Use of novel molecular adhesive

Abstract

The present invention relates to use of a novel compound capable of functioning as a molecular adhesive for preventing or treating brain cancer.

Description

Uses of novel molecular adhesives

[0001] The present invention relates to the use of a novel compound capable of functioning as a molecular adhesive for the prevention or treatment of brain cancer.

[0002]

[0003] Targeted protein degradation (TPD) refers to a mechanism that directly degrades and eliminates a disease-causing factor after it has been translated into a protein. This is represented by a proteolysis-targeting chimera (PROTAC). A PROTAC consists of a protein of interest (POI) binding moiety (corresponding to the substrate protein linking region), a linker, and an E3 ligase binding moiety (corresponding to the enzyme linking region). The core of PROTAC technology is to allow the substrate of the TPD mechanism to directly interact with the E3 ligase enzyme.

[0004] Molecular glues (MGs) are small molecules that interact with a variety of target proteins, often unpredictably, with properties distinct from PROTACs. While PROTACs are divalent molecules composed of two parts, one of which binds to the POI and the other to an E3 ligase, linked by a linker and resulting in a relatively large molecular weight, MGs are smaller than PROTACs and possess superior pharmacological properties, including higher membrane permeability, better cellular uptake, and blood-brain barrier penetration.

[0005] Various types of molecular adhesives have been discovered, including thalidomide-related analogues, lenalidomide, and pomalidomide, which target the E3 ligase cerebral necrosis factor (CRBN).

[0006] However, thalidomide, lenalidomide, and pomalidomide are known to have polar surface areas (tPSA) values ​​approaching or exceeding 90, which affect blood-brain barrier (BBB) ​​penetration, making it difficult for these drugs to cross the BBB.

[0007] Safety and tolerability are important considerations in the development of anticancer agents, thus highlighting the need to develop immunomodulators with favorable safety profiles while minimizing side effects and long-term complications.

[0008]

[0009] The present invention aims to use a novel compound capable of functioning as a molecular adhesive for the prevention or treatment of brain cancer.

[0010]

[0011] [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:

[0012] [Chemical Formula 1]

[0013]

[0014] [Chemical Formula 2]

[0015]

[0016] [2] In the above [1], the cancer may be a recurrent or incurable cancer.

[0017] [3] In the above [1], the cancer may be a drug-resistant cancer.

[0018] [4] In the above [1], the 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 route.

[0019] [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 a step of administering to the subject a compound represented by the chemical formula 1 or chemical formula 2 of [1].

[0020] [6] In another aspect of the present invention, the present invention relates to the use of a compound represented by Chemical Formula 1 or Chemical Formula 2 of [1] for preparing a drug for preventing or treating brain cancer.

[0021] [7] In another aspect of the present invention, the present invention relates to the use of a compound represented by Chemical Formula 1 or Chemical Formula 2 of [1] for preventing or treating brain cancer.

[0022]

[0023] The present invention relates to the use of a novel compound capable of functioning as a molecular adhesive, wherein the compound can be effectively used as a molecular adhesive for the prevention or treatment of brain cancer while minimizing side effects and long-term complications.

[0024]

[0025] Figure 1 shows the cell death effect of the compound of the present invention in brain cancer cell lines (U87, U373, and SK-N-SH).

[0026] Figure 2 shows the results of a colony-formation assay of the compound of the present invention in a brain cancer cell line (U87).

[0027] Figure 3 shows the expression levels of gamma-H2AX and cleaved PARP of the compound of the present invention in a brain cancer cell line (U87).

[0028] Figure 4 shows the results of a PI apoptosis assay of the compound of the present invention in a brain cancer cell line (U87).

[0029] Figure 5 shows the results of evaluating the Sub G1 Population of the compound of the present invention in a brain cancer cell line (U87).

[0030] Figure 6 shows the dose-response curve for each drug.

[0031] Figure 7 shows dose response curves in glioblastoma cell lines (U87 and U373).

[0032] Figure 8 shows the in vivo experimental design and test results of the compound of the present invention.

[0033]

[0034] The terminology used in this specification is intended to adequately express preferred embodiments of the present invention, and definitions of these terms should be based on the content throughout this specification. Throughout this specification, when a part is said to "include" a component, unless otherwise specifically stated, this does not exclude other components, but rather implies the inclusion of additional components.

[0035] Unless otherwise defined, all technical terms used in this invention have the same meaning as commonly understood by those of ordinary skill in the relevant field. While preferred methods and samples are described herein, similar or equivalent methods are also included within the scope of the present invention.

[0036] 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:

[0037] [Chemical Formula 1]

[0038]

[0039] [Chemical Formula 2]

[0040]

[0041] In one embodiment of the present invention, the compound represented by Chemical Formula 1 or Chemical Formula 2 of the present invention can function as a molecular adhesive. Specifically, the compound represented by Chemical Formula 1 or Chemical Formula 2 can be a molecular adhesive substance that binds to the Cereblon (or CRBN) protein and exerts an anticancer effect through an immunomodulatory imide drug (IMiD) mechanism. Molecular adhesives are small molecules that interact with target proteins, and have the advantages of excellent pharmacological properties and membrane permeability, as well as excellent cellular uptake and blood-brain barrier penetration.

[0042] In one embodiment of the present invention, the protein utilized as an endogenous target protein of an immunomodulatory agent may be CRBN. CRBN forms an E3 ubiquitin ligase complex with damaged DNA binding protein 1 (DDB1), Cullin-4A (CUL4A), and regulator of cullin 1 (ROC1), which ubiquitinates numerous other proteins. CRBN is a substrate receptor for CRL4, an E3 ubiquitin ligase, and CRL4 / CRBN can regulate the intracellular homeostasis of several important intracellular proteins through ubiquitin-dependent degradation of these proteins. In addition, CRBN can directly bind to AMPK, which can induce a wide range of metabolic diseases such as cancer. Furthermore, CRBN may also play a negative regulatory role in CD4+ T cell activation. CRBN deficiency increases CD4+ T cell activation and enhances IL-2 secretion, leading to CD4+ T cell differentiation into Th17 cells. Enhanced T cell activation can sometimes lead to T cell-mediated autoimmune diseases such as autoimmune encephalomyelitis and delayed-type hypersensitivity.

[0043] In the present invention, “treatment” means any act of improving or beneficially changing the symptoms of a disease by administering a composition according to the present invention, “prevention” means any act of suppressing or delaying a disease by administering a composition according to the present invention, and “improvement” means any act of improving the bad state of a disease by administering or ingesting a composition of the present invention to a subject.

[0044] The disease targeted for "treatment," "prevention," or "improvement" of the present invention may be cancer. Cancer typically refers to a physiological condition in animals characterized by abnormal or uncontrolled cell growth. Cancer and cancer pathology may be associated with, for example, metastasis, interference with normally functioning surrounding cells, release of cytokines or other secreted products at abnormal levels, suppression or enhancement of inflammatory or immunological responses, neoplasia, premalignancy, or malignancy.

[0045] In one embodiment of the present invention, the cancer may be a recurrent or refractory cancer.

[0046] In one embodiment of the present invention, the cancer may be a drug-resistant cancer.

[0047] The pharmaceutical composition of the present invention may be administered in a therapeutically effective amount of the active ingredient. The therapeutically effective amount refers to a drug dosage that exhibits an effective brain cancer prevention or treatment effect. The appropriate total daily dosage may be determined by the treating physician within the scope of sound medical judgment. The specific therapeutically effective amount for a specific patient may vary depending on various factors, including the type and degree of response to be achieved, the type and amount of drugs administered concomitantly, the specific composition including whether other agents are used in some cases, the patient's age, weight, general health, sex, and diet, the time of administration, the route of administration, the duration of treatment, and similar factors well known in the medical field.

[0048] The pharmaceutical composition of the present invention may be administered to a subject or individual via any route of administration, including intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal injection, oral administration, topical administration, intranasal injection, intrapulmonary injection, or rectal administration. Preferably, the pharmaceutical composition of the present invention may be administered orally or intravenously.

[0049] In another aspect, the present invention relates to a method for preventing or treating brain cancer in a subject, comprising administering to the subject a compound represented by the above chemical formula 1 or chemical formula 2.

[0050] In the present invention, the term “subject” is used synonymously with “individual,” and the subject may be a mammal, for example, a human, a rat, a cow, a horse, a pig, a dog, a sheep, a goat, or a cat.

[0051] Any description that overlaps with the pharmaceutical composition of the present invention described above is omitted.

[0052] In another aspect, the present invention relates to the use of a compound represented by Chemical Formula 1 or Chemical Formula 2 of claim 1 for preparing a medicament for preventing or treating brain cancer.

[0053] Any description that overlaps with the pharmaceutical composition of the present invention described above is omitted.

[0054] In another aspect, the present invention relates to the use of a compound represented by Chemical Formula 1 or Chemical Formula 2 of claim 1 for preventing or treating brain cancer.

[0055] Any description that overlaps with the pharmaceutical composition of the present invention described above is omitted.

[0056] All matters mentioned in the compositions, treatment methods and uses of the present invention apply equally unless they are contradictory.

[0057] Hereinafter, preferred examples are presented to help understand the present invention, but the following examples are only illustrative of the present invention, and it is obvious to those skilled in the art that various changes and modifications are possible within the scope and technical idea of ​​the present invention, and it is also natural that such changes and modifications fall within the scope of the appended patent claims.

[0058] Example

[0059] The novel compound of the present invention can be prepared according to the following reaction scheme, but is not limited thereto.

[0060] [Reaction formula]

[0061]

[0062] Example 1. Synthesis of 4-fluorothalidomide and (S)-penta-2-amine coupling compound (AST-DT-135)

[0063] (S)-penta-2-amine was used as a coupling compound.

[0064] 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (4-fluorothalidomide, 10 g, 0.0362 mmol, purchased from EOS, China) was added DMSO (60 mL), (S)-penta-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). The reaction mixture was stirred at 120°C for 5 h, and then water (300 mL) was added. The precipitate was filtered and washed with water (200 mL), and the obtained solid was The solid was confirmed as the product by recrystallization with isopropyl alcohol and vacuum drying.

[0065] yield: 34%

[0066] Mass: 344(M+1)

[0067] 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)

[0068] Example 2. Synthesis of 4-fluorothalidomide and (R)-1-(4-methoxyphenyl)ethylamine coupling compound (AST-DT-218)

[0069] (R)-1-(4-methoxyphenyl)ethylamine was used as a coupling compound.

[0070] 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (4-fluorothalidomide, 10 g, 0.0362 mmol) was added 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). The reaction mixture was stirred at 120 °C for 5 h, and then water (300 mL) was added. The precipitate was filtered and washed with water (200 mL), and the obtained solid was purified by isopropyl The solid product was confirmed to be a product by recrystallization with alcohol and vacuum drying.

[0071] yield: 68%

[0072] Mass: 408(M+1)

[0073] 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)

[0074] The structure and molecular weight of the compounds prepared through Examples 1 and 2 are described in Table 1 below.

[0075] CodeNameStructureAST-DT-1352-(2,6-dioxopiperidin-3-yl)-4-(((S)-pentan-2-yl)amino)isoindoline-1,3-dione 2-(2,6-dioxopiperidin-3-yl)-4-(((S)-pentan-2-yl)amino)isoindoline-1,3-dione AST-DT-2182-(2,6-dioxopiperidin-3-yl)-4-(((R)-1-(4-methoxyphenyl)ethyl)amino)isoindoline-1,3-dione

[0076]

[0077] Experimental Example 1. In vitro assay

[0078] In vitro experiments were performed to determine whether the compound (AST-DT-218) prepared in Example 2 was effective in killing brain cancer cells. First, brain cancer cell lines (U87, U373, SK-N-SH) were cultured in each flask, and cells that had reached the logarithmic growth phase were prepared. Then, the cells were washed with PBS and harvested with trypsin. The cells were centrifuged to adjust the cell density, 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 conventional glioblastoma treatment, were prepared and diluted to various concentrations (10 points by diluting 1 / 2 from the highest concentration of 50 uM). The wells of the plate were treated with each compound concentration for 48 hours, and the cells were cultured to allow them to react with the compounds. After the culture was completed, the cell viability was evaluated by measuring the cell number through cell nuclear staining (Fig. 1), and IC 50 was calculated (Table 2).

[0079] IC 50 (mM)Brain Cancer Cell LineU87U373SK-N-SHAST-DT-2183.3860.7000.239Temozolomide291.2-24.570

[0080] Through this, it was confirmed that AST-DT-218 showed superior efficacy than temozolomide and induced cell death at very low concentrations.

[0081]

[0082] Experimental Example 2. Colony-formation assay

[0083] U87 cells, a brain cancer cell line, were cultured and, when sufficiently grown, detached using Trypsin-EDTA, uniformly distributed in 12-well plates, and after cell attachment, AST-DT-218 was treated in each well, and the cells were cultured in an environment of 37°C and 5% CO₂ for 2 to 3 weeks. Upon completion of culture, the cells were fixed and colonies were stained using crystal violet solution. After staining, the cells were washed with PBS to remove background staining. The colonies were observed using a microscope, and the number and size of the colonies were measured and quantified (Fig. 2).

[0084] Through this, it was confirmed that AST-DT-218 significantly reduced colony formation of U87 cells.

[0085]

[0086] Experimental Example 3. DNA Fragmentation and Apoptosis

[0087] Experimental Example 3-1. Western blotting

[0088] To determine whether AST-DT-218 induces DNA fragmentation and apoptosis, the following experiments were performed. First, U87 cells were cultured, and once sufficiently grown, cells were detached using Trypsin-EDTA. The cells were evenly distributed in 12-well plates, and after attachment, they were treated with AST-DT-218. Next, the cells were washed with PBS, proteins were extracted using cell lysis buffer, the cell extracts were centrifuged, and the supernatants were collected. Protein samples of equal concentrations were loaded onto SDS-PAGE gels, electrophoresis was performed, and the proteins were transferred to PVDF membranes. The membranes were blocked with 5% skim milk (or BSA) and then treated with antibodies against γ-H2AX (DNA damage marker), cleaved PARP (apoptosis marker), and beta-actin, respectively, at 4°C for 16 hours. Western blotting analysis was used to evaluate the expression levels of γ-H2AX and cleaved PARP. Beta-actin was used as a loading control to confirm the consistency of protein loading. This confirmed that AST-DT-218 induced DNA fragmentation and apoptosis in U87 cells (Fig. 3).

[0089]

[0090] Experimental Example 3-2. PI (Propidium Iodide) apoptosis assay

[0091] To determine whether AST-DT-218 induces DNA fragmentation and apoptosis, as described in Experimental Example 3-1, a PI apoptosis assay was performed. A PI detection kit was prepared, and propidium iodide (PI) was added to AST-DT-218-treated U87 cells. The cells were incubated in a darkened room at room temperature for 10 minutes. PI fluorescence was then measured using a flow cytometer (Fig. 4). This confirmed that AST-DT-218 induced DNA fragmentation and apoptosis.

[0092]

[0093] Experimental Example 3-3. Measurement of sub-G1 population

[0094] To confirm whether AST-DT-218 induces DNA fragmentation and apoptosis, as in Experimental Example 3-1, the sub-G1 population was measured. First, U87 cells were treated with AST-DT-218 at concentrations of 0.5 μM and 1 μM for 48 hours, and the cells were observed under a microscope. The sub-G1 population value of the cells was measured using a flow cytometer (Fig. 5). This confirmed that the sub-G1 population value increased in the cell cycle, confirming that AST-DT-218 induced DNA fragmentation and apoptosis.

[0095]

[0096] Experimental Example 4. Dose Response Curve (DRC)

[0097] To determine whether AST-DT-218 is effective in killing brain cancer cells, a DRC was created. First, glioblastoma (U87 and U373), a brain cancer cell line, was cultured in a flask, and cells that had reached the logarithmic growth phase were prepared. Then, the cells were washed with PBS and harvested with trypsin. The cells were centrifuged to adjust the cell density, resuspended in the appropriate culture medium, and seeded in a 96-well plate. After culturing until the cells attached to the plate and stabilized, AST-DT-218 and controls (lenalidomide, pomalidomide) were prepared and diluted at various concentrations (10-point dilution from the highest concentration of 10 uM, 1 / 2 dilution). Each compound was treated in a well of the plate at each concentration for 48 hours, and the cells were cultured to allow them to react with the compounds. After the culture was completed, the cell number was measured through cell nuclear staining to evaluate cell viability. Data were collected by measuring cell viability at each concentration, then DRC was created (Figs. 6 and 7), and IC 50 was calculated (Table 3).

[0098] IC of DiseaseCell lineAST-DT-218 (μM) 50 GlioblastomaU873.363U3731.239

[0099] Compared with lenalidomide and pomalidomide, AST-DT-218 was confirmed to be effective in killing brain cancer cells used in in vitro experiments.

[0100]

[0101] Experimental Example 5. In vivo assay

[0102] In vivo experiments were conducted using a xenograft model. The experimental design and results are shown in Fig. 8. Specifically, 83NS (a GSC cell line derived from GBM patients) was used, and an orthotopic glioblastoma model was established by intracranial injection of 1x10⁴ cells in a volume of 3μl into each mouse (n=16). Subsequently, the vehicle was administered to mice (n=8), and AST-DT-218 was administered orally (PO) daily for 4 weeks at a concentration of 50mg / kg, corresponding to a dose of approximately 29mM. Meanwhile, the same experiment was conducted with the drug administered instead of AST-DT-135. Kaplan-Meier analysis was performed to compare the survival rates, and it was confirmed that the AST-DT-135 and AST-DT-218 groups showed significantly increased survival rates compared to the vehicle group. This suggests that the compounds of the present invention, AST-DT-135 and AST-DT-218, can exhibit effective anticancer activity in a glioblastoma model.

Claims

1. 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. [Chemical Formula 1] [Chemical Formula 2] 2. A pharmaceutical composition according to claim 1, wherein the cancer is recurrent or refractory cancer.

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

4. A pharmaceutical composition according to claim 1, wherein the composition is administered by intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal injection, oral administration, topical administration, intranasal injection, intrapulmonary injection, or rectal administration.

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

6. Use of a compound represented by Chemical Formula 1 or Chemical Formula 2 of paragraph 1 for manufacturing a drug for preventing or treating brain cancer.

7. Use of a compound represented by Chemical Formula 1 or Chemical Formula 2 of paragraph 1 for preventing or treating brain cancer.

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