Structure-affinity relationship in the interactions of human organic anion transporter 1 with caffeine, theophylline, theobromine and their metabolites

It is well known that human organic anion transporter 1 (hOAT1) transports many kinds of drugs, endogenous compounds, and toxins. However, little is known about the structure-affinity relationship. The aim of this study was to elucidate the structure-affinity relationship using a series of structurally related compounds that interact with hOAT1. Inhibitory effects of xanthine- and uric acid-related compounds on the transport of p-aminohippuric acid were examined using CHO-K1 cells stably expressing hOAT1. The order of potency for the inhibitory effects of xanthine-related compounds on PAH uptake was 1-methyl derivative > 7-methyl derivative > 3-methyl derivative ≒ xanthine > 1,3,7-trimethyl derivative (caffeine). The order of potency of the inhibition was 1,3,7-trimethyluric acid > 1,3-dimethyluric acid > 1,7-dimethyluric acid > 1-methyluric acid > uric acid. A significant correlation between inhibitory potency and lipophilicity of the tested uric acid-related compounds was observed. The main determinant of the affinity of xanthine-related compounds is the position of the methyl group. On the other hand, lipophilicity is the main determinant of the affinity of uric acid-related compounds.     

[3H]SCH 23390 Binding to Human Putamen D-1 Dopamine Receptors: Stereochemical and Structure-Affinity Relationships Among l-Phenyl-1H-3-Benzazepine Derivatives as a Guide to D-l Receptor Topography

Abstract: A series of l-phenyl-1 H -3-benzazepine analogues were assessed for enantiomeric and structure-affinity relationships at human putamen D-1 dopamine receptors labelled with [³H]SCH 23390. Substitution at the 7-position of both 3-H and 3-methyl benzazepine molecules critically affected affinity for these receptors over a 500-fold range. The general rank order of potency of 7-substituents was Cl = Br ≫ CH₃ > OH ≥ H. 3-Methyl substituents increased the affinity of 7-H and 7-OH compounds two- to fivefold compared to desmethyl counterparts. The displacement of [³H]SCH 23390 binding showed substantial enantioselec-tivity; the R-enantiomer of SKF 83566 was 500-fold more potent that its S-antipode. However, the displacement of [³H]spiperone binding from D-2 sites in the same tissue showed negligible enantioselectivity. Through such structure-affinity relationships, these studies may help to define the topography of the human brain D-1 dopamine receptor and guide the design of more selecive agents for functional studies.     

Efficient scavenging of hydroxyl radicals and inhibition of lipid peroxidation by novel analogues of 1,3,7-trimethyluric acid.

New water-soluble analogues of 1,3,7-trimethyluric acid with N-1 methyl replaced by various groups were prepared and evaluated for their ability to scavenge hydroxyl radicals as well as their protective potential against lipid peroxidation in erythrocyte membranes. The deoxyribose degradation method indicates that all the analogues tested effectively scavenge hydroxyl radicals and some of them show better activity than uric acid and methyluric acids. These effects are shown to be concentration dependent and are more potent at low concentrations (10-50 microM). Among the analogues tested, 1-butenyl-, 1-propargyl- and 1-benzyl-3,7-dimethyluric acids show high hydroxyl radical scavenging property with a reaction rate constant (Ks) of 3.2-6.7 x 10(10) M(-1) S(-1), 2.3-3.7 x 10(10) M(-1) S(-1) and 2.4-3.7 x 10(10) M(-1) S(-1), respectively. The effectiveness of these analogues as hydroxyl radical scavengers appears to be better than mannitol (Ks, 1.9-2.5 x 10(9) M(-1) S(-1)). With the exception of 1-pentyl- and 1-(2'-oxopropyl)-3,7-dimethyluric acids, all other analogues tested are effective inhibitors of tert-butylhydroperoxide-induced lipid peroxidation in human erythrocyte membranes. All the analogues tested are susceptible to peroxidation in the presence of hemoprotein and hydrogen peroxide. The present study has pointed out that it is possible to significantly enhance the antioxidant property of 1,3,7-trimethyluric acid by structural modification at N-1 position. Such compounds may be useful as antioxidants in vivo.     

Simultaneous determination of 16 purine derivatives in urinary calculi by gradient reversed-phase high-performance liquid chromatography with UV detection

A reversed-phase high-performance liquid chromatography (HPLC) method with ultraviolet detection has been developed for the analysis of purines in urinary calculi. The method using gradient of methanol concentration and pH was able to separate 16 compounds: uric acid, 2,8-dihydroxyadenine, xanthine, hypoxanthine, allopurinol and oxypurinol as well as 10 methyl derivatives of uric acid or xanthine (1-, 3-, 7- and 9-methyluric acid, 1,3-, 1,7- and 3,7-dimethyluric acid, 1-, 3- and 7-methylxanthine). Limits of detection for individual compounds ranged from 0.006 to 0.035 mg purine/g of the stone weight and precision (CV%) was 0.5-2.4%. The method enabled us to detect in human uric acid stones admixtures of nine other purine derivatives: natural metabolites (hypoxanthine, xanthine, 2,8-dihydroxyadenine) and methylated purines (1-, 3- and 7-methyluric acid, 1,3-dimethyluric acid, 3- and 7-methylxanthine) originating from the metabolism of methylxanthines (caffeine, theophylline and theobromine). The method allows simultaneous quantitation of all known purine constituents of urinary stones, including methylated purines, and may be used as a reference one for diagnosing disorders of purine metabolism and research on the pathogenesis of urolithiasis.     

Structure-activity relationships of rationally designed AMACR 1A inhibitors

α-Methylacyl-CoA racemase (AMACR; P504S) is a promising novel drug target for prostate and other cancers. Assaying enzyme activity is difficult due to the reversibility of the ‘racemisation’ reaction and the difficulties in the separation of epimeric products; consequently few inhibitors have been described and no structure–activity relationship study has been performed. This paper describes the first structure–activity relationship study, in which a series of 23 known and potential rational AMACR inhibitors were evaluated. AMACR was potently inhibited (IC₅₀ = 400–750 nM) by ibuprofenoyl-CoA and derivatives. Potency was positively correlated with inhibitor lipophilicity. AMACR was also inhibited by straight-chain and branched-chain acyl-CoA esters, with potency positively correlating with inhibitor lipophilicity. 2-Methyldecanoyl-CoAs were ca. 3-fold more potent inhibitors than decanoyl-CoA, demonstrating the importance of the 2-methyl group for effective inhibition. Elimination substrates and compounds with modified acyl-CoA cores were also investigated, and shown to be potent inhibitors. These results are the first to demonstrate structure–activity relationships of rational AMACR inhibitors and that potency can be predicted by acyl-CoA lipophilicity. The study also demonstrates the utility of the colorimetric assay for thorough inhibitor characterisation.     

The discovery of YM-60828: a potent, selective and orally-bioavailable factor Xa inhibitor

Since Factor Xa (FXa) is well known to play a central role in thrombosis and hemostasis, inhibition of FXa is an attractive target for antithrombotic strategies. As a part of our investigation of a non-peptide, orally available FXa inhibitor, we found that a series of N-[(7-amidino-2-naphthyl)methyl]aniline derivatives possessed potent and selective inhibitory activities. Structure--activity relationship (SAR) of the substituent (R(1)) on the central aniline moiety suggested that increasing lipophilicity caused a detrimental effect on anticoagulant activity (prothrombin time assay) in plasma. Several compounds bearing a hydrophilic substituent in R(1) showed not only potent FXa inhibitory activities but also high anticoagulant activities. The best compound in this series was sulfamoylacetic acid derivative (YM-60828) which was a potent, selective and orally bioavailable FXa inhibitor and was chosen for clinical development.     

The metabolism of caffeine by a Pseudomonas putida strain

1) A bacterium capable of growing aerobically with caffeine (1,3,7-trimethylxanthine) as sole source of carbon and nitrogen was isolated from soil. The morphological and physiological characteristics of the bacterium were examined. The organism was identified as a strain of Pseudomonas putida and is referred to as Pseudomonas putida C1. 15 additional caffeine-degrading bacteria were isolated, and all of them were also identified as Pseudomonas putida strains. The properties of the isolates are discussed in comparison with 6 Pseudomonas putida strains of the American Type Culture Collection. 2) The degradation of caffeine by Pseudomonas putida C1 was investigated; the following 14 metabolites were identified: 3,7-dimethylxanthine (theobromine), 1,7-dimethylxanthine, 7-methylxanthine, xanthine, 3,7-dimethyluric acid, 1,7-dimethyluric acid, 7-methyluric acid, uric acid, allantoin, allantoic acid, ureidoglycolic acid, glyoxylic acid, urea, and formaldehyde. Formaldehyde has been demonstrated to be the product of oxidative N-demethylation mediated by an inducible demethylase. A pathway of caffeine degradation is proposed.     

Fluorescence-based assay for the interaction of small molecules with the human renal organic anion transporter 1

Secretion of small molecules from the systemic blood circulation into urine is one of the physiologically essential functions of the kidney. The human organic anion transporter (hOAT1) is a key component in the renal tubular secretion of negatively charged molecules including a variety of important therapeutics. In some cases, compounds interacting with hOAT1 may induce pharmacokinetic drug-drug interactions or cause nephrotoxicity. We developed a fluorescence-based, 96-well format assay using CHO cells stably expressing hOAT1, which allows for the evaluation of interactions between small molecules and hOAT1. The assay is based on the inhibition of the transport of 6-carboxyfluorescein, a high-affinity hOAT1 substrate (Km = 3.9 microM), which was identified as one of several fluorescent organic anions. The relative inhibition potency of various known hOAT1 substrates determined using the 6-carboxyfluorescein-based inhibition assay correlated well with their Km values, indicating that the fluorescent assay exhibits a proper specificity. This in vitro assay can be employed to evaluate the mechanism of renal clearance of organic anions, to assess potential drug-drug interactions and/or nephrotoxic effects of various therapeutics, and to screen for novel hOAT1 inhibitors that could serve as efficient nephroprotectants.     

Synthesis, binding and structure-affinity studies of new ligands for the microsomal anti-estrogen binding site (AEBS)

New compounds have been synthesized based on the structure of the anti-tumoral drug tamoxifen and its diphenylmethane derivative, N,N-diethyl-2-[(4-phenyl-methyl)-phenoxy]-ethanamine, HCl (DPPE). These new compounds have no affinity for the estrogen receptor (ER) and bind with various affinity to the anti-estrogen binding site (AEBS). Compounds 2, 10, 12, 13, 20a, 20b, 23a, 23b, 29 exhibited 1.1-69.5 higher affinity than DPPE, and compounds 23a and 23b have 1.2 and 3.5 higher affinity than tamoxifen. Three-dimensional structure analysis, performed using the intersection of the van der Waals volume occupied by tamoxifen in its crystallographic state and the van der Waals volume of these new compounds in their calculated minimal energy conformation, correlated well with their pKi for AEBS (r = 0.84, P<0.0001, n = 18). This is the first structure-affinity relationship (SAR) ever reported for AEBS ligands. Moreover in this study we have reported the synthesis of new compounds of higher affinity than the lead compounds and that are highly specific for AEBS. Since these compounds do not bind ER they will be helpful to study AEBS mediated cytotoxicity. Moreover our study shows that our strategy is a new useful guide to design high affinity and selective ligands for AEBS.     

Characterization of ochratoxin A transport by human organic anion transporters.

The purpose of this study was to investigate the characteristics of ochratoxin A (OTA) transport by multispecific human organic anion transporters (hOAT1 and hOAT3, respectively) using the second segment of proximal tubule (S2) cells from mice stably expressing hOAT1 and hOAT3 (S2 hOAT1 and S2 hOAT3). S2 hOAT1 and S2 hOAT3 exhibited a time- and dose-dependent, and a saturable increase in uptake of [3H]-OTA, with apparent Km values of 0.42 microM (hOAT1) and 0.75 microM (hOAT3). These OTA uptakes were inhibited by several substrates for the OATs. Para-aminohippuric acid (PAH), probenecid, piroxicam, octanoate and citrinin inhibited [3H]-OTA uptake by hOAT1 and hOAT3 in a competitive manner (Ki = 4.29-3080 microM), with the following order of potency: probenecid > octanoate > PAH > piroxicam > citrinin for hOAT1; probenecid > piroxicam > octanoate> citrinin > PAH for hOAT3. These results indicate that hOAT1, as well as hOAT3, mediates a high-affinity transport of OTA on the basolateral side of the proximal tubule, but hOAT1- and hOAT3-mediated OTA transport are differently influenced by the substrates for the OATs. These pharmacological characteristics of hOAT1 and hOAT3 may be significantly related with the events in the development of OTA-induced nephrotoxicity in the human kidney.     

Preparation, 99mTc-labeling, and in vitro characterization of HYNIC and N3S modified RC-160 and [Tyr3]octreotide.

The synthesis of conjugates of two somatostatin analogues, RC-160 and [Tyr3]octreotide with different bifunctional chelators for labeling with Tc-99m, is described. Conjugates with hydrazinonicotinamide (HYNIC) and two N3S compounds (benzoyl MAG3 and a N3S adipate derivative) were prepared on a small scale with high purity allowing evaluation of different bifunctional chelators on the same peptide without extensive peptide synthesis. High in vitro stability and retained binding affinity was found for all conjugates except for the N3S adipate. Peptide conjugates could be labeled at high specific activities (>1 Ci/micromol) with 99mTc, and different coligands were explored for the HYNIC conjugates. The resulting radiolabeled complexes were highly stable and showed binding affinity to somatostatin receptors in the nanomolar range. Varying labeling yield, stability, lipophilicity, and isomerism were found for different coligands used for labeling HYNIC conjugates, with lower lipophilicity, higher stability, and fewer coordination isomers for EDDA and tricine/nicotinic acid as ternary coligand compared to tricine. In particular, HYNIC complexes showed promising results for further in vivo evaluation.     

Inhibition of the human organic anion transporter 1 by the caffeine metabolite 1-methylxanthine

Caffeine (1,3,7-trimethylxanthine) is daily and widely consumed in beverages and food and is mainly metabolized to 1,7-dimethylxanthine and 1-methylxanthine. Indirect clinical evidence suggests that 1-methylxanthine interacts with the organic anion transport system in the human kidney. In this study the effect of caffeine and its main metabolites on the human organic anion transporter 1 (hOAT1) was investigated using CHO cells overexpressing hOAT1. The uptake of 6-carboxyfluorescein into CHO(hOAT) cells was significantly inhibited by > or = 100 microM of 1-methylxanthine. Five hundred micromolar 1-methylxanthine was equieffective to 100 microM probenecid. In contrast, caffeine and 1,7-dimethylxanthine did not inhibit the transport of 6-carboxyfluorescein at concentrations up to 500 microM. In conclusion, the caffeine metabolite 1-methylxanthine inhibits the transport activity of hOAT1 in vitro. The central involvement of hOAT1 in the renal excretion of numerous drugs suggests that this inhibition may alter the pharmacokinetics of a series of clinically important drugs in humans.     

3-Aryl-4-hydroxyquinolin-2(1H)-one derivatives as type I fatty acid synthase inhibitors

A series of 3-aryl-4-hydroxyquinolin-2(1H)-ones with fatty acid synthase inhibitory activity was prepared. Starting from a derivative with an IC(50) = 1.4 microM, SAR studies led to compounds with more than 70-fold increase in potency (IC(50) < 20 nM).     

Discovery, synthesis and biological evaluation of isoquinolones as novel and highly selective JNK inhibitors (2)

3-Metoxycarbonyl isoquinolone derivative 1 has been identified as a potent JNK inhibitor and significantly inhibited cardiac hypertrophy in a rat pressure-overload model. Herein, a series of isoquinolones with an imidazolylmethyl or a pyrazolylmethyl group at the 2-position were designed based on X-ray crystallographic analysis of the complex between the isoquinolone compound and JNK3, as wells as the relationship between compound lipophilicity (logD) and activity in a cell-based assay. The compounds prepared showed potent JNK1 inhibitory activities in a cell-based assay. Among them the isoquinolone derivative possessing 5-[(cyclopropylamino)carbonyl]-1-methyl-1H-pyrazole (16e) exhibited significant anti-hypertrophic activity at doses of more than 1mg/kg (po) in a pressure-overload model.     

Imidazole derivatives as a novel class of hybrid compounds with inhibitory histamine N-methyltransferase potencies and histamine hH3 receptor affinities

In this study, a novel series of imidazole-containing compounds with dual properties, that is, inhibitory potency at the enzyme histamine N(tau)-methyltransferase (HMT) and antagonist potency at histamine H(3) receptors was designed and synthesized. Pharmacologically, these new hybrid drugs were evaluated in functional assays for their inhibitory potencies at rat kidney HMT and for their antagonist activities on synaptosomes of rat cerebral cortex. For selected compounds, binding affinities at recombinant human histamine H(3) receptors were determined. The first compounds (1-10) of the series proved to be H(3) receptor ligands of high potency at rat synaptosomes or of high binding affinity at human H(3) receptors, respectively, but of only moderate activity as inhibitors of rat kidney HMT. In contrast, aminoquinoline- or tetrahydroacridine-containing derivatives 11-17 also displayed HMT inhibitory potency in the nanomolar concentration range. Preliminary data from molecular modeling investigations showed that the imidazole derivative 15 and the HMT inhibitor quinacrine possess identical binding areas. The most interesting compound (14) is simultaneously a highly potent H(3) receptor ligand (K(i)=4.1nM) and a highly potent HMT inhibitor (IC(50)=24nM), which makes this derivative a valuable pharmacological tool for further development.     

Synthesis and cytotoxic activity of carboxamide derivatives of benzo[b][1,6]naphthyridin-(5H)ones

A previous reaction leading to 2-substituted 6-methyl-1-oxo-1,2-dihydrobenzo[b][1,6]naphthyridine-4-carboxylic acids has been extended to encompass a broad range of 2-substituents. Derived carboxamides, particularly 4-N-[2-(dimethylamino)ethyl], were tested for growth inhibitory properties. Potent cytotoxicity against murine P388 leukemia and Lewis lung carcinoma (LLTC) was retained for compounds bearing a remarkably diverse range of 2-substituents with a number having IC50 values <10 nM. Five of the new compounds were tested in vivo against subcutaneous colon 38 tumors in mice; a single dose (1.8 mg/kg) proved curative for the 2-(4-fluorophenyl) derivative, a further increase in potency over the very effective 2-methyl analogue reported previously.     

Lipophilicity plays a major role in modulating the inhibition of monoamine oxidase B by 7-substituted coumarins

A series of coumarin derivatives (1-22), bearing at the 7-position ether, ketone, ester, carbamate, or amide functions of varying size and lipophilicity, were synthesized and investigated for their in vitro monoamine oxidase-A and -B (MAO-A and -B) inhibitory activities. Most of the compounds acted preferentially as MAO-B inhibitors, with IC(50) values in the micromolar to low-nanomolar range. A structure-activity-relationship (SAR) study highlighted lipophilicity as an important property modulating the MAO-B inhibition potency of 7-substituted coumarins, as shown by a linear correlation (n=20, r(2)=0.72) between pIC(50) and calculated log P values. The stability of ester-containing coumarin derivatives in rat plasma provided information on factors that either favor (lipophilicity) or decrease (steric hindrance) esterase-catalyzed hydrolysis. Two compounds (14 and 22) were selected to investigate how lipophilicity and enzymatic stability may affect in vivo MAO activities, as assayed ex vivo in rat. The most-potent and -selective MAO-B inhibitor 22 (=7-[(3,4-difluorobenzyl)oxy]-3,4-dimethyl-1-benzopyran-2(2H)-one) within the examined series significantly inhibited (>60%) ex vivo rat-liver and striatal MAO-B activities 1 h after intraperitoneal administration of high doses (100 and 300 mumol kg(-1)), revealing its ability to cross the blood-brain barrier. At the same doses, liver and striatum MAO-A was less inhibited in vivo, somehow reflecting MAO-B selectivity, as assessed in vitro. In contrast, the metabolically less stable derivative 14, bearing an isopropyl ester in the lateral chain, had a weak effect on hepatic MAO-B activity in vivo, and none on striatal MAO-B, but, surprisingly, displayed inhibitory effects on MAO-A in both peripheral and brain tissues.     

Synthesis and SAR of 8-arylquinolines as potent corticotropin-releasing factor1 (CRF1) receptor antagonists

A series of 4-substituted 8-aryl-2-methylquinolines 4 was designed and synthesized as highly potent antagonists for the human CRF(1) receptor. This series of compounds displayed parallel SAR to other bicyclic systems such as pyrazolo[1,5-a]pyrimidines, with several compounds possessing low nanomolar binding affinity. In addition to the high potency, the basicity of this 4-aminoquinoline core may offer CRF(1) antagonists with lower lipophilicity.     

Carbonic anhydrase inhibitors: aromatic and heterocyclic sulfonamides incorporating adamantyl moieties with strong anticonvulsant activity

A series of aromatic/heterocyclic sulfonamides incorporating adamantyl moieties were prepared by reaction of aromatic/heterocyclic aminosulfonamides with the acyl chlorides derived from adamantyl-1-carboxylic acid and 1-adamantyl-acetic acid. Related derivatives were obtained from the above-mentioned aminosulfonamides with adamantyl isocyanate and adamantyl isothiocyanate, respectively. Some of these derivatives showed good inhibitory potency against two human CA isozymes involved in important physiological processes, CA I, and CA II, of the same order of magnitude as the clinically used drugs acetazolamide and methazolamide. The lipophilicity of the best CA inhibitors was determined and expressed as their experimental log k' IAM and theoretical ClogP value. Their lipophilicity was propitious with the crossing of the blood-brain barrier (log k' > IAM > 1.35). The anticonvulsant activity of some of the best CA inhibitors reported here has been evaluated in a MES test in mice. After intraperitoneal injection (30 mg kg(-1)), compounds A8 and A9 exhibited a high protection against electrically induced convulsions (> 90%). Their ED50 was 3.5 and 2.6 mg kg(-1), respectively.