Molecular modeling approaches in the discovery of new drugs for anti-cancer therapy: the investigation of p53-MDM2 interaction and its inhibition by small molecules.

The mdm2 oncogene product, MDM2, is an ubiquitin protein ligase that inhibits the transcriptional activity of the tumor suppressor p53 and promotes its degradation. About 50% of all human cancers present mutations or deletions in the TP53 gene. In the remaining half of all human neoplasias that express the wild-type protein, aberrations of p53 regulators, such as MDM2, account for p53 inhibition. For this reason, designing small-molecule inhibitors of the p53-MDM2 protein-protein interaction is a promising strategy for the treatment of cancers retaining wild-type p53. The development of inhibitors has been challenging. Although many small-molecule MDM2 inhibitors have shown potent in vitro activity, only a limited number of compounds have demonstrated to possess acceptable pharmacokinetic properties for in vivo evaluation. To date, the most studied chemotypes have been cis-imidazolines (such as nutlins), benzodiazepines, and spiro-oxindoles. The cis-imidazolines were the first discovered potent and selective small-molecule inhibitors of the p53-MDM2 interaction and they continue to show therapeutic potential. This review will focus on recent molecular modeling approaches (molecular dynamics, pharmacophore-based, molecular docking, structure-based design) used with the aim to better understand the behavior of these proteins and to discover new small-molecule inhibitors of the p53-MDM2 protein-protein interaction for the treatment of cancer.

Intercalation of daunomycin into stacked DNA base pairs. DFT study of an anticancer drug.

We have computationally studied the intercalation of the antitumor drug daunomycin into six stacks of Watson-Crick DNA base pairs (i.e., AT-AT, AT-TA, GC-AT, CG-TA, GC-GC, GC-CG) using density functional theory (DFT). The proton affinity of the DNA intercalator daunomycin in water was computed to be 159.2 kcal/mol at BP86/TZ2P, which is in line with the experimental observation that daunomycin is protonated under physiological conditions. The intercalation interaction of protonated daunomycin with two stacked DNA base pairs was studied through a hybrid approach in which intercalation is treated at LDA/TZP while the molecular structure of daunomycin and hydrogen-bonded Watson-Crick pairs is computed at BP86/TZ2P. We find that the affinity of the drug for the six considered base pair dimers decreases in the order AT-AT > AT-TA > GC-AT > GC-TA > GC-CG > GC-GC, in excellent agreement with experimental data on the thermodynamics of the interaction between daunomycin and synthetic polynucleotides in aqueous solution. Our analyses show that the overall stability of the intercalation complexes comes mainly from pi-pi stacking but an important contribution to the computed and experimentally observed sequence specificity comes from hydrogen bonding between daunomycin and hetero atoms in the minor groove of AT base pairs.

Pyrrolo[2,1-d][1,2,3,5]tetrazinones deaza analogues of temozolomide with potent antitumor activity.

The title compounds, that hold the deaza skeleton of temozolomide, exhibited potent in vitro antiproliferative activity. An evaluation of such a biological activity indicates that the mode of action of these compounds differs from that of temozolomide and is also mechanistically unrelated to that of any known antitumor drug.

Indolo[3,2-c]cinnolines with antiproliferative, antifungal, and antibacterial activity.

A series of indolo[3,2-c]cinnoline derivatives was prepared and tested to evaluate their biological activity. Most of them inhibited the proliferation of leukemia, lymphoma and solid tumor-derived cell lines at micromolar concentrations, whereas none of the compounds were active against HIV-1. With the exception of 7g, all title compounds showed antibacterial activity against gram-positive bacteria, being up to 200 times more potent than the reference drug streptomycin. Some of the indolo[3,2-c]cinnolines were also endowed with good antifungal activity, particularly against Criptococcus neoformans.

Synthesis and antiproliferative activity of 2-triazenothiophenes.

A series of 2-triazenothiophene derivatives was prepared and tested to evaluate their biological activity. Two compounds inhibited the proliferation of leukemia, lymphoma and solid tumor-derived cell lines at micromolar concentrations, whereas none of the compounds were active against HIV-1. Compound 3c inhibited DNA, RNA and protein synthesis, and was also effective against KB cells resistant to etoposide and vincristine. The compounds were inactive against fungi and bacteria.

Derivatives of the new ring system indolo[1,2-c]benzo[1,2,3]triazine with potent antitumor and antimicrobial activity.

Derivatives of the new ring system indolo[1,2-c]benzo[1,2,3]triazine 5 were synthesized by diazotization of substituted 2-(2-aminophenyl)indoles followed by an intramolecular coupling reaction of the diazonium group with the indole nitrogen. To obtain the indolobenzotriazine system it was necessary to protect the 3 position of the indole nucleus to avoid cyclization into the indolo[3,2-c]cinnoline system 4. Indolobenzotriazines 5a-g were evaluated in vitro for antitumor activity against a panel of leukemia-, lymphoma-, carcinoma-, and neuroblastoma-derived cell lines. Some compounds inhibited the proliferation of T and B cell lines at submicromolar concentrations, whereas their activity against solid tumor cell lines was in the micromolar range. When evaluated for their antifungal potential 5a,d inhibited some of the fungi tested, although at concentrations very close to those inhibiting the proliferation of human cells. On the contrary, all indolobenzotriazines proved fairly potent and selective inhibitors of Streptococcus and Staphylococcus. In particular 5b,c,g were up to 80 times more potent than the reference drug streptomycin and inhibited the growth of the above Gram-positive bacteria at concentrations far lower than those cytotoxic for animal cells.

Glycosidopyrroles. Part 3. Effect of the benzocondensation on acyclic derivatives: 1-(2-hydroxyethoxy) methylindoles as potential antiviral agents.

The new of 1-(2-hydroxyethoxy)methylindole derivatives 3a-i were prepared in good yields. None of them showed any significant anti-HIV activity and therefore the benzocondensation between the 2 and 3 positions of the pyrrole ring definitely reduced the weak activity found in the analogues 1a-c.

Glycosidopyrroles. Part 1. Acyclic derivatives: 1-(2-hydroxyethoxy)methylpyrroles as potential anti-viral agents.

Acyclic glycosidopyrroles of type 1, synthesized in good overall yields, were evaluated for anti-viral activity. Compound 10i was found to inhibit the HIV-1 replication at concentrations that were very close to those cytotoxic for MT-4 cells. Compounds 10a,f,i inhibited both strains HSV-1 and HSV-2 at concentrations slightly below those cytotoxic for Vero cells. However for this series of glycosidopyrroles some relationship between calculated log P values and the observed cytotoxicity was found.

Acyclic glycosidopyrroles analogues of ganciclovir: synthesis and biological activity.

Acyclic glycosidopyrroles of type 3 were synthetized in good overall yields, according to the Scheme. When evaluated for antiviral activity against DNA and RNA viruses, only compound in which R1 = R2 = Ph, R3 = NH2 was found to inhibit the HIV-1 replication at concentrations that were not cytotoxic for MT-4 cells.

Glycosidopyrroles. Part 2. Acyclic derivatives: 1-(1,3-dihydroxy-2-propoxy)methylpyrroles as potential antiviral agents.

The series of 1-(1,3-dihydroxy-2-propoxy)methylpyrroles 2a-o were prepared in good overall yields according to Scheme I. When evaluated for antiviral activity against HIV-1, only compounds of the triphenyl series (R3 = NH2, N3, Br) were found to inhibit the HIV-1 replication at concentrations that were very not cytotoxic for MT-4 cells, with selectivity index 1.5-9.3. None of these compounds showed antibacterial or antifungal activity.

2-Diazopyrroles: synthesis and antileukemic activity.

The title compounds were synthesised in preparative yields by diazotization of the corresponding 2-aminopyrroles. In preliminary screening tests as antileukemic agents they showed modest activity against the murine and human leukemic cell lines FLC and K562S and their multidrug-resistant daunorubicin selected sublines.

3-Diazopyrroles. Part 6. Mutagenic activity of 3-diazopyrroles in Streptomyces coelicolor A3(2) during various phases of growth.

3-Diazopyrroles, a class of compounds particularly interesting from a chemical and biological point of view, were assayed for their ability to induce gene mutations employing back mutation (his+ reversion) test in the philamentous bacterium Streptomyces coelicolor at various time during life cycle. Our results suggest that in evaluating the mutagenicity and toxicity of chemicals in Streptomyces system it is important to consider factors such as growth phase. Furthermore in this series of diazopyrroles a relationship between toxicity, mutagenicity and chemical structure was found. The observed mutagenic activity can be the molecular basis for the appearance of antitumor activity.

Polycondensed nitrogen heterocycles. Part 27. Indolo[3,2-c]cinnoline. Synthesis and antileukemic activity.

Indolo[3,2-c]cinnolines of type 5, variously substituted either in the indole and in the cinnoline moieties, were prepared in good overall yields, by intramolecular cyclization of indolo derivatives 4. Compounds 5a-d showed a good cytotoxic activity against FLC and K562 leukemic cell lines, both sensitive and multi-drug resistant.

Azolophenanthridines as antineoplastic agents.

Pyrrolo-, pyrazolo- and triazolo-phenanthridines were synthetized by using a Pschorrtype cyclization reaction or an intramolecular cyclization of arylnitrenium ions. By using these synthetic methods several azolo-phenanthridines, variously functionalized either in the azolo ring and in the phenanthridine moiety, were prepared. The title compounds, tested against murine leukemia cell lines, sensible and multidrug resistant, showed moderate activity with IC50 in the range 5-50 microM.

3-Triazenopyrroles: synthesis and antineoplastic activity.

Some 3-triazenopyrroles were prepared by coupling 3-diazopyrroles and secondary amines. In preliminary in vitro screening tests derivatives 3 showed to be cytotoxic and exhibited mutagenic effects.

3-Diazopyrroles. Part 5 (1). Antibacterial activity of 3-diazo-2-phenylpyrroles.

3-Diazo-2-phenylpyrroles 3a-g showed antimicrobial activity against Gram-positive bacteria, whereas against Gram-negative strains the inhibitory activity is limited to derivatives 3a and 3c. The substituents at 4 and 5 positions strongly influence the inhibitory activity, but the presence of the diazo group is crucial for appearance of activity.

Mono and bis(bioreductive) alkylating agents: synthesis and antitumor activities in a B16 melanoma model.

Several potentially bis(alkylating) bis(quinones) (3-5) and 1,4- and 1,3-bis(alkylating) monoquinones (6-13) belonging to general structure 2,2'-ethylenebis[5-[(leaving group)methyl]-1,4-benzoquinone] (3-5) and 2,5- and 2,6-bis[(leaving group)methyl]-1,4-benzoquinone water-soluble and -insoluble classes were prepared by oxidative demethylation of the corresponding tetramethoxydiphenylethanes (17-19) and dimethoxybenzenes (24, 27, 36-39), respectively. Methods employed for the preparation of tetramethoxydiphenylethane intermediates involved (1) arylmethyl bromide coupling and (2) catalytic hydrogenation of stilbene intermediates derived via Wittig reaction of (arylmethyl)phosphonium salts with aryl aldehydes. However, in biological investigations using a subcutaneous B16 (hypoxic) melanoma tumor in BDF1 hybrid mice with cyclophosphamide as positive control the most interesting series of structurally related analogues were the potentially monoalkylating monoquinones of the 2-[(leaving group)methyl]-1,4-benzoquinone type (i.e., 14 and 15) having water-insoluble (acetoxy) and water-solubilizing (succinate) groups. Serial measurements of tumor size, and evaluation of increased life span, in response to drug treatment also revealed potentially 1,4-bis(alkylating) (bromomethyl)-1,4-quinone 7 and 1,3-bis(alkylating) (hydroxymethyl)-1,4-quinone 10 to have variable activity, but none of the potentially bis(alkylating) bis(quinones) showed antitumor properties in this model.