Application of novel molecular glue

Abstract

The invention relates to application of a novel compound capable of serving as molecular glue in prevention or treatment of brain cancer.

Description

Technical Field

[0001] The present invention relates to use of a novel compound capable of acting as molecular glue in preventing or treating brain cancer. Background Art

[0002] Targeted Protein Degradation (TPD) refers to a mechanism that directly decomposes and eliminates pathogenic factors after translation into proteins, represented by targeted protein degradation chimeras (PROTACs). PROTACs consist of a protein of interest (POI) binding moiety (equivalent to the substrate protein connection region), a linker, and an E3 ligase binding moiety (equivalent to the enzyme connection region). The core of PROTAC technology is to allow the corresponding substrate of the TPD mechanism to directly meet the E3 ligase.

[0003] Molecular glue is a substance with different properties from PROTAC. It is a small molecule that can interact with various unpredictable target proteins. PROTAC is a divalent molecule composed of two parts, one part binds to POI and the other part binds to E3 ligase. These parts are connected by a linker and have a relatively large molecular weight, but molecular glue is smaller than PROTAC and has better pharmacological properties, higher membrane permeability, better cell absorption rate and blood-brain barrier penetration than PROTAC.

[0004] Various types of molecular glues have been discovered, representative examples of which include thalidomide-related analogs, lenalidomide and pomalidomide targeting the E3 ligase hydroxycerebroside (CRBN).

[0005] However, it is known that the polar surface area (tPSA) values ​​of thalidomide, lenalidomide, and pomalidomide are close to or exceed about 90, which affects blood-brain barrier (BBB) ​​penetration, and thus these drugs have difficulty in penetrating the BBB.

[0006] Since safety and drug resistance are important considerations in the development of anticancer drugs, there is a need to develop immunomodulatory agents that minimize side effects and long-term complications while having a good safety profile. Summary of the invention

[0007] The purpose of the present invention is to use novel compounds that can act as molecular glue for the prevention or treatment of brain cancer.

[0008] [1] In one aspect of the present invention, the present invention relates to a pharmaceutical composition for preventing or treating brain cancer, comprising a compound represented by the following Chemical Formula 1 or Chemical Formula 2 as an active ingredient:

[0009] Chemical formula 1:

[0010] Chemical formula 2:

[0011] [2] In the above [1], the cancer may be a relapsed or refractory cancer.

[0012] [3] In the above [1], the cancer may be a drug-resistant cancer.

[0013] [4] In the above [1], the above composition can be administered by intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal injection, oral administration, topical administration, intranasal injection, intrapulmonary injection or rectal administration.

[0014] [5] In another aspect of the present invention, the present invention relates to a method for preventing or treating brain cancer in a subject, comprising the step of administering the compound represented by Chemical Formula 1 or Chemical Formula 2 of [1] above to the subject.

[0015] [6] In another aspect of the present invention, the present invention relates to use of a compound represented by Chemical Formula 1 or Chemical Formula 2 of the above [1] in the preparation of a drug for preventing or treating brain cancer.

[0016] [7] In another aspect of the present invention, the present invention relates to use of a compound represented by Chemical Formula 1 or Chemical Formula 2 of the above-mentioned [1] in preventing or treating brain cancer.

[0017] The present invention relates to the use of a novel compound that can act as a molecular glue, which is a molecular glue that can minimize side effects and long-term complications and can be effectively used to prevent or treat brain cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The figure is used to confirm the cell death effect of the compounds of the examples of the present invention in brain cancer cell lines (U87, U373 and SK-N-SH).

[0019] Figure 2 The results of colony formation assay of the compounds of the examples of the present invention in a brain cancer cell line (U87) are shown.

[0020] Figure 3 The expression levels of γ-H2AX and cleaved PARP of the compounds of the examples of the present invention in a brain cancer cell line (U87) are shown.

[0021] Figure 4 The results of PI apoptosis assay of the compounds of the present invention in the brain cancer cell line (U87) are shown.

[0022] Figure 5 The results of evaluating the sub-G1 population (Sub G1 Population) of the compounds of the examples of the present invention in a brain cancer cell line (U87) are shown.

[0023] Figure 6 Dose-response curves for different drugs are shown.

[0024] Figure 7 Shown are dose response curves in glioblastoma cell lines (U87 and U373).

[0025] Figure 8 The in vivo experimental design and experimental results of the compounds of the examples of the present invention are shown.

[0026] Best Mode for Carrying Out the Invention

[0027] The terminology used in this specification is used to appropriately express the preferred embodiments of the present invention, and the definition of these terms should be based on the content of the full text of this specification. Throughout the specification, when a certain part "includes" a certain structural element, this means that it can also include other structural elements rather than excluding other structural elements, unless clearly stated to the contrary.

[0028] Unless otherwise defined, all technical terms used in the present invention have the same meanings as those generally understood by those skilled in the art in the relevant field of the present invention. In addition, preferred methods and samples are described in this specification, but similar or equivalent methods are also included in the scope of the present invention.

[0029] In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating brain cancer, comprising a compound represented by the following Chemical Formula 1 or Chemical Formula 2 as an active ingredient:

[0030] Chemical formula 1:

[0031] Chemical formula 2:

[0032] In one embodiment of the present invention, the compound represented by Chemical Formula 1 or Chemical Formula 2 of the present invention can act as a molecular glue. Specifically, the compound represented by Chemical Formula 1 or Chemical Formula 2 can be a molecular glue substance that binds to hydroxycerebroside (Cereblon or CRBN) protein and exerts an anti-cancer effect through an immunomodulatory imide drug (ImiD) mechanism. Molecular glue is a small molecule that interacts with a target protein and has advantages such as excellent pharmacological properties and membrane permeability, excellent cell absorption rate, and blood-brain barrier penetration.

[0033] In one embodiment of the present invention, the protein used as the endogenous target protein of the immunomodulator can be CRBN protein. CRBN protein forms an E3 ubiquitin ligase complex with DNA damage binding protein 1 (DD B1), Cullin-4A (CUL4A) and Cullin 1 regulatory factor (ROC1), which ubiquitinates many different proteins. CRBN protein is a substrate receptor of CRL4, one of the E3 ubiquitin ligases. CRL4 / CRBN can regulate the intracellular homeostasis of a variety of important intracellular proteins through ubiquitin-dependent degradation, and can directly bind to AMPK, which can cause a variety of metabolic diseases such as cancer. In addition, CRBN protein may also play a negative regulatory role in CD4+T cell activation. CRBN deficiency increases the activation of CD4+T cells and enhances the secretion of IL-2, inducing CD4+T cells to differentiate into Th17 cells. Enhanced T cell activation sometimes leads to T cell-mediated autoimmune diseases such as autoimmune encephalomyelitis and delayed-type hypersensitivity reactions.

[0034] In the present invention, "treatment" refers to all actions to improve or beneficially change the symptoms of a disease by administering the composition according to the present invention, "prevention" refers to all actions to inhibit or delay a disease by administering the composition according to the present invention, and "improvement" refers to all actions to improve the adverse conditions of a disease by administering the composition of the present invention to an individual or allowing the individual to ingest the composition of the present invention.

[0035] The disease to be "treated", "prevented" or "improved" in the present invention may be cancer. Cancer refers to a physiological condition in animals, usually characterized by abnormal or uncontrolled cell growth. Cancer and cancer pathology may be associated with, for example, metastasis, interference with the normal function of surrounding cells, release of cytokines or other secretory products at abnormal levels, suppression or enhancement of inflammation or immune response, neoplasia and premalignant lesions, and malignancies.

[0036] In one embodiment of the present invention, the cancer may be a relapsed or refractory cancer.

[0037] In one embodiment of the present invention, the cancer may be a drug-resistant cancer.

[0038] The pharmaceutical composition of the present invention can be administered in a therapeutically effective amount of the above-mentioned active ingredients. The above-mentioned therapeutically effective amount refers to a drug dosage that exerts an effective brain cancer prevention or treatment effect. The appropriate total daily usage can be determined by the attending physician within the scope of reasonable medical judgment. The specific therapeutically effective amount for a particular patient can be applied differently depending on various factors and similar factors well known in the medical field, including the type and degree of response to be achieved, the type and amount of the drug to be co-administered, the specific composition (including whether other preparations are used as appropriate), the patient's age, weight, general health, sex and diet, administration time, administration route, and treatment cycle.

[0039] The pharmaceutical composition of the present invention can be administered to a subject or individual by any route of administration, for example, by intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration (intradermal injection), oral administration, topical administration, intranasal administration (intranasal injection), intrapulmonary injection (intrapulmonary injection) or intrarectal administration. Preferably, the pharmaceutical composition of the present invention can be used for oral administration or intravenous administration.

[0040] In another aspect, the present invention relates to a method for preventing or treating brain cancer in a subject, comprising the step of administering the compound represented by Chemical Formula 1 or Chemical Formula 2 above to the subject.

[0041] In the present invention, the term "subject" is used synonymously with "individual", and the subject may be a mammal, such as a human, mouse, cow, horse, pig, dog, sheep, goat or cat.

[0042] The contents repeated with the aforementioned pharmaceutical composition of the present invention will be omitted.

[0043] In another aspect, the present invention relates to use of the compound represented by Chemical Formula 1 or Chemical Formula 2 in preparing a drug for preventing or treating brain cancer.

[0044] The contents repeated with the aforementioned pharmaceutical composition of the present invention will be omitted.

[0045] In another aspect, the present invention relates to use of the compound represented by Chemical Formula 1 or Chemical Formula 2 in preventing or treating brain cancer.

[0046] The contents repeated with the aforementioned pharmaceutical composition of the present invention will be omitted.

[0047] All matters mentioned in the compositions, methods of treatment and uses of the present invention are equally applicable unless they are inconsistent with each other.

[0048] Preferred embodiments are presented below to help understand the present invention. However, the following embodiments are merely illustrative of the present invention. It is obvious to those skilled in the art that various deformations and modifications can be made within the scope of the scope and technical idea of ​​the present invention, and these deformations and modifications naturally fall within the scope of the appended claims. DETAILED DESCRIPTION

[0049] Example

[0050] The novel compound of the present invention can be prepared according to the following reaction formula, but is not limited thereto.

[0051] Reaction:

[0052]

[0053] Example 1: Synthesis of 4-Fluthalidomide and (S)-pentan-2-amine coupling compound (AST-DT-135)

[0054] (S)-penta-2-amine was used as a coupling compound.

[0055] DMSO (60 mL), (S)-pentan-2-amine (3.21 g, 0.0369 mmol, purchased from Aldrich) and N,N-diisopropylethylamine (DIPEA, 6.927 ml, 0.0398 mmol, purchased from Aldrich) (5.14 g) were added to 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (4-fluorothalidomide, 10 g, 0.0362 mmol, purchased from EOS, China). The reaction mixture was stirred at 120° C. for 5 hours, and then water (300 mL) was added. After the precipitate was filtered and washed with water (200 mL), the obtained solid was recrystallized with isopropanol and dried in vacuo to confirm a solid product.

[0056] Yield: 34%

[0057] Mass:344(M+1)

[0058] 1 H NMR (400MHz, CDCl3): d 0.94(3H,t),1.24(3H,d),1.40(2H,m),1.60(2H,m),2.14(1H,m),2.80(3H,m),3.64(1H,m),4.91(1H,m),6.13(1H,br s),6.88(1H,d),7.06(1H,d),7.47(1H,dd),8.01(1H,br s)

[0059] Example 2: Synthesis of 4-Fluthalidomide and (R)-1-(4-methoxyphenyl)ethylamine coupling compound (AST-DT-218)

[0060] (R)-1-(4-methoxyphenyl)ethylamine was used as coupling compound.

[0061] DMSO (80 mL), (R)-1-(4-methoxyphenyl)ethylamine (5.58 g, 0.0369 mmol) and N,N-Diisopropylethylamine (DIPEA, 6.927 ml, 0.0398 mmol, purchased from Aldrich) (5.58 g) were added to 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (4-fluorothalidomide, 10 g, 0.0362 mmol). The reaction mixture was stirred at 120° C. for 5 hours, and then water (300 mL) was added. After filtering the precipitate and washing with water (200 mL), the obtained solid was recrystallized from isopropanol and vacuum dried to confirm the solid product.

[0062] Yield: 68%

[0063] Mass:408(M+1)

[0064] 1 H NMR (400MHz, CDCl3):d 1.60(3H,d),2.17(1H,m),2.85(3H,m),3.81(3H,s),4.62(1H,m),4.96(1H,m),6.61( 1H,m),6.68(1H,d),6.89(2H,d),7.09(1H,d),7.28(2H,d),7.36(1H,dd),8.14(1H,br s)

[0065] The structures and molecular weights of the compounds prepared by the above Examples 1 and 2 are listed in Table 1 below.

[0066] Table 1

[0067]

[0068] Experimental Example 1: In vitro assay

[0069] An in vitro experiment was performed to confirm whether the compound (AST-DT-218) prepared in Example 2 is effective in killing brain cancer cells. First, brain cancer cell lines (U87, U373, SK-N-SH) were cultured in each flask to prepare cells that had reached the logarithmic growth phase, then washed with PBS, harvested with trypsin, centrifuged to adjust the cell density, and then resuspended in an appropriate culture medium and dispensed into a 96-well plate. The cells were cultured until they attached to the plate and stabilized. AST-DT-218 and temozolomide, a traditional glioblastoma therapeutic agent, were prepared and diluted to different concentrations (from the highest concentration of 50uM to 1 / 2, 10 points). The wells of the plate were treated with each concentration of each compound for 48 hours, and the cells were cultured to react with the compound. After the culture was completed, the cell number was measured by nuclear staining to evaluate the cell survival rate ( Figure 1 ), and calculate IC 50 (Table 2).

[0070] Table 2

[0071]

[0072] This confirmed that AST-DT-218 exhibited superior efficacy to temozolomide and induced cell death at extremely low concentrations.

[0073] Experimental Example 2: Colony-formation assay

[0074] When the brain cancer cell line U87 cells are fully grown after culturing, trypsin-EDTA is used to separate the cells, and the cells are evenly distributed in a 12-well plate. After the cells adhere to the wall, each well is treated with A ST-DT-218, and the cells are cultured for 2 weeks at 37°C and 5% CO2. After the culture is completed, the cells are fixed and the clones are stained with crystal violet solution, and then washed with PBS to remove background staining. The clones are observed under a microscope, and the number and size of the clones are measured and quantified ( Figure 2 ).

[0075] This confirmed that AST-DT-218 significantly reduced the colony formation of U87 cells ( Figure 3 ).

[0076] Experimental Example 3: DNA fragmentation and cell apoptosis

[0077] Experimental Example 3-1: Western blotting

[0078] The following experiment was performed to confirm whether AST-DT-218 induces DNA breakage and apoptosis. First, when the cells are fully grown after culturing U87 cells, the cells are separated using Trypsin-EDTA, the cells are plated on a 12-well plate and evenly distributed, and after the cells adhere to the wall, they are treated with AST-DT-218. Next, the cells are washed with PBS, the proteins are extracted with a cell lysis buffer, the cell extracts are centrifuged to collect the supernatant, and then the protein samples of the same concentration are loaded on an S DS-PAGE gel for electrophoresis, and the proteins are transferred to a PVDF membrane. After the membrane is blocked with 5% skim milk (or BSA), it is treated with γ-H2AX (DNA damage marker), cleaved PARP (apoptosis marker) and β-actin antibodies at 4°C for 16 hours. Western blot results were analyzed to evaluate the expression levels of γ-H2AX and cleaved PARP. β-actin was used as a loading control to confirm the consistency of protein loading. It was confirmed that AS T-DT-218 induced DNA fragmentation and apoptosis in U87 cells ( Figure 3 ).

[0079] Experimental Example 3-2: Propidium Iodide (PI) apoptosis assay

[0080] As described in Experimental Example 3-1, a PI apoptosis assay was performed to confirm whether AST-DT-218 induces DNA fragmentation and apoptosis. A PI detection kit was prepared, propidium iodide (PI) was added to U87 cells treated with AST-DT-218, and the reaction was carried out at room temperature in the dark for 10 minutes, and then PI fluorescence was measured using a flow cytometer ( Figure 4 ). Thus, it was confirmed that AST-DT-218 induces DNA fragmentation and cell apoptosis.

[0081] Experimental Example 3-3: Sub G1 population measurement

[0082] As described in Experimental Example 3-1, the sub G1 population was measured to confirm AST- Is DT-218 a lure? Induce DNA fragmentation and cell apoptosis. First, 0.5μM and 1U87 cells were treated with AST-DT-218 at a concentration of μM for 48 hours, and then the cell status was observed under a microscope. The sub G1 population value of the cells was measured using a flow cytometer ( Figure 5 ). This confirmed that the sub G1 population value increased in the cell cycle and that AST-DT-218 induced DNA fragmentation and cell apoptosis.

[0083] Experimental Example 4: Dose Response Curve (DRC)

[0084] DRC was created to confirm whether AST-DT-218 effectively kills brain cancer cells. First, glioblastoma (U87 and U373) as brain cancer cell lines were cultured in flasks to prepare cells that reached the logarithmic growth phase, then washed with PBS, harvested with trypsin, centrifuged to adjust the cell density, and then resuspended in an appropriate culture medium and dispensed into a 96-well plate. The cells were cultured until they attached to the plate and stabilized, and then AST-DT-218, control groups (lenalidomide, pomalidomide) were prepared and diluted to different concentrations (from the highest concentration of 10uM to 1 / 2, 10 points (10-point)). The wells of the plate were treated with each concentration of each compound for 48 hours, and the cells were cultured to react with the compounds. After the culture was completed, the cell survival rate was evaluated by measuring the cell number by nuclear staining. Data was collected by measuring the cell survival rate at each concentration, and then a DRC was created ( Figure 6 and 7 ), and calculate IC 50 (Table 3).

[0085] Table 3

[0086]

[0087] AST-DT-218 was confirmed to be effective in killing brain cancer cells used in in vitro experiments compared to lenalidomide and pomalidomide.

[0088] Experimental Example 5: In vivo assay

[0089] Xenograft models were used for in vivo experiments. Figure 8 Specifically, 83NS (GSC cell line derived from GBM patients) was used, and 1×10 4The cells were injected into the brains of mice (n=16) at a dose of 3 μl each to construct an in situ glioblastoma model. Then, the vehicle was administered to mice (n=8), and AST-DT-218 was orally administered (PO) to the remaining mice (n=8) at a concentration of 50 mg / kg per day for 4 weeks, equivalent to a dose of about 29 mM. On the other hand, the administered drug was changed to AST-DT-135, and the same experiment was performed. The results of the survival rate were compared by Kaplan-Meier analysis, and it was confirmed that the survival rates of the AST-DT-135-administered group and the AST-DT-218-administered group were significantly increased compared with the vehicle-administered group. This shows that the compounds AST-DT-135 and AST-DT-218 of the embodiments of the present invention can exhibit effective anti-cancer effects in glioblastoma models.

Claims

1. A pharmaceutical composition for preventing or treating brain cancer, characterized in that it contains a compound represented by the following Chemical Formula 1 or Chemical Formula 2 as an active ingredient: Chemical formula 1: Chemical formula 2:

2. The pharmaceutical composition according to claim 1, wherein the cancer is a relapsed or refractory cancer.

3. The pharmaceutical composition according to claim 1, wherein the cancer is a drug-resistant cancer.

4. The pharmaceutical composition according to claim 1 is characterized in that the composition is administered by intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration or rectal administration.

5. A method for preventing or treating brain cancer in a subject, characterized by comprising the step of administering the compound represented by Chemical Formula 1 or Chemical Formula 2 according to claim 1 to the subject.

6. Use of the compound represented by Chemical Formula 1 or Chemical Formula 2 according to claim 1 in preparing a drug for preventing or treating brain cancer.

7. Use of the compound represented by Chemical Formula 1 or Chemical Formula 2 according to claim 1 in preventing or treating brain cancer.

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