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The reverse transcriptase (RT) was partially purified by a newly developed procedure from the simian immunodeficiency virus TYO-7 isolated from an African green monkey (SIVagmTYO-7). The method comprised lysis of the virus with nonionic detergent followed by two centrifugations in isopycnic sucrose density gradients and one velocity sedimentation in a glycerol gradient. The enzyme exhibited a purity of 70-80% and showed an exceptional high specific activity of 135 nmol incorporation of dTMP per milligram of protein in 1 h with poly(rA).oligo(dT) as template-primer (TP). The molecular weight of the native enzyme was estimated by velocity sedimentation analysis as 120K-130K. Investigation of the RT by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the active enzyme is a heterodimer composed of a 64- and a 50-kDa subunit. The two subunits were identified to be RT specific by Western blot analysis. In activity gels, both subunits exhibited enzymatic activity, whereby the 64-kDa subunit showed the predominant activity. The RT preferred the TP poly(rA).oligo(dT) over poly(rC).oligo(dG). With poly(rCm).oligo(dG), only marginal activity was detected, and no activity was measured with poly(dA).oligo(dT). The TP specificity was influenced by the reaction temperature. The highest activity was measured around the melting temperature of the TP used. Furthermore, the enzyme activity was more thermolabile when measured with poly(rA).oligo(dT) than with poly(rC).oligo(dG). To compare the specificity of RT inhibitors, their inhibition efficiency (IE) was defined as the ratio of the 50% inhibiting concentration (ID50) obtained with the RT in viral lysates to the ID50 of purified RT.

Versuche 5.8-Dimethoxychinolin-2,4-dicarbonsaure 10c, deren Dichlorid 11c oder deren Ester 12c und 13c zu 2.4-bis-Hydroxymethyl-5.8-dimethoxychinolin 6c zu reduzieren waren nicht erfolgreich. Die Chinolin-2-bzw. 4-Mono-carbonsaureester 12d bzw. 12e liesen sich dagegen glatt zu den entsprechenden 2-bzw. 4-Hydroxymethylchinolinen 6d bzw. 6e reduzieren. Antimalarial 6-Aminoquinolines, X: Further Experiments on 2- and 4-Hydroxymethyl Derivatives Attempts to reduce 5,8-dimethoxyquinoline-2,4-dicarboxylic acid 10c, the dichloride 11c or the esters 12c and 13c to 2,4-bis(hydroxymethyl)-5,8-dimethoxyquinoline 6c were not successful. However, the esters 12d and 12c of the 2- and 4-monocarboxylic acids could be easily reduced to yield the 2- and 4-hydroxymethyl derivatives 6d and 6e.

Aus der Umsetzung des als aktive Methylenverbindung fungierenden Malonsaure-diathylesters (2a), Cyanessigsaure-athylesters (2b), Acetessigsaure-athylesters (2d) und Malonitrils (7) mit s-Triazin (1) in Gegenwart eines sekundaren Amins 5 gehen die Dehydro-C-Mannich-Basen 6a--6d und 8 hervor. Abweichend von diesem Reaktionsverhalten bildet sich jedoch aus 2a durch Einwirkung von 1 und Diathylamin (5c) das 5-Athoxycarbonyl-4-hydroxy-pyrimidin (10). Application of Aminomethynylating Three-Component Reaction to CH-Active Compounds Reaction of diethyl malonate (2a), ethyl cyanoacetate (2b), ethyl acetoacetate (2d), or malononitrile (7), which act as CH-active methylene compounds, with s-triazine (1) in the presence of a secondary amine 5 leads to the dehydro-C-Mannich-bases 6a--6d and 8. This course is not followed by the reaction of 2a with 1 and diethylamine (5c) which leads to 5-ethoxycarbonyl-4-hydroxypyrimidine (10).

Die Synthese der 2- und 4-Hydroxymethyl-Derivate li und 1m von 6-(4-Diaathylamino-1-methyl-butylamino)-5,8-dimethoxychinolin (1a) wird beschrieben. 1i und 1m werden als mogliche Metabolite der Methyl-derivate 1b bzw. 1d angesehen. 1i und 1m sind weniger toxisch und vergleichbar wirksam gegen P. vinckei wie 1b bzw. 1d. Antimalarial 6-Aminoquinolines, IX: 2- and 4-Hydroxymethyl Derivatives The 2-and 4-hydroxymethyl derivatives 1i and 1m of 6-(4-diethylamino-1-methylbutylamino)-5,8-dimethoxy-quinoline (1a) were synthesized. 1i and 1m are regarded as potential metabolites of the methyl derivatives 1b and 1d, Hydroxylation of 1i and 1m reduces the toxicity and has little influence on the plasmodicidal activity.

Zwei mono-Chlorderivate, 7a und 7b, und zwei di-Chlor-derivate, 7c und 7d, von 6-(4-Diathylamino-1-methyl-butylamino)-5,8-dimethoxychinolin (1a) wurden synthetisiert, um den Einflus von Chlorsubstituenten auf Malaria-Wirksamkeit und Toxizitat von 1a zu untersuchen. 7a–d besitzen nur eine geringe Wirksamkeit gegen Plasmodium vinckei (in der Maus). Antimalarial 6-Aminoquinolines, VI: 2-, 3- and 4-Chloro Derivatives of 6-(4-Diethylamino-1-methylbutylamino)-5.8-dimethoxyquinoline Two monochloro derivatives, 7a and 7b, and two dichloro derivatives, 7c and 7d, of 6-(4-diethylamino-1-methylbutylamino)-5.8-dimethoxyquinoline (1a) were synthesized with the aim of studying the influence of chloro substituents on the antimalarial activity and toxicity of 1a. 7a–d showed only slight activity against Plasmodium vinckei (in mice).

Zwei Derivate von 5.8-Dimethoxy-2.4-dimethylchinolin mit einer 2-Diethyl-aminoethoxy-(3a) bzw. 3-diethyl-aminopropoxyseitenkette in 6-Stellung (3b) wurden synthetisiert, um zu uberprufen, ob in Verbindungen vom Typ 1 die Wirksamkeit gegen Plasmodien erhalten bleibt, wenn in der basischen Seitenkette der ringstandige Stickstoff durch Sauerstoff ersetzt wird. 3a und 3b zeigen keine Wirkung gegen P. vinckei. Potential Antimalarials, I:6-(Diethylaminoalkoxy)-5,8-dimethoxyquinolines Two derivatives of 5,8-dimethoxy-2,4-dimethylquinoline with a 2-diethylaminoethoxy (3a) and a 3-diethylaminopropoxy side chain (3b) at C-6 were synthesized with the aim to study the influence of the replacement of a nitrogen atom in the side chain of the antimalarial 6-aminoquinoline 1 by an oxygen atom on antimalarial activity. Compounds 3a and 3b are inactive against P. vinckei.

Die sieben moglichen 2-, 3- und 4-Mono-, Di- und Trimethylderivate 2b-h von 6-(4-Di-athylamino-1-methylbutylamino)-5,8-dimethoxychinolin (2a) wurden synthetisiert, um den Einflus von Methylsubstituenten im Pyridinring von 2a auf dessen Malariawirksamkeit und Toxizitat zu untersuchen. Antimalarial 6-Aminoquinolines, V. — 2-, 3-, and 4-Mono-, Di-, and Trimethyl Derivatives of 6-(4-Diethylamino-1-methylbutylamino)-5,8-dimethoxyquinoline The seven possible 2-, 3-, and 4-mono-, di-, and trimethyl derivatives 2b-h of 6-(4-diethyl-amino-1-methylbutylamino)quinoline (2a) were synthesised with the aim of studying the influence of methyl groups in the pyridine ring of 2a on antimalarial activity and toxicity.

Die Synthese der Titelverbindungen wurde versucht, ausgehend von 6-Aminochinolinen uber die 6-Diazoniumverbindung, ausgehend von 6-Bromchinolinen uber eine Grignardverbindung und durch selektive Etherspaltung von 5,6,8-Trialkoxychinolinen. Keiner der eingeschlagenen Wege fuhrte zum Erfolg. Attempted Synthesis of 5,8-Dimethoxy-6-hydroxyquinolines The synthesis of the title compounds was attempted starting from 6-aminoquinolines via diazonium compounds, starting from a 6-bromoquinoline via a Grignard compound or by selective cleavage of an ether group in a 5,6,8-trialkoxyquinoline. None of these ways was successful.

Die Titelverbindung 4 wurde, ausgehend vom 6-Aminochinolin 5, uber das 6-Cyanochinolin 7 und das 6-Aminomethylchinolin 8 synthetisiert. 4 zeigt keine Wirkung gegen Plasmodium vinckei. 4 ist gegen eine Oxidation bestandiger als das 6-Aminochinolin 1.

Neun in 2-, 3-, und/oder 4-Stellung unterschiedlich substituierte 8-Methoxy-chinolin-5,6-dione 3a-i werden durch Oxidation der entspr. 6-Amino-5,8-di-methoxy-chinoline 1a-i erhalten. Diese Oxidation verlauft nur glatt, wenn die 8-Stellung der 6-Aminochinoline substituiert ist. Aus 6-Amino-2,4-di-methyl-5-methoxychinolin (4) entsteht bei der Oxidation 2,4-Dimethyl-8-((2,4-dimethyl-5-methoxy-6-chinolyl)amino)chinolin-5,6-dion (5). 8-Methoxy-quinoline-5,6-diones, II Nine 8-methoxy-quinoline-5,6-diones with varying substituents in the 2-, 3-, and/or 4-position 3a-i were synthesized by oxidation of the corresponding 6-amino-5,8-dimethoxyquinolines 1a-i. By this oxidation quinones of type 3 are obtained only if the 8-position of the quinoline nucleus is substituted. Oxidation of 6-amino-2,4-dimethyl-5-methoxyquinoline (4) yields 2,4-dimethyl-8-((2,4-dimethyl-5-methoxy-6-quinolyl)amino)-quinoline-5,6-dione (5).

Zwolf in 6-Stellung basisch alkylierte 6-Amino-5.8-dimethoxy-chinaldine mit den Seiten-ketten (C2H5)2N(CH2)nNH (n = 2, 3; 15, 16) und H2N(CH2)nCHRNH(n = 19, R H, CH3; 21a–k) wurden synthetisiert, um den Einflus der basischen Seitenkette auf Malaria-Wirksamkeit und Toxizitat der 6-Amino-chinoline zu untersuchen. Antimalarial 6-Aminoquinolines, II. 6-[ω-Diethylaminoalkylamino]- and 6-[ω-Aminoalkyl-amino]-5,8-dimethoxyquinaldines Twelve derivatives of 6-amino-5,8-dimethoxyquinaldine with the basic side chains (C2H5)2N(CH2)nNH (n = 2, 3; 15, 16) and H2N(CH2)nCHRNH (n = 1 – 9, R H, CH3; 21a–k) in the 6-position are prepared with the aim of studying the influence of the basic side chain on malarial activity and toxicity of the 6-aminoquinolines.

Die spektrale Empfindlichkeitsverteilung eines Zeiss-Spektralfluorometers wird ermittelt. Die kauflichen Szintillatorsubstanzen POP, POPOP und Terphenyl werden als Vergleichssubstanzen in der Fluorometrie vorgeschlagen. Die UV-Absorptionsspektren und die korr. Fluoreszenzspektren von 2-Phenylthionaphthen-S-dioxyd und anderen ringgeschlossenen trans-Stilben-Derivaten sowie von ringgeschlossenen Diphenyl-Derivaten in Losung werden ermittelt; relative Quantenausbeuten der praktischen auseren Fluoreszenz werden angegeben. — Das 1.3-Dihydro-2H-1.3.2-benzodiazaborol fluoresziert stark, nicht dagegen die entsprechende Sauerstoffverbindung, o-Phenylen-phenyl-boronat.

Ninety analogues of suramin have been examined for their ability to inhibit the exogenous reverse transcriptase (RT) of human immunodeficiency virus type I (HIV-I). Of these compounds, 57 inhibited the poly(rC).oligo(dG)-dependent RT activity. Three classes of dose-response curves could be discriminated. Allocation of a compound to one class did not correspond with obvious structural features. Twenty-four substances were superior to suramin in our RT inhibition assay. The RT-inhibitory activity of these compounds did not correlate with their effect against filariae or trypanosomes. Preliminary antiviral evaluation in susceptible human T cells inoculated with HIV-I demonstrated in vitro therapeutic efficacy for some compounds with lower drug-related cellular toxicity than suramin. Certain structural features relevant for the RT-inhibitory effect of these compounds were recognized. Predictions are made for the design of more effective RT inhibitors. Such compounds will help to understand the molecular mechanism of reverse transcription and might be useful in the therapy of retroviral infections.

The chromosome of N. gonorrhoeae contains several complete expression genes coding for variant opacity proteins. DNA sequence analysis of two opacity genes derived from the same locus ( opa E1) of two isogenic gonococcal variants reveals common and variable regions in these genes. Genomic blotting experiments using synthetic probes suggest gene conversion as a principle for the assembly of variant sequence information in opacity genes. The 5′ region of opacity genes is composed of identical pentameric pyrimidine units (CTCTT) encoding the hydrophobic portion of the opacity leader peptide. This coding repeat is variable in a given locus with respect of the number of pentameric units. While all expression loci in a single cell are constitutively transcribed, the production of opacity proteins is determined by the coding repeat sequence on the translational level.

In N. gonorrhoeae, the expression of pilus and opacity (Op) proteins can be switched on and off and a single cell apparently has a whole repertoire of genes to express many serologically distinguishable protein types. We describe the isolation of several different Op genes and of nonexpressing gene equivalents, all derived from isogenic gonococcal variants. In the E. coli host, Op proteins identical with those made in the respective N. gonorrhoeae strain are produced. The Op genes map near the pilus expression locus. Genomic blotting experiments with an Op gene probe reveal complex hybridization patterns but little heterogeneity among the genes of Op variants. It appears that colonial variation involving the Op protein of N. gonorrhoeae is based on minor sequence alterations, in contrast to the pilus variation system, in which changes in the expression can be evoked by substantial genomic rearrangements.

Gonococcal virulence is thought to rely on multiple phenomena to which antigenic variation of surface proteins as well as the production of an extracellular protease specific for human IgA1 can be accounted. The expression of pilin and opacity (Op) protein in Neisseria gonorrhoeae can be switched on and off and a single cell apparently has a whole repertoire of genes to express many serologically distinguishable protein types. In order to investigate the molecular mechanisms associated with this surface variability, we have cloned the genes for pilin and Op expression in Escherichia coli. Using these genes as probes in DNA filter hybridizations, we revealed two complex gene systems which undergo genomic reorganizations and/or gene conversions which occur concomitant to changes in the expression of these surface proteins. To study the role of IgA proteases in bacterial pathogenesis, we cloned the IgA protease gene of N. gonorrhoeae MS11. This clone promotes expression and extracellular secretion of active enzyme in E. coli and other gram-negative bacterial species.