Essential structure of orexin 1 receptor antagonist YNT-707, Part I: Role of the 4,5-epoxy ring for binding with orexin 1 receptor

The essential structure of the orexin 1 receptor (OX₁R) antagonist YNT-707 (2) was clarified, particularly the roles to OX₁R antagonist activities of the 3-OMe, the 4,5-epoxy ring, the 14-hydroxy group, and the orientation of the 6-amide side chain.The 3-OMe and 17-sulfonamide group were shown to be essential for the OX₁R antagonistic activity. The 4,5-epoxy ring plays an important role for the active orientation of the 6-amide group. The 14-hydroxy group could lower the activity of the 6β-amide isomer by the interaction of the 14-hydroxy group with the 6-amide group, which could orient the 6-amide group toward the upper side of the C-ring.Finally, we proposed the difference in the active conformation between OX₁R and κ opioid receptor (KOR), especially in the orientation of the 6-amide group which is expected to be a useful guide for medicinal chemists to design OX₁R ligands.     

Essential structure of orexin 1 receptor antagonist YNT-707, Part II: Drastic effect of the 14-hydroxy group on the orexin 1 receptor antagonistic activity

The 14-dehydration- and 14-H derivatives of the orexin 1 receptor (OX₁R) antagonist YNT-707 (2) were synthesized. The obtained derivatives showed higher affinities for OX₁R than the corresponding 14-hydroxy derivatives. The conformational analysis suggested that the 17-sulfonamide groups in the derivatives without the 14-hydroxy group have a greater tendency to be oriented toward the upper side of the D-ring compared with the 14-hydroxy derivatives. Additionally, the 14-dehydration-derivative with 6α-amide side chain showed significantly higher affinity than the 14-hydroxy derivative, while the corresponding 14-H derivative showed only slightly higher affinity. Thus, the 14-hydroxy group strongly affects the affinity of the antagonist for the OX₁R.     

Essential structure of orexin 1 receptor antagonist YNT-707, Part IV: The role of D-ring in 4,5-epoxymorphinan on the orexin 1 receptor antagonistic activity

The orexin 1 receptor (OX₁R) antagonists carrying a morphinan skeleton such as YNT-707 (2) and YNT-1310 (3) showed potent and extremely high selective antagonistic activity against OX₁R. In the course of our study of the essential structure of YNT-707 for high binding affinity against OX₁R, we prepared derivatives of 2 without the D- and 4,5-epoxy rings to clarify the roles of these structural determinants toward OX₁R antagonistic activity. The D- and 4,5-epoxy rings played important roles for the active orientation of the 17-sulfonamide and 6-amide side chains. Finally, we identified the simple structure required for selective OX₁R antagonistic activity in the complex morphinan skeleton, which is expected to be a useful scaffold for further design of OX₁R ligands.     

Essential structure of orexin 1 receptor antagonist YNT-707, part V: Structure-activity relationship study of the substituents on the 17-amino group

The morphinan-type orexin 1 receptor (OX₁R) antagonists such as YNT-707 (2) and YNT-1310 (3) show potent and extremely high selective antagonistic activity against OX₁R. In the course of our studies of the essential structure of 2, we identified new scaffolds by simplification of the morphinan skeleton. However, the new chemical entities carrying the D-ring removed scaffold showed insufficient activity. To improve the activity of these derivatives, we investigated the effect of substituents mainly focused on the 17-nitrogen group. The 17-N-substituted derivatives, as well as the cyclic derivatives, were synthesized and examined the OX₁R antagonistic activity. The assay results showed the interesting relationship between the OX₁R antagonistic activity and the substituents on the 17-nitrogen: the antagonistic activity was increased as the bulkiness of 17-substituents increased. Finally, the 17-N-Boc derivative 14a showed the most potent OX₁R antagonistic activity (K_i = 14.8 nM).     

Discovery of attenuation effect of orexin 1 receptor to aversion of nalfurafine: Synthesis and evaluation of D-nor-nalfurafine derivatives and analyses of the three active conformations of nalfurafine

The D-nor-nalfurafine derivatives, which were synthesized by contraction of the six-membered D-ring in nalfurafine (1), had no affinity for orexin 1 receptors (OX₁Rs). The 17N-lone electron pair in 1 oriented toward the axial direction, while that of D-nor-derivatives was directed in the equatorial configuration. The axial lone electron pair can form a hydrogen bond with the 14-hydroxy group, which could push the 6-amide side chain toward the downward direction with respect to the C-ring. The resulting conformation would be an active conformation for binding with OX₁R. The dual affinities of 1 for OX₁R and κ opioid receptor (KOR) led us to elucidate the mechanism by which only 1 showed no aversion but U-50488H. Actually, 1 selectively induced severe aversion in OX₁R knockout mice, but not in wild-type mice. These results well support that OX₁R suppresses the aversion of 1. This is the elucidation of long period puzzle which 1 showed no aversion in KOR.     

Synthesis and biological evaluation of F-18 labeled tetrahydroisoquinoline derivatives targeting orexin 1 receptor

Orexin 1 receptor (OX₁R) is thought to be involved in various body functions, including arousal maintenance and emotional control, but the full details of its function remain unknown. OX₁R imaging with positron emission tomography (PET) would be useful in elucidating the orexin system including OX₁R, but no PET probes targeting OX₁R have been reported. We, therefore, designed and synthesized tetrahydroisoquinoline (THIQ) derivatives as novel PET probes targeting OX₁R, and evaluated their utility. In an in vitro competitive binding assay, THIQ-1 and THIQ-2 showed significantly higher binding to OX₁R (IC₅₀ = 30 and 31 nM, respectively) than OX₂R (IC₅₀ = 160 and 332 nM, respectively). These features were also observed in a cell binding assay using [¹⁸F]THIQ-1 and [¹⁸F]THIQ-2, demonstrating their OX₁R-specific binding property in vitro. In a biodistribution study using normal mice, the brain uptake of [¹⁸F]THIQ-1 was higher than that of [¹⁸F]THIQ-2, but further improvement is required for in vivo imaging with PET. Taken together, [¹⁸F]THIQ-1 and [¹⁸F]THIQ-2 have the potential to become useful imaging probes for PET targeting the OX₁R, but require additional structural changes to improve their brain uptake.     

Investigation of orexin-2 selective receptor antagonists: Structural modifications resulting in dual orexin receptor antagonists

In an ongoing effort to explore the use of orexin receptor antagonists for the treatment of insomnia, dual orexin receptor antagonists (DORAs) were structurally modified, resulting in compounds selective for the OX₂R subtype and culminating in the discovery of 23, a highly potent, OX₂R-selective molecule that exhibited a promising in vivo profile. Further structural modification led to an unexpected restoration of OX₁R antagonism. Herein, these changes are discussed and a rationale for selectivity based on computational modeling is proposed.     

Involvement of TRPC3 channels in calcium oscillations mediated by OX1 orexin receptors

Oscillations of intracellular Ca²⁺ provide a novel mechanism for sustained activation of cellular processes. Receptor-activated oscillations are mainly thought to occur through rhythmic IP₃-dependent store discharge. However, as shown here in HEK293 cells 1 nM orexin-A (Ox-A) acting at OX₁ receptors (OX₁R) triggered oscillatory Ca²⁺ responses, requiring external Ca²⁺. These responses were attenuated by interference with TRPC3 channel (but not TRPC1/4) function using dominant negative constructs, elevated Mg²⁺ (a blocker of many TRP channels) or inhibition of phospholipase A₂. These treatments did not affect Ca²⁺ oscillations elicited by high concentrations of Ox-A (100 nM) in the absence of external Ca²⁺. OX₁R are thus able to activate TRPC(3)-channel-dependent oscillatory responses independently of store discharge.     

Discovery of novel substituted octahydropyrrolo[3,4-c]pyrroles as dual orexin receptor antagonists for insomnia treatment

A series of octahydropyrrolo[3,4-c]pyrroles were synthesized and evaluated by orexin 1 and 2 receptor (OX_{1 & 2} R) antagonists assays. Compound 14l with potent OXR antagonist activity and suitable pharmacokinetic behavior was chosen to be investigated in an EEG study, which demonstrated effects of sleep promotion comparable to Suvorexant. Furthermore, the di-fluro substituted analogs exhibited reduced hERG inhibition while maintaining moderate potency.     

Discovery of dual orexin receptor antagonists with rat sleep efficacy enabled by expansion of the acetonitrile-assisted/diphosgene-mediated 2,4-dichloropyrimidine synthesis

Recent clinical studies have demonstrated that dual orexin receptor antagonists (OX₁R and OX₂R antagonists or DORAs) represent a novel treatment option for insomnia patients. Previously we have disclosed several compounds in the diazepane amide DORA series with excellent potency and both preclinical and clinical sleep efficacy. Additional SAR studies in this series were enabled by the expansion of the acetonitrile-assisted, diphosgene-mediated 2,4-dichloropyrimidine synthesis to novel substrates providing an array of Western heterocycles. These heterocycles were utilized to synthesize analogs in short order with high levels of potency on orexin 1 and orexin 2 receptors as well as in vivo sleep efficacy in the rat.     

Orexin-1 Receptor Co-Localizes with Pancreatic Hormones in Islet Cells and Modulates the Outcome of Streptozotocin-Induced Diabetes Mellitus

Recent studies have shown that orexins play a critical role in the regulation of sleep/wake states, feeding behaviour, and reward processes. The exocrine and endocrine pancreas are involved in the regulation of food metabolism and energy balance. This function is deranged in diabetes mellitus. This study examined the pattern of distribution of orexin-1 receptor (OX₁R) in the endocrine cells of the pancreas of normal and diabetic Wistar (a model of type 1 diabetes), Goto-Kakizaki (GK, a model of type 2 diabetes) rats and in orexin-deficient (OX^−/−) and wild type mice. Diabetes mellitus (DM) was induced in Wistar rats and mice by streptozotocin (STZ). At different time points (12 h, 24 h, 4 weeks, 8 months and 15 months) after the induction of DM, pancreatic fragments of normal and diabetic rats were processed for immunohistochemistry and Western blotting. OX₁R-immunoreactive nerves were observed in the pancreas of normal and diabetic Wistar rats. OX₁R was also discernible in the pancreatic islets of normal and diabetic Wistar and GK rats, and wild type mice. OX₁R co-localized with insulin (INS) and glucagon (GLU) in the pancreas of Wistar and GK rats. The number of OX₁R-positive cells in the islets increased markedly (p<0.0001) after the onset of DM. The increase in the number of OX₁R-positive cells is associated with a high degree of co-localization with GLU. The number of GLU- positive cells expressing OX₁R was significantly (p<0.0001) higher after the onset of DM. The tissue level of OX₁R protein increased with the duration of DM especially in type 1 diabetes where it co-localized with cleaved caspase 3 in islet cells. In comparison to STZ-treated wild type mice, STZ-treated OX^−/− animals exhibited reduced hyperglycemia and handled glucose more efficiently in glucose tolerance test. The findings suggest an important role for the OX-OX₁R pathway in STZ-induced experimental diabetes.     

An analog of 1α,25-dihydroxy-19-norvitamin D3 with the 1α-hydroxy group fixed in the axial position lacks biological activity in vitro

The relationship between the A-ring chair conformation of vitamin D compounds and their ability to bind the vitamin D receptor (VDR) has long attracted the attention of many researchers. It was established that in the crystalline complexes of hVDRmt with the natural hormone, 1α,25-dihydroxyvitamin D₃ (1), and its side-chain analogs the vitamins exist in β-chair form with an equatorial orientation of 1α-OH. However, with all these ligands the interconversion between both A-ring forms would be possible in solution. In an attempt to verify the conformation of vitamin D compounds required for binding the VDR we prepared analog 4, characterized by the presence of an axial 1α-hydroxy group. Since the additional ring connecting 3β-oxygen and C-2 prevents A-ring conformational flexibility, the synthesized vitamin 4 can exist exclusively in the α-chair form. The geometrical isomer 5 with a free 3β-OH group was also obtained. The analog 5 binds very poorly to VDR, whereas the vitamin 4 is practically devoid of binding ability. Both compounds also show very low HL-60-differentiating activity. When tested in vivo in mice the analogs 4 and 5 exhibit significant calcemic responses with analog 4 showing more activity than analog 5.     

Human orexin/hypocretin receptors form constitutive homo- and heteromeric complexes with each other and with human CB1 cannabinoid receptors

Human OX₁ orexin receptors have been shown to homodimerize and they have also been suggested to heterodimerize with CB₁ cannabinoid receptors. The latter has been suggested to be important for orexin receptor responses and trafficking. In this study, we wanted to assess the ability of the other combinations of receptors to also form similar complexes. Vectors for expression of human OX₁, OX₂ and CB₁ receptors, C-terminally fused with either Renilla luciferase or GFP² green fluorescent protein variant, were generated. The constructs were transiently expressed in Chinese hamster ovary cells, and constitutive dimerization between the receptors was assessed by bioluminescence energy transfer (BRET). Orexin receptor subtypes readily formed homo- and hetero(di)mers, as suggested by significant BRET signals. CB₁ receptors formed homodimers, and they also heterodimerized with both orexin receptors. Interestingly, BRET efficiency was higher for homodimers than for almost all heterodimers. This is likely to be due to the geometry of the interaction; the putatively symmetric dimers may place the C-termini in a more suitable orientation in homomers. Fusion of luciferase to an orexin receptor and GFP² to CB₁ produced more effective BRET than the opposite fusions, also suggesting differences in geometry. Similar was seen for the OX₁–OX₂ interaction. In conclusion, orexin receptors have a significant propensity to make homo- and heterodi-/oligomeric complexes. However, it is unclear whether this affects their signaling. As orexin receptors efficiently signal via endocannabinoid production to CB₁ receptors, dimerization could be an effective way of forming signal complexes with optimal cannabinoid concentrations available for cannabinoid receptors.     

Synthesis,structure–activity relationship studies,and identification of novel 5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine derivatives as dual orexin receptor antagonists. Part 1

A novel series of non-peptidic OX₁R/OX₂R orexin receptor antagonists was prepared by heterocyclic replacement of the dimethoxyphenyl moiety contained in the tetrahydroisoquinoline core skeleton of almorexant. Introduction of substituted imidazole moieties delivered potent dual orexin receptor antagonists with nanomolar potency for hOX₁R and hOX₂R suitable for further fine-tuning. The preparation of these novel orexin receptor antagonists and the outcome of preliminary structure–activity relationship studies are described in this communication.     

Discovery of ORN0829, a potent dual orexin 1/2 receptor antagonist for the treatment of insomnia

Here, we present the design, synthesis, and SAR of dual orexin 1 and 2 receptor antagonists, which were optimized by balancing the antagonistic activity for orexin receptors and lipophilicity. Based on the prototype compound 1, ring construction and the insertion of an additional heteroatom into the resulting ring led to the discovery of orexin 1 and 2 receptor antagonists, which were 3-benzoyl-1,3-oxazinane derivatives. Within these derivatives, (−)-3h enabled a high dual orexin receptor antagonistic activity and a low lipophilicity. Compound (−)-3h exhibited potent sleep-promoting effects at a po dose of 1 mg/kg in a rat polysomnogram study, and optimal PK properties with a rapid T_max and short half-lives in rats and dogs were observed, indicating a predicted human half-life of 0.9–2.0 h. Thus, (−)-3h (ORN0829; investigation code name, TS-142) was selected as a viable candidate and is currently in clinical development for the treatment of insomnia.     

Influence of the peptide chain length on the stability of double-stranded β-helical species of D,L-alternating oligovalines in chloroform solution

The type and distribution of the β-helixes occurring in chloroform solutions of Boc-(L-Val-D-Val)₆-OMe and Boc-(L-Val-D-Val)₈-OMe have been studied by using ¹H-nmr techniques. Right- and left-handed ↑↓β^4.4-helices and left-handed β^5.6-helices occur with the dodecapeptide. β^4.4-Helices of opposite handedness occur also with the hexadecapeptide, but ↑↓β^5.6-helices could not be detected with this oligomer. At equilibrium, at 25°C, the double helix of the dodecapeptide is only moderately populated. These results indicate that increasing the chain length has a destabilizing effect on the ↑↓β^5.6-helices of D ,L -alternating oligovalines in chloroform solution.     

The biological activity of sixteen gibberellin A4 and gibberellin A9 derivatives using seven bioassays

C₂- and C₃-derivatives of GA₄ and GA₉ were tested for biological activity in a range of plant assays. The activity of most of these derivatives was equal to, or less than, that of the parent GAs. However, 2β-methylGA₄ and 2,2-dimethylGA₄ had a higher activity than GA₄ in some assays and the latter derivative was shown to be the most active GA known to date in the Forward oat first leaf, Tan-ginbozu dwarf rice and d₅-maize assays. Two other derivatives, 12,16-cycloGA₉ and 19-desoxyGA₉ had less activity than GA₉.     

Computer-aided de novo ligand design and docking/molecular dynamics study of Vitamin D receptor agonists

1α,25(OH)₂D₃, which is directly mediated by the vitamin D receptor (VDR), exerts a wide variety of biological actions. However, the treatment with 1α,25(OH)₂D₃ is limited because of its side effects. Many analogs and several nonsteroidal mimics with potent biological activity have been reported so far, and our rationale for designing the VDR agonists was on the basis of computer-aided drug design method by de novo design of A-ring and C/D-ring position of 1α,25(OH)₂D₃. Pyrimidine-2,4-diamine was selected as A-ring, and naphthalene and benzene were chosen as C/D-ring. By linking different components, a virtue compound library was obtained. To evaluate the contribution to activity of each component, we performed a series of automated molecular docking operations. Results revealed that the 19-dimethyl derivatives (the C-19 position correspond to C-20 in 1α,25(OH)₂D₃) show the favorable docking affinity to VDR. Moreover, the docking results are quite robust when further validated by molecular dynamics simulations. In addition, by free energy analysis using molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) method, the driving force of the binding between VDR and the ligands is proved to be hydrophobic interactions. Thus, a possible strategy to design new series of VDR agonists is proposed. The strategy can be successfully applied to explain the high potential activities of the 19-dimethyl derivatives. It is anticipated that the findings reported here may provide useful information for designing effective VDR agonists as well as the therapeutic treatment of VDR-related diseases.     

The effect of absolute configuration on activity,subtype selectivity (M3/M2) of 3α-acyloxy-6β-acetoxyltropane derivatives as muscarinic M3 receptor antagonists

Both enantiomers of 3α-acyloxy-6β-acetoxyltropane derivatives 1–4 were prepared respectively and underwent functional studies and radioreceptor binding assays. 6S Enantiomers showed obvious muscarinic M3, M2 antagonistic activity, while the 6R ones elicited little muscarinic activity by functional studies. Besides, the affinity of 6S enantiomers to muscarinic M3 receptors of rat submandibulary gland, M2 receptors of rat left atria was much larger than that of corresponding 6R enantiomers. All these pharmalogical results indicated 6S configuration was favorable for 3α-acyloxy-6β-acetoxyltropane derivatives to bind with muscarinic M3 or M2 receptors and elicited antagonistic activity. Furthermore, the muscarinic M3 activity and subtype selectivity (M3/M2) of 6S enantiomers could be improved by increasing the electron density of carbonyl oxygen or introducing methylene group between the carbonyl and phenyl ring in C-3α position. Understanding the effect of absolute configuration on activity, subtype selectivity (M3/M2) of 3α-acyloxy-6β-acetoxyltropane derivatives will provide the clues for designing muscarinic M3 antagonists with high activity and low side effects or toxicity.