Copyright 2009 by the American Chemical Society
Volume 52, Number 4
February 26, 2009
PerspectiVe Biomedical Application of Orexin/Hypocretin Receptor Ligands in Neuroscience Christoph Boss,* Catherine Brisbare-Roch, and Francois Jenck Drug DiscoVery and Preclinical Research & DeVelopment, Actelion Pharmaceuticals Ltd., CH-4123 Allschwil, Switzerland ReceiVed October 15, 2008
The Orexin/Hypocretin Neuropeptide and Receptor System Orexin/hypocretin neuropeptides (orexin-A or hypocretin1 and orexin-B or hypocretin2) are peptides discovered and published in 1998 by two independent research groups as the outcome of methodical deorphanization programs focusing on brain orphan G-protein-coupled receptors (GPCRa).1,2 Orexins/hypocretins bind to two receptors (orexin1/OX1 or HCRT1 and orexin2/OX2 or HCRT2 receptors) and are proteolytically derived from a single precursor peptide in a discrete population of neurons of the perifornical area of the lateral hypothalamus. OX1 receptors have preferential affinity for orexin-A, whereas OX2 receptors do not discriminate between both neuropeptides in vitro. OX1 or OX2 activation produces intracellular Ca2+ increases via functional coupling involving a Gq or Go mechanism of transduction.3 This ultimately results in slow membrane depolarization and in neuronal activation found to involve different ionic conductance in various brain regions in the rat, such as potassium conductance in the locus coeruleus4 or calcium current in the tuberomammilary nucleus.5 The orexin system is well conserved across mammalian species. Orexin-A is conserved in rat, mouse, pig, dog, and man and contains two disulfide bridges. Orexin-B in rat and mouse differs by only one amino acid (S18N) from porcine, canine, and human orexin-B; it is a linear, nonlipophilic, less stable peptide than orexin* To whom correspondence should be addressed. Phone: +41 61 565 65 61. Fax: +41 61 565 65 00. E-mail:
[email protected]. a Abbreviations: OX1, orexin1; OX2, orexin2; HCRT1, hypocretin 1; HCRT2, hypocretin 2; GPCR, G-protein-coupled receptor; icv, intracerebroventricular; REM, rapid eye movement; NREM, non-rapid-eye movement; CSF, cerebrospinal fluid; Narp, neuronal activity-regulated pentraxin; CNS, central nervous system; GSK, GlaxoSmithKline; HTS, high throughput screening; CHO, Chinese hamster ovary; FLIPR, fluorometric imaging plate reader; nM, nanomolar; ip, intraperitoneal; BBB, blood-brain barrier; h, human; J&J, Johnson and Johnson; SAR, structure-activity relationship; NPY1, neuropeptide Y1; EEG, electroencephalography; EMG, electromyography; P-gp, P-glycoprotein; ADHD, attention deficit hyperactivity disorder.
A. Both orexins are derived from a single precursor peptide coded on human chromosome 17q21-24, which is syntenic with mouse chromosome 11.6 High structural and functional homology is also reported for rat and human OX1 and OX2 receptors. The in vitro pharmacology of human and rat orthologues of OX1 is very similar.7 The human OX1 receptor is coded on chromosome 1p33 and contains seven exons; the human OX2 receptor is coded on chromosome 6p11-q11 containing seven exons over 108 439 base pairs. In the mouse, splice variants of OX2 are distributed in a tissue-specific manner.8 Neurobiological Functions of the Orexin/Hypocretin System Following the original observations that intracerebroventricular (icv) delivery of orexin-A increased feeding in mice, experimental evidence suggested that a prime role for orexins is the maintenance of alertness, with consequences on functionally linked physiologic processes;9 orexins may thus be important in food intake under particular circumstances, e.g., in response to hypoglycemia and/or in the regulation of circadian food intake.10 Since many homeostatic processes in mammals (e.g., food intake, body temperature, hormone release, cognitive processes) are intimately linked to sleep and arousal, it is likely that orexins link sleep and arousal to these processes. Thus, a crucial role for orexin neurons would be in homeostatically sensing the body’s external and internal environments and regulating states of sleep and wakefulness accordingly, which is beneficial for survival.9 Nerve fibers from orexin neurons are widely distributed in the brain except in the cerebellum, suggesting that orexins indeed exert multiple functions, with a particular role as regulators of behavioral arousal, sleep, and wake states. Hypothalamic neurons have dense projections11 to the basal forebrain, limbic structures, and brainstem regions, in particular those related to waking and rapid eye movement (REM) sleep regulation. Enhanced behavioral activity, increased attention, prolonged latency to the first occurrence of
10.1021/jm801296d CCC: $40.75 2009 American Chemical Society Published on Web 01/20/2009
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REM sleep, maintenance of cortical activation, and activated cell firing in thalamic or reticular regions such as the locus coeruleus have been consistently reported following icv infusion of orexins in the rat.12,13 OX1 receptor mRNA is most abundant in the locus coeruleus and OX2 receptor mRNA in the tuberomamillary nucleus, whereas the dorsal raphe nucleus and the ventral tegmental area contain both OX1 and OX2 message.14 A thorough analysis of the phenotypes expressed by mice following disruption of genes coding for the orexin precursor peptide and/or orexin receptors has led to the conclusion that both orexin receptors are involved in the regulation of sleep and wakefulness.9 Orexin/Hypocretin Deficiency in Narcolepsy Significant insight into the physiology of the orexin system was achieved with the discovery that orexin deficiency contributes to the pathophysiology of narcolepsy, a hypersomnolence syndrome.15-18 Mice genetically engineered to lack the orexin precursor peptide, to lack orexin neurons via an ataxin3 transgene, or to lack orexin receptors exhibit similar behavioral phenotypes that are reminiscent of the human narcoleptic condition. Cardinal symptoms of narcolepsy include sleep attacks, sleep onset REM periods, and cataplexy. Genetic analysis of narcolepsy-cataplexy in dogs suggests that deficits in OX2 are sufficient to induce the syndrome.16 Distinct phenotypes of cataplexy and behavioral arrest were also reported for orexin peptide-null mice and OX2 receptor-null mice;19 the latter mice were less severely affected. The mechanisms underlying the various symptoms of the narcoleptic syndrome may thus be complex. Somnolence and cataplexy might be separable phenomena, and orexin deficit alone might not be sufficient to cause cataplexy. Some narcoleptic patients do not develop cataplexy despite low cerebrospinal fluid (CSF) orexins, and loss of hypothalamic factors other than orexins, such as neuronal activity-regulated pentraxin (Narp), dynorphin, and glutamate likely contributes to the narcolepsy.20 Observations made with single administration of the dual OX1/OX2 receptor antagonist almorexant (127, Figure 4), which dose-dependently elicits somnolence without cataplexy, suggest that transient and reversible blockade of both orexin receptors, in contrast to permanent, life-long deactivation of orexin neural function, is not sufficient to evoke cataplexy.21 In addition, partial orexin receptor blockade may also be sufficient to induce somnolence but not cataplexy. Biomedical Applications of Orexin/Hypocretin Receptor Ligands Manipulation of the orexin system using brain-penetrant orexin receptor antagonists or partial or inverse agonists may prove therapeutically useful in the treatment of those medical and psychiatric conditions associated with disturbed vigilance. Orexin circuitry may indeed be disturbed under states such as sleep loss, night or shift work, jetlag, aging, affective disorders, or endocrine diseases. The relative brain distribution of OX1 versus OX2 receptors and their contribution to diverse neurobiological effects imply that different biological effects might be expected from drugs acting in the brain selectively at either OX1 or OX2 receptors or both. Transient and reversible reduction of brain orexin function can be induced by dual orexin receptor antagonists that effectively pass the blood-brain barrier, such as compound 127 which dose-dependently elicits somnolence without cataplexy in healthy rats, dogs, and humans when given acutely during the active phase of their circadian cycle, while endogenous orexin levels are high.7 Dual orexin receptor antagonists thus
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appear to mimic a physiological state of sleep that occurs when endogenous orexin release decreases during the sleep phase. Compound 127 also enhanced sleep efficiency following bedtime administration in patients with primary insomnia.22 In the rat, it does not disrupt the general architecture of sleep; it promotes a natural type of sleep by increasing NREM and REM sleep in physiological proportion, unlike the sleep pattern observed following gabaergic hypnotics which tend to decrease REM sleep while increasing NREM sleep time.7 Similar increase in both REM and NREM sleep was observed with an OX2 receptor antagonist.23 Immunoreactivity for orexin-A shows diurnal variations in several areas of the brain. For example, hypothalamic orexin-A concentrations are significantly higher during active waking than during slow-wave sleep. This has been observed in cats and rats and extends to humans. Current hypotheses on the role of orexins in the central nervous system (CNS) sleep system involve many different types of neurons, including noradrenergic, serotonergic, histaminergic, glutamatergic, and cholinergic neurons (e.g., in basal forebrain, perifornical hypothalamus, locus coeruleus), which are strongly innervated by orexin neurons originating in the hypothalamus.24 Hypothalamic neurons themselves are under the control of incoming circadian signals from suprachiasmatic and other nuclei. Sleep disorders such as primary insomnia are thus possibly related to dysfunctions of the orexin system, as recently confirmed in a clinical trial using compound 127 in primary insomnia patients.22 Studies using the orexin receptor antagonist 20 (SB-334867-A, Figure 1),25 which preferentially blocks the OX1 subtype of receptors,25 have suggested a role for OX1 receptors in feeding behavior and energy homeostasis26-28 (see below). Although orexins stabilize wakefulness, selective OX1 blockade using compound 20 does not modify REM sleep or induce cataplexy in rats.29 Sleep-inducing effects may be mediated by selective blockade of OX2 receptors during the light phase in the rat23 with compound 58 (JNJ-10397049, Table 5).23 Recent studies have also reported that OX1 receptors play a role in drug reward, reinstatement of drug seeking, and psychomotor sensitization.30,31 Orexins play additional roles in modulating the autonomic nervous system, in regulating stress responses, in contributing to pain responsiveness, exploratory locomotion, or other components of the behavioral repertoire of rodents. Orexin receptor antagonists will be particularly useful agents for delineating the physiological and pathophysiological roles of endogenous orexins. The first results of a proof-of-concept study with 127 in patients with primary insomnia suggest a role for orexins in abnormally maintaining wakefulness at night. However, since the full pathophysiology and clinical picture of orexin dysfunctions is still largely unknown, the possibility for therapeutic opportunities with orexin receptor ligands in human or veterinary medicine remains largely open. The relative contributions of OX1 versus OX2 receptors to diverse neurobiological effects and their brain distribution suggest that different effects might be expected from drugs acting in the brain selectively at either OX1 or OX2 receptors or both. Interesting observations and data were generated by using the orexin receptor antagonists of the first generation that are described below. Orexin-1 Receptor Antagonists Researchers at GlaxoSmithKline (GSK) found biarylureas in a functional high throughput screening (HTS) of their compound collection against a Chinese hamster ovary (CHO) cell line expressing the human OX1 receptor by applying fluorometric imaging plate reader (FLIPR) based assay technology. Compound 1, 1-aryl-3-(quinolin-4-yl)urea, is described to show good affinity toward the OX1 receptor, and in general it exhibits good
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Table 1. Indole Ring SAR
Figure 1. Quinoline ring SAR.
Figure 2. OX1 receptor selective compounds.
selectivity over other GPCRs including the OX2 receptor (pKb ) 5.6). The major drawback of this compound was the high binding toward both the 5HT2B and the 5HT2C receptor which seriously limited the potential of compound 1 as a tool.32 Scientists at GSK investigated the SAR by first observing the influence of variations in the indole unit on the activity and selectivity toward the OX1 receptor (Table 1). Compound 2 containing an additional methyl group in position 2 of the indole ring exhibited similar functional affinity when compared to 1. Increasing the size of the N-substituent resulted in significantly lower activity as shown with 3, and entire removal of the N-methyl group led to an only slightly active derivative 4. Replacement of the 1-methylindole substituent by a 1-methylindoline substituent was well tolerated with respect to OX1 receptor affinity, and unfortunately the affinity toward the 5HT2B as well as 5HT2C receptor was strong and prohibitive. As a next step the pyrrole ring from the indole system was removed to give the unsubstituted derivative 6 which was inactive. Introduction of a dimethylamino group in para-position of the phenyl ring gave the highly active OX1 receptor antagonist 7 with reduced susceptibility for 5HT2B and 5HT2C interaction. The shift of the N-dimethyl substituent into the meta-position (8) dramatically reduced the OX1 affinity. Replacing the
dimethylamino group by a diethylamino unit (e.g., 9) was well tolerated, but complete demethylation to an anilinic amino group (e.g., 10) was detrimental with respect to OX1 receptor affinity, whereas the introduction of the lipophilic S-methyl substituent again resulted in a highly active OX1 receptor antagonist (11) unfortunately again lacking selectivity over the 5HT2B and 5HT2C receptors. Although, as seen with 8, 3-monosubstitution resulted in low activity compounds, increased activity was obtained by the introduction of an additional substituent to give 3,4-disubstituted derivatives 12 and 13. Connection of the two substituents into a ring system, for example, an annelated oxazole ring, as shown with 14-16, resulted in a highly potent and selective OX1 receptor antagonist (15) in case the benzoxazole unit was connected to the urea functionality in the 6-position and contained a 2-methyl substituent. Removal of the 2-methyl group gave a less active compound (14), and the regioisomeric benzoxazole contained in 16 was inactive. The next step consisted of an investigation of the influence of the 4-aminoquinoline system on activity and selectivity. Removal of the phenyl part of the quinoline system resulted in a complete loss of OX1 receptor affinity (17), and replacement of the quinoline N-atom as well resulted in a significantly less potent molecule compared to 1. This pointed out the significance of both the annelated biaryl system and the N-atom for substantial OX1 receptor antagonistic activity. In order to change the lipophilicity of 1, a second N-atom was introduced to result in a 1,5-naphthyridine substituent as depicted in 19 with OX1 receptor affinity in the same range as 15. The 1,5-naphthyridine was then combined with the formerly identified 2-methylbenzoxazole substituent to form the potent, selective OX1 receptor antagonist 20. Moving the second N-atom around the heteroaromatic system to the 6- or 8-position resulted in both cases in dramatically less active compounds (21 and 22).
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Compound 20 is the first selective OX1 receptor antagonist described.25,27 In CHO cells, it was shown to inhibit the OX1mediated calcium response at nanomolar (nM) concentrations and to inhibit the OX2-mediated calcium response at considerably higher concentrations (50-fold selectivity for the OX1 over OX2 receptor). It showed selectivity over more than 50 GPCRs and ion channels.25 Following intraperitoneal (ip) administration, it penetrated into the brain with a brain/plasma ratio of 0.74 at 2 h and was rapidly absorbed (Tmax in blood and brain of 0.5 h).32,33 This selective OX1 receptor antagonist has been extensively used as a tool to assess the physiological role of the orexin system. Regarding feeding behavior, compound 20 has been shown to reverse both orexin-A- and fasting-induced food intake.27 Given alone, it reduced food intake and accelerated behavioral satiety.33-36 In genetically obese ob/ob mice, chronic treatment with compound 20 reduced the cumulative food intake and body-weight gain.26 In rats, it reduced food intake in a more effective manner in a strain more susceptible to dietary-induced obesity.37 All those studies suggested a role for the OX1 receptor in feeding behavior and energy homeostasis and also provided evidence that orexin receptor antagonists could be useful to treat disorders related to these systems. In the field of sleep, compound 20 has been shown to reverse the arousal and REM-suppressing effect of orexin-A. However, given alone, the OX1 receptor antagonist did not show any effect on REM sleep.29 In addition, compound 20 has been used to study the role of the orexin system in drug reward and reinstatement of drug seeking.30,31,38 It blocked the conditioned place preference induced by morphine or cocaine.39,40 Concerning reinstatement of drug seeking, compound 20 has been shown to block footshock-stress induced reinstatement of previously extinguished cocaine seeking and locomotor sensitization41,42 as well as cue-induced reinstatement of alcohol seeking behavior.43 Finally, systemic injection of compound 20 had no effect on reinstatement of food seeking in rats induced by orexin-A, pellet priming, and yohimbine (pharmacological stressor).31 In pain, compound 20 reversed the analgesic effects of orexin-A in an animal model of trigeminovascular nociception, showing that the reduction of the neurogenic dural vasodilation produced by orexin-A functions through the OX1 receptor.44 GSK described further OX1 selective compounds such as the substituted quinoline containing ureas 23 (SB-408124) and 24 (SB-410220) or the pyrrolidine-based derivative 25 (SB-674042) (Figure 2) which were used to evaluate potential indications for OX1 selective antagonists in several animal models because of their ability to cross the blood-brain barrier (BBB), at least after ip administration.45,46 Orexin-2 Receptor Antagonists A research team at Banyu Pharmaceuticals developed OX2 receptor selective antagonists based on a 6,7-dimethoxy-1,2,3,4tetrahydroisoquinoline scaffold, starting from compound 26 (Figure 3), showing dual antagonistic activity, which was found as a hit in a HTS.47,48 The authors state that the search for replacements of the 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline moiety was not successful and always ended up in a loss of activity toward both orexin receptors. Therefore, it was decided to keep this structural portion of the antagonists constant and work on modifications in the acyl part of the antagonists in order to optimize the activity and tune other properties. The investigation started by the preparation of N-(3-phenylpropanoyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline analogues. Removing the bromo substituent from 26 to give 27 resulted in a 30-fold more potent antagonist of the OX2 receptor.
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Figure 3. HTS hit based on a 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline scaffold. Table 2. Activity of 1-(3-Phenylpropanoyl)tetrahydroisoquinoline Analogues against hOX1 and hOX2a
IC50, nM 1
2
compd
R
R
hOX1
hOX2
26 27 28 29 30 31 32 33 34 35 36
Br H H H H H H H H H H
tert-butyl tert-butyl H phenyl dimethylamino benzylaminob benzoylamino benzoylaminob 3,5-dichlorobenzoylamino 3,5-dichlorobenzoylaminob 3-bromo-4-fluorobenzoylamino
7000 5500 >10000 >10000 >10000 >10000 >10000 >10000 5300 2300 5900
2000 56 >10000 395 >10000 >10000 900 195 36 30 70
a All compounds were synthesized as racemates unless otherwise indicated. b (S)-Isomer.
Replacing the tert-butyl-substituent by a hydrogen atom, as done in 28, ended with a complete loss of activity toward both orexin receptors. Further replacements for the tert-butyl group in position R2 are summarized in Table 2. A dimethylamino group and a benzylamino group were absolutely not tolerated in this region (30, 31). Introducing a benzoylamino unit as shown in 32 and the enantiomerically pure 33 resulted in compounds exhibiting promising affinity for the OX2 receptor. Introduction of further substituents to the aromatic part, such as in the 3,4dichlorobenzoylamino-containing derivative 34 and its enantiomerically pure derivative 35, resulted in selective, potent OX2 receptor antagonists. Different halogens in other positions were less well tolerated (e.g., 36). In the next step, summarized in Table 3, it was decided to keep the R-tert-butyl substituent fixed and investigate the influence of the introduction of a nitrogen atom, resulting in an amino acid core structure (e.g., tert-butylleucine) and investigate the possibilities for N-substituents to give OX2 receptor selective compounds. It is obvious that only limited possibilities exist. The benzyl group, as depicted in 40 and its enantiomerically pure (S)-derivative 41, was the only substituent resulting in reasonably active compounds. Comparison of 41 and 42 highlights the importance of the (S)-chirality for good OX2 affinity. The obvious continuation was then the variation of the phenyl ring from the N-benzyl substituent in compound 41 as depicted in Table 4. The most active compounds were obtained by the most closely related isosteric replacement of the phenyl ring with either a 2-thienyl ring (48) or a 3-thienyl unit (49) or by a 2-(N-methyl)pyrrolyl system (46). Among the three regioisomeric pyridine replacements, only the 4-pyridyl regioisomer led to a substantially active OX2 receptor antagonist (52). The 4-pyridyl analogue 52 was one of the most promising compounds resulting from this investigation, with a selectivity for the OX2 receptor of >250-fold over the OX1 receptor. The compound in addition showed less than 30% inhibition at 10 µM toward over 50 receptors and ion channels, including GPCRs such as galanin or neuropeptide Y,
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Table 3. Activity of 1-(tert-Leucyl)tetrahydroisoquinoline Analogues against hOX1 and hOX2a
IC50, nM compd
R
hOX1
hOX2
37 38 39 40 41 42 43 44 45
H phenyl benzoyl benzyl benzylb benzylc 2-phenylethyl 3-phenylpropyl cyclohexylmethyl
>10000 >10000 >10000 >10000 3850 >10000 >10000 >10000 >10000
>10000 2100 >10000 910 130 2900 >10000 >10000 >10000
a All compounds were synthesized as racemates unless otherwise indicated. b (S)-Isomer. c (R)-Isomer.
Table 4. Activity of N-Arylmethyl-(S)-tert-leucyltetrahydrosioquinoline Analogues against hOX1 and hOX2
IC50, nM compd
Ar
hOX1
hOX2
41 46 47 48 49 50 51 52
phenyl 2-(N-methyl)pyrrolyl 2-thiazolyl 2-thienyl 3-thienyl 2-pyridyl 3-pyridyl 4-pyridyl
3850 3600 >10000 1130 3670 >10000 >10000 >10000
130 28 59 25 35 1400 240 40
associated with food intake. Because of further beneficial properties (high water solubility (0.81 mg/mL at pH 7), 29 was selected for further pharmacological profiling. So far, no further results have been published. Another class of OX2 selective receptor antagonists was described by Johnson and Johnson (J&J) who found in a HTS 1-aryl3-(4-phenyl[1,3]dioxin-5-yl)urea-based structures such as 53 as hits.49,50 It is stated that OX2 selective antagonists should be used to clarify the pharmacological role and relevance of the OX2 receptor and that compounds with this profile might be potentially useful in future treatments of sleep/wake disorders. Markush structure 54 summarizes the SAR investigations undertaken around hit 53 (Scheme 1). In cases where Z represents -NH- and W represents O, the best results were observed. Table 5 reflects some of the efforts made in this class of OX2 selective antagonists. The synthesis of the urea-containing compounds was very straightforward by reacting commercially available aryl isocyanates with (S,S)-2,2-dimethyl-4phenyl[1,3]dioxan-5-ylamine. Stereochemistry of the dioxanyl subunit seems to be crucial for inhibitory activity. Nothing is said with respect to the influence of substituents on the 4-phenyl unit or replacements by, for example, heteroaryl ring systems attached to the dioxan-5-yl ring on OX2 receptor antagonist activity. Attempts to replace the central urea, which was thought to be the culprit for negative properties such as low water solubility, by thioureas (Z ) -NH- and W ) S), carbamates (Z ) -O- and W ) O), amides (Z ) -O- and W ) absent) or cyanoguanidines (Z ) -NH- and W ) N-CN) resulted in significant losses (>10-fold) of OX2 receptor affinity and usually completely eliminated affinity toward the OX1 receptor.
Receptor specificity was investigated for compounds 53, 58, and 66 in a CEREP screen of 50 receptors. Only compound 58 showed
