2-Arylbenzoxazoles as CETP inhibitors: Substitution of the benzoxazole moiety

A series of 2-arylbenzoxazole inhibitors of the cholesterol ester transfer protein (CETP) is described. Structure–activity studies focused on variation of the substitution of the benzoxazole moiety. Substitution at the 5- and 7-positions of the benzoxazole moiety was found to be beneficial for CETP inhibition. Compound 47 was found to be the most potent inhibitor in this series and inhibited CETP with an IC₅₀ of 28 nM.     

Inhibition of the soluble form of testis adenylate cyclase by catechol estrogens and other catechols

The soluble form of rat germ cell adenylate cyclase was inhibited by compounds with a catechol moiety. Among the naturally occurring catechols tested, catechol estrogens were the most potent inhibitors. Catechol estrogens at 2-6 microM inhibited enzyme activity by 50% and almost completely at 30-100 microM concentration. The inhibitory activity of catechol estrogens depends on the catechol moiety of the molecule. Catechol per se also inhibited the activity of this enzyme, 50% inhibition being achieved at about 11 microM. The two hydroxyls of the catechol moiety are essential for the inhibitory interaction with the enzyme. Thus, aromatic compounds containing only one hydroxyl group in the benzene ring, such as tyrosine, phenylephrine, estradiol, and 6 alpha-hydroxyestradiol were either completely inactive or had marginal inhibitory activity at concentrations up to 0.3-1 mM. Moreover, methylation of the hydroxyl groups of the catechol moiety of the catechol estrogens as in 2-methoxyestradiol 3-methyl ether rendered the catechol estrogens inactive. The inhibitory potency of these compounds varied greatly depending on the structure associated with the catechol ring. Thus, compounds in which catechol is associated with an aliphatic side chain, such as dopamine, L-dopa, norepinephrine, and isoproterenol, were about 11- to 34-fold less potent than catechol. On the other hand, compounds in which catechol is associated either with a hydroaromatic ring system, as in apomorphine, or with an alicyclic ring system, as in catechol estrogens, were about 2- to 5-fold more potent than catechol. The inhibitory effect of dopamine, apomorphine, and catechol estrogens was not affected by specific D-1 or D-2 antagonist, indicating that they do not act via receptors for dopamine.     

3-Substituted-(5-arylfuran-2-ylcarbonyl)guanidines as NHE-1 inhibitors

The C-3 substituents effect on NHE-1 inhibitory activity of (5-arylfuran-2-ylcarbonyl)guanidines, previously identified as potent NHE-1 inhibitors, was investigated. The introduction of amine or alkyl groups at the 3-position of the furan ring, next to the acylguanidine moiety, remarkably improves NHE-1 inhibitory potency. Especially the important finding is that 5-(2,5-dichloro)phenyl and 5-(2-methoxy-5-chloro)phenyl derivatives exhibit high NHE-1 inhibitory activities (IC50 < 0.02 microM) that match those of 3-unsubstituted derivatives.     

Stereospecific inhibition of CETP by chiral N,N-disubstituted trifluoro-3-amino-2-propanols

Chiral N,N-disubstituted trifluoro-3-amino-2-propanols represent a recently discovered class of compounds that inhibit the neutral lipid transfer activity of cholesteryl ester transfer protein (CETP). These compounds all contain a single chiral center that is essential for inhibitory activity. (R,S)SC-744, which is composed of a mixture of the two enantiomers, inhibits CETP-mediated transfer of [(3)H]cholesteryl ester ([(3)H]CE) from HDL donor particles to LDL acceptor particles with an IC(50) = 200 nM when assayed using a reconstituted system in buffer and with an IC(50) = 6 microM when assayed in plasma. Upon isolation of the enantiomers, it was found that the (R,+) enantiomer, SC-795, was about 10-fold more potent than the mixture, and that the (S,-) enantiomer, SC-794, did not have significant inhibitory activity (IC(50) > 0.8 microM). All of the activity of the (S,-)SC-794 enantiomer could be accounted for by contamination of this sample with a residual 2% of the highly potent (R,+) enantiomer, SC-795. The IC(50) of (R,+)SC-795, 20 nM, approached the concentration of CETP (8 nM) in the buffer assay. These chiral N,N-disubstituted trifluoro-3-amino-2-propanols were found to associate with both LDL and HDL, but did not disrupt overall lipoprotein structure. They did not affect the on or off rates of CETP binding to HDL disk particles. Inhibition was highly specific since the activities of phospholipid transfer protein and lecithin cholesterol acyl transferase were not affected. Competition experiments showed that the more potent enantiomer (R)SC-795 prevented cholesteryl ester binding to CETP, and direct binding experiments demonstrated that this inhibitor bound to CETP with high affinity and specificity. It is estimated, based on the relative concentrations of inhibitor and lipid in the transfer assay, that (R)SC-795 binds approximately 5000-fold more efficiently to CETP than the natural ligand, cholesteryl ester. We conclude that these chiral N,N-disubstituted trifluoro-3-amino-2-propanol compounds do not affect lipoprotein structure or CETP-lipoprotein recognition, but inhibit lipid transfer by binding to CETP reversibly and stereospecifically at a site that competes with neutral lipid binding.     

2-Alkynyl-8-aryladenines possessing an amide moiety: their synthesis and structure-activity relationships of effects on hepatic glucose production induced via agonism of the A(2B) adenosine receptor

A series of 2-alkynyl-8-aryladenine derivatives bearing an amide moiety at the 9-position of adenine was synthesized. These analogues were evaluated for inhibitory activity on N-ethylcarboxamidoadenosine (NECA)-induced glucose production in primary cultured rat hepatocytes. The m-primary benzamide derivative 15f was the most potent compound (IC(50)=0.017 microM), being 15-fold more active than the corresponding 9-methyl derivative (1). Compound 15f showed 72- and 5.2-fold selectivity for human A(2B) receptor versus human A(1) and A(2A) receptors, respectively. Structure-activity relationship (SAR) studies of the synthesized compounds indicated that a three-carbon linker, fixed in the form of a benzene ring, between the adenine core and the amide moiety is important for both A(2B) antagonistic activity and selectivity. The IC(50) values in rat hepatocyte glucose assay correlated well with the IC(50) values in cAMP assay using Chinese hamster ovary cells stably transfected with human A(2B) receptors (r(2)=0.94). The A(1) and A(2A) affinities showed no correlation with the potency to inhibit NECA-induced glucose production. These results strongly support our previous conclusion that adenosine agonist-induced hepatic glucose production in rat hepatocytes is mediated through the A(2B) receptor.     

Reconstitution of new inhibitors through mutual exchange of Ile(64)-Met(84) peptides of soybean trypsin inhibitors, Tia and Tib

Singly modified soybean trypsin inhibitors (STIs), Tia* [Tia cleaved at Arg(63)-Ile(64)] and Tib* [Tib cleaved at Arg(63)-Ile(64)], were prepared by limited proteolysis with trypsin at pH 3.0. These singly modified inhibitors were further modified to yield doubly modified inhibitors, Tia** and Tib**, by limited proteolysis with subtilisin BPN', which cleaved the Met(84)-Leu(85) bonds of Tia* and Tib*, respectively. The doubly modified inhibitors could be separated into two parts: protein moiety A and peptide moiety a (IRFIAEGHPLSLKFDS-FAVIM) for Tia**, and protein moiety B and peptide moiety b (IRFIAEGNPLRLKFDS-FAVIM) for Tib**. These protein and peptide moieties showed no trypsin inhibitory activities alone. However, the inhibitors can be reconstituted through the mutual exchange of the protein and peptide moieties isolated from STIs. The reconstituted inhibitor which has tyrosine at position 62 and histidine at position 71 shows the highest inhibitory activity. Its Ki value for bovine trypsin is around 10(-10) M, which is almost the same as that of Tia for bovine trypsin. The inhibitor possessing either tyrosine at position 62 or histidine at position 71 exhibits a Ki value of around 10(-9) M, which is between those of Tia and Tib. The inhibitor having phenylalanine and asparagine at positions 62 and 71, respectively, shows the weakest inhibitory activity of around 10(-8) M similar to that of Tib for bovine trypsin.     

Green tea catechins as a BACE1 (beta-secretase) inhibitor

In the course of searching for BACE1 (beta-secretase) inhibitors from natural products, the ethyl acetate soluble fraction of green tea, which was suspected to be rich in catechin content, showed potent inhibitory activity. (-)-Epigallocatechin gallate, (-)-epicatechin gallate, and (-)-gallocatechin gallate were isolated with IC(50) values of 1.6 x 10(-6), 4.5 x 10(-6), and 1.8 x 10(-6) M, respectively. Seven additional authentic catechins were tested for a fundamental structure-activity relationship. (-)-Catechin gallate, (-)-gallocatechin, and (-)-epigallocatechin significantly inhibited BACE1 activity with IC(50) values of 6.0 x 10(-6), 2.5 x 10(-6), and 2.4 x 10(-6) M, respectively. However, (+)-catechin, (-)-catechin, (+)-epicatechin, and (-)-epicatechin exhibited about ten times less inhibitory activity. The stronger activity seemed to be related to the pyrogallol moiety on C-2 and/or C-3 of catechin skeleton, while the stereochemistry of C-2 and C-3 did not have an effect on the inhibitory activity. The active catechins inhibited BACE1 activity in a non-competitive manner with a substrate in Dixon plots.     

The essential role of a free sulfhydryl group in blocking the cholesteryl site of cholesteryl ester transfer protein (CETP)

Cholesteryl ester transfer protein (CETP) has at least one unpaired sulfhydryl residue, which we have shown previously to be in or near the active site region. We investigated the location of this unpaired cysteine residue(s) of CETP using chemical modification with fluorescent sulfhydryl-specific reagents, limited proteolysis, and amino acid/sequence analysis. The kinetics of labeling CETP by either 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid (MIANS) or acrylodan were followed by observing the increase in fluorescence of the bound probes. Labeling was inhibited strongly by preincubation of the CETP with either PNU-617, a competitive inhibitor of cholesteryl ester (CE) transport, and TP2 antibody. In addition, the transfer activities of the substrate CE by the modified CETP's were also inhibited but not competitively. Finally, preincubation of the native protein with N-ethylmaleimide (NEM) resulted in inhibition of activity that was dependent upon the time of exposure of the protein to the alkylating agent. These results provide further evidence that there is a cysteine residue in the active site region of CETP and ligands that either react or bind to this residue produce steric hindrance to CE transfer activity. Finally, although not conclusive, results of the protein chemistry experiments with the modified CETP suggest that the cysteine residue at position 333 is unpaired.     

Design and synthesis of 4,6-substituted-(diaphenylamino)quinazolines as potent EGFR inhibitors with antitumor activity

A type of novel 4,6-substituted-(diaphenylamino)quinazolines, which designed based on the 4-(phenylamino)quinazoline moiety, have been discovered as potential EGFR inhibitors. These compounds displayed good antiproliferative activity and EGFR-TK inhibitory activity. Especially, 4-((4-(3-bromophenylamino)quinazolin-6-ylamino)methyl)phenol (5b), showed the most potent inhibitory activity (IC(50)=0.28μM for Hep G2, IC(50)=0.59μM for A16-F10 and IC(50)=0.87μM for EGFR) and effectively induces apoptosis in a dose-dependent manner in the Hep G2 cell line. Molecular docking of 5b into EGFR TK active site was also performed. This inhibitor nicely fitting the active site might well explain its excellent inhibitory activity.     

Benzofuran- and furan-2-yl-(phenyl)-3-pyridylmethanols: synthesis and inhibition of P450 aromatase

A series of benzofuran-2-yl-(phenyl)-3-pyridylmethanol derivatives were prepared using an efficient 1-step procedure in good yields. In addition furan-2-yl-(phenyl)-3-pyridylmethanol derivatives were also prepared to determine the effect of the benzene ring in benzofuran with respect to inhibitory activity. The pyridylmethanol derivatives were all evaluated in vitro for inhibitory activity against aromatase (P450(AROM), CYP19), using human placental microsomes. The benzofuran-2-yl-(phenyl)-3-pyridylmethanol derivatives showed good to moderate activity (IC50 = 1.3-25.1 microM), which was either better than or comparable with aminoglutethimide (IC50 = 18.5 microM) but lower than arimidex (IC50 = 0.6 microM), with the 4-methoxyphenyl substituted derivative displaying optimum activity. Molecular modelling of the benzofuran-2-yl-(4-fluorophenyl)-3-pyridylmethanol derivative suggested activity to reside with the (S)-enantiomer. The furan-2-yl-(phenyl)-3-pyridylmethanol derivatives were devoid of activity indicating the essential role of the benzene ring of the benzofuran component for enzyme binding.     

Alcohol absorption inhibitors from bay leaf (Laurus nobilis): structure-requirements of sesquiterpenes for the activity

Through a bioassay-guided separation using inhibitory activity on blood ethanol elevation in oral ethanol-loaded rat, various sesquiterpenes having an alpha-methylene-gamma-butyrolactone moiety, costunolide (1), dehydrocostus lactone (2), zaluzanin D (3), reynosin (4), santamarine (5), 3alpha-acetoxyeudesma-1,4(15),11(13)-trien-12,6alpha-+ ++olide (6) and 3-oxoeudesma-1,4,11(13)-trien-12,6alpha-olide (7), were isolated as the active principle from the leaves of Laurus nobilis (bay leaf, laurel). In order to characterize the structure requirement for the activity, several reduction products (2a-2d) and amino acid adducts (2e, 2f) of the alpha-methylene-gamma-butyrolactone moiety were synthesized from 2 and the inhibitory activities of these sesquiterpenes, together with alpha-methylene-gamma-butyrolactone (12) and its related compounds (13-16), were examined. These results indicated that the gamma-butyrolactone or gamma-butyrolactol moiety having alpha-methylene or alpha-methyl group was essential for the inhibitory activity on ethanol absorption. Since 1, 2 and 12 showed no significant effect on glucose absorption, these sesquiterpenes appeared to selectively inhibit ethanol absorption. In addition, the acute toxicities of 1 and 2 in a single oral administration were found to be lower than that of 12.     

Design, synthesis, and pharmacological evaluation of N-bicyclo-5-chloro-1H-indole-2-carboxamide derivatives as potent glycogen phosphorylase inhibitors

As a result of the various N-bicyclo-5-chloro-1H-indole-2-carboxamide derivatives with a hydroxy moiety synthesized in an effort to discover novel glycogen phosphorylase (GP) inhibitors, 5-chloro-N-(5-hydroxy-5,6,7,8-tetrahydronaphthalen-2-yl)-1H-indole-2-carboxamide (5b) was found to have potent inhibitory activity. The introduction of fluorine atoms both at a position adjacent to the hydroxy group and in the central benzene moiety lead to the optically active derivative 5-chloro-N-[(5R)-1,3,6,6-tetrafluoro-5-hydroxy-5,6,7,8-tetrahydronaphthalen-2-yl]-1H-indole-2-carboxamide (25e(alpha), which was the most potent compound in this series (IC(50)=0.020microM). This compound inhibited glucagon-induced glucose output in cultured primary hepatocytes with an IC(50) value of 0.69microM, and showed oral hypoglycemic activity in diabetic db/db mice at 10mg/kg. Compound 25e(alpha) also had an excellent pharmacokinetic profile, with high oral bioavailability and a long plasma half-life, in male SD rats. The binding mode of 25e(alpha) to this molecule and the role of fluorine atoms in that binding were speculated in an enzyme docking study.     

1-(1-Arylethylidene)thiosemicarbazide derivatives: a new class of tyrosinase inhibitors

A series of 1-(1-arylethylidene)thiosemicarbazide compounds and their analogues were synthesized and characterized by 1H NMR, MS. Their tyrosinase inhibitory activities were investigated by an assay based on the catalyzing ability of tyrosinase for the oxidation of L-DOPA, comparing with 4-methoxycinnamic acid and arbutin. The results showed that (1) all the synthesized compounds could perform a significant inhibitory activity for tyrosinase; (2) for these compounds, the main active moiety interacting with the center of tyrosinase would be thiosemicarbazo group; (3) the inhibitory activity was close related with thiosemicarbazide moieties and the groups attached on the aromatic ring.     

Synthesis of (glycopyranosyl-triazolyl)-purines and their inhibitory activities against protein tyrosine phosphatase 1B (PTP1B)

Development of novel purine derivatives has attracted considerable interest, since both purine and purine-based nucleosides display a wide range of crucial biological activities in nature. We report here a novel expansion of these studies by introducing gluco- or galactopyranosyl scaffold to the N- or 9-position (or both) of 6-Cl purine moiety via Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition. By such an efficient reaction, a series of glycosyl-triazolyl-purines were successfully synthesized in good yields. Biological evaluation showed that the majority of these glycoconjugates were good PTP1B inhibitors with IC(50) values in low micromolar range (1.5-11.1 μM). The benzylated sugar derivatives displayed better inhibitory potency than that of the acetylated ones. Replacement of Cl by MeO at C(6) of the purine moiety decreased the inhibition in the case of benzylated (glycosyl-mono-triazolyl)-purines 11 and 12 (IC(50) >80 μM), whereas MeO-substituted benzylated bis[galactosyl-triazolyl]-purine 16 possessed the best inhibitory activity with an IC(50) value of 1.5 μM. Additionally, these compounds exhibited 2- to 57-fold selectivity over other PTPs (TCPTP, SHP1, SHP2, and LAR).     

Inhibition of bovine dihydrofolate reductase and enhancement of methotrexate sensitivity by N4-(2-acetoxyethoxymethyl)-2-acetylpyridine thiosemicarbazone

N4-(2-Acetoxyethoxymethyl)-2-acetylpyridine thiosemicarbazone (AATSC) belongs to a series of molecules known to have broad antimicrobial inhibitory activity. These molecules contain the 2-acetoxyethoxy moiety which could conceivably take up a conformation analogous to that of the ribosyl group. Moreover, the thiosemicarbazone moiety, when in the presence of a suitable enzymatic site, could mimic the triazine group, which is found in a number of antifolate drugs. AATSC, which has both bacterial inhibitory activity and water solubility, was accordingly evaluated for its antifolate activity against the bovine liver dihydrofolate reductase. AATSC is shown to be a fully uncompetitive inhibitor of that enzyme. Furthermore, AATSC enhances the activity of methotrexate. Such a potentiation could be useful for therapeutic purposes.     

Rapid inactivation of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a potent mutagen in chlorinated drinking water,by sulfhydryl compounds

The mutagenic activity of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), which is formed during chlorination of drinking water, was effectively inhibited by sulfhydryl compounds such as cysteine, cysteamine, glutathione, dithiothreitol and 2-mercaptoethanol. Preincubation of 0.5 μg MX with 15 μg cysteine (molar ratio 1:37) in a phosphate buffer (pH 6.0–8.0) at 37°C for 15 min prior to exposure of bacterial cells depleted the mutagenic activity of MX. Together with the result showing a change in the UV spectra, it is suggested that sulfhydryl compounds inactivate MX by direct chemical interaction before MX induces DNA damage. On the other hand, a variety of antioxidants other than the sulfhydryl compounds showed no inhibitory effects. Investigation using structural analogs of cysteine revealed that the thiol moiety was indispensable for antimutagenic activity and the amino moiety appeared to enhance the MX-inactivating reaction of the SH group.     

Benzofuran- and furan-2-yl-(phenyl)-3-pyridylmethanols: Synthesis and inhibition of P450 aromatase

A series of benzofuran-2-yl-(phenyl)-3-pyridylmethanol derivatives were prepared using an efficient 1-step procedure in good yields. In addition furan-2-yl-(phenyl)-3-pyridylmethanol derivatives were also prepared to determine the effect of the benzene ring in benzofuran with respect to inhibitory activity. The pyridylmethanol derivatives were all evaluated in vitro for inhibitory activity against aromatase (P450_AROM, CYP19), using human placental microsomes. The benzofuran-2-yl-(phenyl)-3-pyridylmethanol derivatives showed good to moderate activity (IC₅₀=1.3–25.1?μM), which was either better than or comparable with aminoglutethimide (IC₅₀=18.5?μM) but lower than arimidex (IC₅₀=0.6?μM), with the 4-methoxyphenyl substituted derivative displaying optimum activity. Molecular modelling of the benzofuran-2-yl-(4-fluorophenyl)-3-pyridylmethanol derivative suggested activity to reside with the (S)-enantiomer. The furan-2-yl-(phenyl)-3-pyridylmethanol derivatives were devoid of activity indicating the essential role of the benzene ring of the benzofuran component for enzyme binding.     

Lipid transfer inhibitor protein defines the participation of high density lipoprotein subfractions in lipid transfer reactions mediated by cholesterol ester transfer protein (CETP)

Cholesterol ester transfer protein (CETP) moves triglyceride (TG) and cholesteryl ester (CE) between lipoproteins. CETP has no apparent preference for high (HDL) or low (LDL) density lipoprotein as lipid donor to very low density lipoprotein (VLDL), and the preference for HDL observed in plasma is due to suppression of LDL transfers by lipid transfer inhibitor protein (LTIP). Given the heterogeneity of HDL, and a demonstrated ability of HDL subfractions to bind LTIP, we examined whether LTIP might also control CETP-facilitated lipid flux among HDL subfractions. CETP-mediated CE transfers from [3H]CE VLDL to various lipoproteins, combined on an equal phospholipid basis, ranged 2-fold and followed the order: HDL3 > LDL > HDL2. LTIP inhibited VLDL to HDL2 transfer at one-half the rate of VLDL to LDL. In contrast, VLDL to HDL3 transfer was stimulated, resulting in a CETP preference for HDL3 that was 3-fold greater than that for LDL or HDL2. Long-term mass transfer experiments confirmed these findings and further established that the previously observed stimulation of CETP activity on HDL by LTIP is due solely to its stimulation of transfer activity on HDL3. TG enrichment of HDL2, which occurs during the HDL cycle, inhibited CETP activity by approximately 2-fold and LTIP activity was blocked almost completely. This suggests that LTIP keeps lipid transfer activity on HDL2 low and constant regardless of its TG enrichment status. Overall, these results show that LTIP tailors CETP-mediated remodeling of HDL3 and HDL2 particles in subclass-specific ways, strongly implicating LTIP as a regulator of HDL metabolism.