Preliminary study on application of impregnated synthetic peptide TLC stationary phases for the pre-screening of 5-HT1A ligands

Chromatographic parameters (deltaR(f)), defined as a difference in the migration of tested compound on the control and peptide impregnated silica gel TLC plates, were determined for 42 arylpiperazine derivatives. An amino acid sequence of the peptide used for impregnation was derived from the III transmembrane segment of the 5-HT(1A) receptor in the close vicinity of aspartic acid (Asp 166) residue. It was found that the deltaR(f) values obtained in a model employing tetrapeptide P4LA (ADVL), as well as the calculated logP correlate with 5-HT(1A) receptor affinity of the studied compounds.     

A new class of arylpiperazine derivatives: the library synthesis on SynPhase lanterns and biological evaluation on serotonin 5-HT(1A) and 5-HT(2A) receptors

An efficient solid-supported method for the synthesis of a new class of arylpiperazine derivatives containing amino acid residues has been developed. A 72-membered library was synthesized on SynPhase Lanterns functionalized by a BAL linker. A one-pot cleavage/cyclization step of aspartic and glutamic acid derivatives yielded succinimide- and pyroglutamyl-containing ligands (chemsets 9 and 10). The library representatives under study showed different levels of affinity for 5-HT(1A) and 5-HT(2A) receptors (estimated K(i) = 24-4000 and 1-2130 nM, respectively). Several dual 5-HT(1A)/5-HT(2A) ligands were found, of which two (9(3,3) and 9(3,5)) displayed high 5-HT(2A) affinity comparable to that of the reference drug ritanserin. A set of individual fragment contributions for the prediction of 5-HT(1A) and 5-HT(2A) affinity of all the library members were defined on the basis of the Free-Wilson analysis of 26 compounds. An alkylarylpiperazine fragment had essentially the same impact on the affinity for both receptors, whereas different terminal amide fragments were preferred by 5-HT(1A) (chemset 17, R(2) = adamantyl) and 5-HT(2A) (chemset 9, R(2) = norborn-2-ylmethyl) binding sites.     

Synthesis, 5-hydroxytryptamine1A receptor affinity and docking studies of 3-[3-(4-aryl-1-piperazinyl)-propyl]-1H-indole derivatives

A series of 3-[3-(4-aryl-1-piperazinyl)-propyl]-1H-indole derivatives (12a-h) was synthesized and evaluated for binding affinity at the human 5-hydroxytryptamine(1A) receptor (5-HT(1A)R) compounds (12b) and (12h) showed the highest 5-HT(1A) receptor affinity (IC(50)=15 nM). Molecular docking studies with all the compounds in a homology model of 5-HT(1A) showed that the main interaction anchoring the ligand in the receptor was a charge-reinforced bond between the protonated nitrogen atom (N-4) of the piperazine ring and Aspartate(3.32).     

The capillary electrophoresis separation of benzodiazepine drug using dextran sulfate and SDS as running buffer

Capillary electrophoresis has been applied the analyses of many clinical drugs due to its rapid, high-resolution separation. In this study, electrokinetic chromatography involving the combination of SDS and dextran sulfate, which are synthetic polymers, was examined in order to obtain high resolution. Use of 2% dextran sulfate (10,000 molecular weight), 20 mm SDS running buffer containing boric acid solution (pH 9.2) and a silica capillary (inner diameter of 75 micro m, effective length of 50 cm, 57 cm overall length) afforded separation of 10 kinds of benzodiazepines. The detection limit was 0.2 micro g/mL; additionally, reproducibilities were de fi ned as the peak height and migration time. The average peak height was 5.92% (2.46-17.61), whereas the average migration time was 0.44% (0.18-0.76; n = 5). This separations system can be applied to the analysis and measurement of other pharmaceuticals as well.     

Quantitative structure-affinity relationship of 5-HT1A receptor ligands by the classification tree method

The influence of molecular structure of 346 ligands on their affinity for 5-HT1A receptors was investigated. It was shown that the effectiveness of the proposed novel approach for interpretation of decision tree models compared favourably with the PLS method. In the context of the proposed approach, molecular fragments and their values of the relative influence on the affinity for 5-HT1A receptors were defined.     

Screening of 5-HT1A receptor antagonists using molecularly imprinted polymers

Molecular imprinting produces network polymers with recognition sites for imprint molecules. The high binding affinity and selectivity in conjunction with the polymers' physical robustness positions molecular imprinted polymers (MIPs) as candidates for use as preliminary screens in drug discovery. As such, MIPs can serve as crude mimics of native receptors. In an effort to evaluate the relationship between MIPs and native receptors, imprinted polymers for WAY-100635, an antagonist of the serotonin (5-HT) receptor subtype 5-HT1A were prepared. The resulting MIP P(WAY) was evaluated as an affinity matrix in the screening of serotonin receptor antagonists with known affinities for the native receptor. Rough correlations in affinity between the synthetic P(WAY) and native receptor 5-HT1A were found. These findings provide some support for the analogy between MIPs and native receptors and their possible use as surrogates.     

New imide 5-HT1A receptor ligands - modification of terminal fragment geometry

Two sets of new o-methoxyphenylpiperazine (MPP; series a) and 1,2,3,4-tetrahydroisoquinoline (THIQ; series b) derivatives, containing various imide moieties derived from NAN190, were synthesized and evaluated in vitro for their ability to bind to the serotonin 5-HT(1A) and 5-HT(2A) receptors. All new derivatives from series a demonstrated high 5-HT(1A) affinities, whereas THIQ analogues were much less active. With respect to 5-HT(2A) receptors, three MPP derivatives presented moderate activity but the rest of the investigated compounds were practically inactive. The influence of changes in terminus geometry on 5-HT(1A) receptor affinity was analyzed in regard to model compounds NAN190and MM199.     

Electrospray mass spectrometric studies of noncovalent complexes of buspirone hydrochloride and other serotonin 5-HT(1A) receptor ligands containing arylpiperazine moieties

Noncovalent complexes consisting of two protonated amines and a chloride anion were observed under electrospray ionization mass spectrometry (ESI-MS) conditions. The observed phenomenon was investigated for the hydrochlorides of buspirone, a well-known anxiolytic drug, and 23 other arylpiperazine derivatives that had been developed as serotonin 5-HT(1A) receptor ligands. Due to the major role of ionic interactions in a vacuum, it was proposed that the detected complexes were formed by NH(+)---Cl(-)---NH(+) bridges. It was found that complexation depended on structural features of the analyzed compounds. For derivatives with a shorter linker (three methylene groups) containing a terminal cyclic amide fragment, complex ions were not observed. It was postulated that, in the latter case, steric hindrance due to a terminal group could disturb ionic bridge formation. Since both the observed complexation and ligand-binding processes are driven by noncovalent forces, and a qualitative relationship between them was found (compounds with a 4-carbon chain always display higher affinity for 5-HT(1A) receptors than do their 3-carbon analogues), such ESI-MS studies may yield valuable information on ligand-receptor interactions.     

Indene-based scaffolds. Design and synthesis of novel serotonin 5-HT6 receptor ligands

A series of novel indene derivatives designed by a scaffold selection gave access to several examples of (Z)-arylmethylideneindenes and indenylsulfonamides that acted as serotonin 5-HT(6) receptor ligands. Different synthetic multistep routes could be applied to these target compounds, each with their own complexity and limitations. A reasonable route involved the (3-indenyl)acetic acids as the key intermediates, and two alternatives were also examined. The first protocol used was a two-step sequence employing a modified Horner-Wadsworth-Emmons reaction, but better results were obtained with a procedure based on the condensation of indanones with the lithium salt of ethyl acetate, followed immediately by dehydration with acid and hydrolysis/isomerization under basic catalysis. (3-Indenyl)acetic acids were transformed to the corresponding acetamides, which were effectively reduced to indenylsulfonamides using an optimized procedure with AlH(3)-NMe(2)Et. The binding at the 5-HT(6) receptor was with moderate affinity (K(i) = 216.5 nM) for the (Z)-benzylideneindenylsulfonamide and enhanced affinity for the simple indenylsulfonamide counterpart (K(i) = 50.6 nM). Selected indenylsulfonamides were then tested, showing K(i) values as low as 20.2 nM.     

Synthesis and structure-activity relationships of 4-amino-5-chloro-N-(1,4-dialkylhexahydro-1,4-diazepin-6-yl)-2-methoxybenzamide derivatives,novel and potent serotonin 5-HT3 and dopamine D2 receptors dual antagonist

In search of a dopamine D2 and serotonin 5-HT3 receptors dual antagonist as a potential broad antiemetic agent, a number of benzamides were prepared from 4-amino-5-chloro-2-methoxybenzoic acid derivatives and 6-amino-1,4-dialkylhexahydro-1,4-diazepines and evaluated for their binding affinity for the dopamine D2 and the serotonin 5-HT3 receptors using rat brain synaptic and rat cortical membranes, respectively. From the results of both in vitro receptor binding and in vivo biological assays for the dopamine D2 receptor, 1-ethyl-4-methylhexahydro-1,4-diazepine ring was selected as an optimum amine moiety. Introduction of one methyl group on the nitrogen atom at the 4-position and/or modification of the substituent at the 5-position of the 4-amino-5-chloro-2-methoxybenzoyl moiety caused a marked increase in the dopamine D2 receptor binding affinity along with a potent 5-HT3 receptor binding affinity. Among the compounds, 5-chloro-N-(1-ethyl-4-methylhexahydro-1,4-diazepin-6-yl)-2-methoxy-4-methylaminobenzamide (82), 5-bromo (110), and 5-iodo (112) analogues exhibited a much higher affinity for the dopamine D2 receptor than that of metoclopramide (IC50=17.5-61.0 nM vs. 483 nM). In particular, 82 showed a potent antagonistic activity for both receptors in vivo tests. Optical resolution of the racemate 82 brought about a dramatic change in the pharmacological profile with the (R)-enantiomer exhibiting a strong affinity for both the dopamine D2 and the 5-HT3 receptors, while the corresponding (S)-enantiomer had a potent and selective serotonin 5-HT3 receptor binding affinity.     

G protein- and agonist-bound serotonin 5-HT2A receptor model activated by steered molecular dynamics simulations

A 5-HT(2A) receptor model was constructed by homology modeling based on the β(2)-adrenergic receptor and the G protein-bound opsin crystal structures. The 5-HT(2A) receptor model was transferred into an active conformation by an agonist ligand and a G(αq) peptide in four subsequent steered molecular dynamics (MD) simulations. The driving force for the transformation was the addition of several known intermolecular and receptor interhelical hydrogen bonds enforcing the necessary helical and rotameric movements. Subsquent MD simulations without constraints confirmed the stability of the activated receptor model as well as revealed new information about stabilizing residues and bonds. The active 5-HT(2A) receptor model was further validated by retrospective ligand screening of more than 9400 compounds, whereof 182 were known ligands. The results show that the model can be used in drug discovery for virtual screening and structure-based ligand design as well as in GPCR activation studies.     

Novel medium-sized di(het)areno-fused 1,4,7-(oxa)thiadiazecines as probes for aminergic receptors

Di(het)areno-fused 1,4,7-(oxa)thiadiazecanes were synthesized by the reduction of the corresponding ten-membered lactams obtained, in turn, via the ‘hydrated imidazoline ring expansion’ (HIRE) methodology. Two of them displayed micromolar agonistic activity towards trace amine-associated receptor 1 (TAAR1) and no affinity towards a panel of dopamine (D2) and serotonin (5-HT1A, 5-HT2A and 5-HT7) receptors. These findings validate compounds of this chemotype as scaffolds for the design of selective aminergic receptor modulators.     

3-D QSAR for intrinsic activity of 5-HT1A receptor ligands by the method of comparative molecular field analysis

The affinity of a ligand for a receptor is usually expressed in terms of the dissociation constant (K_i) of the drug-receptor complex, conveniently measured by the inhibition of radioligand binding. However, a ligand can be an antagonist, a partial agonist, or a full agonist, a property largely independent of its receptor affinity. This property can be quantitated as intrinsic activity (1A), which can range from 0 for a full antagonist to 1 for a full agonist. Although quantitative structure–activity relationship (QSAR) methods have been applied to the prediction of receptor affinity with considerable success, the prediction of IA, even qualitatively, has rarely been attempted. Because most traditional QSAR methods are limited to congeneric series, and there are often major structural differences between agonists and antagonists, this lack of success in predicting IA is understandable. To overcome this limitation, we used the method of comparative molecular field analysis (CoMFA), which, unlike traditional Hansch analysis, permits the inclusion of structurally dissimilar compounds in a single QSAR model. A structurally diverse set of 5-hydroxytryptamine_1A (5-HT_1A) receptor ligands, with literature IA data (determined by the inhibition of 5-HT sensitive forskolin-stimulated adenylate cyclase), was used to develop a 3-D QSAR model correlating intrinsic activity with molecular structure properties of 5HT_1A receptor ligands. This CoMFA model had a crossvalidated r² of 0.481, five components and final conventional r² of 0.943. The receptor model suggests that agonist and antagonist ligands can share parts of a common binding site on the receptor, with a primary agonist binding region that is also occupied by antagonists and a secondary binding site accommodating the excess bulk present in the sidechains of many antagonists and partial agonists. The CoMFA steric field graph clearly shows that agonists tend to be “flatter” (more coplanar) than antagonists, consistent with the difference between the 5-HT_1A agonist and antagonist pharmacophores proposed by Hibert and coworkers. The CoMFA electrostatic field graph suggests that, in the region surrounding the essential protonated aliphatic amino group, the positive molecular electrostatic potential may be weaker in antagonists as compared to agonists. Together, the steric and electrostatic maps suggest that in the secondary binding site region increased hydrophobic binding may enhance antagonist activity. These results demonstrate that CoMFA is capable of generating a statistically crossvalidated 3-D QSAR model that can successfully distinguish between agonist and antagonist 5-HT_1A ligands. To the best of our knowledge, this is the first time this or any other QSAR method has been successfully applied to the correlation of structure with IA rather than potency or affinity. The analysis has suggested various structural features associated with agonist and antagonist behaviors of 5-HT_1A ligands and thus should assist in the future design of drugs that act via 5-HT_1A receptors. © 1993 John Wiley & Sons, Inc.     

Synthesis and evaluation of novel 2-oxo-1,2-dihydro-3-quinolinecarboxamide derivatives as potent and selective serotonin 5-HT4 receptor agonists

A series of 8'-substituted N-(endo-8-azabicyclo[3.2.1]oct-3-yl)-1-isopropyl-2-oxo-1,2-dihydro-3-quinolinecarboxamides were synthesized. The 5-HT4 receptor agonistic activity was evaluated using the isolated guinea pig ileum preparation. Of the compounds synthesized, N-(endo-8-(3-hydroxypropyl)-8-azabicyclo[3.2.1]oct-3-yl)-1-isopropyl-2-oxo-1,2-dihydro-3-quinolinecarboxamide (15a, TS-951) exhibited the most potent serotonin 5-HT4 receptor agonistic activity. This compound had a high affinity for the serotonin 5-HT4 receptor although it had no affinities for other broad spectrum receptors. Furthermore, it remarkably enhanced gastrointestinal motility in conscious fed dogs without unfavorable effects that non-selective serotonin 5-HT4 receptor agonist has. TS-951 may be useful in improving gastrointestinal dysfunction.     

2-(omega-phenylalkyl)phenoxy]alkylamines.II: Synthesis and selective serotonin-2 receptor binding

A series of [2-(omega-phenylalkyl)phenoxy]alkylamines was synthesized and their receptor binding affinity was examined in vitro. These compounds showed an affinity for serotonin-2 (5-HT2) and dopamine-2 (D2) receptors. [2-(2-phenylethyl)phenoxy]alkylamine derivatives with a pyrrolidine or piperidine moiety in the structure showed higher affinity for 5-HT2 receptors but lower affinity for D2 receptors. Among these compounds, (S)-2-[2- [2-[2-(3-methoxyphenyl)ethyl]phenoxy]ethyl]-1-methylpyrrolidine, (S)-27, exhibited the most potent and selective affinity for 5-HT2 receptors. Furthermore, (S)-27 was effective in inhibiting 5-HT-induced vasoconstriction in vitro and platelet aggregation both in vitro and ex vivo.     

Synthesis and serotonin 2 (5-HT2) receptor antagonist activity of 5-aminoalkyl-substituted pyrrolo[3,2-c]azepines and related compounds

A series of 5-aminoalkylpyrrolo[3,2-c]azepine derivatives was synthesized and their serotonin 2 (5-HT2) receptor antagonist and antiplatelet aggregation activities were evaluated. 5-HT2 receptor antagonist activity was largely determined by the nature of the substituent at the 8-position as well as the aminoalkyl group at the 5-position of the pyrrolo[3,2-c]azepine ring. Compound 18a, 5-[3-[4-(4-fluorophenyl)piperazin-1-yl]propyl]-8-hydroxy-1-methyl- 1,4,5,6,7,8-hexahydropyrrolo[3,2-c]azepin-4-one, was recognized as having potent 5-HT2 receptor antagonist activity with weak alpha1 adrenoceptor blocking activity and no significant D2 receptor binding affinity, while the corresponding isomeric pyrrolo[3,4-c]azepine derivative (22) displayed only weak 5-HT2 receptor antagonist activity. After racemic 18a was resolved directly via diastereomeric salt formation, each enantiomer was evaluated precisely. The 5-HT2 receptor antagonist activity of 18a was found to reside primarily in (-)-18a (which was about 14-fold more potent than (+)-18a in isolated guinea pig arteries). Consequently, (S)-(-)-18a (SUN C5174) displayed the overall best profile with potent 5-HT2 receptor antagonist activity (pA2=8.98+/-0.06) and high selectivity versus other receptors. SUN C5174 showed a marked inhibitory effect on the platelet aggregation induced by serotonin in combination with collagen and adenosine diphosphate (ADP) in canine or human platelet-rich plasma (IC50=6.5 to 16 nM). Moreover, this compound significantly inhibited the mortality rate in mouse acute pulmonary thromboembolytic death induced by collagen and serotonin at oral doses of 0.3 mg/kg or higher. SUN C5174 is currently undergoing clinical evaluation.     

Arylpiperazine derivatives of diphenylsulfide:Synthesis and evaluation for dual 5-HT1A/SSRI activities

The design, synthesis and biological evaluation of a novel series of arylpiperazine derivatives of diphenylsulfide with dual 5- HT1A/SSRI activities are reported. The target compounds exhibit low to moderate 5-HT transporter affinity and moderate to high 5- HT1A affinity, Compound 13a shows moderate dual activities and is a promising lead compound for further structure-activity relationships studies.     

Synthesis, docking studies and biological evaluation of benzo[b]thiophen-2-yl-3-(4-arylpiperazin-1-yl)-propan-1-one derivatives on 5-HT1A serotonin receptors

A series of novel benzo[b]thiophen-2-yl-3-(4-arylpiperazin-1-yl)-propan-1-one derivatives 6a-f, 7a-f and their corresponding alcohols 8a-f were synthesized and evaluated for their affinity towards 5-HT(1A) receptors. The influence of arylpiperazine moiety and benzo[b]thiophene ring substitutions on binding affinity was studied. The most promising analogue, 1-(benzo[b]thiophen-2-yl)-3-(4-(pyridin-2-yl)piperazin-1-yl)propan-1-one (7e) displayed micromolar affinity (K(i) = 2.30 μM) toward 5-HT(1A) sites. Docking studies shed light on the relevant electrostatic interactions which could explain the observed affinity for this compound.     

Exploitation of an additional hydrophobic pocket of σ1 receptors: late-stage diverse modifications of spirocyclic thiophenes by C-H bond functionalization

The hypothesis that the σ(1) receptor will tolerate an additional aryl moiety in position 1 of the spirocyclic system was based on spirocyclic pyrazole derivatives, pharmacophore models of σ(1) receptor ligands and DFT calculations. The strategy of introducing the aryl residue at the final step of the synthesis allowed the preparation of a large set of diverse ligands for the exploitation of the hydrophobic pocket of the σ(1) receptor protein. The catalyst system PdCl(2)/2,2'-bipyridyl/Ag(2)CO(3) is able to introduce various aryl groups onto the α-positions of spirocyclic thiophene derivatives 5 and 6 to afford the target aryl-appended spirocyclic thiophenes 3 and 4. Although the σ(1) affinity of the 1-phenyl substituted spirocyclic thiophenes 3a and 4a is slightly reduced compared with the σ(1) affinity of the non-arylated compounds 5 and 6, both compounds represent very potent σ(1) receptor ligands (3a: K(i) = 4.5 nM; 4a: K(i) = 1.0 nM). This result indicates that an aryl moiety in position 1 is well tolerated by the σ(1) receptor protein. The substitution pattern of the additional phenyl moiety has only weak effects on the σ(1) affinity. Even ligands 3f and 4h with extended naphthyl residue show high σ(1) affinity. However, decrease of σ(1) affinity by extension of the π-system to a biphenylyl substituent (4j: K(i) = 30 nM) indicates that the biphenylyl residue is too large for the primary hydrophobic binding pocket of the σ(1) receptor.     

Functional human 5-HT6 receptor assay for high throughput screening of chemical ligands and binding proteins

Continuous identification and validation of novel drug targets require the development of rapid, reliable, and sensitive cell-based high-throughput screening (HTS) methods for proposed targets. Recently, the 5-HT(6) receptor (5-HT(6)R), a member of the class of recently discovered 5-HT receptors, has received considerable attention for its possible implications in depression, cognition, and anxiety. However, the cellular signaling mechanisms of 5-HT(6)R are poorly understood due to the lack of selective 5-HT(6)R ligands. In the present study, we examined functional coupling of the human 5-HT(6)R, 5-HT(7A)R, or 5-HT(7B)R with various Galpha-proteins (Galpha(15), Galpha(qs5), or Galpha(qG66Ds5)) to develop a reliable cell-based HTS method for 5-HT receptors. Among variable couplings between 5-HT receptors and G-proteins, we found that functional coupling of human 5-HT(6)R with Galpha(qG66Ds5) produced the highest levels of Ca(2+) signaling in HEK293 cells as measured by the fluorescence-based HTS plate reader, FDSS6000. After validation of this new 5-HT(6)R HTS system (Z'-factor = 0.56) in 96-well plates and characterization of the pharmacological profile of the 5-HT(6)R, we screened approximately 500 synthetic chemical compounds including butanamide and benzenesulfonamide derivatives. Based on this preliminary screening, we found that the butanamide derivative LSG11104 produced an IC(50) value of 6.3 microM. This compound will serve as a lead structure for further chemical modification to develop novel 5-HT(6)R ligands. Furthermore, we demonstrated that this HTS method can be utilized to identify proteins that modulate 5-HT(6)R function and present Fyn tyrosine kinase as an example, which is already known as a 5-HT(6)R interacting protein. Taken together, these results suggest that the 5-HT(6)R/Galpha(qG66Ds5) FDSS6000 system can be utilized to screen for selective 5-HT(6)R ligands and to examine any functional relationships between 5-HT(6)R and its binding proteins.