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 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).     

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 III: Role of the 14-hydroxy and the 3-methoxy groups in antagonistic activity toward the orexin 1 receptor in YNT-707 derivatives lacking the 4,5-epoxy ring

Morphinan derivatives lacking the 4,5-epoxy ring were synthesized to examine the participation of the 14-OH group, the 3-OMe group, and the aromaticity of the A-ring in the activity and selectivity for the orexin 1 receptor (OX₁R). The assay results and the conformational analyses of the 14-dehydrated and 14-H derivatives suggested that the orientations of the 6-amide side chain and the 17-benzenesulfonyl group would play important roles in the activity for OX₁R. In the 6β-derivatives, removal of the 3-OMe group and the reduction of the A-ring significantly decreased the activity toward the OX₁R, but these changes did not affect the 6α-derivatives. These results indicate that the 3-OMe group and the A-ring would be essential structural moieties for the 6β-derivatives.     

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.     

Hydrolytic instability of the important orexin 1 receptor antagonist SB-334867: Possible confounding effects on in vivo and in vitro studies

SB-334867 has been an important ligand for the study of the orexin 1 (OX1) receptor due to its high OX1/OX2 selectivity and bioavailability. This ligand however, contains a 2-methylbenzoxazole ring system which is known to undergo hydrolysis, particularly under acidic or basic conditions. The possibility that SB-334867 would be susceptible to significant hydrolysis was evaluated in various formulations and in the solid state. SB-334867 was found to be unstable under conditions commonly employed to prepare stock solutions for in vitro and in vivo studies. In addition, and most alarmingly, the hydrochloride salt of SB-334867 was found to quantitatively decompose to an OX1-inactive product even in the solid state. These findings combine to suggest that studies using SB-334867 (and any other 2-methylbenzoxazole-containing compound) should be performed with great care to avoid the confounding effects of the rapid hydrolytic decomposition of this susceptible structure.     

Orexin receptor type-1 antagonist SB-334867 inhibits the development of morphine analgesic tolerance in rats

Herein the effect of orexin receptor type-1 antagonist SB-334867 on the development of tolerance to analgesic effects of morphine was studied in rats. To incite tolerance, morphine sulfate was injected intraperitoneally (i.p., 10mg/kg) once a day for 7 days. The tail flick test was used to evaluate antinociceptive effects of the morphine. A selective OxR1 receptor antagonist, SB-334867, was microinjected (i.c.v.) into the right cerebral ventricle (10 μg/10 μl) immediately before each morphine injection. Repeated morphine application resulted in tolerance to morphine analgesic effects as a decreasing trend during 7 days. Also, repeated administration of SB-334867 (i.c.v.) alone was without significant effect on the nociception as compared to control. Microinjection of SB-334867 prior to each morphine injection inhibited the development of tolerance, so that the analgesic effects of morphine were significantly higher in SB-334867 plus morphine treated rats than that of vehicle plus morphine treated ones on days 4-7. It is concluded that orexin receptor type-1 might be involved in the development of tolerance to morphine analgesic effects.     

N-4-Substituted-benzyl-N'-tert-butylbenzyl thioureas as vanilloid receptor ligands: investigation on the role of methanesulfonamido group in antagonistic activity

A series of N-4-substituted-benzyl-N'-tert-butylbenzyl thioureas were prepared for the study of their agonistic/antagonistic activities to the vanilloid receptor in rat DRG neurons. Their structure-activity relationship reveals that not only the two oxygens and amide hydrogen of sulfonamido group, but also the optimal size of methyl in methanesulfonamido group play an integral role for the antagonistic activity on vanilloid receptor.     

Synthetic studies on himbacine,a potent antagonist of the muscarinic M2 subtype receptor. Part 2: synthesis and muscarinic M2 subtype antagonistic activity of the novel himbacine congeners modified at the C-3 position of lactone moiety

With an aim to disclose the convergency and flexibility of our previously explored synthetic route to natural himbacine 1, and moreover, to clarify some novel aspects of the structure-activity relationships of 1, we prepared various structural types of novel himbacine congeners, 3-demethylhimbacine (3-norhimbacine) 2 and 4-epi-3-demethylhimbacine (4-epi-3-norhimbacine) 4-epi-2 and their enantiomers (ent-2 and ent-4-epi-2), 11-methylhimbacine 3, and 3-epihimbacine 4 in optically pure forms by employing our methodology. All of the synthesized congeners correspond to the compounds modified at the C-3 position of gamma-lactone moiety involved in 1. Among these congeners, 3-demethylhimbacine (3-norhimbacine) 2 was found to exhibit more potent muscarinic M(2) receptor binding affinity than natural 1.     

Bioisosteric replacement of the hydrazide pharmacophore of the cannabinoid-1 receptor antagonist SR141716A. Part I: Potent, orally-active 1,4-disubstituted imidazoles

A new series of CB₁ receptor antagonists incorporating an imidazole-based isosteric replacement for the hydrazide moiety of rimonabant (SR141716) is disclosed. Members of this imidazole series possess potent/selective binding to the rCB₁ receptor and exhibit potent hCB₁ functional activity. Isopropyl analog 9a demonstrated activity in the tetrad assay and was orally-active in a food intake model.     

The emerging role of trace amine-associated receptor 1 in the functional regulation of monoamine transporters and dopaminergic activity

It is now recognized that trace amine associated-receptor 1 (TAAR1) plays a functional role in the regulation of brain monoamines and the mediation of action of amphetamine-like psychostimulants. Accordingly, research on TAAR1 opens the door to a new avenue of approach for medications development to treat drug addiction as well as the spectrum of neuropsychiatric disorders hallmarked by aberrant regulation of brain monoamines. This overview focuses on recent studies which reveal a role for TAAR1 in the functional regulation of monoamine transporters and the neuronal regulatory mechanisms that modulate dopaminergic activity.     

Progesterone reduces the effect of the serotonin 1B/1D receptor antagonist, GR 127935, on lordosis behavior

Ovariectomized rats were hormonally primed with 10 μg estradiol benzoate or with estradiol benzoate plus 500 μg progesterone. Rats received a bilateral infusion with 200 ng of the 5-HT_1B/1D receptor antagonist, N-[4-methoxy-3-(4-methyl-1-piperazinyl)phenyl]-2′-methyl-4′-(5-methyl-1,2,4-oxadiazol-3-yl)-1-1′-biphenyl-4-carboxamide hydrochloride (GR 127935), into the ventromedial nucleus of the hypothalamus (VMN), followed by a 5 min restraint or home cage experience. In estrogen-primed females that had experienced minimal handling between ovariectomy and use in the experiment, infusion with the water vehicle transiently inhibited lordosis behavior, and the 5-HT_1B/1D receptor antagonist amplified this inhibition. There were no effects in rats hormonally primed with estrogen and progesterone. Handling for two days before the experiment reduced the effects of the infusions in estrogen-primed rats. However, when a 5 min restraint experience followed infusion with GR 127935, there was a significant decline in lordosis behavior that persisted for 10 to 15 min after the experience. Regardless of the prior experience or type of infusion, the addition of progesterone to the hormonal priming completely prevented the lordosis inhibition. These findings are consistent with prior evidence that progesterone protects against the inhibitory effects of a 5 min restraint experience on lordosis behavior. Moreover, these are the first experiments to demonstrate an inhibitory effect of a selective 5-HT_1B/1D receptor antagonist in the VMN on lordosis behavior of estrogen primed, but not estrogen and progesterone primed, ovariectomized rats.     

Structure-activity relationship studies on niphimycin, a guanidylpolyol macrolide antibiotic. Part 1: The role of the N-methyl-N'-alkylguanidinium moiety

Several N-methyl-N'-alkylguanidinium derivatives were synthesized and used as simplified analogues of niphimycin (NM), a guanidylpolyol macrolide, in structure-activity relationship studies. The C16-alkylated derivative exerted fungicidal activity by directly damaging the fungal plasma membrane and inducing oxidative stress in a manner similar to niphimycin. These results indicate that the N-methyl-N'-alkylguanidinium moiety is required for antifungal activity by NM.     

Studies on the chemistry of thienoanellated O,N- and S,N-containing heterocycles. Part 30: synthesis and pharmacological properties of thieno[2,3-b][1,4]thiazines with potential vasopressin receptor antagonistic activity

A series of new nonpeptide vasopressin antagonists with a 6-ethyl-thieno[2,3-b][1,4]thiazine or 6-benzyl-thieno[2,3-b][1,4]thiazine skeleton and structural modifications of the aryl side chain were synthesized in this study. The effects on guinea pig heart and smooth muscle preparations were investigated. In the presence of AVP the compounds showed an antagonistic effect. The compounds did not change spontaneous rate in right atria and exerted a slight but not significant negative inotropic effect in papillary muscles. The relaxing effect on vascular smooth muscle and terminal ileum was far more pronounced. Generally the relaxing effect on terminal ilea was more potent maybe due to difference in V1a receptor density. Our results demonstrate that compounds with an ethyl group in position six on the thienothiazine ring (14, 16, 18 and 22) exerted the most potent relaxing activity in terminal ilea, whereas compounds with a phenyl ring in position six reduced this effect.     

A randomized, placebo controlled, double masked phase IB study evaluating the safety and antiviral activity of aprepitant, a neurokinin-1 receptor antagonist in HIV-1 infected adults

Background

Neurokinin-1 receptor (NK1R) antagonists have anti-HIV activity in monocyte-derived macrophages, decrease CCR5 expression and improve natural killer cell function ex vivo. Aprepitant is a NK1R antagonist approved by FDA as an antiemetic.

Methods

We conducted a phase IB randomized, placebo controlled, double masked study to evaluate the safety, antiviral activity, pharmacokinetics and immune-modulatory effects of aprepitant in HIV-infected adults not receiving antiretroviral therapy, with CD4+ cell count ≥350 cells/mm³ and plasma viral load ≥2,000 copies/ml. Subjects were stratified by viral load (< vs. ≥20,000 copies/ml) and randomized within each stratum to receive aprepitant at 125 mg QD(Low), or 250 mg QD(High), or placebo(PL) for 14 days, and followed for 42 days.

Results

Thirty subjects were randomized and 27 completed treatment (9, 8, 10 subjects in 125 (Low), 250 (High), and PL groups). 63% were male; 37% white; mean (SD) age 43 (9.3) years. Geometric mean baseline viral load (copies/ml) for Low, High, and PL was 15,709, 33,013, and 19,450, respectively. Mean (95%CI) change in log10 viral load at day 14 for Low, High, and PL was −0.02(−0.24,+0.20), −0.05(−0.21,+0.10), and +0.04(−0.08,+0.16), respectively. The number of subjects with AEs was 4(44.4%), 5(62.5%), and 1(10%) for Low, High, and PL. No Grade 4 AEs occurred.

Conclusions

Adverse events of aprepitant were more common in the treated groups. At the dose used in this two-week phase IB study, aprepitant showed biological activity, but no significant antiviral activity.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00428519","term_id":"NCT00428519"}}NCT00428519     

The androgen receptor antagonist enzalutamide induces apoptosis,dysregulates the heat shock protein system,and diminishes the androgen receptor and estrogen receptor β1 expression in prostate cancer cells

Enzalutamide's accepted mode of action is by targeting the androgen receptor's (AR) activity. In clinical practice, enzalutamide demonstrates a good benefit-risk profile for the treatment of advanced prostate cancer (PC), even after poor response to standard antihormonal treatment. However, since both, well-established antiandrogens and enzalutamide, target AR functionality, we hypothesized that additional unknown mechanisms might be responsible for enzalutamide's superior anticancer activity. In the current study, PC cells were incubated with enzalutamide and enzalutamide-dependent modulation of apoptotic mechanisms were assessed via Western blot analysis, TDT-mediated dUTP-biotin nick end-labeling assay, and nuclear morphology assay. Alterations of heat shock protein (HSP), AR, and estrogen receptor (ER) expression were examined by Western blot analysis. Enzalutamide attenuated the proliferation of PC cells in a time- and dose-dependent manner. In the presence of enzalutamide, apoptosis occurred which was shown by increased BAX expression, decreased Bcl-2 expression, nuclear pyknosis, and genomic DNA fragmentation. Moreover, enzalutamide inhibited the expression of HSPs primarily involved in steroid receptor stabilization and suppressed AR and ERβ1 expression. This study demonstrates for the first time that enzalutamide treatment of PC cells triggers varying molecular mechanisms resulting in antiproliferative effects of the drug. In addition to the well-characterized antagonistic inhibition of AR functionality, we have shown that enzalutamide also affects the intracellular synthesis of steroid receptor-associated HSPs, thereby diminishing the expression of AR and ERβ1 proteins and inducing apoptotic pathways. According to an indirect attenuation of HSP-associated factors such as steroid receptors, endometrial carcinoma, uterine leiomyosarcoma, and mamma carcinoma cells also demonstrated inhibited cell growth in the presence of enzalutamide. Our data, therefore, suggest that enzalutamide's high efficacy is at least partially independent of AR and p53 protein expression, which are frequently lost in advanced PC.     

Chemical modification of islet-activating protein,pertussis toxin: Essential role of free amino groups in its lymphocytosis-promoting activity

Chemical modification of amino groups in the molecule of islet-activating protein (IAP), pertussis toxin, resulted in differential modification of biological activities of the toxin estimated in vivo with rats. Acetamidination of ε-amino groups of 50% (or more) of lysine residues in the IAP molecule totally abolished the lymphocytosis-promoting activity, but exerted no effects on the epinephrine-hyperglycemia inhibitory activity, of the toxin. Both activities were abolished by acylation of 50% or more of the amino groups probably due to the destruction of the toxin's quarternary structure. In contrast, the subunit assembly of IAP was maintained after exhaustive acetamidination of its lysine residues. The ADP-ribosyltranferase (or NAD-glycohydrolase) activity of the A-protomer (the biggest subunit) of IAP, which is responsible for the principal action of the toxin, enhancing insulin secretory responses and thereby inhibiting epinephrine hyperglycemia, was not affected by acetamidination of lysine residues. Thus, the A-protomer moiety of IAP is not directly involved in, but the amino groups of lysine residues in other subunits are selectively essential for, the development of the toxin-induced lymphocytosis.     

The role of tyrosine kinase activity in endocytosis, compartmentation, and down-regulation of the epidermal growth factor receptor.

Occupancy-induced down-regulation of cell surface epidermal growth factor (EGF) receptors attenuates signal transduction. To define mechanisms through which down-regulation of this class of growth factor receptors occurs, we have investigated the relative roles of ligand-induced internalization and recycling in this process. Occupied, kinase-active EGF receptors were internalized through a high affinity, saturable endocytic system at rates up to 10-fold faster than empty receptors. In contrast, full length EGF receptors lacking tyrosine kinase activity underwent internalization at a rate independent of occupancy. This "kinase-independent" internalization rate appeared to reflect constitutive receptor internalization since it was similar to the internalization rate of both receptors lacking a cytoplasmic domain and of antibodies bound to empty receptors. EGF internalized by either kinase-active or kinase-inactive receptors was efficiently recycled and was found within endosomes containing recycling transferrin receptors. However, targeting of internalized receptors to lysosomes did not require receptor kinase activity. All receptors that displayed ligand-induced internalization also underwent down-regulation, indicating that the proximal cause of down-regulation is occupancy-induced endocytosis. Tyrosine kinase activity greatly enhances this process by stabilizing receptor association with the endocytic apparatus.