Antiviral properties of 3-quinolinecarboxamides: a series of novel non-nucleoside antiherpetic agents.

Novel antiherpetic 3-quinolinecarboxamides were discovered as part of a drug discovery program at Sterling Winthrop Inc. A major goal of this research was to identify novel non-nucleoside agents possessing activity against acyclovir resistant herpes simplex virus. From screening compound libraries in an HSV-2 plaque reduction assay, 1-ethyl-1,4-dihydro-4-oxo-7-(4-pyridinyl)-3-quinolinecarboxamide (1) emerged as an attractive lead structure. By modifying the quinoline ring at the 1-, 2-, 3-, 4-, and 7-positions, analogues were identified that have up to 5-fold increased in vitro potency relative to acyclovir. In a single dose mouse model of infection the 1-(4-FC6H4) analogue 17, one of the most potent derivatives in vitro, displayed comparable oral antiherpetic efficacy to acyclovir at 1/16 the dose; in a multiple dose regimen, however, it was 2-fold less potent. Mechanism of action studies indicate that these new compounds interact with a different, as yet undefined, molecular target than acyclovir.

Indication of an involvement of interleukin-1 beta converting enzyme-like protease in intramedullary apoptotic cell death in the bone marrow of patients with myelodysplastic syndromes.

Our previous studies using in situ end labeling (ISEL) of fragmented DNA revealed extensive apoptotic cell death in the bone marrows (BM) of patients with myelodysplastic syndromes (MDS) involving both stromal and hematopoietic cells. In the present report we show greater synthesis of interleukin-1 beta (IL-1 beta) in 4 hour cultures of density separated BM aspirate mononuclear (BMAM) cells from MDS patients as compared to the cultures of normal BM from healthy donors or lymphoma patients (1.7 +/- 0.37 pg/10(5) cells, n = 29 v 0.42 +/- 0.24 pg/10(5) cells, n = 11, respectively, P = .049). Further, these amounts of IL-1 beta in MDS showed a significant correlation with the extent of apoptosis detected by ISEL in corresponding plastic embedded BM biopsies (r = .480, n = 30, P = .007). In contrast normal BMs did not show any correlation between the two (r = .091, n = 12, P = .779). No significant correlation was found between the amounts of IL-1 beta and % S-phase cells (labeling index; LI%) in MDS determined in BM biopsies using immunohistochemistry following in vivo infusions of iodo- and/or bromodeoxyuridine. Neither anti-IL-1 beta antibody nor IL-1 receptor antagonist blocked the apoptotic death of BMAM cells in 4 hour cultures (n = 5) determined by ISEL (apoptotic index; AI%), although the latter led to a dose-dependent accumulation of active IL-1 beta in the culture supernatants. On the other hand, a specific tetrapetide-aldehyde inhibitor of ICE significantly retarded the apoptotic death of BMAM cells at 1 mumol/L in 5/6 MDS cases studied (AI% = 2.99 +/- 0.30 in controls v 1.58 +/- 0.40 with ICE-inhibitor, P = .05) and also reduced the levels of active IL-1 beta synthesized (5.59 +/- 2.63 v 2.24 +/- 0.93 pg/10(6) cells, respectively). In normal cells, neither IL-1 blockers nor the ICE inhibitor showed any effect on the marginal increase in apoptosis observed in 4 hour cultures. Our data thus suggest a possible involvement of an ICE-like protease in the intramedullary apoptotic cell death in the BMs of MDS patients.

A column-switching high-performance liquid chromatographic assay for a novel cytotoxic thioxanthone derivative (WIN 33377) in mouse plasma with toxicokinetic results from a mouse LD10 study.

WIN 33377 (I) is a member of a novel class of cytotoxic antitumor agents, 4-aminomethyl thioxanthone derivatives. A simple, rapid and reproducible method has been developed for the assay of I in mouse plasma using a high-performance liquid chromatographic method utilizing a column-switching technique. The method involves direct injection of buffered plasma to the extraction column for sample clean-up followed by switching onto an analytical column for analysis with UV detection at 256 nm. The method has demonstrated accuracy and precision over the range 10-2500 ng/ml using a 100-microliters plasma sample with a minimum quantifiable level at 10 ng/ml. Stability of mouse plasma samples was demonstrated after storage for 4 weeks at -15 to -20 degrees C, as was the ability of samples to be accurately quantified after a maximum of three freeze-thaw cycles. Recovery was greater than 87% for the compound and the internal standard. The assay was accurate and reproducible with measured values lying within the limits of defined acceptance criteria. The utility of the method was demonstrated by analyzing plasma samples obtained from mice dosed with I as part of a pre-clinical safety study intended to assist in the design of a pharmacokinetically guided dose escalation strategy.

Cyclic variations of 3-quinolinecarboxamides and effects on antiherpetic activity.

Supported by the antiherpetic properties of 3-quinolinecarboxamides and the importance of the planar intramolecular H-bonded beta-keto amide pharmacophore, a series of novel conformationally rigid analogues that contain a heterocyclic bridge between the 3- and 4-positions of the quinoline ring have been evaluated. Two isoxazolo-fused derivatives 17 and 23 displayed good in vitro antiherpetic potency that was similar to that of 1, the 3-quinolinecarboxamide that served as the comparison structure for this study. The pyrazolo, pyrrolo, and pyrimido derivatives showed considerably less or no activity. In vitro activity did not translate to in vivo efficacy. For 17, the lack of in vivo activity is likely a consequence of insufficient plasma drug levels (both Cmax and duration) in mice relative to the MIC versus HSV-2.

Antinociceptive (aminoalkyl)indoles.

The (aminoalkyl)indole (AAI) derivative pravadoline (1a) inhibited prostaglandin (PG) synthesis in mouse brain microsomes in vitro and ex vivo and exhibited antinociceptive activity in several rodent assays. In vitro structure-activity relationship studies of this new class of PG synthesis inhibitors revealed a correspondence in three respects to those reported for the arylacetic acids: (1) "alpha-methylation" caused an increase in PG inhibitory potency, (2) the (R)-alpha-methyl isomer was more active than the S isomer, (3) the hypothesized aroyl group conformation of the 2-methyl derivatives corresponded to the proposed and reported "active" conformations of the aroyl and related aromatic acetic acid derivatives. The 1H NMR chemical shift of the C-4 hydrogen of pravadoline in comparison to the deshielding seen with 50, which lacks a substituent at C-2, suggested that the carbonyl group of pravadoline is located near C-2 but is located near C-4 in 50. Associated with this conformational change of the carbonyl group of 1a is a diminution of PG synthetase inhibitory activity. The results of UV and difference nuclear Overhauser studies of the two compounds were consistent with these conformational assignments. The low eudismic ratios of the alpha-methyl derivatives and the observation that the side chain may be extended by three methylene groups without significant loss of PG inhibitory potency suggests that this class of inhibitors bound less strongly and less selectively to the active site of PG synthetase than do the arylacetic acids. Two AAIs, 1a and 30, were found to be metabolized to the corresponding acetic acid derivatives, both of which inhibited PG synthesis. An exception to the observation that the antinociceptive activity of the AAIs was associated with PG synthetase inhibitory activity was the 1-naphthoyl derivative 67 since neither it nor its acetic acid metabolite 74 inhibited PG synthesis. Yet 67 was antinociceptive in four different rodent assays. This naphthoyl derivative, like opioids, also inhibited electrically stimulated contractions in the mouse vas deferens (MVD) preparation. Unlike opioids, however, the inhibition was not antagonized by naloxone. A subseries of AAIs was identified, of which 67 was prototypic. These compounds lacked PG synthetase inhibitory activity, but their inhibitory potency in MVD preparations correlated roughly with their antinociceptive potency in vivo. Pravadoline was also inhibitory in the MVD. Its antinociceptive activity, therefore, may be a consequence of both its PG synthetase inhibitory potency and another antinociceptive mechanism, the latter associated with its inhibitory potency in the MVD.(ABSTRACT TRUNCATED AT 400 WORDS)

Exposure to carcinogenic chemicals and smoking increases urinary excretion of mutagens in humans.

Urine samples from a control population and from a population of chemical workers from two chemical plants near Florence, Italy, were analyzed for the presence of mutagenic chemicals by the Salmonella/microsome test. When tested with strain TA1538, the urine of nonsmoking chemical workers showed higher mutagenic activity than that of controls in the presence of in vitro metabolic activation, but no difference was found between controls and chemical workers who both smoked. Increased mutagenic activity was observed in the group of control smokers compared to control nonsmokers, but the same effect was not observed for chemical workers. When TA100 was used as the tester strain, the chemical workers, both smoking and nonsmoking, had significantly higher mutagenic activity than controls. The mutagenic activity fell to control levels in some workers' urine after 20 d leave. Although some perturbing effects of smoking habits were observed, the results seemed to indicate the usefulness of the Salmonella/microsome test for detection of mutagens in human urine. The results also suggest that people exposed to potentially carcinogenic chemicals may show high enough traces of those chemicals and/or their metabolites in their body fluids to be detected with current mutagenesis techniques.

5-fluorouracil, adriamycin, and mitomycin-C (Hi-FAM) chemotherapy for adenocarcinoma of the lung.

Thirty patients with unresectable adenocarcinoma of the lung were treated with high doses of 5-fluorouracil, Adriamycin, and mitomycin-C (Hi-FAM). Objective responses were seen in ten patients (one complete and nine partial remissions). No patient with pleural disease responded to treatment. Responses were seen in all other sites of involvement including liver. In a subgroup of patients younger than 65 years, who had not had prior treatment, and who had a performance status of greater than 60 (Karnofsky), an overall response rate of 50% was realized. The overall median survival for responding patients was 10+ months while nonresponders had a median survival of 5.21 months. Patients who had had prior irradiation had a median survival of 4.81 months compared with patients who had not had any prior treatment, whose median survival was 8.45 months. Toxicity was substantial and included primarily bone marrow suppression and stomatitis. Elderly patients with poor performance status and prior treatment tolerated therapy less well. These results indicate that Hi-FAM is useful in selected groups of patients with advanced adenocrcinoma of the lung.

Isolation, synthesis, and biological activity of five metabolites of danazol.

Metabolites of danazol (17 alpha-pregna-2,4-dien-20-yno[2,3-d]isoxazol-17-ol), an orally effective pituitary gonadotropin inhibitory agent devoid of estrogenic and progestational activites, were isolated from urine of a female subject who had taken danzol orally at a dose of 800 mg/day for 7 days, The metabolites isolated were 17-hydroxy-17alpha-pregn-4-en-20-yn-3-one (11), 17-hydroxy-2alpha-(hydroxymethyl)-17alpha-pregn-4-3n-20-yn-3-one (5), 17-hydroxy-2-(hydroxymethyl)-17alpha-pregna-1,4-dien-20-yn-3-one(7), 6beta,17-dihydroxy-2alpha-(hydroxymethyl)-17alphapregn-4-en-20-yn-3-one(8), and 6beta, 17-dihydroxy-2(hydroxymethyl)-17alphapregna-1,4-dien-20yn-3-one(10). None of these metabolites exhibited pituitary inhibiting activity comparable to danazol.

PIP: Urinary metabolites of danazol (17alpha-pregna-2,4-dien-20-yno (2,3-delta)isoxazol-17-01), and inhibitor of pituitary gonadotropins, were isolated from a woman who had been taking the drug orally at a dose of 800 mg/day for 7 days. The isolated metabolites were 17-hydroxy-17alpha-pregn-4-en-20-yn-3-one, 17-hydroxy-2alpha-(hydroxymethyl)-17alpha-pregn-4-en-20-yn-3-one, 47-hydroxy-2-(hydroxymethyl)-17alpha-pregna-1,4-dien-20-yn-3-one, 6beta,17-dihydroxy-2alpha-(hydroxymethyl)-17alpha-pregn-4-en-20-yn-3-one and 6beta,17-dihydroxy-2-(hydroxymethyl)-17alpha-pregna-1,4-dien-20-yn-3-one. The pituitary inhibiting activity of these metabolites was less than that of danazol.

Mutational biosynthesis by idiotrophs of Micromonospora purpurea. I. Conversion of aminocyclitols to new aminoglycoside antibiotics.

By mutation and strain improvement techniques idiotrophs of Micromonospora purpurea, the gentamicin-producing organism, were obtained which require an exogenous source of 2-deoxystreptamine in order to produce gentamicin. Streptamine incorporation afforded a mixture of 2-hydroxygentamicin C as a complex of essentially the C1 and C2 components whereas 2-deoxystreptamine when incorporated by the same idiotroph afforded the same mixture of C1, C2 and C1a gentamicins as the parent (m1) organism. The 2-hydroxygentamicin C complex exhibited broad-spectrum antibiotic activity with an in vitro potency less than that for the gentamicin C complex, but with greater activity against selected gentamicin C resistant organisms. The LD 50 (i.v.) in mice of the 2-hydroxygentamicin C complex indicated that it had approximately half the toxicity of the gentamicin C complex. 2, 5-Dideoxystreptamine affordeda C1, C2, and C1a mixture of 5-deoxygentamicins, which also had broad spectrum activity, and exhibited improved activity against several gentamicin-acetylating strains of resistant bacteria. The LD50 (i.v.) in mice of the 5-deoxygentamicin C complex indicated that it was about 2.5 times more toxic than the gentamicin C complex. Two derivatives of 2,5-dideoxystreptamine afforded the same mixture of 5-deoxygentamicins. 2-Epistreptamine upon supplementation to a broth containing growing cultures of these idiotrophs also produced antibiotic.

Mutational biosynthesis by idiotrophs of Micromonospora purpurea. II. Conversion of non-amino containing cyclitols to aminoglycoside antibiotics.

A mutant of Micromonospora purpurea, which produces the gentamicin complex only when 2-deoxystreptamine is added to the fermentation medium, produces a new antibiotic complex, 2-hydroxygentamicin, when streptamine or 2,4,6/3,5-pentahydroxycyclohexanone is added to the fermentation medium. This mutant also produces the gentamicin complex when 2,4/3,5-tetrahydroxycyclohexanone is added to the fermentation medium. The C1 and C2 components of 2-hydroxygentamicin have broad spectrum in vitro antibacterial activity similar to the gentamicin C1 and C2 components, but with greater activity against some gentamicin-resistant strains.

Microbiological metabolism of naphthyridines.

Penicillium adametzi and seven other species convert nalidixic acid, 1,4-dihydro-1-ethyl-7-methyl-4-oxo-1,8-naphthyridine-3-carboxylic acid, to 1,4-dihydro-1-ethyl-7-hydroxymethyl-4-oxo-1,8-naphthyridine-3-carboxylic acid. Forty-seven other species from six orders of fungi seem to achieve the same conversion as judged by chromatographic and spectral evidence. Under special conditions, P. adametzi also produces a second metabolite which was identified as the corresponding 7-carboxylic acid. The metabolic attack on the ring substituent is identical with the pathway previously established with humans. No evidence was obtained for metabolic attack on the naphthyridine nucleus itself.