Synthesis of α3β4 Nicotinic Acetylcholine Receptor Modulators Derived from Aristoquinoline that Reduce Reinstatement of Cocaine-Seeking Behavior

Abstract

Growing evidence suggests that inhibition of the α3β4 nicotinic acetylcholine receptor (nAChR) represents a promising therapeutic strategy to treat cocaine use disorder. Recently, aristoquinoline (1), an alkaloid from Aristotelia chilensis, was identified as an α3β4-selective nAChR inhibitor. Here, we prepared 22 derivatives of 1 and evaluated their ability to inhibit the α3β4 nAChR. These studies revealed structure-activity trends and several compounds with increased potency compared to 1 with few off-target liabilities. Additional mechanistic studies indicated that these compounds inhibit the α3β4 nAChR non-competitively, but do not act as channel blockers, suggesting they are negative allosteric modulators. Finally, using a cocaine-primed reinstatement paradigm, we demonstrated that 1 significantly attenuates drug-seeking behavior in an animal model of cocaine relapse. The results from these studies further support a role for the α3β4 nAChR in the addictive properties of cocaine and highlight the possible utility of aristoquinoline derivatives in treating cocaine use disorder.

Graphical Abstract

Introduction

Cocaine is a highly addictive stimulant, with 1.4 million Americans meeting the clinical definition of having a cocaine use disorder (CUD).¹ Furthermore, since 2019, more overdose deaths have involved cocaine than heroin or prescription opioids, with 24,486 cocaine-related deaths occurring in 2021 in the United States.² Unlike other addictive substances like nicotine, alcohol, and opioids, no approved pharmacotherapies are currently available to treat CUD. Due in part to this lack of treatment options, relapse rates for CUD patients are higher than those for alcohol or opioids.³ Clearly, a medication that assists in reducing these relapse rates would tremendously improve the outcomes of CUD.

Recently, the nicotinic acetylcholine receptors (nAChRs) have emerged as promising targets for CUD treatment due to their apparent involvement in modulating the addictive properties of multiple drugs of abuse, including cocaine.^4,5 The nAChRs are cation-permeable members of the Cys-loop family of ligand-gated ion channels.⁶ In mammalian neurons, eight α subunits (α2–7, α9, and α10) and three β subunits (β2–4) assemble into pentameric complexes to make up the various nAChR subtypes. As their name implies, nAChRs are activated by the endogenous agonist acetylcholine (ACh) and exogenous agonists such as nicotine and epibatidine. While multiple lines of evidence suggest that α4β2 is the major subtype responsible for nicotine addiction,^7–9 the α3β4 nAChR subtype also appears to play a significant role in the effects of cocaine and other drugs of abuse.⁴

The α3β4 nAChR is the predominant subtype in the medial habenula (MHb) and interpeduncular nucleus (IPN).^10,11 Notably, the MHb and IPN form functional interactions with the regions of the brain associated with drug reward: the MHb receives input from the nucleus accumbens (NAc) and sends efferent projections to the IPN, which in turn innervates the ventral tegmental area (VTA).¹² When low doses of cocaine are introduced, the extracellular concentration of ACh increases in the IPN, much like how stimulants produce a rapid release of dopamine in the nucleus accumbens.¹³ Similarly, neurons in the MHb are activated during cocaine-primed reinstatement of conditioned place preference (CPP), a mouse model of cocaine relapse. Moreover, chemogenic activation of MHb neurons is sufficient to induce cocaine-relapse-like behavior.¹⁴ Taken together, these studies indicate that the MHb and IPN constitute significant brain regions involved in cocaine addiction.¹² As a result, the pharmacological inhibition of the α3β4 nAChR found within the MHb-IPN axis represents a promising strategy for developing treatments for CUD. In fact, several α3β4 ligands, including the ibogaine derivative 18-MC, the synthetic partial agonist AT-1001, and the non-selective nAChR channel blocker mecamylamine (Figure 1), provide compelling support for the effectiveness of α3β4 inhibition in modulating the behavioral effects of cocaine in rodent models of CUD.^4,15–18 However, their limited α3β4 potency or off-target liabilities have thus far restricted their translation into the clinic. On the other hand, the α-conotoxin AuIB (Figure 1) is a highly potent and selective α3β4 antagonist that reduces the self-administration of nicotine, methamphetamine, and opioids.^15,19,20 However, in vivo studies have required the direct introduction of AuIB into the MHb or IPN to overcome its poor pharmacokinetic stability and blood-barrier permeability.

Figure 1.

Structures of α3β4-targeting ligands that have been investigated for the treatment of substance use disorders (SUDs).

Recognizing the need to explore additional α3β4 nAChR-targeting compounds, we were intrigued by the recent discovery that aristoquinoline (1), an alkaloid isolated from the maqui tree (Aristotelia chilensis), inhibits the α3β4 nAChR preferentially over the α4β2 and α7 subtypes.^{21 22}Natural products like the Aristotelia alkaloids continue to serve as excellent starting points and sources of inspiration in the drug discovery process.²³ Moreover, the structural complexity inherent to many natural product scaffolds often imparts unique pharmacological and signaling properties that make them attractive chemical probes for studying biological systems.^23,24 In light of these advantages, we developed a streamlined chemical synthesis of 1.²⁵ Building off this result, here we report a series of analogues evaluating how modifications to the aromatic ring and alkyl amine of 1 influence activity at the α3β4 receptor, other nAChR subtypes, and central nervous system (CNS) receptors. We also demonstrate that 1 effectively attenuates drug-seeking behavior in an animal model of cocaine relapse, thereby further supporting that inhibition of the α3β4 nAChR represents a promising approach to treat CUD.

Results

Chemistry.

To investigate the structure-activity relationships (SAR) of 1, a series of derivatives was prepared by adapting our previously reported synthesis of 1.²⁵ Employing an aza-Prins reaction to construct the 3-azabicyclo[3.3.1]nonene scaffold, we focused on modifying the quinoline group of 1, anticipating that these changes would allow us to examine how π system, steric bulk, and orientation of the nitrogen impacts activity at the α3β4 nAChR. Generally, we observed that this aza-Prins reaction of various aryl aldehydes with primary amine 2, which can be prepared on a gram-scale in good enantiopurity (e.r. > 95:5) from (+)-limonene (Scheme 1),²⁵ provided the desired analogue as a single diastereomer. This broad reaction scope allowed us to develop a library of 22 analogues exploring ligand-receptor interactions with the α3β4 nAChR (Scheme 1).

Scheme 1.

Synthesis of 1 and its derivatives.^a

^aPercentages represent isolated yields.^bNo product observed. ^cUncyclized imine intermediate (19a) isolated as the only product.

We began the SAR study by scanning the quinoline nitrogen at the various positions on the A- and B-rings to generate five quinoline analogues (3-7). By starting with the quinoline-nitrogen scan, we probed potential interactions between the nitrogen atom and the α3β4 binding pocket without significantly changing the steric or electronic properties of the heterocycle. The X-ray crystal structure of 1 indicates that the quinoline B-ring is located proximal to the C17 methyl, which may restrict rotation about the C4-C9 bond.^25,26 Therefore, to assess whether this restricted rotation influences activity, we synthesized the truncated pyridine analogues 8–11. To test for tolerability to other heterocycles and to determine whether the nitrogen was essential for activity, the quinoline was completely replaced by an imidazole (12) and a benzofuran (13). Unfortunately, attempts to synthesize an indole analogue were unsuccessful, as we observed rapid decomposition of the compound upon purification.

During our initial evaluation of the pyridine analogues, we determined that while the B-ring appeared important, it was not essential to inhibit α3β4 nAChR. We, therefore, synthesized a series of analogues containing substituted phenyl rings to study the effects of modulating the electron density of the aromatic ring and probe the steric tolerance of the binding pocket. Analogues 14–19, bearing nitro, trifluoromethyl, dimethylamino, and phenol moieties, were included to test how electron density in the aromatic system would influence activity. However, synthesis of analogues with a C4’ or C2’ hydroxyl group (17–18) was unsuccessful. Given the otherwise broad substrate scope of the aza-Prins cyclization, this suggests that after condensation between 2 and the hydroxybenzaldehydes, the resulting imine intermediates tautomerize to the corresponding quinomethides, which may be unable to cyclize. Attempts to synthesize 19, bearing a dimethylamino group at the C4’ position, were similarly unsuccessful and in this case, we were able to isolate the imine intermediate 19a from the reaction mixture. Finally, to better understand the binding site tolerance towards steric bulk and hydrophobic groups at C4’, the alkyl-substituted analogues 20 and 21 were also synthesized.

After assessing the activity of this initial series of phenyl-containing analogues, we observed that 16, bearing a trifluoromethyl group in the C4’ position, possessed increased potency almost 3-fold relative to 1. This improved activity does not appear to arise solely from the decreased electron density in the ring, as the nitro-containing analogues (14-15) are less potent than 1. Similarly, since including alkyl groups at this position (20-21) does not produce similar increases in activity, the potency of 16 is also unlikely to be due simply to hydrophobic interactions. Thus, we prepared compounds 22–26 to probe these apparent fluorine-specific effects further.

Azabicyclic Amine Modifications

In addition to modifying the aromatic ring of 1, we also sought to test how changing the secondary amine would influence its activity. Many nAChR ligands that bind to the orthosteric site possess alkyl amines that are positively charged at physiological pH. It is now well-established that these cationic nitrogen atoms form multiple cation-pi interactions with an aromatic box motif that is highly conserved across multiple nAChR subtypes.^27–29 To investigate whether these interactions are required in these compounds, we replaced the secondary amine in the azabicyclo[3.3.1]non-6-ene core of 23, which is one of the more potent derivatives, with an ether. The ether 27 was synthesized using a BF₃•Et₂O catalyzed oxy-Prins reaction between 4-fluoro-3-(trifluoromethyl)-benzaldehyde and (+)-limonene (Scheme 2).

Scheme 2.

Modification of the secondary amine through replacement as an ether.

In Vitro Evaluation

With this focused library of analogues, we sought to determine how these structural modifications would impact the compounds’ functional activity at the nAChRs. To do so, each analogue was evaluated in a calcium flux assay using HEK293 cells that stably express the rat α3β4 (rα3β4) nAChR. As previously described by our lab and others, these assays were performed using a protocol that sequentially measures receptor activation and inhibition.^25,30 In this assay, solutions of compounds (0.3 nM-100 μM) are added to a 96-well plate containing cells preincubated with an intracellular calcium dye (Calcium 6, Molecular Devices) using a robotic pipettor integrated into the plate reader (FlexStation 3 Multi-Mode Microplate Reader, Molecular Devices). Subsequent increases in fluorescence intensity correspond to increases in intracellular [Ca²⁺] arising from channel opening. In this assay, the agonist epibatidine and partial agonist AT-1001 both produce the expected concentration-dependent increases in fluorescence, with potencies consistent to previously reported values (Figure 2A).³¹ However, as we previously observed with 1, treating cells with its analogues resulted in no significant changes in fluorescence, indicating that the binding any of these compounds to the α3β4 nAChR does not induce channel opening.

Figure 2.

Functional activity of compounds at the rat α3β4 nAChR expressed in HEK293 cells was measured using a Calcium 6 calcium flux assay. Error bars indicate SEM (n≥3). (A) Activation of receptor produced by epibatidine (EPI), AT-1001, and 1. (B) Inhibition of epibatidine-induced (100 nM) activation of the receptor. All curves represent average values of at least three independent experiments.

To measure the compounds’ abilities to inhibit channel activation, a solution of epibatidine (100 nM, final concentration) was added to each well after a 10-minute incubation with the test compounds. As expected, epibatidine, AT-1001, and 1 all produced concentration-dependent inhibition (Figure 2B). In the case of epibatidine and AT-1001, this inhibition likely reflects desensitization of the receptor following activation. Like 1, all analogues tested reduced epibatidine-evoked activation of the rα3β4 nAChR. Using each compound’s respective potency (IC₅₀), we compared their activity to identify SAR trends and determine how structural modifications impacted their ability to inhibit the rα3β4 nAChR (Table 1).

Table 1.

Inhibitory Potencies of 1 and its Derivatives at the rα3β4 nAChR.

Compound	X	R	pIC50	α3β4 IC50 (μM)a	Fold Changec
1	NH	4-quinoline	6.0 ± 0.1	0.92	--
3	NH	4-isoquinoline	5.9 ± 0.2	1.1	0.9
4	NH	5-quinoline	6.2 ± 0.1	0.64	1.5
5	NH	5-isoquinoline	5.6 ± 0.2	2.6	0.4*
	NH	8-isoquinoline	5.4 ± 0.2	4.4	0.2**
7	NH	2-quinoline	6.1 ± 0.1	0.7	1.3
8	NH	4-pyridine	4.4 ± 0.3	38	0.02***
9	NH	3-pyridine	4.6 ± 0.4	26	0.04***
10	NH	2-pyridine	5.8 ± 0.2	1.6	0.6
11	NH	4-chloro-3-pyridine	5.8 ± 0.1	1.7	0.5
12	NH	imidazole	6.4 ± 0.2	0.39	2.4
13	NH	2-benzofuran	5.4 ± 0.2	4.1	0.2*
14	NH	4-nitrobenzene	5.6 ± 0.1	2.8	0.4*
15	NH	3-nitrobenzene	5.2 ± 0.1	6.9	0.1***
16	NH	4-trifluoromethylbenzene	6.5 ± 0.1	0.34	2.7**
20	NH	4-isopropylbenzene	6.0 ± 0.1	0.98	0.9
21	NH	4-t-butylbenzene	5.9 ± 0.1	1.1	0.8
22	NH	3-fluoro-4-(trifluoromethyl)benzene	5.6 ± 0.1	2.8	0.3*
23	NH	4-fluoro-3-(trifluoromethyl)benzene	6.4 ± 0.1	0.43	2.1*
24	NH	3,4-difluorobenzene	6.4 ± 0.1	0.44	2.1
25	NH	3,5-difluorobenzene	5.5 ± 0.1	3.4	0.3**
26	NH	2,5-difluorobenzene	5.4 ± 0.1	3.8	0.2**
27	O	4-fluoro-3-(trifluoromethyl)benzene	n.ab	n.a.b	--

^aInhibition of response produced by epibatidine (100 nM) measured using a calcium flux assay against rat α3β4 expressed in HEK293. Data are presented as mean ± SEM (n≥3). All compounds produce <5% receptor activation.

^bNo inhibition activity observed.

^cFold change is calculated as IC₅₀ of 1 divided by IC₅₀ of analogue. Asterisk(s) indicate significant difference from pIC₅₀ of 1

(* = p<0.05, ** = p<0.01, *** - p<0.001).

As detailed above, the analogues can be divided into two major generations of derivatization. The first round included five quinoline analogues (3-7), where we found that repositioning the nitrogen around both the A- and B-rings yielded a range of IC₅₀ values, with a general preference for the quinoline nitrogen to be in the 4’- or 5’-positions. This suggests that the nitrogen may form directional polar interactions with the receptor, most likely acting as a hydrogen bond acceptor. Moving the attachment point to the 1’ position, such as in 7, also improved activity (pIC₅₀: 6.1 ± 0.1, IC₅₀: 0.7 μM).

Our initial derivatization also included the removal of the B-ring to generate pyridine analogues (8-11), which resulted in significant reductions in potency compared to 1. The reduced activity of these derivatives indicates that, while not strictly required, the quinoline B-ring is important for maintaining antagonist potency, perhaps by filling additional space in the binding pocket. Replacement of the quinoline with the electron-rich imidazole (12) improved potency more than 2-fold compared to 1, but electron-rich benzofuran (13) reduced potency by more than 4-fold, highlighting the importance of the aromatic nitrogen. Reducing the electron density by appending a chlorine to the pyridine ring, as in 11, produced only marginal changes in potency, despite its structural similarities to the high-affinity nAChR ligand epibatidine.

To further explore how the electronics of the π-system may influence activity, the quinoline of 1 was replaced with substituted phenyl rings. When nitro groups were added (14–15), they showed activity significantly less potent than 1. Probing this aspect of the SAR, strongly electron-withdrawing analogues were then synthesized, beginning with the 4-trifluoromethyl in 16, which demonstrated almost 3-fold improvement in potency to 1 (pIC₅₀: −6.5 ± 0.1, IC₅₀: 0.34 μM), and proved to be our most potent analogue. Considering the increased hydrophobicity inherent with a trifluoromethyl group, similarly hydrophobic compounds (20-21) were synthesized but maintained similar activity to 1.

These results indicated that the trifluoromethyl group may be forming important ligand-receptor interaction. Adding an additional fluorine to 16 yielded 22, which is three times less potent than 1 and 8-fold less potent than 16. However, reversing the position of trifluoromethyl and fluorine substituents of 22, as in 23, rescued this activity (pIC₅₀: −6.4 ± 0.1, IC₅₀: 0.43 μM). These results led us to consider whether decreasing steric bulk while maintaining electron-withdrawing properties could improve potency, thus leading to the synthesis of 24, which maintained potency (pIC₅₀: 6.4 ± 0.1, IC₅₀: 0.44 μM). However, moving the fluorines to other positions, as seen in 25 and 26, again decreased potency, indicating substitutions in the 3- and 4-positions on the ring were preferred.

Finally, having evaluated how alterations to the aromatic ring of 1 impacted activity, modifications were made to the secondary amine in the azabicyclic core. Converting the amine to an ether (27) completely abolished activity, indicating a cationic nitrogen is likely required for activity.

Mechanism of Action Studies.

After identifying several compounds with improved or comparable potency relative to 1, we sought to gain additional insight into how these compounds inhibit the α3β4 nAChR. Generally, compounds that inhibit nAChRs can be categorized as competitive antagonists that bind to the orthosteric site, located at the α3(+)-β4(−) interface (Figure 3A), as allosteric inhibitors that bind elsewhere on the receptor, or as channel blockers that occlude the ion pore. To differentiate between the first two potential mechanisms, concentration-response curves were generated for epibatidine in the presence and absence of 1. Under this paradigm, a competitive antagonist would be expected to produce a rightward shift in the epibatidine concentration-response curves. However, pre-incubation with 1 (10 μM) had a minimal effect on epibatidine potency (Figure 3B). Instead, we observed a significant lowering of the maximal effect (E_max) produced by epibatidine, resulting in a downward shift in the curve. This insurmountable antagonism suggests that 1 does not bind to the orthosteric binding site and instead acts as an allosteric inhibitor. Alternatively, the observed insurmountable inhibition can also arise from compounds that bind irreversibly to the receptor. However, in patch clamp electrophysiology studies, α3β4 nAChR recovers its ability to respond to epibatidine agonism following a 30-second wash-out after exposure to 1 (Figure 3C). Together, these data are consistent with 1 inhibiting α3β4 by binding reversibly to an allosteric site.

Figure 3.

Mechanism of action studies suggest 1 operates as nAChR NAM. A) Diagram depicting the orthosteric and channel blocker sites. B) Concentration-response curves for epibatidine in the presence and absence of 1 indicate a non-competitive mode of inhibition. Error bars indicate SEM (n=3). C) 1 (1 μM) inhibition is reversible following a 30-second wash. D) Inhibition of epibatidine by 1 is voltage-independent, indicating 1 does not bind within the membrane-spanning pore of the channel. Data for B-D were collected using whole-cell patch clamp electrophysiology using rα3β4 nAChR expressed in HEK293 cells.

Channel blockers that occlude the ion pore could give rise to non-competitive inhibiton.³² However, because pore blockers interact within the voltage field of the nAChR pore domain, they exhibit different potencies depending on the transmembrane voltage.³³ Conversely, negative allosteric modulators (NAMs) that bind to the extracellular domains of the nAChR and stabilize the resting or desensitized states of the receptor would be expected to confer voltage-independent inhibition. To distinguish between these mechanisms, we co-applied 1 and epibatidine to α3β4-HEK293 cells and varied the membrane holding potential used to drive inward currents from −80 mV to −20 mV. In these experiments, we observed similar levels of inhibition at all membrane potentials (Figure 3D), suggesting that 1 is not blocking within the voltage field that spans the transmembrane pore but instead acts as a NAM within the ligand binding domain.

Assessment at other nAChR Subtypes.

Previous studies indicated that 1 inhibits the α3β4 nAChR more potently than the α4β2 and α7 subtypes.²¹ To determine whether the analogues of 1 possessed similar or improved subtype-selectivity, we compared 1 and seven analogues (4, 16, 20, 22-25) at different nAChR subtypes using radioligand binding and functional calcium flux assays. To determine whether activity at other subtypes would be influenced by the nature of the aryl ring, we selected derivatives that contained modifications that impacted α3β4 activity including placement of the nitrogen (4), substitutions at the C4’ position (20), and fluorine-containing groups (16, 22-25). While many of these compounds have increased or decreased potency relative to 1, they all maintain their ability to inhibit rα3β4 nAChR. Radioligand binding assays were conducted by the Psychoactive Drug Screening Program (PDSP) and assessed radioligand binding against seven human nAChR subtypes (hα3β4, hα4β2, hα7, hα2β2, hα2β4, hα3β2, and hα4β4). Six of the compounds, including 1, failed to inhibit [³H]-epibatidine binding from the nAChRs (Table S1). These data are consistent with our proposed allosteric mechanism of action, wherein 1 and most of its derivatives do not prevent orthosteric ligand binding. In contrast to the other ligands, compounds 4 and 24 did inhibit [³H]-epibatidine binding at the hα3β4 nAChR. Most notably, compound 4 showed a strong binding affinity for the hα3β4 subtype (K_i = 37 nM, Table 3), along with some affinity at the hα2β4 subtype (K_i = 1.3 μM, Table 3), but lacked affinity at all other tested subtypes. These data suggest that despite its structural similarity to 1, compound 4 may act as a competitive orthosteric antagonist.

Table 3:

Binding Affinities (K_i, nM) of Compounds at CNS Receptors and Transporters

Receptor	1	4	16	20	22	23	24	25
α2A	2,300	--	--	--	--	500	--	--
α2C	1,400	660	--	--	--	280	1,200	--
σ1	--	6,800	2,700	--	--	--	--	4,500
σ2	--	--	70	17*	63	280	210	810
D2	--	--	--	--	570	--	--	--
D3	--	--	--	--	520	--	--	--
D4	--	--	--	--	1,000	--	--	--
M2	--	--	1,500	2,400	2,800	--	--	--
M4	--	--	1,600	1,900	--	--	--	--
BZP	--	--	9,000*	--	--	--	--	4,800*
DAT	--	--	1,400	--	--	--	--	230
GABAA	--	8,600	>10,000	--	--	--	--	4,500
α3β4	--	37	--	--	--	--	2,600	--
α2β4	--	1,300	--	--	--	--	--	--

Determined through the Psychoactive Drug Screening Programing using radioligand binding assays (n=3). “--” indicates compound produced less than 50% displacement of radioligand at 10 μM.

^*Did not produce >50% displacement, but triggered a secondary binding assessment.

We also employed functional assays to compare the analogues’ ability to inhibit the three most prevalent nAChR subtypes in the human CNS: hα3β4, hα4β2, and hα7. A similar calcium flux assay protocol was employed with three notable differences: all receptors were transiently expressed in HEK293 cells, nicotine was used as a test agonist, and compounds were only evaluated for antagonist activity. Despite these differences, compounds 1, 4, 16, 20, and 22-25 possess similar potency at the hα3β4 nAChR (Table 2) as seen in the rα3β4 nAChR (Table 2). Consistent with previous reports, 1 was nearly 10-fold less potent at the α4β2 than α3β4 and only inhibited α7 at the highest concentration tested (Figure S1). Compared to 1, compound 4 possessed reduced potency at the hα7 subtype, resulting in improved subtype-selectivity. In contrast to 1 and 4, derivatives lacking the B-ring (16, 20, and 22–25) possessed considerably less subtype-selectivity. Most notably, derivatives 20 and 22–25 inhibited α7 more potently than the α3β4 subtype. Thus, while the B-ring of 1 can be removed and retain activity at hα3β4, the quinoline ring appears to be important for maintaining subtype-selectivity.

Table 2:

Inhibitory Potencies of 1 and its Derivatives at the hα3β4, hα4β2, and hα7 nAChRs

Compound	α3β4 IC50 (μM)	α4β2 IC50 (μM)	α7 IC50 (μM)	α4β2 IC50/ α3β4 IC50	α7 IC50/ α3β4 IC50
1	1.47 ± 0.35	10.86 ± 2.45	115.7 ± 109.15	7.3	78
4	2.12 ± 0.46	22.45 ± 7.02	19.74 ± 8.5	10.6	9.31
16	0.56 ± 0.12	2.99 ± 0.46	8.48 ± 2.77	5.3	15.06
20	1.21 ± 0.39	8.73 ± 3.35	0.38 ± 0.102	7.2	0.31
22	1.05 ± 0.21	4.05 ± 1.44	0.63 ± 0.26	3.9	0.59
23	1.10 ± 0.28	12.31 ± 3.73	0.58 ± 0.15	11.19	0.53
24	1.57 ± 0.41	5.59 ± 1.42	1.24 ± 0.47	3.57	0.79
25	1.46 ± 0.44	1.91 ± 0.51	0.63 ± 0.32	1.31	0.43
SR16584	2.13 ± 0.83	--	--	--	--
DHβE	--	0.88 ± 0.314	--	--	--
MLA	--	--	0.012 ± 0.004	--	--

Inhibition of response produced by nicotine was measured using a calcium flux assay against human α3β4, α4β2, or α7 nAChRs expressed in HEK293. Data are presented as mean ± SEM (n=3). All compounds produce <5% receptor activation. SR16584, DhβE, and MLA were used as control antagonists for the α3β4, α4β2, and α7 nAChRs, respectively.

Receptor Binding Profiling.

We also sought to determine the extent to which 1 and its analogues were selective for the nAChRs and screened the eight compounds for affinity to 52 G-protein coupled receptors (GPCRs), ion channels, and transporters through the PDSP.³⁴ Using radioligand binding assays, all eight compounds were first assessed for their ability to inhibit radioligands from binding to their respective receptors at a single concentration (10 μM). Any analogues demonstrating >50% inhibition of radioligand binding at 10 μM were further investigated with complete concentration-response curves to determine binding affinities (K_i) (Table 3). Notably, 1 showed interactions with only two non-nAChR targets, possessing low affinity at both these anti-targets: α2A (K_i 2.3 μM) and α2C (K_i 1.4 μM). In general, similarly low levels of off-target binding were also observed for the analogues; where affinity was noted, it also tended to be low. The σ2 receptor, however, was a notable exception, with six of the seven analogues possessing significant affinity (K_i = 17–810 nM). Interestingly, both quinoline compounds (1 and 4) lack any measurable affinity for σ2, providing further support that the additional B-ring is important for α3β4 selectivity.

In vivo – Pharmacokinetics and Cocaine Relapse Model

Encouraged by the in vitro activity and receptor profile of 1 and its derivatives, we sought to investigate how inhibition of α3β4 by these compounds would affect drug taking and seeking, using animal models of CUD. Although our SAR studies identified several derivatives with comparable or improved potency compared to 1, these derivatives possess reduced subtype-selectivity and bound to more off-target receptors, most notably the σ2 receptor. Based on this selectivity and the unique allosteric mechanism of action, we elected to focus these in vivo studies on the natural product 1.

Prior to evaluating 1 in models of CUD, we first conducted an initial pharmacokinetic study. Following intraperitoneal administration of 1 (10 mg/kg), peak plasma concentration (C_max = 723.3 ± 50.6 ng/mL) was observed within 10 minutes (Figure S2, Table S3). Compound 1 showed significant systemic exposure of 602.4 ± 182.3 ng•h/mL (AUC) with an elimination half-life (T_½) of 1.7 ± 0.2 hours. The pharmacokinetic properties indicate that a single 10 mg/kg dose of 1 should provide adequate exposure throughout a 2-hour self-administration and relapse operant session. To determine whether 1 can cross the blood-brain barrier and reach the site of action in the brain, we also compared concentrations 1 in the brain and plasma. Thirty minutes following intraperitoneal administration of 1 (1.5 mg/kg), 1 reached a concentration of 232.7 ± 21.0 ng/g and 36.4 ± 4.7 ng/mL in the brain and plasma, respectively. This brain-to-plasma concentration ratio of 6.4 ± 0.6 indicates that 1 is able to cross the blood-brain barrier and act in the CNS.

Previous studies have used a variety of behavioral paradigms, including self-administration and drug reinstatement, to demonstrate that α3β4 antagonists modulate the addictive properties of nearly all drugs of abuse, including cocaine. To determine whether 1 possesses similar effects, we assessed its anti-addictive properties using cocaine self-administration and cocaine-primed reinstatement models of CUD. In the self-administration paradigm, Sprague-Dawley rats were trained to self-administer cocaine (0.5 mg/kg/0.1 ml infusion) under an FR-1 schedule in 2-hour operant sessions. After a robust and stable response for cocaine was established, rats (n=8) were intraperitoneally treated with two doses of 1 (1, 10 mg/kg) or vehicle (5% DMSO, 5% Tween 80 and 90% 0.9% Saline) 15 minutes before FR-1 cocaine self-administration sessions, using a Latin Square counterbalanced design. However, at both doses, 1 was found to have no effect on cocaine self-administration (F_(2,14) = 1.0, p = not significant, Figure 4A).

Figure 4.

A. Active lever responses for rats trained to self-administer cocaine (FR-1) following treatment with 1 (i.p.). Error bars indicate SEM (n=8); B. Pattern of active lever responses during self-administration training and extinction periods. Error bars indicate SEM (n=6). C. Active lever responses on the final day of extinction and after cocaine-prime reinstatement of drug-seeking behavior following treatment with 1 or vehicle (5% DMSO, 5% Tween 80, and 90% saline, i.p.). Error bars indicate SEM (n=6), * = p<0.05 difference from period of extinction (EXT), # = p<0.05 difference from vehicle-treated rats.

Despite the inability of 1 to modulate the rate of cocaine self-administration, we also evaluated its effects on attenuating cocaine-seeking behavior using cocaine prime-induced reinstatement, a model of cocaine relapse. Rats that stably self-administered cocaine (n=6) underwent an extinction period with daily operant sessions in which cocaine was unavailable, significantly reducing active lever pressing (Figure 4B). Rats were then treated with a priming injection of cocaine (10 mg/kg, i.p.) immediately before an extinction session, inducing a reinstatement of cocaine-seeking behavior as measured by an increase in active lever pressing despite no cocaine infusions (p<0.05, Figure 4C). Conversely, pretreating rats with 1 (10 mg/kg, i.p.) 15 minutes prior to the cocaine-prime significantly reduced drug-seeking behavior during the reinstatement period (p<0.05). The observed effect of 1 in decreasing reinstatement seems to be behaviorally specific, as the same dose failed to block the rate of cocaine taking. Thus, while 1 appears ineffective at affecting cocaine self-administration behavior, it remarkably reduces cocaine-seeking behavior in a model of cocaine relapse.

Discussion

The high prevalence of CUD and the staggering number of cocaine-associated overdose deaths underscores the urgent need to develop a medication to treat CUD. Previously, the α3β4 partial agonist AT-1001 was shown to suppress the acquisition of cocaine CPP in mice trained to self-administer cocaine. Similarly, 18-MC induces a dose-dependent reduction in cocaine self-administration.¹⁵ Together, these studies with AT-1001 and 18-MC have demonstrated the usefulness and safety of targeting α3β4 as a treatment for CUD. However, there are noteworthy differences between these compounds and 1. In particular, AT-1001 and 18-MC possess significant affinities for other nAChR subtypes and CNS receptors. 18-MC is only 4-fold more potent at the α3β4 subtype compared to the α4β2 nAChR and has affinity for μ, δ, and κ opioid receptors.⁴ While AT-1001 is considerably more subtype-selective, it, too, has affinity for non-nAChR targets. Most notably, AT-1001 has affinity for the σ2 receptor (K_i = 418 nM) comparable to that measured for compounds 23–25, and even greater affinity at the σ1 receptor (K_i = 85 nM). Thus, while it is unclear if the activity at these off-target receptors impacts the behavioral effects of AT-1001 and 18-MC, by comparison, 1 has fewer off-target liabilities.

The azabicyclic scaffold is a common motif found in many nAChR ligands that bind to the orthosteric site, including epibatidine, AT-1001, atropine, and methyllycaconitine. Conversely, our electrophysiology and radioligand binding data indicate 1, and presumably most of its analogues, do not bind to the orthosteric site. This NAM mechanism of action may be responsible for the observed α3β4-preference of 1, but opens up additional SAR studies to investigate the α7-preference seen in 20, 22, 23, and 25. Additionally, the >50% [³H]-epibatidine displacement of 4 and 24 indicates that some aristoquinoline scaffold compounds do bind to the orthosteric site, yet still display this subtype selectivity, which also warrants further study.

Because of the challenges associated with developing subtype-selective ligands that target the highly conserved orthosteric pocket, several classes of nAChR allosteric modulators have been developed.³² As exemplified by the highly α7-selective positive allosteric modulator (PAM) PNU-120,596, developed for the treatment of Alzheimer’s disease and schizophrenia, this approach of targeting allosteric sites can lead to small molecules with complete subtype-selectivity.³⁵ However, the development of α3β4-selective NAMs has thus far proven more challenging; previously reported α3β4 NAMs such as COB-3, IB2, and UCI-30002 are equally effective at α4β2. As such, 1 and the other Aristotelia alkaloids appear to represent a unique opportunity to develop α3β4-selective NAMs, providing yet another example of a natural product scaffold serving as a valuable chemical tool to probe receptor pharmacology. Moreover, while the ligand-based design approach employed in this study yielded compounds that maintain and improve potency compared to 1, identifying their binding site will likely lead to further improvements in potency and subtype-selectivity. In this regard, the SAR revealed in this study — namely, the use of fluorine substitutions to improve potency and the importance of the B-ring for selectivity — will provide useful insight to inform computational studies to identify putative allosteric binding sites and allow for future structure-based drug design.

The effectiveness of 1 in reducing cocaine-seeking behavior adds to the growing body of literature demonstrating that inhibition of the α3β4 nAChR represents a promising approach to treating CUD. Interestingly, the effects of 1 appear to be specific for relapse, as it only reduces drug-seeking behavior in the cocaine-prime reinstatement paradigm without reducing cocaine self-administration. Although drug cessation is an important translational focus for developing CUD treatments, relapse rates of stimulant use disorders, which include CUD and amphetamine use disorder, are among the highest for any SUDs.³ Almost 75% of patients with a stimulant use disorder will relapse in two to five years.^36,37 Furthermore, the risk of overdose and overdose death is highest at the point of relapse when patients’ previous tolerance has diminished, thereby making them more sensitive to a low dose of stimulant.³⁸ In the past decade, cocaine-associated overdose deaths have nearly tripled.² Therefore, the effectiveness of 1 in preventing drug-seeking behavior in a model of cocaine relapse suggests there is great clinical utility as a novel pharmacotherapy for CUD. Furthermore, α3β4 inhibitors have proven effective in animal models for nearly all SUDs, implying targeting the α3β4 nAChR may be a promising approach to treating polysubstance use disorders.⁴ With the “fourth wave” of the opioid epidemic witnessing a significant rise in the number of deaths caused by both stimulants and opioids, the development of drugs that can treat multiple co-morbid SUDs could prove to be a particularly effective solution.³⁹

Experimental Section

General.

All reactions were performed within a nitrogen atmosphere in oven-dried round bottom flasks. Solvents for anhydrous reactions were degassed with argon and passed through two drying columns using an Inert Solvent Purification System. Reagents were purchased from commercially available sources and used without further purification. Reactions were monitored using Macherey-Nagel silica gel 60 F254 TLC plates and visualized under a UV Lamp (254 nm) and I₂ chamber. Compounds were purified via column chromatography using silica gel from Macherey-Nagel of 40–63 μm mesh size. Bruker 400 MHz, Bruker 400 MHz HD, and Bruker 600 MHz nuclear magnetic resonance (NMR) spectrometers were used to record ¹H and ¹³C NMR spectra with reference to the residual solvent peaks (CHCl₃: ¹H δ = 7.26, ¹³C δ = 77.16 ppm). High-resolution mass spectrometry was conducted on a Shimadzu LCMS-IT-TOF and observed values are within 5.0 ppm of calculated exact masses of the indicated ions. All analogues were converted to hydrochloride salts before biological evaluation by the careful addition of etheral HCl to a solution of the freebase. The purity of each HCl salt was determined using a high-performance liquid chromatography (HPLC) performed on an Agilent 1260 Infinity II fitted with a DAD detector and Phenomenox Luna Omega PS-C18 column (100 × 4.6 mm). A gradient of acetonitrile/water (20–45%) with 0.1% formic acid with a flow rate of 1 mL/min was used. All analogues tested for biological evaluation were determined to be >95% by HPLC.

General Procedure A: Aza-Prins Synthesis of Analogues.

In an oven-dried round bottom flask under nitrogen containing 3 Å molecular sieves, 2 (1.0 equiv.) was dissolved in 5 mL of anhydrous CH₂Cl₂ at room temperature. The corresponding aldehyde (1.1 equiv.) was added in a single portion at room temperature. After stirring for 16 h, trifluoroacetic acid (2 equiv.) was added dropwise and the reaction was allowed to stir for 14 h. The reaction was quenched by the addition of aqueous 20% NaOH until basic. The aqueous layer was extracted with CH₂Cl₂ (3 × 30 mL) and the combined organic phases were dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting crude residue was purified via silica gel chromatography to afford the free base of the corresponding analogue. To convert each analogue into its corresponding HCl salt, the free base was dissolved in Et₂O and an ethereal HCl solution was added dropwise until no additional precipitation was observed. The Et₂O and excess HCl solution were removed in vacuo to yield a white solid.

4-((1R,2R,5R)-4,4,8-trimethyl-3-azabicyclo[3.3.1]non-7-en-2-yl)isoquinoline (3).

Prepared from 2 (68.7 mg, 0.448 mmol) and isoquinoline-4-carbaldehyde (74 mg, 0.471 mmol) according to General Procedure A. The crude product was purified by silica gel chromatography (25–100% EtOAc/Hexanes) to afford 10.5 mg of 3 (8% yield). ¹H NMR (400 MHz, CDCl₃) δ 9.14 (s, 1H), 8.29 (s, 1H), 8.07 (dd, J = 8.7, 1.1 Hz, 1H), 7.98 (dt, J = 8.2, 1.0 Hz, 1H), 7.76 (ddd, J = 8.4, 6.9, 1.4 Hz, 1H), 7.61 (ddd, J = 8.0, 6.8, 1.0 Hz, 1H), 5.61 (ddq, J = 4.3, 2.9, 1.4 Hz, 1H), 4.95 (d, J = 2.5 Hz, 1H), 2.55 (q, J = 3.0 Hz, 1H), 2.48 (dtd, J = 12.4, 3.1, 1.2 Hz, 1H), 2.42 – 2.31 (m, 1H), 2.20 – 2.08 (m 1H), 1.79 (dt, J = 12.5, 3.2 Hz, 1H), 1.61 (dt, J = 6.6, 3.3 Hz, 1H), 1.41 (s, 3H), 1.29 (s, 3H), 0.69 (s, 3H).¹³C NMR (101 MHz, CDCl₃) δ 151.74, 139.63, 133.48, 132.94, 132.06, 130.38, 128.78, 128.18, 126.81, 125.07, 122.44, 54.47, 52.67, 40.52, 34.76, 30.41, 29.44, 27.91, 25.69, 24.28. HRMS calculated for C₂₀H₂₅N₂: [M + H]⁺: 293.2020 (found); 293.2018 (calcd).

5-((1R,2R,5R)-4,4,8-trimethyl-3-azabicyclo[3.3.1]non-7-en-2-yl)quinoline (4).

Prepared from 2 (86.1 mg, 0.56 mmol) and 5-quinoline carboxaldehyde (92.7 mg, 0.59 mmol) according to General Procedure A. The crude product was purified by silica gel chromatography (25–100% EtOAc/Hexanes) to afford 11.5 mg of 4 (7% yield).¹H NMR (400 MHz, CDCl₃) δ 8.93 (dd, J = 4.2, 1.6 Hz, 1H), 8.45 (dt, J = 8.7, 1.3 Hz, 1H), 7.98 (dd, J = 8.5 Hz, 1H), 7.63 (t, J = 7.3 Hz, 1H), 7.46 (dd, J = 8.7, 4.2 Hz, 1H), 7.33 (dd, J = 7.4, 1.1 Hz, 1H), 5.57 (ddq, J = 3.0, 3.0, 1.6 Hz, 1H), 4.95 (d, J = 2.2 Hz, 1H), 2.50 – 2.44 (m, 2H), 2.35 (d, J = 19.9 Hz, 1H), 2.22 – 2.08 (m, 1H), 1.82 – 1.73 (m, 1H), 1.61 (dd, J = 6.4, 3.4 Hz, 1H), 1.41 (s, 3H), 1.29 (s, 3H), 0.64 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 149.95, 148.79, 139.93, 133.22, 131.74, 129.22, 128.52, 125.75, 124.71, 122.90, 120.74, 54.48, 53.31, 41.24, 34.77, 30.45, 29.49, 27.91, 25.82, 24.11. HRMS calculated for C₂₀H₂₅N₂: [M + H]⁺: 293.2020 (found); 293.2018 (calcd).

5-((1R,2R,5R)-4,4,8-trimethyl-3-azabicyclo[3.3.1]non-7-en-2-yl)isoquinoline (5).

Prepared from 2 (67.7 mg, 0.44 mmol) and isoquinoline-5-carbaldehyde (72.9 mg, 0.46 mmol) according to General Procedure A. The crude product was purified by silica gel chromatography (25–100% EtOAc/Hexanes) to afford 6.4 mg of 5 (5% yield). ¹H NMR (400 MHz, CDCl₃) δ 9.24 (s, 1H), 8.57 (d, J = 6.0 Hz, 1H), 7.85 (d, J = 6.1 Hz, 1H), 7.83 (dd, J = 7.8, 1.5 Hz, 1H), 7.52 (t, J = 7.5 Hz, 1H), 7.48 (dt, J = 7.1, 1.3 Hz, 1H), 5.56 (ddt, J = 4.3, 2.9, 1.5 Hz, 1H), 4.92 (d, J = 2.5 Hz, 1H), 2.54 (q, J = 3.0 Hz, 1H), 2.47 (dd, J = 12.5, 1.2 Hz, 1H), 2.34 (dd, J = 19.2, 1.6 Hz, 1H), 2.20 – 2.06 (m, 1H), 1.77 (dt, J = 12.5, 3.2 Hz, 1H), 1.60 (dt, J = 6.5, 3.3 Hz, 1H), 1.40 (s, 3H), 1.28 (s, 3H), 0.61 (s, 3H).¹³C NMR (101 MHz, CDCl₃) δ 153.68, 143.13, 138.85, 133.44, 133.08, 128.94, 127.02, 126.62, 126.50, 124.81, 116.44, 54.46, 53.26, 40.64, 34.71, 30.41, 29.40, 27.88, 25.78, 24.06. HRMS calculated for C₂₀H₂₅N₂: [M + H]⁺: 293.2021 (found); 293.2018 (calcd).

8-((1R,2R,5R)-4,4,8-trimethyl-3-azabicyclo[3.3.1]non-7-en-2-yl)isoquinoline (6).

Prepared from 2 (52.9 mg, 0.345 mmol) and 7 quinoline carboxaldehyde (57.0 mg, 0.363 mmol) according to General Procedure A. The crude product was purified by silica gel chromatography (25–100% EtOAc/Hexanes) to afford 21.2 mg of 6 (21% yield). ¹H NMR (400 MHz, CDCl₃) δ 9.60 (s, 1H), 8.54 (d, J = 5.7 Hz, 1H), 7.68 (d, J = 7.3 Hz, 1H), 7.66 (d, J = 6.8 Hz, 1H), 7.59 (t, J = 7.3 Hz, 1H), 7.38 (dt, J = 7.2, 1.1 Hz, 1H), 5.58 (tt, J = 2.9, 1.5 Hz, 1H), 5.13 (d, J = 2.5 Hz, 1H), 2.62 (q, J = 3.0 Hz, 1H), 2.51 (dtd, J = 12.5, 3.1, 1.3 Hz, 1H), 2.35 (d, J = 19.0 Hz, 1H), 2.21–2.08 (m, 1H), 1.78 (dt, J = 11.7, 2.8 Hz, 1H), 1.61 (dt, J = 6.8, 3.3 Hz, 1H), 1.42 (s, 3H), 1.29 (s, 3H), 0.64 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 148.50, 142.69, 140.86, 136.40, 133.10, 130.18, 125.61, 124.86, 123.65, 121.51, 54.55, 53.00, 41.82, 34.73, 30.43, 29.45, 27.92, 25.80, 24.09. HRMS calculated for C₂₀H₂₅N₂: [M + H]⁺: 293.2020 (found); 293.2018 (calcd).

2-((1R,2R,5R)-4,4,8-trimethyl-3-azabicyclo[3.3.1]non-7-en-2-yl)quinoline (7).

Prepared from 2 (74.3 mg, 0.48 mmol) and 2-quinoline carboxaldehyde (80.0 mg, 0.51 mmol) according to General Procedure A. The crude product was purified by silica gel chromatography (25–100% EtOAc/Hexanes) to afford 40.7 mg of 7 (29% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.06 (d, J = 7.1 Hz, 1H), 8.04 (d, J = 6.3 Hz, 1H),7.74 (dd, J = 8.1, 1.4 Hz, 1H), 7.64 (ddd, J = 8.5, 6.8, 1.5 Hz, 1H), 7.46 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 7.37 (d, J = 8.5 Hz, 1H), 5.53 (ddt, J = 4.4, 2.9, 1.5 Hz, 1H), 4.54 (d, J = 2.8 Hz, 1H), 2.58 (q, J = 3.0 Hz, 1H), 2.41 (dt, J = 12.4, 1.3 Hz, 1H), 2.37 – 2.27 (m, 1H), 2.18 – 2.04 (m, 1H), 1.77 (dt, J = 12.4, 3.3 Hz, 1H), 1.55 (dt, J = 6.5, 3.3 Hz, 1H), 1.34 (s, 3H), 1.29 (s, 3H), 0.63 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 162.24, 147.66, 135.86, 132.75, 129.74, 129.19, 127.52, 127.17, 125.93, 124.73, 119.96, 77.52, 77.20, 76.88, 59.61, 53.45, 41.23, 34.36, 30.05, 29.61, 27.81, 26.23, 23.78. HRMS calculated for C₂₀H₂₅N₂: [M + H]⁺: 293.2019 (found); 293.2018 (calcd).

(1R,4R,5R)-2,2,6-trimethyl-4-(pyridin-4-yl)-3-azabicyclo[3.3.1]non-6-ene (8).

Prepared from 2 (62.4 mg, 0.41 mmol) and 4-pyridine carboxaldehyde (45.8 mg, 0.43 mmol) according to General Procedure A. The crude product was purified by silica gel chromatography (89:1:10 CH₂Cl₂/MeOH/NH₄OH) to afford 22.9 mg of 8 (23% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.47 (d, J = 5.1 Hz, 2H), 7.18 (d, J = 5.9 Hz, 2H), 5.54 (ddd, J = 4.3, 3.0, 1.6 Hz, 1H), 4.22 (d, J = 2.7 Hz, 1H), 2.37 (q, J = 3.1 Hz, 1H), 2.33–2.20 (m, 2H), 2.15–2.02 (m, 1H), 1.72 (dt, J = 12.7, 3.2 Hz, 1H), 1.51 (dt, J = 6.6, 3.3 Hz, 1H), 1.27 (s, 3H), 1.21 (s, 3H), 0.88 (s, 3H).¹³C NMR (101 MHz, CDCl₃) δ 152.44, 149.60, 132.50, 125.23, 121.64, 56.78, 54.34, 41.55, 34.45, 30.12, 29.46, 27.67, 25.49, 24.33. HRMS calculated for C₁₆H₂₃N₂: [M + H]⁺: 243.1859 (found); 243.1861 (calcd).

(1R,4R,5R)-2,2,6-trimethyl-4-(pyridin-3-yl)-3-azabicyclo[3.3.1]non-6-ene (9).

Prepared from 2 (60.0 mg, 0.39 mmol) and 3-pyridine carboxaldehyde (44.03 mg, 0.41 mmol) according to General Procedure A. The crude product was purified by silica gel chromatography (89:1:10 CH₂Cl₂/MeOH/NH₄OH) to afford 27.4 mg of 9 (29% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.55 (s, 1H), 8.45 (d, J = 4.8 Hz, 1H), 7.55 (dt, J = 7.9, 2.0 Hz, 1H), 7.20 (dd, J = 7.9, 4.7 Hz, 1H), 5.59 (s, 1H), 4.32 (d, J = 2.4 Hz, 1H), 2.36 (d, J = 3.2 Hz, 1H), 2.35 – 2.26 (m, 3H), 2.12 (dt, J = 19.1, 2.9 Hz, 1H), 1.75 (dt, J = 12.2, 2.7 Hz, 1H), 1.54 (dt, J = 6.8, 3.2 Hz, 1H), 1.32 (s, 3H), 1.24 (s, 3H), 0.92 (s, 3H).¹³C NMR (101 MHz, CDCl₃) δ 148.50, 147.97, 138.84, 133.54, 132.53, 125.32, 123.18, 55.70, 54.39, 41.86, 34.37, 30.10, 29.35, 27.76, 25.55, 24.53. HRMS calculated for C₁₆H₂₃N₂: [M + H]⁺: 243.1859 (found); 243.1861 (calcd).

(1R,4R,5R)-2,2,6-trimethyl-4-(pyridin-2-yl)-3-azabicyclo[3.3.1]non-6-ene (10).

Prepared from 2 (60.0 mg, 0.39 mmol) and 2-pyridine carboxaldehyde (44.03 mg, 0.41 mmol) according to General Procedure A. The crude product was purified by silica gel chromatography (89:1:10 CH₂Cl₂/MeOH/NH₄OH) to afford 20.8 mg of 10 (22% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.50 (ddd, J = 4.9, 1.8, 0.9 Hz, 1H), 7.58 (td, J = 7.7, 1.8 Hz, 1H), 7.22 (d, J = 7.9 Hz, 1H), 7.10 (ddd, J = 7.5, 4.9, 1.2 Hz, 1H), 5.54 (tt, J = 3.0, 1.5 Hz, 1H), 4.41 (d, J = 2.9 Hz, 1H), 2.47 (q, J = 3.1 Hz, 1H), 2.39–2.31 (m, 1H), 2.27 (t, J = 3.0 Hz, 1H), 2.11 (ddt, J = 18.9, 5.9, 3.0 Hz, 1H), 1.74 (dt, J = 12.6, 3.3 Hz, 1H), 1.54 (dt, J = 6.7, 3.3 Hz, 1H), 1.35 (s, 3H), 1.28 (s, 3H), 0.73 (s, 3H).¹³C NMR (101 MHz, CDCl₃) δ 148.70, 136.13, 132.57, 124.62, 121.85, 121.27, 58.43, 54.20, 40.79, 34.19, 29.44, 28.93, 27.48, 25.46, 23.48.HRMS calculated for C₁₆H₂₃N₂: [M + H]⁺: 243.1858 (found); 243.1861 (calcd).

(1R,4R,5R)-4-(6-chloropyridin-3-yl)-2,2,6-trimethyl-3-azabicyclo[3.3.1]non-6-ene (11).

Prepared from 2 (50.0 mg, 0.33 mmol) and 2-chloropyridine-2-carboxaldehyde (48.1 mg, 0.34 mmol) according to General Procedure A. The crude product was purified by silica gel chromatography (89:1:10 CH₂Cl₂/MeOH/NH₄OH) to afford 22.8 mg of 11 (25% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.30 (d, J = 2.5 Hz, 1H), 7.52 (dd, J = 8.3, 2.5 Hz, 1H), 7.23 (d, J = 8.2 Hz, 1H), 5.58 (ddd, J = 4.4, 3.2, 1.7 Hz, 1H), 4.27 (d, J = 2.5 Hz, 1H), 2.36 – 2.22 (m, 3H), 2.10 (dt, J = 19.1, 2.9 Hz, 1H), 1.73 (dt, J = 13.2, 3.8 Hz, 1H), 1.52 (dd, J = 6.4, 3.6 Hz, 1H), 1.28 (s, 3H), 1.20 (s, 3H), 0.95 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 149.57, 148.28, 138.32, 136.68, 132.22, 125.62, 123.70, 55.24, 54.22, 41.84, 34.31, 30.20, 29.35, 27.79, 25.63, 24.74. HRMS calculated for C₁₆H₂₂N₂Cl: [M + H]⁺: 277.1471 (found); 277.1472 (calcd).

(1R,4R,5R)-4-(1H-imidazol-2-yl)-2,2,6-trimethyl-3-azabicyclo[3.3.1]non-6-ene (12).

Prepared from 2 (150 mg, 0.978 mmol) 1H-imidazole-2-carbaldehyde (103 mg, 1.08 mmol) according to General Procedure A. The crude product was purified by silica gel chromatography (1–20% MeOH/CH₂Cl₂) to afford 22.6 mg of 12 (10% yield). ¹H NMR (400 MHz, CDCl₃) δ 6.90 (s, 2H), 5.54 (s, 1H), 4.41 (d, J = 2.9 Hz, 1H), 2.65 (q, J = 3.1 Hz, 1H), 2.30 – 2.20 (m, 2H), 2.17 – 2.07 (m, 1H), 1.72 (dt, J = 12.7, 3.2 Hz, 1H), 1.52 (dt, 1H), 1.29 (s, 3H), 1.26 (d, J = 4.9 Hz, 1H), 1.18 (s, 3H), 0.97 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 149.77, 133.57, 124.87, 54.40, 53.20, 39.80, 34.79, 30.08, 28.44, 27.92, 25.81, 23.03. HRMS calculated for C₁₄H₂₂N₃: [M + H]⁺: 232.1815 (found); 232.1814 (calcd).

(1R,4R,5R)-4-(benzofuran-2-yl)-2,2,6-trimethyl-3-azabicyclo[3.3.1]non-6-ene (13).

Prepared from 2 (50 mg, 0.326 mmol) and 2-benzofuran carboxaldehyde (0.043 mL, 1.21 mmol) according to General Procedure A. The crude product was purified by silica gel chromatography (1% MeOH/CH₂Cl₂) to afford 3.7 mg of 13 (4% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.46 – 7.42 (m, 2H), 7.21 – 7.16 (m, 2H), 6.45 (dt, J = 2.1, 1.1 Hz, 1H), 5.57 (bs, 1H), 4.40 (d, J = 2.5 Hz, 1H), 2.65 (q, J = 2.8 Hz, 1H), 2.33 – 2.22 (m, 2H), 2.19 – 2.04 (m, 1H), 1.80 – 1.70 (m, 1H), 1.56 – 1.48 (m, 1H), 1.31 (s, 3H), 1.22 (s, 3H), 1.01 (s, 3H).¹³C NMR (151 MHz, CDCl₃) δ 160.02, 154.34, 133.21, 128.72, 124.69, 123.51, 122.66, 120.71, 111.05, 101.35, 54.00, 53.18, 39.20, 34.64, 30.02, 28.54, 27.78, 25.78, 23.40. HRMS calculated for C₁₉H₂₄NO: [M + H]⁺: 282.1864 (found); 282.1858 (calcd).

(1R,4R,5R)-2,2,6-trimethyl-4-(4-nitrophenyl)-3-azabicyclo[3.3.1]non-6-ene (14).

Prepared from 2 (50 mg, 0.326 mmol) and 4-nitro benzaldehyde (54.1 mg, 0.358 mmol) according to General Procedure A. The crude product was purified by silica gel chromatography (1% MeOH/CH₂Cl₂) to afford 48.5 mg of 14 (52% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.14 (d, J = 8.8 Hz, 2H), 7.42 (d, J = 8.6 Hz, 2H), 5.56 (s, 1H), 4.35 (d, J = 2.7 Hz, 1H), 2.40 (q, J = 3.1 Hz, 1H), 2.36 – 2.29 (m, 2H), 2.17 – 2.05 (m, 1H), 1.75 (dt, J = 12.6, 3.2 Hz, 1H), 1.53 (dt, J = 6.6, 3.4 Hz, 1H), 1.29 (s, 3H), 1.23 (s, 3H), 0.85 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 151.69, 146.72, 132.40, 127.13, 125.43, 123.45, 57.44, 54.27, 42.10, 34.32, 30.20, 29.54, 27.75, 25.61, 24.44. HRMS calculated for C₁₇H₂₃N₂O₂: [M + H]⁺: 287.1759 (found); 287.1760 (calcd).

(1R,4R,5R)-2,2,6-trimethyl-4-(3-nitrophenyl)-3-azabicyclo[3.3.1]non-6-ene (15).

Prepared from 2 (50 mg, 0.326 mmol) 3-nitro benzaldehyde (54.1 mg, 0.358 mmol) according to General Procedure A. The crude product was purified by silica gel chromatography (1% MeOH/CH₂Cl₂) to afford 44.8 mg of 15 (48% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.12 (s, 1H), 8.06 (dd, J = 8.1, 1.0 Hz, 1H), 7.60 (dt, J = 7.7, 0.9 Hz, 1H), 7.44 (t, J = 7.7 Hz, 1H), 5.57 (ddq, J = 4.5, 3.0, 1.5 Hz, 1H), 4.35 (d, J = 2.7 Hz, 1H), 2.41 (q, J = 3.0 Hz, 1H), 2.33 (dtd, J = 12.7, 3.1, 1.3 Hz, 1H), 2.29 – 2.25 (m, 1H), 2.17 – 2.05 (m, 1H), 1.75 (dt, J = 12.6, 3.2 Hz, 1H), 1.53 (dt, J = 6.5, 3.2 Hz, 1H), 1.29 (s, 3H), 1.23 (s, 3H), 0.85 (dt, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 148.31, 146.25, 132.68, 132.21, 129.04, 125.55, 121.67, 121.21, 57.06, 54.28, 41.91, 34.29, 30.22, 29.48, 27.77, 25.65, 24.43. HRMS calculated for C₁₇H₂₃N₂O₂: [M + H]⁺: 287.1760 (found); 287.1760 (calcd).

(1R,4R,5R)-2,2,6-trimethyl-4-(4-(trifluoromethyl)phenyl)-3-azabicyclo[3.3.1]non-6-ene (16).

Prepared from 2 (20 mg, 0.130 mmol) 4-trifluoromethyl benzaldehyde (0.020 mL, 0.143mmol) according to General Procedure A. The crude product was purified by silica gel chromatography (1–3% MeOH/CH₂Cl₂) to afford 13.3 mg of 16 (33% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.53 (d, J = 8.1 Hz, 2H), 7.43 (d, J = 8.0 Hz, 2H), 5.59 (s, 1H), 4.36 (d, J = 2.7 Hz, 1H), 2.38 – 2.33 (m, 3H), 2.20 – 2.08 (m, 1H), 1.75 (dt, J = 12.6, 3.2 Hz, 1H), 1.57 (dd, J = 6.7, 3.6 Hz, 1H), 1.35 (s, 3H), 1.31 (s, 3H), 0.89 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 132.55, 129.11 (q, J = 32.7 Hz), 126.82, 126.71, 125.44, 125.21 (q, J = 3.8 Hz), 124.41 (q, J = 271.7 Hz), 57.51, 41.79, 34.51, 29.89, 29.68, 29.30, 27.67, 25.24, 24.44. HRMS calculated for C₁₈H₂₃NF₃: [M + H]⁺: 310.1779 (found); 310.1783 (calcd).

(R,E)-N,N-dimethyl-4-(((2-(4-methylcyclohex-3-en-1-yl)propan-2-yl)imino)methyl)aniline (19b).

Prepared from 2 (50 mg, 0.326 mmol) and 4-(dimethylamine) benzaldehyde (53.4 mg, 0.358 mmol) according to General Procedure A. The crude product was purified by silica gel chromatography (1–5% MeOH/CH₂Cl₂) to afford 13.0 mg of 19b (14% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.08 (s, 1H), 7.68 (s, 1H), 6.71 (d, J = 8.6 Hz, 2H), 5.17 (s, 1H), 3.04 (s, 6H), 2.17 – 2.04 (m, 2H), 2.04 – 1.91 (m, 1H), 1.25 (s, 4H), 0.94 (s, 2H), 0.81 (s, 4H). HRMS calculated for C₁₉H₂₉N₂: [M + H]⁺: 285.2327 (found); 285.2331 (calcd).

(1R,4R,5R)-4-(4-isopropylphenyl)-2,2,6-trimethyl-3-azabicyclo[3.3.1]non-6-ene (20).

Prepared from 2 (100 mg, 0.652 mmol) 4-cuminaldehyde (0.108 mL, 0.717 mmol) according to General Procedure A. The crude product was purified by silica gel chromatography (1% MeOH/CH₂Cl₂) to afford 72.1 mg of 20 (39% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.12 (s, 4H), 5.53 (bs, 1H), 4.24 (d, J = 2.5 Hz, 1H), 2.85 (q, J = 6.9 Hz, 1H), 2.36 – 2.24 (m, 3H), 2.16 – 2.02 (m, 1H), 1.70 (dt, J = 13.3, 5.7 Hz, 1H), 1.51 (dt, J = 18.1, 11.9 Hz, 1H), 1.30 (s, 3H), 1.23 (s, 3H), 1.20 (d, J = 6.9 Hz, 6H), 0.90 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 147.23, 140.72, 133.80, 126.18, 126.03, 124.19, 57.16, 42.04, 34.59, 33.86, 30.19, 29.53, 27.85, 25.69, 24.44, 24.30, 24.22. HRMS calculated for C₂₀H₃₀N : [M + H]⁺: 284.2377 (found); 284.2378 (calcd).

(1R,4R,5R)-4-(4-(tert-butyl)phenyl)-2,2,6-trimethyl-3-azabicyclo[3.3.1]non-6-ene (21).

Prepared from 2 (100 mg, 0.652 mmol) and 4-tert-butyl benzaldehyde (116 mg, 0.717 mmol) according to General Procedure A. The crude product was purified by silica gel chromatography (1% MeOH/CH₂Cl₂) to afford 46.6 mg of 21 (24% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.31 – 7.26 (m, 2H), 7.13 (d, J = 8.1 Hz, 2H), 5.54 (s, 1H), 4.24 (d, J = 2.9 Hz, 1H), 2.38 – 2.25 (m, 2H), 2.15 – 2.06 (m, 1H), 1.85 (td, J = 5.9, 3.3 Hz, 1H), 1.70 (dt, J = 12.2, 3.1 Hz, 1H), 1.51 (d, J = 6.5 Hz, 1H), 1.31 (s, 3H), 1.29 – 1.25 (m, 9H), 1.24 (s, 3H), 0.91 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 149.60, 133.72, 125.78, 125.06, 124.35, 123.99, 57.13, 41.88, 34.62, 31.57, 29.89, 29.40, 27.80, 25.47, 24.45. HRMS calculated for C₂₁H₃₂N: [M + H]⁺: 298.2538 (found); 298.2535 (calcd).

(1R,4R,5R)-4-(3-fluoro-4-(trifluoromethyl)phenyl)-2,2,6-trimethyl-3-azabicyclo[3.3.1]non-6-ene (22).

Prepared from 2 (20 mg, 0.130 mmol) 3-fluoro-4-trifluoromethyl benzaldehyde (0.036 mL, 0.358 mmol) according to General Procedure A. The crude product was purified by silica gel chromatography (1% MeOH/CH₂Cl₂) to afford 26.4 mg of 22 (62% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.50 (t, J = 7.7 Hz, 1H), 7.14 (s, 1H), 7.12 (s, 1H), 5.56 (s, 1H), 4.28 (d, J = 2.7 Hz, 1H), 2.38 (q, J = 3.0 Hz, 1H), 2.33 (dt, J = 4.2, 2.1 Hz, 1H), 2.29 (dt, J = 3.1, 1.2 Hz, 1H), 2.26 (s, 1H), 2.17 – 2.04 (m, 1H), 1.74 (dt, J = 12.6, 3.2 Hz, 1H), 1.28 (s, 3H), 1.21 (s, 3H), 0.91 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 159.91 (d, J = 255.5 Hz), 151.50 (d, J = 7.0 Hz), 132.44, 126.78 (q, J = 5.3 Hz), 125.34, 122.92 (q, J = 271.2 Hz), 121.98 (d, J = 3.6 Hz), 116.34 (dd, J = 32.8, 12.5 Hz), 114.74 (d, J = 21.1 Hz), 57.05, 54.23, 41.89, 34.37, 30.22, 29.52, 27.78, 25.66, 24.46. HRMS calculated for C₁₈H₂₂NF₄: [M + H]⁺: 328.1689 (found); 328.1688 (calcd).

(1R,4R,5R)-4-(4-fluoro-3-(trifluoromethyl)phenyl)-2,2,6-trimethyl-3-azabicyclo[3.3.1]non-6-ene (23).

Prepared from 2 (156 mg, 1.02 mmol) and 3-trifluoromethyl-4-fluoro benzaldehyde (0.112 mL, 1.12 mmol) according to General Procedure A. The crude product was purified by silica gel chromatography (1–2% MeOH/CH₂Cl₂) to afford 173.6 mg of 23 (52% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.46 (dd, J = 6.9, 2.3 Hz, 1H), 7.42 (dd, J = 7.4, 2.2 Hz, 1H), 7.09 (t, J = 9.3 Hz, 1H), 5.56 (s, 1H), 4.26 (d, J = 2.6 Hz, 1H), 2.31 – 2.27 (m, 2H), 2.09 (dt, J = 19.0, 2.9 Hz, 1H), 1.72 (dt, J = 12.4, 3.0 Hz, 1H), 1.51 (dt, J = 6.8, 3.3 Hz, 2H), 1.28 (s, 3H), 1.21 (s, 3H), 0.88 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 158.52 (d, J = 252.8 Hz), 140.26 (d, J = 3.6 Hz), 132.39, 131.64 (d, J = 8.1 Hz), 125.32, 124.74 (q, J = 4.8 Hz), 122.91 (q, J = 272.4 Hz), 117.92 (qd, J = 32.6, 12.2 Hz), 116.52 (d, J = 20.3 Hz), 56.68, 54.23, 41.93, 34.32, 30.19, 29.40, 27.76, 25.63, 24.35. HRMS calculated for C₁₈H₂₂NF₄: [M + H]⁺: 328.1688 (found); 328.1688 (calcd).

(1R,4R,5R)-4-(3,4-difluorophenyl)-2,2,6-trimethyl-3-azabicyclo[3.3.1]non-6-ene (24).

Prepared from 2 (50 mg, 0.326 mmol) 3,4-difluoro benzaldehyde (0.039 mL, 0.358 mmol) according to General Procedure A. The crude product was purified by silica gel chromatography (1% MeOH/CH₂Cl₂) to afford 31.6 mg of 24 (35% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.10 – 7.02 (m, 2H), 6.99 – 6.93 (m, 1H), 5.55 (bs, 1H), 4.20 (d, J = 2.5 Hz, 1H), 2.35 – 2.21 (m, 3H), 2.15 – 2.04 (m, 1H), 1.71 (dt, J = 12.3, 3.0 Hz, 1H), 1.51 (dt, J = 6.4, 3.4 Hz, 1H), 1.28 (s, 3H), 1.20 (s, 3H), 0.93 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 150.49 (dd, J = 191.8, 12.5 Hz), 148.86 (dd, J = 191.1, 12.6 Hz), 141.14, 132.80, 124.94, 122.00, 116.82 (d, J = 16.8 Hz), 115.19 (d, J = 17.5 Hz), 56.56, 54.16, 42.00, 34.40, 30.25, 29.43, 27.79, 25.72, 24.41. HRMS calculated for C₁₇H₂₂NF: [M + H]⁺: 278.1720 (found); 278.1720(calcd).

(1R,4R,5R)-4-(3,5-difluorophenyl)-2,2,6-trimethyl-3-azabicyclo[3.3.1]non-6-ene (25).

Prepared from 2 (100 mg, 0.652 mmol) and 3,5-difluoro benzaldehyde (0.38 mL, 0.358 mmol) according to General Procedure A. The crude product was purified by silica gel chromatography (1% MeOH/CH₂Cl₂) to afford 52.4 mg of 25 (58% yield). ¹H NMR (600 MHz, CDCl₃) δ 6.79 (d, J = 8.6 Hz, 2H), 6.62 (td, J = 8.9, 2.4 Hz, 1H), 5.55 (bs, 1H), 4.21 (d, J = 2.7 Hz, 1H), 2.34 (q, J = 3.0 Hz, 1H), 2.31 – 2.23 (m, 2H), 2.13 – 2.05 (m, 1H), 1.72 (dt, J = 12.6, 3.2 Hz, 1H), 1.50 (dt, J = 6.4, 3.2 Hz, 1H), 1.27 (s, 3H), 1.20 (s, 3H), 0.95 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 163.08 (dd, J = 247.5, 13.0 Hz), 148.32 (t, J = 8.6 Hz), 132.67, 125.04, 109.20 (dd, J = 20.4, 4.6 Hz), 101.79 (t, J = 25.4 Hz), 56.97 (t, J = 2.3 Hz), 54.17, 41.93, 34.40, 30.22, 29.49, 27.77, 25.68, 24.34. HRMS calculated for C₁₇H₂₂NF₂: [M + H]⁺: 278.1723 (found); 278.1732 (calcd).

(1R,4R,5R)-4-(2,5-difluorophenyl)-2,2,6-trimethyl-3-azabicyclo[3.3.1]non-6-ene (26).

Prepared from 2 (100 mg, 0.652 mmol) and 2,5-difuloro benzaldehyde (0.038 mL, 0.358 mmol) according to General Procedure A. The crude product was purified by silica gel chromatography (1% MeOH/CH₂Cl₂) to afford 59.7 mg of 26 (33% yield). ¹H NMR (400 MHz, CDCl₃) δ 6.99 – 6.90 (m, 1H), 6.89 – 6.80 (m, 2H), 5.59 (s, 1H), 4.47 (d, J = 1.8 Hz, 1H), 2.45 (q, J = 3.3 Hz, 1H), 2.32 (dt, J = 12.2, 3.1 Hz, 1H), 2.28 – 2.23 (m, 1H), 2.17 – 2.05 (m, 1H), 1.69 (dt, J = 12.7, 3.3 Hz, 1H), 1.52 (dt, J = 6.5, 3.3 Hz, 1H), 1.30 (s, 3H), 1.21 (s, 3H), 0.97 (s, 3H).¹³C NMR (151 MHz, CDCl₃) δ159.08 (d, J = 239.6 Hz), 156.27 (d, J = 240.6 Hz), 133.04 (d, J = 7.1 Hz), 132.93, 125.04, 115.89 (dd, J = 25.4, 8.6 Hz), 113.97 (dd, J = 24.2, 8.4 Hz), 113.81 (dd, J = 25.2, 6.4 Hz), 54.33, 51.53, 39.50, 34.48, 30.29, 29.19, 27.79, 25.61, 24.22. HRMS calculated for C₁₇H₂₂NF₂: [M + H]⁺: 278.1719 (found); 278.1720 (calcd).

(1R,4R,5R)-4-(4-fluoro-3-(trifluoromethyl)phenyl)-2,2,6-trimethyl-3-oxabicyclo[3.3.1]non-6-ene (27).

In an oven-dried round bottom flask under nitrogen, (+)-limonene (1.47 mL, 9.09 mmol, 1.0 equiv.) and BF₃ • Et₂O (0.22 mL, 1.81 mmol, 0.2 equiv.) were dissolved in 10 mL of anhydrous CH₂Cl₂ and cooled to 0°C. 4-fluoro-3-trifluoromethylbenzaldehyde (1.36 mL, 10 mmol, 1.1 equiv.) was added dropwise. After stirring for 5 h, the reaction was concentrated in vacuo and quenched by the addition of NaHCO₃ until basic. The aqueous layer was extracted with EtOAc (3 × 30 mL), then washed with brine (3 × 30 mL). The combined organic phases were dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting crude residue was purified via silica gel chromatography (5% EtOAc/Hexanes) to afford 126 mg of 27 (4% yield). H NMR (400 MHz, CDCl₃) δ 7.55 (dd, J = 6.9, 1.7 Hz, 1H), 7.50 (dd, J = 8.7, 5.3 Hz, 1H), 7.10 (t, J = 9.0 Hz, 1H), 5.48 (bs, 1H), 4.88 (d, 1H), 2.40 (d, J = 6.8 Hz, 1H), 2.36 (dt, J = 12.1, 7.8 Hz, 1H), 2.17 (q, J = 3.1 Hz, 1H), 2.14 – 2.04 (m, 1H), 1.75 (dt, J = 12.5, 3.3 Hz, 1H), 1.61 – 1.55 (m, 1H), 1.39 (s, 3H), 1.34 (s, 3H), 0.86 (d, J = 2.4 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 158.74 (d, J = 254.3 Hz), 139.39 (d, J = 3.8 Hz), 132.07, 131.34 (d, J = 8.3 Hz), 124.62 (q, J = 4.9 Hz), 124.47, 122.94 (q, J = 272.2 Hz), 116.93 (dq, J = 32.4, 12.2 Hz), 116.34 (d, J = 20.6 Hz), 116.28, 75.94, 73.49, 41.50, 33.88, 28.72, 28.24, 27.76, 24.23, 24.07. HRMS calculated for C₁₈H₂₀F₄O: [M + H]⁺: 327.1375 (found); 327.1372 (calcd).

Cell culture.

HEK-293 cells expressing rat α3β4 nicotinic acetylcholine receptors were obtained from Kenneth Kellar (Georgetown University) and were cultured in Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal bovine serum, G-418 (0.6 mg/mL), penicillin (100U/mL), streptomycin (100 μg/mL), and maintained in an atmosphere of 5% CO₂ at 37°C. For fluorescence assays, the cells were seeded into 96-well flat bottom, black wall plates at a density of approximately 50,000 cells per well in 75 μL of media. Cells seeded at this density grew into a confluent monolayer in 24 h. The cell lines were established by co-transfecting HEK-293 cells with a 1:1 ratio of the α3 and β4 genes,⁴⁰ which predominantly results in the (α3)₃(β4)₂ stoichiometry.⁴¹

Ca²⁺ fluorescence assays.

Ca²⁺ uptake was determined using a FLIPR Calcium-6 assay (Molecular Devices). Per the manufacturer’s protocol, the fluorescent dye was reconstituted in 100 mL assay buffer and used in 10 mL aliquots. For dye loading, 75 μL of the dye solution was added to each well, and the plate was incubated at 37°C for 1 h. Plates were then transferred to a FlexStation 3 (Molecular Devices) maintained at 37°C.

Dual agonist/antagonist assay.

For calcium measurements, fluorescence measurements (Ex: 485 nM, Em: 525, Cutoff: 515 nM) were taken for 20 seconds prior to the addition of compounds (50 μL of HBSS + 0.1% DMSO solution at 5X final concentration) using a FlexStation 3 followed by 90 seconds of continuous measurements. For agonist assays, 12 concentrations of test compounds were added to the cells (300 pM – 100 μM, final concentrations). Epibatidine (30 pM – 10 μM) and mecamylamine (150 pM – 50 μM) were used as positive controls for agonists and antagonists, respectively. For subsequent antagonist assays, epibatidine (100 nM, final concentration in HBSS) was added to each well 10 min after the initial agonist assay. Once again, the fluorescence was measured for 20 seconds prior to addition and 90 seconds afterward.

Data reduction.

Ligand response for both agonist and antagonist data was calculated by subtracting the average of the pre-addition fluorescence (F_o) from the maximum response (F_max) for each well, correcting to a DMSO control, and normalizing to the maximal response produced by epibatidine. Each concentration-response point represents a minimum of two technical replicates and three biological replicates. EC₅₀, E_max, IC₅₀, and pIC₅₀ values were calculated from corresponding concentration-response curves fitted using the logistical curve on GraphPad Prism software. Differences in pIC₅₀ values between 1 and analogues were compared by Student’s T-test on GraphPad Prism software.

Patch clamp electrophysiology.

Cell preparation.

Human α3 and β4 nAChR cDNA clones in a pcDNA3.1(−) vector were provided by Ryan Hibbs (UT Southwestern). HEK293T cells at 50–60% confluency on 35 mm culture dishes were lipofected with 3 plasmids containing α4 (0.5 μg/well), β2 (0.5 μg/well) and eGFP (0.2 μg/well) using Xtremegene HP (Sigma-Aldrich) and cells were incubated at 30°C post-transfection. After 48 h, cells were lifted from culture dishes using Accutase (GeminiBio), plated onto 12 mm glass coverslips coated with poly-L-lysine, and returned to the 30°C incubator. After 2–3 h, 10 μM nicotine ditartrate dihydrate (Thermo Fisher) in DMSO was added to the plated cells (resulting [DMSO] was 0.1%). Cells were then incubated at 30°C degrees overnight and patch-clamped the next day.

Patch clamp electrophysiology.

Whole-cell patch clamp electrophysiology was performed on HEK293 cells using an Axopatch 200B patch clamp amplifier (Molecular Devices) connected to a Dendrite digitizer (Sutter Instrument) driven by SutterPatch recording software. GΩ seals were established at 0 mV, and cells were held at −80 mV after patch rupture for the duration of recording except where otherwise specified. Due to the high expression levels of α3β4 channels in the stable cells, the holding potential was set to −60 mV to stabilize the seal and maintain the health of the cell for the duration of the recording. The extracellular bath solution contained (mM): 140 NaCl, 2.4 KCl, 4 CaCl₂, 2 MgCl₂, 10 HEPES, 0.1 D-glucose. The internal solution contained (mM): 140 KCl, 10 TEA-Cl, 10 HEPES, 5 EGTA, 2 MgCl₂. The pH for both solutions was calibrated to 7.3 using HCl, and osmolality was adjusted to 310 and 294 mOsm/L for the extracellular and the internal solution, respectively, by addition of D-mannitol. Thick-wall borosilicate glass with filaments (Sutter Instrument #BF150–86-10) were pulled to a tip resistance between 3–4 MΩ. For competition studies, the bath solution contained either 0, 1 μM, or 10 μM of 1. External solutions containing increasing concentrations of epibatidine (10 nM - 10 mM) were applied by positive perfusion exchange using an Octaflow II system (ALA Scientific). Analysis of patch clamp data was performed using Wavemetrics IgorPro 9 and GraphPad Prism software.

Subtype-selectivity Assays.

Cell culture.

HEK cells stably expressing human nAChR α3β4, α4β2 and α7 were cultured in DMEM (high glucose, no pyruvate or glutamine; Corning 15-013-CM) supplemented with 10% FBS (Corning 35011CV), PenStrep. Cells were routinely passed every 48 to 72 hours when they reached around 70% confluence. For fluorescence assays, the cells were trypsinized, counted, and seeded in black, clear-bottomed 96 well plates at a density of 25,000 cells per well and incubated for 24 hours. After 24 hours, cells were induced with 1 μg/mL tetracycline and incubated overnight. Next day, media was removed from cell plates before adding 25 μL assay buffer (137 mM NaCl, 4 mM KCl, 1 mM MgCl2, 10 mM HEPES, 5 mM Glucose pH7.4 supplemented with 2 mM CaCl₂).

Antagonist Ca²⁺ fluorescence assays.

Ca²⁺ uptake was determined using a FLIPR Calcium-5 assay (Molecular Devices: R8186). Per the manufacturer’s protocol, the fluorescent dye was reconstituted in assay buffer and 10 μL of the dye solution was added to the wells and incubated at 37 ⁰C for 45 minutes. Test compounds ([100, 10, 3, 1, 0.3, 0.1 and 0.01 μM], final concentrations) were added to the wells and incubated at room temperature for 10 minutes. SR 16584, DHβE, and MLA, were used as control antagonists against α3β4, α4β2 and α7 respectively. The plates were then placed in the FLIPR Tetra and fluorescence monitored every 1 second. After 20 seconds, 10 μL of nicotine corresponding to approximate EC₈₀ for each subtype (50 μM, 15 μM and 10 μM nicotine was used for nAChR α3β4, α4β2 and α7, respectively) was added and the fluorescence (Ex: 488 nM, Em: 510–570 nM) monitored for 5 minutes.

Data reduction.

FLIPR reads max (peak) fluorescence and background was normalized to zero. IC₅₀ values of the test compounds, where possible, and reference compounds were determined using GraphPad Prism software.

Competitive and nAChR radioligand binding assays.

Binding assays were performed using tritiated radioligand on membranes isolated from stably transfected cells (CHO, HEK, fibroblast, MDCK) and transiently transfected cells (HEK-T) expressing the indicated receptors as previously described. The detailed experimental protocols are available on the NIMH PDSP website (https://pdsp.unc.edu/pdspweb/content/PDSP%20Protocols%20II%202013-03-28.pdf).³⁴

Animals.

Male Sprague‐Dawley rats (200‐225 g at their arrival), purchased from Charles River Laboratories (Raleigh, NC), were housed two per cage and placed in a room with a reverse 12‐hour light/12‐hour dark cycle (lights off at 7:00 AM) of an AAALAC accredited facility. Self-administration experiments were conducted during the dark phase of the cycle. Animals were acclimated for 7 days with water and chow (5L0D PicoLab Laboratory Rodent Diet) provided ad libitum and handled for 3 days before the experiments were conducted. All experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All methods used were preapproved by the Institutional Animal Care and Use Committee at Florida Atlantic University and the University of Florida.

Pharmacokinetics.

A pharmacokinetic study of aristoquinoline was performed after intraperitoneal administration (10 mg/kg) in male Sprague Dawley rats (250 ± 25 g of body weight). During the pharmacokinetic study, animals were placed individually inside a Basi CulexNxT^® automated blood sampling system (West Lafayette, IN) with ad libitum access to food and water. Blood (80 μL) was collected in heparinized tubes at pre-dose, and 0.083, 0.166, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 12, 18 and 24 h, post-dose. Plasma samples (20 μL) were processed by a simple protein precipitation method using acetonitrile (80 μL) containing internal standard (verapamil; IS, 5 ng/mL). After quenching, samples were vortex mixed, filtered through a multiscreen Solvinert (Millipore, St. Louis, MO) 96-well filter plate (0.45 μm), centrifuged (1500g, 5 min, 4°C), and injected onto Waters Acquity UPLC coupled with Xevo^® TQ-S micro triple quadruple mass spectrometer. Chromatographic separation was achieved in gradient mode using a Waters Acquity UPLC BEH C18 column (1.7 μm, 2.1 × 100 mm) with a VanGuard pre-column of a similar chemistry (Waters Co, Milford, MA). The optimized linear gradient program for quantitation of aristoquinoline was as follows: 0–0.5 min, 5% B; 0.5–2.5 min, 5–95% B; 2.5–3.0 min, 95% B; 3.0–3.1 min, 95–5% B; 3.1–3.5 min, 5% B, where mobile phase A was aqueous ammonium acetate buffer (2.5 mM; pH: 3.5) and mobile phase B was acetonitrile. Calibration and quality control standards were prepared by spiking known concentrations of aristoquinoline in blank plasma. The concentration-time data was subjected to a non-compartmental analysis using Phoenix^® software (Certara USA, Inc., Princeton, NJ).

Cocaine self-administration.

Rats were implanted with intravenous catheters, as we previously described.^42,43 Self‐administration experiments began 1 week after surgery and were conducted in operant conditioning chambers (Med Associates Inc.) equipped with two retractable levers, auditory stimuli, and visual stimuli (cue lights). Infusions occurred by means of syringe pumps (Med Associates Inc.) and liquid swivels (Instech Solomon), connected to plastic tubing protected by a flexible metal sheath for attachment to the external catheter terminus. An infusion pump was activated by responses on one (active) lever and resulted in the delivery of cocaine, whereas responses on the second (inactive) lever were recorded but did not result in any programmed consequences. Rats were trained to self-administer cocaine under a fixed ratio 1 (FR-1) schedule of reinforcement in daily 2-h sessions. Each active lever press resulted in the delivery of one cocaine dose (0.5 mg/kg/0.1 mL infusion). A 20-s timeout (TO) period followed each cocaine infusion. During the TO period, responses on the active lever did not have programmed consequences. This TO period occurred concurrently with the illumination of a cue light that was located above the active lever to signal delivery of the reinforcer. An intermittent tone (7 kHz, 70 dB) was on throughout sessions. The rats were trained to self-administer cocaine under these conditions across 18 sessions. 1 was then tested as described in Figure 4A. Cocaine self-administration data were analyzed by means of Repeated measures ANOVA. After drug testing, responses were extinguished over 16 consecutive 60 min daily sessions. Extinction sessions were identical to cocaine self-administration sessions, except that cocaine was no longer available. After the last extinction session, animals were pretreated with 1 or vehicle according to a Latin Square counterbalanced order 15 min prior to i.p. administration of 10 mg/kg cocaine dose, which occurred 5 min prior to placing the animals in the operant chambers under the same conditions as extinction sessions (cocaine prime-induced reinstatement sessions). Reinstatement data were analyzed by Wilcoxon test.