6-[2-(4-Aryl-1-piperazinyl)ethyl]-2H-1,4-benzoxazin-3(4H)-ones: dual-acting 5-HT1 receptor antagonists and serotonin reuptake inhibitors

Investigation of a series 6-[2-(4-aryl-1-piperazinyl)ethyl]-2H-1,4-benzoxazin-3(4H)-ones has led to the discovery of potent 5-HT(1A/1B/1D) receptor antagonists with and without additional SerT affinity. Modulation of the different target activities gave compounds with a range of profiles suitable for further in vivo characterization.     

Role of 5-HT1B/D receptors in canine gastric accommodation: effect of sumatriptan and 5-HT1B/D receptor antagonists

The 5-HT1B/D receptor agonist sumatriptan has been proposed to treat dyspeptic symptoms, because it facilitates gastric accommodation. It is unknown whether stimulation of 5-HT1B/D receptors is involved. Thus, in four conscious dogs, we compared the effects of sumatriptan alone or combined with 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 hydrocloride (GR-127935), N-[3-[3 (dimethylamino)-ethoxy]-4-methoxyphenyl]-2'-[methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)]-[1,1-biphenyl]-4-carboxamide hydrocloride (SB-216641 hydrochloride), or 3-[4-(4-chloro-phenyl)piperazin-1-yl]-1,1-diphenyl-2-propanol hydrochloride (BRL-15572 hydrochloride) (respectively, nonselective 5-HT1B/D, selective 5-HT1B, and selective 5-HT1D receptor antagonists) on gastric accommodation to isobaric distensions performed with a barostat. An exponential and a linear model were used to fit the pressure-volume relationship. An exponential equation fitted the data better than a linear equation. Sumatriptan (800 nmol/kg iv) induced an immediate gastric relaxation (Deltavolume: 112 +/- 44 ml, P < 0.05). After sumatriptan, the pressure-volume curve was shifted toward significantly higher volumes. This effect was fully reversed by GR-127935 or SB-216641 but not by BRL-15572. In conclusion, 5-HT1B receptors seem to play an important role in modulating gastric accommodation to a distending stimulus. An exponential model for pressure-volume curves fits well with the concept of gastric adaptive relaxation.     

Dual 5-HT1A agonists and 5-HT re-uptake inhibitors by combination of indole-butyl-amine and chromenonyl-piperazine structural elements in a single molecular entity

The dual serotonin (5-HT) re-uptake inhibitor and 5-HT(1A) receptor agonist vilazodone was found to increase central serotonin levels in rat brain. In the course of structural modifications of vilazodone 3-[4-[4-(2-oxo-2H-1-benzopyran-6-yl)-1-piperazinyl]-butyl]-1H-indole-5-carbonitrile 8i and its fluorine analogue 6-[4-[4-(5-fluor-3-indolyl)-butyl]-1-piperazinyl]-2H-1-benzopyran-2-one have been identified. These unsubstituted chromenones are equally potent at the 5-HT(1A) receptor and 5-HT transporter. The implementation of nitrogen functionalities in position 3 of the chromenones resulted in compounds acting as agonists at the 5-HT(1A) receptor and as 5-HT re-uptake inhibitors like vilazodone. Ex vivo 5-HT re-uptake inhibition and in vitro 5-HT agonism were determined in the PCA- and GTPgammaS-assay, respectively. The potential of these chromenones to increase central 5-HT levels was measured in microdialysis studies and especially the derivatives 3-[4-[4-(3-amino-2-oxo-2H-chromen-6-yl)-piperazin-1-yl]-butyl]-1H-indole-5-carbonitrile 8f, ethyl (6-[4-[4-(5-cyano-1H-indol-3-yl)-butyl]-piperazin-1-yl]-2-oxo-2H-chromen-3-yl)-carbamate 8h and N-(6-[4-[4-(5-cyano-1H-indol-3-yl)-butyl]-piperazin-1-yl]-2-oxo-2H-chromen-3-yl)-acetamide 8k give rise to rapid development of increased serotonin levels in rat brain cortex, lasting longer than 3h.     

Identification and role of serotonin 5-HT1A and 5-HT1B receptors in primary cultures of rat embryonic rostral raphe nucleus neurons

Autoregulatory mechanisms affecting serotonin [5-hydroxytryptamine (5-HT)] release and synthesis during the early period of development were investigated in dissociated cell cultures raised from embryonic rostral rat rhombencephalon. The presence of 5-HT1A and 5-HT1B receptors in serotoninergic neurons was assessed using binding assays. The involvement of 5-HT1A and 5-HT1B receptors in the control of the synthesis and release of [3H]5-HT was studied using biochemical approaches with several serotoninergic receptor ligands. A mean decrease of 30% in [3H]5-HT synthesis and release was observed in the presence of 5-HT (10(-8) M), the 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), the 5HT1B/1A agonist 5-methoxy-3-(1,2,5,6-tetrahydro-4-pyridinyl)-1H-indole (RU 24969), the 5-HT1B agonist 3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrolo[3,2-b]pyrid-5-one (CP-93,129), and the 5-HT(1D/1B) agonist sumatriptan. Inhibition of 5-HT synthesis and release induced by 8-OH-DPAT was blocked by chiral N-tert-butyl-3-[1-[1-(2-methoxy)phenyl]piperazinyl]-1-phenylpropionam ide dihydrochloride quaternary-hydrate (WAY 100135) (10(7) M) or methyl 4-[4-[4-(1,1,3-trioxo-2H-1,2-benzoisothiazol-2-yl)butyl]-1-p iperazinyl]-1Hindole-2-carboxylate (SDZ 216-525) (10(-7)M), and that of CP-93,129 was blocked by methiothepin (10(-7) M). Paradoxically, extracellular levels of [3H]5-HT increased in the presence of 8-OH-DPAT and RU 24969 at 10(-6) M. 5-HT uptake experiments showed that these two agonists interacted with the 5-HT transporter. 5-HT1 binding sites (620 fmol/mg of protein) and 5-HT1A (482 fmol/mg of protein) and 5-HT1B (127 fmol/mg of protein) receptors were detected in 12-day in vitro cell cultures. Experiments carried out with tetrodotoxin suggested that 5-HT1A receptors are located on nerve cell bodies, whereas 5-HT1B receptors are located on the nerve terminals. We concluded that autoregulatory mechanisms involving 5-HT1A and 5-HT1B autoreceptors are functionally mature in cells from rostral raphe nuclei during the early period of development.     

New 5-HT1A receptor agonists possessing 1,4-benzoxazepine scaffold exhibit highly potent anti-ischemic effects

A series of new 3-substituted-4-(4-aminobutyl)-1,4-benzoxazepin-5(4H)-one derivatives (1-5) which showed a very high affinity for 5-HT1A receptor with good selectivity over dopamine D2 receptor was synthesized. Among these compounds, 3-chloro-4-[4-[4-(2-pyridinyl)-1,2,3,6-tetrahydropyridin-1-yl]butyl]-1,4-benzoxazepin-5(4H)-one (5: SUN N4057) exhibited remarkable neuroprotective activity in a transient middle cerebral artery occlusion (t-MCAO) model.     

gamma-Carbolines: binding at 5-HT5A serotonin receptors

Screening of various agents resulted in the identification of 5-methyl-1,2,3,4-tetrahydro-gamma-carboline (1; K(i)=5,300 nM) as a compound with modest affinity for mouse 5-HT(5A) receptors. Structure-affinity studies were conducted resulting in 5-methyl-2-[3-(4-fluorophenoxy)propyl]-1,2,3,4-tetrahydro-gamma-carboline (17; K(i)=13 nM). Although 17 also binds at 5-HT(2) receptors, it serves as a novel lead for the further development of 5-HT(5A) ligands.     

Influence of the terminal amide fragment geometry in some 3-arylideneindolin-2(1H)-ones on their 5-HT1A/5-HT2A receptor activity

Several 1,4-disubstituted arylpiperazine derivatives of 3-arylideneindolin-2(1H)-one (Z and E isomers) were tested for their 5-HT1A and 5-HT2A receptor activity in vitro and in vivo. It was shown that introduction of 3-arylidene substituents to indolin-2(1H)-one moiety allowed to change the mixed 5-HT1A/5-HT2A receptor ligands to 5-HT2A ones with antagonistic in vivo activity.     

Quinoline- and isoquinoline-sulfonamide derivatives of LCAP as potent CNS multi-receptor-5-HT1A/5-HT2A/5-HT7 and D2/D3/D4-agents: the synthesis and pharmacological evaluation

Two series of arylpiperazinyl-alkyl quinoline-, isoquinoline-, naphthalene-sulfonamides with flexible (13-26) and semi-rigid (33-36) alkylene spacer were synthesized and evaluated for 5-HT(1A), 5-HT(2A), 5-HT(6), 5-HT(7) and selected compounds for D(2), D(3), D(4) receptors. The compounds with a mixed 5-HT and D receptors profile 16 (N-{4-[4-(3-chlorophenyl)-piperazin-1-yl]-butyl}-3-quinolinesulfonamide) and 36 (4-(4-{2-[4-(4-chloro-phenyl)-piperazin-1-yl]-ethyl}-piperidine-1-sulfonyl)-isoquinoline), displaying antagonistic activity at 5-HT(7), 5-HT(2A), D(2) postsynaptic sites, produced antidepressant-like effects in the forced swim test in mice and showed significant anxiolytic activity in the plus-maze test in rats. The lead compound 36, a multi-receptor 5-HT(2A)/5-HT(7)/D(2)/D(3)/D(4) agent, also displayed significant antipsychotic properties in the MK-801-induced hyperlocomotor activity in mice.     

Benzoxazinoid glucosides from Acanthus ilicifolius

From the aerial parts of Acanthus ilicifolius, two benzoxazinoid glucosides, 7-chloro-(2R)-2-O-beta-D-glucopyranosyl-2H-1,4-benzoxazin-3(4H)-one and (2R)-2-O-beta-D-glucopyranosyl-5-hydroxy-2H-1,4-benzoxazin-3(4H)-one have been isolated, together with six known compounds. The structural elucidations were based on the analyses of spectroscopic data.     

In vivo efflux of serotonin in the dorsal raphe nucleus of 5-HT1A receptor knockout mice

In the dorsal raphe nucleus (DR), extracellular serotonin (5-HT) regulates serotonergic transmission through 5-HT1A autoreceptors. In this work we used in vivo microdialysis to examine the effects of stressful and pharmacological challenges on DR 5-HT efflux in 5-HT1A receptor knockout (5-HT1A-/-) mice and their wild-type counterparts (5-HT1A+/+). Baseline 5-HT concentrations did not differ between both lines of mice, which is consistent with a lack of tonic control of 5-HT1A autoreceptors on DR 5-HT release. (R)-(+)-8-Hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT, 0.5 mg/kg) reduced 5-HT levels to 30% of basal values in 5-HT1A+/+ mice, but not in 5-HT1A-/- mice. The selective 5-HT1B receptor agonist 1,4-dihydro-3-(1,2,3,6-tetrahydro-4-pyridinyl)-5H-pyrrolo[3,2-b]pyridin-5-one dihydrochloride (CP 93129, 300 micro m) reduced dialysate 5-HT to the same extent (30-40% of baseline) in the two genotypes, which suggests a lack of compensatory changes in 5-HT1B receptors in the DR of such mutant mice. Both a saline injection and handling for 3 min increased DR dialysate 5-HT in mutants, but not in 5-HT1A+/+ mice. Fluoxetine (5 and 20 mg/kg) elevated 5-HT in a dose-dependent manner in both genotypes. However, this effect was markedly more pronounced in the 5-HT1A-/- mice. The increased responsiveness of the extracellular 5-HT in the DR of 5-HT1A receptor knockout mice reflects a lack of the autoinhibitory control exerted by 5-HT1A autoreceptors.     

Protein kinase C induces phosphorylation and desensitization of the human 5-HT1A receptor

The effects of short-term phorbol ester treatment of CHO cells that stably express 900 fmol of recombinant human serotonin 5-HT1A receptor/mg of protein on coupling to the inhibition of adenylyl cyclase and on phosphorylation of the receptor were studied. Pretreatment of cell monolayers with phorbol 12-myristate 13-acetate (PMA) caused a dose- and time-dependent shift of the half-maximal dose of serotonin (5-HT) required to inhibit membrane adenylyl cyclase (from IC50 approximately 100 nM to approximately 400 nM). This desensitization (shift in IC50) was rapid, occurring with 5 min of pretreatment and being maximal by 10-15 min; it was also dose-dependent, being half-maximal at approximately 300 nM PMA. Desensitization was also induced by sn-dioctanoylglycerol (DiC8) and blocked by the protein kinase C (PKC) inhibitors sphingosine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7). In detached permeabilized cells, PMA pretreatment caused a rapid phosphorylation of immunoprecipitated 5-HT1A receptors, with an approximately 3-4-fold increase that was maximal after 15 min and persisted for 90 min. The phosphorylation occurred at a similar dose of PMA as that which induced desensitization (half-maximal at approximately 300 nM, maximal at 500 nM to 1 microM), could be reproduced by pretreatment with the PKC activators DiC8 or phorbol 12,13-dibutyrate (PDBu), and could be blocked by the PKC inhibitors sphingosine or H-7. The stoichiometry of the phosphorylation was approximately 2 mol of [32P]ATP/mol of receptor, suggesting the involvement at least two of three putative PKC sites within the 5-HT1A receptor. The close concordance between the PKC-induced desensitization and phosphorylation suggests a potential causative link between these two effects of PKC on the human 5-HT1A receptor.     

Synthesis, SAR studies, and evaluation of 1,4-benzoxazepine derivatives as selective 5-HT1A receptor agonists with neuroprotective effect: Discovery of Piclozotan

A new series of 1,4-benzoxazepine derivatives was designed, synthesized, and evaluated for binding to 5-HT1A receptor and cerebral anti-ischemic effect. A lot of compounds exhibited nanomolar affinity for 5-HT1A receptor with good selectivity over both dopamine D2 and alpha1-adrenergic receptors. Among these compounds, 3-chloro-4-[4-[4-(2-pyridinyl)-1,2,3,6-tetrahydropyridin-1-yl]butyl]-1, 4-benzoxazepin-5(4H)-one (50: SUN N4057 (Piclozotan) as 2HCl salt) showed remarkable neuroprotective activity in a transient middle cerebral artery occlusion (t-MCAO) model.     

Synthesis, in vitro and in vivo evaluation of [O-methyl-11C] 2-{4-[4-(3-methoxyphenyl)piperazin-1-yl]-butyl}-4-methyl-2H-[1,2,4]-triazine-3,5-dione: a novel agonist 5-HT1A receptor PET ligand

Synthesis and in vivo evaluation of 2-{4-[4-(3-methoxyphenyl)piperazin-1-yl]-butyl}-4-methyl-2H-[1,2,4]triazine-3,5-dione (5 or MMT), a high affinity and selective serotonin 5-HT1AR agonist PET tracer, are described. GTPgammaS assay shows that MMT is an agonist with an EC50 comparable to 5-HT. Radiolabeling of 5 was achieved in 30% yield (EOS) from desmethyl-MMT (4) with >99% chemical and radiochemical purities and a specific activity >1000 Ci/mmol. PET studies in baboon show that [11C]5 penetrates the blood-brain barrier but, because of low specific binding and fast clearance of radioactivity it is not a suitable PET tracer for the in vivo quantification of 5-HT1AR.     

Nad-299 antagonises 5-HT-stimulated and spiperone-inhibited [35S]GTPgammaS binding in cloned 5-HT1A receptors

In Chinese Hamster Ovary (CHO) cells expressing cloned human 5-hydroxytryptamine1A A (5-HT1A) receptors, (R)-3-N,N-dicyclobutylamino-8-fluoro-[6-3H]-3,4-dihydro-2H-1-benzopyan-5-carboxamide ([3H]NAD-299) exhibited high affinity (Kd = 0.16 nM) and labeled 34% more receptors than 8-hydroxy-2-([2,3-3H]di-n-propylamino)tetralin ([3H]8-OH-DPAT). NAD-299 behaved as a silent antagonist in [35S]GTPgammaS binding similar to N-tert-butyl-3-(4-(2-methoxyphenyl)-piperazin-1-yl)-2-phenylpropanamide (WAY-100635) and (S)-5-fluoro-8-hydroxy-2-(di-n-propylamino)tetralin ((S)UH-301). 5-HT and 5-carboxamidotryptamine (5-CT) stimulated [35S]GTPgammaS binding 2.5-fold while spiperone and methiothepin inhibited [35S]GTPgammaS binding 1.4-fold. Furthermore, NAD-299 antagonised both the 5-HT stimulated and the spiperone inhibited [35S]GTPgammaS binding to basal levels. The KiL/KiH ratios for spiperone (0.66), methiothepin (0.39), WAY-100635 (0.32), (S)UH-301 (0.94), NAD-299 (1.29), NAN-190 (1.23), (S)pindolol (5.85), ipsapirone (13.1), buspirone (24.6), (+/-)8-OH-DPAT (47.3), flesinoxan (55.8), 5-HT (200) and 5-CT (389) correlated highly significantly with the intrinsic activity obtained with [35S] GTPgammaS (r = 0.97).     

Relative selectivity of some conformationally constrained tryptamine analogs at 5-HT1, 5-HT1A and 5-HT2 recognition sites

In an attempt to define pharmacophoric differences between 5-HT1, 5-HT1A and 5-HT2 recognition sites, a number of rigid analogs were studied and compared to analogous free chain tryptamines. Racemic partial ergolines RU 27849 and RU 28306 showed reduced potency at all 5-HT1 sites, but were at least equipotent to analogous tryptamines at the 5-HT2 site. A nonergoline-like constrained analog of tryptamine was similar in potency to RU 27849 at all 5-HT1 sites, but showed a 4-fold enhancement in potency over RU 27849 and tryptamine at the 5-HT2 site. At all 3 sites, 3-(tetrahydropyridyl) indoles (unless substituted at the indole 2-position) were the most potent rigid analogs studied and represent the most promising class for the development of selective compounds.     

Effect of hypothyroidism on 5-HT1A-, 5-HT2A-receptors and serotonin transporter in the rat brain

Effects of thyroid hormone deficiency on 5-HT1A receptors, 5-HT2A receptors and serotonin transporter in the brain were studied in thyroidectomised Wistar rats receiving an iodine-free diet and receiving 15 micrograms/kg of thyroxine for 21 days. Binding of 3H-8-OH-DPAT to 5-HT1A receptors and 3H-cytalopram to serotonin transporter were unchanged in hypothyroid rats as compared to the control. 3H-ketanserin binding to 5-HT2A receptors was significantly decreased in the frontal cortex in hypothyroid rats. The cortical 3H-ketanserin binding in thyroidectomised rats was normalised after thyroxine replacement. The data suggest that the decrease in the cortical 5-HT2A receptors is the main consequence of impairing effect of hypothyroidism on serotonin neurotransmission.     

Synthesis and biological evaluation of a novel structural type of serotonin 5-HT3 receptor antagonists

A series of novel 3-substituted quinoxalin-2-carboxamides were designed as per the pharmacophoric requirement for 5-HT(3) receptor antagonists and prepared by microwave irradiation and also by conventional method. The compounds were characterized by spectral data (IR, (1)H NMR, and MS) and the purity was ascertained by microanalysis. The synthesized compounds were evaluated for 5-HT(3) antagonisms in longitudinal muscle-myenteric plexus preparation from guinea pig ileum against 5-HT(3) agonist, 2-methyl-5-HT. Among the test compounds, N-{3-[(4-methylpiperazin-1-yl)methyl]-4-hydroxyphenyl}-3-methoxyquinoxalin-2-carboxamide 4e showed most favorable 5-HT(3) receptor antagonism.     

2,3,4,5-tetrahydro- and 2,3,4,5,11,11a-hexahydro-1H-[1,4]diazepino[1,7-a]indoles: new templates for 5-HT(2C) agonists

The design and synthesis of the novel 2,3,4,5-tetrahydro-1H-[1,4]diazepino[1,7-a]indole 5 is described. This azepinoindole has excellent affinity for 5-HT(2C) (K(i) 4.8 nM) and modest selectivity over 5-HT(2A) ( approximately 4-fold). Several N- and C(11)-substituted analogues of 5 were prepared, as were a number of biaryl indoline derivatives. The anxiolytic potential for the azepinoindole template 5 is demonstrated by activity in a mouse shock-aggression assay.     

Serotonin 5-HT1D and 5-HT1A Receptors Respectively Mediate Inhibition of Glutamate Release and Inhibition of Cyclic GMP Production in Rat Cerebellum In Vitro

Abstract: The K⁺-evoked overflow of endogenous glutamate from cerebellar synaptosomes was inhibited by serotonin [5-hydroxytryptamine (5-HT); pD₂ = 8.95], 8-hydroxy-2-(di- n -propylamino)tetralin (8-OH-DPAT; pD₂ = 7.35), and sumatriptan (pD₂ = 8.43). These inhibitions were prevented by the selective 5-HT_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 (GR-127935). The three agonists tested also inhibited the cyclic GMP (cGMP) response provoked in slices by K⁺ depolarization; pD₂ values were 9.37 (5-HT), 9.00 (8-OH-DPAT), and 8.39 (sumatriptan). When cGMP formation was elevated by directly activating glutamate receptors with NMDA or α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), the inhibition of the cGMP responses displayed the following pattern: 5-HT (pD₂ values of 8.68 and 8.72 against NMDA and AMPA, respectively); 8-OH-DPAT (respective pD₂ values of 9.15 and 9.00); sumatriptan (0.1 µ M ) was ineffective. The 5-HT_1A receptor antagonist ( S )-(+) N-tert -butyl-3-[4-(2-methoxyphenyl)piperazin-1-yl]-2-phenylpropionamide dihydrochloride [(+)-WAY 100135] did not prevent the inhibition of glutamate release by 5-HT but blocked the inhibition by 8-OH-DPAT of the NMDA/AMPA-evoked cGMP responses. It is suggested that presynaptic 5-HT_1D receptors mediate inhibition directly of glutamate release and indirectly of the cGMP responses to the released glutamate; on the other hand, activation of (postsynaptic) 5-HT_1A receptors causes inhibition of the cGMP responses linked to stimulation of NMDA/AMPA receptors.     

Chemical constituents from the roots of Acanthus ilicifolius var. xiamenensis

Chemical investigation of Acanthus ilicifolius var. xiamenensis led to the isolation of eleven compounds, and their structures were identified to be 2-benzoxazolinone (1), 2-hydroxy-2H-1,4-benzoxazin-3(4H)-one (2), (2R)-2-O-β-d-glucopyranosyl-2H-1,4-benzox azin-3(4H)-one (3), (2R)-2-O-β-d-glucopyranosyl-4-hydroxy-2H-1,4-benzoxazin-3(4H)-one (4), (2R)-2-O-β-d-glucopyranosyl-7-hydroxy-2H-1,4-benzoxazin-3(4H)-one (5), lyoniside (6), 3′-methoxy-luteolin-7-O-β-d-glucopyranoside (7), β-sitosterol-3-O-β-d-glucopyranoside (8), stigmasterol octadecanoate (9), β-sitosterol octadecanoate (10), stigmasterol-3-O-β-d-glucopyranoside (11) on the basis of mass and NMR spectra. This is the first report on the occurrence of compound 6 and 7 in Acanthaceae. This work also represents the first phytochemical work on the roots of A. ilicifolius var. xiamenensis.