Synthesis and antimicrobial activity of new 3-(1-R-3(5)-methyl-4-nitroso-1H-5(3)-pyrazolyl)-5-methylisoxazoles

A number of new 3-(1-R-3(5)-methyl-4-nitroso-1H-5(3)-pyrazolyl)-5-methylisoxazoles 6a–g (7b–f) were synthesized and tested for antibacterial and antifungal activity. Some of these compounds displayed antifungal activity at non-cytotoxic concentrations. Derivative 6c was 9 times more potent in vitro than miconazole and 20 times more selective against C. neoformans. 6c was also 8- and 125-fold more potent than amphotericin B and fluconazole, respectively. None of the compounds was active against bacteria. Preliminary structure–activity relationship (SAR) studies showed that the NO group at position 4 of the pyrazole ring is essential for the activity. Lipophilicity of the pyrazole moiety, N-alkyl chain length and planarity of the two heterocyclic rings appear to play a decisive role in modulating cytotoxicity and antifungal activity.     

Novel aryl and heteroaryl substituted N-[3-(4-phenylpiperazin-1-yl)propyl]-1,2,4-oxadiazole-5-carboxamides as selective GSK-3 inhibitors

Synthesis, biological evaluation, and SAR dependencies for a series of novel aryl and heteroaryl substituted N-[3-(4-phenylpiperazin-1-yl)propyl]-1,2,4-oxadiazole-5-carboxamide inhibitors of GSK-3beta kinase are described. The inhibitory activity of the synthesized compounds is highly dependent on the character of substituents in the phenyl ring and the nature of terminal heterocyclic fragment of the core molecular scaffold. The most potent compounds from this series contain 3,4-di-methyl or 2-methoxy substituents within the phenyl ring and 3-pyridine fragment connected to the 1,2,4-oxadiazole heterocycle. These compounds selectively inhibit GSK-3beta kinase with IC(50) value of 0.35 and 0.41 microM, respectively.     

New azole antagonists with high affinity for the P2Y1 receptor

Five-membered-ring heterocyclic urea mimics have been found to be potent and selective antagonists of the P2Y₁ receptor. SAR of the various heterocyclic replacements is presented, as well as side-chain SAR of the more potent thiadiazole ring system which leads to thiadiazole 4c as a new antiplatelet agent.     

7-(aryl/heteroaryl-2-ylethynyl)-4-phenylamino-3-quinolinecarbonitriles as new Src kinase inhibitors: addition of water solubilizing groups

New 4-phenylamino-3-quinolinecarbonitriles with a 7-ethynyl group substituted by a pyridine, phenyl or thiophene ring containing basic water solubilizing groups were prepared and evaluated as Src kinase inhibitors. Of these new analogs, potent activity was observed with compounds having a (2,4-dichloro-5-methoxyphenyl)amino group at C-4, a methoxy or ethoxy group at C-6, and a pyridyl group bearing a dimethylamine or N-methylpiperazine on the ethynyl group at C-7.     

Selective inhibition of trypsin by (2R,4R)-4-phenyl-1-[Nalpha-(7- methoxy-2-naphthalenesulfonyl)-L-arginyl]-2-piperidinecarboxylic acid

Evidence is accumulating indicating that trypsin stimulates divergent cellular reactions through the proteinase-activated receptor, in addition to its role as the digestive enzyme. In this report, we introduce (2R,4R)- 4-phenyl-1-[N(alpha)-(7-methoxy-2-naphthalenesulfonyl)-l-arginyl]- 2-p iperidinecarboxylic acid as a potent and selective trypsin inhibitor. The agent inhibited trypsin competitively with the K(i) value of 0. 1 micrometer. It inhibited thrombin weakly (K(i) = 2 micrometer) and did not inhibit plasmin, plasma kallikrein, urokinase, and mast cell tryptase (K(i) values for these enzymes are >60 micrometer). Comparative studies with several established proteinase inhibitors revealed that the compound was the first small molecular weight trypsin inhibitor without tryptase inhibitory activity. A docking study has provided a plausible explanation for the molecular mechanism of the selective inhibition showing that the agent fits into the active site of trypsin without any severe collision but that it comes into clash at the 4-phenyl group of piperidine ring against the "60-insertion loop" of thrombin and at the 7-methoxy-2-naphthalenesulfonyl group against Gln(98) of tryptase.     

Investigation of various N-heterocyclic substituted piperazine versions of 5/7-{[2-(4-aryl-piperazin-1-yl)-ethyl]-propyl-amino}-5,6,7,8-tetrahydro-naphthalen-2-ol: Effect on affinity and selectivity for dopamine D3 receptor

Here we report on the design and synthesis of several heterocyclic analogues belonging to the 5/7-{[2-(4-aryl-piperazin-1-yl)-ethyl]-propyl-amino}-5,6,7,8-tetrahydro-naphthalen-2-ol series of molecules. Compounds were subjected to [³H]spiperone binding assays, carried out with HEK-293 cells expressing either D2 or D3 dopamine receptors, in order to evaluate their inhibition constant (K_i) at these receptors. Results indicate that N-substitution on the piperazine ring can accommodate various substituted indole rings. The results also show that in order to maintain high affinity and selectivity for the D3 receptor the heterocyclic ring does not need to be connected directly to the piperazine ring as the majority of compounds included here are linked either via an amide or a methylene linker to the heterocyclic moiety. The enantiomers of the most potent racemic compound 10e exhibited differential activity with (?)-10e (K_i; D2 = 47.5 nM, D3 = 0.57 nM) displaying higher affinity at both D2 and D3 receptors compared to its enantiomer (+)-10e (K_i; D2 = 113 nM, D3 = 3.73 nM). Additionally, compound (?)-10e was more potent and selective for the D3 receptor compared to either 7-OH-DPAT or 5-OH-DPAT. Among the bioisosteric derivatives, the indazole derivative 10g and benzo[b]thiophene derivative 10i exhibited the highest affinity for D2 and D3 receptors. In the functional GTPγS binding study, one of the lead molecules, (?)-15, exhibited potent agonist activity at both D2 and D3 receptors with preferential affinity at D3.     

(3R)-4-[(3R)-3-Amino-4-(2,4,5-trifluorophenyl)butanoyl]-3-(2,2,2-trifluoroethyl)-1,4-diazepan-2-one, a selective dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes

Replacement of the triazolopiperazine ring of sitagliptin (DPP-4 IC(50)=18nM) with 3-(2,2,2-trifluoroethyl)-1,4-diazepan-2-one gave dipeptidyl peptidase IV (DPP-4) inhibitor 1 which is potent (DPP-4 IC(50)=2.6nM), selective, and efficacious in an oral glucose tolerance test in mice. It was selected for extensive preclinical development as a potential back-up candidate to sitagliptin.     

SAR studies of 4-pyridyl heterocyclic anilines that selectively induce autophagic cell death in von Hippel-Lindau-deficient renal cell carcinoma cells

We recently identified a class of pyridyl aniline thiazoles (PAT) that displayed selective cytotoxicity for von Hippel-Lindau (VHL) deficient renal cell carcinoma (RCC) cells in vitro and in vivo. Structure-activity relationship (SAR) studies were used to develop a comparative molecular field analysis (CoMFA) model that related VHL-selective potency to the three-dimensional arrangement of chemical features of the chemotype. We now report the further molecular alignment-guided exploration of the chemotype to discover potent and selective PAT analogues. The contribution of the central thiazole ring was explored using a series of five- and six-membered ring heterocyclic replacements to vary the electronic and steric interactions in the central unit. We also explored a positive steric CoMFA contour adjacent to the pyridyl ring using Pd-catalysed cross-coupling Suzuki-Miyaura, Sonogashira and nucleophilic displacement reactions to prepare of a series of aryl-, alkynyl-, alkoxy- and alkylamino-substituted pyridines, respectively. In vitro potency and selectivity were determined using paired RCC cell lines: the VHL-null cell line RCC4 and the VHL-positive cell line RCC4-VHL. Active analogues selectively induced autophagy in RCC4 cells. We have used the new SAR data to further develop the CoMFA model, and compared this to a 2D-QSAR method. Our progress towards realising the therapeutic potential of this chemotype as a targeted cytotoxic therapy for the treatment of RCC by exploiting the absence of the VHL tumour suppressor gene is reported.     

Synthesis and biological evaluation of new disubstituted analogues of 6-methoxy-3-(3',4',5'-trimethoxybenzoyl)-1H-indole (BPR0L075), as potential antivascular agents

6-Methoxy-3-(3',4',5'-trimethoxybenzoyl)-1H-indole (BPR0L075) (1) is a potent inhibitor of tubulin polymerization which exhibits both in vitro and in vivo activities against a broad spectrum of solid tumors. This compound was designed as a heterocyclic analogue of combretastatin A4 (CA-4), a natural stilbene derivative that disrupts the tumor vasculature and causes tumor regression. In the present work, we describe the design and synthesis of several new disubstituted analogues of 1, along with their biological evaluation as potential antivascular agents. Compound 13, bearing a hydroxyl group at the 7-position of the indole nucleus that mimics the hydroxyl group at the 3-position of the B-ring of CA-4, was identified as a potent inhibitor of tubulin polymerization and also as a cytotoxic agent against B16 melanoma cells at sub-micromolar concentration. In addition, compound 13 displayed marked morphological activity (rounding up) at nanomolar concentrations on endothelial cells (EA.hy 926 cells), which is indicative of potential antivascular activity. Interestingly, the corresponding 7-O-mesylate derivative 28 (an intermediate in the synthesis of 13), was also found active in cellular assays, although it was moderately active in the tubulin polymerization inhibition test. Finally, in order to better understand the SAR of disubstituted analogues of 1, two other position isomers (10 and 14), were synthesized and evaluated for their biological activities. It was noted that the 7-hydroxysubstituted analogue 13 was more potent than the 5-hydroxysubstituted analogue 10. In conclusion, this work has allowed the identification of biologically potent CA-4 analogues (13 and 28) and also contributes to a better understanding of the SAR of 1.     

Identification of trans-4-[1-[[7-fluoro-2-(1-methyl-3-indolyl)-6-benzoxazolyl]acetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylic acid as a potent, orally active VLA-4 antagonist

For the purpose of obtaining orally potent VLA-4 inhibitors, we have carried out structural modification of the (N'-phenylureido)phenyl group in compound 1, where the group was found to be attributed to poor pharmacokinetic profile in our previous research. Through modification, we have identified several compounds with both potent in vitro activity and improved oral exposure. In particular, compound 7e with 7-fluoro-2-(1-methyl-1H-indol-3-yl)-1,3-benzoxazolyl group as a novel replacement of the (N'-phenylureido)phenyl group significantly inhibited eosinophil infiltration into bronchoalveolar lavage fluid at 15mg/kg in an Ascaris-antigen-induced murine bronchial inflammatory model, and its efficacy was comparable to that of the anti-mouse α(4) antibody (R1-2).     

Potential antitumour and pro-oxidative effects of (E)-methyl 2-(7-chloroquinolin-4-ylthio)-3-(4-hydroxyphenyl) acrylate (QNACR)

Context: Characterization of the pro-oxidant activity of QNACR.

Objectives: Reactive oxygen species (ROS) induce cellular damage and represent unique opportunities to kill malignant cells. In this study, we synthesized and evaluated the new compound, (E)-methyl 2-(7-chloroquinolin-4-ylthio)-3-(4-hydroxyphenyl) acrylate (QNACR) as potential pro-oxidative agent against breast cancer.

Methods: Oxidative stress biomarkers such as ROS, thiobarbuturic acid reactive species (TBARs) and different antioxidant enzyme activities were determined in cell lysates.

Results: QNACR showed cytotoxic and more selective effects to tumour MCF7 cells (IC₅₀ < 25 µM) compared to antitumour controls, inducing ROS and TBARs parallel to inhibitions of catalase (CAT), glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH). Longer exposures to QNACR triggered adaptive effects increasing the overall activities of CAT, glutathione reductase, G6PDH and 6PGDH, but eventually the adaptation changes faded and cells died.

Conclusion: QNACR led to remarkable modifications in the oxidative status of tumour cells, proposing this compound as potential alternative for antitumour therapy.     

Synthesis and antiplasmodial activity of new heteroaryl derivatives of 7-chloro-4-aminoquinoline

With the aim to investigate the effect of different heterocyclic rings linked to the 4-aminoquinoline nucleus on the antimalarial activity, a set of 7-chloro-N-(heteroaryl)-methyl-4-aminoquinoline and 7-chloro-N-(heteroaryl)-4-aminoquinoline was synthesized and tested in vitro against D-10 (CQ-S) and W-2 (CQ-R) strains of Plasmodium falciparum. All compounds exhibited from moderate to high antiplasmodial activities. The activity was strongly influenced both by the presence of a methylenic group, as a spacer between the 4-aminoquinoline and the heterocyclic ring, and by the presence of a basic head. The most potent molecules inhibited the growth of both CQ-S and CQ-R strains of P. falciparum with IC₅₀ < 30 nM and were not toxic against human endothelial cells. These results confirm that the presence of an heteroaryl moiety in the side chain of 7-chloro-4-aminoquinoline is useful for the design and development of new powerful antimalarial agents.     

In Vitro antifungal activity of 2-(4-substituted phenyl)-3(2H)-isothiazolones

The in vitro antifungal activity of several N2-phenyl-3(2H)-isothiazolones substituted at C4 of the phenyl moiety with heterocyclic nucleus or groups of different physico-chemical properties against four human pathogenic fungi was determined by broth macrodilution method; results were compared with those obtained with itraconazole and ketoconazole. These isothiazolones showed moderate to high activity against some or all tested strains and in comparison with the reference drugs, 5-chloro-2-(4-nitrophenyl)isothiazol-3-one (1g), 5-chloro-2-phenylisothiazol-3-one (1c), 4-[4-(5-chloro-3-oxo-3H-isothiazol-2-yl)phenyl]-1,4-dihydrotriazol-5-one (1s) and 2-(4-nitrophenyl)isothiazol-3-one (2g) against Aspergillus niger, 5-chloro-2-(4-nitrophenyl)isothiazol-3-one (1g) and 4-[4-(5-chloro-3-oxo-3H-isothiazol-2-yl)phenyl]piperazine-1-carboxamide (1q) against Trichophyton mentagrophytes had comparable activity, compounds 1g and 2g showing higher activity against Microsporum canis. Antifungal activity was favored by the presence of chlorine at C5 of the isothiazolone and/or the presence of nitro group or heterocyclic nucleus at C4 of the phenyl ring and proper hydrophilicity of the molecule.     

Heterocyclic derivatives of 2-(3,5-dimethylphenyl)tryptamine as GnRH receptor antagonists

A series of heterocyclic 2-(3,5-dimethylphenyl)tryptamine derivatives was prepared and evaluated on a rat gonadotropin releasing hormone receptor assay. The carbon tether length and heterocyclic ring attached to the amino group of 2-(3,5-dimethylphenyl)tryptamine were varied. Several of these derivatives were potent GnRH antagonists with the most potent compound having an IC50 of 16 nM.     

Imidazopyridines as VLA-4 integrin antagonists

We describe a novel series of imidazopyridine substituted phenylalanines which are potent VLA-4 antagonists. A wide variety of substituents are tolerated as replacements for the pendant 3-pyridyl ring. A clear structure–activity relationship was identified around the substitution of the 3-amino-cyclobut-2-enone portion of the molecule.     

Part 3: synthesis and biological evaluation of some analogs of the antitumor agents, 2-{4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid, and 2-{4-[(7-bromo-2-quinolinyl)oxy]phenoxy}propionic acid

2-{4-[(7-Chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid (X469) and 2-{4-[(7-bromo-2-quinolinyl)oxy]phenoxy}propionic Acid (SH80) are among the most highly and broadly active antitumor agents to have been developed in our laboratories. However, the mechanism(s) of action of these agents remain to be elucidated, which prompted our continued endeavor to delineate a pharmacophoric pattern, from which a putative target might be deduced. Herein, we provide additional evidence that intact quinoxaline and quinoline rings in XK469 and SH80, respectively, are fundamental to the activities of these structures against transplanted tumors in mice. The consequence of further modification of the heterocyclic ring system in XK469 and SH80, leading to [1,8]naphthyridine; pyrrolo[1,2-a]; imidazo[1,2-a]; and imidazo[1,5-a] derivatives, all deprive the parent structures of antitumor activity. Introduction of CH3, CF3, CH3O, CO2H, or C6H5 substituents at C4 of the quinoline ring of SH80 led to weakly active antitumor agents. Similarly, the phenanthridine analog of SH80 manifested only modest cytotoxicity. Lastly, XK469 and SH80 are both significantly more active than the corresponding regioisomeric structures, 2-{4-[(7-halo-4-quinolinyl)oxy]phenoxy)propionic acids.     

Design,synthesis, and evaluation of 2-aryl-7-(3′,4′-dialkoxyphenyl)-pyrazolo[1,5-a]pyrimidines as novel PDE-4 inhibitors

Described herein is design, synthesis, and biological evaluation of novel series of 2-aryl-7-(3′,4′-dialkoxyphenyl)-pyrazolo[1,5-a]pyrimidines acting as inhibitors of type 4 phosphodiesterase (PDE4) which is known as a good target for the treatment of asthma and COPD. For this purpose, structure optimization was conducted with the aid of structure-based drug design using the known X-ray crystallography. Also, biological effects of these compounds on the target enzyme were evaluated by using in vitro assays, leading to the potent and selective PDE-4 inhibitor (IC₅₀ < 10 nM).     

Chemical modification of aryl-1,2,3,6-tetrahydropyridinopyrimidine derivative to discover corticotropin-releasing factor(1) receptor antagonists: aryl-1,2,3,6-tetrahydropyridino-purine, -3H-1,2,3-triazolo[4,5-d)pyrimidine, -purin-8-one, and -7H-pyrrolo[2,3-d]pyrimidine derivatives

Structure-affinity relationships (SARs) of non-peptide CRF(1) antagonists suggest that such antagonists can be constructed of three units: a hydrophobic unit (Up-Area), a proton accepting unit (Central-Area), and an aromatic unit (Down-Area). Recently, various non-peptide corticotropin-releasing factor(1) (CRF(1)) receptor antagonists obtained by modification of the Central-Area have been reported. In contrast, we modified the Up-Area and presented 4- or 5-aryl-1,2,3,6-tetrahydropyridinopyrimidine derivatives including potent CRF receptor ligands 1a-c, and proposed that the 4- or 5-aryl-1,2,3,6-tetrahydropyridino moiety might be useful as a substituent in the Up-Area. Our interest shifted to the chemical modification in which the pyrimidine ring of 1a-c was replaced by other heterocycles, purine ring of 2, 3H-1,2,3-triazolo[4,5-d]pyrimidine ring of 3, purin-8-one ring of 4 and 7H-pyrrolo[2,3-d]pyrimidine ring of 5. Among them, 5-aryl-1,2,3,6-tetrahydropyridinopurine compound 6j (CRA0186) had the highest affinity for CRF(1) receptors (IC(50)=20nM). We report here the synthesis and SARs of derivatives 6-9.     

Structural study of porcine pancreatic elastase complexed with 7-amino-3-(2-bromoethoxy)-4-chloroisocoumarin as a nonreactivatable doubly covalent enzyme-inhibitor complex

The complex of porcine pancreatic elastase (PPE) with 7-amino-3-(2-bromoethoxy)-4-chloroisocoumarin, a potent mechanism-based inhibitor, was crystallized and the crystal structure determined at 1.9-A resolution with a final R factor of 17.1%. The unbiased difference Fourier electron density map showed continuous density from O gamma of Ser 195 to the benzoyl carbonyl carbon atom and from N epsilon 2 of His 57 to the carbon atom at the 4-position of the isocoumarin ring in the inhibitor. This suggested unambiguously that the inhibitor was doubly covalently bound to the enzyme. It represents the first structural evidence for irreversible binding of an isocoumarin inhibitor to PPE through both Ser 195 and His 57 in the active site. The PPE-inhibitor complex is only partially activated in solution by hydroxylamine and confirms the existence of the doubly covalently bound complex along with the acyl enzyme. The benzoyl carbonyl oxygen atom of the inhibitor is not situated in the oxyanion hole formed by the amide (greater than NH) groups of Gly 193 and Ser 195. The complex is stabilized by the hydrogen-bonding interactions in the active site (from the N epsilon 2 of Gln 192 to the bromine atom in the inhibitor and the amino group at the 7-position of the isocoumarin ring to the carbonyl oxygen of Thr 41) and by van der Waals interactions. The inhibition rates of several 7-substituted 4-chloro-3-(bromoalkoxy)isocoumarins toward PPE were measured.(ABSTRACT TRUNCATED AT 250 WORDS)     

2-(4-carbonylphenyl)benzoxazole inhibitors of CETP: scaffold design and advancement in HDLc-raising efficacy

The development of 2-phenylbenzoxazoles as inhibitors of cholesteryl ester transfer protein (CETP) is described. Initial efforts aimed at engineering replacements for the aniline substructures in the benchmark molecule. Reversing the connectivity of the central aniline lead to a new class of 2-(4-carbonylphenyl)benzoxazoles. Structure-activity studies at the C-7 and terminal pyridine ring allowed for the optimization of potency and HDLc-raising efficacy in this new class of inhibitors. These efforts lead to the discovery of benzoxazole 11v, which raised HDLc by 24 mg/dl in our transgenic mouse PD model.