Design, synthesis and biological evaluation of novel stilbene-based antitumor agents.

A series of novel stilbene derivatives has been synthesized and studied with the main goal to investigate SAR of the amino compound 1a, as well as to improve its water solubility, a potentially negative aspect of the molecule that could be a serious obstacle for a pre-clinical development. We have obtained derivatives with good cytotoxic activity, in particular, the derivatives 5c and 6b could represent two novel leads for further investigation. Compound 8b, a morpholino-carbamate derivative, prodrug of 1a, has a very good solubility in water, and is active in suppressing growth of tumor cells at a concentration of 5000 nM, which is a concentration 100 times higher than the parent stilbene 1a.

Galangin increases the cytotoxic activity of imatinib mesylate in imatinib-sensitive and imatinib-resistant Bcr-Abl expressing leukemia cells.

Resistance to imatinib mesylate is an emergent problem in the treatment of Bcr-Abl expressing myelogenous leukemias and additional therapeutic strategies are required. We observed that galangin, a non-toxic, naturally occurring flavonoid was effective as anti-proliferative, and apoptotic agent in Bcr-Abl expressing K562 and KCL22 cells and in imatinib mesylate resistant K562-R and KCL22-R cells. Galangin induced an arrest of cells in G0-G1phase of cell cycle and a decrease in pRb, cdk4, cdk1, cycline B levels; moreover, it was able to induce a monocytic differentiation of leukemic Bcr-Abl+ cells. Of note, galangin caused a decrease in Bcl-2 levels and markedly increased the apoptotic activity of imatinib both in sensitive or imatinib-resistant Bcr-Abl+ cell lines. In contrast, flavonoids unable to modify the Bcl-2 intracellular levels, such as fisetin and chrysin, did not increase the apoptotic effect of imatinib. These data suggest that galangin is an interesting candidate for a combination therapy in the treatment of imatinib-resistant leukemias.

Antitumor effects of curcumin and structurally beta-diketone modified analogs on multidrug resistant cancer cells.

Using concepts of bioisostery a series of curcumin analogs were synthesized: the diketonic system of the compound was elaborated into enaminones, oximes, and the isoxazole heterocycle. The cell growth inhibitory and apoptosis inducing effects of the new analogs were evaluated by in vitro assays in the hepatocellular carcinoma HA22T/VGH cells, as well as in the MCF-7 breast cancer cell line and in its multidrug resistant (MDR) variant MCF-7R. Increased antitumor activity on all cell lines was found with the isoxazole analog and especially with the benzyl oxime derivative; in the HA22T/VGH cell model, the latter compound inhibited constitutive NF-kappaB activation.

The synergistic apoptotic effects of thiophenfurin, an inosine monophosphate dehydrogenase inhibitor, in combination with retinoids in HL60 cells.

New effective cytotoxic agents and combinations are urgently needed in cancer treatment. The enzyme inosine monophosphate dehydrogenase is a potentially useful target for drug development, since its activity has been shown to be amplified in malignant cells. Thiophenfurin, an inhibitor of the enzyme synthesized by us, is endowed with a significant apoptotic activity in promyelocytic leukaemia HL60 cells. Since retinoids were successfully employed in the treatment of patients with leukaemia, demonstrating significant differentiation-inducing and apoptotic effects, we carried out this study to evaluate the effects of the combination of thiophenfurin and several retinoid molecules, acting in different phases of the cell cycle in vitro. The results show that thiophenfurin is capable of eliciting significant S phase-specific antiproliferative effects in different sensitive and resistant cell lines with the IC50s ranging from 6.7 to 26 microM. When HL60 cells were treated with thiophenfurin in combination with retinoids, the effects on cell growth were additive or synergistic, depending on the kind of retinoid used and the sequence of treatment. In particular, we observed additive effects when the cells were exposed to thiophenfurin and all-transretinoic acid either simultaneously or sequentially. Instead, when the new heterocyclic retinoid isoxazole benzoic acid was used, synergism was obtained in the cells treated sequentially. The combination of thiophenfurin and isoxazole benzoic acid determined synergistic apoptotic effects through a mitochondrion-dependent mechanism, suggesting the possible usefulness of this combination in the treatment of leukaemia.

Pterostilbene and 3'-hydroxypterostilbene are effective apoptosis-inducing agents in MDR and BCR-ABL-expressing leukemia cells.

Pterostilbene and 3,5-hydroxypterostilbene are the natural 3,5-dimethoxy analogs of trans-resveratrol and piceatannol, two compounds which can induce apoptosis in tumor cells. In previous studies we demonstrated the importance of a 3,5-dimethoxy motif in conferring pro-apoptotic activity to stilbene based compounds so we now wanted to evaluate the ability of pterostilbene and 3,5-hydroxypterostilbene in inducing apoptosis in sensitive and resistant leukemia cells. When tested in sensitive cell lines, HL60 and HUT78, 3'-hydroxypterostilbene was 50-97 times more potent than trans-resveratrol in inducing apoptosis, while pterostilbene appeared barely active. However, both compounds, but not trans-resveratrol and piceatannol, were able to induce apoptosis in the two Fas-ligand resistant lymphoma cell lines, HUT78B1 and HUT78B3, and the multi drug-resistant leukemia cell lines HL60-R and K562-ADR (a Bcr-Abl-expressing cell line resistant to imatinib mesylate). Of note, pterostilbene-induced apoptosis was not inhibited by the pancaspase-inhibitor Z-VAD-fmk, suggesting that this compound acts through a caspase-independent pathway. On the contrary, 3'-hydroxypterostilbene seemed to trigger apoptosis through the intrinsic apoptotic pathway: indeed, it caused a marked disruption of the mitochondrial membrane potential delta psi and its apoptotic effects were inhibited by Z-VAD-fmk and the caspase-9-inhibitor Z-LEHD-fmk. Moreover, pterostilbene and 3'-hydroxypterostilbene, when used at concentrations that elicit significant apoptotic effects in tumor cell lines, did not show any cytotoxicity in normal hemopoietic stem cells. In conclusion, our data show that pterostilbene and particularly 3'-hydroxypterostilbene are interesting antitumor natural compounds that may be useful in the treatment of resistant hematological malignancies, including imatinib, non-responsive neoplasms.

Heterocyclic and phenyl double-bond-locked combretastatin analogues possessing potent apoptosis-inducing activity in HL60 and in MDR cell lines.

Two new series of combretastatin (CA-4) analogues have been prepared. The alkenyl motif of CA-4 was replaced either by a five-membered heterocyclic (isoxazoline or isoxazole) or by a six-membered ring (pyridine or benzene). The new compounds have been evaluated for their effects on tubulin assembly and for cytotoxic and apoptotic activities. Five compounds (18b, 20a, 21a, 34b, and 35b) demonstrated an attractive profile of cytotoxicity (IC50 < 1 microM) and apoptosis-inducing activity but poor antitubulin activity. The isoxazoline derivatives 18b, 20a, and 21a, demonstrated potent apoptotic activity different from that of natural CA-4. Their ability to block most cells in the G2 phase suggests that these compounds could act on targets different from the mitotic spindle. This would indicate activation of both the intrinsic and the extrinsic apoptotic pathways. The data suggest unambiguously that structural alteration of the stilbene motif of CA-4 can be extremely effective in producing potent apoptosis-inducing agents.

Studies on the apoptotic activity of natural and synthetic retinoids: discovery of a new class of synthetic terphenyls that potently support cell growth and inhibit apoptosis in neuronal and HL-60 cells.

New terphenyl derivatives have been synthesized and tested for their effect on cell survival in serum-free cultures. These compounds protected HL60 cells from death and supported their growth with an activity higher than that of the natural 14-hydroxy-retro-retinol. Terphenyls 26 and 28 also possess antiapoptotic activity on neuronal cells, proving them as possible candidates for the treatment of neurodegenerative and ischemic diseases.

Synthesis and pharmacology of 6-substituted benztropines: discovery of novel dopamine uptake inhibitors possessing low binding affinity to the dopamine transporter.

A series of 6alpha- and 6beta-substituted benztropines were synthesized. A marked enantioselectivity was observed for the 6beta-methoxylated benztropines, the (1R)-isomers being more potent than the corresponding (1S) compounds. The racemic 6alpha-methoxy-3-(4',4' '-difluorodiphenylmethoxy)tropane (5 g) was the most potent compound. It has been found that modifications at the 6-position of benztropine might reduce the DAT binding affinity, maintaining otherwise a significant dopamine uptake inhibitory activity. A reinvestigation of the absolute configuration of 6beta-methoxytropinone proved the 6R configuration for the (+)-enantiomer.

Novel terphenyls and 3,5-diaryl isoxazole derivatives endowed with growth supporting and antiapoptotic properties.

A new study on terphenyl and diaryl-isoxazole and -isoxazoline derivatives, maintaining a common 3-adamantyl-4-hydroxyphenyl moiety, has been conducted to find compounds with growth supporting and antiapoptotic properties. Unexpectedly, diphenyisoxazole derivatives bearing a nitro group replacing the carboxylic function have been found with the highest cell protective activity within the series, in complete and in serum-free conditions. Inhibition of apoptosis induced by daunorubicin has also been observed for the most active compound.

Design, synthesis, and biological evaluation of novel aminobisphosphonates possessing an in vivo antitumor activity through a gammadelta-T lymphocytes-mediated activation mechanism.

A small series of aminobisphosphonates (N-BPs) structurally related to zoledronic acid was synthesized with the aim of improving activity toward activation of human gammadelta T cells and in turn their in vivo antitumor activity. The absence of the 1-OH moiety, together with the position and the different basicity of the nitrogen, appears crucial for antitumor activity. In comparison to zoledronic acid, compound 6a shows a greater ability to activate gammadelta T cells expression (100 times more) and a proapoptotic effect that is better than zoledronic acid. The potent activation of gammadelta T cells, in addition to evidence of the in vivo antitumor activity of 6a, suggests it may be a new potential drug candidate for cancer treatment.

Stilbene-based anticancer agents: resveratrol analogues active toward HL60 leukemic cells with a non-specific phase mechanism.

Several stilbenes, related to known resveratrol, have been synthesized and tested for their anticancer effect on HL60 leukemia cell line, taking particular care of the cell cycle analysis. The most potent compound was the known (Z)-3,4',5-trimethoxystilbene (6b) which was active as apoptotic agent at 0.24 microM. Differently from other stilbenes (including resveratrol) that induced a prevalent recruitment of cells in S phase of cell cycle, we found a peculiar behavior of 6b that caused a decrease of cells in all phases of cell cycle (G0-G1, S, and G2-M) and a proportional increase of apoptotic cells. The potent pro-apoptotic activity shown by compound 6b and its effects on cell cycle make this compound of great interest for further investigations.