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Background: In order to extend the search for tumour-targeting compounds, this study performed a quantitative structure-activity relationship (QSAR) analysis of 26 newly synthesised trihaloacetylazulenes., Materials and Methods: The value of 50% cytotoxic concentration (CC(50)) of trihaloacetylazulenes against human oral squamous cell carcinoma (HSC-2, HSC-3, HSC-4) and human promyelocytic leukaemia (HL-60) cell lines was calculated from the dose-response curve by the MTT method. CONFLEX/CAChe PM5 was used for the calculation of 11 physico-chemical features for each compound., Results: When all 26 compounds were analysed together, the CC(50) values correlated well with the dipole moment, the lowest unoccupied molecular orbital energy and the reactivity index, and somewhat with the heat of formation, the stability of hydration and the absolute electron negativity, but not with other features. When these 26 compounds were separated into two groups with or without substituents of the 7-membered ring, the correlation coefficient was increased. When the 26 compounds were separated into a different set of two groups with different halogen atoms in the 5-membered ring, no improvement of correlation coefficient was observed., Conclusion: The present study suggests that appropriate grouping of test compounds may further increase the correlation coefficient. The QSAR approach was useful in the design of azulene compounds that are expected to show higher potency.

We have previously reported the cytotoxicity and type of cell death induced by twenty trihaloacetylazulenes in human tumor cell lines. We determined for the first time the most-stable chemical structures from their reported structures, using the semiempirical molecular-orbital method (CONFLEX/PM5), and then delineated the relationship between their cytotoxicity (evaluated by 50% cytotoxic concentration, CC(50)) and a total of twelve parameters. The parameters used are the molecular weight and eleven chemical descriptors: the heat of formation (COSMO, non-COSMO), stability of hydration (=COSMO - nonCOSMO (DeltaH)), dipole moment (D), hydrophobicity (log P), highest occupied molecular orbital energy (E(HOMO)), lowest unoccupied molecular orbital energy (E(LUMO)), absolute hardness [eta=(E(LUMO)-E(HOMO))/2], absolute electron negativity [chi=-(E(LUMO)+E(HOMO))/2], reactivity index (omega=chi(2)/2eta) and surface area (A(2)), and volume of the molecule (A(3)). There was good correlation between the CC(50) value and all descriptors except for absolute hardness in HL-60 cells. There was also a good correlation between the CC(50) value and EHOMO, chi, omega, surface area, volume and molecular weight in HSC-2, HSC-3 and HSC-4 cells. The descriptors determined by the present method are useful in evaluating the biological activity of trihaloacetylazulenes.

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).

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.

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.

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.

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.

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

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.