Synthesis and structure-activity relationship of the isoindolinyl benzisoxazolpiperidines as potent,selective, and orally active human dopamine D4 receptor antagonists

A new class of potent dopamine D(4) antagonists was discovered with selectivity over dopamine D(2) and the alpha-1 adrenoceptor. The lead compound was discovered by screening our compound collection. The structure-activity relationships of substituted isoindoline rings and the chirality about the hydroxymethyl side chain were explored. The isoindoline analogues showed modest differences in potency and selectivity. The S enantiomer proved to be the more potent enantiomer at the D(4) receptor. Several analogues with greater than 100-fold selectivity for D(4) over D(2) and the alpha-1 adrenoreceptor were discovered. Several selective analogues were active in vivo upon oral or intraperitoneal administration. A chiral synthesis starting from either D- or L-O-benzylserine is also described.     

Mapping the Melatonin Receptor. 8. Selective MT2 Agonists Derived from 5,6-Dihydroindolo[2,1-a]isoquinolines and Related Systems

A series of substituted indolo[2,1-a]isoquinolines and indolo[1,2-a]benzoxazines have been prepared, as melatonin analogues, to investigate the nature of the binding site of the melatonin receptor. Agonist and antagonist potency of all the analogues was measured using the [35S]GTPγS binding assay protocol. The binding affinity of the analogues were measured by competition binding studies against the human MT1 (hMT1) and MT2 (hMT2) receptors stably transfected in Chinese Hamster Ovarian (CHO) cells, using 2-[¹²⁵I]-iodomelatonin, as a ligand. N-Acetyl 2-(10-methoxy-5,6-dihydroindolo[2,1-a]isoquinolin-12-yl)propyl-1-amine (12 a) binds strongly to both the hMT1 and hMT2 receptors, and shows a preference for the hMT2, as does its propanamido counterpart 12 b . The introduction of two methyl groups into their side chain, analogues 15 a and 1 5 b , leads to antagonism, in the case of the former, and drastically diminishes its hMT1 binding; an analogous profile is seen for 15 b , which, however, is a partial agonist. Introduction of chlorine or methoxy groups into ring 4 gives compounds, that are weakly binding, with a preference for MT2. Substitution of oxygen for carbon at position 5 gives the indolo[1,2-c]benzoxazines 33 , 36 a and b , that bind strongly to the human receptors, 33 , 36 b being potent agonists at the melatonin receptors, but do not discriminate between hMT1 and hMT2.     

High‐Throughput Virtual Screening Using Quantum Mechanical Probes: Discovery of Selective Kinase Inhibitors

A procedure based on semi‐empirical quantum mechanical (QM) calculations of interaction energy is proposed for the rapid screening of compound poses generated by high‐throughput docking. Small molecules (consisting of 2–10 atoms and termed “probes”) are overlapped with polar groups in the binding site of the protein target. The interaction energy values between each compound pose and the probes, calculated by a semi‐empirical Hamiltonian, are used as filters. The QM probe method does not require fixed partial charges and takes into account polarization and charge‐transfer effects which are not captured by conventional force fields. The procedure is applied to screen ～100 million poses (of 2.7 million commercially available compounds) obtained by high‐throughput docking in the ATP binding site of the tyrosine kinase erythropoietin‐producing human hepatocellular carcinoma receptor B4 (EphB4). Three QM probes on the hinge region and one at the entrance pocket are employed to select for binding affinity, while a QM probe on the side chain of the so‐called gatekeeper residue (a hypervariable residue in the kinome) is used to enforce selectivity. The poses with favorable interactions with the five QM probes are filtered further for hydrophobic matching and low ligand strain. In this way, a single‐digit micromolar inhibitor of EphB4 with a relatively good selectivity profile is identified in a multimillion‐compound library upon experimental tests of only 23 molecules.     

Synthesis of conformationally constrained glutamic acid homologues and investigation of their pharmacological profiles

Homologation of the glutamic acid chain together with conformational constraint is a commonly used strategy to achieve selectivity towards different types of glutamate receptors. We investigated the effects of a further increase in the distance between the amino acid moiety and the distal carboxylate group of model compounds (+/-)-1 and (+/-)-2 on their activity/selectivity profiles. We therefore synthesized new derivatives (+/-)-3-(+/-)-6, which are homologues of glutamic acid containing three additional carbon units. Moreover, because the potency of NMDA antagonists can be markedly increased by replacing the distal carboxylate with the bioisosteric phosphonate group, we also prepared the corresponding phosphonate derivatives (+/-)-7-(+/-)-10. All new compounds were submitted to binding assays with iGluRs, and derivatives (+/-)-3-(+/-)-6 were also tested in second messenger assays at representative mGluR subtypes. All the applied structural modifications were detrimental to the interaction with NMDA receptors. Conversely, structural variation of the nonselective mGluR ligand (+/-)-2 led to derivative (+/-)-5, which behaved as a selective group I metabotropic receptor antagonist. Notably, upon i.c.v. administration in DBA/2 mice, amino acid (+/-)-5 produced a significant protection against audiogenic seizures, whereas it was inactive after i.p. administration.     

Conformationally constrained CCK8 analogues obtained from a rationally designed peptide library as ligands for cholecystokinin type B receptor

A library of 14 cyclic peptide analogues derived from the octapeptide C-terminal sequence of the human cholecystokinin hormone (CCK(26-33), or CCK8) was designed, synthesized, and characterized. The 14 peptide analogues were rationally designed to specifically interact with the CCK type B receptor (CCK(B)-R) on the basis of the structure of the bimolecular complex between CCK8 and the third extracellular loop of CCK(B)-R, namely CCK(B)-R(352-379). The rational design of new ligands for CCK(B)-R has relied on stabilization by cyclic constraints of the structural motifs that bring the key residues of the ligand (especially Trp 30, Met 31, and Phe 33) in the proper spatial orientation for optimal interaction with the receptor. The binding affinity of the new ligands for CCK(B)-R was assessed by displacement experiments of (111)In-radiolabeled CCK8 in cells that overexpress the CCK(B) receptor. The new ligands generally showed binding affinities lower than that of parent CCK8, with the best compounds having IC50 values around 10 microM. Structure-activity relationship data show that preservation of the Trp 30-Met 31 motif is essential and that the Phe 33 side chain must be present. NMR conformational studies of the compound with maximal binding affinity (cyclo-B11, IC50=11 microM) in DPC micelles shows that this compound presents a turn-like conformation centered at the Trp 30-Met 31 segment, as planned by rational design. Such a conformation is stabilized by its interaction with the micelle rather than by the cyclic constraint.     

Improving potency, selectivity, and water solubility of adenosine A1 receptor antagonists: xanthines modified at position 3 and related pyrimido[1,2,3-cd]purinediones

The structure-activity relationships of xanthine derivatives related to the adenosine A(1) receptor antagonists 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and 1,3-dipropyl-8-(3-noradamantyl)xanthine (KW3902) were investigated by focusing on variations of the 3-substituent. Aromatic residues were well tolerated by the A(1) receptor in that position. A moderate effect of stereochemistry was found for the 3-(1-phenylethyl)-substituted analogue of DPCPX (S>R) at A(1) and A(3) receptors, whereas the opposite stereoselectivity was observed at the A(2) receptor subtypes. A 3-hydroxypropyl substituent was found to be optimal for high A(1) affinity and selectivity. The most potent compound of the present series was 1-butyl-3-(3-hydroxypropyl)-8-(3-noradamantyl)xanthine (10 c), which exhibits a K(i) value of 0.124 nM at rat, and 0.7 nM at human adenosine A(1) receptors, combined with high selectivity (>200-fold) versus the other receptor subtypes. The similarly potent 8-cyclopentyl-3-(3-hydroxypropyl)-1-propylxanthine was converted into a water-soluble phosphate prodrug, which may become a useful pharmacological tool for in vivo studies. 8-Alkyl-2-(3-noradamantyl)pyrimido[1,2,3-cd]purine-8,10-diones, which can be envisaged as xanthine analogues with a fixed 3-propyl substituent, were identified as a new class of potent, selective adenosine A(1) receptor antagonists. For example, compound 14 (8-butyl-substituted) exhibits a K(i) value of 13.8 nM at human A(1) receptors. A selection of the most potent compounds was investigated in [(35)S]GTPgammaS binding assays and showed inverse agonistic activity. Their efficacy was generally lower than that of the full inverse agonist DPCPX, and depended on subtle structural changes. Some of the new compounds belong to the most potent and selective A(1) antagonists described to date.     

Synthesis,Characterization, and Metabolism Studies of Fluspidine Enantiomers

The enantiomers of the potent σ₁ ligand fluspidine ( 1 ) were prepared by using chiral preparative HPLC. Synthesis of racemic tosylate 2 and subsequent separation of enantiomers yielded (R)‐ 2 and (S)‐ 2 in excellent enantiomeric purities. The fluspidine enantiomers (R)‐ 1 and (S)‐ 1 were synthesized from (R)‐ 2 and (S)‐ 2 by nucleophilic substitution with tetra‐n‐butylammonium fluoride, affording (R)‐ 1 with 99.6 % ee and (S)‐ 1 with 96.4 % ee. Tosylates (R)‐ 2 and (S)‐ 2 can also serve as precursors for the radiosynthesis of enantiomerically pure radiotracers [¹⁸F](R)‐ 1 and [¹⁸F](S)‐ 1 . The absolute configuration of the pure enantiomers was elucidated by comparison of their CD spectra with a calculated CD spectrum of a simplified model compound. In receptor binding studies, both enantiomers displayed very high σ₁ receptor affinity and selectivity against the σ₂ receptor. (R)‐Fluspidine ((R)‐ 1 ) is the eutomer, with a K_i value of 0.57 nM and a eudysmic ratio of 4. Incubation of (R)‐ 1 and (S)‐ 1 with rat liver microsomes led to the identification of seven and eight metabolites, respectively. Although the S‐configured enantiomer formed additional metabolite (S)‐ 1‐3 , it is metabolically more stable than (R)‐ 1 .     

Pyrrolo[1,2-a]quinoxalines: Insulin Mimetics that Exhibit Potent and Selective Inhibition against Protein Tyrosine Phosphatase 1B

PTP1B dephosphorylates insulin receptor and substrates to modulate glucose metabolism. This enzyme is a validated therapeutic target for type 2 diabetes, but no current drug candidates have completed clinical trials. Pyrrolo[1,2-a]quinoxalines substituted at positions C1–C4 and/or C7–C8 were found to be nontoxic to cells and good inhibitors in the low- to sub-micromolar range, with the 4-benzyl derivative being the most potent inhibitor (0.24 μm ). Some analogues bearing chlorine atoms at C7 and/or C8 kept potency and showed good selectivity compared to TCPTP (selectivity index >40). The most potent inhibitors behaved as insulin mimetics by increasing glucose uptake. The 4-benzyl derivative inhibited insulin receptor substrate 1 and AKT phosphorylation. Molecular docking and molecular dynamics simulations supported a putative binding mode for these compounds to the allosteric α3/α6/α7 pocket, but inconsistent results in enzyme inhibition kinetics were obtained due to the high tendency of these inhibitors to form stable aggregates. Computational calculations supported the druggability of inhibitors.     

Enantiomers of methyl substituted analogs of (Z)-5-decenyl acetate as probes for the chirality and complementarity of its receptor inAgrotis segetum 1: Synthesis and structure-activity relationships

The enantiomers of analogs of (Z)-5-decenyl acetate, a pheromone component ofAgrotis segetum, substituted by a methyl group in the 2, 3, 4, 7, and 8 positions and dimethyl substituted in the 4,7 positions, have been synthesized and studied by an electrophysiological single-cell technique and by molecular mechanics calculations. The results demonstrate that the electrophysiological activity as well as the ability of the (Z)-5-decenyl acetate receptor to differentiate between enantiomers depends on the position of the methyl substituent. For analogs methyl substituted in the 2, 4, or 8 position, no differences in the activities of the enantiomers could be observed. In contrast, the enantiomers of the 3- and 7-methyl analogs display a significant difference in the activities, theR-enantiomers being more active than theS-enantiomers. From an analysis of the structure-activity results of the enantiomers of the 4,7-dimethyl-substituted analogs, the chiral sense of the alkylchain of the natural pheromone component on binding to its receptor could be deduced.Schiff., Lepidoptera: Noctuidae.     

Structure–Activity Relationships of Quinoxaline‐Based 5‐HT3A and 5‐HT3AB Receptor‐Selective Ligands

Until recently, discriminating between homomeric 5‐HT₃A and heteromeric 5‐HT₃AB receptors was only possible with ligands that bind in the receptor pore. This study describes the first series of ligands that can discriminate between these receptor types at the level of the orthosteric binding site. During a recent fragment screen, 2‐chloro‐3‐(4‐methylpiperazin‐1‐yl)quinoxaline (VUF10166) was identified as a ligand that displays an 83‐fold difference in [³H]granisetron binding affinity between 5‐HT₃A and 5‐HT₃AB receptors. Fragment hit exploration, initiated from VUF10166 and 3‐(4‐methylpiperazin‐1‐yl)quinoxalin‐2‐ol, resulted in a series of compounds with higher affinity at either 5‐HT₃A or 5‐HT₃AB receptors. These ligands reveal that a single atom is sufficient to change the selectivity profile of a compound. At the extremes of the new compounds were 2‐amino‐3‐(4‐methylpiperazin‐1‐yl)quinoxaline, which showed 11‐fold selectivity for the 5‐HT₃A receptor, and 2‐(4‐methylpiperazin‐1‐yl)quinoxaline, which showed an 8.3‐fold selectivity for the 5‐HT₃AB receptor. These compounds represent novel molecular tools for studying 5‐HT₃ receptor subtypes and could help elucidate their physiological roles.     

Designing Multitarget Anti-inflammatory Agents: Chemical Modulation of the Lumiracoxib Structure toward Dual Thromboxane Antagonists-COX-2 Inhibitors

A series of lumiracoxib derivatives were designed to explore the influence of isosteric substitution on balancing COX-2 inhibition and thromboxane A(2) prostanoid (TP) receptor antagonism. The compounds were synthesized through a copper-catalyzed coupling procedure and characterized for their pK(a) values. TP receptor antagonism was assessed on human platelets; COX-2 inhibition was determined on human isolated monocytes and human whole blood. TPα receptor binding of the most promising compounds was evaluated through radioligand binding assays. Some of the isosteric substitutions at the carboxylic acid group afforded compounds with improved TP receptor antagonism; of these, a tetrazole derivative retained good COX-2 inhibitory activity and selectivity. The identification of this tetrazole acting as a balanced dual-acting compound in human whole blood, along with SAR analysis of the synthesized lumiracoxib derivatives, might contribute to the rational design of a new class of cardioprotective anti-inflammatory agents.     

Synthesis of stable analogues of geranylgeranyl diphosphate possessing a (Z,E,E)-geranylgeranyl side chain, docking analysis, and biological assays for prenyl protein transferase inhibition

Herein, we report the synthesis of novel stable analogues of geranylgeranyl diphosphate (GGPP), in which the "natural" all-trans geranylgeranyl portion has been replaced by a (Z,E,E)-geranylgeranyl chain. The change in configuration and consequent change in the relative position of the polar portion with the lipophilic side chain did not improve the properties of the E,E,E analogues in their inhibition of geranylgeranyl protein transferase I (GGTase I). However, a significant level of GGTase I inhibition and selectivity for GGTase I over farnesyl transferase (FTase) was maintained the unsubstituted phosphonoacetamidoxy derivative 4 a. This has shed light on the relative importance of the configuration at the C2=C3 double bond among GGPP derivatives. Moreover, the biological activities of all the compounds reported herein, in particular the preferential FTase inhibitory activity shown by compound 6, were in good agreement with the results of docking analysis.     

Synthesis and Pharmacological Evaluation of a series of the Agomelatine Analogues as Melatonin MT1/MT2 Agonist and 5‐HT2C Antagonist

Agomelatine is a naphthalenic analogue of melatonin that is in clinical use for the treatment of major depressive disorders. Interestingly, while agomelatine exhibits potent affinity for melatonin receptors, it binds with only moderate affinity to the serotonin 5‐HT_2C receptor. Optimization of agomelatine toward this target could further potentiate its clinical efficacy. To explore this hypothesis and to access derivatives in which a key point of agomelatine metabolism is blocked, a series of naphthalenic derivatives was designed and synthesized as novel analogues of agomelatine. Most of the prepared compounds exhibited good binding affinity at the melatonin MT₁ and MT₂ receptor subtypes. Two compounds, an acetamide and an acrylamide derivative, exhibited good binding affinities at both the human melatonin (MT) receptors and the serotonin 5‐HT_2C receptor subtype, with pK_i values of 7.96 and 7.95 against MT₁, 7.86 and 8.68 against MT2, and 6.64 and 6.44 against 5‐HT_2C, respectively.     

Characterization of the Stereochemical Structures of 2H‐Thiazolo[3,2‐a]pyrimidine Compounds and Their Binding Affinities for Anti‐apoptotic Bcl‐2 Family Proteins

In a previous study we reported a class of compounds with a 2H‐thiazolo[3,2‐a]pyrimidine core structure as general inhibitors of anti‐apoptotic Bcl‐2 family proteins. However, the absolute stereochemical configuration of one carbon atom on the core structure remained unsolved, and its potential impact on the binding affinities of compounds in this class was unknown. In this study, we obtained pure R and S enantiomers of four selected compounds by HPLC separation and chiral synthesis. The absolute configurations of these enantiomers were determined by comparing their circular dichroism spectra to that of an appropriate reference compound. In addition, a crystal structure of one selected compound revealed the exocyclic double bond in these compounds to be in the Z configuration. The binding affinities of all four pairs of enantiomers to Bcl‐x_L, Bcl‐2, and Mcl‐1 proteins were measured in a fluorescence‐polarization‐based binding assay, yielding inhibition constants (K_i values) ranging from 0.24 to 2.20 μM . Interestingly, our results indicate that most R and S enantiomers exhibit similar binding affinities for the three tested proteins. A binding mode for this compound class was derived by molecular docking and molecular dynamics simulations to provide a reasonable interpretation of this observation.     

The Binding Mode of Side Chain‐ and C3‐Modified Epothilones to Tubulin

The tubulin‐binding mode of C3‐ and C15‐modified analogues of epothilone A (Epo A) was determined by NMR spectroscopy and computational methods and compared with the existing structural models of tubulin‐bound natural Epo A. Only minor differences were observed in the conformation of the macrocycle between Epo A and the C3‐modified analogues investigated. In particular, 3‐deoxy‐ (compound 2 ) and 3‐deoxy‐2,3‐didehydro‐Epo A ( 3 ) were found to adopt similar conformations in the tubulin‐binding cleft as Epo A, thus indicating that the 3‐OH group is not essential for epothilones to assume their bioactive conformation. None of the available models of the tubulin–epothilone complex is able to fully recapitulate the differences in tubulin‐polymerizing activity and microtubule‐binding affinity between C20‐modified epothilones 6 (C20‐propyl), 7 (C20‐butyl), and 8 (C20‐hydroxypropyl). Based on the results of transferred NOE experiments in the presence of tubulin, the isomeric C15 quinoline‐based Epo B analogues 4 and 5 show very similar orientations of the side chain, irrespective of the position of the nitrogen atom in the quinoline ring. The quinoline side chain stacks on the imidazole moiety of β‐His227 with equal efficiency in both cases, thus suggesting that the aromatic side chain moiety in epothilones contributes to tubulin binding through strong van der Waals interactions with the protein rather than hydrogen bonding involving the heteroaromatic nitrogen atom. These conclusions are in line with existing tubulin polymerization and microtubule‐binding data for 4 , 5 , and Epo B.     

Design, synthesis, and pharmacological characterization of novel spirocyclic quinuclidinyl-Δ2-isoxazoline derivatives as potent and selective agonists of α7 nicotinic acetylcholine receptors

A set of racemic spirocyclic quinuclidinyl‐Δ²‐isoxazoline derivatives was synthesized using a 1,3‐dipolar cycloaddition‐based approach. Target compounds were assayed for binding affinity toward rat neuronal homomeric (α7) and heteromeric (α4β2) nicotinic acetylcholine receptors. Δ²‐Isoxazolines 3 a (3‐Br), 6 a (3‐OMe), 5 a (3‐Ph), 8 a (3‐OnPr), and 4 a (3‐Me) were the ligands with the highest affinity for the α7 subtype (K_i values equal to 13.5, 14.2, 25.0, 71.6, and 96.2 nM , respectively), and showed excellent α7 versus α4β2 subtype selectivity. These compounds, tested in electrophysiological experiments against human α7 and α4β2 receptors stably expressed in cell lines, behaved as partial α7 agonists with varying levels of potency. The two enantiomers of (±)‐3‐methoxy‐1‐oxa‐2,7‐diaza‐7,10‐ethanospiro[4.5]dec‐2‐ene sesquifumarate 6 a were prepared using (+)‐dibenzoyl‐L ‐ or (?)‐dibenzoyl‐D ‐tartaric acid as resolving agents. Enantiomer (R)‐(?)‐ 6 a was found to be the eutomer, with K_i values of 4.6 and 48.7 nM against rat and human α7 receptors, respectively.     

Synthesis, antiproliferative activity, and structure-activity relationships of 3-aryl-1H-quinolin-4-ones

The antiproliferative activities of 36 3-aryl-1H-quinolin-4-ones were determined against two cancer cell lines (Hep G2 and KB) in vitro. The results indicate that most of these compounds show good cytotoxic activity against human cancer cell lines, but no cytotoxicity against a human normal cell line (L02). The positive control compounds genistein and 5-fluorouracil show no selectivity at inhibiting the growth of the above three cell lines. Generally, compounds that bear a halogen atom at the 8 position and a methoxy group at the 3' position exhibited remarkable cytotoxicity toward human cancer cell lines. Electron-withdrawing substituents at the 6 position decrease the antiproliferative activity significantly. We also put forward a pharmacophore model for 3-aryl-4-quinolinones binding with epidermal growth factor receptor protein tyrosine kinases (EGFR PTK). Out of the 36 synthetic compounds, 34 are reported for the first time.     

Chemoenzymatic synthesis and antimicrobial and haemolytic activities of amphiphilic bis(phenylacetylarginine) derivatives

Novel bis(N(alpha)-phenylacetyl-L-arginine)-alpha,omega-alkanediamide dihydrochloride (bis(PhAcArg)) derivatives with antimicrobial activity were designed and synthesised by a chemoenzymatic strategy. The new structures consist of two N(alpha)-phenylacetyl-L-arginine moieties connected by an alkanediamine spacer chain of 6, 8, 10, 12, and 14 methylene units through amide bonds. The key step in the chemoenzymatic strategy is the double aminolysis of the N(alpha)-phenylacetyl-L-arginine methyl ester by the corresponding alpha,omega-alkanediamine catalyzed by papain in ethanolic media. The compounds synthesised were tested as antimicrobials against 15 bacterial and 8 fungal species. The antimicrobial activity and selectivity depend strongly on the spacer chain length. The bis(PhAcArg) derivative with the spacer chain of 12 methylene groups gave the lowest MIC values against Gram-positive bacteria, whereas that with 14 methylene units was the best against Gram-negative bacteria. Interestingly, these novel compounds showed enhanced antibacterial activity relative to the lead compound, bis(N(alpha)-caproyl-L-arginine)-1,3-propanediamide dihydrochloride (C(3)(CA)(2)), and moderate antifungal activity. Moreover, tests of haemolytic activity toward human erythrocytes revealed that haemolysis increases with spacer chain length. Importantly, the compounds were classified as not irritating to eyes, with the exception of the compound with the spacer chain of 14 methylene groups, which was a slight eye irritant.