New derivatives of kanamycin B obtained by modifications and substitutions in position 6". 1. Synthesis and microbiological evaluation.

The clinical use of the potent, wide-spectrum aminoglycoside antibiotics is limited by oto- and nephrotoxicities. The latter is related to the binding of these polycationic drugs to negatively charged phospholipids and to the subsequent inhibition of lysosomal phospholipases. In order to explore the influence of a modification of the hydrophobic/hydrophilic balance at a specific site of an aminoglycoside, kanamycin B has been chemically modified in position 6" by substitution of the hydroxyl group with a halogen atom (or a pseudohalogen group), or an amino, an amido, a thioalkyl, or an alkoxy group, each series containing increasingly bulkier chains. Examination of the antibacterial activity of the synthesized compounds revealed a negative correlation between the size of the 6"-substituent and the antibacterial activity against kanamycin B sensitive Gram-positive and -negative organisms. Only derivatives with small substituents in position 6", namely chloro, bromo, azido, amino, methylcarbamido, acetamido, methylthio, methylsulfinyl, O-methyl, O-ethyl, and O-isopropyl, showed acceptable activity (geometric mean of minimum inhibitory concentrations for Gram-negative strains less than or equal to 2.5 mg/L; value for kanamycin B, 0.5 mg/L). In vitro toxicological evaluation of all derivatives and computer-aided conformational analysis of selected compounds inserted in a phosphatidylinositol monolayer are presented in the following paper in this issue.

New derivatives of kanamycin B obtained by combined modifications in positions 1 and 6". Synthesis, microbiological properties, and in vitro and computer-aided toxicological evaluation.

Substitution of the C-1 atom in the 2-deoxystreptamine moiety of gentamicin C2, a broad-spectrum aminoglycoside antibiotic, by an axial hydroxymethyl group has been reported to confer protection against most clinically important bacterial enzymes inactivating aminoglycosides, while simultaneously reducing the nephrotoxic potential of this drug. We report here on a similar modification of kanamycin B. Microbiological evaluation, however, revealed no useful protection, as established by the almost complete lack of activity of 1-C-(hydroxymethyl)kanamycin B against an array of organisms producing defined types of aminoglycoside-inactivating enzymes and against which 1-C-(hydroxymethyl)gentamicin C2 and amikacin (1-N-[(S)-2-hydroxy-4-aminobutyryl]kanamycin A) are active. Moreover, toxicological evaluation, based on the in vitro measurement of the drug inhibitory potential toward lysosomal phospholipases, a predictive test of the intrinsic nephrotoxic potential of aminoglycosides, showed not decreased but rather increased toxicity. Comparative conformational analysis of the interactions of the drug with a phosphatidylinositol monolayer explained the lack of protective effect, since no significant change of the mode of insertion of the derivative in this monolayer was detected compared to that of kanamycin B. Combination of a 1-C-(hydroxymethyl) substituent with a 6"-chloro, 6"-acetamido substituent resulted in a partial improvement of the toxicological behavior with no loss of activity for the 6"-chloro and the 6"-azido derivatives, but not to the extent of obtaining better derivatives than kanamycin B itself. We, therefore, suggest that the advantages of an axial hydroxymethyl substituent at C-1 are probably restricted to the gentamicin family and do not extend to kanamycins. It might be concluded that the structural differences between gentamicins and kanamycins play an important, still undescribed role both in their effective recognition by aminoglycoside-inactivating enzymes, which are responsible for most of the clinically important cases of resistance to aminoglycosides, and also in the interactions with phospholipids, which in turn cause nephrotoxicity.

New derivatives of kanamycin B obtained by modifications and substitutions in position 6''. 2. In vitro and computer-aided toxicological evaluation with respect to interactions with phosphatidylinositol.

In a companion paper (previous paper in this issue), we report on the synthesis and microbiological evaluation of new derivatives of the aminoglycoside antibiotic kanamycin B carrying substitutions in 6" (halogeno, or amino, amido, thioalkyl, and alkoxy groups, each series with increasingly bulkier chains). These modifications were intended to potentially modulate the interactions of kanamycin B with phospholipids since these are related to inhibition of lysosomal phospholipase activities and lysosomal phospholipidosis, an early and predictive index of the nephrotoxic potential of aminoglycosides. The new derivatives were therefore examined for inhibitory potency in vitro toward lysosomal phospholipase A1 acting on phosphatidylcholine included in negatively charged liposomes. No simple correlation was observed between the nature or the size of the 6''-substituent and the inhibitory potencies of the corresponding derivatives, although certain groups (diethylamino, isopropylthio) caused a significant increase in inhibitory potency, whereas an N-acetyl-N-methylamino substituent had the opposite effect. 6''-Deoxy-6''-chlorokanamycin B, however, was the only derivative showing both a decrease (albeit limited) of inhibitory potency toward phospholipase A1 associated with the maintenance of a satisfactory microbiological activity (actually equal or slightly better than that of kanamycin B). Computer-aided conformational analysis showed that this chloro substituent did not allow the molecule to insert itself very differently compared to kanamycin B or 6''-deoxykanamycin B in a monolayer of phosphatidylinositol, all three drugs adopting an orientation largely parallel to the hydrophobic-hydrophilic interface and being largely "embedded" in the bilayer at that level. In contrast, the N-acetyl-N-methylamino and isopropylthio substituents caused the corresponding derivatives to adopt an orientation largely perpendicular to the interface, because of the attraction of this substituent, and therefore of the 3''-amino sugar moiety of kanamycin B into the hydrophobic domain of the monolayer, whereas the opposite part of the drug (2',6'-diamino sugar) protruded into the aqueous phase. No simple correlation, however, could be drawn between these changes of conformation and the relative inhibitory potencies of the derivatives.

[Nontraditional beta-lactam antibiotics]. / Niet-traditionele beta-lactam antibiotica.

In recent years several beta-lactam derivatives have been obtained, which differ markedly from the traditional penicillins and cephalosporins, some of which have been introduced in the clinic. Clavulanic acid has an oxygen atom instead of sulfur but differs also markedly on C2 and C6. This inhibitor of penicillinase is used in association with amoxicillin and ticarcillin. Sulbactam and tazobactam are other beta-lactamase inhibitors, which are also used in association with penicillins. In the carbapenems sulfur is replaced by a carbon atom and a double bond is also present. Thienamycin, which is rather labile, has been transformed into a more stable product, imipenem, which is used in the treatment of infections with gram-positive and -negative bacteria. Other carbapenems are being studied. Several synthetic penems also present an interesting activity and may be used in medicine. Monobactams are monocyclic beta-lactams isolated from bacteria. By modification of the natural products aztreonam was obtained, a substance which is used in the treatment of infections with gram-negative bacteria. Other monobactams, prepared by total synthesis like aztreonam, are being studied.

Quantitative analysis of chlortetracycline and related substances by high-performance liquid chromatography.

Isocratic high-performance liquid chromatography on Zorbax C8 7 microns allows quantitative determination of chlortetracycline, 4-epichlortetracycline, tetracycline, demethylchlortetracycline and isochlortetracycline using a mobile phase containing dimethylsulphoxide, 1 M perchloric acid and water (35:5:60). The minor impurities anhydrochlortetracycline and 4-epianhydrochlortetracycline, which are more strongly retained can be determined using a second isocratic system with a mobile phase containing more organic modifier. The method has been used for the comparison of official standards and for the analysis of a number of commercial samples.

A collaborative study of the analysis of doxycycline hyclate by high-performance liquid chromatography on polystyrene-divinylbenzene packing materials.

An improved method for the analysis of doxycycline hyclate by high-performance liquid chromatography using polystyrene-divinylbenzene column packings is described. The separation obtained with this method was compared with that of other, recently described methods. The improved method was examined in a collaborative study involving five separate laboratories, using 11 different columns and four discrete samples. The main component and impurities were determined. An analysis of variance showed absence of consistent laboratory bias and presence of significant laboratory-sample interaction. Estimates for the repeatability and reproducibility of the method, expressed as relative standard deviations (RSD) of the result of the determination of doxycycline, were found to be 0.9 and 1.2%, respectively.

Tissue distribution and excretion of radioactively labelled compounds in the Wistar rat after administration of [N-methyl-14C]-erythromycin A.

Tissue distribution and excretion of radioactively labelled compounds was studied in the Wistar rat after i.v. administration of [N-methyl-14C]-erythromycin A. Whole-body autoradiography and liquid scintillation counting was used to investigate the tissue localization of radioactivity in pregnant and non-pregnant rats. Tissue levels were maximal within 20 min, except for lachrymal glands, thymus and brain. Large amounts of radioactively labelled compounds, partly originating from active secretion, were present in the small intestine and caecum. Marked concentration of radioactively labelled compounds was also observed in the liver, spleen, lachrymal and salivary glands, lymph nodes, mammary glands, skin, bone marrow, and, to a lesser extent, in the lung, kidney and skeletal muscle. During six hours of experimental follow-up, plasma levels remained lower than corresponding tissue levels. At 1 h the radioactivity in fetuses was about three times lower than that in maternal blood. Within 48 h, more than 90% of the administered radioactivity was excreted. The amounts of radioactivity recovered in urine, faeces and expired air were about 19%, 48% and 24% respectively. After 48 h, 8% of the administered radioactivity was found in the carcass.

Thin-layer chromatographic study of the metabolites of erythromycins in the Wistar rat.

The metabolites of erythromycin A, anhydroerythromycin A, N-demethylerythromycin A and erythromycin B in the Wistar rat were studied by thin-layer chromatography. In some experiments germ-free rats, rats with a cannulated bile duct and a gastrectomized rat were used. The erythromycins examined were shown to undergo two principal changes, N-demethylation and acid-catalysed degradation. It was demonstrated that the stomach and the liver are not the sole sites of acid degradation and demethylation of erythromycins, respectively. Erythromycin A gives three principal metabolites, anhydroerythromycin A, anhydro-N-demethylerythromycin A and N-demethylerythromycin A, and erythromycin A enol ether and N-demethylerythromycin A enol ether are present to a minor extent. 5-O-Desosaminylerythronolide A was also identified, suggesting the presence of an erythromycin glycosidase.

Optimization of the separation of erythromycin and related substances by high-performance liquid chromatography.

An improved high-performance liquid chromatographic method for analysis of erythromycin is described. The separation can be performed under mild conditions of pH (6.5) and temperature (35 degrees C) on C8 and C18 silica-based reversed-phase materials of different origins. The mobile phase, with a flow-rate of 1.5 ml/min, contained various amounts of acetonitrile (25-40%, v/v), 5% (v/v) 0.2 M ammonium phosphate buffer pH 6.5, 20% (v/v) 0.2 M tetramethylammonium phosphate and water. UV detection at 215 nm allows quantitation of erythromycins A, B and C, N-demethylerythromycin A, erythromycin A enol ether and anhydroerythromycin A. The column history plays a major role, older columns often giving better separations.

Quantitative analysis of oxytetracycline and related substances by high-performance liquid chromatography.

Isocratic high-performance liquid chromatography on PLRP-S 8-microns poly(styrene-divinylbenzene) copolymer allows complete separation of oxytetracycline, 4-epioxytetracycline, tetracycline, anhydrooxytetracycline, alpha- and beta-apooxytetracycline. The mobile phase was tert.-butanol-0.2 M phosphate buffer pH 8.0-0.02 M tetrabutylammonium sulphate pH 8-0.0001 M sodium ethylenediaminetetraacetate pH 8.0-water (5.9:10:5:10:78.1, m/v/v/v/v). With this isocratic method, 2-acetyl-2-decarboxamidooxytetracycline is only partly resolved from oxytetracycline. The separation and the detection limits can be improved by the use of gradient elution. Gradient elution was used for the comparison of official standards and for the analysis of a number of commercial samples, and to monitor the stability of oxytetracycline hydrochloride during storage in the solid state for about 6 years at various temperatures.

3'-substituted 2',3'-dideoxynucleoside analogues as potential anti-HIV (HTLV-III/LAV) agents.

A series of 2',3'-unsaturated and 3'-substituted 2',3'-dideoxynucleoside analogues of purines and pyrimidines have been synthesized and evaluated for their inhibitory activity against human immunodeficiency virus (HIV). The 2',3'-unsaturated analogues of 2',3'-dideoxycytidine (ddeCyd) and 2',3'-dideoxythymidine (ddeThd), 3'-azido-2',3'-dideoxythymidine (AzddThd), 3'-fluoro-2',3'-dideoxythymidine, 2',3'-dideoxycytidine (ddCyd), and 2',3'-dideoxyadenosine (ddAdo) emerged as the most potent inhibitors of HIV-induced cytopathogenicity in the human T lymphocyte cell lines ATH8 and MT4. In ATH8 cells ddCyd, ddeCyd, and ddAdo had the highest therapeutic index whereas in MT4 cells AzddThd, ddThd, ddCyd, and ddAdo were the most selective. Derivatives from ddThd in which the substituent group was linked to the 3'-carbon atom via a thio, sulfonyl, or oxygen bridge were far less inhibitory to HIV than was AzddThd.

Sensitive and rapid assay on MT-4 cells for detection of antiviral compounds against the AIDS virus.

Human immunodeficiency virus (HIV) infection of MT-4 cells, an HTLV-I-transformed T-cell line, proved to be a rapid and sensitive assay system for the detection of potential antiviral drugs effective against the acquired immune deficiency syndrome (AIDS). Four days after HIV inoculation of the MT-4 cells, viral antigen expression was monitored in parallel with indirect immunofluorescence microscopy and laser flow cytofluorography. When 3'-azido-2',3'-dideoxythymidine (AzddThd, AZT) and 2',3'-dideoxycytidine (ddCyd) were evaluated under these conditions, they inhibited viral antigen expression at a minimum (33% inhibitory) concentration of 0.0004 and 0.02 microM, respectively. Similar minimum effective concentrations were found for AzddThd and ddCyd in assays where inhibition of viral cytopathogenicity was based on cell survival. While laser flow cytofluorography could be best adapted for quantitative measurements, cell survival and reconstitution of disrupted cell aggregates gave an equally rapid and sensitive endpoint; and the latter may be ideally suited for preliminary drug screening.

Influence of conversion of penicillin G into a basic derivative on its accumulation and subcellular localization in cultured macrophages.

beta-Lactam antibiotics do not accumulate in phagocytes, probably because of their acidic character. We therefore synthesized a basic derivative of penicillin G, namely, 14C-labeled N-(3-dimethylamino-propyl)benzylpenicillinamide (ABP), and studied its uptake and subcellular localization in J774 macrophages compared with that of 14C-labeled penicillin G. Whereas the intracellular concentration (Ci) of penicillin G remained lower than its extracellular concentration (Ce), ABP reached a Ci/Ce ratio of 4 to 5. Moreover, approximately 50% of intracellular ABP was found associated with lysosomes after isopycnic centrifugation of cell homogenates in isoosmotic Percoll or hyperosmotic sucrose gradients. The behavior of ABP was thus partly consistent with the model of de Duve et al. (C. de Duve, T. de Barsy, B. Poole, A. Trovet, P. Tulkens, and A. Van Hoof, Biochem. Pharmacol. 23:2495-2531, 1974), in which they described the intralysosomal accumulation of weak organic bases in lysosomes. Although ABP is microbiologically inactive, our results show that beta-lactam antibiotics can be driven into cells by appropriate modification. Further efforts therefore may be warranted in the design of active compounds or prodrugs that may prove useful in the chemotherapy of intracellular infections.

Identification of novel erythromycin derivatives in mother liquor concentrates of Streptomyces erythraeus.

The identification of five novel compounds, pseudo-erythromycin A-6,9-hemiketal, 8,9-anhydro-pseudo-erythromycin A-6,9-hemiketal, 8,9-anhydro-pseudo-N-demethylerythromycin A-6,9-hemiketal, 5-O-beta-D-desosaminylerythronolide A and 15-nor-erythromycin C, in mother liquor concentrates of Streptomyces erythraeus is described. The pseudo-erythromycin derivatives are characterized by a 12-membered macrocyclic ring as a result of C13----C11 trans-lactonization. The five compounds have very little antimicrobial activity.

Determination of the composition of gentamicin sulfates by 1H and 13C nuclear magnetic spectroscopy.

The British Pharmacopoeia test controlling the composition of gentamicin sulfate is based on CW 60 MHz magnetic resonance spectroscopy. Application of this method to FT 90 MHz spectra was evaluated. Results clearly show the limitations of this technique and point out the need for more reliable assay methods. Thus a 13C nuclear magnetic resonance (NMR) procedure for quantitative analysis of gentamicin sulfate was developed. Ratios of 4 gentamicin components (C1, C2, C1a, and C2a) were obtained from peak height measurements of selected resonance signals in spectra recorded under steady-state conditions. Relative response factors were determined from spectra of a reference mixture or, alternatively, from spectra of the individual pure components. Results obtained by the 13C NMR method were in agreement with those obtained by liquid chromatography using pre-column derivatization.

Synthesis of delta 3-1-methylene-1-carbacephems.

The total synthesis of (+/-)-1-methylene-2,2- dimethyl-7-amino-1-carbacephem-4-carboxylic acid (1) is described. The reaction scheme was essentially that described by Christensen et al. for the synthesis of (+/-)-1-carbacephems. In vitro antibacterial activities of the 7-phenoxyacetyl and 7-D-alpha-phenylglycyl derivatives of 1 were compared with those of 7-(phenoxyacetamido)desacetoxycephalosporanic acid and cefalexin. Derivatives of 1 were 2-4 times less active against most of the sensitive organisms than the corresponding 7-aminodesacetoxycephalosporanic acid analogues. The activity of the 7-D-alpha-phenylglycyl derivative of 1 however was about 20 times lower than that of cefalexin when measured against Staphylococcus aureus ATCC 6538P.

Antibacterial activities of erythromycins A, B, C, and D and some of their derivatives.

The MICs of erythromycins A, B, C, and D and some of their derivatives were determined against 21 gram-positive and 15 gram-negative microorganisms. Antibacterial activity was confined to gram-positive and very few gram-negative bacteria. Erythromycin B was somewhat less active than erythromycin A, and erythromycin C and D showed about half that activity or even less. Most other derivatives had negligible activity. Determination of potency by diffusion and turbidimetric assays were in line with MICs. The examination of the results of these assays, however, revealed that there are differences between the data of different laboratories, depending on the microorganisms and conditions used.