3,5-Disubstituted indole derivatives as selective human neuronal nitric oxide synthase (nNOS) inhibitors

A series of 3,5-disubstituted indole derivatives was designed, synthesized and evaluated as inhibitors of human nitric oxide synthase (NOS). Various guanidine isosteric groups were explored at the 5-position of the indole ring, while keeping the basic amine side chain such as N-methylpiperidine ring, fixed at the 3-position of the indole ring. Compounds having 2-thiophene amidine and 2-furanyl amidine groups (7, 8, 10 and 12) showed increased activity for human neuronal NOS and good selectivity over endothelial and inducible NOS isoforms. Compound 8 was shown to reverse (10mg/kg, ip) thermal hyperalgesia in the L(5)/L(6) spinal nerve ligation (neuropathic pain) model and was devoid of any significant drug-drug interaction potential due to cytochrome P450 inhibition or cardiovascular liabilities associated with the inhibition of endothelial NOS.     

Comparison of the inhibitory effect of isothiourea and mercapto-alkylguanidine derivatives on the alternative pathways of arginine metabolism in macrophages

Novel, non-arginine based compounds have been identified as potent inhibitors of nitric oxide synthase (NOS). Members of the isothiourea and mercapto-alkylguanidine classes have generated much interest, as some members of these classes show selectivity towards the inducible isoform of NOS (iNOS), which plays a role in inflammation and shock. Here we compared the effect of a number of these compounds as well as L-arginine based NOS inhibitor reference compounds on macrophage-derived and liver arginase and macrophage iNOS activities. From the nonarginine based NOS inhibitors studied only S-aminoethyl-isothiourea (AETU) caused a slight inhibition of arginase activity. This inhibition was kinetically competitive and due to the rearrangement of AETU to mercapto-ethylguanidine (MEG). The weak inhibitory effect of non-arginine based iNOS inhibitors on arginase activity further supports the view that such compounds may be of practical use for inhibition of NO production in cells simultaneously expressing iNOS and arginase.     

Illuminating the binding interactions of galactonoamidines during the inhibition of β-galactosidase (E. coli)

Several galactonoamidines were previously identified as very potent competitive inhibitors that exhibit stabilizing hydrophobic interactions of the aglycon in the active site of β-galactosidase (Aspergillus oryzae). To elucidate the contributions of the glycon to the overall inhibition ability of the compounds, three glyconoamidine derivatives with alteration in the glycon at C-2 and C-4 were synthesized and evaluated herein. All amidines are competitive inhibitors of β-galactosidase (Escherichia coli) and show significantly reduced inhibition ability when compared to the parent. The results highlight strong hydrogen-bonding interactions between the hydroxyl group at C-2 of the amidine glycon and the active site of the enzyme. Slightly weaker H-bonds are promoted through the hydroxyl group at C-4. The inhibition constants were determined to be picomolar for the parent galactonoamidine, and nanomolar for the designed derivatives rendering all glyconoamidines very potent inhibitors of glycosidases albeit the derivatized amidines show up to 700-fold lower inhibition activity than the parent.     

A cellular model for screening neuronal nitric oxide synthase inhibitors

Nitric oxide synthase (NOS) inhibitors are potential drug candidates because it has been well demonstrated that excessive production of nitric oxide critically contributes to a range of diseases. Most inhibitors have been screened in vitro using recombinant enzymes, leading to the discovery of a variety of potent compounds. To make inhibition studies more physiologically relevant and bridge the gap between the in vitro assay and in vivo studies, we report here a cellular model for screening NOS inhibitors. Stable transformants were generated by overexpressing rat neuronal NOS in HEK 293T cells. The enzyme was activated by introducing calcium ions into cells, and its activity was assayed by determining the amount of nitrite that was formed in culture medium using the Griess reagent. We tested a few NOS inhibitors with this assay and found that the method is sensitive, versatile, and easy to use. The cell-based assay provides more information than in vitro assays regarding the bioavailability of NOS inhibitors, and it is suitable for high-throughput screening.     

Synthesis and evaluation of trans 3,4-cyclopropyl L-arginine analogues as isoform selective inhibitors of nitric oxide synthase

Four optically pure conformationally restricted L-arginine analogues syn- 1 and anti- 2 trans-3,4-cyclopropyl L-arginine, and syn- 3 and anti-trans-3,4-cyclopropyl N-(1-iminoethyl) L-ornithine 4 were synthesized. These compounds were tested as potential inhibitors against the three isoforms of nitric oxide synthase (NOS). Compound 1 was determined to be a poor substrate of NOS, while compound 2 was determined to be a poor mixed type inhibitor and did not exhibit any isoform selectivity. Syn- 3 and anti-trans-3,4-cyclopropyl N-(1-iminoethyl) L-ornithine 4 were found to be competitive inhibitors of NOS. These compounds were time dependent inhibitors of inducible NOS (iNOS), but not of neuronal NOS (nNOS) or endothelial NOS (eNOS). Compound 3 was 10- to 100-fold more potent an inhibitor than 4, exhibited a 5-fold increase in nNOS/iNOS and eNOS/iNOS selectivity over 4, and displayed tight binding characteristics against iNOS. These results indicate that the relative configuration of the cyclopropyl ring in the L-arginine analogues significantly affects their inhibitory potential and NOS isoform selectivity.     

Identification of orally bioavailable,non-amidine inhibitors of Urokinase Plasminogen Activator (uPA)

In this Letter we report the synthesis and evaluation of a series of non-amidine inhibitors of Urokinase Plasminogen Activator (uPA). Starting from compound 1, a significant change provided compounds in which the amidine, binding in the S1 pocket, was replaced with a primary amine. Further modifications led to the identification of potent, selective, and orally bioavailable uPA inhibitors.     

C17,20-lyase inhibitors I. Structure-based de novo design and SAR study of C17,20-lyase inhibitors

Novel nonsteroidal C(17,20)-lyase inhibitors were synthesized using de novo design based on its substrate, 17 alpha-hydroxypregnenolone, and several compounds exhibited potent C(17,20)-lyase inhibition. However, in vivo activities were found to be short-lasting, and in order to improve the duration of action, a series of benzothiophene derivatives were evaluated. As a result, compounds 9h, (S)-9i, and 9k with nanomolar enzyme inhibition (IC(50)=4-9 nM) and 9e (IC(50)=27 nM) were identified to have powerful in vivo efficacy with extended duration of action. The key structural determinants for the in vivo efficacy were demonstrated to be the 5-fluoro group on the benzothiophene ring and the 4-imidazolyl moiety. Superimposition of 9k and 17 alpha-hydroxypregnenolone demonstrated their structural similarity and enabled rationalization of the pharmacological results. In addition, selected compounds were also identified to be potent inhibitors of human enzyme with IC(50) values of 20-30 nM.     

Structure and activity of NO synthase inhibitors specific to the L-arginine binding site

Synthesis of compounds containing a fragment similar to the guanidine group of L-arginine, which is a substrate of nitric oxide synthase (NOS), is the main direction in creating NOS inhibitors. The inhibitory effect of such compounds is caused not only by their competition with the substrate for the L-arginine-binding site and/or oxidizing center of the enzyme (heme) but also by interaction with peptide motifs of the enzyme that influence its dimerization, affinity for cofactors, and interaction with associated proteins. Structures, activities, and relative in vitro and in vivo specificities of various NOS inhibitors (amino acid and non-amino acid) with linear or cyclic structure and containing guanidine, amidine, or isothiuronium group are considered. These properties are mainly analyzed by comparison with effects of the inhibitors on the inducible NOS.Translated from Biokhimiya, Vol. 70, No. 1, 2005, pp. 14–32.Original Russian Text Copyright © 2005 by Proskuryakov, Konoplyannikov, Skvortsov, Mandrugin, Fedoseev.     

Evaluation of analogues of furan-amidines as inhibitors of NQO2

Inhibitors of the enzyme NQO2 (NRH: quinone oxidoreductase 2) are of potential use in cancer chemotherapy and malaria. We have previously reported that non-symmetrical furan amidines are potent inhibitors of NQO2 and here novel analogues are evaluated. The furan ring has been changed to other heterocycles (imidazole, N-methylimidazole, oxazole, thiophene) and the amidine group has been replaced with imidate, reversed amidine, N-arylamide and amidoxime to probe NQO2 activity, improve solubility and decrease basicity of the lead furan amidine. All compounds were fully characterised spectroscopically and the structure of the unexpected product N-hydroxy-4-(5-methyl-4-phenylfuran-2-yl)benzamidine was established by X-ray crystallography. The analogues were evaluated for inhibition of NQO2, which showed lower activity than the lead furan amidine. The observed structure-activity relationship for the furan-amidine series with NQO2 was rationalized by preliminary molecular docking and binding mode analysis. In addition, the oxazole-amidine analogue inhibited the growth of Plasmodium falciparum with an IC₅₀ value of 0.3?μM.     

Design and synthesis of factor Xa inhibitors and their prodrugs

In addition to our previously reported fluoro acrylamides Xa inhibitors 2 and 3, a series of potent and novel cyclic diimide amidine compounds has been identified. In efforts to improve their oral bioavailability, replacement of the amidine group with methyl amidrazone gives compounds of moderate potency (14, IC(50)=0.028 microM). In the amidoxime prodrug approach, the amidoxime compounds show good oral bioavailability in rats and dogs. High plasma level of prodrug 26 and significant concentration of active drug 26a were obtained upon oral administration of prodrug 26 in rats.     

Insulin-like growth factor-II, phosphatidylinositol 3-kinase, nuclear factor-kappaB and inducible nitric-oxide synthase define a common myogenic signaling pathway.

Insulin-like growth factors (IGFs) are potent inducers of skeletal muscle differentiation and phosphatidylinositol (PI) 3-kinase activity is essential for this process. Here we show that IGF-II induces nuclear factor-kappaB (NF-kappaB) and nitric-oxide synthase (NOS) activities downstream from PI 3-kinase and that these events are critical for myogenesis. Differentiation of rat L6E9 myoblasts with IGF-II transiently induced NF-kappaB DNA binding activity, inducible nitric-oxide synthase (iNOS) expression, and nitric oxide (NO) production. IGF-II-induced iNOS expression and NO production were blocked by NF-kappaB inhibition. Both NF-kappaB and NOS activities were essential for IGF-II-induced terminal differentiation (myotube formation and expression of skeletal muscle proteins: myosin heavy chain, GLUT 4, and caveolin 3), which was totally blocked by NF-kappaB or NOS inhibitors in rat and human myoblasts. Moreover, the NOS substrate L-Arg induced myogenesis in the absence of IGFs in both rat and human myoblasts, and this effect was blocked by NOS inhibition. Regarding the mechanisms involved in IGF-II activation of NF-kappaB, PI 3-kinase inhibition prevented NF-kappaB activation, iNOS expression, and NO production. Moreover, IGF-II induced, through a PI 3-kinase-dependent pathway, a decrease in IkappaB-alpha protein content that correlated with a decrease in the amount of IkappaB-alpha associated with p65 NF-kappaB.     

Discovery and evaluation of 3-phenyl-1H-5-pyrazolylamine-based derivatives as potent, selective and efficacious inhibitors of FMS-like tyrosine kinase-3 (FLT3)

Preclinical investigations and early clinical trial studies suggest that FLT3 inhibitors offer a viable therapy for acute myeloid leukemia. However, early clinical data for direct FLT3 inhibitors provided only modest results because of the failure to fully inhibit FLT3. We have designed and synthesized a novel class of 3-phenyl-1H-5-pyrazolylamine-derived compounds as FLT3 inhibitors which exhibit potent FLT3 inhibition and high selectivity toward different receptor tyrosine kinases. The structure-activity relationships led to the discovery of two series of FLT3 inhibitors, and some potent compounds within these two series exhibited comparable potency to FLT3 inhibitors sorafenib (3) and ABT-869 (4) in both wt-FLT3 enzyme inhibition and FLT3-ITD inhibition on cell growth (MOLM-13 and MV4;11 cells). In particular, the selected compound 12a exhibited the ability to regress tumors in mouse xenograft models using MOLM-13 and MV4;11 cells.     

Factor VIIa inhibitors: a prodrug strategy to improve oral bioavailability

We have developed a series of potent and selective factor VIIa inhibitors based on the 2-[5-(5-carbamimidoyl-1H-benzoimidazol-2-yl)-6-hydroxy-biphenyl-3-yl]-succinic acid scaffold. These amidine-containing compounds have low oral bioavailability. Herein, we describe our efforts to improve the oral bioavailability of the parent amidine via a prodrug strategy where the amidine basicity and polarity were reduced with either an alkoxy-amidine or a carbamate prodrug.     

Modification of Bischler-Möhlau indole derivatives through palladium catalyzed Suzuki reaction as effective cholinesterase inhibitors,their kinetic and molecular docking studies

Due to the immense importance of aryl indole nucleus, herein we report the palladium-catalyzed arylation of N-substituted 2-aryl indole utilizing Suzuki-Miyaura cross coupling methodology. The biological screening for cholinesterase inhibition of the resulted biaryl indole moieties was carried out to evaluate their pharmacological potential, expecting to involve the development of new therapeutics for various inflammatory, cardiovascular, gastrointestinal and neurological diseases. This research work also involved the use of utilization of microwave-assisted organic synthesis (MAOS) for the synthesis of Bischler-Möhlau indole which is further biarylated via palladium-catalyzed cross coupling reaction. All the synthetic compounds (3a-n) were tested for cholinesterase inhibition and exhibited high level of AChE inhibitory activities. Interestingly, compounds 3m and 3n were found to be dual inhibitors, however, remaining compound exhibited no inhibitory activity against BChE. The biological potential of the resulted compounds was explained on the basis of molecular docking studies, performed against AChE and BChE, exploring the probable binding modes of most potent inhibitors.     

Inhibition of alpha, betaI, delta, eta, and zeta protein kinase C isoforms by xanthonolignoids

The effect of the xanthonolignoids trans-(+/-)-kielcorin C, cis-(+/-)-kielcorin C, trans-(+/-)-kielcorin D, trans-(+/-)-isokielcorin D and trans-(+/-)-kielcorin E on isoforms alpha, betaI, delta, eta and zeta of protein kinase C (PKC) was studied using the yeast phenotypic assay. All the compounds tested revealed an effect compatible with PKC inhibition, similar to that exhibited by the well established PKC inhibitor chelerythrine, and with differences in their potency towards the distinct isoforms tested, being, in general, potent inhibitors of the atypical PKC isoform (PKC-zeta). PKC inhibition caused by these kielcorins was confirmed using an in vitro kinase assay. The present study constitutes the first attempt to unravel the molecular mechanism of kielcorins activity, and shows that xanthonolignoids are a promising group of compounds to investigate for isoform selective PKC inhibitors.     

Antioxidation and tyrosinase inhibition of polyphenolic curcumin analogs

A series of polyphenolic curcumin analogs were synthesized and their inhibitory effects on mushroom tyrosinase and the inhibition of 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical formation were evaluated. The results indictated that the analogs possessing m-diphenols and o-diphenols exhibited more potent inhibitory activity on tyrosinase than reference compound rojic acid, and that the analogs with o-diphenols exhibited more potent inhibitory activity of DPPH free-radical formation than reference compound vitamin C. The inhibition kinetics, analyzed by Lineweaver-Burk plots, revealed that compounds B(2) and C(2) bearing o-diphenols were non-competitive inhibitors, while compounds B(11) and C(11) bearing m-diphenols were competitive inhibitors. In particular, representative compounds C(2) and B(11) showed no side effects at a dose of 2,000 mg/kg in a preliminary evaluation of acute toxicity in mice. These results suggest that such polyphenolic curcumin analogs might serve as lead compounds for further design of new potential tyrosinase inhibitors.     

Discovery of 2-ureidophenyltriazines bearing bridged morpholines as potent and selective ATP-competitive mTOR inhibitors

Incorporation of bridged morpholines in monocyclic triazine PI3K/mTOR inhibitors gave compounds with increased mTOR selectivity relative to the corresponding morpholine analogs. Compounds with ureidophenyl groups gave highly potent and selective mTOR inhibitors. Potency and selectivity was demonstrated both in vitro and in vivo through biomarker suppression studies. Select compounds exhibited potent inhibition of tumor growth in nude mouse xenograft assays upon PO and IV administration.     

Synthesis,topoisomerase-targeting activity and growth inhibition of lycobetaine analogs

The plant alkaloid lycobetaine has potent topoisomerase-targeting properties and shows anticancer activity. Based on these findings, several lycobetaine analogs were synthesized mainly differing in their substituents at 2, 8 and 9 position and their biological activities were evaluated. The topoisomerase-targeting properties and cytotoxicity of these structural analogs were assessed in the human gastric carcinoma cell line GXF251L. Performing a plasmid relaxation assay, an increased inhibition of topoisomerase I was found with N-methylphenanthridinium chlorides bearing a 8,9-methylenedioxy moiety or a methoxy group in 2-position. Furthermore, quaternized phenanthridinium derivatives bearing either a 2-methoxy or a 8,9-methylenedioxy moiety in conjunction with a 2-hydroxy or 2-methoxy group display potent topoisomerase II inhibition as shown by decatenation of kinetoplast DNA. In general, the N-methylphenanthridinium chlorides possess more potency in inhibiting topoisomerase I than topoisomerase II. All quaternized derivatives also exhibited potent inhibition of tumor cell growth in the low micromolar concentration range. Hence, N-methylphenanthridinium compounds were found to represent a promising class of compounds, potently inhibiting both, topoisomerases I and II, and may be further developed into clinically useful topoisomerase inhibitors.     

A potent inhibitor of inducible nitric oxide synthase, ONO-1714, a cyclic amidine derivative

(1S,5S,6R,7R)-7-Chloro-3-imino-5-methyl-2-azabicyclo[4.1.0]heptane hydrochloride (ONO-1714), a novel cyclic amidine analogue, inhibits human inducible nitric oxide (iNOS) with a K(i) of 1.88 nM and rodent iNOS with similar potency in vitro. ONO-1714 was found to be 10-fold selective for human iNOS over human endothelial NOS (ecNOS). When the inhibitory activity of ONO-1714 was compared for iNOS, it was found to be 451-fold and >20,000-fold more potent than L-NMMA and aminoguanidine (AG), respectively. In terms of human iNOS selectivity, ONO-1714 was approximately 34- and 2-fold more selective for iNOS than L-NMMA and AG, respectively. ONO-1714 inhibited the LPS-induced elevation of plasma nitrate/nitrite in mice with an ID(50) value of 0.010 mg/kg, s.c. The maximum tolerated dose of ONO-1714 was 30 mg/kg, i.v. Thus, ONO-1714 represents one of the most potent iNOS inhibitors in vitro and in vivo to date and has great potentials for use as an inhibitor for clarifying the pathophysiological roles of iNOS and for use as a therapeutic agent.     

Identification of Redox Partners and Development of a Novel Chimeric Bacterial Nitric Oxide Synthase for Structure Activity Analyses

Production of nitric oxide (NO) by nitric oxide synthase (NOS) requires electrons to reduce the heme iron for substrate oxidation. Both FAD and FMN flavin groups mediate the transfer of NADPH derived electrons to NOS. Unlike mammalian NOS that contain both FAD and FMN binding domains within a single polypeptide chain, bacterial NOS is only composed of an oxygenase domain and must rely on separate redox partners for electron transfer and subsequent activity. Here, we report on the native redox partners for Bacillus subtilis NOS (bsNOS) and a novel chimera that promotes bsNOS activity. By identifying and characterizing native redox partners, we were also able to establish a robust enzyme assay for measuring bsNOS activity and inhibition. This assay was used to evaluate a series of established NOS inhibitors. Using the new assay for screening small molecules led to the identification of several potent inhibitors for which bsNOS-inhibitor crystal structures were determined. In addition to characterizing potent bsNOS inhibitors, substrate binding was also analyzed using isothermal titration calorimetry giving the first detailed thermodynamic analysis of substrate binding to NOS.