Amine Prodrugs Which Utilize Hydroxy Amide Lactonization. II. A Potential Esterase-Sensitive Amide Prodrug

In an effort to develop esterase-sensitive pro-prodrugs for amines, an amide derivative of 3-(2-acetoxy-4,6-dimethylphenyl)-3,3- dimethylpropionic acid (4-methoxyaniline amide (8) was synthesized and its stability investigated. This esterifled hydroxy amide was found under all conditions to degrade via a two-step process initiated by acetyl ester hydrolysis generating the hydroxy amide intermediate 9a. The lactonization of this intermediate 9a in the second step resulted in the formation of 4-methoxyaniline (10) and 4,4,5,7-tetramethyl-3,4-dihydrocoumarin (la). The pro-prodrug 8 was observed to possess the following half-lives at 37°C under various conditions: 4030 min in phosphate buffer (50 mM, µ = 0.15) fixed to pH 7.4, 11.9 min in the same buffer containing a porcine liver esterase, 53.7 min in plasma, and 475 min in plasma containing diisopropylfluorophosphate. These results suggest that in a biological milieu the ester hydrolysis will occur by the enzymic hydrolysis rather than the chemical hydrolysis and that the enzymic hydrolysis of 8 in plasma is due, in part, to the action of serine-dependent esterases.     

The in vitro/in vivo comparative metabolism of 4-aminobiphenyl using isolated hepatocytes

The metabolism of 4-aminobiphenyl by isolated hepatocytes from various species was compared with urinary metabolite profiles in the same species. Radioactive compounds in concentrates of ether extracts from hepatocytes or urine following hydrolysis were analysed by TLC and reversed phase HPLC in conjunction with radioactivity monitoring and synthetic standards.The major metabolites from hepatocytes and in urine were 4-acetamidobiphenyl, 3-hydroxy-4-aminobiphenyl 4-hydroxy-4-aminobiphenyl and 4-hydroxy-4-acetamidobiphenyl. Oxidation of the amine nitrogen gave hydroxylamino, nitroso and nitro compounds. Minor metabolites were 2-hydroxy amine and amide, the hydroxamic acid and the oxamic acid. The urinary metabolite profiles correlated well with those from hepatocytes for each species.Abbreviations Used 4-ABP 4-aminobiphenyl - AA 4-acetamidobiphenyl - A3-OH 3-hydroxy-4-aminobiphenyl - A4-OH 4-hydroxy-4-aminobiphenyl - A2-OH 2-hydroxy-4-aminobiphenyl - AA4-OH 4-hydroxy-4-acetamidobiphenyl - AA3-OH 3-hydroxy-4-acetamidobiphenyl - AA2-OH 2-hydroxy-4-acetamidobiphenyl - AAN-OH N-hydroxy-4-acetamidobiphenyl - NBP 4-nitrobiphenyl - AN-OH 4-hydroxylaminobiphenyl - NOBP 4-nitrosobiphenyl Dedicated to Professor Dr. med. Herbert Remmer on the occasion of his 65th birthday     

Relationship of metabolism of 2′-, 3′- and 5′-adenine nucleotides to presynaptic inhibition of transmitter release in rat vas deferens

Summary In the isolated rat vas deferens stimulated at 0.2 Hz, a series of 2, 3-, and 5-substituted adenine nucleotides all inhibited the twitch responses, their actions being potentiated by the nucleoside transport inhibitors, HNBTGR, NBMPR and dipyridamole.The metabolism of these nucleotides was examined utilising HPLC analysis of the bathing medium after exposure to 30 M nucleoside or nucleotide for 5 min. 5-AMP, 5-ADP, 5-ATP, and NAD⁺ were all partially hydrolysed to adenosine, the relative extent of this being 5-AMP>5-ADP=5-ATPNAD⁺. However, the other nucleotides examined were not detectably converted to adenosine or to adenosine deamination products.These results indicate that the 2-, 3- and 5-substituted nucleotides studied act at a P₁-purinoceptor in rat vas deferens to inhibit neurotransmission and, with the exception of 5-AMP, 5-ADP, 5-ATP and NAD⁺, all appear to act directly at this receptor. However, the 5-adenine nucleotides (AMP, ADP and ATP) and NAD⁺ all appear to act at least partially indirectly subsequent to their hydrolysis to adenosine.Abbreviations. The following abbreviations are used ADA adenosine deaminase (EC 3.5.4.4) - 5-ADP adenosine 5-diphosphate - 2,5-ADP adenosine 2,5-diphosphate - 3 5-ADP, adenosine 3,5-diphosphate - 2-, 3 or 5-AMP adenosine 2-, 3-, or 5-monophosphate - 5-ATP adenosine 5-triphosphate - cNADP⁺ -nicotinamide dinucleotide 2,3-cyclic monophosphate - CoA coenzyme A - HNBTGR 6-(2-hydroxy-5-nitrobenzyl)-thioguanosine - NAD⁺ -nicotinamide adenine dinucleotide - NADP⁺ -nicotinamide adenine dinucleotide phosphate - NBMPR 6-(4-nitrobenzylthio)-purine riboside     

Comparative studies on distribution and covalent tissue binding of 2,4,2′,4′- and 3,4,3′,4′-tetrachlorobiphenyl isomers in the rat

The ¹⁴C-labeled tetrachlorobiphenyl (TCB) isomers 2,4,2,4-tetrachlorobiphenyl (2,4,2,4-TCB) and 3,4,3,4-tetrachlorobiphenyl (3,4,34-TCB) were administered orally to rats, and distribution and covalent binding were measured in several organs. Marked differences in distribution and covalent binding of the two TCBs were observed. The accumulation and retention of 2,4,2,4-TCB in adipose tissue were much higher than those of 3,4,3,4-TCB, although the level of radioactivity in the blood was consistently higher in 3,4,3,4-TCB treated rats. The radioactivity bound in covalent linkages with cellular macromolecules in several tissues was also measured. The data obtained indicated that covalent binding was higher in 3,4,3,4-TCB treated rats than in those treated with 2,4,2,4-TCB, particularly in liver and blood components. These results suggest that the two TCB isomers have different pharmacokinetic properties in rats, and the association of covalent binding with 3,4,3,4-TCB-induced toxicities might be important. In addition, we found that repeated oral dosing with the two TCB isomers caused an increase in in vitro liver microsomal generation of reactive metabolites of TCBs, indicating that the microsomal enzyme system is likely to play an important role in the in vivo covalent binding of TCB.     

Identification of the Diastereomers of Pentobarbital N-Glucosides Excreted in Human Urine

A study was undertaken to determine if humans excreted pentobarbital N-glucosides as urinary metabolites following oral administration of pentobarbital. (lRS,5RS)-l--D-Glucopyranosyl) pentobarbital ((lRS,5RS)-PTBG) was isolated from the urine of one subject. The two diastereomers, (lRS,5R)-PTBG and (lRS,5S)-PTBG were separated and found to be identical to synthetic standards when compared using HPLC retention times coupled with UV (with and without post-column ionization) and mass spectrometry (HPLC/ MS). A HPLC method was developed for detecting and quantifying (lRS,5R)-PTBG, (lRS,5S)-PTBG and pentobarbital in urine. Following a single oral dose of sodium pentobarbital to male subjects (n = 6), 1.6–6.2% of the pentobarbital dose was excreted as (lRS,5S)-PTBG over 60 hours. (lRS,5R)-PTBG was also detected in one subject and accounted for 0.3% of the pentobarbital dose. Using a modified HPLC system, the four pentobarbital N-glucosides were resolved and analysis of a partially purified pentobarbital N-glucoside extract from one subject indicated that only (lR,5R)-PTBG and (lS,5S)-PTBG could be detected as urinary excretion products. These results indicate that the side chain chirality of pentobarbital may influence the observed enantioselectivity for the formation and/or urinary excretion of the pentobarbital N-glucosides.     

Relaxation of hormonally stimulated smooth muscular tissues by the 8-bromo derivative of cyclic GMP

Summary Substances that cause contraction or relaxation of smooth muscle have been shown to increase intracellular levels of cyclic GMP. Because of the unclear role of cyclic GMP in the control of smooth muscle tone, cyclic GMP derivatives were exogenously applied to various smooth muscle preparations and their effects on tissue tone were studied.Whereas the basal tone of the rat ductus deferens was not affected by exogenous cyclic GMP or its dibutyryl or 8-bromo derivatives, the contractile responses of this tissue to noradrenaline and acetylcholine were depressed by preincubation with 10 M 8-bromo cyclic GMP (Br-cGMP). The 8-bromo derivatives of 2:3-cyclic GMP, 5-GMP and guanosine were without effects. Cyclic AMP levels were not changed by Br-cGMP. The frequency of oxytocin-stimulated rat uteri was also depressed by Br-cGMP (10 M). In helical strips of rat and rabbit aortae, Br-cGMP (1–100 M) caused a concentration-dependent, rapid decrease in noradrenaline-stimulated tissue tension. Br-2:3-cyclic GMP was ineffective. Noradrenaline-stimulated strips from hog spleen arteries were less sensitive to Br-cGMP than aortic tissue. In ductus deferentes and aortic strips stimulated by K⁺ at a depolarizing concentration, Br-cGMP caused less relaxation than under hormonal stimulation.These findings support the concept that cyclic GMP is involved in the control of smooth muscle tone and that hormone- and drug-induced elevations of the cyclic GMP level can reduce contractile responses to neurotransmitters and hormones.Abbreviations cGMP Guanosine 3:5-monophosphate, cyclic GMP - dibutyryl cGMP N², 2-O-dibutyryl guanosine 3:5-monophosphate - Br-cGMP 8-bromo guanosine 3:5-monophosphate - Br-2:3-cGMP 8-bromo guanosine 2:3-monophosphate - Br-GMP 8-bromo guanosine 5-monophosphate - Br-Guo 8-bromo guanosine, Br-guanosine - cAMP adenosine 3:5-monophosphate, cyclic AMP - dibutyryl cAMP N⁶, 2-O-dibutyryl adenosine 3:5-monophosphate - Br-cAMP 8-bromo adenosine 3:5-monophosphate This work was supported by grants from the Deutsche Forschungsgemeinschaft. Preliminary reports were presented (Schultz, 1977b; Schultz et al., 1978).     

Metabolism and presynaptic inhibitory activity of 2′,3′ and 5′-adenine nucleotides in rat vas deferens

Summary In the isolated rat vas deferens stimulated at 0.2 Hz, [¹⁴C]labelled 5-AMP, 5-ADP and 5-ATP (10 M) inhibited twitch responses, were broken down to [¹⁴C]adenosine in the medium and incorporated into [¹⁴C]adenine ribonucleotides in the tissue. Pretreatment of tissues with 6-(2-hydroxy-5-nitrobenzyl)-thioguanosine (NBTGR), a potent inhibitor of adenosine transport, potentiated the presynaptic inhibitory action of these 5 nucleotides and reduced their incorporation in [¹⁴C]adenine nucleotides, but did not alter the appearance of [¹⁴C]adenosine in the medium.A series of 2, 3 and 5-substituted adenine nucleotides (10 M) inhibited the twitch responses of the vas deferens stimulated at 0.2 Hz. This effect was potentiated by NBTGR. Addition of exogenous adenosine deaminase very significantly reduced the inhibitory actions of adenosine, 5-AMP, 5-ADP and 5-ATP and also reduced those of 2, 5-ADP, NAD⁺ and dePCoA. The inhibitory actions of the other 2, 3 and 5 adenine nucleotides studied were not altered by exogenous adenosine deaminase.These results indicated that the presynaptic inhibitory actions of 5-AMP, 5-ADP and 5-ATP in rat vas deferens predominantly result from their prior hydrolysis to adenosine whereas the 2, 3 and 5-substituted adenine nucleotides appear to act mainly directly to inhibit transmitter release.Abbreviations. The following abbreviations are used 5-ADP 5-adenosine diphosphate - 2,5-ADP 2,5-adenosine diphosphate - 3,5-ADP 3,5-adenosine diphosphate - 2,3 or 5-AMP 2,3 or 5-adenosine monophosphate - 5-ATP 5-adenosine triphosphate - CoA coenzyme A - 2,3-cAMP 2,3-cyclic adenosine monophosphate - cNADP⁺ -nicotinamide dinucleotide 2,3-cyclic monophosphate - dePCoA dephosphocoenzyme A - NAD⁺ -nicotinamide adenine dinucleotide - NADP⁺ -nicotinamide adenine dinucleotide phosphate - NBTGR 6-(2-hydroxy-5-nitrobenzyl)-thioguanosine - oxid CoA oxidized-coenzyme A     

Viscoelastic Properties of Bioadhesive,Chlorhexidine-Containing Semi-Solids for Topical Application to the Oropharynx

Purpose. This study examined the viscoelastic properties of bioadhesive, chlorhexidine-containing semi-solid formulations, designed for topical application to the oropharynx. Methods. Oscillatory rheometry was performed using a Carri-Med CSL²-100 rheometer at 20.0 ± 0.1° C in conjunction with parallel plate geometry (2 cm diameter, 0.5 mm sample thickness). Samples were subjected to a constant strain (6.5 × 10^–3 rad) and defined viscoelastic parameters, namely storage modulus (G), loss modulus (G), loss tangent (tan ) and dynamic viscosity (), measured over a defined frequency range (0.01-1.0 Hz). Results. As the oscillatory frequency was increased, G G of all formulations increased, whereas both and tan significantly decreased. The magnitude of increase of G and G as a function of frequency was relatively small, indicating that, in general, the formulations were non-cross-linked elastic systems. Increasing concentrations of HEC, PVP and PC significantly increased G, G, yet decreased tan observations that may be attributed to the physical state of each polymer in the formulations. Formulation elasticity increased (i.e. tan decreased) as a result of increased entanglement of polymeric chains of dissolved components (i.e. HEC and PVP) and the restrained extension of swollen, cross-linked chains of PC. Additionally, in formulations where the saturation solubility of PVP was exceeded and/or insufficient 'free-water' was available for maximal swelling of PC, formulation elasticity increased as a result of the increasing mass of dispersed solid particles of PVP and/or PC. Formulation increased due to the attendent effects of polymer chain entanglement and polymer state on overall formulation viscosity. Conclusions. Following application to the oropharynx, the formulations will behave as elastic systems. Thus, these formulations would be expected to offer advantageous clinical properties, e.g., prolonged drug release, increased bioadhesion. However, it is noteworthy that the final choice of formulation for clinical evaluation will involve a compromise between viscoelastic characteristics and acceptable textural properties, e.g. ease of product application. This study has shown the applicability of oscillatory rheometry for both the characterisation and selection of candidate, topical bioadhesive formulations for clinical evaluation.     

Dose-dependent excretion of DDE(1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene) in rats

Dose-dependent excretion of p,pDDE in rats was investigated. p,pDDE itself was the major excreta in rats. But some o,p'isomer of DDE was detected in feces by GC-MS analysis. The excretion of p,p'DDE after a single administration was modified by its dose level.The time pattern of p,pDDE excretion agrees well with the modified Hill equation. The value of the equilibrium constant (K) increases in proportion to time t after p,pDDE administration.Using the modified Hill equation and the linear K equation, the excretion rate of p,pDDE during the experimental time t can be estimated. The estimated p,pDDE excretion rate in feces agrees well with the measurements.     

Synthesis and Anti-inflammatory Effect of Chalcones and Related Compounds

Purpose. Mast cell and neutrophil degradations are the important players in inflammatory disorders. Combined with potent inhibition of chemical mediators released from mast cells and neutrophil degranulations, it could be a promising anti-inflammatory agent. 2,5-Dihydroxychalcone has been reported as a potent chemical mediator and cyclooxygenase inhibitor. In an effort to continually develop potent anti-inflammatory agents, a novel series of chalcone, 2- and 3-hydroxychalcones, 2,5-dihydroxychalcones and flavanones were continually synthesized to evaluate their inhibitory effects on the activation of mast cells and neutrophils and the inhibitory effect on phlogist-induced hind-paw edema in mice. Methods. A series of chalcones and related compounds were prepared by Claisen-Schmidt condensation of appropriate acetophenones with appropriate aromatic aldehyde and the anti-inflammatory activities of these synthetic compounds were studied on inhibitory effects on the activation of mast cells and neutrophils. Results. Some chalcones showed strong inhibitory effects on the release of -glucuronidase and histamine from rat peritoneal mast cells stimulated with compound 48/80. Almost all chalcones and 4-hydroxyflavanone exhibited potent inhibitory effects on the release of -glucuronidase and lysozyme from rat neutrophils stimulated with formyl-Met-Leu-Phe (fMLP). Some chalcones showed potent inhibitory effects on superoxide formation of rat neutrophils stimulated with fMLP/cytochalasin B (CB) or phorbol myristate acetate (PMA). 2,3-Dihydroxy-, 2,5-dihydroxy-4-chloro-, and 2,5-dihydroxychalcone showed remarkable inhibitory effects on hind-paw edema induced by polymyxin B in normal as well as in adrenalectomized mice. Conclusions. These results indicated that the anti-inflammatory effects of these compounds were mediated, at least partly, through the suppression of chemical mediators released from mast cells and neutrophils.     

Toxicity and 7-ethoxyresorufin O-deethylase-inducing potency of coplanar polychlorinated biphenyls (PCBs) in chick embryos

The toxicities of the coplanar polychlorinated biphenyls 3,3,4,4-tetrachlorobiphenyl (TCB), 3,3,4,4,5-pentachlorobiphenyl (PeCB) and 3,3,4,4,5,5hexachlorobiphenyl (HCB) were compared in a 72-h study on chick embryos. The substances were injected into the air sacs of hens' eggs preincubated for 7 days. Mortality was measured 72 h later and corresponding LD₅₀ values were calculated. The rank order of toxicity was PeCB> TCB>HCB. Using the same injection procedure, the potencies of these chlorobiphenyls with regard to their induction of hepatic 7-ethoxyresorufin O-deethylase activity were compared. The ranking order of the substances as inducers was the same as their order when ranked according to toxicity. The three coplanar chlorobiphenyls were considerably more toxic and potent as inducers than the nonplanar 2,2,4,4,5,5-hexachlorobiphenyl. In a 2-week toxicity study, PeCB and HCB were injected into the yolks of hens' eggs preincubated for 4 days. PeCB was about 50-fold more potent than HCB in causing embryonic death. Both substances caused abnormalities, including edema, liver lesions, microphthalmia and beak deformities.     

Synthesis and transformations of furan derivatives

By the reactions of 2-phenyl-4-(2-furfuryliden)-, 2-phenyl-4-(5-nitro-2-furfuryliden)-, and 2-methyl-4-(5-nitro-2-furfuryliden)-5-oxazolones with primary and secondary amines, a series of N-mono- and N,N-disubstituted amides of the corresponding-benzamido--(2-furyl)-acrylic and-benzamido- and-acetamido--(5-nitro-2-furyl)acrylic acids was synthesized. 1-Alkyl(aryl) substituted 2-phenyl-4-(5-nitro-2-furfuryliden)-5-imidazolones were synthesized from the reaction of phosphorus oxychloride and the monosubstituted amides of-benzamido--(5-nitro-2-furyl)acrylic acid.Translated from Khimiko-Farmatsevticheskii Zhurnal, No. 2, pp. 21–27, February, 1967.     

Effects of N-chlorobenzyl analogues of amiloride on myocardial contractility,Na-Ca-exchange carrier and other cardiac enzymatic activities

Summary 1. In electrically driven guinea pig left atria, micromolar concentrations (2 mol/l to 80 mol/l) of N-chlorobenzyl derivatives of amiloride (o-chlorobenzamil and 3,4-dichlorobenzamil) produced quantitatively similar positive inotropic effects. Contracture developed with 3,4-dichlorobenzamil. Endogenously released catecholamines contributed 30% to the positive inotropic effect of ochlorobenzamil but did not contribute at all to the effect of 3,4-dichlorobenzamil. When tested in the presence of the inhibitor of phosphodiesterase isobutylmethylxanthine, ochlorobenzamil antagonized its positive inotropic effect, whereas 3,4-dichlorobenzamil potentiated it. o-Chlorobenzamil also antagonized the positive inotropic effect of ouabain in that it shifted its concentration-effect curve to the right. Moreover, o-chlorobenzamil prevented the appearance of ouabain toxicity in terms of a rise in the resting force. 2. Also, in electrically driven guinea pig papillary muscle, micromolar concentrations (5 mol/l to 30 mol/l) of both N-chlorobenzyl derivatives of amiloride produced a positive inotropic effect. This effect was more marked with 3,4-dichlorobenzamil than with o-chlorobenzamil and was associated for both compounds with lengthening of relaxation time. 3. o-Chlorobenzamil and 3,4-dichlorobenzamil influenced, though not to the same extent, several systems involved in the onset and in the control of cardiac contractility. 3,4-Dichlorobenzamil inhibited with the same potency Na-K-ATPase, sarcotubular Ca-ATPase, Na-Ca-exchange carrier, cAMP-dependent phosphodiesterase isolated from bovine heart and oxidative phosphorylation of mitochondria isolated from rat liver. Low micromolar concentrations of o-chlorobenzamil mainly inhibited Na-Ca-exchange carrier and cAMP-dependent phosphodiesterase. 4. The results suggest that 3,4-dichlorobenzamil is a quite unspecific compound and its cardiac effects are the result of an interference with several enzymatic and transport systems. In contrast, both the inhibition of the Na-Ca-exchange carrier and cAMP-dependent phosphodiesterase can contribute to the increase in the force of contraction induced by o-chlorobenzamil. Finally, the antagonism of o-chlorobenzamil against the cardiac effects of ouabain can be explained by the inhibition of the Na-Ca-exchange carrier. Send offprint requests to M. Floreani     

Constituents of Laurus nobilis L. inhibit recombinant human lanosterol synthase

Extracts from 37 kinds of foods and foodstuffs were tested for inhibitory activity against recombinant human lanosterol synthase. Among them, extracts from five samples showed significant inhibition. Potent activity (55%) was found in 95% ethanol extract of Laurus nobilis L. Therefore, large-scale methanol extraction of the plant was carried out, and the constituents were separated by partition and fractionation by silica gel chromatography and HPLC. Four flavonoids, kaemperol 3-O-[2,4-O-di-E-p-coumaroyl--l-pyranorhamnoside] (1); 3,3,4,5,6,7,8-heptamethoxyflavone (2); 3,4,5,6,7,8-hexamethoxyflavone (nobiletin) (3); and 4,5,6,7,8-pentamethoxyflavone (tangeretin) (4); and six sesquiterpens, eremanthine (5), dehydrocostus lactone (6), costunolide (7), zaluzanin C (8), zaluzanin D (9) and reynosin (10) were isolated. Eremanthine (5) showed the most potent activity, 70% inhibition, at the concentration of 500 M.     

Trennung und Bestimmung der Nucleotide des Gehirns

Ohne ZusammenfassungFolgende Abkürzungen werden in der Arbeit verwendet AMP Adenosin-5-monophosphat - ADP Adenosin-5-diphosphat - ATP Adenosin-5-triphosphat - GMP Guanosin-5-monophosphat - GDP Guanosin-5-diphosphat - GTP Guanosin-5-triphosphat - IMP Inosin-5-monophosphat - UMP Uridin-5-monophosphat - UDP Uridin-5-diphosphat - UTP Uridin-5-triphosphat - UDPAG Uridin-5-diphosphat-N-acetylglucosamin - UDPG Uridin-5-diphosphat-glucose - DPN Diphosphopyridinnucleotid - TPN Triphosphopyridinnucleotid Mit 10 TextabbildungenMit Unterstütznng der Deutschen Forschungsgemeinschaft.     

Effects of adenosine derivatives on cAMP accumulation and lipolysis in rat adipocytes and on adenylate cyclase in adipocyte plasma membranes

Summary N⁶-monosubstituted adenosine (Ad)-derivatives and Ad-derivatives altered in the adenine-or ribose-moiety have been compared with Ad in their effects on noradrenaline (NA)-stimulated cAMP accumulation, on lipolysis stimulated by NA or theophylline (THEO) and on adenylate cyclase (AC) activity of adipocyte plasma membranes.In isolated adipocytes about 0.01 M Ad caused a 50% inhibition of cAMP accumulation stimulated maximally by 1M NA. Depending upon the structure of substituent, the Ad-N⁶-derivatives were up to one order of magnitude either more or less active than Ad itself. 2-fluoro-Ad was nearly as active as Ad, whereas 2,5-dideoxy-Ad and 2-deoxy-Ad were practically ineffective as inhibitors of NA-stimulated cAMP accumulation. All compounds showed the same order of potency relative to Ad, when tested against lipolysis stimulated maximally by 1 mM THEO or submaximally by 0.3 M NA.In adipocyte plasma membranes a 50% inhibition of AC activity stimulated by 10M NA was observed at about 10 M Ad. None of the N⁶-substituted derivatives had any effect on either basal or NA-stimulated AC activity, whereas 2,5-dideoxy-Ad proved to be about 40 times more potent than Ad. 2-deoxy-Ad and 2-fluoro-Ad were nearly equipotent to Ad. Similar results were obtained, if AC was stimulated with 5-guanylylimidodiphosphate or NaF. Neither the N⁶-derivatives nor THEO could reverse the inhibitory effect of Ad on AC in plasma membranes.It is concluded, that different mechanisms are involved in the inhibitory effects of Ad on cAMP accumulation and lipolysis in intact cells and on AC activity of adipocyte plasma membranes.     

Hemmung von Adenyl-Cyclase-Präparationen aus der Rattenniere durch Calciumionen und verschiedene Diuretica

Summary Antidiuretic hormone (ADH) increases the permeability to water of certain epithelial membranes. This effect, found in the urinary bladder of the toad and in the distal tubules and the collecting ducts of kidney, is mediated intracellularly by adenosine 35-monophosphate (Ado-35-P). Calcium ions and the diuretic ethacrynic acid are known to inhibit the ADH-induced increase in water permeability of the toad bladder. In adenyl cyclase preparations from rat renal cortex and medulla, the influence of these substances as well as of other diuretics added in vitro has been studied. Adenyl cyclase activity has been determined, excepted as noted, by measuring Ado-35-P formed from 1 mM ¹⁴C-ATP in the presence of 10 mM Mg⁺⁺, an ATP regenerating system, and 5 mM unlabeled Ado-35-P to reduce the enzymatic degradation of the labeled Ado-35-P.Calcium ions reduced the rate of Ado-35-P formation by particles from renal cortex and medulla when the activity was measured in the presence of either Mg⁺⁺ or Mn⁺⁺. With 10 mM Mg⁺⁺, 1 mM Ca⁺⁺ decreased adenyl cylase activity by about 50%. Activities of cortical adenyl cyclase stimulated by parathyroid hormone, thyrocalcitonin or ADH and of medullary adenyl cyclase stimulated by ADH were also reduced by about 50% in the presence of 1 mM Ca⁺⁺. The inhibition was independent of the ATP concentration, but was influenced by the Mg⁺⁺ content of the incubation medium.Adenyl cyclase activities of cortical and medullary membrane preparations were reduced by about 50% by 0.2 mM ethacrynic acid. The extent of this inhibition was essentially the same whether the enzymatic activity was determined in the absence or presence of stimulating hormones. The inhibitory action of ethacrynic acid was partially prevented by simultaneous addition of dithioerythritol (DTE). A derivative of ethacrynic acid, L 589420-0-2, also inhibited renal adenyl cyclase, but its action was not influenced by the addition of DTE. Adenyl cyclase from both parts of the kidney was inhibited by about 90% by 0.2 mM mersalyl. This action was almost completely prevented by the addition of 1 mM DTE. The pharmacological significance of adenyl cyclase inhibition by these diuretics is still uncertain since the role of Ado-35-P in the regulation of sodium transport is as yet unclear.Other diuretics, hydrochlorothiazide, furosemide, mefruside, amiloride, and the non-diuretic benzothiadiazine, diazoxide, had essentially no effect on cortical and medullary adenyl cyclase preparations when they were added in 0.1–0.5 mM concentration.The methylxanthines, theophylline and caffeine, which are known to inhibit nucleoside 35-monophosphate phosphodiesterase, reduced the rate of Ado-35-P formation. The unstimulated and the hormone-stimulated adenyl cyclases were inhibited to the same extent by theophylline. When adenyl cyclases was stimulated by fluoride, however, we found only a very small inhibition by theophylline. Inhibition of the medullary adenyl cyclase was greater than that of the enzyme prepared from renal cortex. At a concentration of 1 mM these methylxanthines significantly inhibited the medullary enzyme, but the inhibition became asymptotic at about 50% when concentrations up to 20 mM were used. Therefore, it is likely that inhibition by these substances varies in different cell types and tissues.Instead of phosphodiesterase inhibitors, unlabeled Ado-35-P can be used in the assay of adenyl cyclase activity to reduce the degradation of enzymatically formed labeled Ado-35-P. This addition, though, can also influence adenyl cyclase activity. In a medullary enzyme preparation 0.2 mM Ado-35-P reduced the adenyl cyclase activity by 13%, 5 mM Ado-35-P by 35%.

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Abkürzungen Ado-35-P Adenosin-35-monophosphat - Guo-35-P Guanosin-35-monophosphat - ADH antidiuretisches Hormon, Vasopressin - PTH Parathormon - TCT Thyreocalcitonin - DTE Dithioerythrit - EDTA Äthylendiamintetraessigsäure Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.Über einen Teil der Ergebnisse wurde auf der 11. Frühjahrstagung der Deutschen Pharmakologischen Gesellschaft berichtet (Jakobs et al., 1970). Einige der vorliegenden Ergebnisse sind der Inauguraldissertation von K. H. J. (Medizinische Fakultät der Universität Heidelberg, 1971) entnommen.     

DNA damage induced by doxorubicin, 4′-epidoxorubicin and their copper(II) complexes

The DNA-damaging activities of doxorubicin (DXR) and 4-epidoxorubicin (4epiDXR) were tested on a covalently closed circular plasmid. In the presence of a reducing agent (sodium borohydride), DXR and 4epiDXR produced similar dose-dependent alterations of the electrophoretic pattern of DNA fragments. Since transition metal ions are known to catalyze this effect, the effects of Cu(II)DXR and Cu(II)epiDXR were also tested. When the Cu(II)-anthracycline complexes were formed at a drug: metal ratio of 21, the effect on DNA was more severe than in the case of the free drugs; since copper ions alone were found to be devoid of activity at the concentrations present in the complexes, the effect is attributed to a direct interaction between the complexes and DNA. Cu(II)4epiDXR proved to be more potent than Cu(II)DXR; this is probably due to the different structures of the two complexes.     

Studies on the constituents of Scutellaria species (XXI): constituents of the leaves of Scutellaria strigillosa Hemsley

From the leaves of Scutellaria strigillosa, 14 compounds, chrysin, apigenin, 5,7,2-trihydroxyflavone, norwogonin, ursolic acid, 6-hydroxy-4-stigmasten-3-one, 6-hydroxy-4,22-stigmastadien-3-one, 2 R,4 R,8 R--tocopherol, (S)-5,5 -bi--tocopheryl, (R)-5,5 -bi--tocopheryl, solanachromene, tocopherylquinone, jodrellin T, and 14,15-dihydrojodrellin T were isolated. The structures were determined on the basis of chemical and spectral data.     

Adenosine 3′,5′-monophosphate in cultured neuroblastoma cells: Effect of adenosine,phosphodiesterase inhibitors and benzazepines

Summary The effect of various neurohormones on intracellular levels of adenosine 3,5-monophosphate were evaluated in a neuroblastoma cell line both, in the presence and in the absence of the phosphodiesterase inhibitors isobutylmethylxanthine and papaverine. Without the phosphodiesterase inhibitors only prostaglandin E₁ increased intracellular adenosine 3,5-monophosphate levels. In the presence of isobutylmethylxanthine and/or papaverine, however, adenosine stimulated adenosine 3,5-monophosphate formation and the effect of prostaglandin E₁ was greatly potentiated. Treatment of the cells with dopamine, 5-hydroxytryptamine, noradrenaline, adrenaline, histamine and prostaglandin F₁ was without effect on adenosine 3,5-monophosphate levels either in the presence or absence of the phosphodiesterase inhibitors. The adenosine concentration for a half maximal effect was about 75 M. The effect of 0.1 mM adenosine was not antagonized by 1 mM theophylline. Several adenosine analogs were tested and found to have little or no effect on adenosine 3,5-monophosphate levels in neuroblastoma N4TG3. Diazepam and to a lesser extent chlordiazepoxide act like phosphodiesterase inhibitors when incubated together with prostaglandin E₁.Part of this work was done during a visit of the authors to NIH, U.S.A., J. S. being a fellow of the Deutsche Forschungsgemeinschaft and B. H. of the Max-Planck-Gesellschaft.