Tetrazoles as carboxylic acid surrogates in the suosan sweetener series

The structure-activity relationship (SAR) in the suosan series of sweeteners has been extended to include additional replacements for the carboxyl group. Tetrazole analogues have been prepared and were found to be sweet. However, both the urea and thiourea tetrazolyl analogues exhibited reduced potency when compared with the carboxyl compounds. Because of the larger size of tetrazole compared with carboxylic acid, chain-shortened tetrazolyl analogues were prepared and found to not be sweet. Some important aspects about the requirements for promoting a sweet taste in vivo can be gleaned from these results. The importance of the degree of delocalization and the orientation of charge density in the anionic group are discussed.     

Comparison of metabolism and toxicity to the structure of the anticancer agent sulofenur and related sulfonylureas.

The metabolic formation of p-chloroaniline from the oncolytic agent sulofenur [N-(5-indanesulfonyl)-N'-(4-chlorophenyl)urea, LY186641,] and from similar diaryl-substituted sulfonylureas, and its possible relevance to the compound's toxicity, was studied. In previous studies it was found that significant amounts of metabolites such as 2-amino-5-chlorophenyl sulfate (II), which is also a metabolite of p-chloroaniline, are formed from sulofenur in mice, rats, monkeys, and humans. The metabolism of N-(4-tolyl)-N'-(2-hydroxy-4-chlorophenyl)-urea (V) was studied, and V was not found to be an intermediate in the metabolic formation of II from the sulfonylurea N-(4-tolyl)-N'-(4-chlorophenyl)urea (LY181984, III). The amounts of this p-chloroaniline metabolite (II) formed in C3H mice from a series of diarylsulfonylureas were found to correlate with the compound's propensities to form methemoglobin, one notable toxicity of p-chloroaniline. This metabolism was also found to correlate with the structure of the arylsulfonyl moiety of the sulfonylurea. Other evidence supports the hypothesis that p-chloroaniline is directly formed by metabolism of sulfofenur and similar diarylsulfonylureas as well. Metabolic formation of p-chloroaniline thus appears to be a plausible explanation for the methemoglobinemia and anemia found to be dose-limiting toxicities of sulofenur in Phase I trials.     

Improvement of physicochemical properties of N-4472. Part II: characterization of N-4472 microemulsion and the enhanced oral absorption

The optimized formulation of N-4472, N-[2-(3,5-di-tert-butyl-4-hydroxyphenethyl)-4,6-difluorophenyl]-N'-[4-(N-benzylpiperidyl)] urea, which was a poorly water-soluble drug, was developed by utilizing the complexation between N-4472 and L-ascorbic acid (VC). It was found that the formulation with Gelucire((R)) 44/14, HCO-60((R)) and sodium dodecyl sulfate provided a self-microemulsifying system consisting of fine droplets in approximately 18 nm size with a narrow distribution. 1H-NMR spectroscopic study indicated that the N-4472/VC complex was molecularly incorporated into surfactant molecular assembly in the microemulsion droplets. It was found that the N-4472 microemulsion was stable at the pH range from 2.0 to 7.0, suggesting the stability in the gastrointestinal tract. When the microemulsion containing N-4472/VC complex was orally administrated in rats, high AUC value of N-4472 (2 to 4-fold) was observed in comparison with the aqueous solution containing N-4472/VC complex.     

Synthesis, anti-HIV and anticancer activities of new 4-(2-mercaptobenzenesulfonyl)perhydro-1,2,4-triazin-3-ones

Syntheses of N-(6-chloro-1,1-dioxo-7-R1-1,4,2-benzodithiazyn-3-yl)-N'-(2- hydroxyethyl)hydrazines (IIa-d], N-(6-chloro-1,1-dioxo-7-methyl-1,4,2-benzodithiazyn-3-yl)-N'-(2- chloroethyl)hydrazine [IV], 4-(4-chloro-5-R1-2-mercaptobenzenesulfonyl)perhydro-1,2,4-triaz in-3-ones [IIIa-d] and 5-chloro-4-methyl-2-(3-oxoperhydro-1,2,4-triazin-4- ylsulfonyl)phenylthioacetic acid [V] have been described. Preliminary screening data have indicated that compounds [IIIa-d] exhibit either a moderate or a high anti-HIV activity and a moderate anticancer activity in some human tumor cell lines.     

Species differences in the in vitro metabolism of methapyrilene

1. The metabolism of N,N-dimethyl-N'-(2-pyridyl)-N'-(2-thienylmethyl)-1,2- ethanediamine(methapyrilene, I) by liver microsomes from rat, guinea pig, and rabbit has been examined. 2. Methapyrilene-N-oxide, (III), normethapyrilene, (II), 2-thiophene methanol, (VI), 2-thiophene carboxylic acid, (VII), N-(2-pyridyl)-N',N'-dimethylethylenediamine, (IX), and methapyrilene amide, (XIV) were found in all species. 3. N-(2-Thienylmethyl)-2-amino pyridine, (VIII), 2-aminopyridine, (X), and (5-hydroxypridyl)-methapyrilene, (XII), were detected in rat and rabbit only. 4. N-Hydroxynormethapyrilene, (XXI), was tentatively identified by mass spectral fragmentation patterns only in rabbit liver microsomes incubations; however, it was found in 9000 g supernatant fraction incubations of rabbit, rat and guinea pig. 5. The formation of IX and XII was quantitatively more important in the rat than in either rabbit or guinea pig.     

Peptide sweeteners. 8. Synthesis and structure-taste relationship studies of L-aspartyl-D-alanyl tripeptides

Several L-aspartyl-D-alanyl tripeptides have been synthesized to investigate the structural requirements of the C-terminal amino acid needed to elicit a taste response. Following our suggestion that a rigid, hydrophobic residue is required, both alpha, alpha-dialkane and cycloalkane alpha-amino acid methyl esters were incorporated into the tripeptide. The L-aspartyl-D-alanine-based tripeptide derivatives of alpha-aminoisobutyric acid methyl ester, alpha, alpha-diethylglycine methyl ester, and alpha-aminocycloalkanecarboxylic acid methyl esters from three- to six-membered rings are sweet. The higher analogues of the cycloalkane series containing alpha-aminocycloheptanecarboxylic acid methyl ester and alpha-aminocyclooctanecarboxylic acid methyl ester are bitter. It is important to note that this series of tripeptides (analogous to the previously reported dipeptides) goes from sweet to bitter to tasteless as the ring size of the C-terminal amino acid increases. The relationships between effective volume of the C-terminal residue, size requirements of the sweet receptor, and taste are discussed.     

N,N'-disubstituted guanidine high-potency sweeteners.

The role and function of the aryl group in the highly potent trisubstituted guanidine sweeteners 7a-d was investigated. Four disubstituted guanidines, lacking the aryl group, were prepared. These guanidines contain a hydrophobic substituent on one nitrogen and a carboxymethyl group substituted on one of the other nitrogens. They were found to be tasteless or to have a significantly lower sweetness potency than the corresponding trisubstituted compounds. Possible rationales for the effects of structure on the sweet taste activity are discussed.     

Synthesis and structure-activity relationships of (aryloxy)quinazoline ureas as novel, potent, and selective vascular endothelial growth factor receptor-2 inhibitors

In our continuing search for medicinal agents to treat proliferative diseases, quinazoline derivatives were synthesized and evaluated pharmacologically as epithelial growth factor receptor and vascular endothelial growth factor receptor 2 (VEGFR-2) tyrosine kinase inhibitors. A quantitative structure-activity relationship analysis was conducted to rationalize the structure-activity relationship and to predict how similar the inhibitor-binding profiles of two protein kinases are likely to be on the basis of the docking of lead coumpounds into the ATP-binding site. This model was used to direct the synthesis of new compounds. A series of N-(aromatic)-N'-{4-[(6,7-dimethoxyquinazolin-4-yl)oxy]phenyl}urea were identified as potent and selective inhibitors of the tyrosine kinase activity of VEGFR-2 (fetal liver kinase 1, kinase insert domain-containing receptor). An efficient route was developed that enabled the synthesis of a wide variety of analogues with substitution on several positions of the template. Substitution of diarylurea, competitive with ATP, afforded several analogues with low nanomolar inhibition of enzymatic activity of VEGFR-2. In this paper, we describe the synthesis, structure-activity relationships, and pharmacological characterization of the series.     

Synthesis of new benzimidazole derivatives as potential antimicrobial agents.

Barbiturates 3 as possible antimicrobial agents were obtained by reacting the N,N'-disubstituted urea 1a or the thiourea analogues 1b,c with the magic malonates 2a,b. On the other hand, reaction of 1a with ethoxycarbonyl isocyanate (4) yielded the substituted s-triazine-2,4,6(1H,3H,5H)-trione 5. The reaction of 4 with 2-aminomethyl-benzimidazole (6) gave the allophanate 7 which upon treatment with Na2CO3 yielded N-(1H-benzimidazol-2-yl)urea 8.     

From small sweeteners to sweet proteins: anatomy of the binding sites of the human T1R2_T1R3 receptor

The sweet taste receptor, a heterodimeric G protein coupled receptor (GPCR) protein, formed by the T1R2 and T1R3 subunits, recognizes several sweet compounds including carbohydrates, amino acids, peptides, proteins, and synthetic sweeteners. Its similarity with the metabotropic glutamate mGluR1 receptor allowed us to build homology models. All possible dimers formed by combinations of the human T1R2 and T1R3 subunits, modeled on the A (closed) or B (open) chains of the extracellular ligand binding domain of the mGluR1 template, yield four ligand binding sites for low-molecular-weight sweeteners. These sites were probed by docking a set of molecules representative of all classes of sweet compounds and calculating the free energy of ligand binding. These sites are not easily accessible to sweet proteins, but docking experiments in silico showed that sweet proteins can bind to a secondary site without entering the deep cleft. Our models account for many experimental observations on the tastes of sweeteners, including sweetness synergy, and can help to design new sweeteners.     

Peptide sweeteners. 6. Structural studies on the C-terminal amino acid of L-aspartyl dipeptide sweeteners

Stereochemical and structural aspects of the variations in the C-terminal residue of L-aspartyl-L-phenylalanine methyl ester have been investigated. Novel configurational analogues such as L-aspartyl-D-alanine benzyl ester and L-aspartyl-D-alpha-aminobutyric acid benzyl ester were found to be sweet. In addition, chiral and achiral alpha, alpha-dialkylglycine and alpha-aminocycloalkanecarboxylic acids were incorporated into the dipeptides. The L-aspartic acid based dipeptide derivatives of alpha-aminoisobutyric acid methyl ester, alpha-aminocyclopropanecarboxylic acid methyl ester, alpha-aminocyclobutanecarboxylic acid methyl ester, and alpha-aminocyclopentanecarboxylic acid methyl ester are sweet. Dipeptides with alpha-aminocyclohexanecarboxylic acid methyl ester and alpha-aminocycloheptanecarboxylic acid methyl ester are bitter, whereas the analogues with alpha-aminocyclooctanecarboxylic acid methyl ester, alpha, alpha-diethylglycine methyl ester, and alpha-aminoisobutyric acid benzyl ester are tasteless. Aspects on chirality and effective volume of the C-terminal residue are discussed and correlated with taste.     

Potential inhibitors of L-asparagine biosynthesis. 5. Electrophilic amide analogues of (S)-2,3-diaminopropionic acid

Three electrophilic amide analogues of (S)-2,3-diaminopropionic acid (1, DAP) have been prepared as potential inhibitors of L-asparagine synthetase (ASase, from Novikoff hepatoma, EC 6.3.5.4). DAP was selectively blocked by the carbobenzoxy (Cbz) group to give 3-N-Cbz-DAP (2a). Esterification of 2a with isobutylene afforded tert-butyl 3-N-carbobenzoxy-(S)-2,3-diaminopropionate (3a), which was then blocked at the 2 position with the tert-butoxycarbonyl (Boc) group to give tert-butyl 2-[(S)-(tert-butoxycarbonyl)amino]-3-[(carbobenzoxy)amino]propionate (4). Selective cleavage of the Cbz group by H2/Pd gave the key intermediate tert-butyl 2-N-(tert-butoxycarbonyl)-(S)-2,3-diaminopropionate (5), which was acylated, via the N-hydroxysuccinimide esters, with bromoacetic acid, dichloroacetic acid, and fumaric acid monoethyl ester to give tert-butyl 2-[(S)-(tert-butoxycarbonyl)-amino]-3-(2-bromoacetamido)propionate (6a), tert-butyl 2-[(S)-(tert-butoxycarbonyl)amino]-3-(2,2-dichloroacetamido)propionate (6b), and tert-butyl 2-[(S)-(tert-butoxycarbonyl)amino]-3-(ethoxycarbonyl)acrylamido]-propionate (6c), respectively. Deblocking of 6a-c gave the corresponding amino acids (S)-2-amino-3-(2-bromoacetamido)propionic acid hydrobromide (7a), (S)-2-amino-3-(2,2-dichloroacetamido)propionic acid (7b), and ethyl N-[(S)-2-amino-2-carboxyethyl]fumarate (7c). By a slightly different procedure, 5 was converted in two steps to (S)-2-amino-3-acetamidopropionic acid hydrobromide (7d). The inhibition of ASase by 7a-c at 1 mM was 93, 19, and 37%, respectively, while 7d was without inhibition at 2 mM. Compounds 7a-c failed to increase the life span of mice infected with B16 melanoma.     

Peptide sweeteners. 7. Taste relationships of trifluoroacetyl-L-aspartylanilides

A series of analogues of trifluoroacetyl-alpha-L-aspartylanilides substituted at various positions on the aromatic ring was synthesized and tasted. The position of the substitution is essential for the nature of the taste response. The results clearly establish the close relationship between sweet and bitter taste for these compounds. Combined electronic and topochemical contributions are discussed.     

Sulfonylureas: a new class of cancer chemotherapeutic agents.

This study summarizes the antitumor properties of a number of sulofenur thiophene analogs against subcutaneously implanted 6C3HED lymphosarcoma with structural modification of the aryl moiety of the sulfonamide portion of the diarylsulfonylureas. The spectrum of activity of N-(p-chlorophenyl)-N'- [(5-methoxy-2-thienyl)sulfonyl]urea in the HXGC3, VRC5, CX-1, and LX-1 cell lines is also presented.     

High pressure liquid chromatographic analysis of in vivo metabolites of N-(substituted phenyl)-N'-(1,3,5-trimethyl pyrazole-4-yl)thioureas in rats.

The aim of this study was to investigate the in vivo metabolism of N-(substituted phenyl)-N'-(1,3,5-trimethylpyrazole-4-yl)thioureas (substrate) as model compounds in rats via HPLC. The substrates, N-(4-fluoro/chlorophenyl)-N'-(1,3,5-trimethylpyrazole-4-yl)thioureas (T2 and T3), and their possible metabolites were synthesized and the structures of the compounds were elucidated both by spectral and elemental analysis. Substrates were dissolved in 5% gum arabic and administered 100 mg/kg intraperitoneally (i.p.) in a volume of 0.1 ml/100 g. Blood samples were withdrawn before and at 30 min, 1, 2, 4, 8, 12 and 24 h post-dose. Chromatographic separation of the substrate and its metabolites was performed using a Hichrom chromasil C18 column (150 mm x 4.6 mm i.d., 5 microm particle size). The optimal composition of the mobile phase was achieved by using different mixtures of pure methanol and water. From the biotransformation of these thiourea compounds, N-dealkylation metabolites N-(4-fluoro/chloro-phenyl)-N'-(3,5-dimethylpyrazole-4-yl)thioureas (TI and T4) were identified together with unchanged substrate (T2 and T3) in the plasma by comparing them to reference standards via HPLC UV/DAD.     

Synthesis and anti-HIV-1 activity of 4,5,6,7-tetrahydro-5-methylimidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-on e (TIBO) derivatives. 2

In the first paper of this series a new structure with anti-HIV-1 activity was disclosed and analogues were synthesized to explore the structure-activity relationship of changes in the substituent (R) attached at the N-6 position of 9. This study describes the syntheses and anti-HIV-1 testing of analogues with variations of the five-membered urea ring of the 4,5,6,7-tetrahydro-5-methylimidazo[4,5,1-jk] [1,4]benzodiazepin-2(1H)-one (TIBO) structures. Although many different rings were synthesized to replace the cyclic urea of TIBO, most were found to be inactive in inhibiting the replication of the HIV-1 virus in MT-4 cells. The exceptions were replacement of the urea oxygen with sulfur or selenium to give the corresponding thio- or selenoureas. These were found to be more active than the oxygen counterparts. A small series of analogues was synthesized and tested which allowed direct comparison of urea and thiourea derivatives. Without exception, the latter were always more active than the former. The most active compound of this series (8d) was found to inhibit the HIV-1 virus with an IC50 of 0.012 microM which is comparable to that of AZT.     

The heterodimeric sweet taste receptor has multiple potential ligand binding sites

The sweet taste receptor is a heterodimer of two G protein coupled receptors, T1R2 and T1R3. This discovery has increased our understanding at the molecular level of the mechanisms underlying sweet taste. Previous experimental studies using sweet receptor chimeras and mutants show that there are at least three potential binding sites in this heterodimeric receptor. Receptor activity toward the artificial sweeteners aspartame and neotame depends on residues in the amino terminal domain of human T1R2. In contrast, receptor activity toward the sweetener cyclamate and the sweet taste inhibitor lactisole depends on residues within the transmembrane domain of human T1R3. Furthermore, receptor activity toward the sweet protein brazzein depends on the cysteine rich domain of human T1R3. Although crystal structures are not available for the sweet taste receptor, useful homology models can be developed based on appropriate templates. The amino terminal domain, cysteine rich domain and transmembrane helix domain of T1R2 and T1R3 have been modeled based on the crystal structures of metabotropic glutamate receptor type 1, tumor necrosis factor receptor, and bovine rhodopsin, respectively. We have used homology models of the sweet taste receptors, molecular docking of sweet ligands to the receptors, and site-directed mutagenesis of the receptors to identify potential ligand binding sites of the sweet taste receptor. These studies have led to a better understanding of the structure and function of this heterodimeric receptor, and can act as a guide for rational structure-based design of novel non-caloric sweeteners, which can be used in the fighting against obesity and diabetes.     

Improvement of physicochemical properties of N-4472 part I formulation design by using self-microemulsifying system

The optimization of oral dosage form formulation has been developed for N-4472, N-[2-(3,5-di-tert-butyl-4-hydroxyphenethyl)-4,6-difluorophenyl]-N'-[4-(N-benzylpiperidyl)] urea, which was a poorly water-soluble drug having a lipid-lowering effect. Formulations that contained various surfactants and water-soluble polymers were prepared and the solubility of N-4472 was evaluated in JP XIV first fluid (pH 1.2), JP XIV second fluid (pH 6.8), and distilled water. The highest solubility of N-4472 was achieved when L-ascorbic acid (VC), Gelucire 44/14, and HCO-60 were used as additives. It was confirmed that this formulation could create microemulsion droplets with a mean droplet size of approximately 20 nm and a sharp droplet distribution pattern in JP XIV first fluid, JP XIV second fluid, and distilled water. When JP XIV second fluid was used as a dissolution medium, however, an eventual decrease of solubility was observed, that is, the fluid became white and cloudy as time passed. It was found that the addition of sodium dodecyl sulfate (SDS) was effective to prevent the lowering of solubility, and that a weight ratio of 1.0/1.5/11.4/4.9/3.8 for N-4472/VC/Gelucire 44/14/HCO-60/SDS was optimum for the self-microemulsifying formulation. It was assumed that electrostatic repulsion of microemulsion droplets and an increase of the cloud point by the addition of SDS were responsible for the successful formation of a stable microemulsion.     

Novel potent inhibitors of lipid peroxidation with protective effects against reperfusion arrhythmias.

A series of new compounds that contain lipoic acid and trolox connected through spacers were synthesized and examined for their antioxidant activity and their protective effects against reperfusion arrhythmias in isolated heart preparations. All compounds tested are strong inhibitors of lipid peroxidation in rat liver microsomal membranes induced by ferrous ions and ascorbate. N-(3,4-Dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-carbonyl)-N'-(1,2-dithiolane-3-pentanoyl)-1,2-phenylenediamine (13) exhibits anti-lipid peroxidation activity at the nanomolar range. N-(3,4-Dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-carbonyl)-N'-(1,2-dithiolane-3-pentanoyl)ethylenediamine (10) and 13 totally suppressed reperfusion arrhythmias.     

An intensely sweet dihydroflavonol derivative based on a natural product lead compound

The dihydroflavonol dihydroquercetin 3-acetate (1) was isolated as a sweet constituent of the young shoots of Tessaria dodoneifolia (Hook. & Arn.) Cabrera (Compositae). Compound 1 and dihydroquercetin 3-acetate 4'-(methyl ether) (2), a novel synthetic analogue of this natural product lead compound, were rated by a taste panel as being 80 and 400 times sweeter than a 2% w/v sucrose solution, respectively. Synthetic dihydroquercetin 4'-(methyl ether) (3) showed a reduced sweetness intensity when compared to 2, while (+)-dihydroquercetin (4) was devoid of sweetness. Dihydroflavonol derivatives 1-3 represent a new class of potentially noncaloric and noncariogenic intense sweeteners.