Tumor-specific cytotoxicity and type of cell death induced by benzo[b]cyclohept[e][1,4]oxazine and 2-aminotropone derivatives.

A total of twenty benzo[b]cyclohept[e] [1,4]oxazines and their S-analogs, and 2-aminotropone derivatives were investigated for their cytotoxicity against three human normal cells and four tumor cell lines. These compounds showed moderate tumor-specific cytotoxicity. The cytotoxicity was enhanced by bromination at the tropone ring and replacement by formylbenzene. The cytotoxicity of 2-(2-hydroxyanilino) tropone was enhanced by introduction of bromine or isopropyl group to the tropone ring. The presence of a hydroxyl group at ortho or para-position should be necessary for the appearance of cytotoxicity and tumor-specificity. The highly active derivatives, 7-bromo-2-(4-hydroxyanilino)tropone [16] and 4-isopropyl-2-(2-hydroxyanilino)tropone [20], induced internucleosomal DNA fragmentation and caspase-3, -8 and -9 activation in human promyelocytic leukemia HL-60 cells, but only at concentrations twice or four times higher than CC(50) values. These compounds induced no discernible DNA fragmentation, and activated caspases much more weakly in human oral squamous cell carcinoma HSC-2 cells. Both [16] and [20] failed to induce the production of acidic organelles, a marker of autophagy, in contrast to the nutritional starvation. These data demonstrated that 2-aminotropones showed relatively higher tumor-specificity than benzo[b]cyclohept[e] [1,4]oxazine, and that 2-aminotropones induced little or no apoptotic cell death in oral squamous cell carcinoma, in contrast to HL-60 cells.

Effect of tropolone, azulene and azulenequinone derivatives on prostaglandin E2 production by activated macrophage-like cells.

We have previously reported that tropolone (T-3), 2,4-dibromo-7-methoxytropone (T-21), diethyl 2-chloroazulene-1,3-dicarboxylate (A-9), 1,3-difluoroazulene (A-11), 3-morpholino-1,5-azulenequinone (AQ-8) and 3,7-dibromo-1,5-azulenequinone (AQ-13) inhibited the nitric oxide (NO) production of lipopolysaccharide (LPS)-activated mouse macrophage-like RAW264.7 cells, with or without the inhibition of inducible NO synthase (iNOS) mRNA and protein expression. In order to confirm the anti-inflammatory potency, possible effects on prostaglandin (PG) E2 production and the expression of enzymes involved in the arachidonic acid pathway were investigated. Among these six compounds, only A-9 effectively inhibited the PGE2 production of the LPS-stimulated RAW264.7 cells. Western blot analysis demonstrated that A-9 inhibited phospholipase A2 (PLA2), cyclooxygenase (COX)-2 and iNOS proteins only by 12, 45 and 42%, respectively. These data demonstrate the lack of correlation between the extent of inhibition of iNOS protein expression by tropolone or azulene derivatives and that of PGE2, and suggest the possible antiinflammatory potency of A-9.

Tumor-specific cytotoxicity and type of cell death induced by naphtho[2,3-b]furan-4,9-diones and related compounds in human tumor cell lines: relationship to electronic structure.

A total of thirty-nine naphtho[2,3-b]furan-4,9-diones and related compounds were tested for their cytotoxicity against three human normal oral cells (gingival fibroblast, HGF, pulp cell, HPC, periodontal ligament fibroblast, HPLF) and four human tumor cell lines (oral squamous cell carcinoma HSC-2, HSC-3, HSC-4, promyelocytic leukemia HL-60). 2-Acetylnaphtho[2,3-b]furan-4,9-dione [1] was highly cytotoxic to both normal and tumor cells, yielding low tumor-specificity. 2-Acetyl-4,9-dimethoxynaphtho[2,3-b]furan [4], the 2-(3-furanoyl) benzoic acids [5, 6] and the 1,4-naphthoquinones [7, 8] showed much reduced cytototoxicity and low tumor-specificity. The introduction of phenoxy [18], isopropylamino [23] or 2-methylpiperidino [33] groups to the 2-position of naphtho[2,3-b]furan-4,9-dione yielded compounds that showed the greatest tumor-specificity. These compounds, at twice or four times higher concentrations than CC50, induced the activation of caspase-3, caspase-8 and caspase-9 in the HSC-2 and HL-60 cells, but not so apparently in the HSC-4 cells. However, they did not induce internucleosomal DNA fragmentation in the HSC-2 and HSC-4 cells even after 24 hours incubation and only slightly induced DNA fragmentation in the HL-60 cells. Compound [18] induced the production of annexin-positive cells, but did not induce microtubule-associated protein light chain 3 (LC3) accumulation in autophagosomes in LC3-green fluorescent protein (GFP)-transfected HSC-2 cells. These data suggested that naphtho[2,3-b]furan-4,9-diones may induce the early apoptotic marker, without induction of caspase activation and DNA fragmentation in oral squamous cell carcinoma cell lines. Quantitative structure-activity relationship (QSAR) analysis suggests the applicability of the theoretical calculations such as frontier molecular orbital, dipole moments and hydrophobicity in predicting their cytotoxic activity.

Inhibition of LPS-stimulated NO production in mouse macrophage-like cells by benzocycloheptoxazines.

Twenty-six benzocycloheptoxazine derivatives were investigated for their effect on nitric oxide (NO) production by lipopolysaccharide (LPS)-stimulated mouse macrophage-like RAW 264.7 cells. Benzo[b]cyclohepta[e][1,4]thiazine most effectively inhibited the LPS-stimulated NO production at noncytotoxic concentrations. 6H-Benzo[b]cyclohepta[e][1,4]-diazine cation, and benzo[b]cyclohepta[e][1,4]oxazine and its 6-bromo derivative also efficiently inhibited the LPS-stimulated NO production. Another sixteen benzo[b]cyclohepta[e]-[1,4]oxazine derivatives, 14H-[1,4]benzoxazino[3',2' :3,4]-cyclohepta[1,2-b][1,4]benzoxazine and its 7-bromo- and 7-isopropyl derivatives were slightly less active (selectivity index (SI)=83-66). Bromination of benzo[b]cyclohepta[e][1,4]-thiazine, benzo[b]cyclohepta[e][1,4]oxazine and 2-methylbenzo[b]cyclohepta[e][1,4]oxazine at C-6, C-8 or C-10 positions resulted in the significant reduction of the inhibitory activity. The observed inhibitory activity of benzo[b]cyclohepta-[e][1,4]thiazine and its 6,8-dibromo derivatives were not due to the reduction of the intracellular level of inducible NO synthase protein (based on Western blot analysis), nor to NO scavenging activity (based on ESR spectroscopy). These results suggest the possible anti-inflammatory action of benzocyclo-heptoxazines via inhibition of LPS-activated macrophages.

Tumor-specific cytotoxicity and type of cell death induced by benzocycloheptoxazines in human tumor cell lines.

Twenty-six benzocycloheptoxazine derivatives were investigated for their tumor-specific cytotoxicity and apoptosis-inducing activity against three human normal cells (gingival fibroblast HGF, pulp cell HPC, periodontal ligament fibroblast HPLF) and four human tumor cell lines (squamous cell carcinoma HSC-2, HSC-3, HSC-4, promyelocytic leukemia HL-60). Benzo[b]cyclohepta[e][1,4]thiazine [1] exhibited very weak cytotoxicity, whereas its 6,8,10-tribromo derivative [3] exhibited higher cytotoxicity and tumor specificity (TS = 5.6). 6H-Benzo[b]cyclohepta[e][1,4]diazine [4] and its cation [5] exhibited no tumor specificity. Among eighteen benzo[b]cyclohepta[e][1,4]oxazine derivatives [6-23], 6,8,10-triboromo- [9], 6-bromo-2-methyl- [20], and 6-bromo-2-chloro- [21] derivatives showed the highest tumor-specific cytotoxicity (TS = 12.5, 9.1 and 11.5, respectively). 14H-[1,4]Benzoxazino[3',2':3,4]cyclohepta[1, 2-b][1,4]benzoxazine [24] and its 7-bromo- [25] and 7-isopropyl- [26] derivatives had much lower cytotoxicity and tumor-specificity. Compounds [9, 20, 21] at 50% cytotoxic concentration (CC50) induced internucleosomal DNA fragmentation and caspase activation in HL-60 cells. On the other hand, these compounds induced apoptosis only at concentrations higher than CC50 in HSC-2 cells and failed to induce apoptosis in HSC-4 cells. Compounds [9, 20, 21] induced the formation of acidic organelles as measured by acridine orange staining. Transmission electron microscopy demonstrated the induction of moderate enlargement of mitochondria, the endoplasmic reticulum and nuclear membrane, and the vacuolation of the endoplasmic reticulum and the presence of a number of lamellar body-like organelles. These results indicate the diversity of the type of cell death induced by benzocycloheptoxazine derivatives in human tumor cell lines.

Inhibition of NO production in LPS-stimulated mouse macrophage-like cells by trihaloacetylazulene derivatives.

The effect of 20 trihaloacetylazulene derivatives with one halogen atom, on nitric oxide (NO) production by mouse macrophage-like cells Raw 264.7 was investigated. 2-Methoxyazulenes and 2-ethoxyazulenes exhibited comparable cytotoxicity. Trichloroacetylazulenes generally exhibited higher cytotoxicity, as compared with the corresponding trifluoroacetylazulenes. Substitution of chloride, bromide or iodine at the C-3 position further enhanced their cytotoxicity. All of these compounds failed to stimulate the Raw 264.7 cells to produce detectable amounts of NO, but did inhibit NO production by LPS-activated Raw 264.7 cells to different extents. 1-Trichloroacetyl-2-methoxyazulene and 1-trichloroacetyl-2-ethoxyazulene, with less cytotoxic activity, inhibited NO production to the greatest extent, producing the highest selectivity index (SI) of >24.7 and >28.7, respectively. This was accompanied by the efficient inhibition of inducible NO synthase (iNOS) mRNA expression, but not by iNOS protein abundance. Electron spin resonance (ESR) spectroscopy showed that neither of these compounds produced radicals, nor scavenged NO, superoxide anion or diphenyl-2-picrylhydrazyl radicals. The present study suggests that the inhibitory effects of trifluoroacetylazulenes and trichloroacetylazulenes on NO production by activated macrophages might be derived from the perturbation of NO anabolism (inhibition of iNOS mRNA expression and possibly the inactivation of iNOS protein) rather than NO catabolism (NO scavenging).

Relationship between electronic structure and cytotoxic activity of azulenequinones and trihaloacetylazulenes.

The relationship between the structure and cytotoxic activity of azulenequinones and trihaloacetylazulenes was investigated based on theoretical calculations. Four different dipole moments (mu(G), mu(ESP-G), mu(W) and mu(ESP-W)) and heats of formation (DeltaH(f)) of the azulenequinones [1-27] and trihaloacetylazulenes [28a,b-40a,b] were separately calculated in gas phase and aqueous solution using the conductor-like screening model/parametric method 3 (COSMO/PM3) method. The cytotoxic activity of azulenequinones was well correlated to DeltaDeltaH(f) HOMO energy and mu(ESP-w). The cytotoxic activity of trihaloacetylazulenes was correlated to DeltaDeltaH(f) LUMO energy and mu(ESP-W). QSAR may be applicable to predict the cytotoxicity of azulenequinones and trihaloacetylazulenes.

Factors that affect the type of cell death induced by chemicals.

Surveying about 1000 compounds, we found that several low molecular weight alpha, beta-unsaturated ketones induced non-apoptotic cell death characterized by the formation of autophagosomes, occasionally accompanied by mitochondrial shrinkage. The cytotoxic activity of these compounds was significantly reduced by the addition of N-acetyl-L-cysteine, suggesting their interaction with SH groups of intracellular targeted molecules (the so-called "non-sterically hindered Michael acceptor"). This suggests that the nature of the chemical structure as well as the type of target cells is another factor that determines the type of cell death induced by chemicals.

Tumor-specificity and type of cell death induced by trihaloacetylazulenes in human tumor cell lines.

Twenty trihaloacetylazulene derivatives with one atom of fluorine, chlorine, bromine or iodine was investigated for their tumor-specific cytotoxicity and apoptosis-inducing activity against three human normal cells (gingival fibroblast, HGF; pulp cell, HPC; periodontal ligament fibroblast, HPLF) and four human tumor cell lines (squamous cell carcinoma, HSC-2, HSC-3, HSC-4; promyelocytic leukemia, HL-60). There was no apparent difference in the cytotoxic activity between 2-methoxyazulenes [1a-1e, 2a-2e] and 2-ethoxyazulenes [3a-3e, 4a-4e]. Trichloroacetylazulenes [2a-2e, 4a-4e] generally showed higher cytotoxicity and tumor-specificity (expressed as a TS value) as compared with the corresponding trifluoroacetylazulenes [1a-1e, 3a-3e]. Substitution of chloride [1c, 2c, 3c. 4c], bromide [1d, 2d, 3d, 4d] or iodine [1e, 2e, 3e, 4e] at the C-3 position further enhanced cytotoxic activity against four tumor cell lines, especially HL-60 cells. Among twenty trihaloacetylazulene derivatives, two compounds [2d] and [4c] showed the highest tumor specificity (TS = > 3.5 and > 2.5, respectively). Compounds [2d] and [4c] induced apoptotic cell death characterized by caspase-3, -8 and -9 activation and internucleosomal DNA fragmentation in HL-60 cells. On the other hand, compounds [2d] and [4c] induced autophagic cell death characterized by lower activation of caspases, lack of DNA fragmentation, vacuolization and autophagosome formation detected by acridine orange and LC3-GFP fluorescence, without the decline of the intracellular concentration of three major polyamines in HSC-4 cells. The cytotoxic activity of [4c], but not [2d], was slightly reduced by 3-methyladenine, an inhibitor of autophagy. These results suggest the diversity of cell death type induced in human tumor cell lines by trihaloacetylazulene derivatives.

Semisynthesis of isetexane diterpenoid analogues and their cytotoxic activity.

Isetexane diterpene analogues were semisynthesized from demethylsalvicanol isolated from Perovskia abrotanoides (Labiatae). The structure and cytotoxic activity relationships (SAR) of the natural parent diterpene, demethylsalvicanol, and its semisynthetic analogues were studied by using P388 murine leukemia cells.

Inhibition of NO production in LPS-stimulated mouse macrophage-like cells by trihaloacetylazulenes.

The effects of 26 trihaloacetylazulene derivatives on nitric oxide (NO) production by the mouse macrophage-like Raw 264.7 cells was investigated. The trichloroacetylazulenes [1b-13b] generally showed higher cytotoxicity as compared with the corresponding trifluoroacetylazulenes [1a-13a]. All the compounds inhibited NO production by lipopolysaccharide (LPS)-activated Raw 264.7 cells to various extents. 3-Trifluoroacetylguaiazulene [8a], 1-trifluoroacetyl-4,6,8-trimethylazulene [10a], 3-methyl-l-trichloroacetylazulene [2b] and 3-ethyl-1-trichloroacetylazulene [3b] showed lower cytotoxic activity and most effectively inhibited NO production. Western blot analysis revealed that compounds [8a, 1Oal dose-dependently reduced the intracellular concentration of inducible NO synthase (iNOS), whereas compounds [2b, 3b] only marginally affected the iNOS protein expression. RT-PCR analysis showed that compounds [8a, 2b] reduced the iNOS mRNA expression by approximately 50%. These compounds affected cyclooxygenase-2 protein and mRNA expression, depending on the concentrations. ESR spectroscopy revealed that compounds [8a, 10a, 2b, 3b] neither produced radical, nor scavenged NO, superoxide anion or diphenyl-2-picrylhydrazyl radicals. The present study showed the inhibitory effects of trifluoroacetylazulenes and trichloroacetylazulenes on NO production by activated macrophages.

Apoptosis-inducing activity of trihaloacetylazulenes against human oral tumor cell lines.

Twenty-six trihaloacetylazulene derivatives were investigated for their tumor-specific cytotoxicity and apoptosis-inducing activity against three human normal cells (HGF, HPC, HPLF) and four human tumor cell lines (HSC-2, HSC-3, HSC-4, HL-60). The trichloroacetylazulenes [1b-13b] generally showed higher cytotoxicity as compared to the corresponding trifluoroacetylazulenes [1a-13a]. The trichloroacetylazulenes [1b-13b] also showed higher tumor-specific cytotoxicity (expressed as TS value) than the corresponding trifluoroacetylazulenes [1a-13a]. Especially, 2,3-dimethyl-1-trichloroacetylazulene [5b] and 1,3-ditrichloroacetyl-4,6,8-trimethylazulene [11b] showed the highest cytotoxicity and tumor specificity (TS > 35.6 and > 44.1, respectively). These compounds induced internucleosomal DNA fragmentation in HL-60 cells, but not in HSC-2 and HSC-3 cells, but activated caspase-3, -8 and -9 in all of these cells, suggesting the activation of both mitochondria-independent (extrinsic) and dependent (intrinsic) pathways. Western blot analysis showed that two compounds [5b, 11b] slightly increased the intracellular concentration of pro-apoptotic proteins (Bad, Bax) in HSC-2 cells. None of the 26 compounds showed anti-HIV activity. These results suggest [5b] and [11b] as possible candidates for future cancer chemotherapy.

Relationship between electronic structure and cytotoxic activity of azulenes.

The structure-activity relationship of the cytotoxic activity of azulene and azulene derivatives was discussed, using theoretically calculated results. In order to clearly divide the azulenes into three groups according to their functional groups, the CC50, four different dipole moments (muG, muESP-G, muwand muESP-W) and heats of formation (deltaHf) of the azulenes [1-24] were separately calculated in two states, gas-phase and water, by the conductor-like screening model/parametric method 3 (COSMO/PM3). For the halogenated azulenes and isopropyl azulenes, the cytotoxic activity might follow the three quantitative structure-activity relationship (QSAR) parameters: deltadeltaHf, HOMO energy and muw Whereas, for the other ten compounds [3-5, 7-8, 10, 15-18], the cytotoxic activity might be related to the three QSAR parameters, deltadeltaHf, LUMO energy and muG

Relationship between electronic structure and cytotoxic activity of tropolones.

A structure-activity relationship of the cytotoxic activity of tropolone derivatives was discussed, using theoretical calculations. In order to clearly divide the tropolones into two structurally analogous groups, four different dipole moments (muG, muESP-G, muW and muESP-W) and heats of formation (deltaHf) of the tropolones [1-21] were calculated in the gas-phase and in water-solution by the conductor-like screening model/parametric method 3 (COSMO/PM3). The cytotoxic activities of the tropolones and 2-methoxytropones seem to be related to the three QSAR parameters deltadeltaHf, HOMO energy (EH) and muw. The cytotoxic activity of the five tropone derivatives [17-21] might depend on the QSAR parameters deltadeltaHf, LUMO energy (EL) and muESP-G. The results of the present study suggest the applicability of theoretical calculations such as frontier molecular orbital, dipole moments and deltadeltaHf in the prediction of the cytotoxic activity of tropolone derivatives.

Cytotoxicity of abietane diterpenoids from Perovskia abrotanoides and of their semisynthetic analogues.

Seven known abietane diterpenoids and 11-O- and 12-O-acetylcarnosic acids were isolated from a methanol extract of Perovskia abrotanoides (Labiatae). Structure and cytotoxic activity relationships (SAR) of the natural and semisynthetic analogues of the presently isolated abietane diterpenoids were studied by using P388 murine leukemia cells.

Inhibition of LPS-stimulated NO production in mouse macrophage-like cells by azulenequinones.

Azulenequinone derivatives have been reported to display a broad spectrum of biological activities, but study at the cellular level has been limited. The effect of twenty-seven azulenequinone derivatives on nitric oxide (NO) production by mouse macrophage-like cells Raw 264.7 was investigated in this study. All of these compounds failed to stimulate the Raw 264.7 cells to produce detectable amounts of NO, but did inhibit NO production by lipopolysaccharide (LPS)-activated Raw 264. 7 cells to varying extents. Compounds [7, 8, 9, 13, 16, 25, 27], which showed lesser cytotoxic activity (CC50 = 425, 381, 482, 179, 119, 235, 225 microM, respectively), inhibited NO production to the greatest extent [selectivity index (SI) = 15.4, 26.2, 3.9, 21.6, 3.1, 6.0, 8.4, respectively]. Western blot and RT-PCR analyses demonstrated that the most active derivatives, 3-morpholino-1, 5-azulenequinone [8] and 3,7-dibromo-1, 5-azulenequinone [13], significantly reduced both the intracellular concentration of iNOS protein and the expression of iNOS mRNA. ESR spectroscopy showed that compounds [8, 13] weakly scavenged NO produced by NOC-7, possibly via their general reducing activity. These data suggest that the inhibitory effect of NO production by compounds [8, 13] might be generated mostly via the inhibition of iNOS expression, rather than the radical-mediated mechanism.

Cytotoxic activity of azulenequinones against human oral tumor cell lines.

We investigated twenty-seven azulenequinone derivatives for their relative cytotoxicity against three human normal cell lines (HGF, HPC, HPLF) and four human tumor cell lines (HSG, HSC-2, HSC-3, HL-60). Parent 1,5-azulenequinone showed potent and some tumor-specific cytotoxicity. Halogenated derivatives of 1,5- and 1,7-azulenequinone showed potent cytotoxicity, but lower tumor-specific cytotoxicity. In contrast to other azulenequinones, amino derivatives such as 3-amino-1,5- and 1, 7-azulenequinones showed relatively lower cytotoxic activity. The 3-Phenoxy-1,5-azuleneqinone derivative showed higher cytotoxicity than the 3-phenoxy-1, 7-azulenequinone derivative. 1,5- and 1,7-Azulenequinones generally showed higher cytotoxicity, as compared with tropolones and azulene derivatives. 3- (3-Guaiazulenyl)-1, 5-azulenequinone [12] and 7-isopropyl-3- (4-methylanilino)-2-methyl- 1, 5-azulenequinone [24] showed a relatively higher TS value and induced apoptosis (internucleosomal DNA fragmentation, activation of caspases 3, 8 and 9) in HL-60 and HSC-2 cells, possibly via the activation of both mitochondria-independent (extrinsic) and -dependent (intrinsic) pathways. Western blot analysis showed that [24] slightly increased the intracellular concentration of pro-apoptotic proteins (Bad, Bax) in HSC-2 cells, whereas [12] was much less active. None of the twenty-seven azulenequinones showed anti-HIV activity. These results suggest [12] and [24] as possible candidates for future cancer chemotherapy.

Relationship between electronic structure and cytotoxic activity of dopamine and 3-benzazepine derivatives.

A structure-activity relationship of dopamine and 3-benzazepine derivatives is discussed, using theoretically calculated results. In order to clearly divide dopamines and 3-benzazepines into a strongly active and a weakly active group, the CC50, two different dipole moments (microESP-G and microESP-W) and heat of formation (deltaHf) of dopamine [1-13] and 3-benzazepine derivatives [14-23] were separately calculated in two states of gas-phase and water-solution by the COSMO/PM3 method. It was found that ten derivatives [1-3, 9, 12-13 and 20-23] (CC50: 0.056 to 2.5 mM) showed the strongest cytotoxic activity with small delta(deltaHf) values, whereas thirteen derivatives [4-8, 10-11, 14-19] (CC50: > 3.6 mM) showed the weakest cytotoxic activity with large delta(deltaHf) values.

Biological activity of barbados cherry (acerola fruits, fruit of Malpighia emarginata DC) extracts and fractions.

Fractionation of barbados cherry (acerola fruit, a fruit of Malpighia emarginata DC.) extracts were performed by organic solvent extractions and column chromatographies, using two extraction methods. Higher cytotoxic activity was concentrated in fractions A4 and A6 (acetone extract), and H3 and HE3 (hexane extract). These four fractions showed higher cytotoxic activity against tumor cell lines such as human oral squamous cell carcinoma (HSC-2) and human submandibular gland carcinoma (HSG), when compared with that against normal cells such as human periodontal ligament fibroblasts (HPLF) and human gingival fibroblasts (HGF). HE2 (hexane extract), AE2 (ethyl acetate extract), AE3, AE4, AE5, A8, A9 and A10 showed some relatively higher anti-bacterial activity on the Gram-positive Staphylococcus epidermidis ATCC 1228 but were ineffective on the representative Gram-negative species E. coli and Ps. aeruginosa. The fractions were inactive against Helicobacter pylori, two representative Candida species, and human immunodeficiency virus (HIV). H3, H4 and HE3, which displayed higher tumor-specific cytotoxicity also showed higher multidrug resistance (MDR) reversal activity, than (+/-)-verapamil as positive control. ESR spectroscopy shows that the radical-mediated oxidation is not involved in the induction of tumor-specific cytotoxic activity. The tumor specific cytotoxic activity and MDR reversal activity of barbados cherry may suggest its possible application for cancer therapy.

Inhibition of LPS-stimulated NO production in mouse macrophage-like cells by tropolones.

We investigated the effect of 27 tropolones on nitric oxide (NO) production by mouse macrophage-like Raw 264.7 cells. All of these compounds failed to stimulate the Raw 264. 7 cells to produce detectable amounts of NO, but inhibited NO production by lipopolysaccharide (LPS)-activated Raw 264.7 cells to various extents. Generally, the ability of tropolones to inhibit LPS-stimulated NO production was inversely related to their cytotoxic activity. Western blot and RT-PCR analyses demonstrated that the most active compound, 2,4-dibromo-7-methoxytropone [21], significantly reduced both the intracellular concentration of iNOS protein and the expression of iNOS mRNA. ESR spectroscopy showed that [21] did not produce radicals under alkaline condition, nor scavenged NO, produced by NOC-7. These data suggested that the inhibitory effect of [21] on NO production might be generated via the inhibition of iNOS expression, rather than a radical-mediated mechanism.