CN111840298A

CN111840298A - Application of small molecule inhibitor in preparing medicine for inhibiting interaction of ornithine decarboxylase and ornithine decarboxylase antitase 1

Info

Publication number: CN111840298A
Application number: CN201910346073.9A
Authority: CN
Inventors: 刘森; 何梦溪; 柴晓颖; 占景琼
Original assignee: Hubei University of Technology
Current assignee: Hubei University of Technology
Priority date: 2019-04-26
Filing date: 2019-04-26
Publication date: 2020-10-30

Abstract

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.

Description

Application of small molecule inhibitor in preparing medicine for inhibiting interaction of ornithine decarboxylase and ornithine decarboxylase antitase 1

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

Example 1

Small molecule inhibitors: a chemical synthesis way of 6- [ N' - (4-dimethylamino benzylidene) -hydrazino- [1,3,5] triazole-2, 4-diamine.

5g of D24-1-1 (purchased from Shanghai Aladdin Biotechnology Co., Ltd.), 11g of hydrazine hydrate (purchased from Shanghai Aladdin Biotechnology Co., Ltd.) (80 wt%) and 50ml of water are sequentially added into a 100ml single-mouth bottle, the temperature is raised to 85 ℃, the reaction is carried out for 7h under the protection of nitrogen, the temperature is lowered to room temperature, the filtration and the vacuum drying are carried out (45 ℃, 24h), 3.85g of white D24-1-2 solid is obtained, and the yield is 79.4%.

In a 100ml single-neck bottle, D24-1-23.5 g, D24-1-3 (purchased from Shanghai Aladdin Biotechnology Co., Ltd.) 3.8g and methanol 40ml are added in sequence, nitrogen protection is carried out, reaction is carried out for 18h at 30 ℃, cooling to room temperature, filtration is carried out, the obtained solid is pulped by DMSO 20ml, nitrogen protection is carried out, filtration is carried out, vacuum drying is carried out for 12h at 45 ℃, and white D24-1-4 solid compound 2.1g (yield 31.1%) is obtained.

The purity of D24-1-4 was determined by HPLC to be 98% or more, as shown in FIG. 13.

The molecular weight of D24-1-4 was determined by mass spectrometry, which is consistent with the theoretical molecular weight, as shown in FIG. 14.

The NMR results for D24-1-4 also agreed with the theoretical values, as shown in FIG. 15.

FIG. 19: d24-1-4 nuclear magnetic hydrogen spectrum analysis results.¹H NMR(400MHz,DMSO)10.23(s,1H),7.94(s,1H),7.42(d,J＝8.9Hz,2H),6.73(d,J＝8.9Hz,2H),6.27(br,4H),2.95(d,J＝4.1Hz,6H).

In conclusion, the synthesis method can effectively synthesize the high-purity D-24-1-4 compound.

For the 6- [ N' - (4-dimethylaminobenzylidene) -hydrazino- [1,3,5] triazole-2, 4-diamine small molecule, in order to verify the inhibition of ODC-OAZ1 interaction, a Fluorescence Resonance Energy Transfer (FRET) based method was used. ODC protein labeled with YPET fluorescent protein (YPET-ODC) and OAZ1 labeled with CyPET fluorescent protein (CyPET-OAZ1) were purified by expression and then incubated together. Under normal conditions, when ODC and OAZ1 are bonded to each other, YPET and CyPET can be brought into close proximity, and FRET occurs. If the small molecule is capable of inhibiting the interaction of ODC and OAZ1, the FRET signal may be reduced to some extent. The inhibition of the small molecule to ODC-OAZ1 interaction based on Fluorescence Resonance Energy Transfer (FRET) detection as shown in FIG. 5, the small molecule of 6- [ N' - (4-dimethylamino benzylidene) -hydrazino- [1,3,5] triazole-2, 4-diamine can obviously inhibit FRET signal. This means that the 6- [ N' - (4-dimethylaminobenzylidene) -hydrazino- [1,3,5] triazole-2, 4-diamine small molecule can inhibit the interaction of ODC and OAZ 1.

In the previous patent, 6- [ N '- (4-dimethylaminobenzylidene) -hydrazino- [1,3,5] triazole-2, 4-diamine was tested for its inhibition of ODC enzyme activity, and in order to further test whether 6- [ N' - (4-dimethylaminobenzylidene) -hydrazino- [1,3,5] triazole-2, 4-diamine molecules can stabilize ODC homodimers, the oligomeric state of ODC was analyzed using an analytical size exclusion gel column. As shown in FIG. 6 (FIG. 6 shows that ODC differ in their oligomeric state at different salt concentrations, the earlier the time of elution represents the easier the formation of ODC homodimers under the conditions), ODC differ in their oligomeric state at different salt (NaCl) concentrations, and differ in their elution time from the gel column (the earlier the time, the larger the molecular weight): under 50mM NaCl, the ODC exists mainly in a dimer form (calculated molecular weight: 105.1kDa), under 300mM NaCl, the ODC exists mainly in a monomer form (calculated molecular weight: 53.0kDa), and under 150mM NaCl, the monomer and dimer in the ODC are in a coexisting and rapid exchange state, with a corresponding efflux peak therebetween.

As shown in FIG. 7 (FIG. 7 shows the effect of 6- [ N ' - (4-dimethylaminobenzylidene) -hydrazino- [1,3,5] triazole-2, 4-diamine molecules and DFMO on the oligomerization state of ODC under 50mM NaCl, without adding any small molecule solution as a control. the three flow curves almost completely coincide, meaning that neither the 6- [ N ' - (4-dimethylaminobenzylidene) -hydrazino- [1,3,5] triazole-2, 4-diamine molecules nor DFMO would destroy ODC homodimers under the conditions), under 50mM NaCl, the 6- [ N ' - (4-dimethylaminobenzylidene) -hydrazino- [1,3,5] triazole-2, 4-diamine molecules, The high coincidence of the efflux peak heights of both DFMO and the no small molecule control means that neither A molecule nor DFMO destabilizes the ODC homodimer. As shown in FIG. 8 (FIG. 8 is a graph showing the effect of 6- [ N ' - (4-dimethylaminobenzylidene) -hydrazino- [1,3,5] triazole-2, 4-diamine molecules and DFMO on the oligomerization state of ODC under 150mM NaCl, without adding any small molecule solution as a control.) the curve of the addition of 6- [ N ' - (4-dimethylaminobenzylidene) -hydrazino- [1,3,5] triazole-2, 4-diamine molecules shows an advancing efflux peak, while the efflux peaks of DFMO and a blank control overlap, which means that 6- [ N ' - (4-dimethylaminobenzylidene) -hydrazino- [1,3,5] triazole-2, 4-diamine molecules can stabilize ODC homodimers, while DFMO cannot.), under the condition of 150mM NaCl, the 6- [ N' - (4-dimethylaminobenzenemethylene) -hydrazino- [1,3,5] triazole-2, 4-diamine molecule can obviously lead the outflow peak to be moved forward, which means that the content of ODC homodimer is increased. While DFMO was consistent with the blank, indicating that DFMO did not promote ODC homodimer formation.

From the above results, it can be concluded that the small 6- [ N' - (4-dimethylaminobenzenemethylene) -hydrazino- [1,3,5] triazole-2, 4-diamine molecule described in this example is a novel small molecule compound capable of inhibiting ODC enzyme activity, stabilizing ODC homodimer, and inhibiting ODC-OAZ1 interaction.

In order to further study the potential of the 6- [ N' - (4-dimethylaminobenzylidene) -hydrazino- [1,3,5] triazole-2, 4-diamine small molecule used as a living drug, the stability of liver microsome (microsome) of the small molecule is evaluated. The stability of the liver microsomes refers to evaluating whether a compound is metabolized by liver drug metabolizing enzymes so as to influence the bioavailability of the compound and provide parameters for the design of identification of metabolites and pathways of the metabolites. The remaining percentage of control drug is high over the test period and can be considered to be stable.

A transplantation tumor model was constructed by inoculating a549 cells in nude mice. Then, the compound is continuously administrated through the abdominal cavity for 10 days, and the inhibition effect of the 6- [ N' - (4-dimethylamino benzylidene) -hydrazino- [1,3,5] triazole-2, 4-diamine compound on the transplanted tumor is evaluated. As shown in FIG. 9 and FIG. 10, the 6- [ N' - (4-dimethylaminobenzenemethylene) -hydrazino- [1,3,5] triazole-2, 4-diamine compound can obviously inhibit the increase of the tumor volume and the tumor weight under the administration dosage of 3.6mg/kg and 0.36 mg/kg. As shown in fig. 11, the body weight of the nude mice was not significantly affected at the above concentrations. As shown in FIG. 12, there was a significant difference in the tumor tissues exfoliated from the nude mice administered with the drug.

In conclusion, the 6- [ N' - (4-dimethylaminobenzenemethylene) -hydrazino- [1,3,5] triazole-2, 4-diamine compound is a safe and effective compound for inhibiting tumor growth.

Example 2

The small molecule inhibitor 1, 3-dimethyl-8- (2-thienyl) -3, 7-dihydro-1H-purine-2, 6-dione, available from SPECS (http:// www.specs.net).

In order to verify whether the 1, 3-dimethyl-8- (2-thienyl) -3, 7-dihydro-1H-purine-2, 6-dione small molecule can inhibit the enzymatic activity of ODC, the enzyme catalysis product putrescine is detected by High Performance Liquid Chromatography (HPLC), and the half inhibition concentration of the C small molecule on the enzymatic activity of ODC is about 31.8 mu M (figure 16). FIG. 16 is a graph showing the detection of putrescine, a catalytic product of ODC, by HPLC, and the efficiency of ODC enzyme activity inhibition by small molecules was calculated by comparison with the case where no small molecules were added.

To verify the inhibition of the ODC-OAZ1 interaction by 1, 3-dimethyl-8- (2-thienyl) -3, 7-dihydro-1H-purine-2, 6-dione small molecule, a Fluorescence Resonance Energy Transfer (FRET) -based method was used. ODC protein labeled with YPET fluorescent protein (YPET-ODC) and OAZ1 labeled with CyPET fluorescent protein (CyPET-OAZ1) were purified by expression and then incubated together. Under normal conditions, when ODC and OAZ1 are bonded to each other, YPET and CyPET can be brought into close proximity, and FRET occurs. If the small molecule is capable of inhibiting the interaction of ODC and OAZ1, the FRET signal may be reduced to some extent. The inhibition of ODC-OAZ1 interaction by small molecules based on Fluorescence Resonance Energy Transfer (FRET) assay, as depicted in fig. 17, 1, 3-dimethyl-8- (2-thienyl) -3, 7-dihydro-1H-purine-2, 6-dione small molecules are able to significantly suppress FRET signals. This means that small 1, 3-dimethyl-8- (2-thienyl) -3, 7-dihydro-1H-purine-2, 6-dione molecules are able to inhibit the interaction of ODC and OAZ 1.

The inhibition of ODC enzyme activity by 1, 3-dimethyl-8- (2-thienyl) -3, 7-dihydro-1H-purine-2, 6-dione was verified as described above, and in order to further verify whether 1, 3-dimethyl-8- (2-thienyl) -3, 7-dihydro-1H-purine-2, 6-dione molecules can stabilize ODC homodimers, the oligomeric state of ODC was analyzed using an analytical size exclusion gel column. As shown in FIG. 18 (FIG. 18 shows the oligomeric state of ODC at different salt concentrations, the earlier the time of elution represents the easier the formation of ODC homodimers under the conditions), the oligomeric state of ODC is different at different salt (NaCl) concentrations, and the time of elution from the gel column is different (the earlier the time, the larger the molecular weight): under 50mM NaCl, the ODC exists mainly in a dimer form (calculated molecular weight: 105.1kDa), under 300mM NaCl, the ODC exists mainly in a monomer form (calculated molecular weight: 53.0kDa), and under 150mM NaCl, the monomer and dimer in the ODC are in a coexisting and rapid exchange state, with a corresponding efflux peak therebetween.

As shown in FIG. 19 (FIG. 19 is a graph showing the effect of 1, 3-dimethyl-8- (2-thienyl) -3, 7-dihydro-1H-purine-2, 6-dione molecules and DFMO on the oligomerization state of ODC under 50mM NaCl conditions, without adding any small molecule as a control. the three flow curves almost completely coincide, meaning that neither 1, 3-dimethyl-8- (2-thienyl) -3, 7-dihydro-1H-purine-2, 6-dione molecules nor DFMO damages ODC homodimers under these conditions), 1, 3-dimethyl-8- (2-thienyl) -3, 7-dihydro-1H-purine-2 under 50mM NaCl conditions, the high coincidence of the efflux peaks for the 6-dione molecule, DFMO and the no small molecule control means that neither the 1, 3-dimethyl-8- (2-thienyl) -3, 7-dihydro-1H-purine-2, 6-dione molecule nor DFMO destroyed the stability of the ODC homodimer.

As shown in FIG. 20 (FIG. 20 is a graph showing the effect of 1, 3-dimethyl-8- (2-thienyl) -3, 7-dihydro-1H-purine-2, 6-dione and DFMO on the oligomerization state of ODC under 150mM NaCl conditions, without adding any small molecule solution as a control. the curve efflux peaks of the 1, 3-dimethyl-8- (2-thienyl) -3, 7-dihydro-1H-purine-2, 6-dione molecule added are shifted forward, while the efflux peaks of DFMO and the blank control overlap, which means that the 1, 3-dimethyl-8- (2-thienyl) -3, 7-dihydro-1H-purine-2, 6-dione molecule can stabilize ODC homodimer, while DFMO cannot. ) The ability of the 1, 3-dimethyl-8- (2-thienyl) -3, 7-dihydro-1H-purine-2, 6-dione molecule to significantly shift the efflux peak at 150mM NaCl suggests an increased ODC homodimer content. While DFMO was consistent with the blank, indicating that DFMO did not promote ODC homodimer formation.

From the above results, it can be concluded that the small 1, 3-dimethyl-8- (2-thienyl) -3, 7-dihydro-1H-purine-2, 6-dione molecule described in this example is a novel small molecule compound capable of inhibiting ODC enzyme activity, stabilizing ODC homodimer, and inhibiting ODC-OAZ1 interaction.

Example 3

Small molecule inhibitors: 1-butyl-N- (4-chlorophenyl) -1H-benzo [ d ] imidazol-2-amine, purchased from SPECS (http:// www.specs.net).

In order to verify whether the small molecule 1-butyl-N- (4-chlorophenyl) -1H-benzo [ d ] imidazole-2-amine can inhibit the enzymatic activity of ODC, the enzyme catalysis product putrescine is detected by High Performance Liquid Chromatography (HPLC), and the half inhibition concentration of the small molecule C on the enzymatic activity of ODC is about 31.8 mu M (figure 21). FIG. 21 shows the high performance liquid chromatography detection of the ODC catalytic product putrescine, and the inhibition efficiency of small molecules on the ODC enzyme activity is calculated by comparing with that without the addition of small molecules.

To verify the inhibition of the ODC-OAZ1 interaction by the 1-butyl-N- (4-chlorophenyl) -1H-benzo [ d ] imidazol-2-amine molecule, a Fluorescence Resonance Energy Transfer (FRET) -based method was used. ODC protein labeled with YPET fluorescent protein (YPET-ODC) and OAZ1 labeled with CyPET fluorescent protein (CyPET-OAZ1) were purified by expression and then incubated together. Under normal conditions, when ODC and OAZ1 are bonded to each other, YPET and CyPET can be brought into close proximity, and FRET occurs. If the small molecule is capable of inhibiting the interaction of ODC and OAZ1, the FRET signal may be reduced to some extent. As shown in fig. 22 (fig. 22 is a graph showing that the small molecule can significantly inhibit FRET signal by detecting the inhibition of ODC-OAZ1 interaction by small molecule based on Fluorescence Resonance Energy Transfer (FRET) —, 1-butyl-N- (4-chlorophenyl) -1H-benzo [ d ] imidazol-2-amine. This means that small molecules of 1-butyl-N- (4-chlorophenyl) -1H-benzo [ d ] imidazol-2-amine are able to inhibit the interaction of ODC and OAZ 1.

To further verify whether the 1-butyl-N- (4-chlorophenyl) -1H-benzo [ d ] imidazol-2-amine molecule can stabilize ODC homodimers, the oligomeric state of ODCs was analyzed using an analytical size exclusion gel column. As shown in FIG. 23 (FIG. 23 shows that ODC differ in their oligomeric state at different salt concentrations, the earlier the efflux time, the easier the formation of ODC homodimers under the conditions), ODC differ in their oligomeric state and the time of efflux from the gel column differs at different salt (NaCl) concentrations (the earlier the time, the larger the molecular weight): under 50mM NaCl, the ODC exists mainly in a dimer form (calculated molecular weight: 105.1kDa), under 300mM NaCl, the ODC exists mainly in a monomer form (calculated molecular weight: 53.0kDa), and under 150mM NaCl, the monomer and dimer in the ODC are in a coexisting and rapid exchange state, with a corresponding efflux peak therebetween.

As shown in FIG. 24 (FIG. 24 shows the effect of 1-butyl-N- (4-chlorophenyl) -1H-benzo [ d ] imidazol-2-amine and DFMO on the oligomeric state of ODC measured under 50mM NaCl conditions without adding any small molecule solution as a control. the three efflux curves almost completely coincide, meaning that neither 1-butyl-N- (4-chlorophenyl) -1H-benzo [ d ] imidazol-2-amine nor DFMO were able to destroy ODC homodimers under these conditions), and the efflux peaks of 1-butyl-N- (4-chlorophenyl) -1H-benzo [ d ] imidazol-2-amine, DFMO and the control without small molecule were highly coincident under 50mM NaCl conditions, meaning that 1-butyl-N- (4-chlorophenyl) -1H-benzo [ d ] imidazol-2-amine, DFMO and the control without small molecule were able to flow out, meaning that 1-butyl-N- (4-chlorophenyl) -1H-benzo [ d ] m Neither the imidazol-2-amine molecule nor DFMO destabilizes ODC homodimers.

As shown in FIG. 25 (FIG. 25 is a graph showing the effect of 1-butyl-N- (4-chlorophenyl) -1H-benzo [ d ] imidazol-2-amine molecules and DFMO on the oligomerization state of ODC under 150mM NaCl, without adding any small molecule solution as a control.) the curve efflux peak of 1-butyl-N- (4-chlorophenyl) -1H-benzo [ d ] imidazol-2-amine molecules advances, while the efflux peaks of DFMO and blank control overlap, meaning that 1-butyl-N- (4-chlorophenyl) -1H-benzo [ d ] imidazol-2-amine molecules can stabilize ODC homodimers and DFMO cannot- The amine molecule was able to significantly shift the efflux peak forward, meaning that the ODC homodimer content was increased. While DFMO was consistent with the blank, indicating that DFMO did not promote ODC homodimer formation.

From the above results, it can be concluded that the 1-butyl-N- (4-chlorophenyl) -1H-benzo [ d ] imidazol-2-amine small molecule is a novel small molecule compound capable of inhibiting ODC enzyme activity, stabilizing ODC homodimer, and inhibiting ODC-OAZ1 interaction.

Claims

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.