Modified ezetimibe drug for cancer treatment

Abstract

Disclosed is a new compound of formula (I) or a pharmaceutically acceptable salt thereof, which binds tightly to the hydrophobic binding pocket of Mdm2, thereby preventing Mdm2 from binding to the tumor suppressor p53 and increasing p53 levels. Also disclosed is the use of the compound or a pharmaceutically acceptable salt thereof for the treatment of Mdm2 cancer and its use in the manufacture of a medicament.

Description

Technical Field

[0001] The present invention relates to an anticancer drug, which is a modified version of the drug ezetimibe. The modified drug has improved drug-like properties and is less sensitive to metabolic enzymes than ezetimibe. Background Art

[0002] The tumor suppressor protein p53 is involved in many important biological processes crucial for cancer development, including the cell cycle, apoptosis, DNA repair, angiogenesis, glucose metabolism, and innate immunity. It is a transcription factor that acts as a regulator of the oncogene Mouse Double Minute (Mdm2) in an autoregulatory feedback loop. p53 activates expression of the Mdm2 gene, and Mdm2 regulates p53 by controlling its trafficking from the nucleus, rendering it unavailable for its gene targets. This inhibits p53's transcriptional function or promotes its degradation by the proteasome, which utilizes ubiquitin ligase activity.

[0003] Mdm2 has a hydrophobic binding pocket to which p53 binds via a peptide in its transactivation domain. This pocket is a key target for drugs that inhibit the p53-Mdm2 interaction. Small molecule drug design attempts to create small classes of drug molecules that can competitively target the Mdm2 p53 binding domain, disrupting the formation of the Mdm2-p53 complex, thereby increasing the level of reactive p53 in cancer cells to promote p53-dependent cell death. These drug design studies led to the synthesis of nutrins (nutrin-2, nutrin-3a, and MI-219), of which nutrin-3a is the most effective with very low toxicity (IC 50 0.09 μM).

[0004] The applicant has previously shown that ezetimibe binds more strongly to the hydrophobic pocket of Mdm2 than nutrin. In addition, the applicant has demonstrated that ezetimibe is toxic to cancer cell lines, especially those that overexpress Mdm2. Although ezetimibe is not structurally similar to nutrin, based on molecular docking simulations, ezetimibe accurately mimics the binding of p53 to the hydrophobic cleft of Mdm2. However, ezetimibe has structural defects, such as its conversion by intestinal metabolic enzymes, which hinder its use as an anticancer drug. This results in negligible bioavailability of ezetimibe in the intestine, making it unsuitable for the treatment of colon cancer.

[0005] To address this issue, the applicant was prompted to modify ezetimibe to reduce its degradation by metabolic enzymes in the intestine. This was achieved by replacing the hydroxyl group (glucuronidation site) in the ezetimibe lead molecule with fluorine. This new drug, MC011019, has improved drug-like properties. The modification prevents the drug from being metabolically converted in the intestine and improves its bioavailability in the intestine, making it suitable for the treatment of colon and colorectal cancers, as well as other cancers that may overexpress Mdm2. Summary of the Invention

[0006] According to a first aspect of the present invention, there is provided a compound having a structure of formula (I)

[0007]

[0008] or a pharmaceutically acceptable salt thereof.

[0009] The compound or a pharmaceutically acceptable salt thereof can tightly bind to Mdm2, particularly to the hydrophobic binding pocket of Mdm2 to which p53 binds. Therefore, the compound or a pharmaceutically acceptable salt thereof can prevent the binding of Mdm2 to p53, thereby increasing the level of p53 in cells.

[0010] The compound or a pharmaceutically acceptable salt thereof can have good bioavailability in the intestine because it is resistant to degradation, particularly glucuronidation.

[0011] According to a second aspect of the present invention, there is provided a use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in a method for treating cancer.

[0012] The cancer can be one in which Mdm2 levels are elevated or Mdm2 is overexpressed, and can include colon cancer, colorectal cancer, sarcoma, glioma, lymphoma, breast cancer, lung cancer, liver cancer, esophagogastric cancer, and gynecological cancer.

[0013] The compound or a pharmaceutically acceptable salt thereof can bind to the hydrophobic binding pocket of Mdm2 and increase the level of active p53 in cells. This can increase p53-mediated cell death in cancer cells.

[0014] According to a third aspect of the present invention, there is provided a use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating cancer, wherein the cancer may be a cancer with high Mdm2 levels or overexpression of Mdm2.

[0015] The cancer may include colon cancer, colorectal cancer, sarcoma, glioma, lymphoma, breast cancer, lung cancer, liver cancer, esophageal gastric cancer, and gynecological cancer.

[0016] The compound or a pharmaceutically acceptable salt thereof can bind to the hydrophobic binding pocket of Mdm2 to prevent Mdm2 from inhibiting p53, thereby increasing p53-mediated cell death of cancer cells.

[0017] According to a fourth aspect of the present invention, there is provided a method of treating cancer by administering a compound of formula (I) or a pharmaceutically acceptable salt thereof to a patient in need thereof, wherein the cancer may be characterized by high levels or overexpression of Mdm2.

[0018] The compound or a pharmaceutically acceptable salt thereof can bind to the hydrophobic binding pocket of Mdm2 and increase the level of p53 in cells. The compound or a pharmaceutically acceptable salt thereof can promote p53-mediated cell death of cancer cells.

[0019] The cancer may be selected from the group consisting of colon cancer, colorectal cancer, sarcoma, glioma, lymphoma, breast cancer, lung cancer, liver cancer, esophagogastric cancer, and gynecological cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Example

[0021] The present invention will now be described in more detail with reference to the following examples and accompanying drawings.

[0022] In the accompanying drawings

[0023] Figure 1 An overview of the structure-guided computer-aided drug design method used in the embodiment is shown;

[0024] Figure 2 Shown are the residue attribute surfaces and good spatial contact networks of (A) the interaction between ezetimibe and Mdm2 and (B) the ezetimibe-Mdm2 complex for the examples;

[0025] Figure 3 The structural composition and interaction analysis of Mdm2 and ezetimibe in the embodiment are shown, wherein (A) is a ball-and-stick model of ezetimibe (ZINC03810860), and (B) is a 2D ligand interaction map, including a collection of nearby residues and a single hydrogen bond coordination with VAL93;

[0026] Figure 4 The simulated surface representation and interaction analysis of the Mdm2-MC011019 complex are shown, where (A) is the Mdm2-MC011019 complex, where Mdm2 is shown in a gray surface area with a deep hydrophobic pocket and MC011019 is presented in a ball-and-stick model; (B) is a 2D ligand interaction diagram including The set of nearby residues at the axis and the single hydrogen bond coordination with VAL93; the p-fluorophenyl group of MC011019 is circled and labeled, which is predicted to be the three key residues Phe19, Trp23 and Leu26 that competitively inhibit the p53 transactivation domain;

[0027] FIG5 shows the pharmacokinetic (PK) properties of MC011019 (A) compared to ezetimibe (B);

[0028] FIG6 shows target prediction analysis for the embodiment, wherein (A) is an overview of ezetimibe protein targets, and (B) is an overview of possible MC011019 protein targets;

[0029] Figure 7 The figure shows the pharmacokinetic curve of the plasma concentration (plasma concentration, cp)-time curve analysis of MC011019 in the embodiment, describing the prediction of possible plasma concentration curves (cp-time curves), the percentage of absorbed portion (%Fa) and bioavailability (%Fb), as well as C-max and its corresponding T-max when MC011019 is administered at different doses of 0.01 mg (C), 0.1 mg (B) and 1.0 mg (A) using a mouse model;

[0030] Figure 8 The pharmacokinetic curve of the cp-time curve analysis of MC011019 in the embodiment is shown, which describes the prediction of the possible plasma concentration curve (cp-time curve), the percentage of the absorbed portion (%Fa) and the bioavailability (%Fb), as well as the C-max and its corresponding T-max of MC011019 administered at different doses of 1 mg (C), 10 mg (B) and 100 mg (A) based on a human model; and

[0031] FIG9 shows (A) the probability of off-target interaction of MC011019 with various cellular proteins, and (B) the probability of off-target interaction of ezetimibe with various cellular proteins for the Examples. DETAILED DESCRIPTION

[0032] Materials and methods

[0033] Protein and drug structures

[0034] The Mdm2 protein structure was downloaded from the Protein Database (PDB) in pdb format and analyzed using PyMol, with the p53 peptide removed prior to docking studies. The 2D structures of the drug ligands were retrieved from the Zinc Drug Database (Zdd). The structure of the nutrin-3a drug was obtained in sdf format using the PubChem database. The structure of ezetimibe was obtained from the DrugBank database in SMILE format.

[0035] Compound screening and molecular docking

[0036] The Mdm2 p53-binding domain (Mdm2 p53BD) was used as a Schrödinger Maestro 2019-4: Template on the Glide standard precision (SP) application to screen compounds in Zdd that can target Mdm2 p53BD. Zdd constructs FDA-approved commercial drugs that are available globally as pure compounds. In this study, the entire Zdd database of 2924 structures was screened. The use of Glide enables virtual screening and molecular docking studies to be performed simultaneously. This application takes as input key residues in Mdm2 p53BD and Zdd and generates a collection of compounds that dock into the specified pocket with different docking scores. The receptor-based ligand docking protocol uses a multi-step procedure that involves preparing and manipulating the Mdm2 p53BD and ligands prior to screening and docking studies. These steps are performed sequentially as follows: protein domain preparation, ligand preparation, grid generation, and receptor-based ligand docking.

[0037] Protein preparation

[0038] PDB structures are not suitable for direct use in molecular modeling calculations because they are usually composed of heavy atoms only. They may also include core crystallographic ligands, water molecules, metal ions, and cofactors. In addition, some structures are multimeric and need to be simplified to single units, and due to the limited resolution of X-ray experiments, it is difficult to distinguish the carbonyl oxygen of the amide and the secondary amine nitrogen in the crystal structure, so the position of the group must be checked. PDB structures may also be missing atoms or connectivity information, which must be specified together with the bond order and formal charge. Therefore, in this study, the Maestro v10.7 Protein Preparation Wizard. This takes the Mdm2 p53BD from its original state (with missing atoms or incorrect bond order assignments, incorrect charge states, and orientations of various groups) and prepares it for use with Glide. The wizard includes a graphical user interface (GUI) with the system's functional programs.

[0039] Ligand preparation

[0040] Zdd, downloaded in SMILE and SDF formats (http: / / zinc12.docking.org / browse / subsets / special), contains compounds with 2D structures. This configuration is not applicable when performing molecular docking calculations or simulations using computational docking algorithms. Proteins exist in three-dimensional space, so drugs that successfully target these proteins should also exhibit this configuration. The Maestro v10.7 Ligand Preparation Wizard was used to convert 2,924 2D structures into 4,909 lowest-energy possible 3D structures in Maestro format. This program allows for the expansion of each input structure by generating variations in ionization states, tautomers, stereochemistry, and ring conformation. Possible ionization states of the ligands were generated over a target pH range of 7.0 + / - 2 using Epik, a built-in application within Glide that predicts ionization states and their associated losses. Epik also predicts different tautomeric forms and calculates the energy loss for each predicted ligand state. In Glide, Epik state losses are also used to distinguish active from inactive compounds during docking; indeed, the use of Epik is known to improve virtual screening enrichment.

[0041] Mesh generation

[0042] The external scoring grid is divided into two parts: to The meshes were generated at different sizes. Generally, it is important to conform the outer mesh to the shape of the protein active site, so this was done only to mask the active site volume of the Mdm2p53 binding hydrophobic cleft. The residues of the Mdm2 p53BD were used as a prerequisite for accurately mapping the key residues, thereby facilitating the binding coordination of the p53 transactivation domain. A ligand center box (inner mesh) was generated to define acceptable ligand center positions during the side point search, providing a true measure of the effective search space size. The ligand center box helps the ligand find a conventional or asymmetric binding mode in the active site, or constrain its center point to a smaller box to save computational time. The "center of mass of selected residues" option was also used, which specifies the residues that best define the active site and then centers the inner mesh on the center of mass of these selected residues.

[0043] Receptor-based ligand docking

[0044] The final docking algorithm used in this study was the Glide SP algorithm, also known as standard precision. The nature of the docking simulations employed by this algorithm is identical to that of High Throughput Virtual Screening (HTVS), except that HTVS reduces the number of intermediate conformations, the final torsional refinement, and the thoroughness of the sampling throughout the docking funnel. During the docking process, the domain structures remain rigid (even the hydroxyl and thiol groups cannot rotate), while flexibility is induced in all docked ligands. This is achieved using a ligand preparation wizard, which generates a collection of multiple poses from the ligand database (Zdd). The entire work was performed using a Core i7 with 4 cores, 8 processors, and 8GB of RAM.

[0045] result

[0046] Screening with Zdd revealed that ezetimibe binds well to the Mdm2-p53 binding pocket, including hydrogen bonding to VAL93. Further molecular docking studies yielded Figure 2 Shown is the interaction element where ezetimibe docks into the p53-binding domain of Mdm2, the hydrophobic pocket. Figure 2 In A, a single hydrogen bond and possible hydrophobic interaction with VAL93 were observed. Figure 2 In Figure B, a surface representation of the residue properties and a well-defined spatial contact network of the Mdm2-ezetimibe complex is observed. This surface representation shows the pocket-like nature of the binding site and how ezetimibe accurately mimics three key p53-binding residues (PHE19, TRP23, and LEU26).

[0047] Ligand interaction diagram ( Figure 3) shows that ezetimibe binds tightly to the hydrophobic pocket of Mdm2-p53BD. In addition, it shows the Ionic interactions at the axis ( Figure 2 This finding may have significant implications for cancers in which Mdm2 is highly expressed. Figure 3 As shown in the square in the figure, fluorine was introduced and pharmacokinetic studies were performed in silico. This substitution is crucial because it blocks the conversion of the new drug to the new drug by glucuronidation in the intestine. This modification also improves the drug-likeness of the lead compound. One evidence of this is the increase in the drug's lipophilicity (ClogP) to 5.04. For ezetimibe, lipophilicity does not improve oral bioavailability because, after glucuronidation, ezetimibe is excreted through the digestive tract.

[0048] exist Figure 4 In Figure 2, the structural interactions of MC011019 docked into the hydrophobic pocket of MDM2 are depicted, along with a 2D ligand interaction map showing This binding mode accurately mimics the p53 transactivation domain, as the three p-fluorophenyl groups of MC011019 directly bind to and occupy three key residues (Phe19, Trp23, and Leu26) that facilitate p53 binding coordination. Furthermore, MC011019 also forms hydrogen bonds with VAL93, similar to the hydrogen bonds that p53 forms when binding to the MDM2 hydrophobic pocket. Furthermore, the docking score for this binding mode is slightly higher at -7.89 kJ / mol compared to the docking score of -7.76 kJ / mol for ezetimibe.

[0049] In FIG5 , MC011019 ( Figure 5A ) and ezetimibe ( Figure 5B ) Pharmacokinetics (PK) and drug-like properties of . This figure was calculated using Swiss ADME.

[0050] Target prediction experiments showed that MC011019 and ezetimibe have similar biological targets (Figure 6, Figure 9A and Figure 9B Notably, MC011019 is not predicted to bind to the Niemann-Pick C1-like protein, the cholesterol-associated receptor for ezetimibe. This suggests that MC011019 may not replicate the current application of ezetimibe. Alternatively, cholesterolemia is unlikely to be an indication for MC011019, as this depends on binding to Niemann-Pick C1-like proteins. Furthermore, MC011019 has a significantly lower probability of binding to cannabinoid receptor 1 compared to ezetimibe.

[0051] exist Figure 7 and Figure 8 , the relationship between the plasma concentration of MC011019 and time is depicted. Figure 7 and Figure 8 Calculated using ADMET PREDICTORv9.5 from SimulationPlus. These plots describe the predicted pharmacokinetic profiles of MC011019 administered at different doses of 0.01 mg or 1 mg (C), 0.1 mg or 10 mg (B), and 1.0 mg or 100 mg (A) using mouse and human models, respectively. The simulation also calculated the percentage of absorbed portion (%Fa) and bioavailability (%Fb) as well as C-max and its corresponding T-max. It is also noteworthy that in both models, MC011019 showed high absorption and elimination rates, as well as good bioavailability (%Fb) in humans. These observations are shown in Figures 6, Figure 9A and Figure 9B This is also supported by the findings of , which showed that MC011019 does not bind to Niemann-Pick C1-like proteins and also has a very low probability of off-target interactions with other cellular proteins.

[0052] discuss

[0053] The above-mentioned computer simulation studies showed that MC011019 binds strongly in the hydrophobic pocket of Mdm2-p53. o / w ) suggest that MC011019 will have improved bioavailability compared to the parent molecule, ezetimibe, due to the addition of fluorine. Replacing the hydroxyl group with fluorine is necessary to prevent metabolic transformation of the drug in the small intestine. Another important observation is that MC011019 may not interact with the Niemann-Pick C1 protein, which promotes cholesterol absorption.

[0054] These studies suggest that MC011019 blocks the binding of Mdm2 to the tumor suppressor protein p53, thereby activating p53's positive effects on cancer cells. MC011019 is expected to be effective against cancers with wild-type p53 and those that overexpress Mdm2. In particular, MC011019 is expected to be effective against colon and colorectal cancers because it is not easily degraded by intestinal metabolism.

Claims

1. A compound having a structure of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating cancers with elevated Mdm2 levels or overexpression of Mdm2, wherein The cancer is selected from the group consisting of colon cancer, colorectal cancer, and esophagogastric cancer.

2. The use according to claim 1, wherein Compared with unmodified ezetimibe, the drug has better oral bioavailability.

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