Synthesis and biological evaluation of benzoisothiazole derivatives possessing N,N-dimethylformimidamide group as 5-HT₆ receptor antagonists

A series of novel N,N-dimethyl-N'-(5-(Ar-sulfonamido) benzo[d]isothiazol-3-yl)formimidamides was designed and synthesized as 5-HT(6) ligands. Here N,N-dimethyl formimidamides was used as a replacement for an aminoethyl moiety. In vitro functional assays demonstrated compounds 9b and 9i significantly inhibited the 5-HT-induced Ca(2+) increases (9b; IC(50)=0.36 μM and 9i; IC(50)=0.44 μM), indicating that 9b and 9i were potent 5-HT(6) receptor antagonists. Compounds 9i also showed good selectivity on the 5-HT(6) over 5-HT(4) and 5-HT(7) receptors.     

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.     

Discovery of 3-aryl-3-methyl-1H-quinoline-2,4-diones as a new class of selective 5-HT6 receptor antagonists

A 5,7-dichloro-3-phenyl-3-methyl-quinoline-2,4-dione (11a) has been identified in a random screen as a lead for 5-HT(6) antagonist. During the lead optimization process, several analogs were synthesized and their biological activities were investigated. Within this series, several compounds display high binding affinity and selectivity for the 5-HT(6) receptor. In particular, 3-(4-hydroxyphenyl)-3-methyl-quinoline-2,4-dione (12f) exhibits high affinity (K(i)=12.3 nM) for 5-HT(6) receptor with good selectivity over other serotonin and dopamine (D(1)-D(4)) receptor subtypes. In a functional adenylyl cyclase stimulation assay, this compound exhibited considerable antagonistic activity (IC(50)=0.61 microM).     

Arene- and quinoline-sulfonamides as novel 5-HT7 receptor ligands

Novel arene- and quinolinesulfonamides were synthesized using different solutions and a solid-support methodology, and were evaluated for their affinity for 5-HT(1A), 5-HT(2A), 5-HT(6), and 5-HT(7) receptors. Compound 54 (N-Ethyl-N-[4-(1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinolin-2-yl)butyl]-8-quinolinesulfonamide) was identified as potent 5-HT(7) antagonist (K(i)=13 nM, K(B)=140 nM) with good selectivity over 5-HT(1A), 5-HT(2A), 5-HT(6) receptors. In the FST in mice, it reduced immobility in a manner similar to the selective 5-HT(7) antagonist SB-269970.     

Cyclothiazide binding to functionally active AMPA receptor reveals genuine allosteric interaction with agonist binding sites

The agonist, [3H](-)[S]-1-(2-amino-2-carboxyethyl)-5-fluoro-pyrimidine-2,4-dione ([3H](S)F-Willardiine) binding to functional alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors of resealed plasma membrane vesicles and nerve endings freshly isolated from the rat cerebral cortex displayed two binding sites (K(D1)=33+/-7 nM, B(MAX1)=1.6+/-0.3 pmol/mg protein, K(D2)=720+/-250 nM and B(MAX2)=7.8+/-4.0 pmol/mg protein). The drug which impairs AMPA receptor desensitisation, 6-chloro-3,4-dihydro-3-(2-norbornene-5-yl)-2H-1,2,4-benzothiadiazine-7-sulphonamide-1,1-dioxide (cyclothiazide, CTZ) fully displaced the [3H](S)F-Willardiine binding at a concentration of 500 microM. In the presence of 100 microM CTZ (K(I(CTZ))=60+/-6 microM), both the antagonist [3H]-1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo(F)quinoxaline-7-sulfonamide ([3H]NBQX: K(D)=24+/-4 nM, B(MAX)=12.0+/-0.1 pmol/mg protein) and the high-affinity agonist binding showed similar affinity reduction ([3H](S)F-Willardiine: K(D)=140+/-19 nM, B(MAX)=2.9+/-0.5 pmol/mg protein; [3H]NBQX: K(D)=111+/-34 nM, B(MAX)=12+/-3 pmol/mg protein). To disclose structural correlates underlying genuine allosteric binding interactions, molecular mechanics calculations of CTZ-induced structural changes were performed with the use of PDB data on extracellular GluR2 binding domain dimeric crystals available by now. Hydrogen-bonding and root mean square (rms) values of amino acid residues recognising receptor agonists showed minor alterations in the agonist binding sites itself. Moreover, CTZ binding did not affect dimeric subunit structures significantly. These findings indicated that the structural changes featuring the non-desensitised state could possibly occur to a further site of the extracellular GluR2 binding domain. The increase of agonist efficacy on allosteric CTZ binding may be interpreted in terms of a mechanism involving AMPA receptor desensitisation sequential to activation.     

Studies toward the discovery of the next generation of antidepressants. Part 6: Dual 5-HT1A receptor and serotonin transporter affinity within a class of arylpiperazinyl-cyclohexyl indole derivatives

Based on the previously reported discovery lead, 3-(cis-4-(4-(1H-indol-4-yl)piperazin-1-yl)cyclohexyl)-5-fluoro-1H-indole (2), a series of related arylpiperazin-4-yl-cyclohexyl indole analogs were synthesized then evaluated as 5-HT transporter inhibitors and 5-HT(1A) receptor antagonists. The investigation of the structure-activity relationships revealed the optimal pharmacophoric elements required for activities in this series. The best example from this study, 5-(piperazin-1-yl)quinoline analog (trans-20), exhibited equal binding affinities at 5-HT transporter (K(i)=4.9nM), 5-HT(1A) receptor (K(i)=6.2nM) and functioned as a 5-HT(1A) receptor antagonist.     

Binding of isotryptamines and indenes at h5-HT6 serotonin receptors

To determine if the indolic nitrogen atom is required for the binding of N(1)-benzyltryptamines at h5-HT(6) serotonin receptors, several isotryptamines and indene analogs were examined. The affinity of 3-benzyl-N(1)-(N,N-dimethylaminoethyl)indole (5, K(i)=32nM) and 1-benzyl-3-(N,N-dimethylaminoethyl)indene (11, K(i)=3nM) indicates that the indolic nitrogen atom is not essential for binding.     

N1-benzenesulfonylgramine and N1-benzenesulfonylskatole: novel 5-HT6 receptor ligand templates

1-Benzenesulfonyl-5-methoxy-N,N-dimethyltryptamine (3; K(i)=2.3 nM) is a 5-HT(6) receptor antagonist; removal of the 5-methoxy group (i.e., 6; K(i)=4.1 nM) has little impact on receptor affinity. In the present study, it is shown that the aminomethyl portion of 6 can be shortened to gramine analogue 10a (K(i)=3.1 nM); a related skatole derivative 11b (K(i)=12 nM) also binds with high affinity indicating that the aminoethyl portion of the tryptamines is not required for binding. Compounds 10a and 11b represent members of novel classes of 5-HT(6) antagonists.     

The influence of substitution at aromatic part of 1,2,3,4-tetrahydroisoquinoline on in vitro and in vivo 5-HT(1A)/5-HT(2A) receptor activities of its 1-adamantoyloaminoalkyl derivatives.

Further structure-activity relationship (SAR) studies with the 1,2,3,4-tetrahydroisoquinoline (THIQ) class of 5-HT(1A) ligands led to the synthesis of new 1-adamantoyloaminoalkyl derivatives. The impact of substituent variations in the aromatic part of THIQ moiety on 5-HT(1A) and 5-HT(2A) receptor affinities, as well as in vivo functional properties of the investigated compounds were discussed. It was found that those modifications reduced the binding affinity for 5-HT(1A) receptors (in comparison with unsubstituted THIQ derivatives); however, the majority of new compounds still remained potent 5-HT(1A) ligands (K(i)=4.9-46 nM) and most of them showed features of partial agonists of postsynaptic 5-HT(1A) receptors. At the same time, their 5-HT(2A) receptor affinity was slightly increased (K(i)=40-1475 nM), which resulted in a loss of 5-HT(2A)/5-HT(1A) selectivity. 5-Br,8-OCH3 derivative-the most potent, mixed 5-HT(1A)/5-HT(2A) ligand-produced activation of presynaptic 5-HT(1A) receptors and showed properties of a 5-HT(2A) receptor antagonist.     

Benzimidazole derivatives. Part 5: design and synthesis of new benzimidazole-arylpiperazine derivatives acting as mixed 5-HT1A/5-HT3 ligands

A series of new mixed benzimidazole-arylpiperazine derivatives were designed by incorporating in general structure III the pharmacophoric elements of 5-HT(1A) and 5-HT(3) receptors. Compounds 1-11 were synthesized and evaluated for binding affinity at both serotoninergic receptors, all of them exhibiting high 5-HT(3)R affinity (K(i)=10-62nM), and derivatives with an o-alkoxy group in the arylpiperazine ring showing nanomolar affinity for the 5-HT(1A)R (K(i)=18-150nM). Additionally, all the synthesized compounds were selective over alpha(1)-adrenergic and dopamine D(2) receptors (K(i)>1000-10,000nM). Compound 3 was selected for further pharmacological characterization due to its interesting binding profile as mixed 5-HT(1A)/5-HT(3) ligand with high affinity for both receptors (5-HT(1A): K(i)=18.0nM, 5-HT(3): K(i)=27.2nM). In vitro and in vivo findings suggest that this compound acts as a partial agonist at 5-HT(1A)Rs and as a 5-HT(3)R antagonist. This novel mixed 5-HT(1A)/5-HT(3) ligand was also effective in preventing the cognitive deficits induced by muscarinic receptor blockade in a passive avoidance learning test, suggesting a potential interest in the treatment of cognitive dysfunction.     

1,2,4-Benzothiadiazine derivatives as alpha1 and 5-HT1A receptor ligands

A series of new 1,2,4-benzothiadiazine derivatives with an arylpiperazine mojety linked at position 3 of the heterocyclic ring were synthesized and assessed for their pharmacological profiles at alpha(1)-adrenoceptor subtypes (alpha(1A), alpha(1B) and alpha(1D)) by functional experiments and by in vitro binding studies at human cloned 5-HT(1A) receptor. Compound 1 was identified as a novel alpha(1D) antagonist (pK(b)alpha(1D)=7.59; alpha(1D)/alpha(1A)>389; alpha(1D)/alpha(1B)=135) with high selectivity over 5-HT(1A) receptor (5-HT(1A)/alpha(1D)<0.01), while compound 6, a 3,4-dihydro-derivative, was characterized as a novel 5-HT(1A) receptor ligand, highly selective over alpha(1D)-adrenoceptor subtype (pK(i)5-HT(1A)=8.04; 5-HT(1A)/alpha(1D)=1096). Further pharmacological studies demonstrated that 6 is a partial agonist at 5-HT(1A) receptor (E(max)=23, pD(2)=6.92).     

Binding of methoxy-substituted N1-benzenesulfonylindole analogs at human 5-HT6 serotonin receptors

Comparison of several amine-substituted and methoxy-substituted analogs of N1-(4-aminobenzene)sulfonylindole suggests that these substituents might contribute to the 5-HT6 serotonin receptor affinity of these agents via their electronic effect on the indolic nucleus. Their 1,2,3,4-tetrahydrocarbazole counterparts behave differently.     

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).     

Structure-activity relationships of 2-chloro-N6-substituted-4'-thioadenosine-5'-N,N-dialkyluronamides as human A3 adenosine receptor antagonists

On the basis of potent and selective A(3) adenosine receptor (AR) antagonist, 2-chloro-N(6)-(3-iodobenzyl)-4'-thioadenosine-5'-N,N-dimethyluronamide, structure-activity relationships were studied for a series of 5'-N,N-dialkyluronamide derivatives, synthesized from D-gulonic gamma-lactone. From this study, it was revealed that removal of the hydrogen bond-donating ability of the 5'-uronamide was essential for the pure A(3)AR antagonism. 5'-N,N-Dimethyluronamide derivatives exhibited higher binding affinity than larger 5'-N,N-dialkyl or 5'-N,N-cycloalkylamide derivatives, indicating that steric factors are crucial in binding to the human A(3)AR. A N(6)-(3-bromobenzyl) derivative 6c (K(i)=9.32 nM) exhibited the highest binding affinity at the human A(3)AR with very low binding affinities to other AR subtypes.     

Cloning, functional expression, and mRNA tissue distribution of the rat 5-hydroxytryptamine1A receptor gene

G protein-coupled receptors comprise a family of genes that share significant sequence similarity. We have screened a rat genomic library under low stringency hybridization conditions with the coding portion of the hamster beta 2-adrenergic receptor gene to isolate new members of this gene family. We show that one of these clones, clone D, codes for a 5-hydroxytryptamine1A (5-HT1A) binding site since: 1) it possesses an intronless open reading frame encoding a protein with seven putative transmembrane domains and 89% amino acid identity with the human 5-HT1A receptor (G21); 2) when transfected into Ltk- cells, it expresses a ligand-binding site with the pharmacology of the 5-HT1A receptor subtype, including 5-HT- and spiroxatrine-displaceable binding of 8-hydroxy-(2-(N,N-di[2,3-3H]propylamino)-1,2,3,4-tetrahydronaphthalene (KH = 0.8 nM). We further show that clone D encodes a functional receptor because its binding site interacts with G proteins and because it mediates agonist-induced inhibition of basal and stimulated cAMP accumulation in transfected GH4C1 pituitary cells. Finally, we have analyzed the tissue distribution of 5-HT1A receptor mRNA in rat brain and have found that 5-HT1A mRNA is present with the expected distribution of the 5-HT1A receptor (highest in septum and hippocampus) but is present as three RNA species (3.9, 3.6, and 3.3 kilobases). These studies represent the first characterization of receptor function and brain distribution of the cloned rat 5-HT1A receptor.     

Novel 5-HT(1A/1B/1D) receptors antagonists with potent 5-HT reuptake inhibitory activity

Novel 2-methyl-5-quinolinyl-1-piperazinylalkyl-3,4-dihydro-2H-1,4-benzoxazin-3-ones showing high affinities for the 5-HT(1A/1B/1D) receptors coupled with potent 5-HT reuptake inhibitory activity have been discovered. This is the first report describing docking of the lead compound 6-{2-[4-(2-methyl-5-quinolinyl)-1-piperazinyl]ethyl}-2H-1,4-benzoxazin-3(4H)-one 1, into a model of the 5-HT transporter and the 5-HT(1A) receptor model.     

Serotonergic regulation of the central heart auricles of Sepia officinalis L. (Mollusca, Cephalopoda)

In pharmacological bioassays on isolated isotonically suspended auricles of Sepia officinalis, the regulatory action of the neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) on these autonomous contractile compartments was demonstrated. 5-HT induced concentration-dependent positive effects on frequency and tone, whereas the concentrations/response curve for the amplitude showed a biphasic course. All applied antagonists inhibited mainly the effect of 5-HT on frequency and amplitude. The chronotropic effects of 5-HT were blocked mainly by the 5-HT(1,2) antagonist methiothepin (pA(2)=8.01), the 5-HT(1a) antagonist NAN-190 (pA(2)-) and in lesser extent by the 5-HT(1,2) antagonist mianserin (pA(2)=6.81). In the presence of each antagonist applied the 5-HT action on amplitude was transformed into a positive inotropic effect with the maximum under influence of NAN-190 and the 5-HT(2,1c)-antagonist ketanserin. The auricular tone was also influenced by the antagonists and in combination with methiothepin it turned into strong negative tonotropic effect. In addition to the pharmacological bioassays, the presence of 5-HT in nerve endings within the auricle wall was demonstrated by immunohistochemical and fluorescence microscopic findings. Altogether the findings presented here confirm that 5-HT evokes excitatory effects on the autonomous contractile auricle of S. officinalis and acts obviously over different receptors, whereby a 5-HT(1)- and a 5-HT(2)-like seem to be involved.     

Discovery of 7-arylsulfonyl-1,2,3,4, 4a,9a-hexahydro-benzo[4,5]furo[2,3-c]pyridines: identification of a potent and selective 5-HT₆ receptor antagonist showing activity in rat social recognition test

Serotoninergic neurotransmission has been implicated in modulation of learning and memory. It has been demonstrated that 5-hydroxytryptamine(6) (5-HT(6)) receptor antagonists show beneficial effect on cognition in several animal models. Based on a pharmacophore model reported in the literature, we have designed and successfully identified a 7-benzenesulfonyl-1,2,3,4-tetrahydro-benzo[4,5]furo[2,3-c]pyridine (3a) scaffold as a novel class of 5-HT(6) receptor antagonists. Despite good activity against 5-HT(6) receptor, 3a exhibited poor liver microsome stability in mouse, rat and dog. It was demonstrated that the saturation of the double bond of the tetrahydropyridine ring of 3a enhanced metabolic stability. However the resulting compound, 4a (7-phenylsulfonyl-1,2,3,4,4a,9a-hexahydro-benzo[4,5]furo[2,3-c] pyridine-HCl salt) exhibited ～30-fold loss in potency along with introduction of two chiral centers. In our optimization process for this series, we found that substituents at the 2 or 3 positions on the distal aryl group are important for enhancing activity against 5-HT(6). Separation of enantiomers and subsequent optimization and SAR with bis substituted phenyl sulfone provided potent 5-HT(6) antagonists with improved PK profiles in rat. A potent, selective 5-HT(6)R antagonist (15k) was identified from this study which showed good oral bioavailability (F=39%) in rat with brain penetration (B/P=2.76) and in vivo activity in a rat social recognition test.     

Synthesis and biodistribution of [11C]R107474, a new radiolabeled alpha2-adrenoceptor antagonist

R107474, 2-methyl-3-[2-(1,2,3,4-tetrahydrobenzo[4,5]furo[3,2-c]pyridin-2-yl)ethyl]-4H-pyrido[1,2-a]pyrimidin-4-one, was investigated using in vitro and in vivo receptor assays and proved to be a potent and relatively selective alpha(2)-adrenoceptor antagonist. Performed assays in vitro were inhibition of binding to a large number of neurotransmitter receptor sites, drug receptor binding sites, ion channel binding sites, peptide receptor binding sites, and the monoamine transporters in membrane preparations of brain tissue or of cells expressing the cloned human receptors. The compound has subnanomolar affinity for halpha(2A)- and halpha(2C)-adrenoceptors (K(i) = 0.13 and 0.15 nM, respectively) and showed nanomolar affinity for the halpha(2B)-adrenoceptors and 5-hydroxytryptamine(7) (h5-HT(7)) receptors (K(i) = 1 and 5 nM, respectively). R107474 interacted weakly (K(i) values ranging between 81 and 920 nM) with dopamine-hD(2L), -hD(3) and -hD(4), h5-HT(1D)-, h5-HT(1F)-, h5-HT(2A)-, h5-HT(2C)-, and h5-HT(5A) receptors. The compound, tested up to 10 microM, interacted only at micromolar concentrations or not at all with any of the other receptor or transporter binding sites tested in this study. In vivo alpha(2A)- and alpha(2C)-adrenoceptor occupancy was measured by ex vivo autoradiography 1h after subcutaneous (sc) administration of R107474. It was found that R107474 occupies the alpha(2A)- and alpha(2C)-adrenoceptors with an ED(50) (95% confidence limits) of 0.014 mg/kg sc (0.009-0.019) and 0.026 mg/kg sc (0.022-0.030), respectively. Radiolabeled 2-methyl-3-[2-([1-(11)C]-1,2,3,4-tetrahydrobenzo[4,5]furo[3,2-c]pyridin-2-yl)ethyl]-4H-pyrido[1,2-a]pyrimidin-4-one ([(11)C]R107474) was prepared and evaluated as a potential positron emission tomography (PET) ligand for studying central alpha(2)-adrenoceptors. [(11)C]R107474 was obtained via a Pictet-Spengler reaction with [(11)C]formaldehyde in 33 +/- 4% overall decay-corrected radiochemical yield. The total synthesis time was 55 min and the specific activity was 24-28 GBq/micromol. The biodistribution of [(11)C]R107474 in rats revealed that the uptake of [(11)C]R107474 after in vivo intravenous administration is very rapid; in most tissues (including the brain) it reaches maximum concentration at 5 min after tracer injection. In agreement with the known distribution of alpha(2)-adrenoceptors in the brain, highest uptake of radioactivity was observed in septum (3.54 +/- 0.52 ID/g, 5 min pi) and entorhinal cortex (1.57 +/- 0.10 ID/g, 5 min pi). Tissue/cerebellum concentration ratios for septum (5.38 +/- 0.45, 30 min pi) and entorhinal cortex (3.43+/-0.24, 30 min pi) increased with time due to rapid uptake followed by a slow washout. In vivo blocking experiments using the non-selective alpha(2)-adrenoceptor antagonist mirtazapine demonstrated specific inhibition of [(11)C]R107474 binding in selective brain areas. The receptor binding profile of mirtazapine is reported and the selectivity of inhibition of binding is discussed. These results suggest that [(11)C]R107474 deserves further investigation as a potential radioligand for studying alpha(2)-adrenoceptors using PET.     

5-Substituted 2-aminothiophenes as A1 adenosine receptor allosteric enhancers

Two series of 5-substituted 2-amino-4-(3-trifluoromethylphenyl)thiophenes were prepared and evaluated as allosteric enhancers at the A(1) adenosine receptor (A(1)AR). In the 3-benzoyl series, a 5-phenyl group was found to confer the greatest potency (9a: ED(50)=2.1 microM, AE score=18%). However, the analogue with no 5-substituent (6b: ED(50)=15.8 microM, AE score=77%) proved to be the most efficacious. In the 3-ethoxycarbonyl series, the 5-(4-chlorophenyl) analogue was clearly the most potent and efficacious (9l: ED(50)=6.6 microM, AE score=57%). The antagonist activity of all compounds was measured using a [(3)H]CPX competitive binding assay.