5320 J. Med. Chem. 2010, 53, 5320–5332 DOI: 10.1021/jm100541c
Discovery of the Dual Orexin Receptor Antagonist [(7R)-4-(5-Chloro-1,3-benzoxazol-2-yl)7-methyl-1,4-diazepan-1-yl][5-methyl-2-(2H-1,2,3-triazol-2-yl)phenyl]methanone (MK-4305) for the Treatment of Insomnia
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Christopher D. Cox,*,† Michael J. Breslin,† David B. Whitman,† John D. Schreier,† Georgia B. McGaughey,‡ Michael J. Bogusky,† Anthony J. Roecker,† Swati P. Mercer,† Rodney A. Bednar,§ Wei Lemaire,§ Joseph G. Bruno,§ Duane R. Reiss, C. Meacham Harrell, Kathy L. Murphy, Susan L. Garson, Scott M. Doran, Thomayant Prueksaritanont,^ Wayne B. Anderson,^ Cuyue Tang,^ Shane Roller,^ Tamara D. Cabalu,^ Donghui Cui,^ George D. Hartman,† Steven D. Young,† Ken S. Koblan, Christopher J. Winrow, John J. Renger, and Paul J. Coleman† †
Departments of Medicinal Chemistry, ‡Chemistry Modeling and Informatics, §In Vitro Sciences, Depression and Circadian Disorders, and Drug Metabolism, Merck Research Laboratories, P.O. Box 4, Sumneytown Pike, West Point, Pennsylvania 19486
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Received May 5, 2010
Despite increased understanding of the biological basis for sleep control in the brain, few novel mechanisms for the treatment of insomnia have been identified in recent years. One notable exception is inhibition of the excitatory neuropeptides orexins A and B by design of orexin receptor antagonists. Herein, we describe how efforts to understand the origin of poor oral pharmacokinetics in a leading HTSderived diazepane orexin receptor antagonist led to the identification of compound 10 with a 7-methyl substitution on the diazepane core. Though 10 displayed good potency, improved pharmacokinetics, and excellent in vivo efficacy, it formed reactive metabolites in microsomal incubations. A mechanistic hypothesis coupled with an in vitro assay to assess bioactivation led to replacement of the fluoroquinazoline ring of 10 with a chlorobenzoxazole to provide 3 (MK-4305), a potent dual orexin receptor antagonist that is currently being tested in phase III clinical trials for the treatment of primary insomnia.
Introduction Insomnia is characterized by difficulty initiating and/or maintaining sleep, waking early, or finding sleep nonrestorative. In a recent poll of American adults, over 50% reported having at least one symptom of insomnia during the previous year, and a third reported symptoms almost every night.1 Insomnia is considered primary if no known underlying mental or physical condition leads to its development, and has been directly linked to significant daytime consequences such as irritability, inability to concentrate, reduced energy levels, poor job performance, work absenteeism, and a greater risk of traffic and work-related accidents as well as a higher incidence of health problems.1,2 Insomnia is also commonly experienced as a secondary symptom of comorbid disorders. The direct and indirect costs to the US economy related to insomnia have been estimated to be well in excess of $100 billion per year.3 It is believed that a significant number of cases go undiagnosed and/or untreated due to an opinion by many sufferers that sleep problems are inconsequential and a pervasive belief by physicians that currently available treatments have questionable safety and efficacy.1 Notwithstanding, one-quarter of American adults take sleep medications during the course of a year, and the U.S. market for sleep drugs is estimated to reach $5 billion per year by 2010.3a Despite the physical, psychological, and economic toll of insomnia, treatments that improve total sleep time and subjective sleep quality without risk of next-day impairment, *To whom correspondence should be addressed. Phone: þ 1-215-6522411. Fax: þ 1-215-652-7310. E-mail:
[email protected].
pubs.acs.org/jmc
Published on Web 06/21/2010
dependence, rebound insomnia, or safety concerns remain elusive.4 The most widely prescribed agents for treatment of insomnia are central nervous system depressants that enhance signaling of the inhibitory neurotransmitter γ-aminobutyric acid (GABAa). Developed beginning in the early 1960s, benzodiazepines decrease sleep onset time by binding to GABAA receptors on postsynaptic neurons in the central nervous system but are associated with issues such as daytime sleepiness, dizziness, impaired memory, and risk of dependence.1 The subtype selective benzodiazepine and nonbenzodiazepine GABAA modulators developed more recently, such as zolpidem, zaleplon, and eszopiclone, address some of the issues associated with earlier agents but questions remain about the overall safety and efficacy of these agents.3 The FDA recently mandated black-box warnings related to the safety profile of all sedative hypnotics, underscoring the agency’s concern with the currently available treatments.5 In 2005, the FDA approved the melatonin receptor antagonist ramelteon for the treatment of insomnia, representing the first novel mechanism to be approved in 35 years and the only hypnotic not classified as a a Abbreviations: BDC, bile-duct cannulated; Boc, tert-butoxycarbonyl; BSA, bovine serum albumin; CSF, cerebrospinal fluid; DORA, dual orexin receptor antagonist; ECoG, electrocorticogram; EMG, electromyogram; FLIPR, fluorometric imaging plate reader; GABA, γ-aminobutyric acid; GSH, glutathione; ICV, intracerebroventricular; LPS, latency to persistent sleep; NSB, nonspecific binding; OTC, over-thecounter; OX1R, orexin receptor 1; OX2R, orexin receptor 2; PEG, polyethylene glycol; REM, rapid-eye movement; SORA, selective orexin receptor antagonist; SWS, slow-wave sleep; TB, total binding; TPGS, D-R-tocopheryl polyethylene glycol-100-succinate; WASO, wake after sleep onset.
r 2010 American Chemical Society
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Figure 1. DORAs studied to date in clinical trials.
controlled substance.6 However, questions remain about the effectiveness of ramelteon relative to GABAA modulators.7 Clearly, novel mechanisms for the treatment of insomnia that simultaneously address the need for improved safety and efficacy are of great interest, and an increased understanding of the neuronal circuitry involved in the sleep/wake cycle has spurred a rational search for such targets. One of the most promising discoveries in this regard is the orexin (or hypocretin) system that is highly conserved across species and intricately linked to the regulation of sleep and wake states.8 Orexins A and B are neuropeptides derived from the same prepro-orexin peptide that is produced in neurons located in the lateral hypothalamus. These neurons project widely to regions of the brain involved in regulation of arousal. Discovered in 1998 by independent research groups, the orexin peptides were found to bind to the previously identified orphan G protein-coupled receptors, orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R).9 Whereas OX1R binds orexin A with greater affinity than orexin B, OX2R binds to both peptides with equal affinity. A number of genetic and pharmacological studies have confirmed the central role that orexin signaling plays in regulation of the sleep/wake cycle. Mice engineered to be deficient in orexin peptides demonstrate a robust phenotype consistent with human narcolepsy, a sleep disorder characterized by excessive daytime sleepiness, sleep fragmentation, and abnormal transitions to REM sleep.10 Selective knockout of OX1R in mice revealed a mild phenotype characterized by sleep fragmentation, whereas knockout of OX2R produced a narcoleptic phenotype but one that is less severe than the peptide knockout. Simultaneous knockout of both OX1R and OX2R produced an acute narcoleptic phenotype similar to the peptide-deficient animals.11 Additionally, a colony of dogs that naturally suffer from narcolepsy possess a mutation in the gene coding for the OX2R,12 whereas the disorder in humans has been linked to a reduction of orexin-producing neurons, resulting in low or undetectable levels of orexin A in the cerebrospinal fluid (CSF) of narcoleptics.13 Consistent with these observations, direct intracerebroventricular (ICV) infusion of the orexin peptides into rat CSF leads to an increase in arousal and a decrease in REM and non-REM sleep.14 Because hypofunction of the orexin system leads to decreased wakefulness, orexin receptor antagonists have emerged as a potential target for the treatment of insomnia as well as conditions resulting from disruption of the natural circadian cycle such as shift work or jet lag.15 However, on the basis of genetic studies, the potential for orexin receptor blockade to cause cataplexy, a condition closely linked to a subset of narcolepsy sufferers and characterized by sudden loss of muscle tone, was identified as a potential concern. A number of laboratories therefore embarked on a search for selective (OX1R or OX2R) as well as dual (OX1R/OX2R) orexin
receptor antagonists to better characterize their therapeutic potential. These efforts have resulted in identification of potent dual orexin receptor antagonists (DORAs) as well as selective orexin receptor antagonists (SORAs) that have enabled pharmacological studies of their in vivo activity.15c,d,16 Most work to date has focused on DORAs, likely due to the fact that genetic studies suggested a more robust effect on the sleep/wake cycle when both receptors are affected;11 however, a recent publication revealed that an OX2R-selective SORA promotes sleep in rats.17 The most fully characterized DORA studied to date is (2R)2-[(1S)-6,7-dimethoxy-1-{2-[4-(trifluoromethyl)phenyl]ethyl}3,4-dihydroisoquinolin-2(1H)-yl]-N-methyl-2-phenylethanamide (1, Almorexant), a compound currently in phase III clinical trials for the treatment of primary insomnia (Figure 1).18 Developed by Actelion Pharmaceuticals, it has been shown to significantly decrease wakefulness in rats, dogs, and humans, with no evidence of cataplexy.19 In a phase II trial, Almorexant provided clinical proof of concept for the orexin antagonist mechanism in the treatment of insomnia, improving sleep efficiency in primary insomniacs, and showing no significant next-day effects on motor function or reaction time.20 The second entry into the clinic was the DORA N-{[(2S)-1-{[5-(4fluorophenyl)-2-methyl-1,3-thiazol-4-yl]carbonyl}piperidin2-yl]methyl}-1-benzofuran-4-carboxamide (2, SB-649868) from GlaxoSmithKline. It showed positive results in terms of efficacy without residual next day effects; however, preclinical toxicology findings have delayed advancement of this compound.21 Importantly, both compounds have been shown to induce a natural sleep architecture characterized by increases in the time spent in REM and non-REM sleep, differentiating them from GABAA modulators such as zolpidem that reduce time spent in these sleep stages believed to be important for consolidation of memory and the restorative functions of sleep.15c In December 2008, Merck presented preliminary phase I results indicating that DORA [(7R)-4-(5chloro-1,3-benzoxazol-2-yl)-7-methyl-1,4-diazepan-1-yl][5-methyl-2-(2H-1,2,3-triazol-2-yl)phenyl]methanone (3, MK4305) decreased latency to persistent sleep (LPS) and wake after sleep onset (WASO), with no disruption of sleep architecture, in healthy volunteers.22 The favorable clinical results obtained with three structurally diverse DORAs underscores the promise that orexin receptor antagonism holds for the treatment of sleep disorders. In this article, we describe the discovery and preclinical profile of 3, a potent brain-penetrant DORA that recently entered phase III clinical trials for the treatment of primary insomnia.23 Results and Discussion A recent publication from our group described the discovery of 4, a novel diazepane-based DORA identified by hitto-lead efforts following high throughput screening of the
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Merck sample collection (Figure 2).24 Compound 4 demonstrates low nanomolar affinity for the human OX1R and OX2R as measured by a radioligand-displacement binding assay (expressed as Ki), as well as excellent potency in a fluorometric imaging plate reader (FLIPR) assay that provides a functional readout of orexin receptor antagonism in CHO cells engineered to overexpress the human receptors (expressed as IC50). It was found to promote sleep in rats when dosed orally at 100 mg/kg by increasing the amount of time spent in both REM and delta sleep.25 This observation is consistent with the profile of clinically studied DORAs 1 and 215c and opposite to the effect seen following ICV administration of the native peptide.14 Though 4 has excellent brain penetration, good physical properties, a clean ancillary profile, and produces desired effects on sleep architecture, it suffers from high clearance and low bioavailability in the rat (Table 1). In the dog, 4 displays moderate plasma clearance (11.6 mL/min/kg) but low bioavailability (16%). In light of the observed clearance (calculated blood clearance accounts for ∼50% of hepatic blood flow), maximal bioavailability is anticipated to be ∼50% in dog assuming complete absorption and primary elimination via the liver. Thus, the observed low bioavailability was unexpected and suggested the possibility of poor absorption in this species. However, screening a number of vehicles did not result in any improvement over polyethylene glycol (PEG) 200 used in the initial study.26 Bile-duct cannulated (BDC) dog and portal/cephalic vein infusion studies were therefore carried out to better understand the reason for poor bioavailability. These studies revealed that 4 is well absorbed when dosed in PEG 200, with 78% of the total dose recovered in bile and urine as products of oxidative metabolism. Hepatic extraction, determined by comparing plasma AUCs during infusion via the portal or cephalic vein, was 80%. Therefore, despite the moderate clearance of 4 in dog, hepatic first-pass metabolism was the major factor limiting its bioavailability. As the dog was predicted to be most like human in pharmacokinetic profile based on the similar intrinsic clearance in these two species, improved dog pharmacokinetics (reduced clearance, increased bioavailability) was required to advance a compound into development. We therefore began a rational approach to identify molecules with this potential.
Figure 2. Previously reported diazepane-based DORA 4.
Cox et al.
In vitro metabolism studies of 4 in microsomal preparations indicated several sites of metabolism that we sought to eliminate or modify by synthetic modifications. A major metabolic pathway identified was oxidation of the tolyl methyl group on the 5-methyl-2-(triazolyl)-benzamide portion of the molecule to form the benzyl alcohol and further oxidation to the benzoic acid. Additionally, complex metabolic patterns were observed on the remainder of the molecule that indicated oxidation was occurring on both the core and the quinazoline heterocycle. Interestingly, when 4 was incubated in microsomal preparations fortified with semicarbazide, mass spectral adducts consistent with structure 4b were identified (Scheme 1). Mechanistically, this is suggestive of oxidation at one of the four positions on the core adjacent to a nitrogen atom to form intermediate 4a, followed by ringopening to reveal an aldehyde, which is subsequently trapped by semicarbazide.27 Taken as a whole, the metabolism data in hand suggested that removal of the benzylic methyl group, alteration of the heterocycle, and chemical modification of the core, especially blocking positions adjacent to the nitrogen atoms, were logical tactics to employ in an effort to improve pharmacokinetics in the diazepane series. With this knowledge, we began an intensive synthetic investigation focused on identification of novel diazepane cores that had substitution at one or more carbon atoms. During this effort, we identified over 15 mono- or disubstituted diazepane cores that provided potent DORAs (Ki < 5 nM on both OX1R and OX2R). In general, substitution at positions 3 and 5 of the diazepane ring was not tolerated by the receptors, whereas substitution at positions 2, 6, and 7 was not only tolerated but often potency enhancing (Figure 3).28 With a large number of potent diazepane cores in hand, we combined these with the optimal triazolyl-benzamide substituent and various heterocycles to assemble a large number of potent DORAs for pharmacokinetic evaluation. Our expectation was that reduced IV clearance would predict improved bioavailability as a result of reduced metabolism, so we studied many compounds in parallel by dosing IV in the dog. Promising analogues were then followed up by PO dosing to determine bioavailability.29 On the basis of the overall potency, ancillary, and pharmacokinetic profiles of analogues made with a methyl substituent at the 7-position of the diazepane core, these analogues were targeted for further investigation. Surprisingly, a search of the literature revealed a dearth of practical synthetic strategies to access a simple orthogonally protected 7-methyldiazepane.30 The most promising route
Figure 3. General scaffold for examining mono- and disubstituted diazepane cores.
Table 1. Pharmacokinetics of 4 in Rat and Dog IVa
POb
species
dose (mg/kg)
Cl (mL/min/kg)
Vdss (L/kg)
T1/2 (h)
dose (mg/kg)
AUC ( μM 3 h)
Cmax ( μM)
F (%)
rat dog
2 0.5
52.6 11.6
1.08 0.86
0.35 1.28
100 3
1.50 1.81
0.65 1.44
2 16
a
Vehicle = DMSO; n = 2 in rat, n = 3 in dog. b Rat: dosed as the HCl salt in 20% TPGS; n = 3; dog: dosed as free base in PEG 200, n = 2.
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Scheme 1. Proposed Mechanism of Semicarbazide Trapping When 4 Is Incubated in Liver Microsomes Fortified with Semicarbazide
Scheme 2. Previously Reported Synthesis of the 7-Methyldiazepane Core30ba
a
Reagents and conditions: (a) Et2O (70%); (b) PPh3, Et2O (72%); (c), LAH, Et2O (91%).
Scheme 3. Synthesis of 7-Methyldiazepane DORA Analoguesa
a Reagents and conditions: (a) Et2O, then TEA and benzyl chloroformate; (b) HCl(g), EtOAc; (c) NaBH(OAc)3, HOAc, CH2Cl2; (d) Boc2O, TEA, CH2Cl2 (38% for 4 steps); (e) Chiralpak AD, 60% EtOH in hexanes (0.1% DEA as modifier), first isomer to elute is the desired (R)-isomer; (f ) HCl(g), EtOAc; (g) 2-(2H-1,2,3-triazol-2-yl)-5-methylbenzoic acid, EDC, HOAT, NMM, DMF (87% for 2 steps); (h) H2, Pd(OH)2, MeOH/EtOAc; (i) 2-chloroquinazoline, K2CO3, DMF (75% for 2 steps).
involved conjugate addition of a 1,2-amino azide to methyl vinyl ketone, followed by an intramolecular aza-Wittig cyclization and subsequent reduction (Scheme 2).30b Although short and convergent, this route is unattractive for large scale synthesis because it necessitates the synthesis and use of a potentially explosive alkyl azide.31 As illustrated in Scheme 3, this route did, however, provide inspiration for a related strategy utilizing the commercially available N-Boc-1,2-diaminoethane as a surrogate for the azide. In our modified route, conjugate addition of the monoprotected diamine to methyl vinyl ketone followed by in situ trapping with benzyl chloroformate provides ketone 5.
Boc deprotection and subsequent intramolecular reductive amination with NaBH(OAc)3 forms the diazepane ring. A Boc group is reinstalled to simplify purification, resulting in a 38% overall yield of 6 for the four steps from methyl vinyl ketone. Importantly, this chemistry can be readily carried out on large scale to provide access to quantities of 6 for analogue synthesis and in vivo evaluation of leading compounds. Subsequent studies indicated that the (R)-antipode of analogues in this series had superior potency for the orexin receptors.32 A chiral stationary phase HPLC resolution was therefore carried out on racemic 6 to provide the desired (R)-enantiomer as the first eluting isomer. To complete the synthesis of analogues in this series, Boc deprotection followed by amide coupling to 2-(2H-1,2,3-triazol-2-yl)-5methylbenzoic acid provided 7 in excellent yield. Removal of the benzyl carbamate followed by reaction with 2-chloroquinazoline provided 8, the 7-(R)-methyldiazepane analogue of 4 in 75% yield from 7. As illustrated in Table 2, 8 displayed excellent potency for OX1R and OX2R receptors in both binding and FLIPR assays, but a reduction in dog clearance did not lead to improved oral bioavailability relative to 4. Synthesis of 9 which lacks the benzylic methyl group of 8 provided an analogue with similar IV clearance but with diminished potency on OX1R as measured in the binding assay.33 Installation of a fluorine atom at the 6-position of the quinazoline, a substitution we found to impart improved pharmacokinetics to a number of analogues in the diazepane series,25b provided 10 with improved potency, a further reduction in IV clearance, and favorable bioavailability of 37% in dog. Similar to compound 4, 10 is not a substrate for Pgp efflux (BA/AB = 0.6), has excellent passive permeability (38 10-6 cm/s), a plasma free fraction of ∼3% in rat, and possesses a clean ancillary profile as determined by an MDS Pharma offtarget screen. In vivo analysis in rat indicates 10 demonstrates reasonable rat pharmacokinetics with a clearance of 33 mL/ min/kg and oral bioavailability of 47% when dosed at 10 mg/ kg in 20% D-R-tocopheryl polyethylene glycol-100-succinate (TPGS)34 and maintains a favorable brain/plasma ratio of 0.4-0.6 and a CSF/plasma ratio of 0.02-0.03.35 Compound 10 possesses the overall profile we were seeking in a development compound, and it was therefore advanced into a rat sleep assay to assess its effects on sleep architecture. The preclinical sleep laboratory at Merck makes use of radiotelemetric recording of electrocorticogram (ECoG) and electromyogram (EMG) signals to assess the time spent by animals in various states of arousal, including active wake, light sleep, REM sleep, and delta or slow wave sleep (SWS). Upon dosing 10 orally at 10 mg/kg/day to telemetry-implanted rats during their active phase (lights off), the amount of time spent in each state of arousal was measured and binned into 30 min intervals and compared to vehicle treated rats (Figure 4). Similar to the effects seen with compound 4, 10
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Table 2. Profile of 7-(R)-Methyldiazepane Analogues 8-10
compd
R1
R2
R3
OX1R Ki, FLIPRa (nM)
OX2R Ki, FLIPRa (nM)
dog Clb (mL/min/kg)
dog F c (%)
4 8 9 10
H Me Me Me
Me Me H H
H H H F
1.2, 29 0.39, 30 6.2, 26 1.8, 27
0.59, 27 0.36, 30 0.47, 18 0.17, 27
11.6 6.0 6.4 5.2
16
