3,5-Bis-benzylidene-4-piperidones and related compounds with high activity towards P388 leukemia cells.

3,5-Bis(benzylidene)-4-piperidones and related compounds were prepared and found to have between 100 and 9700 times the activity of N,N'-bis(2-chloroethyl)-N-nitrosourea towards P388 leukemia cells. The shapes of six of these molecules--determined by X-ray crystallography--were compared, but no correlation between the stereochemistry of the molecules or their electronic properties and cytotoxicity was apparent. Molecular modification of the compounds by forming two mono-benzylidene compounds, a related acyclic derivative and an N-acyl compound resulted in diminished but retained high cytotoxicity. Two representative compounds lowered glutathione levels of liver following their intraperitoneal injection into mice. Two quaternary ammonium compounds were shown to bind in the minor groove of DNA, while four related non-quaternary ammonium derivatives did not demonstrate this property. We conclude that the modes of action of these highly cytotoxic compounds include alkylation of cellular thiols and DNA binding, but interference with other biochemical processes is also probably involved.

Effect of some 4-alkyl-5-dimethylamino-1-phenyl-1-penten-3-one hydrochlorides and related compounds on respiration in mouse liver mitochondria.

5-Dimethylamino-1-phenyl-1-penten-3-one hydrochloride (1a) has high inhibiting properties in mitochondria isolated from mice liver. Substitution at the 4-methylene group of 1a by different alkyl substituents lead to compounds with wide variation in respiration-inhibiting properties. No correlations were found between either the Charton steric parameter (v), Taft inductive value (f*) or fragmental constant (f) of the substituents at position 4 of the Mannich bases with inhibition of respiration. However the biological results suggested two receptor sites were present in the mitochondria which may interact with the Mannich bases namely a common receptor for all compounds and in addition a narrow hydrophobic binding area which can accommodate a n-butyl or higher alkyl groups which are present in some of the compounds.

bis-Mannich bases of styryl ketones as antileukemic agents.

A novel series of bis-Mannich bases have been synthesized and evaluated against P388 lymphocytic leukemia in mice. Two compounds showed a perceptible beneficial response in this screen and all the compounds displayed marked murine toxicity. A representative compound inhibited respiration in mitochondria isolated from rat and mouse liver cells by 90% approximately at a dose of 2.5 mumol and it caused a small elevation in mouse liver glutathione equivalent concentrations at 5 mg/kg.

Evaluation of some Mannich bases derived from substituted acetophenones against P-388 lymphocytic leukemia and on respiration in isolated rat liver mitochondria.

Series of 3-dimethylamino-1-aryl-1-propanone hydrobromides (IV) and 3-dimethylamino-2-dimethylaminomethyl-1-aryl-1-propanone dihydrobromides (V) were synthesized. Evaluation of these derivatives against P-388 lymphocytic leukemia growth revealed that two compounds show promise as antineoplastic agents. Compounds of the V series were unstable in phosphate buffer (in contrast to series IV), and when the same nuclear substituent was present in both series of compounds, V was approximately 100 times more active than IV in both the stimulation and inhibition of respiration of mitochondria isolated from rat liver cells. Representatives from both series showed that respiration in mitochondria was affected by changing the pH of the aqueous buffer from 7.4 to 6.9 or 6.4 and by reducing the temperature from 37 degrees to 20 degrees. The compounds showed reactivity toward a biomimetic thiol.

Effect of 4-dimethylaminomethyl-1-(3-hydroxyphenyl)-1-nonen-3-one hydrochloride and related compounds on respiration in rat liver mitochondria.

4-Dimethylaminomethyl-1-(3-hydroxyphenyl)-1-nonen 3-one hydrochloride (II) was shown to stimulate respiration in rat liver mitochondria at levels of 2.5 mumoles or less; but at levels higher than 5.0 mumoles, respiration was inhibited when succinate and 3-hydroxybutyrate were the substrates. Compound II caused inhibition of respiration in the presence of glutamate over the dose range studied. The stimulating effect of II was attributed to its functioning as an uncoupling agent. Its inhibiting properties were considered to be due to its behaving like antimycin A in blocking transport of electrons between cytochromes b and c1. The effect of II on respiration in mitochondria varied with the pH of the medium. A conjugated styryl ketone, which contained a nuclear hydroxy function and was structurally related to II, also stimulated respiration at low doses while inhibiting respiration at higher concentrations. Etherification of the hydroxy group led to compounds in which only stimulation of respiration was noted.

Nuclear-substituted styryl ketone analogs: effects on neoplasms, microorganisms, and mitochondrial respiration of tumorous and normal cells.

Analogs of some antineoplastic and cytotoxic Mannich bases derived from conjugated styryl ketones were prepared and evaluated for activity in the P-388 lymphocytic leukemia screen. Most of the new compounds had lower antineoplastic and murine toxicity than the parent compounds. Antimicrobial evaluation of some oximes and alcohols related to the Mannich bases revealed activity against certain Gram-positive bacteria and fungi. Primary pharmacological evaluation showed that some compounds containing a dimethylaminomethyl group displayed analgesic and antihistaminic properties. Five of the Mannich bases were evaluated as respiratory inhibitors in mitochondria derived from hepatic tumors, liver tissue from tumor-bearing animals, and normal rat liver. No statistical difference between the sensitivity of the three tissues to the compounds was obtained.

Effect of antineoplastic and cytotoxic Mannich Bases derived from conjugated styryl ketones on mitochondrial respiration in rat liver cells.

Five cytotoxic Mannich bases (5-dimethylamino-1-substituted phenyl-1-penten-3-ones), three having antineoplastic activity, were evaluated for respiratory-inhibiting properties in rat liver mitochondria in the presence of four substrates: succinate, glutamate, 3-hydroxybutyrate, and palmitylcarnitine. Four compounds (Ib--Ie) showed significant inhibiting properties which, on occasion, were reversed partially by coenzyme Q10. Evaluation of the spectra of the mitochondrial cytochromes indicated that Ib--Ie blocked the electron transport chain prior to the sequence of cytochromes. Since inhibition occurred when different substrates were used, a common site of action for Ib--Ie is likely; competition of Ib--Ie with coenzyme Q10 probably occurs. Compounds Ia--Ie inhibited RNA polymerase from Swiss mouse kidney cells but were virtually bereft of activity versus RNA polymerase from L-1210 leukemia cells. Polarography of the Mannich bases and the related styryl ketones showed that antineoplastic activity was associated with higher half-wave potentials.

Effect of diphenhydramine on methaqualone metabolism: an in vitro study.

A GLC assay to quantitate the methaqualone metabolite 2-methyl-3-(2-hydroxymethylphenyl)-4(3H)-quinazolinone was developed. Standard curves were linear, and recovery of the metabolite from tissue homogenates averaged 89%. In vitro metabolism of methaqualone by the 10,000Xg supernatant fraction of rat liver homogenate was measured by monitoring metabolite formation with the GLC assay. Diphenhydramine inhibited the in vitro metabolism of methaqualone. The percentage inhibition increased with increasing diphenhydramine concentration. The significance of this inhibition in relation to use and abuse of methaqualone--diphenhydramine combinations is discussed.

Simultaneous identification and quantitation of diphenhydramine and methaqualone.

Methods for the simulataneous identification and quantitation of diphenhydramine and methaqualone in urine have been investigated. Suitable thin-layer and gas chromatographic techniques have been developed. The extraction techniques were evaluated by extracting human urine after the ingestion of therapeutic dosages of the two drugs. A charcoal cartridge technique proved to be superior to direct solvent extraction. Although enzyme hydrolysis gave slightly better recoveries than the charcoal cartridge technique, it is a time-consuming procedure.

Evaluation of Mannich bases and related compounds as inhibitors of mitochondrial function in yeast and inhibition of blood platelet aggregation, blood clotting, and in vitro metabolism of 5-dimethylamino-1-phenyl-1-penten-3-one hydrochloride.

Dimethylamino-1-phenyl-1-penten-3-one hydrochloride (Ia) and 32 analogs were tested for inhibition of respiratory-dependent growth in Saccharomyces cerevisiae. Thirteen of the 33 compounds tested appeared to affect mitochondrial function, since the inhibition of respiratory-dependent growth was statistically greater than the inhibition of growth on fermentable energy sources. Inhibition of mitochondrial function in yeast and growth inhibition of an in vitro culture of human epidermoid carcinoma (KB) were positively correlated since 83% of the compounds tested either had mitochondrial-inhibiting properties and significant activity in the KB test or were inactive in both tests. Similarly, 78% of compounds tested showed murine toxicity and mitochondrial inhibition or had no effect on murine toxicity and yeast mitochondrial function. Injection of Ia into rats resulted in the appearance of blood in the urine and feces. Compound Ia inhibited adenosine diphosphate and collagen-induced aggregation of rat platelets but had no effect on blood clotting. TLC, following incubation of Ia with a rat liver extract, showed that the structure of Ia was not enzymatically modified and indicated activity per se on platelet aggregation and mitochondrial function.

Evaluation of 2-benzylidenecyclohexanones and 2,6-bis(benzylidene)cyclohexanones for antitumor and cytotoxic activity and as inhibitors of mitochondrial function in yeast: metabolism studies of (E)-2-benzylidenecyclohexanone.

Some 2-benzylidenecyclohexanones, 2,6-bis(benzylidene)cyclohexanones, and related compounds were evaluated for antitumor and cytotoxic activities; (E)-2-benzylidenecyclohexanone (Ia) was shown to have significant cytotoxic properties and a potent inhibitory effect on yeast mitochondria. After intraperitoneal injection of Ia, unchanged drug and a metabolite, tentatively identified as 2-(p-hydroxybenzyl)cyclohexanol, were found in the urine. No metabolites were found in the feces. Oral administration of Ia afforded three unidentified metabolites in the urine and three unidentified metabolites in the feces.