Introduction to
experimental section
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Introduction to experimental section.
One of the main negative regulator of p53 is the ubiquitin E3 ligase Murine Double Minute 2 protein, MDM2, which is able to interact with p53 and mediate its degradation by proteosoma, thus blocking its activity. MDM2 downregulates p53 transcption factor activity by binding to its transactivation domain (TAD). As many kinds of tumors show an inactivation of p53 pathway, nongenotoxic therapeutic strategies that activate p53 in one way or another are highly desirable. The inactivation of p53 pathway may depends on nucleotide mutation of TP53 gene (p53 mutated) or altered regulation of its functions (p53 non mutated). When p53 is mutated, a valid strategy to promote mitochondria-mediated apoptosis seems to be the activation of the apoptotic factors Bax and Bak. On the contrary, when p53 is not mutated, but its altered functionality is related to the overexpression of MDM2 protein, the disruption of the p53-MDM2 interaction may represent a valuable strategy for the therapeutic treatment of cancer7.
The MDM2-p53 complex is characterized by an MDM2 hot spot occupied by the critical p53 residues Phe19, Trp23 and Leu26, which are inserted into a deep hydrophobic pocket on the surface of MDM2. The hydrophobic contacts are augmented by two hydrogen bonds, one between the Phe19 backbone amide NH of p53 and the carbonyl of the Glu72 side chain of MDM2 at one end of the cleft, and another between the p53 Trp23 indole NH and the MDM2 Leu54 backbone carbonyl group inside the cleft25.
Historically, it has been difficult to develop small-molecule inhibitors of non-enzyme protein-protein interactions, but the existence of such a well-defined pocket on the MDM2 ourface raised the expectation that compounds with low molecular weight could be found that would block the interaction of MDM2 with p5331.
Structural data suggest that a synthetic molecule featuring three hydrophobic groups in an orientation that mimics the side chains of the three key aminoacids by p53 should occupy the MDM2 cleft and inhibit the p53-MDM2 interaction. The first reported potent and selective small molecule MDM2 antagonists were a class of cis-imidazolines, the nutlins (Figure 19). Crystal structure studies demonstrated that the nutlins bind to the p53 pocket of MDM2 mimicking the molecular interactions between MDM2 and the crucial amino acid residues from p53. Other small molecules have been developed including calchone-based inhibitors, benzodiazepinones,
spiro-oxindoles, isoindolinones, piperidone derivatives,imidazolines and chromenotriazolpyrimidines (Figure 19).
Most of these small molecules share the common features of a rigid heterocyclic scaffold, decorated with p-halophenyl groups projected toward the phenylalanine and tryptophan binding pockets on the MDM2 surface. The aim of this research is to develop novel indole-based scaffold small molecules as potential inhibitors of the MDM2-p53 interaction on the basis of the structure of chalchone inhibitors. We designed a series a anilide derivatives by sostitution of the chetonic alfa beta insature system with a glyoxylamide moiety.
Figure 20. Structure of anilides and dipeptide derivatives.
In addition the phenyl directly linked to the carbonyl group in the calchones is replaced by the indole ring, reasonably maintaining the π-π interactions with the target protein and the H-binding ability (important in the interaction between the Trp23 and the Leu54 of MDM2). Finally the phenyl ring of the side chain could reproduce the other phenyl group of calchones. Further development of these derivatives will be the introduction of a lipophilic group at the 2-position of the indole ring, that is a methyl or a phenyl group which could be able to engage lipophilic interaction with specific areas in the cleft of MDM2. Another possible modification of the
2-phenylindolyoxylamide scaffold aimed to identify to design novel p53-MDM2 inhibitors may be represented by the introduction on the amide side chains of a dipeptide moiety, in order to mimic p53 residues Phe19, Trp23, and Leu26, critical for the interaction with MDM2. In this vein, a number of aminoacids featuring side chain of different lipophilic and elettronic properties will be inserted on the glyoxyl bride to create a small library.
The synthesis of anilide derivatives 7, 8 and 9 was reported in Scheme 1. The commercially available indoles (indole 1, 2-methylindole 2 and 2-phenylindole 3) were acylated with oxalyl chloride in anhydrous ethyl ether at 0°C to obtain the corresponding indolylglyoxylyl chlorides
4, 5 and 6 which were allowed to react at 0°C with the 4-chloroaniline in the presence of
triethylamine in dry toluene solution, to give products 7, 8 and 9.
SCHEME 1
The synthesis of the target compounds 13 and 14 is reported in Scheme 2. The indole (1) and 5-nitro-indole (10) are acylated with oxalyl chloride, in anhydrous ethyl ether at 0°C to obtain the corrisponding 2-phenylindolylglyoxylyl chlorides 4 and 11 which were allowed to react with L-Glycine-L-Phenylalanine ethyl ester 12 ( commercially available) in the presence of triethylamine in dry toluene solution at 0°C, to give compounds 13 and 14, which were purified by flash chromatography.
SCHEME 2
The synthesis of compounds 23 and 24 is reported in Scheme 3. Compound 17 is prepared with the condensation between the BOC-L-Leucine and the L-Phenylalanine ethyl ester. Then the BOC group is removed with trifluoroacetic acid (TFA) at room temperature and the dipeptide obtained (18) is reacted with the acyl chloride 6 and 22, obtained through the acylation of 2-phenyl indol 3, commercially available and the 2-(4'-cloro2-phenyl)indole 21 preparated with a one-step Fischer indole synthesis. Coumpound 23 was finally hydrolized to the corresponding acid 25 by treatment with solution of lithium hydroxide in an aqueous solution of methanol.
