阅读说明：本技术 小分子抑制剂在制备抑制鸟氨酸脱羧酶与鸟氨酸脱羧酶抗酶1的相互作用的制药用途 (Application of small molecule inhibitor in preparing medicine for inhibiting interaction of ornithine decarboxylase and ornithine decarboxylase antitase 1 ) 是由 刘森 何梦溪 柴晓颖 占景琼 于 2019-04-26 设计创作，主要内容包括：本发明提供小分子抑制剂在制备抑制鸟氨酸脱羧酶与鸟氨酸脱羧酶抗酶1的相互作用的制药用途,以及在制备稳定鸟氨酸脱羧酶非活性二聚体的药物上的应用。所述的小分子抑制剂包括6-[N’-(4-二甲基氨基苯甲基)-肼基-[1,3,5]三氮唑-2,4-二胺、或1-丁基-N-(4-氯苯基)-1H-苯并[d]咪唑-2-胺、或1,3-二甲基-8-(2-噻吩基)-3,7-二氢-1H-嘌呤-2,6-二酮中的任意一种。(The invention provides a pharmaceutical application of a small molecule inhibitor in preparation of a medicament for inhibiting the interaction of ornithine decarboxylase and ornithine decarboxylase antitase 1 and an application in preparation of a medicament for stabilizing an ornithine decarboxylase inactive dimer. The small molecule inhibitor comprises any one of 6- [ N' - (4-dimethylaminobenzoyl) -hydrazino- [1,3,5] triazole-2, 4-diamine, or 1-butyl-N- (4-chlorphenyl) -1H-benzo [ d ] imidazole-2-amine, or 1, 3-dimethyl-8- (2-thienyl) -3, 7-dihydro-1H-purine-2, 6-diketone.)

1. The application of the small molecule inhibitor in preparing the medicines for inhibiting the interaction of the ornithine decarboxylase and the ornithine decarboxylase antithrombase 1 is characterized in that the small molecule inhibitor comprises any one of 6- [ N' - (4-dimethylaminobenzoyl) -hydrazino- [1,3,5] triazole-2, 4-diamine, 1-butyl-N- (4-chlorphenyl) -1H-benzo [ d ] imidazole-2-amine, or 1, 3-dimethyl-8- (2-thienyl) -3, 7-dihydro-1H-purine-2, 6-dione.

2. The use of a small molecule inhibitor according to claim 1 for the preparation of a medicament for stabilizing an ornithine decarboxylase inactive dimer.

Technical Field

The invention relates to a small molecule inhibitor and pharmaceutical application thereof, in particular to pharmaceutical application of- [ N' - (4-dimethylaminobenzoyl) -hydrazino- [1,3,5] triazole-2, 4-diamine, or 1-butyl-N- (4-chlorphenyl) -1H-benzo [ d ] imidazole-2-amine, or 1, 3-dimethyl-8- (2-thienyl) -3, 7-dihydro-1H-purine-2, 6-diketone and similar compounds thereof.

Background

Proteins are one of the major components of living organisms and are the main substances that perform various vital activities. Among various proteins, proteases are essential for life activities, and almost all biochemical reaction processes in organisms are catalyzed by proteases. The activity of various proteases in organisms has strict regulation mechanism, and once the regulation mechanism has problems, the corresponding diseases can be caused if the activity of the proteases is too high, too low or completely inactivated. Therefore, the regulation and control of the activity of the protease by the medicament to recover and maintain the protease at a normal level have very important theoretical significance and practical significance. The structure-based drug design is a very important means for designing a protein-targeted drug.

Polyamines (polyamines) are positively charged cationic small molecules produced from amino acid metabolism, which are present in all organisms and essential for cell growth, differentiation, survival and normal biological functions. The multiple positive charge nature of polyamines allows them to regulate a very wide range of biological processes including chromosome structure formation, DNA synthesis and stabilization, DNA replication, transcription and translation, protein phosphorylation, ribosome production, regulation of ion channels and membrane surface receptors, free radical scavenging, etc. by electrostatic interaction with negatively charged biological macromolecules (DNA, RNA, proteins, cell membranes, etc.). There are many natural polyamines. In mammals, there are three naturally occurring species, putrescine (putrescine), speramine (spermidine), speramine (spermine), which are essential for the normal growth and development of mammals. Since polyamines have important biological functions, their intracellular levels are tightly regulated. Polyamine levels and ODC expression levels are also elevated and deregulated in rapidly proliferating cells, such as tumor cells. The increase of polyamine level is accompanied by the acceleration of cell proliferation, the decrease of apoptosis, the increase of expression level of tumor infiltration and metastasis related genes and the like. Therefore, the regulation and control of polyamine become an important means in tumor treatment and drug development.

The initial substrate for polyamine metabolism is ornithine (ornithine), which is the reaction product of arginine catalyzed by arginase (arginase) in the urea cycle (urea). ODC is the first enzyme in the polyamine synthesis pathway, catalyzing the reaction from ornithine (ornithline) to putrescine, a step which is also a rate limiting step in the polyamine synthesis pathway. Therefore, synthesizing ODC inhibitor, inhibiting putrescine generation, is a tumor treatment approach which is very much concerned at present. Also, because pathogenic microorganisms also require normal polyamine levels, ODC inhibitors are also important targets for pathogenic microorganisms (e.g., trypanosoma brucei causing africana trypanosomiasis).

Currently, the ODC inhibitor DFMO (α -difluoromethylornithine) has been used clinically to assist cancer chemotherapy. But the binding capacity of the inhibitor and ODC is weak, the action concentration is high, and the toxic and side effects are very large due to the suicide inhibitor forming covalent bonds with ODC. Therefore, there is a great need to develop novel ODC inhibitors having better effects.

Disclosure of Invention

The invention aims to obtain a multifunctional small molecule compound which can inhibit the activity of Ornithine Decarboxylase (ODC), stabilize an Ornithine Decarboxylase (ODC) inactive dimer and inhibit the interaction of the ornithine decarboxylase and ornithine decarboxylase antitase 1(ODC-OAZ1) by utilizing computer-aided screening.

The technical scheme of the invention is that a binding pocket which can be used for drug design is found and determined by analyzing the crystal structure of ODC and utilizing protein drug pocket analysis software, and the pocket is subjected to small molecule drug screening and verification.

According to the scheme, firstly, the substrate and the PLP ligand binding Pocket of the human ODC are analyzed by using Pocket protein drug Pocket analysis software based on the crystal structure of the human ODC. With the help of Pocket software, it was determined that the region where the substrate and PLP ligand on ODC homodimer interface are located is a drug design Pocket. In order to verify the feasibility of the pocket as a drug pocket, drug screening and experimental verification were performed.

The experimental subjects were human ODCs, but due to the homology between ODCs of different origins, the inhibitors may be able to act equally well on other ODCs of non-human origin, as well as on proteins that are highly homologous to the substrates and PLP binding pockets of the ODCs.

Based on the work, the technical scheme of the invention screens and synthesizes a small molecule inhibitor compound, in particular to a synthesis method of 6- [ N' - (4-dimethylamino benzylidene) -hydrazino- [1,3,5] triazole-2, 4-diamine, which comprises the following steps:

Sequentially adding D24-1-1, hydrazine hydrate and water into a reaction vessel, heating to 70-90 ℃ under the protection of nitrogen, reacting for 5-8h, cooling to room temperature, filtering, and drying in vacuum to obtain a white D24-1-2 solid;

d24-1-2, D24-1-3 and methanol are sequentially added into a reaction container, the mixture reacts for 15-20h at the temperature of 25-40 ℃ under the protection of nitrogen, the mixture is cooled to room temperature and filtered, the obtained solid is pulped by DMSO, the obtained solid is filtered under the protection of nitrogen, and a white D24-1-4 solid compound is obtained after vacuum drying, so that a product 6- [ N' - (4-dimethylaminobenzylidene) -hydrazino- [1,3,5] triazole-2, 4-diamine is obtained, and the synthetic route is as follows:

the mass concentration of the hydrazine hydrate is 75-90%.

The mass ratio of D24-1-1, hydrazine hydrate with the mass concentration of 75-90% and water is 1: 1.5-2.5: 8-15.

The mass ratio of D24-1-2 to D24-1-3 to methanol is 1: 0.9-1.1: 8-18.

The invention relates to application of the prepared micromolecular compound 6- [ N' - (4-dimethylamino benzylidene) -hydrazino- [1,3,5] triazole-2, 4-diamine as an inhibitor in preparation of a medicament for inhibiting interaction of ornithine decarboxylase and ornithine decarboxylase antitase 1.

Or the 6- [ N' - (4-dimethylamino benzylidene) -hydrazino- [1,3,5] triazole-2, 4-diamine prepared by the method is applied to the preparation of medicaments for stabilizing the ornithine decarboxylase inactive dimer.

Furthermore, the invention also provides the application of the 1-butyl-N- (4-chlorphenyl) -1H-benzo [ d ] imidazole-2-amine in preparing medicines for inhibiting the interaction of ornithine decarboxylase and ornithine decarboxylase antitase 1.

Furthermore, the invention also provides the application of the 1, 3-dimethyl-8- (2-thienyl) -3, 7-dihydro-1H-purine-2, 6-diketone in preparing drugs for inhibiting the interaction of the ornithine decarboxylase and the ornithine decarboxylase antitinase 1.

Furthermore, the invention also provides the application of the 1-butyl-N- (4-chlorphenyl) -1H-benzo [ d ] imidazole-2-amine in preparing the medicament for stabilizing the inactive dimer of the ornithine decarboxylase.

Furthermore, the invention also provides the application of the 1, 3-dimethyl-8- (2-thienyl) -3, 7-dihydro-1H-purine-2, 6-diketone in preparing the medicament for stabilizing the non-active dimer of the ornithine decarboxylase.

In the technical scheme of the invention, in order to obtain the micromolecules with 3 functions simultaneously by screening, the following processes and strategies are adopted: first, based on the structural analysis of ODC homodimers (see fig. 1, wherein fig. 1 is a structural diagram of human ODC homodimers, one strand is shown as surface and the other is shown as cartoon.), a pocket (see fig. 2, wherein fig. 2 is a schematic diagram of substrate and PLP binding sites of ODC, one strand is shown as surface and the other is shown as cartoon, and substrate (putrescine) and PLP are shown as sticks) composed by co-factor PLP and substrate binding sites thereof is determined as a screening target for small molecule screening. To obtain molecules that inhibit ODC activity, the following 3 steps were used.

In the first step, residues contributing a smaller number of residues to the pocket (see FIG. 3) are first removed, followed by molecular docking, and the binding score of small molecules is required to be lower than that of PLP.

In the second step, in order to obtain a small molecule capable of inhibiting the ODC enzyme activity and stabilizing the ODC inactive homodimer, the small molecule obtained in the first step was further docked into the dimer common pocket of FIG. 3, and it was required that the small molecule had a similar binding conformation as the first step and a lower binding score than the first step. FIG. 3 is a schematic representation of the dimer interface pocket, shown as a semi-transparent surface, with surrounding rods of the side chains of amino acid residues on the ODC homodimer, with the residues making up the pocket being Phe65, Ala67, Lys69, Cys70, Asp88, Ala90, Ala111, Asn112, Pro113, Thr132, Arg154, Cys164, Arg165, Leu166, Phe170, Phe196, His197, Gly199, Ser200, Gly201, Gly235, Gly236, Gly237, Phe238, Pro239, Glu274, Pro275, Gly276, Arg277, Tyr278, Asn327, Cys328, Tyr331, Asp332, His, Ala388, Tyr389, and Tyr323, Thr359, 333, Asp361, Gly362, Leu 39397, Asn on one of the protein monomers; the thin stick in the pocket was the known inhibitors DFMO of PLP, putrescine and ODC, respectively.

And thirdly, in order to further screen molecules capable of inhibiting the ODC-OAZ1 interaction from the molecules obtained in the second step, the small molecules are butted to a binding pocket part formed by the two proteins together at the binding interface of the ODC-OAZ1 complex (figure 4, figure 4: ODC-OAZ1 complex pocket for butting screening, wherein the black color is ODC, the light gray color is OAZ1, the sphere shows PLP.PLP binding sites and the surrounding space is a small molecule butting region). It is further required that small molecules cannot bind to the pocket in a similar conformation as the first and second steps.

Small molecules that satisfy the above-described rules at the same time are considered to be small molecules that can simultaneously inhibit ODC activity, stabilize ODC-inactive dimers, and inhibit ODC-OAZ1 interaction.

Small molecules that bind to the pocket of figure 3 of an ODC, while generally inhibiting the enzymatic activity of the ODC, do not naturally serve the latter two functions. For example, the specific inhibitor of ODC, DFMO, has not been reported and experimental evidence suggests the latter two functions; in contrast, DFMO is able to promote to some extent the interaction of ODC and OAZ1, which is also one of the major factors in the limited clinical function of DFMO. Therefore, the obtained small molecules have great innovative value through the innovative screening approach of the technical scheme of the invention.

Drawings

FIG. 1 is a schematic diagram of the structure of a human ODC homodimer, one strand being shown as a surface and the other as a cartoon.

FIG. 2 is a schematic representation of the substrate and PLP binding sites of ODC, one strand shown as a surface, the other as a cartoon, and the substrate (putrescine) and PLP shown as sticks.

FIG. 3 is a schematic diagram of the dimer interface pocket, with the thin stick in the pocket being DFMO, a known inhibitor of PLP, putrescine and ODC, respectively.

FIG. 4: ODC-OAZ1 complex pocket for docking screening. In the figure, ODC is black, OAZ1 is light gray, and PLP is shown as a sphere.

FIG. 5: the small molecule inhibitor 6- [ N' - (4-dimethylaminobenzylidene) -hydrazino- [1,3,5] triazole-2, 4-diamine of example 1 was tested for its inhibition of ODC-OAZ1 interaction based on Fluorescence Resonance Energy Transfer (FRET).

FIG. 6 is the oligomeric state of ODC at different salt concentrations.

FIG. 7 shows the effect of the small molecule inhibitors 6- [ N' - (4-dimethylaminobenzylidene) -hydrazino- [1,3,5] triazole-2, 4-diamine of example 1, DFMO and a control solution without any small molecule on ODC oligomerization status under 50mM NaCl.

FIG. 8 shows the effect of the small molecule inhibitors 6- [ N' - (4-dimethylaminobenzylidene) -hydrazino- [1,3,5] triazole-2, 4-diamine of example 1, DFMO and a control solution without any small molecule addition on ODC oligomerization status under 150mM NaCl.

FIG. 9 is a graph showing the effect of the small molecule inhibitor 6- [ N' - (4-dimethylaminobenzylidene) -hydrazino- [1,3,5] triazole-2, 4-diamine of example 1 on the volume growth of transplanted tumors.

FIG. 10 is a graph showing the effect of the small molecule inhibitor 6- [ N' - (4-dimethylaminobenzylidene) -hydrazino- [1,3,5] triazole-2, 4-diamine of example 1 on the weight gain of transplanted tumors.

FIG. 11 is a graph showing the effect of the small molecule inhibitor 6- [ N' - (4-dimethylaminobenzylidene) -hydrazino- [1,3,5] triazole-2, 4-diamine of example 1 on body weight in nude mice.

FIG. 12 is a graph showing the effect of various concentrations of the small molecule inhibitor 6- [ N' - (4-dimethylaminobenzylidene) -hydrazino- [1,3,5] triazole-2, 4-diamine of example 1 on tumor tissue morphology.

FIG. 13 is a high performance liquid chromatogram of the small molecule inhibitor 6- [ N' - (4-dimethylaminobenzylidene) -hydrazino- [1,3,5] triazole-2, 4-diamine of example 1.

FIG. 14 is a mass spectrum of 6- [ N' - (4-dimethylaminobenzylidene) -hydrazino- [1,3,5] triazole-2, 4-diamine, a small molecule inhibitor of example 1.

FIG. 15 is the nuclear magnetic hydrogen spectrum of the small molecule inhibitor 6- [ N' - (4-dimethylaminobenzylidene) -hydrazino- [1,3,5] triazole-2, 4-diamine of example 1.

FIG. 16 is a graph showing the half inhibitory concentration of the inhibitor 1, 3-dimethyl-8- (2-thienyl) -3, 7-dihydro-1H-purine-2, 6-dione on ODC enzyme activity.

FIG. 17 is a graph of the inhibition of ODC-OAZ1 interaction by the small molecule inhibitor 1, 3-dimethyl-8- (2-thienyl) -3, 7-dihydro-1H-purine-2, 6-dione of example 2 based on Fluorescence Resonance Energy Transfer (FRET) assay.

FIG. 18 is the oligomeric state of ODC at different salt concentrations.

FIG. 19 is a graph showing the effect of the small molecule inhibitors 1, 3-dimethyl-8- (2-thienyl) -3, 7-dihydro-1H-purine-2, 6-dione, DFMO of example 2 and a solution control without any small molecule on ODC oligomerization status under 50mM NaCl.

FIG. 20 is a graph showing the effect of the small molecule inhibitors 1, 3-dimethyl-8- (2-thienyl) -3, 7-dihydro-1H-purine-2, 6-dione, DFMO of example 2 and a solution control without any small molecule on ODC oligomerization status under 150mM NaCl.

FIG. 21 shows the half inhibitory concentration of the inhibitor 1-butyl-N- (4-chlorophenyl) -1H-benzo [ d ] imidazol-2-amine on ODC enzyme activity.

FIG. 22 is a graph of the inhibition of ODC-OAZ1 interaction by the small molecule inhibitor 1-butyl-N- (4-chlorophenyl) -1H-benzo [ d ] imidazol-2-amine of example 3, detected based on Fluorescence Resonance Energy Transfer (FRET).

FIG. 23 is the oligomeric state of ODC at different salt concentrations.

FIG. 24 shows the effect of the small molecule inhibitors 1-butyl-N- (4-chlorophenyl) -1H-benzo [ d ] imidazol-2-amine of example 3, DFMO and a solution control without any small molecule addition on ODC oligomerization status under 50mM NaCl.

FIG. 25 shows the effect of the small molecule inhibitors 1-butyl-N- (4-chlorophenyl) -1H-benzo [ d ] imidazol-2-amine of example 3, DFMO and a control solution without any small molecule addition on ODC oligomerization status under 150mM NaCl.

Detailed Description