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Small-molecule inhibitor of Mdm<X>/Mdm<2>, as well as preparation method and applications

A technology of small molecule inhibitors and chemical structural formulas, which is applied in the field of medicinal chemistry technology and can solve the problems of no cis-imidazoline framework, no reversible inhibitors, and no such problems

Active Publication Date: 2014-07-16
HUBEI UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Before the present invention, there was no patent report on the high-affinity MdmX reversible inhibitor based on the cis-imidazoline framework, no patent report on the high-affinity Mdm2 / MdmX dual inhibitor based on the cis-imidazoline framework, and no There are reports of high-affinity MdmX inhibitors and Mdm2 / MdmX dual inhibitors combining cis-imidazolines and indole

Method used

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  • Small-molecule inhibitor of Mdm&lt;X&gt;/Mdm&lt;2&gt;, as well as preparation method and applications
  • Small-molecule inhibitor of Mdm&lt;X&gt;/Mdm&lt;2&gt;, as well as preparation method and applications
  • Small-molecule inhibitor of Mdm&lt;X&gt;/Mdm&lt;2&gt;, as well as preparation method and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1: A preparation method of a small molecule inhibitor of MdmX / Mdm2:

[0057] The small molecule inhibitors of MdmX / Mdm2 are cis-imidazolines, and their synthesis routes are as follows: figure 1 As shown, the principle is to use a diastereoselective and stereoselective catalyst to catalyze the addition reaction of arylnitromethane and Schiff base (Davis, T.A. & Johnston, J.N. Chem. Sci.2, 1076-1079, 2011).

[0058] R 1 The functional group was introduced using nitrobenzene (compound 8). Using halogenated benzene as raw material, prepared according to the method described by Kornblum (JACS,1956,78:1497), functional group R 1 (4) Hydroxyl, R 1 Carboxyl and R in (5) 1 The amides in (6) are respectively protected by methyl ether, methyl ester and benzyl, and deprotected by hydrolysis in the later stage of synthesis.

[0059] R 2 The functional group was introduced using aryl imine (compound 7). Using α-aminophenyl sulfone or α-aminoindole sulfone as raw materi...

Embodiment 2

[0063] Embodiment 2: MdmX and Mdm2 protein sample preparation method:

[0064] The amino-terminal amino acid 22-110 sequence fragment of human MdmX (N-MdmX) and the Mdm2 amino-terminal amino acid 22-110 (N-Mdm2) sequence fragment were synthesized according to the codon preference of Escherichia coli. The artificially synthesized DNA fragments were respectively used Restriction endonucleases BamH I and EcoR I were treated for use.

[0065] The commercialized pET28a plasmid (Novagen) was used for protein expression, and its original thrombin cleavage site (Leu-Val-Pro-Arg-Gly-Ser) was replaced with the TEV protease cleavage site (Glu -Asn-Leu-Tyr-Phe-Gln-Gly), the new plasmid was named pSD. pSD was treated with the same restriction endonuclease for use.

[0066] The N-Mdm2 and MdmX DNA fragments treated with BamH I and EcoR I were respectively connected to the pSD vector treated with the same restriction enzymes to construct the N-Mdm2 and MdmX protein expression plasmids, pSD...

Embodiment 3

[0070] Embodiment 3, high-throughput MdmX inhibitor screening method:

[0071] MdmX inhibitor screening was determined by fluorescence polarization (FP) method. Performed in assay buffer containing 10 mM Tris (pH 8.0), 200 mM NaCl and 0.01% Tween-20. The human p53 protein amino acid 15-29 sequence fragment peptide (p53p) was labeled with fluorescein (fluorescein-GSGSSQETFSDLWKLLPEN, Flu-p53p). Mutant p53 peptides (fluorescein-GSGSSQETASDLAKLAPEN, Flu-p53pAAA) were used as negative controls.

[0072]FP detection was performed using 15nM fluorescein and 1μM N-MdmX in a buffer containing 10mM Tris (pH 8.0), 200mM NaCl and 0.01% Tween-20. In the N-MdmX / p53p inhibitor assay, the nutlin analog was premixed with the protein for 30 minutes, then the labeled peptide was added and mixed for 30 minutes. The (Corning) FP assay was then performed in 384-well black microplates. FP assay analysis was performed using an EnVision multilabel microplate reader with 555nm excitation filter, 6...

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Abstract

The invention relates to the technical field of medicinal chemistry, particularly discloses a small-molecule inhibitor of Mdm<X> / Mdm<2>, and also relates to a preparation method of the small-molecule inhibitor of Mdm<X> / Mdm<2>. The small-molecule inhibitor compound can inhibit the interaction of Mdm<X> protein and p53 protein and also can inhibit the interaction of Mdm<2> protein and p53 protein, has antiproliferative activity on cancer cells, and cannot generate toxic and side effects on patients. The small-molecule inhibitor compound can be combined with other therapies for use.

Description

technical field [0001] The present invention relates to the technical field of medicinal chemistry, more specifically to a small molecule inhibitor of MdmX / Mdm2, and also to a preparation method of a small molecule inhibitor of MdmX / Mdm2, the small molecule inhibitor compound can inhibit Mdmx protein and The interaction of p53 protein can also inhibit the interaction between Mdm2 protein and p53 protein, and the small molecule inhibitor compound has anti-proliferation activity on cancer cells. Background technique [0002] Inactivation of p53 protein's tumor suppressive effect is associated with the occurrence of most human tumors. Muller et al. evaluated the p53 gene mutation or the inhibition by Mdm2 and MdmX proteins, which is the inactivation of p53 function and the mechanism related to tumorigenesis (Muller, P.A. & Vousden, K.H. Nat. Cell Biol. 15, 2-8 (2013). Analysis by Wade et al. Finding more and more evidences that the expression of Mdm2 and / or MdmX is increased t...

Claims

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Application Information

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IPC IPC(8): C07D403/06C07D403/14A61P35/00
CPCC07D403/06C07D403/14
Inventor 苏正定张华山陈瑶王伟平
Owner HUBEI UNIV OF TECH
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