Piperidine-Based Nocaine/Modafinil Hybrid Ligands as Highly Potent Monoamine Transporter Inhibitors: Efficient Drug Discovery by Rational Lead Hybridization

Abstract

Some piperidine-based nocaine/modafinil hybrid ligands have been designed, synthesized, and found to display an improved potency at all three monoamine transporters and particularly for DAT and/or NET. Some highly active and selective monoamine transporter inhibitors with low nanomolar to subnanomolar potency were identified. Ligands of this type may find important applications as positron emission tomography imaging tools and in the treatment of central nervous system disorders such as depression and sleep apnea.

In the central nervous system (CNS), monoamines such as dopamine (DA), serotonin (5-HT), and norepinephrine (NE) have an important modulatory role in neurotransmission and are intimately involved in a variety of physiological functions and pathological conditions.^1,2 Selective monoamine transporter (DAT, SERT, or NET) inhibitors (e.g., GBR 12909, mazindol, fluoxetine, paroxetine, reboxetine, amoxapine, desipramine, imipramine, bupropion, and nisoxetine) have been developed to treat a number of psychological and neurological disorders such as depression, drug abuse, attention deficit hyperactivity disorder (ADHD), anxiety, mood problems, Parkinson’s disease, Alzheimer’s disease, schizophrenia, bipolar disorder, chronic pain, migraine, epilepsy, multiple sclerosis, stroke, trauma, mania, obesity, and narcolepsy.^3–8 However, important issues regarding the selectivity and mechanisms of action of these drugs remain unresolved. Research with novel ligands that vary in their selectivity profiles and potency at each of these three transporter sites may be of value to unraveling the relevant pharmacological mechanisms and thus aid in the discovery of new medications with fewer side effects.

Previously, we reported on the synthesis and pharmacology of some 3,4-substituted piperidine-based cocaine analogues.⁹ After extensive in vitro studies, together with assays of locomotor activity, and self-administration and drug discrimination studies, the DAT/NET selective ligand (+)-methyl 4β-(4-chlorophenyl)-1-methylpiperidine-3α-carboxylate [(+)-CPCA, 1, nocaine] was chosen for advancement to human clinical studies for the treatment of cocaine addiction^10,11 Nocaine has lower potency and efficacy compared with cocaine in increasing locomotor activity in rodents and produces partial methamphetamine-like discriminative stimulus effects, although it is fully cocaine-like in cocaine-trained animals. Moreover, it shows reduced reinforcing effects in nonhuman primates. Nocaine dose-dependently antagonizes cocaine-induced locomotor activation and potentiates the discriminative stimulus effects of a low dose of cocaine. On the other hand, modafinil (provigil) 2, a non-amphetamine-like wake-promoting agent, has been approved recently for the treatment of narcolepsy. However, modafinil has been linked to side effects that include nausea, infection, nervousness, anxiety, and insomnia. It shows a low but selective affinity to the DAT in canine brain membranes.¹² Modafinil’s mode of action is complex and still uncertain, although studies suggest that it increases wakefulness by activating α-1 noradrenergic transmission¹³ or hypothalamic cells that contain the peptide hypocretin¹⁴ or that it may work by modulating GABAergic tone.¹⁵ Other research suggests that the presynaptic activation of DA transmission is a key pharmacological event in mediating the wake-promoting effects of currently available CNS stimulants and that it is critical for the pharmacological control of wakefulness, while activation of the NE system is critical for rapid eye movement sleep regulation.¹⁶ Experiments using DAT knock-out mice appear to support an important role for the DAT in sleep regulation and thus in the wake-promoting action of modafinil.¹⁷

Since valuable therapies have emerged from compounds exhibiting varying levels of transporter selectivity, we sought to examine the effect of creating hybrid molecules combining structural features of nocaine and modafinil. Specifically, we decided to explore the effect of replacement of the hydrolyzable ester function of nocaine with the same type of sulfur-containing side chain as found in modafinil. This alteration to one of the key pharmacophore elements of nocaine was anticipated to further reduce its reinforcing properties while possibly improving its half-life (Chart 1).

Chart 1.

Rational Design of Nocaine/Modafinil Hybrid Ligands

Our synthetic approach to these hybrid structures starting from arecoline⁹ is outlined in Schemes 1 and 2. The requisite steps are quite straightforward and based in part on the methods devised for the synthesis of nocaine itself. In the course of oxidation of the sulfide intermediates to the corresponding sulfoxides, an additional chiral center is created at the sulfur atom. Typically, two diastereoisomers are generated in a ratio of approximately 1:1, which is readily observable by NMR. Possible separation of the sulfoxides by HPLC was attempted but was unsuccessful. However, in light of the fact that the sulfoxides have substantially lower activity compared with the sulfides (see Table 1) and that the commercial drug modafinil itself is used in racemic form, further efforts to bring about the preferential formation of one stereoisomer over the other by optimizing the oxidation conditions using selective oxidizing agents have not been made.

Scheme 1^a.

Scheme 2^a.

Table 1.

Inhibition at Monoamine Transporters (K_i ± SEM (nM))^a

compd	[3H]-DA uptake	[3H]-5-HT uptake	[3H]-NE uptake
Cocaine	423 ± 147	155 ± 0.4	108 ± 3.5
(+)-CPCAb	233 ± 62	8490 ± 1430	252 ± 43
modafinilc	3800		>10000
4b	497 ± 58	1550 ± 360	198 ± 53
5	376 ± 54	238 ± 49	424 ± 53
6	80 ± 23	208 ± 47	25 ± 6
7	231 ± 93	809 ± 36	53 ± 16
8	16 ± 5	158 ± 5	0.94 ± 0.27
9	12 ± 1	2183 ± 373	42 ± 16
10	50 ± 15	191 ± 57	6 ± 2
11	15 ± 5	469 ± 36	25 ± 6
12	35 ± 11	57 ± 18	3.6 ± 1.5
13	9 ± 3	87 ± 17	29 ± 4
14	68 ± 22	6.7 ± 1.5	4.5 ±1.2
15	32 ± 12	199 ± 14	18 ± 6
16a	159 ± 19	557 ± 150	39 ± 5
16b	85 ± 19	227 ± 7	15 ± 2
16c	13 ± 3	110 ± 45	25 ± 2
16d	116 ± 46	88 ± 22	27 ± 7
16e	1.0 ± 0.2	1.1 ± 0.4	0.8 ± 0.1
16f	83 ± 1	4.5 ± 0.8	0.68 ± 0.25
17a	209 ± 25	5790 ± 1712	178 ± 49
17b	55 ± 8	1795 ± 67	12 ± 1
17c	164 ± 28	2033 ± 573	86 ± 2
17d	2884 ± 654	924 ± 139	1249 ± 19
17e	248 ± 78	473 ± 60	20 ± 3
17f	379 ± 90	825 ± 99	460 ± 11
18	126 ± 15	221 ± 46	32 ± 10
19	1653 ± 56	1079 ± 269	218 ± 14
20	51 ± 16	13 ± 3	0.56 ± 0.09
21	114 ± 32	170 ± 6	10 ± 0.1
22	108 ± 29	775 ± 96	31 ± 3

^aData are the mean ± standard error of at least three experiments as described in ref 6a.

^bData taken from ref 18.

^cData taken from ref 16.

The newly synthesized ligands were evaluated for the ability to inhibit high affinity uptake of DA, 5-HT, and NE using rat synaptosomal nerve endings.^6a From the uptake inhibition data (Table 1), it is surmised that the replacement of the hydrolyzable ester function of nocaine with the sulfur appendage found in modafinil leads in general to a substantial enhancement in DAT/NET inhibitory potency for many of the ligands. In addition, some of the ligands display unique levels of transporter selectivity and potency.

The conversion of the carboxylic ester of nocaine into the intermediate alcohol 4 and iodide 5 improves activity at the SERT without causing any significant alteration in potency at the DAT or NET. However, the introduction of the sulfur appendage into nocaine significantly enhances the inhibitory activity at the DAT and NET. Thus, the key intermediate 6, a hybrid ligand, displays fairly good activity at the NET (K_i = 25 nM) and at the DAT (K_i = 80 nM) while at the same time exhibiting moderate activity at the SERT (K_i = 208 nM). The oxidation of sulfide 6 to sulfoxide 7 leads to a reduced transporter activity. Alcohol 8 exhibits a remarkable potency at the NET (K_i = 0.94 nM) and at the DAT (K_i = 16 nM) as well as a 170-fold and 10-fold selectivity vs the SERT, respectively. The corresponding alcohol 9 containing a sulfoxide function has a lower potency (K_i = 42 nM) at the NET and a 50-fold selectivity vs the SERT, but it displays a higher potency (K_i = 12 nM) at the DAT and a 180-fold selectivity vs the SERT. In comparison to alcohol 8, the methyl ether analogues 10 and 11 exhibit a lower but still fairly good DAT/NET activity. Among the inverse acyl and benzoyl esters, ligands 12, 13, and 15 are good DAT/NET inhibitors while ligand 14 is interestingly a good SERT and NET inhibitor with potencies of 6.7 and 4.5 nM, respectively. Among the amide analogues, ligands 16a–d display a similar and fairly potent NET activity while 16c is also a good DAT inhibitor. Interestingly, ligand 16e exhibits outstanding activity with approximately 1 nM potency at all three monoamine transporters. Ligand 16f is an outstanding SERT and NET inhibitor with potencies of 4.5 and 0.68 nM, respectively. In comparison to the sulfide ligands 16a–f, the corresponding sulfoxide-amide analogues 17a–f generally display a reduced activity except compound 17b, which exhibits a slightly improved potency at the NET (K_i = 12 nM) and the DAT (K_i = 55 nM) and a much better selectivity vs the SERT (K_i = 1795 nM). While oxadiazole analogues are of interest because of their anticipated prolonged action and high metabolic stability, ligands 18 and 19 display only a moderate to low activity at the NET. Interestingly, the N-demethylated ligands 20–22 exhibit a remarkable activity at the NET with K_i down to 0.56 nM (20), a result that likely reflects the change from a tertiary to a secondary amine.

In regard to their druglike properties, many of these ligands have ClogP values (see the Supporting Information) in the range deemed suitable for penetration of the blood–brain barrier, as per Lipinski’s “rule of five”.¹⁹ On the basis of results in hand, we intend to focus on ligands displaying improved NET activity with K_i values less than 1 nM. Specifically, given the fact that ligands 8 and 16f exhibit remarkable NET inhibitory activity(K_i values for NET are 0.94 and 0.68 nM, respectively) while also exhibiting suitable lipophilicity (ClogP in the range of 2–4), we believe that they may be good NET-selective ligands primed for C-11 labeling for advancement to the small-molecule-directed brain imaging and therapy program. To assess the bioavailability and initial cocaine-like behavioral activity of 8, we compared two doses of cocaine and 8 in an open-field locomotor activity study (Figure 1). Ligand 8 was more potent than cocaine and appeared to be somewhat more efficacious as well. Although much more extensive testing is needed to fully characterize the behavioral effects of 8, these differences are consistent with its 17-fold NET vs DAT selectivity and its virtual lack of activity at SERT.²⁰ Further behavioral testing of some of the ligands in this series is currently underway.

Figure 1.

Effect of cocaine and ligand 8 (JZ-III-84) on horizontal locomotor activity. Following a 30 min habituation period, saline (0.9%), cocaine, and ligand 8 were injected ip (1 cm³/kg) in the doses indicated and total locomotor activity was automatically recorded for 90 min as infrared beam breaks in a 16 in. × 16 in. photobeam activity system (San Diego Instruments). Cocaine and ligand 8 significantly increased total activity counts as indicated by one-way analysis of variance (F_4,14 = 28.4, p < 0.001). The asterisk (*) indicates that the individual treatment group is significantly different from saline control (p < 0.05, Tukey’s post hoc test).

In summary, some piperidine-based nocaine/modafinil hybrid ligands have been synthesized and found to display an improved potency at all three monoamine transporters but particularly for DAT and/or NET. Some highly active and selective monoamine transporter inhibitors with low nanomolar to subnanomolar potency were identified including 8 and 20 (NET), 9, 11, 13, 15, 16c, and 17b (DAT/NET), 14 and 16f (SERT/NET), and 16e (DAT/SERT/NET). Because many of these ligands have ClogP values in the range deemed suitable for penetration of the blood–brain barrier, they can be examined for their in vivo actions and radiolabeled as possible positron emission tomography (PET) imaging tools. The potency and selectivity profiles of these hybrid molecules may allow us to gain a better understanding of the relation between transporter activity and biological effect. This knowledge may allow for an improved design of CNS therapeutics.