Research program
Design, synthesis and biological evaluation of new antitumor agents
University Co-ordinator
Università degli Studi di FERRARA -
SCIENZE FARMACEUTICHE - FERRARA(FE)
Research Unit Leader
Daniele SIMONI
Description
Preliminary Results.20-36 Simoni et al. (J. Med. Chem. 1997, 40, 3581; Med. Chem. Res. 1998, 8, 291; J. Med. Chem. 1999, 42, 4961) have started a study aimed to design retinoid compounds endowed with potent apoptotic activity and selectivity toward neoplastic cells. They observed that isoxazole arotinoids bearing the cis stereochemistry at the alkenyl portion were more efficient inducers of apoptosis than the correspondent trans stereoisomers. Additionally, they discovered that the cis-TTNPB, which still retains some structural features of 9-cis-RA, is endowed with potent apoptotic activity, while the trans isomer has only moderate effects in inhibiting cell proliferation. In addition, TTNPB-retinoid analogs bearing amino moieties replacing the carboxylic group proved to be particularly potent apoptosis-inducing agents active in multidrug resistant (MDR) cell lines, in cell lines resistant to drug-induced apoptosis and in cell lines expressing the BCR-ABL oncogene. Therefore, they have learned that simple modifications at the stilbene motif of arotinoids may greatly influence their activity and compounds possessing various differentiating or apoptototic activities can be obtained. Since the stereochemistry of the C9 alkenyl portion of natural 9-cis-RA, as well as of TTNPB, seemed of particular importance for their apoptotic activity, Simoni et al. (J. Med. Chem. 2001, 44, 2308; Org. Lett. 2000, 2, 3765) have recently planned the synthesis of new retinoid analogs using a sterically restricted flexibility in this region. The alkenyl basic motif of TTNPB was replaced by an isoxazole or an isoxazoline moiety which may stiffen the molecule; vice versa, a more flexible connecting amino- or oxymethyl moiety, which may enable the system to better "fit" the receptor, was introduced in others derivatives. Among the synthesized isoxazole arotinoids, a compound 4-[3-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethylnaphtalen-2-yl)-4,5-isoxazol-3-yl]benzoic acid (1) showed an apoptosis-inducer activity toward MDR cells and cell lines expressing the anti-apoptotic BCR-ABL oncogene similar or greater than that observed with the TTNPB amino derivative. Interestingly, a compound bearing the isoxazoline heterocyclic system 4-[3-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethylnaphtalen-2-yl)-4,5-dihydroisoxazol-5-yl]benzoic acid (2) was devoid of any apoptotic activity, but having potent RXR affinity. More recently, the structure of the known arotinoid CD437 has been combined with the isoxazole portion of the previously synthesized compounds. Very surprisingly a novel class of antiapoptotic agents has been discovered with a mechanism of action that seems related to the release of calcium ions from mitochondria (Simoni D. et al. Blood, 2004, 103, 194). Terphenyl compounds structurally related to CD437 demonstrated also for the first time potent antiapoptotic activity (Tetrahedron Lett. 2003, 44, 3009). As the stilbene motif represents the key structural feature of the natural combretastatin A-4 (CA-4) and trans-resveratrol, two well-known antitumor agents, Simoni et al. have synthesized analogues of these natural stilbenoids with the aim to discover potent apoptotic agents. They found that amino derivatives of combretastatin AC7739 and compounds structurally related to trans-resveratrol possess potent apoptosis-inducing activity (Simoni et al. J. Med Chem. 2003, 46, 3546; Blood, 2002, 44, 445). Additionally, some of the heterocyclic-bridged combretastatins demonstrated an attracting cytoxicity profile and were able to induce apoptosis at concentrations lower than 0,1 _icromolar_in HL60 cells (Simoni et al. J. Med. Chem. 2004, submitted for publication, patent RM2003A000355). The 2-methoxy-5-[3-methoxy-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-4-isoxazolyl]-phenol (3) and its regioisomer 4 appear as intriguing leads for the discovery of potent apoptotic agents. The ability of 3 and 4 to block most cells in the G2 phase of the cell cycle suggests that these compounds could act on targets different from the mitotic spindle. This may confer on these molecules a wider spectrum of action than the parent CA-4, which arrests cells in the M phase of the cell cycle. Hypothesis. Structural alterations of the structure of the leads 1, 2 and CD437 will afford compounds endowed with both differentiating and apoptotic properties. Substitution of the benzene ring of CD437 with alkene moieties will afford potent apoptosis inducing agents. Introduction of a double bond-locked motif confers on the structurally restricted combretastatins a potent apoptotic activity different from that of natural CA-4. Structural alteration of the stilbene motif of CA-4 can be extremely effective in producing potent apoptosis-inducing agents, by activating both the intrinsic and the extrinsic pathways. Objectives. Since the simplicity of trans-resveratrol, CA-4 and synthetic arotinoids, associated with their interesting anticancer activity, offers promises for the rational design of new chemotherapeutic agents, an objective of this proposal will be a detailed study of the structure-activity relationships (SAR) of these natural and synthetic stilbenoids as a logical starting point in the quest for novel anticancer chemotherapeutics. With this aim, the design and synthesis of novel stilbenes structurally related to arotinoids endowed with differentiating and apoptotic properties will be carried out. To obtain biological information on apoptosis induced by new synthesized compounds, we rely on collaboration with Dr. Manlio Tolomeo and Dr. Luisa Dusonchet, by Palermo Polyclinic (Palermo, Italy). Objective of the project is also the design of new stilbenes with, selectively, the different biological activities of CA-4. The novel combretastatins will be endowed with potent apoptotic activity thus providing an innovative class of antitumor agents. Aim of the project is the design of improved combretastatins targeted to specific cell-cycle proteins, as well as the study of their mechanism of action inside the apoptotic machinery. Specific objectives. Apoptosis and differentiating agents structurally related to arotinoids. a) Derivatives of the leads compounds 1, 2 and CD437 will be synthesized and tested for retinoid receptor binding and activation in order to verify whether substitutions already known for retinoids will exert similar effects in the isoxazole derivatives. The information gained about structure-function relationships will be used to design new improved retinoids targeted to specific receptors. b) The effects of rigidity and hydrophilicity of the linking group on receptor binding and activation will be determined. The starting heterocyclic rings of 1 and 2 will be opened, resulting in structures that have the same number of carbon atoms as TTNPB, but with different rigidity. These compounds, containing a beta aminoketone or a beta hydroxyketone linking group, will possess the same number of heteroatoms as their parent structures, thus allowing additional possibilities for hydrogen bonding in comparison to TTNPB and the starting lead compounds 1 and 2. These novel compounds will be tested for receptor binding and activation. If they activate the retinoid receptors, then the hydrogen atoms of the amine group will be substituted to evaluate the effects of hydrogen bonding on receptor binding. c) Analogues of CD437 will be synthesized by means of the substitution of the different benzene rings of CD437 with alkenyl portion linkages. d) Novel analogs of the leads 1 and 2 bearing a heteroatom (N, O, S) will be synthesized, as other known retinoids bearing such substitution have been shown endowed with less toxicity. The receptor selective compounds developed in this project will be tested for growth inhibition and modulation of endothelial cell proliferation in different cancer cell lines in order to evaluate the roles of the individual receptors in these biological activities. The informations gained can be used to design improved retinoids targeted to individual receptors or subsets of receptors and eventually having specific biological activities expected to improve disease states without significant toxicity.CA-4 derivatives with apoptotic activity. a) New derivatives of the two lead compounds 1 and 2 will be synthesized and tested for their cytotoxicity and apoptotic activity. The novel compounds will be obtained by substitution of the isoxazoline bridge of 1 and 2 with other pentaatomic aromatic rings. The information gained about structure-function relationships will be used to design improved combretastatinss targeted to specific cell-cycle proteins. b) Novel combretastatins will be obtained by substitution of the trimethoxy-benzene ring residue of the natural CA-4 with an aromatic heterocyclic residue such as benzofuran, benzothiophene or simple furan and thiophene based heterocycles with possibly functionalizing substituents. c) Substitution of one or more methoxyl residues on the trimethoxyphenyl group with other substituents (alkyl groups, halogens, amines, phenols and others). d) Derivatives of CA-4 bearing a benzothiophene or a benzofurane ring will be prepared as such compounds could resemble known CA-4 analogs endowed with potent apoptotic activity. e) The effects of rigidity and hydrophilicity of the linking group of 1 and 2 will be determined. The starting heterocyclic rings of 1 and 2 will be opened, resulting in structures that have the same number of carbon atoms as the parent compounds, but with different rigidity. These compounds containing an aminoalcohol or an hydroxyketone posses additional possibilities for hydrogen bonding than the natural combretastatin and the starting leads 1 and 2. In case of compounds with improved activity, then hydrogen atoms on the amine group will be substituted to evaluate the effects of hydrogen bonding on receptor binding. f) Novel analogs of CA-4 constrained at the alkenyl bridge will be synthesized. Because it is well appreciated that replacement of the alkenyl bridge portion of combretastatin by five-membered heterocyclic systems leads to compounds of potent cytotoxyc and apoptotic activity, it is possible that six-membered heterocyclic-bridged combretastatins, which are less investigated, may have improved activity. These new modifications will provide novel restricted analogues of CA-4. f) The alkenyl bridge of CA-4 and compounds obtained as in point 2 and 3 (first year) will be replaced by an isosteric ketone moiety. g) Combretastatin and its analogs show a typical structure (the stilbene motif) characterized by two polysubstituted aromatic rings linked by an olefinic function. The presence of a phenolic group into one ring may serve as a handle for attachment to a suitable linker, making this class of molecules suitable both for a solid phase syntethic approach as well as for a parallel synthesis to construct libraries of compounds structurally related to CA-4. In the first phase, it will be possible to link a 3-hydroxybenzoic moiety to the resin as a precursor of the corresponding aldehyde. An olefination reaction between the resin-bound aldehyde derivatives and a polysubstituted aromatic phosphoric ylide could allow the synthesis of the desired targets. The ready availability of substituted aromatic ylides can envisage the application to the combinatorial chemistry.
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