were transfected with DsRED expressing plasmid using Fugene6 as transfection reagent. After transfection, the culturing media was supplemented with 1μg/mL of doxycycline to induce the expression of the transporters. After three days the cells were visualized in an Olympus IX70 microscope equipped with a monochromator-based lamp (Polychrome V), an EMCCD camera (Andor), and a pressurized perfusion system (Automate Scientific). DsRED fluorescence was used to identify the focal plane of the culture monolayer, and the APP⁺ was visualized using an excitation wavelength of 460 nm and an emission of 535/50 nm at an acquisition frequency of 10Hz. The imaging experiments were performed using the following imaging buffer: 130 NaCl, 4 KCl, 2 CaCl₂, 1 MgCl₂, 10 HEPES, 10 glucose, pH 7.3, in mM. Experiments were performed under constant perfusion and solution switching was computer-controlled using electronic valves. First, cells were exposed to imaging buffer for 10s, then exposed for 40s to a test compound, and then for 30s to APP⁺ in combination with the compound. The concentration of APP⁺ used was 3 μM and 5 μM in hDAT and hSERT experiments, respectively. The concentration of APP⁺ used was slightly higher in hSERT experiments to compensate for the slower rate of APP⁺ uptake in hSERT cells compared to hDAT cells. The concentration of the test compounds were different for each well to construct the dose-response curve. Each experimental day few wells were measured omitting test compounds to define the 100% uptake control condition. Movies were recorded for offline analysis, the maximal fluorescence was measured for the cells (30–60 cells per well) at the end of the recording, and the mean maximal fluorescence was assigned as the uptake value for that well. Each well value was normalized to the maximal intensity of the control wells. Several wells were tested per concentration in each experimental day, and data from at least 2 experimental days were used to construct a dose-response curve using GraphPad Prism 5.0 software (Figure 2 shows curves for DAT experiments, and data for both transporters are included in Table 1). The pEC₅₀ ± SEM, 95% confidence interval (CI) and EC₅₀ values for each compound tested are shown in Table 1.