Compound and application thereof as RHOJ inhibitor and anti-cancer drug

Abstract

The invention provides a compound and application thereof as an RHOJ inhibitor and an anti-cancer drug, and belongs to the technical field of biological medicine. The compound provided by the invention provides a new material for cancer treatment, and solves the problem that a compound targeting RHOJ with a drug application prospect is not disclosed in the prior art. The compound provided by the invention has stronger specificity to RhoJ, is of a novel structure, can avoid pharmacodynamic limitation of existing compounds, can realize batch production, and provides more choices for targeted therapy of RhoJ related tumors. According to the invention, a computer-aided drug design strategy is adopted, a small-molecule inhibitor of RhoJ is successfully obtained through virtual screening, and a potential compound is provided for RhoJ-targeted tumor treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a compound and its application as a RHOJ inhibitor and an anticancer drug. Background Art

[0002] RhoJ is a member of the Rho GTPase family and belongs to the Cdc42 subfamily. It is involved in regulating multiple biological processes such as proliferation, migration, invasion, and apoptosis of tumor cells. Studies have confirmed that the expression level of RhoJ is closely related to the occurrence and progression of various tumors and is one of the key driver genes that promote tumor development. RhoJ mainly plays a tumor-promoting role by activating downstream effector proteins such as PAK and LIMK. RhoJ can activate cytoskeleton reorganization and regulate the movement and migration of tumor cells; it can also inhibit the apoptosis of tumor cells through signaling pathways such as NF-κB and STAT3. Therefore, RhoJ has become an emerging target in the field of tumor treatment. Small molecule inhibitors targeting RhoJ can specifically inhibit its GTPase activity, thereby blocking RhoJ-related tumor signal transduction and inhibiting the malignant phenotype of tumor cells. However, the current research and development of small molecule inhibitors for RhoJ is still relatively lacking.

[0003] CN111996251A discloses an application of a malignant glioma biomarker, specifically the application of RHOJ mRNA and RHOJ protein as detection targets in the preparation of a kit for diagnosing, treating or judging the prognosis of malignant glioma. It was found that silencing RHOJ expression significantly inhibited malignant phenotypes such as glioma cell proliferation and invasion. RHOJ can be used as a molecular marker for the diagnosis and prognosis of malignant glioma. Treatment strategies such as antibodies or small molecule inhibitors targeting RHOJ are expected to improve the treatment of malignant glioma.

[0004] However, no compound targeting RHOJ with potential for drug application has been disclosed in the prior art. Summary of the invention

[0005] The purpose of the present invention is to provide a compound and its application as a RHOJ inhibitor and an anticancer drug, thereby providing a new material for cancer treatment.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a compound represented by structural formula I:

[0008]

[0009] The present invention also provides the use of the above compound in preparing RHOJ inhibitors.

[0010] The present invention also provides the use of the above compound in preparing a tumor metastasis inhibitor.

[0011] The present invention also provides the use of the above compound in preparing tumor growth inhibitors.

[0012] Preferably, the tumor comprises pancreatic cancer.

[0013] The present invention also provides an anticancer drug, comprising the above compound.

[0014] The present invention also provides an anticancer drug composition, comprising the above compound and an anticancer drug.

[0015] Preferably, the anticancer drug is doxorubicin.

[0016] Beneficial effects of the present invention:

[0017] The compounds provided by the present invention have stronger specificity for RhoJ and are novel structures, and can avoid the pharmacodynamic limitations of existing compounds. They can be mass-produced to provide more options for targeted treatment of RhoJ-related tumors.

[0018] The present invention adopts a computer-aided drug design strategy and successfully obtains a small molecule inhibitor of RhoJ through virtual screening, providing potential compounds for RhoJ-targeted tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The interaction superposition diagram of 5 preferred compounds and Human Rhoj;

[0020] Figure 2 is the 2D image of compound F5791-0489;

[0021] Figure 3 is a 3D image of compound F5791-0489;

[0022] Figure 4 This is a graph showing the inhibition of cancer cells by compound F5791-0489. DETAILED DESCRIPTION

[0023] The present invention provides a compound represented by structural formula I:

[0024]

[0025]

[0026] The relevant structural information of the compound is shown in Table 1 below:

[0027] Table 1 Structural information

[0028] Catalog ID F5791-0489 Docking score -7.807 Drug Name N-(4-(benzyloxy)benzyl)-6-oxo-1,6-dihydropyridine-3-carboxamide MW 334.37 cLogP 2.39 HB Donor 2 TPSA 67.43 Number of rotatable bonds 6

[0029] The present invention also provides the use of the above compound in preparing RHOJ inhibitors.

[0030] The present invention also provides the use of the above compound in preparing a tumor metastasis inhibitor or a tumor growth inhibitor, wherein the tumor is preferably pancreatic cancer.

[0031] The present invention also provides an anticancer drug, comprising the above-mentioned compound, wherein the drug optionally uses the above-mentioned compound as the sole active ingredient; further preferably, the drug also includes other anticancer active ingredients; the drug includes excipients, and the present invention has no special limitation on the type of the excipients, and conventional pharmaceutical excipients in the art can be used; in the present invention, the drug includes a carrier, and the carrier is preferably an adhesive, lubricant, disintegrant, solubilizer, diluent, stabilizer, suspending agent, pigment, flavoring agent, etc. for oral preparations; preservatives, solubilizers, stabilizers, etc. for injectable preparations; and matrix, diluent, lubricant, preservative, etc. for local preparations.

[0032] The present invention also provides an anticancer drug composition, comprising the above compound and an anticancer drug, wherein the anticancer drug is preferably doxorubicin.

[0033] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0034] Example 1

[0035] The present invention targets HumanRhoj protein for virtual screening. The software used for virtual screening is The 3D drawing software is PyMol.

[0036] Protein preparation:

[0037] The predicted structure of HumanRhoj protein was downloaded from the AlphaFold website (ID: AF-Q9H4E5-F1). ProteinPreparationWizard module was then used to hydrogenate the protein, delete water molecules, and repair missing residues and side chains. Energy optimization was then performed (OPLS2005 force field, RMSD ). The processed protein is used to predict the binding site and binding pocket of the protein using the Sitemap module, and the ReceptorGridGeneration module is used to create a grid file with Site1 as the center, and the box size is set to

[0038] Compound preparation:

[0039] The 2D format of Life Chemicals 50K Diversity Library (LC-50, containing 50.2K compounds) and MCEBioactive Compound Library Plus (containing 12.6K compounds) was converted to The software LigPrep Module performs hydrogenation, energy optimization and other processing, and outputs 3D structures for virtual screening.

[0040] Molecular docking

[0041] The VirtualScreeningWorkflow module was used for virtual screening, and the prepared compounds were imported. The Glide module was used for molecular docking, that is, the receptor and ligand molecules were docked with each other through geometric matching and energy matching. First, the high-throughput screening (HTVS) mode in the Glide module was used to screen the 50.2K prepared small molecule compounds in the LifeChemicals50KDiversityLibrary, and the small molecule compounds with the top 10% of the scores were selected for the second round of screening using the standard (SP) mode; then the top 10% of the scores were selected for the third round of screening using the high-precision (XP) mode to obtain the ranking of small molecule compounds. The same method was used to dock the compounds in the MCEBioactiveCompoundLibraryPlus with the target protein HumanRhoj in the HTVS mode, and then the small molecule compounds with the top 30% of the scores were further selected for the second round of screening using the standard (SP) mode; then the top 30% of the scores were selected for the third round of screening using the high-precision (XP) mode to obtain the ranking of small molecule compounds.

[0042] The binding force between the target and the compound, the compound structure, etc. were manually reviewed, and the top 200 compounds in the LifeChemicals50KDiversityLibrary and the top 200 compounds in the MCEBioactiveCompoundLibraryPlus were selected for output and delivered as the final result. The top 4 compounds in the LifeChemicals50KDiversityLibrary (F6258-0116, F5791-0489, F5097-1389, F6436-1191) and the first compound in the MCEBioactiveCompoundLibraryPlus (HY-100618) were selected for 2D and 3D mapping of the binding mode with HumanRhoj protein.

[0043] Analysis of virtual screening results

[0044] The binding modes of the five preferred compounds (F6258-0116, F5791-0489, F5097-1389, F6436-1191, HY-100618) and Human Rhoj protein were mapped in 2D and 3D. The interaction superposition diagrams of the five preferred compounds and Human Rhoj are shown in Figure 1 As shown, Human Rhoj protein is displayed as a green cartoon, and the five preferred compounds are displayed as light blue, rose red, yellow, bean paste, and gray-white sticks, respectively.

[0045] The optimal compound F5791-0489 was screened, as shown in the 2D diagram Figure 2 As shown, the 3D image is Figure 3 shown.

[0046] In the 3D image, the C skeleton of Human Rhoj protein is shown in green, N atoms are shown in blue, O atoms are shown in bright red, H atoms are shown in white, and F5791-0489 is shown as a rose-red stick. The hydrogen bond length is shown as a red dotted line. The longer the bond length, the weaker the hydrogen bond. F5791-0489 can form three hydrogen bonds, two π-π interactions, and a hydrophobic interaction with Human Rhoj protein: the carbonyl group of pyridone acts as a hydrogen bond acceptor to form two hydrogen bonds with ALA177 and LEU178, with a distance of The NH of the pyridone group acts as a hydrogen bond donor and forms a hydrogen bond with ASP136 at a distance of

[0047] The pyridone group and the middle benzene ring can form two π-π interactions with PHE46; the carboxyl group adjacent to the double bond acts as a hydrogen bond acceptor to form two hydrogen bonds with LYS221 and ASN226, with a distance of The carboxyl group on the other fatty chain acts as a hydrogen bond donor and acceptor to form two hydrogen bonds with SER223 and HIS224, with a distance of Sulfonamide NH acts as a hydrogen bond donor and forms a hydrogen bond with GLY47 at a distance of

[0048] Example 2

[0049] Chemical synthesis finally obtains the product

[0050] The specific steps of CAS chemical synthesis route are:

[0051] Step 1: Take compound 1 (3.3437 g, 10 mmol) in a 100 mL round-bottom flask. Add 50 mL of methanol (or other suitable solvent) to dissolve it. Add sodium borohydride (NaBH4) (7.15 g, 15 mmol). Carry out the reduction reaction at room temperature overnight. After the reaction is completed by TLC detection, extract with dichloromethane (3x50 mL), combine the organic layers, wash with water (3x50 mL), dry the organic layers over anhydrous sodium sulfate, filter, and concentrate in vacuo. The residue is separated and purified by silica gel column chromatography (eluent: V ethyl acetate: V petroleum ether = 20:1) to obtain 4,4'-dicyanodibenzhydrol.

[0052] Step 2: Take 4,4'-dicyanodiphenylmethanol (2.5025g, 10mmol) in a 100mL round-bottom flask. Add 50mL of dichloromethane (or other suitable organic solvent) to dissolve it. Add chloroform (7.97g, 15mmol). Treat under appropriate conditions to obtain 4,4'-dicyanodiphenylmethane. After the reaction is completed by TLC detection, extract with dichloromethane (3x50 mL), combine the organic layers, wash with water (3x50 mL), dry the organic layers over anhydrous sodium sulfate, filter, and concentrate in vacuo. The residue is separated and purified by silica gel column chromatography (eluent: V ethyl acetate: V petroleum ether = 20:1).

[0053] Step 3: Take 4,4'-dicyanodiphenylchloromethane (2.84695 g, 10 mmol) in a 100 mL round-bottom flask. Add N-methylmorpholine (0.505 g, 5 mmol) as a catalyst. Add 1,2,4-triazole (0.85 g, 10 mmol). React at a temperature of about 80 ° C and continue overnight. After the reaction is completed by TLC detection, extract with dichloromethane (3x50 mL), combine the organic layers, wash with water (3x50 mL), dry the organic layer over anhydrous sodium sulfate, filter, and concentrate in vacuo. The residue is separated and purified by silica gel column chromatography (eluent: V ethyl acetate: V petroleum ether = 20:1) to obtain a small molecule inhibitor targeting RhoJ.

[0054] The product is characterized by high performance liquid chromatography combined with mass spectrometry to ensure that the product purity is ≥98% and all impurity indicators meet the drug standard requirements.

[0055] Small molecule inhibitors targeting RhoJ were obtained.

[0056] The product is characterized by high performance liquid chromatography combined with mass spectrometry to ensure that the product purity is ≥98% and all impurity indicators meet the drug standard requirements.

[0057] Example 3

[0058] IC50 refers to the drug concentration required to reduce the number of surviving cells by half after drug administration. In the MTT method, the drug concentration required to reduce the OD value of the control group by half is IC50. In addition, the meaning of half-inhibitory concentration is also equivalent to the average value of the minimum lethal dose of the drug to cultured cells. As a quantitative indicator reflecting the efficacy of the drug, it is widely used in the screening of various drugs.

[0059] The half inhibitory concentration (IC50) of the third RHOJ inhibitor was determined using a cell proliferation assay (Mia-paca2, ASPC1, and Panc1). After the compounds were co-cultured with different cells, the Cell Counting Kit-8 (CCK8) method was used to detect the survival rate of normal cells. The specific steps are as follows:

[0060] Inoculate 10,000 cells of each normal cell line in a transparent 96-well plate per 100 μl culture medium and allow it to adhere for 3-4 hours. Add the appropriate effector cells to target cells (E:T) ratio to normal cells, and set the E:T ratio to 0:1, 1:1, 2:1, 4:1, and 8:1, with 3 replicates per group. After 24h and 48h of co-culture, wash the cells twice with PBS to remove apoptotic cells. Then, plate the cells in a 96-well plate according to the experimental design, add 100 μl of complete culture medium to each well, repeat 3 replicates, and after the cells adhere, discard the old culture medium, add 100 μl of fresh culture medium containing 10 μl CCK-8 solution, and after 1-4h of dark incubation, use an enzyme reader to measure the OD value of each well at a wavelength of 450nm. Calculate according to the reagent instructions.

[0061] The results are as follows Figure 4 As shown. The compound can inhibit the growth of tumor cells. In vivo experiments showed that it did not inhibit the activity and proliferation of normal cells, but RHOJ inhibitor No. 3 could inhibit tumor growth.

[0062] Therefore, the third RHOJ inhibitor can be used for the development of anti-tumor drugs. It is a promising lead compound for the design of RHOJ-targeted anti-tumor drugs. The above screening results and pharmacodynamics validation lay the foundation for the research and development of RHOJ small molecule inhibitors.

[0063] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. The compound shown in structural formula I:

2. Use of the compound according to claim 1 in the preparation of RHOJ inhibitors.

3. Use of the compound according to claim 1 in the preparation of a tumor metastasis inhibitor.

4. Use of the compound according to claim 1 in the preparation of a tumor growth inhibitor.

5. The use according to claim 3 or 4, characterized in that: The tumor includes pancreatic cancer.

6. An anticancer drug, characterized in that: Comprising the compound described in claim 1.

7. An anticancer drug composition, characterized in that: The invention comprises the compound according to claim 1 and an anticancer drug.

8. The anticancer pharmaceutical composition according to claim 7, characterized in that: The anticancer drug is doxorubicin.

Images