Species differences in the metabolism and excretion of sulphasomidine and sulphamethomidine

1. The excretion of 2,4-dimethyl-6-sulphanilamidopyrimidine (sulphasomidine; Elkosin) and 4-methoxy-2-methyl-6-sulphanilamidopyrimidine (sulphamethomidine) given orally was examined in man, rhesus monkey, rabbit and rat. 2. About 70% of sulphasomidine (0.1g./kg.) is excreted mainly unchanged in the urine by these species in 24hr.; less than 15% of the dose is acetylated and there is no marked species difference in the fate of this drug. 3. Sulphamethomidine is excreted more slowly than sulphasomidine, and in the rat, rabbit and monkey the main metabolite is the N(4)-acetyl derivative. In man, only 20-30% of the dose is excreted in 24hr. and nearly 70% of this is sulphamethomidine N(1)-glucuronide, which is also excreted by the monkey but not by the rat or rabbit. There is therefore a marked species difference in the metabolism of sulphamethomidine. 4. Sulphamethomidine N(1)-glucuronide was synthesized and shown to be identical with the glucuronide isolated from monkey urine. 5. Sulphasomidine, sulphamethomidine and sulphadimethoxine (2,4-dimethoxy-6-sulphanilamidopyrimidine) were acetylated by rabbit or monkey liver homogenates. Although sulphasomidine is poorly acetylated in vivo, it is acetylated in vitro at rates comparable with those of the other two drugs. 6. The solubilities, partition coefficients and plasma-protein-binding of the drugs were measured. 7. The results are discussed.     

The fate of sulphadimethoxine in primates compared with other species

1. The metabolism of sulphadimethoxine (2,4-dimethoxy-6-sulphanilamidopyrimidine) was examined in nine species of primates and nine species of non-primates. 2. The main metabolite of the drug in the urine in man, rhesus monkey, baboon, squirrel monkey, capuchin, bushbaby, slow loris and tree shrew was sulphadimethoxine N(1)-glucuronide. In the green monkey, although the main metabolite was N(4)-acetylsulphadimethoxine, the N(1)-glucuronide was also a major metabolite. 3. In the dog, rat, mouse, guinea pig, Indian fruit bat and hen the N(1)-glucuronide was a minor metabolite in the urine, whereas in the cat, ferret and rabbit this glucuronide was not found in the urine. 4. All the species examined except the dog excreted some N(4)-acetylsulphadimethoxine, which was the major metabolite in the green monkey, rabbit and guinea pig. 5. In the tree shrew, a doubtful primate, N(1)-glucuronide formation was similar to that in the other primates. 6. It is suggested that the slow excretion of the drug by the rat may be due partly to strong binding of the drug to tissue proteins and that the strength of binding may vary with species. 7. In the rat the amount of N(1)-glucuronide found in the urine is not a true indication of the extent of this conjugation since much more of the conjugate was found in the bile (7% of the dose) than in the urine (1%). In the rabbit, no N(1)-glucuronide was found in the bile or urine, but a small amount of sulphadimethoxine N(4)-glucuronide was found in the bile of the rat (0.5% of dose) and rabbit (0.8%).     

The discovery of a lipid-linked glucuronide and its synthesis by chicken liver

Upon incubation with uridine diphosphate-[14C]glucuronic acid, membrane fractions from adult and phenobarbital-induced embryonic liver synthesize a single glucuronide, which is soluble in chloroform:methanol (2:1). The compound is completely hydrolyzed and glucuronic acid released by either mild acid or beta-glucuronidase, whereas mild base hydrolysis results in a mixture of glucuronic acid and glucuronic acid-1,2-cyclic phosphate. These data and the behavior of the lipid-linked glucuronide on DEAE-cellulose chromatography indicate that the compound contains a monophosphate diester of glucuronic acid, which is beta-linked to a lipid. The synthesis of the lipid-linked glucuronide in uninduced normal embryonic liver is very low (5-15 pmol product/mg/5 min) at all developmental ages up to hatching, but the introduction of phenobarbital into the air space of a 9-10-day-old embryo causes a premature increase of activity (75-150 pmol products/mg/5 min) within 7 days. The glucuronyltransferase in adult and induced embryonic liver has a Km for UDPGlcUA of 0.17 x 10(-3) M and a broad pH optimum between pH 6 and 7. Glucuronic acid is released from the lipid-linked glucuronide by a beta-glucuronidase in liver that is active at neutral pH and is not inhibited by saccharolactone. This glycosidase activity appears, therefore, to be distinct from the previously characterized lysosomal beta-glucuronidase. Fractionation of adult chicken liver membranes by differential centrifugation indicates that over 70% of the glucuronyltransferase is associated with the nuclear and mitochondrial fractions. The endogenous beta-glucuronidase capable of hydrolyzing the lipid-linked glucuronide was not separated from the glucuronyl-transferase activity during fractionation. The data available suggests that the lipid-linked glucuronide is involved directly in the generation of free glucuronic acid for further metabolism.     

The metabolism of four sulphonamides in cows

1. The metabolism of sulphanilamide, sulphadimidine (4,6-dimethyl-2-sulphanilamidopyrimidine), sulphamethoxazole (5-methyl-3-sulphanilamidoisoxazole) and sulphadoxine (5,6-dimethoxy-4-sulphanilamidopyrimidine) given by intravenous injection has been examined in cows. 2. The sulphonamides were present mainly as unchanged drugs in blood samples collected 2h after administration. 3. The sulphonamides were excreted in the milk partly as unchanged drugs and partly as conjugated metabolites whereas only small amounts were excreted as the N(4)-acetyl derivatives. 4. The unchanged drug and the N(4)-acetyl derivative were the major constituents in urine samples after administration of sulphanilamide, sulphamethoxazole and sulphadoxine. 5. Besides the unchanged drug, the N(4)-acetyl derivative and the conjugated metabolites, three further metabolites of sulphadimidine were isolated from urine samples and identified. They were 5-hydroxy-4,6-dimethyl-2-sulphanilamidopyrimidine, 4-hydroxymethyl-6-methyl-2-sulphanilamidopyrimidine and sulphaguanidine.     

The metabolism of tetralin

1. [1-(14)C]Tetralin was synthesized and fed to rabbits. 2. Of the radioactivity, 87-90% was excreted in the urine within two days and 0.5-3.7% on the third day. The faeces contained 0.6-1.8%. No radioactivity was found in the breath and negligible amounts were retained in the tissues. About 90-99% of an administered dose was accounted for. 3. The main metabolite in the urine was the glucuronide of alpha-tetralol (52.4%). Other conjugated metabolites were beta-tetralol (25.3%), 4-hydroxy-alpha-tetralone (6.1%), cis-tetralin-1,2-diol (0.4%) and trans-tetralin-1,2-diol (0.6%). 4. beta-Tetralone, alpha-naphthol, 1,2-dihydronaphthalene and naphthalene, previously reported as metabolites, are artifacts, and tetralin, alpha-tetralone, beta-naphthol, 5-hydroxytetralin, and 6-hydroxytetralin are not metabolites. 5. The major metabolite of tetralin, alpha-tetralol and alpha-tetralone is the glucuronide of alpha-tetralol, which was isolated as methyl (1,2,3,4-tetrahydro-1-naphthyl tri-O-acetyl-beta-d-glucosid)uronate; the major metabolite of beta-tetralol and beta-tetralone is the glucuronide of beta-tetralol, which was characterized as methyl (1,2,3,4-tetrahydro-2-naphthyl tri-O-acetyl-beta-d-glucosid)uronate. 5-Hydroxytetralin is conjugated with glucuronic acid, and was characterized as methyl (5,6,7,8-tetrahydro-1-naphthyl tri-O-acetyl-beta-d-glucosid)uronate. 6-Hydroxytetralin is conjugated with glucuronic acid, and was characterized as methyl (5,6,7,8-tetrahydro-2-naphthyl tri-O-acetyl-beta-d-glucosid)uronate. 6. A metabolic sequence accounting for the observed biological transformation products is proposed.     

Species differences in the metabolism of sulphadimethoxine

1. The fate of sulphadimethoxine (2,4-dimethoxy-6-sulphanilamidopyrimidine) was studied in man, rhesus monkey, dog, rat, guinea pig and rabbit. 2. About 20–46% of the dose (0·1g./kg.) of the drug is excreted in the urine in 24hr. in these species, except the rat, in which only 13% is excreted. 3. In man and the monkey sulphadimethoxine N¹-glucuronide is the major metabolite in the urine. In the rabbit and guinea pig N⁴-acetylsulphadimethoxine is the main metabolite. In the dog the drug is excreted mainly unchanged. In the rat equal amounts of the unchanged drug and its N⁴-acetyl derivative are the main products. 4. Small amounts of sulphadimethoxine N⁴-glucuronide are found in the urine of all the species. Sulphadimethoxine N¹-glucuronide occurs in small amounts in the urine of rat, dog and guinea pig; none is found in rabbit urine. 5. Sulphadimethoxine N⁴-sulphate was synthesized and found to occur in small amounts in rat urine. 6. Monkey liver homogenates fortified with UDP-glucuronic acid are able to synthesize sulphadimethoxine N¹-glucuronide with the drug as substrate. Rat liver has also this ability to a slight extent, but rabbit liver is unable to do so. 7. Sulphadimethoxine N⁴-glucuronide is formed spontaneously when the drug is added to human urine. 8. The biliary excretion of the drug and its metabolites was examined in rats. The drug is excreted in rat bile mainly as the N¹-glucuronide. The N¹- and N⁴-glucuronides administered as such are extensively excreted in the bile by rats.     

Pryogalloloestrogens -- a new group of oestrogen metabolites.

After incubation of radioactive catecholoestrogen monomethyl ethers with rat liver slices the following well known metabolic pathways were observed: 1) demethylation, 2) 16alpha-hydroxylation, 3) oxidoreduction at C-atom 17, and 4) conjugation with glutathione, sulphuric acid and glucuronic acid. In addition, for the first time a further aromatic ortho-hydroxylation, leading to pyrogalloloestrogen derivatives, was detected. Thus, the incubation of 2-methoxyoestrone yielded 2,4-dihydroxyoestrone 2-methyl ether as the main metabolite of the lipophile fraction. Under the same conditions, 4-methoxyoestrone was converted to 2,4-dihydroxyoestrone 4-methyl ether and 2,4-dihydroxyoestradiol-17beta 4-methyl ether; these compounds were the quantitatively most important metabolites not only in the lipophile but also in the sulphate and glucuronide fractions. The identity of these new metabolic products was established by chromatography, microchemical reactions and recrystallisation to constant specific radioactivity.     

Derivatives of methanopterin, a coenzyme involved in methanogenesis

Degradational studies of methanopterin, a coenzyme involved in methanogenesis, are reported. The results of these studies are in full accordance with the proposed structure of methanopterin as N-[1'-(2'-amino-4'-hydroxy-7' -methyl-6'-pteridinyl)ethyl]-4-[2', 3', 4', 5'-tetrahydroxypent-1'-yl(5'-1' )O-alpha-ribofuranosyl-5'-phosphoric acid] aniline in which the phosphate group is esterified with alpha-hydroxyglutaric acid. Acid hydrolysis of methanopterin cleaved the 5'----1' glycosidic bond and yielded a 'hydrolytic product' which was identified as N-[1'-(2'-amino-4'-hydroxy-7' -methyl-6'-pteridinyl)ethyl]-4-[2', 3', 4', 5'-tetrahydroxypent-1'-yl]aniline. Alkaline permanganate oxidation of methanopterin yielded 7-methylpterin-6-carboxylic acid. Catalytic (or enzymatic) hydrogenation of methanopterin gave a mixture of 6-ethyl-7-methyl-7,8-dihydropterin, 6-ethyl-7-methylpterin and a third compound, named methaniline which was identified as 4-[2', 3', 4', 5'-tetrahydroxypent-1'-yl(5'----1')O-alpha -ribofuranosyl-5'-phosphoric acid]aniline, in which the phosphate group is esterified with alpha-hydroxyglutaric acid. Methanosarcina barkeri contains a closely related coenzyme called sarcinapterin, which was identified as a L-glutamyl derivative of methanopterin, where the glutamate moiety is attached to the alpha-carboxylic acid group of the alpha-hydroxyglutaric acid moiety of methanopterin via an amide linkage.     

Zinc(II), cadmium(II) and mercury(II) complexes of the vitamin B1 antagonist oxythiamine

The reaction of oxythiamine chloride hydrochloride (HOxTCl x HCl) with ZnCl2, CdCl2 and HgCl2 in ethanol yielded the complexes [ZnCl3(HOxT)], [CdCl3(HOxT)] and [HgCl3(HOxT)]. In water, the reaction with CdCl2 afforded [CdCl2(OxT)], but reaction with ZnCl2 or HgCl2 yielded unidentified products. The four new complexes were characterized by mass spectrometry and IR spectroscopy in the solid state and by 1H, 13C and 15N nuclear magnetic resonance (NMR) spectroscopy in hexadeuterated dimethylsulfoxide (DMSO-d6), and three were also studied by X-ray diffractometry. In [ZnCl3(HOxT)] and [HgCl3(HOxT)] the oxythiamine ligand is bound to the metal via N(1') and adopts the V conformation exhibited by thiamine in biological contexts. The infrared (IR) spectrum of [CdCl3(HOxT)] suggests a similar coordination mode. In [CdCl2(OxT)] each OxT zwitterion coordinates to one Cd(II) ion via its N(1') atom and to another via its N(3') and O atoms, giving rise to a polymeric chain along the x-axis. The coordination number of the metal is made up to six by Cdc...Cl interactions, two of which link the polymeric chains in pairs. This seems to be the first metal complex containing the oxythiamine ligand as a zwitterion, with the N(3')-H/O(4'alpha)-H group deprotonated. Neither HOxTCl nor its zinc(II) complex showed any significant activity in vitro against HeLa cells.     

Stimulation of microsomal uridine diphosphate glucuronyltransferase by glucuronic acid derivatives

The glucuronic acid adducts of 1-naphthol, 2-naphthol and 4-methylumbelliferone activate microsomal UDP-glucuronyltransferase (EC 2.4.1.17) when the enzyme is assayed with p-nitrophenol as aglycone. Phenyl glucuronide and oestriol 3beta-glucuronide also activate UDP-glucuronyltransferase. but to a lesser extent. Activation by glucuronides is not dependent on metal ions, but is blocked by prior treatment of microsomal fractions with p-chloromercuribenzoate. The kinetic mechanism of activation is concluded to be an increase in the affinity of the enzyme for UDP-glucuronic acid. Activation by 1-naphthyl glucuronide, at high concentrations of p-nitrophenol, is not affected by 1-naphthol. Apparently 1-naphthyl glucuronide activates the preparation by binding at a site that is separate from the site of glucuronidation of 1-naphthol. Further evidence for the existence of distinct effector sites for the glucuronides was provided by the finding that activation by glucuronides is inhibited competitively by aglycone glucosides. These glucosides do not inhibit the rate of glucuronidation of p-nitrophenol in the absence of glucuronide adducts, nor do they alter the rate of glucuronidation of 1-naphthol. When UDP-glucuronyltransferase is assayed with 1-naphthol as aglycone it is activated by p-nitrophenyl glucuronide, 4-methyl-umbelliferyl glucuronide and under appropriate conditions by its own glucuronide. These activations are similarly inhibited by aglycone glucosides. p-Nitrophenyl glucuronide also stimulates the rate of glucuronidation of o-aminophenol, o-aminobenzoate and bilirubin.     

The copolymeric structure of pig skin dermatan sulphate. Characterization of d-glucuronic acid-containing oligosaccharides isolated after controlled degradation of oxydermatan sulphate

Selective periodate oxidation of unsubstituted l-iduronic acid residues in copolymeric dermatan sulphate chains was followed by reduction-hydrolysis or alkaline elimination. By this procedure the glucuronic acid-containing periods were isolated in oligosaccharide form; general formula: [Formula: see text] Further degradation of these oligosaccharides with chondroitinase-AC yielded three types of products: (a) sulphated trisaccharide containing an unsaturated uronosyl moiety in the non-reducing terminal and a C(4) fragment in the reducing terminal, DeltaUA-GalNAc-(-SO(4))-R; (b) monosulphated, unsaturated disaccharide, DeltaUA-GalNAc-SO(4) when n is greater than or equal to 2; and (c) N-acetylgalactosamine with or without sulphate. Oligosaccharides containing a single glucuronic acid residue (n=1) comprised more than half of the glucuronic acid-containing oligosaccharides. The terminal N-acetylgalactosamine moiety of the shortest oligosaccharide was largely 4-sulphated, whereas higher oligosaccharides primarily contained 6-sulphated or unsulphated hexosamine moieties in the same position. Moreover, IdUA-SO(4)-containing oligosaccharides were encountered. These oligosaccharides were resistant to the action of chondroitinase-ABC.     

Biliary excretion in foreign compounds. Species difference in biliary excretion

1. The biliary excretion of injected [¹⁴C]aniline, [¹⁴C]benzoic acid, 4-amino-hippuric acid and 4-acetamidohippuric acid in six or eight species of animal (rat, dog, hen, cat, rabbit, guinea pig, rhesus monkey and sheep) was studied. 2. These compounds, with molecular weights in the range 93–236, are poorly excreted in the bile in all the species examined and, in effect, there is little significant species difference in the extent of their biliary excretion. 3. Compounds of higher molecular weight (355–495) were also studied, namely succinylsulphathiazole, [¹⁴C]stilboestrol glucuronide, sulphadimethoxine N¹-glucuronide and phenolphthalein glucuronide. 4. With these compounds a clear species difference in the extent of biliary excretion was found, the rat, dog and hen being good excretors, the rabbit, guinea pig and monkey poor excretors, and the cat and sheep taking an intermediary position. 5. There was a general trend for biliary excretion to be higher in all species when the compounds were of higher molecular weight. 6. These results are discussed in their relation to species differences in drug metabolism.     

High-performance liquid chromatographic determination and identification of acyl migration and photodegradation products of furosemide 1-O-acyl glucuronide

Stability of furosemide glucuronide, the major metabolite of furosemide, was studied in order to accurately assess the glucuronidation of furosemide. Furosemide glucuronide was purified by high-performance liquid chromatography, and the mass spectrum of furosemide glucuronide showed the molecular ion peaks [M−H]⁻ at 505 and 507 (m/z). Furosemide glucuronide was photodegraded to the compound, which was shown more hydrophilic than furosemide glucuronide by high-performance liquid chromatography assay. The photodegradation product of furosemide glucuronide was hydrolyzed to one of the photodegradation products of furosemide by β-glucuronidase, indicating that the photodegradation product of furosemide glucuronide possessed a glucuronic acid moiety. Furthermore, the mass spectrum of the photodegradation product of furosemide glucuronide exhibited molecular ion peaks [M−H]⁻ at 487 and [M−2H+2Na]⁻ at 509, indicating the chlorine displacement of furosemide glucuronide by a hydroxyl group. Furosemide glucuronide was unstable in an aqueous solution (pH=7.4), and presumed acyl migration isomers of furosemide glucuronide (furosemide glucuronide-isomers) were detected by high-performance liquid chromatography equipped with photodiode array UV detector. The UV spectra of seven furosemide glucuronide-isomers were closely similar to that of furosemide glucuronide but not furosemide. Exposing a mixture of furosemide glucuronide and furosemide glucuronide-isomers to light resulted in the production of new compounds. UV spectra of photodegradation products of furosemide glucuronide-isomers were closely similar to those of photodegradation product of furosemide glucuronide. These results suggested that furosemide glucuronide-isomers were also photodegraded, resulting in the displacement of chlorine by a hydroxyl group as in furosemide glucuronide.     

Duocarmycin-based prodrugs for cancer prodrug monotherapy

The synthesis and biological evaluation of novel prodrugs based on the cytotoxic antibiotic duocarmycin SA (1) for a selective treatment of cancer using a prodrug monotherapy (PMT) are described. Transformation of the phenol 8 with the glucuronic acid benzyl ester trichloroacetimidate 9b followed by reaction with DMAI x HCl (10) gives the glucuronide 11b, which is deprotected to afford the desired prodrug 4a containing a glucuronic acid moiety. In addition, the prodrug 4b with a glucuronic methyl ester unit is prepared. The cytotoxicity of the glucuronides is determined using a HTCFA-assay with IC(50) values of 610 nM for 4a and 3300 nM for 4b. In the presence of beta-glucuronidase, 4a expresses an IC(50) value of 0.9 nM and 4b of 2.1 nM resulting in QIC(50) values of about 700 for 4a and 1600 for 4b.     

Mass spectrometric analysis of catechol-histidine adducts from insect cuticle

Adducts of catechols and histidine, which are produced by reactions of 1,2-quinones and p-quinone methides with histidyl residues in proteins incorporated into the insect exoskeleton, were characterized using electrospray ionization mass spectrometry (ESMS), tandem electrospray mass spectrometry (ESMS-MS, collision-induced dissociation), and ion trap mass spectrometry (ITMS). Compounds examined included adducts obtained from acid hydrolysates of Manduca sexta (tobacco hornworm) pupal cuticle exuviae and products obtained from model reactions under defined conditions. The ESMS and ITMS spectra of 6-(N-3')-histidyldopamine [6-(N-3')-His-DA, pi isomer] isolated from M. sexta cuticle were dominated by a [M + H]+ ion at m/z 308, rather than the expected m/z 307. High-resolution fast atom bombardment MS yielded an empirical formula of C14H18N3O5, which was consistent with this compound being 6-(N-1')-histidyl-2-(3, 4-dihydroxyphenyl)ethanol [6-(N-1')-His-DOPET] instead of a DA adduct. Similar results were obtained when histidyl-catechol compounds linked at C-7 of the catechol were examined; the (N-1') isomer was confirmed as a DA adduct, and the (N-3') isomer identified as an (N-1')-DOPET derivative. Direct MS analysis of unfractionated cuticle hydrolysate revealed intense parent and product ions characteristic of 6- and 7-linked adducts of histidine and DOPET. Mass spectrometric analysis of model adducts synthesized by electrochemical oxidative coupling of N-acetyldopamine (NADA) quinone and N-acetylhistidine (NAcH) identified the point of attachment in the two isomers. A prominent product ion corresponding to loss of CO2 from [M + H]+ of 2-NAcH-NADA confirmed this as being the (N-3') isomer. Loss of (H2O + CO) from 6-NAcH-NADA suggested that this adduct was the (N-1') isomer. The results support the hypothesis that insect cuticle sclerotization involves the formation of C-N cross-links between histidine residues in cuticular proteins, and both ring and side-chain carbons of three catechols: NADA, N-beta-alanyldopamine, and DOPET.     

The metabolism of 2-chloro-1-(2′,4′-dichlorophenyl)vinyl diethyl phosphate (Chlorfenvinphos) in the dog and rat

1. A single oral dose of [(14)C]Chlorfenvinphos to rats is quantitatively eliminated in 4 days. Rats do not show a sex difference in the elimination pattern and show only a small degree of biological variation in the total excretion data. Of the label 87.2% is excreted in the urine (67.5% in the first day after dosage), 11.2% in the faeces and 1.4% in the expired gases; less than 0.9% of (14)C is present in the gut and contents after 4 days. 2. After oral administration of [(14)C]Chlorfenvinphos to dogs, 94.0% (91.8-97.6%) of the (14)C is excreted in the urine and faeces during 4 days. Dogs do not show a sex difference in the pattern of elimination, and excretion of radioactivity in the urine is very rapid: 86.0% of (14)C during 0-24hr. 3. Chlorfenvinphos is completely metabolized in rats and dogs: unchanged Chlorfenvinphos is absent from the urine and from the carcass, when elimination is complete. In rats, 2-chloro-1-(2',4'-dichlorophenyl)vinyl ethyl hydrogen phosphate accounts for 32.3% of a dose of Chlorfenvinphos, [1-(2',4'-dichlorophenyl)ethyl beta-d-glucopyranosid]uronic acid for 41.0%, 2,4-dichloromandelic acid for 7.0%, 2,4-dichlorophenylethanediol glucuronide for 2.6% and 2,4-dichlorohippuric acid for 4.3%; in dogs, 2-chloro-1-(2',4'-dichlorophenyl)vinyl ethyl hydrogen phosphate accounts for 69.6%, [1-(2',4'-dichlorophenyl)ethyl beta-d-glucopyranosid] uronic acid for 3.6%, 2,4-dichloromandelic acid for 13.4% and 2,4-dichlorophenylethanediol glucuronide for 2.7%. 4. Dogs and rats show a species difference in the rate of excretion of (14)C in the urine, and in the proportions of the metabolites, with the exception of 2,4-dichlorophenylethanediol glucuronide, that are excreted in the urine. Alternative explanations for the latter species difference are suggested. 5. 2-Chloro-1-(2',4'-dichlorophenyl)vinyl ethyl hydrogen phosphate and 2,4-dichlorophenacyl chloride probably lie on the main metabolic pathway of Chlorfenvinphos, since, in common with that insecticide, they give rise to [1-(2',4'-dichlorophenyl)ethyl beta-d-glucopyranosid]uronic acid and 2,4-dichloromandelic acid as major metabolites in the urine. 6. The proposed scheme for the metabolism of Chlorfenvinphos represents a detoxication mechanism.     

Diprenylated chalcones and other constituents from the twigs of Dorstenia barteri var. subtriangularis

The twigs of Dorstenia barteri var. subtriangularis yielded three diprenylated chalcones: (-)-3-(3,3-dimethylallyl)-5'-(2-hydroxy-3-methylbut-3-enyl)-4,2',4'-trihydroxychalcone, (+)-3-(3,3-dimethylallyl)-4',5'-[2'-(1-hydroxy-1-methylethyl)-dihydrofurano]-4,2'-dihydroxychalcone and 3,4-(6",6"-dimethyldihydropyrano)-4',5'-[2',-(1-hydroxy-1-methylethyl)-dihydrofurano]-2'-hydroxychalcone for which the names bartericins A, B and C, respectively, are proposed. Stipulin, beta-sitosterol and its 3-beta-D-glucopyranosyl derivative were also isolated. The structures of these secondary metabolites were determined on the basis of spectroscopic analysis, especially, NMR spectra in conjunction with 2D experiments, COSY, HMQC and HMBC. The structural relationship of bartericins B and C was further established by the chemical cyclization of one to the other.     

Trinitrophenylation of nucleic acids and their constituents.

1. Under relatively mild conditions, nucleic acids and their constituents were trinitrophenylated with 2,4,6-trinitrobenzenesulfonate (TNBS) in aqueous solution (pH 8-11), yielding reddish-orange trinitrophenyl (TNP) derivatives. Guanine residues were trinitrophenylated on the base residues at the 2-amino group (N2-TNP derivatives), and in addition, 2'- and 3'-hydroxyl groups of the ribose moieties of nucleosides or nucleotides were trinitrophenylated to form Meisenheimer complexes. 2. The preparation of TNP derivatives (N2-TNP-guanine, -guanosine, N2, O-bis-TNP-guanosine, O-TNP-guanosine, -adenosine, -cytidine , and -uridine), their rates of formation, absorption spectra (UV, visible, and infrared), molar extinction coefficients, Rf value, electrophoretic mobilities, and stability in acid or alkaline solution, are presented. 3. Trinitrophenylation of several kinds of nucleic acid was investigated. Calf thymus DNA and yeast transfer RNA showed a resistance to trinitrophenylation compared to guanosine 3'(2')-phosphate, yeast RNA or denatured calf thymus DNA. TNP-RNA showed resistance to the action of ribonucleases T1 and T2 [EC 3.1.4.8 and 3.1.4.23]. 4. Trinitrophenylation reactions using 2,4,6-trinitrochlorobenzene and 2,4,6-trinitrofluorobenzene were compared with that using TNBS as regards specificity and reaction rate.     

Bilirubin glucuronyltransferase. Specific assay and kinetic studies

1. Bilirubin glucuronide was synthesized in vitro in a system containing a rat liver microsomal fraction, UDP-glucuronic acid, Mg(2+) and bilirubin. The enzymic synthesis was accomplished without the addition of a bilirubin carrier. 2. Azobilirubin and azobilirubin glucuronide were separated by t.l.c. and paper chromatography and the measurement of the conjugate provided a specific assay for bilirubin UDP-glucuronyltransferase (EC 2.4.1.17). 3. This diazo compound was labelled when [U-(14)C]UDP-glucuronic acid was employed in the transglucuronidation reaction. 4. Identity of the glucuronide nature of the product was further confirmed by hydrolysis with beta-glucuronidase prepared from limpets and Helix pomatia. In each instance azobilirubin and glucuronic acid were liberated. 5. There was a close correlation between the bilirubin glucuronyl-transferase activity as measured by two procedures, colorimetric and radioisotopic. The specific activities so measured were 19nmol of bilirubin ;equivalents' conjugated/h per mg of protein and 16.9-18.4nmol of UDP-glucuronic acid incorporated/h per mg of protein, respectively. On this basis, it was concluded that the major product formed in vitro was bilirubin monoglucuronide; this represents about 77% of the total products formed. 6. The K(m) values for bilirubin and UDP-glucuronic acid at pH8.2 are 3.3x10(-4)m and 1.67x10(-3)m, respectively. 7. The addition of Mg(2+) at a final concentration of 5mm to the reaction mixture increased the rate of conjugation by 5.6-fold in the microsomal preparation that had been subjected to overnight dialysis against 10mm-EDTA (disodium salt). 8. Diethyl-nitrosamine at a final concentration of 1-20mm has no effect on the glucuronidation of bilirubin in vitro.     

Reaction of acid-activated mitomycin C with calf thymus DNA and model guanines: elucidation of the base-catalyzed degradation of N7-alkylguanine nucleosides

Mitomycin C (MC, 1) forms covalent adducts under acidic activating conditions (pH approximately 4) with deoxyguanosine, d(GpC), and guanine residues of calf thymus DNA. In the case of deoxyguanosine, five adducts arise from a common precursor, N7-(2' beta, 7'-diaminomitosen-1'-yl)-2'-deoxyguanosine (10a; not isolated), which hydrolyzes spontaneously via two pathways: scission of the glycosidic bond to form N7-(2' beta, 7'-diaminomitosen-1' alpha-yl)guanine (5) and its 1' beta-isomer (6) and imidazolium ring opening to generate three 2,6-diamino-4-hydroxy-5-(N-formyl-2' beta, 7'-diaminomitosen-1' beta-yl)pyrimidine (FAPyr) derivatives that are substituted at N6 by isomeric 2'-deoxyribose units [i.e., 1' beta-furanose (7), 1' alpha-furanose (8), and 1' beta-pyranose (9)]. The structures of 5-9 were determined by spectroscopic methods. The same five adducts were obtained from d(GpC), but only the guanine adducts 5 and 6 were formed in DNA. Adducts 7-9 interconvert during high-performance liquid chromatography (HPLC). The unexpected isomerization of the deoxyribose moiety of the initially formed 1' beta-furanose adduct 7 to those of 8 and 9 occurs upon imidazolium ring opening, as discerned by the course of imidazolium cleavage of the simple models N7-ethyl- and N7-methylguanosine and N7-methyl-2'-deoxyguanosine. All ring-opened N7-alkylguanosine derivatives studied here exist as a mixture of distinct N-formyl rotamers, manifested by multiple interconverting peaks on HPLC and in the 1H NMR spectra. In the UV spectra of such derivatives, a new and diagnostic maximum at 218 nm (at pH 7) is observed. Acid-activated MC is found to alkylate preferentially the Gua-N7 position in deoxyguanosine or d(GpC), in contrast to reductively activated MC, which preferentially alkylates the Gua-N2 position. This finding is explained by the different electronic structures of acid- and reduction-activated MC. In DNA, the N7 specificity of acid-activated MC is partially offset by steric factors.