Synthetic and application perspectives of azapodophyllotoxins: alternative scaffolds of podophyllotoxin.

Podophyllotoxin (1) has been known to possess anti-tumor activity and is still considered an important lead for research and development of antineoplastic agents. Derivatives of podophyllotoxin, namely etoposide (2), etopophos (3) and teniposide (4) have been developed and are currently used in clinic for the treatment of a variety of malignancies. These agents are also used in combination therapies with other drugs. Due to the drug resistance developed by cancer cells as well as side effects associated with the use of these agents in clinic, the search for new effective anticancer analogues of podophyllotoxin remains an intense area of research. The structural complexity of podophyllotoxin, arising from the presence of four stereogenic carbons in ring C has restricted most of the structural activity relationship (SAR) studied by derivatization of the parent natural product rather than by de novo multi-step chemical synthesis. These issues provide strong impetus to a search for analogues of 1 with simplified structures, which can be accessible via short synthetic sequences from simple starting materials. Even if such initial compounds might have diminished cytotoxic potencies compared with the parent cyclolignan, the ease of preparation of carefully designed libraries of analogues would lead to more informative SAR studies and expeditious structure optimization. In this regard, during the last two decades considerable efforts have been made to synthesize aza- analogs of podophyllotoxin, i. e. aza-podophyllotoxins, with hetero atoms at different positions of the podophyllotoxin skeleton, while keeping the basic podophyllotoxin structure. Recently, there have been significant efforts towards the convenient synthesis of aza-analogs of 1. The use of multicomponent reactions (MCRs) and the synergies of ultrasound and microwave irradiations have increased the synthetic speed and variety of azapodophyllotoxins which are and will be available to be tested against a diverse population of carcinomas and other diseases. It has been reported that several aza-podophyllotoxins retain a great fraction of the cytotoxicity associated with the parent lignan. This review focuses on the strategies towards synthesis of various aza-podophyllotoxin analogues and their biological activities.

Role of membrane charge and semiquinone structure on naphthosemiquinone derivatives and 1,4-benzosemiquinone disproportionation and membrane-buffer distribution coefficients.

Semiquinone membrane/buffer partition coefficients have been determined for 1,2-naphthosemiquinone (ONQ.-), 1,4-naphthosemiquinone (NQ.-) and two of its hydroxylated derivatives, 5,8-dihydroxy-1,4-naphthosemiquinone (NZQ.-) and 5-hydroxy-1,4-naphthosemiquinone (JQ.-) as a function of membrane charge in multilamellar vesicles of phosphatidylcholine (PC) and equimolar mixtures of this lipid and phosphatidic acid (PC:PA) and cetyltrimethylammonium bromide (PC:CTAB) at physiological pH with the exception of values corresponding to PC:PA mixtures which were obtained at pH 9. These coefficients follow the order PC:PA < PC < PC:CTAB in agreement with the negative charge of the semiquinones. The disproportionation equilibria of the naphthosemiquinone derivatives are shifted to the semiquinone in the presence of neutral and positive membranes, being more pronounced in the latter. However, very low partition coefficients as well as small shifts in the semiquinone disproportionation equilibrium were observed for ONQ.- as compared to the other semiquinones. No partition of 1,4-benzosemiquinone (BQ.-) into the lipid phase was detected for either charged or neutral lipid membranes. The presence of lipid membranes decreases the BQ.- equilibrium concentration in the presence of all the types of membranes considered here.

Sonochemistry of quinones in argon-saturated aqueous solutions: enhanced cytochrome c reduction.

Sonolysis of argon-saturated aqueous quinone solutions resulted in an enhancement in ferricytochrome c (Cyt c) reduction. Upon addition of superoxide dismutase, Cyt c reduction was partially inhibited, thus implying a role of superoxide ion in this reduction process. Neither quinone hydrophobicity nor reduction potential exclusively controls the Cyt c reduction enhancement, although a preference for hydrophobicity versus reduction potential is noted.

The photobiological differences of gilvocarcins V and M are not related to their transient intermediates and triplet yields.

The transient absorption spectra of the intermediates produced by the 355 nm laser excitation of gilvocarcin derivatives have been investigated in various solvents. The spectra consist of a triplet-triplet absorption in the visible region and a residual absorption observed between 340 and 700 nm due to a long-lived species, assigned to the radical cation. A broad-fast decaying band with a maximum at around 700 nm attributed to the solvated electron is also seen in solutions containing a low DMSO/water volume ratio and at 266 nm irradiation of a 50% methanol/water solvent mixture. The molar absorption coefficient of the triplet state of gilvocarcin V (GV) and gilvocarcin M (GM), determined by the energy transfer method, is independent of the solvent properties and has a value of 3.0 x 10(4)/Mcm. The triplet decay rate constants for both drugs are between 1 and 5 x 10(4)/s. A similar initial yield and triplet decay rate constant of GV were observed in the presence of 3.4 mM thymine. Thus, a quenching rate constant of the GV's triplet state by thymine is estimated to be lower than 10(6)/Ms. The triplet quantum yields of both antibiotics determined by using the comparative method are higher in dimethylsulfoxide (DMSO) (0.18) than are those corresponding to 25% DMSO/water (0.06). The decrease in phi T in the presence of water could be attributed to an enhanced internal conversion rate constant from the S1 state or to an increase in the photoionization yield. The similarity of the transient intermediates and their yields for GV and GM suggest that their photobiological differences are due to other factors such as DNA binding constants, preferential localization of the drugs in the cell or the enhanced reactivity of the vinyl group toward cellular components.

Adriamycin and daunomycin semiquinone membrane/buffer partition constants using the spin-broadening technique.

Membrane/buffer partition constants have been determined for the semiquinones of adriamycin, Adrsq, and daunomycin, Daunosq, as a function of ionic strength and cholesterol content in phosphatidylcholine multilamellar vesicles using the spin-broadening technique. The partition constants corresponding to Adrsq were essentially similar to those of Daunosq under any of the conditions studied. These constants increase with ionic strength increase and decrease with an increase in cholesterol concentration at the membrane. These observations could have implications in the Adr and Dauno cytotoxicities where their corresponding semiquinones are postulated as important intermediates and consequently their interactions with biological membranes should also be considered as important.

Photochemistry of water-soluble quinones. Production of a water-derived spin adduct.

The photolyses of phosphate-buffered (pH 7) air- and nitrogen-saturated solutions containing the water-soluble quinones, 1,4-benzoquinone (BQ), 2-methyl-1,4-benzoquinone (MBQ), sodium 1,4-naphthoquinone-2-sulfonate (NQ2S), 9,10-anthraquinone-2-sulfonate (AQ2S) or 9,10-anthraquinone-1,5-disulfonate (AQDS), and the spin trap 5,5-dimethylpyrroline-1-oxide (DMPO) produce a DMPO-OH adduct. Electron paramagnetic resonance spectroscopy of the photolyzed samples in 17O-enriched water demonstrates that this adduct derives almost exclusively from water. With the exception of BQ, quantum yields for the formation of DMPO-OH are larger in air than in nitrogen-saturated samples, thus supporting the idea of the formation of air-oxidized intermediates that enhance the DMPO hydroxylation reaction rate. Evidence has been obtained which suggests that BQ and MBQ, but not AQDS, are able to photooxidize water, with the consequent production of the free OH radical.

Photophysical properties of gilvocarcins V and M and their binding constant to calf thymus DNA.

Absorption and emission techniques were used to characterize the ground (S0), singlet (S1) and triplet states (T1) of gilvocarcin V (GV) and gilvocarcin M (GM) in different solvents. Aggregation of GV with dimerization constant equal to 7800 M-1 is observed in 10% dimethyl-sulfoxide (DMSO)/water. The photophysical properties of the S1 state of these molecules are more sensitive to changes in solvent characteristics than the corresponding ground states. The absorption of visible light by GV and GM results in a higher dipole moment of the excited state causing a red shift in the fluorescence spectra with increasing solvent polarity. The fluorescence quantum yield remains practically unchanged with changes in solvent properties unless water is present as a co-solvent. Both phi f and tau f values corresponding to GV in DMSO are larger than those of GM, whereas in 10% DMSO/H2O the opposite is observed. Thus, GV is more susceptible to other deactivation pathways besides emission in the presence of water than GM. The relative phosphorescence quantum yield (phi p = 0.03) and the triplet energy (ET = 52 kcal/mol) of GV and GM are similar. The S0-S1 energy difference is 63 kcal/mol for GV, whereas for GM it is 67. Thus, the singlet-triplet energy difference is 11 and 15 kcal/mol, respectively. The PM3/CI calculated electronic structures of these compounds are consistent with the observed photophysical properties. The dark binding constants of GV to calf thymus DNA ([1.1-0.08] x 10(6) M-1) are about an order of magnitude larger than those of GM ([0.24-0.018] x 10(6) M-1) at different ionic strengths (0-2.00 M NaCl). Also, the number of gilvocarcin molecules bound per base pair is smaller for GM than for GV. These differences in dark DNA binding parameters between GV and GM could have implications in the large photocytotoxic ability of GV as compared to GM.

A comparative study of the visible light photochemistry of gilvocarcins V and M.

Quantum yields for the formation of superoxide ions, O2-., and singlet oxygen, 1O2, were determined during the photolyses of gilvocarcin M (GM) in air-saturated dry dimethylsulfoxide (DMSO) and in 45:55 (vol/vol) DMSO-water mixtures. The quantum yield for the photoreduction of methyl viologen by GM in nitrogen-saturated dry DMSO was also determined. These values are not different, within experimental error, from those corresponding to gilvocarcin V (GV). Because GV is a strong photocytotoxic agent and GM is not, these results imply that Type I and Type II mechanisms are not important pathways in the cytotoxicity of GV.

Reductive activation of benzazolo[3,2-a]-quinolinium chlorides.

Initial ferricytochrome c (Cyt(III)c) reduction rates occurring in aerobic or anaerobic solutions containing either 3-nitrobenzothiazolo[3,2-a]-(NBQCl), 1-ethyl-3-nitrobenzimidazolo[3,2-a]-(ENBIQCl), 7-ethylbenzimidazolo[3,2-a]quinolinium chloride (EHBIQCL), or nitrofurantoin (NFT) and xanthine/xanthine oxidase were measured. Maximum rates in nitrogen-saturated solutions follow the order NFT > NBQCL > ENBIQCL > EHBIQCL. These rates correlate linearly with the half-wave reduction potentials (E1/2) of these compounds. With the exception of EHBIQCl, smaller rates of Cyt(III)c reduction were obtained in air-saturated than in nitrogen-saturated solutions at the quinolinium salt concentrations used. Larger concentrations of superoxide dismutase (SOD) are needed for 50% inhibition of the Cyt(III)c reduction reaction for heterocyclic compounds with larger E1/2 values. Thus, measurement of the portion of the Cyt(III)c reduction rate under air that is inhibited by SOD does not account solely for the production of superoxide. These observations suggest that NBQCL, ENBIQCl, and less probably EHBIQCl may interfere with mitochondrial energy metabolism or induce DNA damage through reduced intermediates.

Semiquinones derived from anthraquinone-containing antitumor drugs can partition into phosphatidylcholine bilayers.

Semiquinones derived from anthraquinone-containing antitumor drugs (doxorubicin, daunorubicin and 4'-epidoxorubicin) were generated by the hypoxanthine/xanthine oxidase system in argon-saturated phosphate buffer (pH 7.4) in the presence of egg-yolk phosphatidylcholine multilamellar vesicles (MLVs) containing 1 mol% of a doxylstearic acid (DSA) isomer. The destruction of the electron spin resonance signal corresponding to 5-, 12- and 15-DSA included in the MLVs follows pseudo-first-order kinetics. Higher rates of destruction are obtained for the 12-DSA isomer which indicates that these semiquinones can localize preferentially about the depth of the 12th position of stearic acid in membranes. It is demonstrated that DSA destruction is due to a reversible reduction of DSA to the hydroxylamine species. This work shows that anthracycline semiquinones can partition into phosphatidylcholine bilayers under anoxic conditions which may imply another pathway in their cytotoxic action.

Free radicals induced by adriamycin-sensitive and adriamycin-resistant cells: a spin-trapping study.

The radicals generated by adriamycin-sensitive (CHO-AB) and adriamycin-resistant (CHO-C5) Chinese hamster ovary cells as well as by adriamycin-sensitive and -resistant human breast cancer cells (MCF7-WT and MCF7-ADR) have been studied with spin-trapping and ESR spectroscopy. During anoxic exposure to adriamycin (ADR) both pairs of cell lines produced the broad ESR singlet characteristic of ADR semiquinone (AQ.). By use of tris(oxalato)chromate (CrOx) as an extracellular line-broadening agent, the distribution of AQ. between the intra- and extracellular compartments was studied. For cell densities of (1-3) X 10(7) cells/mL, CrOx eliminated most, though not all, of the ESR signal, indicating that the AQ. radicals freely diffuse and partition between the intra- and extracellular compartments proportionally to their respective volumes. Similar behavior was exhibited by all four cell lines studied. Upon introduction of oxygen to anoxic cells in the presence of the spin trap 5,5-dimethylpyrroline N-oxide (DMPO), the AQ. signal was replaced by that of the DMPO-OH spin adduct. Metal chelators such as desferrioxamine had no effect on DMPO-OH or AQ. formation. Superoxide dismutase, not catalase, totally eliminated the ESR signal, indicating that DMPO-OH produced by ADR-treated cells originates from superoxide rather than from .OH produced from H2O2. In the presence of CrOx, the DMPO-OH signal was not distinguishable from the background noise, thus excluding any contribution to the signal by intracellular spin adducts.(ABSTRACT TRUNCATED AT 250 WORDS)

An ESR study of the visible light photochemistry of gilvocarcin V.

Photolysis of gilvocarcin (GV) at 405 nm in argon saturated dimethylsulfoxide (DMSO) or 50% DMSO-water solutions in the presence of the sodium salt of 3,5-dibromo-2,6-dideutero-4-nitrosobenzene sulfonic acid (DBNBS-d2) generates the CH3-DBNBS-d2.spin adduct. It is postulated that this spin adduct is produced by photoreduction of DMSO by GV and the consequent formation and trapping of the generated methyl radicals. Gilvocarcin V also photoreduces oxygen and methyl viologen with quantum yields of 0.019 and 0.0012 respectively. The quantum yield for singlet oxygen formation by GV in DMSO, determined by measuring the rate of production of the nitroxyl radical produced by the reaction of 2,2,6,6-tetramethylpiperidinol with singlet oxygen, was found to be 0.15. Thus, GV photochemistry proceeds by both Type I and Type II pathways which could contribute to the reported GV phototoxicity in biological systems.

Photochemistry of aqueous solutions of benzazolo[3,2-a]quinolinium salts. A spin-trapping study using 17O-enriched water and oxygen.

The photolysis of buffered aqueous solutions containing the quinolinium salts, 3-nitro-7-ethyl-benzimidazolo[3,2-a]quinolinium perchlorate (NEBQClO4) and 3-nitrobenzothiazolo[3,2-a]quinolinium chloride (NBQCl), at 344 and 365 nm, respectively, was studied in the presence of the spin trap 5,5-dimethyl-1-pyrroline-1-oxide (DMPO). Only a water-derived DMPO-OH.spin-adduct was obtained for both of these salts, at a DMPO concentration of 14 mM, as confirmed by H2 17O-enriched water experiments. A photosolvated intermediate is postulated as the OH donating species. Lower steady-state concentrations of the spin adduct were obtained in argon-saturated solutions, implying that oxygen gas is at least partially necessary in the water-derived DMPO-OH formation. Evidence for superoxide ion formation was obtained by the DMPO-17OH spin-adduct formation during the photolysis of NBQCl in an 17O-enriched oxygen atmosphere in the presence of 150 mM DMPO. An increase in the DMPO-OH steady-state concentration was observed if the photolysis of NBQCl was performed in the presence of superoxide dismutase (SOD). Our results suggest that this effect is due to the SOD inhibition of the destruction of DMPO-OH.by superoxide ion.