Synthesis of alkoxy-substituted diaryl compounds and correlation of ring separation with inhibition of tubulin polymerization: differential enhancement of inhibitory effects under suboptimal polymerization reaction conditions.

A number of cytostatic compounds (2-4, 7, and 8), which can be described as "diaryl", inhibit tubulin polymerization, cause cells to accumulate in mitotic arrest, and competitively inhibit the binding of colchicine to tubulin. They differ, however, in the separation of the two aryl moieties. To attempt to understand this variability we prepared a series of analogues modeled on 3 and 4 ("benzodioxole series") and on 7 and 8 ("combretastatin series") which differed only in the number of methylene units (ranging from none to four) separating the aryl moieties. These compounds were evaluated for their effects on tubulin polymerization, colchicine binding, and the growth of L1210 murine leukemia cells. In terms of inhibitory effects on tubulin polymerization, for the combretastatin series there was an optimal separation of the two phenyl rings by a two-carbon bridge (compound 24), with progressively decreasing inhibitory activity when the separation was by one carbon (20), three carbons (25), or four carbons (28) (the biphenyl analogue 16 was inactive). The benzodioxole series, however, did not permit us to generalize this finding, because the least active agents prepared (39 and 40) had a two-carbon bridge, while those with one- (5 and 6) and three-carbon (46 and 47) bridges were nearly equivalent in potency. Submicromolar IC50 values for inhibition of L1210 cell growth were only obtained for compounds 20 (IC50, 0.2 microM), 24 (0.07 microM), and 25 (0.4 microM). While evaluating the effects of these agents on tubulin polymerization, we noted with the combretastatin series and with several standard agents that apparent potency (in terms of IC50 values) was always lower if the reaction was performed at 30 degrees C, with 0.25 mM MgCl2, than at 37 degrees C, with 1.0 mM MgCl2. This enhancement of IC50 values in the former system as compared with the latter was particularly dramatic for the less active agents (e.g., 28) as compared with the more active (e.g. 24).

Methylenedioxy-benzopyran analogs of podophyllotoxin, a new synthetic class of antimitotic agents that inhibit tubulin polymerization.

A new class of compounds was synthesized and, based on structural analogy to podophyllotoxin, examined as potential microtubule inhibitors and evaluated for in vivo antineoplastic activity. These agents are derivatives of methylenedioxy-benzopyran bearing a phenyl substituent at position 8. The hydrogen atoms at positions 7 and 8 are in a trans configuration, in contrast to the cis configuration of analogous hydrogen atoms at positions 1 and 2 in podophyllotoxin. Compounds with a variety of substituents at positions 6 and 7 were examined, as well as compounds with varying methoxy substituent patterns on the phenyl ring attached at position 8. The most active compounds inhibited tubulin polymerization at concentrations approximately stoichiometric with tubulin, competitively inhibited the binding of colchicine to tubulin, and caused mitotic arrest at cytotoxic drug concentrations. No structure-activity correlations were obvious for the substituents at positions 6 and 7, but optimal activity was only observed when the phenyl substituent at position 8 was a trimethoxybenzene ring identical to the analogous ring in podophyllotoxin (i.e. methoxy groups at positions 3', 4' and 5'). Despite their structural and functional similarities to podophyllotoxin, however, the methylenedioxy-benzopyran derivatives subtly differ from the natural product in their interaction with tubulin, for they stimulated rather than inhibited tubulin-dependent GTP hydrolysis.

In vivo antitumor activity of 6-benzyl-1,3-benzodioxole derivatives against the P388, L1210, B16, and M5076 murine models.

A series of 6-benzyl-1,3-benzodioxoles have been synthesized and evaluated against the in vivo ip P388 murine lymphocytic leukemia. Selected activities against this system were tested against the additional in vivo systems L1210, B16, M5076, and MX1. The most active of the 6-benzyl-1,3-benzodioxoles tested were as effective as podophyllotoxin as experimental antitumor agents in vivo, but larger doses were required. Three of the P388-active series members were active against the in vitro astrocytoma assay, which detects compounds that interfere with or bind to tubulin.

The effects of 6-benzyl-1,3-benzodioxole derivatives on the alkylation of tubulin.

Derivatives of 6-benzyl-1,3-benzodioxole are known to bind to tubulin and inhibit tubulin polymerization. For a better understanding of the mechanism of action of the 6-benzyl-1,3-benzodioxole derivatives, we have examined their effect on the alkylation of tubulin sulfhydryls by iodo[14C]acetamide and N,N'-ethylene(bis)iodoacetamide. We have found that the 6-benzyl-1,3-benzodioxole derivatives with an intact dioxole ring affect alkylation to an extent proportional to their ability to inhibit tubulin polymerization. Those derivatives with the strongest resemblance to podophyllotoxin have the weakest effects. However, derivatives with a disrupted dioxole ring show little or no ability to inhibit polymerization, but their effect on alkylation is directly related to the degree of resemblance they bear to the trimethoxy ring of podophyllotoxin. It thus appears that the relatively simple approach of using alkylating agents can generate a significant amount of information on the mechanism by which various drugs interact with tubulin.

Assessment of six benzyl-1,3-benzodioxole compounds for anti-juvenile hormone activity in Culex pipiens.

Fourth instar larvae of Culex pipiens were exposed to six benzyl-1,3-benzodioxole derivatives to assess the effectiveness of these compounds as anti-juvenile hormone agents. Mortality ranging from between 18 and 99% was observed in larvae and early pupae but the surviving adults showed no clearly defined anti-juvenile hormone effects. Adult effects included a reduction in number of eggs developed and the presence of degenerating eggs 4 days after the blood meal.

Lack of chemical factors in host-plant resistance of alfalfa towards alfalfa weevil: Ovicidal activity of some coumarin derivatives.

A resistant line of alfalfa was studied in an attempt to identify possible chemical factors responsible for alfalfa weevil (Hypera brunneipennis) resistance. Bioassays were developed to measure ovicidal, larval development, pupation, and adult development rates. Serial alfalfa extracts of a weevil-resistant line showed no effect in the bioassays when compared to those of a weevil-susceptible line. It was concluded that resistance substances are not present but that resistant lines are simply more tolerant to weevil attack. Alfalfa constitutents and synthetic analogs were also screened in the egg hatch bioassay. Among these were a series of 3-acyl-4-hydroxycoumarins. 3-Acyl-4-hydroxycoumarins with short-chain acyl groups showed good ovicidal rates, but activity decreased with increasing length of the acyl group.

Feeding deterrency of some 4-hydroxycoumarins and related compounds: Relationship to host-plant resistance of alfalfa towards pea aphid (Acyrthosiphon pisum).

A series of 3-acyl-4-hydroxycoumarins, structurally related to dicoumarol, as well as several alfalfa constituents including coumestrol were tested for their feeding deterrency towards the pea aphid. Feeding deterrency of the 3-acyl-4-hydroxycoumarins decreased as the size of the 3-acyl group increased.

Morpholino derivatives of benzyl-benzodioxole, a study of structural requirements for drug interactions at the colchicine/podophyllotoxin binding site of tubulin.

We performed a structure-activity evaluation of the effects of methoxy substituents in the benzyl moiety of a series of morpholinyl Mannich base derivatives of 6-benzyl-1,3-benzodioxol-5-ol ("morpholino compounds") on the ability of these compounds to inhibit tubulin polymerization in vitro. Structurally these agents are most similar to the natural product podophyllotoxin and, like podophyllotoxin, they inhibited in vitro tubulin polymerization, tubulin-dependent GTP hydrolysis, and the binding of colchicine to tubulin. The benzyl ring (C ring) of these compounds appeared to be analogous to the trimethoxybenzene ring (E ring) of podophyllotoxin (with its methoxy substituents at the 3', 4' and 5' positions), but the morpholino compound superficially most similar to podophyllotoxin (with 3', 4' and 5' methoxy substituents) was the least active in the series. The most potent methoxy-substituted morpholino compounds bear these substituents either at the 2' and 4' positions (NSC 370277) or at the 2', 4' and 6' positions (NSC 381577). NSC 370277 and NSC 381577 were essentially identical in their inhibitory effects on tubulin polymerization, but the latter compound was considerably more effective as an inhibitor of the binding of colchicine to tubulin. The most active of the monomethoxy substituted compounds bore this group at position 4'. A number of compounds with alternative substituents at this position (in particular, alkyl-substituted amines) also had significant in vitro inhibitory effects on tubulin polymerization. Although the morpholino compounds appear to possess only limited cytotoxicity, these findings suggest possible modifications of the antimitotic benzyl-benzodioxole compounds described previously [Batra et al., Molec. Pharmac. 27, 94 (1985)] to enhance their antineoplastic activity.

Structure-function studies with derivatives of 6-benzyl-1,3-benzodioxole, a new class of synthetic compounds which inhibit tubulin polymerization and mitosis.

A new class of synthetic antineoplastic compounds, derivatives of 6-benzyl-1,3-benzodioxole, has significant antimitotic activity. These compounds inhibit microtubule assembly and are competitive inhibitors of the binding of colchicine to tubulin. Both their structure and their partial inhibition of tubulin-dependent GTP hydrolysis indicate that they are most comparable to podophyllotoxin of all known antimitotic drugs. Maximum activity required an intact dioxole ring, a methoxy or ethoxy substituent at position 5, and, on the benzyl moiety at position 6, a para-methoxy group. Additional methoxy groups on the benzyl substituent, to increase the apparent structural similarity to podophyllotoxin, resulted in major reduction of the antitubulin activity of these drugs.

In vivo exposure to plant flavonols. Influence on frequencies of micronuclei in mouse erythrocytes and sister-chromatid exchange in rabbit lymphocytes.

No consistent increases in the micronucleus frequency were observed in bone marrow or peripheral blood erythrocytes from mice treated with quercetin, rhamnetin, neohesperidin dihydrochalcone, or hesperetin dihydrochalcone under various exposure and sampling conditions. Over the dose range of 100-1000 mg/kg, quercetin failed to increase significantly erythrocyte micronucleus frequencies either (1) in bone marrow of male mice at 6 h after the second of 2 i.p. or oral doses given 24 h apart, or at 48, 96 or 192 h after a single i.p. or oral dose, or (2) in peripheral blood of male or female mice sampled for 7 consecutive days following a single i.p. dose. Feeding 5% or 10% quercetin for 8 days also failed to increase the micronucleus frequency in bone marrow erythrocytes of female or male mice. Hesperetin dihydrochalcone and neohesperidin dihydrochalcone, at p.o. doses of 100-1000 mg/kg, did not increase the micronucleus frequency in bone marrow erythrocytes 6 h after the second of 2 doses 24 h apart, nor did rhamnetin at 48 or 96 h after a single i.p. dose of 1000 mg/kg. Galangin, in contrast, did significantly increase the micronucleus frequency in bone marrow and blood erythrocytes under certain conditions, but the largest increases were only between 2 and 3 times control values and these were observed at highly toxic doses. Rabbits given up to 250 mg/kg quercetin i.p. showed no treatment-related increase in the sister-chromatid-exchange frequency in peripheral blood lymphocytes sampled at 1 and 7 days after treatment. These results fail to confirm published data which report a markedly increased frequency of micronuclei in bone marrow erythrocytes from quercetin-treated mice, show no quercetin-related alterations in the sister-chromatid-exchange frequency in rabbit lymphocytes, and indicate that clastogenesis in bone marrow erythroblasts due to oral or i.p. administration of the flavonols studied is at most very weak.

Mutagenicity of plant flavonoids: structural requirements for mutagenic activity in Salmonella typhimurium.

40 compounds structurally related to the plant flavonol quercetin were tested for mutagenic activity in Salmonella typhimurium strain TA98. 10 flavonols, quercetin, myricetin, rhamnetin, galangin, kaempferol, tamarixetin, morin, 3'-O-methylquercetin, 7,4'-di-O-methylquercetin and 5,7-di-O-methyl-quercetin, exhibited unequivocal mutagenic activity. 4 compounds, quercetin, myricetin, rhamnetin and 5,7-di-O-methylquercetin, were active without metabolic activation, although metabolic activation markedly enhanced their activity. All 4 have free hydroxyl groups at the 3' and 4' positions of the B ring. The other active compounds required an in vitro rat-liver metabolizing system for significant activity. Structural features which appear essential for mutagenic activity in this strain are a basic flavanoid ring structure with (1) a free hydroxyl group at the 3 position, (2) a double bond at the 2, 3 position, (3) a keto group at the 4 position, and (4) a structure which permits the proton of the 3-hydroxyl group to tautomerise to a 3-keto compound. The data are consistent with the requirement for a B ring structure that permits oxidation to quininoid intermediates. Free hydroxyl groups in the B ring are not essential for activity if a rat-liver metabolic activating system is employed. Data from 12 compounds which differ only at the essential sites described above indicate that the structural requirements for mutagenicity in strain TA100 are the same as those for activity in strain TA98. Based on the above structural requirements, a metabolic pathway for flavonol activation to DNA-reactive species is proposed.

Antimicrobial protection of moisturized deglet noor dates.

The growth of Saccharomyces rouxii and Saccharomyces mellis, which are two of the main spoilage organisms of dates, can be inhibited by various treatments. The most effective treatment found in this study that did not affect flavor consisted of a predip of the dates in 2% potassium sorbate solution followed by injection of methyl bromide into the sealed package.

Antimicrobial properties of natural phenols and related compounds: obtusastyrene and dihydro-obtusastyrene.

Factors influencing the antimicrobial properties of obtusastyrene and dihydro-obtusastyrene were studied. Both of these compounds were soluble in acetone, alcohol, and olive oil. In water, they were soluble at concentrations of 34 and 53 mug/ml, respectively. The minimal inhibitory concentrations against gram-positive bacteria and yeast were below 100 mug/ml. The compounds were not effective against gram-negative bacteria at 200 mug/ml or lower concentrations. With initial populations of cells greater than 10(6)/ml, the concentrations of these compounds required to prevent growth were greater than with lower cell populations. Changing the pH of the growth medium did not decrease the effectiveness of these two compounds in the pH range of 3 through 8. Both obtusastyrene and dihydro-obtusastyrene were rapidly bactericidal to Staphylococcus aureus and Bacillus cereus at 25 mug/ml.