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A series of brusatol, bisbrusatol, and bruceantin esters were examined for their ability to inhibit protein synthesis in P-388 lymphocytic leukemia cells. Compounds which produced high T/C % values (170-272) resulted in ID50 of 5.4-15.5 microM for inhibition of whole cell protein synthesis, ID50 of 1.3-13 microM for inhibition of endogenous protein synthesis in cell homogenates, and ID50 of 1.9-6 microM for inhibition of polyuridine directed polyphenylalanine synthesis using "runoff" ribosomes and a "pH 5" enzyme preparation. The polyuridine directed polyphenylalanine synthesis requires neither initiation nor termination factors, suggesting that quassinoids are exclusively elongation inhibitors. Bruceantin, brusatol, and bisbrusatolyl malonate allowed a runoff of the polyribosomes to 80S free ribosomes. However, formation of the ternary complex and 80S initiation complex were not inhibited by the quassinoids. Thus, these agents do not affect the individual steps leading to the formation of a stable 80S initiation complex in P-388 cells. Brusatol, bruceantin, and bisbrusatolyl malonate inhibited the formation of the first peptide bond between puromycin and [3H]methionyl-transfer RNA bound to the initiation complex, indicating peptidyl transferase activity is inhibited by the quassinoids in P-388 cells. These studies also suggest that the free 80S ribosome is the site of binding by the quassinoid. Ribosomes actively conducting protein synthesis will continue protein synthesis and terminate before the quassinoids bind. This proves quassinoids are elongation inhibitors of tumor cells. A strong correlation was observed between potent antileukemic activity and the ability to inhibit protein synthesis in P-388 lymphocytic leukemia cells.

The quassinoids bruceantin, brucein D, brucein E, bruceoside A, and brusatol significantly inhibited P-388 lymphocytic leukemic cell RNA and protein synthesis in tissue culture. However, DNA synthesis inhibition seemed to correlate more directly with the anti-neoplastic activity of these compounds in the in vivo P-338 survival system. In vitro, brusatol and bruceoside A marginally inhibited 10-day P-388 lymphocytic leukemia DNA polymerase, RNA polymerase, thymidylate synthetase, dihydrofolate reductase, phosphoribosyl pyrophosphate aminotransferase, and cathepsin protease activities. In vivo studies demonstrated similar inhibition and elevated cyclic AMP levels, correlating positively with the antineoplastic activity of individual compounds. Purine synthesis was inhibited drastically by brusatol in vivo, and one key inhibition site in purine synthesis was at phosphoribosyl pyrophosphate aminotransferase, the regulatory enzyme. Histone phosphorylation and ribonucleotide reductase activity also were inhibited marginally by brusatol.

Bisbrusatolyl malonate, which was shown previously to be active against P-388 lymphocytic leukemia cell growth, was investigated for inhibitory effects on nucleic acid and protein synthesis. DNA and RNA synthesis as well as protein synthesis were markedly inhibited at 10,25, and 50 mu mole final concentrations in vitro. The major sites of inhibition of nucleic acid synthesis appeared to be DNA polymerase, messenger and transfer RNA polymerases, orotidine-5'-monophosphate decarboxylase, phosphoribosyl pyrophosphate amino transferase, and dihydrofolate reductase. Moderate inhibition of nucleotide kinase activities and oxidative phosphorylation processes occurred after drug treatment. Cyclic adenosine monophosphate levels were reduced. Protein synthesis was inhibited during the elongation step of peptide synthesis. The data suggested that bisbrusatolyl malonate interfered with the peptide bond formation. However, the ongoing polypeptide synthesis must be completed before the drug can bind to the ribosome effectively.

A series of new bisbrusatolyl and brusatolyl esters and related compounds were synthesized and tested for in vivo antileukemia activity against a quassinoid sensitive strain of P-388 lymphocytic leukemia in BDF1 mice. The bisbrusatolyl malonate, succinate, glutarate, adipate, and sebacate were as active or more active than brusatol. The C-3 esters of brusatol and bruceantin were also found to be as active or more active than brusatol or bruceantin in general. The free hydroxyl groups at C-11 and C-12 as well as the enone double bond in ring A of both bisbrusatolyl and brusatolyl esters are required for antileukemic activity. The presence of a double bond in the ester side chain contributes to the enhanced activity of these esters.

Based on the fact that some known antineoplastic agents possess an ester moiety within their structure, the esters of helenalin, a sesquiterpene lactone, and of brusatol and bruceantin, quassinoids, were synthesized and tested for antileukemic activity in the P-388 screen. These agents gave different T/C % values dependent on the P-388 lymphocytic leukemia strain and the host strain of mice used. Later studies demonstrated that the agents caused different degrees of inhibition of nucleic acid and protein synthesis in the various P-388 strains. The higher the degree of inhibition of precursor incorporation into the nucleic acid or protein, the higher was the T/C % value obtained in a given P-388 strain. The study demonstrates the lack of consistency of P-388 lymphocytic leukemia cell lines used in various laboratories and indicates that the inbred strain of mice is a critical factor in the tolerance of drug toxicity and, thus, T/C % obtained.

A series of esters of brusatol, bisbrusatol, and bruceantin were shown to have potent antileukemic activity. Antineoplastic activity was correlated with the ability of the compounds to suppress DNA and protein synthesis in P-388 lymphocytic leukemia cells. Compounds with high T/C% values successfully inhibited DNA polymerase activity and purine synthesis. The ability to inhibit protein synthesis during the elongation process also correlated positively with high antileukemic activity in this series of quassinoids. Dihydrofolate reductase activity and basal and adenosine diphosphate stimulated respiration of P-388 cells were also inhibited.

The quassinoids (brusatol, bruceantin, bisbrusatolyl esters, and bisbruceantinyl esters of succinic and malonic acids) were observed not to be universal protein synthesis inhibitors. Rather, they were selective for both the types of cancers, e.g., P-388 lymphocytic leukemia, Ehrlich and hepatoma carcinoma and L-1210 lymphoid leukemia, as well as types of normal tissues (e.g., lymphocytes), in which they demonstrated protein synthesis inhibition. The data suggest that the observed difference in the magnitude of protein synthesis inhibition of two P-388 lymphocytic leukemia cell lines by the quassinoids was at the ribosomal levels, whereas the observed difference in normal livers from various strains of mice involve differences in cell membrane transport of the quassinoids into the various tissues. Detailed studies indicated that the mode of action of the quassinoids as protein synthesis inhibitors was identical in all of the cells where inhibition was observed; i.e., the elongation step of protein synthesis was blocked by the quassinoids. The data derived from assays for polyuridine-directed polyphenylalanine synthesis of isolated ribosomes demonstrated that the ID50 values obtained were consistent with the observed inhibition of whole cell protein synthesis inhibition for P-388 cells, as well as for BDF1 and DBA/2 liver cells. The ID50 values obtained from various cells, e.g., P-388 cells and normal liver, were all in the microM range and are consistent with previously published values for the quassinoids in the rabbit reticulocyte and yeast systems.

Brusatol, a quassinoid with potent antineoplastic activity against P-388 lymphocytic leukemia cell proliferation, significantly inhibited P-388 cell hexokinase, phosphofructokinase, malic dehydrogenase, and succinic dehydrogenase. Mitochondrial oxidative phosphorylation, basal, and adenosine diphosphate-stimulated respiration, utilizing succinate and alpha-ketoglutarate as the substrate, was suppressed significantly by in vivo treatment with brusatol. However, brusatol treatment had no effect on liver oxidative phosphorylation. Brusatol greatly increased P-388 cyclic AMP levels but had no effect on liver cyclic nucleotides. Similar inhibitory effects on P-388 cell oxidative phosphorylation were found in vitro with brusatol, bruceoside A, and bruceantin. Brusatol had no effect on adenosine triphosphatase activity or on uncoupling of oxidative phosphorylation. Rather, brusatol appeared to increase the concentration of reduced mitochondrial electron-transport cofactors, thereby blocking aerobic respiration. A proposed mechanism of action is discussed.

A series of quassinoids were observed to be potent inhibitors of induced inflammation and arthritis in rodents. Brusatol afforded the most potent activity followed by brucein-D. A 3-hydroxy-delta 3-2-oxo moiety in brusatol or a 1-hydroxy-delta 3-2-oxo moiety in brucein-D, as well as a C-15 ester-bearing delta-lactone ring in brusatol and C-11 and C-12 free hydroxyl groups are required in both quassinoids for potent anti-inflammatory activity. Preliminary studies indicate that one of the modes of action of quassinoids as anti-inflammatory agents is to stabilize lysosomal membranes, reducing the release of hydrolytic enzymes that cause damage to surrounding tissues.