In vitro inhibition of lens aldose reductase by (2-benzyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole-8-yl)-acetic acid in enzyme preparations isolated from diabetic rats

(2-benzyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole-8-yl)-acetic acid (compound 1), a novel aldose reductase inhibitor, was assayed for efficacy and selectivity to inhibit rat lens aldose reductase under in vitro conditions by using enzyme preparations obtained from diabetic animals. The inhibitory efficiency was characterized by IC(50) in micromolar region. Enzyme kinetics analysis revealed uncompetitive type of inhibition, both in relation to the D,L-glyceraldehyde substrate and to the NADPH cofactor. In testing for selectivity, comparisons to rat kidney aldehyde reductase, an enzyme with the highest homology to aldose reductase, was used. The inhibition selectivity of the compound tested was characterized by selectivity factor around 20 and was even slightly improved under conditions of prolonged experimental diabetes. These findings were identical with those in the control rats. To conclude, the inhibitory mode, efficacy and selectivity of compound 1, a novel aldose reductase inhibitor, was preserved even under the conditions of prolonged STZ-induced experimental diabetes of rats.     

(2-Benzyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indol-8-yl)-acetic acid: an aldose reductase inhibitor and antioxidant of zwitterionic nature

Novel carboxymethylated pyridoindoles, characterized by antioxidant activity combined with the ability to inhibit aldose reductase, represent an example of a multitarget approach to the treatment of diabetic complications - severe diabetes-related health disorders of multifunctional nature. One of the novel carboxymethylated pyridoindoles, (2-benzyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indol-8-yl)-acetic acid (compound 1), was found to inhibit aldose reductase with the IC(50) value 18.2 ± 1.2 μM. Owing to aldose reductase pharmacophore requirements for an acidic proton, most aldose reductase inhibitors contain an acetic acid moiety, ionized at physiological pH, resulting in poor bioavailability of the drugs. The presence of a basicity center at the tertiary nitrogen of the carboxymethylated pyridoindoles, in addition to the acidic carboxylic function, predisposes these compounds to form double-charged zwitterionic species. The zwitterionic nature of compound 1 may remarkably affect its pH-lipophilicity profile allowing for increased membrane penetration in the pH region around its isoelectric point, which lies close to the physiological pH 7.4. In the first part of this study, the presence of zwitterionic species was experimentally proved by the concentration-dependent effect of sodium 1-hexanesulphonate on the distribution profile of compound 1. Then a series of experiments was performed in the cellular system of isolated erythrocytes in vitro. Isolated rat erythrocytes exposed to peroxyl radicals, generated in the solution by decomposition of the hydrophilic azoinitiator AAPH or intracellularly by decay of lipophilic t-BuOOH, underwent progressive hemolysis. The onset of the hemolysis was shifted from the starting zero point by the time interval assigned as a lag period. In the presence of compound 1 the lag period was significantly prolonged. Finally, under conditions of a short-term experiment in STZ-diabetic rats in vivo, increase in sorbitol levels in erythrocytes was recorded. Compound 1 administered in the dose 50mg/kg/day (i.g.) significantly decreased the sorbitol level in the erythrocytes. To conclude, the physico-chemical proof of the zwitterionic nature of compound 1 was established and the results obtained in isolated red blood cells indicated good cellular availability of the compound. In addition, in diabetic rats, sorbitol accumulation in red blood cells was significantly inhibited by compound 1 administered intra-gastrically, suggesting its ready uptake into the central compartment. The zwitterionic principle thus may have significant consequences for increased bioavailability of drugs bearing an acidic function.     

Substituted indole-1-acetic acids as potent and selective CRTh2 antagonists-discovery of AZD1981

Novel indole-3-thio-, 3-sulfonyl- and 3-oxy-aryl-1-acetic acids are reported which are potent, selective antagonists of the chemoattractant receptor-homologous expressed on Th2 lymphocytes receptor (CRTh2 or DP2). Optimization required maintenance of high CRTh2 potency whilst achieving a concomitant reduction in rates of metabolism, removal of cyp p450 inhibition and minimization of aldose reductase and aldehyde reductase activity. High quality compounds suitable for in vivo studies are highlighted, culminating in the discovery of AZD1981 (22).     

Evolution of novel tricyclic CRTh2 receptor antagonists from a (E)-2-cyano-3-(1H-indol-3-yl)acrylamide scaffold

(E)-2-(3-(3-((3-Bromophenyl)amino)-2-cyano-3-oxoprop-1-en-1-yl)-1H-indol-1-yl)acetic acid (1) was discovered in a HTS campaign for CRTh2 receptor antagonists. An SAR around this hit could be established and representatives with interesting activity profiles were obtained. Ring closing tactics to convert this hit series into a novel 2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole based CRTh2 receptor antagonist series is presented.     

Dihydrobenzoxazinone derivatives as aldose reductase inhibitors with antioxidant activity

Dihydrobenzoxazinone based design and synthesis produced two series of compounds as aldose reductase (ALR2) inhibitor candidates. In particular, phenolic residues were embodied into the compounds for the combination of strengthening the inhibitory acitvity and antioxidant ability to retard the progression of diabetic complications. Most of the derivatives with styryl side chains exhibited excellent activities on selective ALR2 inhibition with IC₅₀ values ranging from 0.082 to 0.308 μM, and {8-[2-(4-hydroxy-phenyl)-vinyl]-2-oxo-2,3-dihydro-benzo[1,4]oxazin-4-yl}-acetic acid (3a) was the most potent. More significantly, most of dihydrobenzoxazinone compounds revealed not only good inhibitory effect on ALR2, but also showed powerful antioxidant activity. Notably, phenolic compound 3a was even comparable to the well-known antioxidant Trolox, confirming that the C8 p-hydroxystyryl substitution was key structure of lowering oxidative stress. Therefore, these results provided an achievement of multifunctional ALR2 inhibitors possessing capacities for both ALR2 inhibition and as antioxidants.     

Synthesis and positive inotropic evaluation of N-(1-oxo-1,2,4,5-tetrahydro-[1,2,4]triazolo[4,3-a]quinolin-7-yl)acetamides bearing piperazine and 1,4-diazepane moieties

Two series of N-(1-oxo-1,2,4,5-tetrahydro-[1,2,4]triazolo[4,3-a]quinolin-7-yl)acetamides bearing piperazine and 1,4-diazepane moieties were synthesized and screened for their positive inotropic activity by measuring left atrium stroke volume on isolated rabbit heart preparations. Most of the derivatives exhibited better in vitro positive inotropic activity than the existing drug, milrinone, among which 2-(4-(4-chlorobenzyl)-1,4-diazepan-1-yl)-N-(1-oxo-1,2,4,5-tetrahydro-[1,2,4]triazolo[4,3-a]quinolin-7-yl)acetamide 6c proved to be the most potent with 15.48 ± 0.27% increased stroke volume (milrinone: 2.46 ± 0.07%) at a concentration of 3 × 10(-5) M. The chronotropic effects of the compounds that exhibited inotropic effects were also evaluated.     

Quantitative structure-activity analysis of 5-arylidene-2,4-thiazolidinediones as aldose reductase inhibitors

Design of aldose reductase (ALR2) inhibitors has received considerable attention. Aldose reductase inhibitors, when administered from the onset of hyperglycemia, prevent the progression of polyol accumulation-linked complications. The feasibility that inhibition of aldose reductase provides a pharmacologically direct treatment for diabetic complications that is independent of the control of blood sugar levels has spurred the development of structurally diverse aldose reductase inhibitors. In this work, we report quantitative structure-activity relationship (QSAR) analysis performed by 3D-QSAR analysis, Hansch analysis, and Fujita-Ban analysis on a series of 5-arylidene-2,4-thiazolidinediones as aldose reductase inhibitors. The 2D & 3D-QSAR models were generated using 18 compounds and Fujita-Ban analysis models were obtained using 23 compounds. The predictive ability of the resulting 2D and 3D models was evaluated against a test set of 5 compounds. Analyses of results from the present QSAR study inferred that 3rd position of the phenyl ring and acetic acid substitution at N-position of thiazolidinediones play a key role in the aldose reductase inhibitory activity.     

3-Mercapto-5H-1,2,4-Triazino[5,6-b]Indole-5-Acetic Acid (Cemtirestat) Alleviates Symptoms of Peripheral Diabetic Neuropathy in Zucker Diabetic Fatty (ZDF) Rats: A Role of Aldose Reductase

Peripheral neuropathy is the most prevalent chronic complication of diabetes mellitus. Good glycemic control can delay the appearance of neuropathic symptoms in diabetic patients but it is not sufficient to prevent or cure the disease. Therefore therapeutic approaches should focus on attenuation of pathogenetic mechanisms responsible for the nerve injury. Considering the role of polyol pathway in the etiology of diabetic neuropathy, we evaluated the effect of a novel efficient and selective aldose reductase inhibitor, 3-mercapto-5H-1,2,4-triazino[5,6-b]indole-5-acetic acid (cemtirestat), on symptoms of diabetic peripheral neuropathy in Zucker Diabetic Fatty (ZDF) rats. Since the age of 5 months, male ZDF rats were orally administered cemtirestat, 2.5 and 7.5 mg/kg/day, for two following months. Thermal hypoalgesia was evaluated by tail flick and hot plate tests. Tactile allodynia was determined by a von Frey flexible filament test. Two-month treatment of ZDF rats with cemtirestat (i) did not affect physical and glycemic status of the animals; (ii) partially inhibited sorbitol accumulation in red blood cells and the sciatic nerve; (iii) markedly decreased plasma levels of thiobarbituric acid reactive substances; (iv) normalized symptoms of peripheral neuropathy with high significance. The presented findings indicate that inhibition of aldose reductase by cemtirestat is not solely responsible for the recorded improvement of the behavioral responses. In future studies, potential effects of cemtirestat on consequences of diabetes that are not exclusively dependent on glucose metabolism via polyol pathway should be taken into consideration.

     

A practical synthesis of (S) 3-tert-butoxycarbonylamino-2-oxo-2,3,4,5-tetrahydro-1,5-benzodiazepine-1-acetic acid methyl ester as a conformationally restricted dipeptido-mimetic for caspase-1 (ICE) inhibitors

A simple and versatile method for the synthesis of (S) 3-tert-butoxycarbonylamino-2-oxo-2,3,4,5-tetrahydro-1,5-benzodiazepine-1-acetic acid methyl ester (4), a dipeptide mimetic, has been developed. The regioselective functionalization of the N1 and N5 ring nitrogens and the C3 amino group is demonstrated in the synthesis of an interleukin-1beta converting enzyme inhibitor 13.     

Structure-activity relationships of ganoderma acids from Ganoderma lucidum as aldose reductase inhibitors

A series of lanostane-type triterpenoids, known as ganoderma acids were isolated from the fruiting body of Ganoderma lucidum. Some of these compounds were identified as active inhibitors of the in vitro human recombinant aldose reductase. To clarify the structural requirement for inhibition, some structure–activity relationships were determined. Our structure–activity studies of ganoderma acids revealed that the OH substituent at C-11 is an important feature and the carboxylic group in the side chain is essential for the recognition of aldose reductase inhibitory activity. Moreover, double bond moiety at C-20 and C-22 in the side chain contributes to improving aldose reductase inhibitory activity. In the case of ganoderic acid C2, all of OH substituent at C-3, C-7 and C-15 is important for potent aldose reductase inhibition. These results provide an approach to understanding the structural requirements of ganoderma acids from G. lucidum for aldose reductase inhibitor. This understanding is necessary to design a new-type of aldose reductase inhibitor.     

Indole RSK inhibitors. Part 1: discovery and initial SAR

A series of inhibitors for the 90 kDa ribosomal S6 kinase (RSK) based on an 1-oxo-2,3,4,5-tetrahydro-1H-[1,4]diazepino[1,2-a]indole-8-carboxamide scaffold were identified through high throughput screening. An RSK crystal structure and exploratory SAR were used to define the series pharmacophore. Compounds with good cell potency, such as compounds 43, 44, and 55 were identified, and form the basis for subsequent kinase selectivity optimization.     

On-bead combinatorial techniques for the identification of selective aldose reductase inhibitors

Aldose reductase (AKR1B1; ALR2; E.C. 1.1.1.21) is an NADPH-dependent carbonyl reductase which has long been associated with complications resulting from the elevated blood glucose often found in diabetics. The development of effective inhibitors has been plagued by lack of specificity which has led to side effects in clinical trials. To address this problem, a library of bead-immobilized compounds was screened against fluorescently labeled aldose reductase in the presence of fluorescently labeled aldehyde reductase, a non-target enzyme, to identify compounds which were aldose reductase specific. Picked beads were decoded via novel bifunctional bead mass spec-based techniques and kinetic analysis of the ten inhibitors which were identified using this protocol yielded IC50 values in the micromolar range. Most importantly, all of these compounds showed a preference for aldose reductase with selectivities as high as approximately 7500-fold. The most potent of these exhibited uncompetitive inhibition versus the carbonyl-containing substrate D/L-glyceraldehyde with a Ki of 1.16 microM.     

Design and synthesis of 3-(4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridin-2-yl)-1H-quinolin-2-ones as VEGFR-2 kinase inhibitors

A series of 3-(4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridin-2-yl)-1H-quinolin-2-ones have been identified as a new class of VEGFR-2 kinase inhibitors. A variety of (4,5,6,7-tetrahydro-imidazo[5,4-c]pyridin-2-yl)-acetic acid ethyl esters were synthesized, and their VEGFR-2 inhibitory activity was evaluated. Described herein are the preparation of the series and the effects of the compounds on VEGFR-2 kinase activity.     

Quinazolinone-based rhodanine-3-acetic acids as potent aldose reductase inhibitors: Synthesis,functional evaluation and molecular modeling study

A series of quinazolinone-based rhodanine-3-acetic acids was synthesized and tested for in vitro aldose reductase inhibitory activity. All the target compounds displayed nanomolar activity against the target enzyme. Compounds 3a, 3b, and 3e exhibited almost 3-fold higher activity as compared to the only marketed reference drug epalrestat. Structure-activity relationship studies indicated that bulky substituents at the 3-phenyl ring of the quinazolinone moiety are generally not tolerated in the active site of the enzyme. Insertion of a methoxy group on the central benzylidene ring was found to have a variable effect on ALR-2 activity depending on the nature of peripheral quinazolinone ring substituents. Removal of the acetic acid moiety led to inactive or weakly active target compounds. Docking and molecular dynamic simulations of the most active rhodanine-3-acetic acid derivatives were also carried out, to provide the basis for further structure-guided design of novel inhibitors.     

[11C]Dimebon,radiosynthesis and lipophilicity of a new potential PET agent for imaging of Alzheimer’s disease and Huntington’s disease

[¹¹C]Dimebon (2-[¹¹C]methyl-8-methyl-5-(2-(6-methylpyridin-3-yl)ethyl)-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole), a new potential PET agent for imaging of Alzheimer’s disease and Huntington’s disease, was prepared by N-[¹¹C]methylation of desmethyl-Domebon precursor with [¹¹C]CH₃OTf and purified with a semi-preparative HPLC method in 30–40% decay corrected radiochemical yield and 222–296 GBq/μmol specific activity at EOB. The measured lipophilicity coefficient (Log P) value of [¹¹C]Dimebon was 2.53.     

Novel 4,1-benzoxazepine derivatives with potent squalene synthase inhibitory activities.

A series of (3,5-trans)-2-oxo-5-phenyl-1,2,3,5-tetrahydro-4,1-benzoxazepine derivatives were synthesized and evaluated for squalene synthase inhibitory and cholesterol biosynthesis inhibitory activities. Through modification of substituents of the lead compounds 1a and 1b, it was found that 4,1-benzoxazepine-3-acetic acid derivatives with isobutyl and neopentyl groups at the 1-position, the chloro atom at the 7-position, and the chloro and methoxy groups at the 2'-position on the 5-phenyl ring, had potent squalene synthase inhibitory activity. Among such compounds, the 5-(2,3-dimethoxyphenyl) derivative 2t exhibited potent inhibition of cholesterol biosynthesis in HepG2 cells. As a result of optical resolution study of 2t, the absolute stereochemistry required for inhibitory activity was determined to be 3R,5S. In vivo study showed that the sodium salt of (3R,5S)-7-chloro-5-(2,3-dimethoxyphenyl)-1-neopentyl-2-oxo-1,2,3,5-tetrahydro-4,1-benzoxazepine-3-acetic acid 20 effectively reduced plasma cholesterol in marmosets.     

Iron-chlorophyllin-mediated conversion of 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2(NHOH)) into its nitroso derivative

Early work from our laboratory has shown that the mutagenicity of heterocyclic amines in Salmonella can be inhibited by hemin and chlorophyllins. We have speculated that the inhibition is a result of complex formation between heterocyclic amines and the pigments, and the speculation has been given a line of experimental evidence. We have now found that ferric-chlorophyllin (Fe-chlorophyllin) can modify the mutagenicity of 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2(NHOH)), a metabolically activated form of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). The mutagenicity of Trp-P-2(NHOH)) in Salmonella typhimurium TA 98 (without S9) was strongly inhibited by an addition of an equimolar Fe-chlorophyllin in the pre-incubation mixture. Fe-chlorophyllin also inhibited the mutagenicity of 2-hydroxyamino-6-methyldipyrido[1,2-a:3′,2′-d] imidazole (Glu-P-1(NHOH)). A rapid change in the UV spectrum of a mixture of Trp-P-2(NHOH) and Fe-chlorophyllin was observed. Analysis by high performance liquid chromatography showed that Trp-P-2(NHOH) was converted into 3-nitroso-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2(NO)), the mutagenic potency of which is a quarter of that of Trp-P-2(NHOH). Furthermore, the mutagenicity of Trp-P-2(NO), in turn, was inhibited by Fe-chlorophyllin. We conclude that the suppression of the mutagenicity of Trp-P-2(NHOH) is ascribable to the oxidative function of Fe-chlorophyllin, coupled with its ability to form complex formation with the planar surface of the heterocyclic amine molecules.     

Novel agents combining platelet activating factor (PAF) receptor antagonist with thromboxane synthase inhibitor (TxSI)

Compounds 1 or 2 which possess dual-acting PAF antagonist/TxSI in a previous paper were modified and evaluated for the dual-acting activity. It was found that several compounds were potent dual-acting PAF antagonist/TxSI in and ex vivo. 6-(2-Chlorophenyl)-3-[4-[(E/Z)-6-ethoxycarbonyl-1-(3-pyridyl)-1-hexenyl]phenylmethyl]-8,11-dimethyl-2,3,4,5-tetrahydro-8H-pyrido[4',3': 4,5]thieno[3,2-f]triazolo[4,3-a]diazepine (12) is excellent orally dual-acting PAF antagonist/TxSI.     

Free radical scavengers, antioxidants and aldose reductase inhibitors from Camptosorus sibiricus Rupr

Flavonoids and organic acids were recommended in the literature as the main active constituents of Camptosorus sibiricus Rupr. Assay-guided fractionation led to the isolation of 9 flavonoids and 8 phenolic acids. All compounds were tested for DPPH scavenging activity, SOD-like and aldose reductase inhibition. Among them, compounds 1, 2, 3, 5, 6, 7, 8, 9, 11, 15 showed activities. The most active free radical scavenger and antioxidant was compound 8, while compound 1 exhibited strong inhibiting activity of aldose reductase. The structure-activity relation was dicussed briefly.     

Inhibitory effect of rhetsinine isolated from Evodia rutaecarpa on aldose reductase activity

Aldose reductase inhibitors have considerable potential for the treatment of diabetic complications, without increased risk of hypoglycemia. Search for components inhibiting aldose reductase led to the discovery of active compounds contained in Evodia rutaecarpa Bentham (Rutaceae), which is the one of the component of Kampo-herbal medicine. The hot water extract from the E. rutaecarpa was subjected to distribution or gel filtration chromatography to give an active compound, N2-(2-methylaminobenzoyl)tetrahydro-1H-pyrido[3,4-b]indol-1-one (rhetsinine). It inhibited aldose reductase with IC₅₀ values of 24.1 μM. Furthermore, rhetsinine inhibited sorbitol accumulation by 79.3% at 100 μM. These results suggested that the E. rutaecarpa derived component, rhetsinine, would be potentially useful in the treatment of diabetic complications.