Cytotoxic responses to aromatic ring and configurational variations in alpha-conidendrin, podophyllotoxin, and sikkimotoxin derivatives.

Derivatives of alpha-conidendrin, podophyllotoxin, and sikkimotoxin were prepared to evaluate the cytotoxic contributions of C-4 configuration and pendant and fused arene substitutions. Dimethyl-alpha-conidendryl alcohol (5), 9-deoxypodophyllol (6), and 9-deoxysikkimol (17) were dehydrated to their respective oxolane derivatives 4, 3, and 9. Diols 5 and 6 were converted via oxabicyclo[3.2.1]octanols 10 and 14 to target oxolanes 8 and 7 where C-4 had been inverted relative to that in 3 and 4. Cytotoxicities of the five oxolanes were determined in two drug-sensitive human leukemia and two multidrug-resistant cell lines expressing P-glycoprotein or multidrug-resistance associated protein (MRP). Changing the pendant arene configuration or replacing a m-methoxy by hydrogen resulted in a 100-fold cytotoxicity loss. Replacing a methylenedioxy group in the fused arene by two methoxy substituents reduced cytotoxicity by 10-fold. Drug-resistant cell lines were equally resistant to compounds 3, 4, 8, and 9 indicating that these four compounds do not serve as substrates of the transport proteins P-glycoprotein and MRP.

Mucochloric acid action on phi X174 DNA: a comparison to other chlorine-substituted 2(5H)-furanones.

The action of the drinking water mutagen 3,4-dichloro-5-hydroxy-2(5H)-furanone (MCA) on the plasmid phi X174 converted this initially closed circular, supercoiled (SC) DNA to its relaxed (R) and linear (L) forms. Kinetic analysis of this process gave results coinciding with a sequential, two-step cleavage model whereby the SC was cleaved to the R form, which in its turn was cleaved to the L form. The actions of two additional chlorine-substituted 2(5H)-furanones, reduced MCA (RMCA) and the C-5 isopropyl ether of MCA (MCA-IPE), were compared to that of MCA. Incubation of SC-phi X174 with RMCA gave R form only while MCA-IPE had virtually no effect. Likewise, neither methyl methanesulfonate nor sodium azide cleaved SC-phi X174 to its L form, under conditions whereby MCA caused the formation of L form. Also, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) caused formation of R form, but no apparent L form. Increasing concentration of glutathione diminished cleavage of SC phi X174 by MCA, but increased cleavage for MX. The DNA cleavage action of MCA was unique, among the several stronger and weaker mutagens investigated, in its action phi X174.

Bromine-, chlorine-, and mixed halogen-substituted 4-methyl-2(5H)-furanones: synthesis and mutagenic effects of halogen and hydroxyl group replacements.

The versatility of 4-(hydroxymethyl)-2(5H)-furanone as a starting point for the synthesis of several bromine and mixed halogen analogues of the potent water mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) has been demonstrated. However, in some preparations the yields of desired products were lower for bromine- than chlorine-substituted counterparts. A total of 12 bromine-, chlorine-, and mixed halogen-substituted 4-methyl-2(5H)-furanones were tested repeatedly in 10 independent experiments for levels of Salmonella typhimurium (TA100) mutagenicities. The purpose of these experiments was to determine the mutagenic response to changing halogen content, type, and position as well as to learn the measure of these responses in the presence and absence of the C-5 OH group. Mutagenicities reached levels of 10(3) and 10(2) rev/nmol for all trihalo- and dihalo-4-methyl-5-hydroxy-2(5H)-furanones, respectively, notwithstanding substitutions by bromine or chlorine. Trihalides lacking the C-5 hydroxyl group possessed mutagenicities of the order of 10(2) rev/nmol, while hydroxyl group absence in the dihalides resulted in potency levels of slightly less than 10 rev/ nmol. Pairwise comparisons of compound mutagenicities showed that overall the C-5 H-by-OH replacement and, next in importance, increasing the number of C-6 halogens from one to two resulted in the greatest enhancements of mutagenicities. However, in comparing compound pairs within two different sets of four di- and trihalides, it was observed that replacement of a C-5 H by OH enhanced mutagenicity more for the dihalides than the trihalides indicating that increasing the C-6 halogen number simultaneously with replacing C-5 H by OH results in a nonlinear, additive enhancement. For fewer than half of the compound pairs compared, changing the C-6 halogen from chlorine to bromine resulted in small increases in mutagenicity, and for the remaining compound pairs, no increase could be discerned. This result points to the relative unimportance of only C-6 halogen type as a determinant of mutagenicity. Similarly, no impact on mutagenicity was observed for changing only the halogen type attached to C-3.

Interactive chlorine-by-bromine and hydrogen-by-hydroxyl group replacement effects in 2(5H)-furanone mutagenicity.

Both bromine- and chlorine-substituted 2(5H)-furanones are produced by the chlorination of ligno-humic waters containing bromide ion. The molar mutagenicities of four bromine- and chlorine-substituted 2(5H)-furanones were determined by the Salmonella typhimurium (TA100) assay to explore Cl-by-Br and H-by-OH replacement effects on mutagenicity. Each of these two replacements was expected to enhance mutagenicity based on earlier work showing that lower LUMO energy levels and greater radical anion stability correlated with elevated TA100 mutagenicity. The four compounds investigated were the following: 2,3-dibromo-5-hydroxy-2(5H)-furanone (mucobromic acid, MBA); 2,3-dibromo-2(5H)-furanone (reduced mucobraomic acid, RMBA); 2,3-dichloro-5-hydroxy-2(5H)-furanone (mucochloric acid, MCA); and 2,3-dichloro-2(5H)-furanone (reduced mucochloric acid, RMCA). Mean molar mutagenicities were found to be 5.54, 1.18, 2.92 and 0.105 revertants/nmol for the four compounds in the order named. Mutagenicity enhancements resulting from Cl-by-Br and H-by-OH replacements were analyzed by simple ratios of mean molar mutagenicity and by multiple regression analysis. The effect of the Cl-by-Br replacement on mutagenicity amounted to a 1.9-fold enhancement in the presence of C-5 OH, but an 11-fold enhancement in the presence of C-5 H. This demonstrated that the two replacement effects were interactive. Higher mutagenicity values corresponded to lower AM1 computed LUMO energy levels and greater radical anion stabilities.

Oxidation and radical intermediates associated with the glutathione conjugation of mucochloric acid.

The inactivation of the drinking water mutagen mucochloric acid (MCA) by reduced glutathione (GSH) was linked to the formation of an MCA-GSH conjugate, a nonmutagen in the Salmonella typhimurium (TA100) plate incorporation assay. Anaerobic formation of MCA-GSH is found now to be associated with oxidized glutathione (GSSG) and unconverted MCA. The anaerobic reaction of GSH with MCA in the presence of the radical trap 2-methyl-2-nitrosopropane (tNB; "tert-nitrosobutane") gives rise to an electron paramagnetic resonance (EPR) resulting from the overlapping spectra of two radical adducts. The first species exhibited hyperfine coupling constants of aN = 13.65 G and aH beta = 0.73 G. The second radical adduct exhibited a three-line signal of aN = 12.8 G. The first species is assigned to an adduct of the MCA radical because deuteration of MCA (5-deuterio-MCA) caused the beta-hydrogen hyperfine coupling to collapse. The second radical adduct is unaffected by the deuteration of MCA. Thus, the involvement of both GSSG and a carbon-centered MCA radical in the action of MCA on GSH is indicated.

Glutathione and N-acetylcysteine inactivations of mutagenic 2(5H)-furanones from the chlorination of humics in water.

The mutagenic 2(5H)-furanones resulting from the chlorination of lignohumic substances in water disinfection and paper pulp bleaching are known to be inactivated by thiols. The objectives of the present study were to characterize the kinetics of an inactivating reaction, isolate and characterize products, and determine their mutagenicity in relation to the starting, mutagenic 2(5H)-furanones. The Salmonella typhimurium (TA100) mutagenicity of mucochloric acid (MCA) had a mean value of 2800 revertants/mumol from four assays and was twice as potent as the C-5 isopropyl ether of MCA (MCA-IPE), whose mutagenicity was determined in the same four assays. A second-order reaction of MCA with GSH at pH 7 was observed. The major product, making up 70% of the total product mixture, was identified as a 1.5:1 mixture of two diastereomers formed by sulfur displacement of the C-4 Cl atom from MCA. The major diastereomer was isolated from the 1.5:1 mixture. Connectivity of GSH to the MCA moiety in the product was established by 2D long-range coupling NMR and fully coupled 13C NMR. On the basis of circular dichroism, the major diastereomer had the S configuration at the hydroxyl-bearing, C-5 ring carbon. MCA-IPE reacted with GSH and N-acetylcysteine (NAC), giving 1:1 mixtures of two diastereomers, again by displacement of the C-4 Cl atom from MCA. A single diastereomer was isolated from the 1:1 MCA-IPE plus NAC reaction. Its structure, determined by X-ray crystallography, had the 5R,8R configuration and was in agreement with the ross structure deduced from the NMR analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Test of chiral recognition in the Salmonella typhimurium (TA100) mutagenicity of mucochloric acid-cysteine adducts.

A difference in biological response to enantiomers is not an uncommon observation and is, therefore, to be expected in various manifestations of genotoxicity. The bacterial mutagen mucochloric acid (2,3-dichloro-5-hydroxy-2(5H)-furanone) has one chiral center, at C-5, but this mutagen exists in racemic form because of the facile stereoisomerization occurring by the mechanism of ring-chain tautomerism. Two readily synthesized enantiomeric analogs of mucochloric acid, as well as the racemic form of the two, were prepared from mucochloric acid and (R)-(+)-, (S)-(-)-, and (R,S)-(+/-)-cysteine. Using Salmonella typhimurium (TA100), the enantiomeric compounds were assayed together in four dose/response assays along with mucochloric acid, the reference mutagen. In three of the same four assays, the racemic form was also assayed. Neither statistically significant differences in mutagenicity, as determined in slope responses, nor distinctions from the plotted curves were observed among the two enantiomers and their racemic form. Therefore, no enantiospecific interaction between enantiomers and chiral DNA or enzymes involved in repair or replication could be concluded.

A study of inactivation reactions of N-acetylcysteine with mucochloric acid, a mutagenic product of the chlorination of humic substances in water.

The Salmonella typhimurium (TA100) mutagenic compound, mucochloric acid [3,4-dichloro-5-hydroxy-2(5H)-furanone (MCA)], was inactivated by in vitro N-acetylcysteine (NAC). The reaction of MCA with NAC at pH7 was second order and gave products 4, 5, and 6a that resulted from the displacement of chlorine from C-3 or C-4 of MCA. The sodium borohydride treatment of product 4 gave the same product (7) as was obtained by treating 3,4-dichloro-2(5H)-furanone with NAC. The treatment of MCA with (R)-(+)-cysteine gave the bicyclic product 9a, in which the two chlorine atoms of MCA were still present. This product was slightly more mutagenic than MCA, whereas product 5 was less mutagenic than MCA and product 4 was nonmutagenic in the Salmonella typhimurium (TA100) assay.

Associations of the bacterial mutagenicity of halogenated 2(5H)-furanones with their MNDO-PM3 computed properties and mode of reactivity with sodium borohydride.

Electrophilicity as a general basis for both the mutagenicity and nucleophile inactivation of halogen-substituted 2(5H)-furanones was tested. Lowest unoccupied molecular orbital (LUMO) energy levels and stabilities of 2(5H)-furanone radical anions and C-2, C-3, and C-4 anionic hydride adducts were computed with MNDO-PM3 for each of 10 compounds. These three computed sets of values were considered electrophilicity indicators. Each individual value from a given indicator set was plotted against the logarithm of the Salmonella typhimurium (TA100) mutagenicities (log Mm) for each of the corresponding compounds. Highest occupied molecular orbital (HOMO) energy levels for the 10 compounds were also computed. Strong negative correlations were obtained from the plots of LUMO and radical anion stability against log Mm of the 10 compounds. Also, a negative correlation was observed for the plot of the stability of the C-4 anionic hydride adduct for a smaller set of six compounds possessing a 4-(chloromethyl)-2(5H)-furanone structure and having the same HOMO characteristics. HOMO energies failed to correlate with mutagenicity. Neither computed atomic charge nor 13C chemical shift values for the larger compound group of 10 correlated well with mutagenicity or with each other. Sodium borohydride and borodeuteride reductions of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone demonstrated experimentally that hydride became attached to C-4.

Structure-activity relationships of bacterial mutagens related to 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone: an emphasis on the effect of stepwise removal of chlorine from the dichloromethyl group.

The Salmonella typhimurium (TA100) mutagenicities of six structural analogues of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) were determined and compared. These were also compared to previously determined mutagenicities for another four analogues. This study was conducted for the primary purpose of ascertaining the effect of C-6 chlorine-by-hydrogen replacement on mutagenicity. The compounds assayed were 3-chloro-4-(chloromethyl)-5-hydroxy-2(5H)-furanone (3), 3-chloro-4-(chloromethyl)-2(5H)-furanone (4), 3-chloro-4-methyl-5-hydroxy-2(5H)-furanone (7), 3-chloro-4-methyl-2(5H)-furanone (8), 4-methyl-5-hydroxy-2(5H)-furanone (9), and 4-methyl-2(5H)-furanone (10). Compounds 3, 4, and 7 were mutagenic whereas 8-10 were not. All six compounds were stable under assay conditions. Mutagenicity data for the three active compounds were combined with data of another four active compounds studied previously to obtain an expanded data set. Mutagenicities of the seven compounds were compared, pairwise, in 21 comparisons and then by multiple regression analysis. On the average, chlorine-by-hydrogen replacement of a single chlorine located at a chloromethyl group (C-6) had a markedly greater effect in reducing mutagenicity than a similar replacement at C-3 or a hydroxyl-by-hydrogen replacement at C-5. The chlorine-by-hydrogen replacement at C-6 of compound 3 resulted in the greatest mutagenicity reduction of any single replacement and amounted to a 10(3)-fold diminished mutagenicity.

Salmonella typhimurium (TA100) mutagenicity of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone and its open- and closed-ring analogs.

The mutagenicities of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX, compound 1), 3-chloro-4-(dichloromethyl)-2(5H)-furanone (RMX, compound 6), and 2-(dichloromethyl)-3,3-dichloropropenal (TCB, compound 7) were determined in the same assay and in repetitive determinations using Salmonella typhimurium (TA 100) without microsomal fraction activation. In addition, the mutagenicity of 2-methyl-3,3-dichloropropenal (compound 8) was assayed in the same manner although not simultaneously with MX, RMX, and TCB. This study was undertaken to ascertain the role of open- and closed-ring forms of MX in the mutagenicity of MX. MX proved to be roughly 100 times more mutagenic than the open-ring analogue TCB and 10 times more mutagenic than the closed-ring analogue RMX. Compound 8 was inactive. Assay stability of the three active compounds in Vogel-Bonner medium at 38 degrees C was estimated as the chemical half-life values by following the change in UV absorbance at selected wave lengths. Half-life values were 10.7, 2.6, and 2.8 hr, respectively, for MX, RMX, and TCB. The enhanced mutagenicity of MX relative to RMX and TCB is attributed to the intrinsic mutagenicity of MX and its greater stability is judged to play only a minor role. Moreover, the greater mutagenicity of the closed-ring analogue RMX relative to the open-ring analogue TCB points to the ring form of MX as the active species even though the open form of MX is predominant under assay conditions.

Effect on mutagenicity of the stepwise removal of hydroxyl group and chlorine atoms from 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone: 13C NMR chemical shifts as determinants of mutagenicity.

The response of mutagenicity to the stepwise replacement of chlorine atoms and the hydroxyl group by hydrogen in 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX, 1) was determined in several assays by using Salmonella typhimurium tester strain (TA100). In all, eight MX derivatives were assayed. Several were studied together in at least one assay. In addition to MX, the seven included 3-chloro-4-(dichloromethyl)-2(5H)-furanone (RMX, 2), 3-chloro-4-(chloromethyl)-5-hydroxy-2(5H)-furanone (3), 3-chloro-4-(chloromethyl)-2(5H)-furanone (4), 4-(chloromethyl)-5-hydroxy-2(5H)-furanone (5), 4-chloromethyl-2(5H)-furanone (6), and 4-(dichloromethyl)-2(5H)-furanone (8). Compounds 1-6 were mutagenic. Compound 8 gave erratic results. 4-(Acetoxymethyl)-2(5H)-furanone (11) was nonmutagenic. The largest drop in mutagenicity amounted to a factor of about 10(2) for the replacement of the hydroxyl group or a C-3 chlorine atom from 3. Other replacements of the hydroxyl group or a C-3 or C-6 chlorine atom amounted to mutagenicity diminished by a factor of only 10. On the basis of the rates of UV spectral changes under assay conditions, chemical half-life values (ct 1/2) for 1-6 and 8 were estimated as indicators of compound stability. However, mutagenicity differences were shown to result principally from the intrinsic mutagenicities of the six compounds 1-6 rather than from differences in stability.(ABSTRACT TRUNCATED AT 250 WORDS)

Preparation and in vitro antifungal activities of some quinolizidine derived hemiaminals and beta-tert-amino sulfides.

New alpha-thiohemiaminals, 7alpha-phenylthio-7-epideoxynupharidin-6-ol, the diasteriomeric 7beta-phenylthiodeoxy-nupharidin-6-ol, and 3-methyl-3-methylthio-4-hydroxyquinolizidine, were prepared and reduced to the corresponding beta-tert-amino sulfides. The configuration at C-7 of the beta-tert-amino sulfides was determined by observing the direction of the solvent-induced shift of the C-7 methyl proton resonance. The configuration at C-7 of the new alpha-thiohemiaminals was established by correlations with the beta-tert-amino sulfides and confirmed, in the case of the deoxynupharidine derivatives, by circular dichroism and ascertaining the sterochemistry of deuteride incorporation upon sodium borodeuteride reduction of the alpha-thiohemiaminal. The in vitro antifungal activities of six compounds possessing the quinolizidine skeleton, including all the newly synthesized compounds as well as some previously reported ones, and amphotericin B were tested against several human pathogenic fungi. Besides amphotericin B, only the two deoxynupharidine alpha-thiohemiaminals were active, especially against Histoplasma capsulatum and Blastomyces dermatitidis. The observations indicate activity is derived from the 3-furyl group and the functionality from which alpha-thioimmonium ions can be produced.