Proteasome inhibitor comprising lactone structure

Abstract

The present invention relates to a novel proteasome inhibitor comprising a digoxigenin-derived lactone ring-based compound as an active ingredient, and can simultaneously exhibit high 20S proteasome inhibitory activity and low normal cell toxicity. The compound has excellent cancer cell selectivity, has pharmacological properties with improved anticancer effects and therapeutic indices, and can be useful for various carcinomas such as multiple myeloma, breast cancer, and hepatocellular carcinoma.

Description

Proteasome inhibitors containing lactone structures

[0001] The present invention relates to a pharmaceutical composition exhibiting an anticancer effect by inhibiting the activity of proteasome, and more specifically, to an anticancer composition comprising a compound having a lactone structure, which effectively inhibits the activity of 20S proteasome and has low toxicity to human-derived normal cells, or a pharmaceutically acceptable salt thereof, as an active ingredient, and a use thereof.

[0002] The composition of the present invention can be usefully utilized in the prevention or treatment of tumors dependent on the proteasome pathway, such as multiple myeloma.

[0003]

[0004] The proteasome is a protein degradation complex that plays a key role in maintaining intracellular protein homeostasis. It selectively removes unnecessary or damaged proteins within cells through the ubiquitin-proteasome system (UPS). In particular, cancer cells have a metabolic profile that relies on protein synthesis and degradation, and therefore strategies that selectively inhibit proteasome activity are being utilized as an effective mechanism of anticancer treatment. Bortezomib, a representative proteasome inhibitor developed against this backdrop, binds to the β5 subunit of the 20S proteasome, inhibiting its chymotrypsin-like activity and thereby inducing apoptosis. Bortezomib has been approved for the treatment of multiple myeloma and mantle cell lymphoma, and is considered the first clinically applicable proteasome inhibitor.

[0005] However, bortezomib presents several structural, pharmacological, and industrial challenges. Bortezomib exists in the solid state as a trimeric boroxine, and exposure to moisture can hydrolyze it into monomeric boronic acid, potentially reducing its efficacy. Furthermore, its formulation instability necessitates frozen storage, and room temperature or refrigerated conditions can result in appearance changes and loss of active ingredients. The manufacturing process requires special conditions, including the use of hydrochloric acid gas, which poses safety and cost challenges for mass production. Furthermore, non-target toxicities, such as peripheral neuropathy, frequently occur with long-term use, and drug resistance issues have also been reported.

[0006] Therefore, there is a growing need for novel proteasome inhibitors that maintain the efficacy of existing bortezomib-based treatments while also offering structural stability, toxicity safety, and ease of manufacturing. To address these issues, the present invention provides a lactone-based compound that is structurally distinct from existing bortezomib-based compounds.

[0007]

[0008] The present invention relates to a pharmaceutical composition for preventing or treating cancer and a food composition for preventing or improving cancer, and more specifically, to a pharmaceutical composition and a food composition comprising, as an active ingredient, a compound that suppresses the proliferation of cancer cells and induces apoptosis by selectively inhibiting 20S proteasome activity, or a pharmaceutically acceptable salt thereof.

[0009] The compound of the present invention has a digoxigenin-derived skeleton and, unlike existing proteasome inhibitors, does not contain structures such as boron-based structures or sugar linkers that may induce cardiotoxicity, thereby reducing the toxic burden on the body while exhibiting excellent proteasome inhibitory activity. In particular, in an experiment targeting fibroblast cells (L929), it showed a cell viability rate of over 80%, confirming low toxicity to normal cells and excellent safety.

[0010] Therefore, the present invention aims to provide a new anticancer strategy targeting the proteasome while improving the problems of existing anticancer drugs such as serious side effects, structural instability, and complexity of the manufacturing process, thereby providing a safer and more effective means of cancer prevention and treatment.

[0011]

[0012] The technical problem to be solved by the present invention is not limited to the above-mentioned contents, and should be interpreted to include all technical problems that can be understood by a person skilled in the art from the description of this specification.

[0013]

[0014] As an embodiment for achieving the above technical task, the present invention relates to a pharmaceutical composition for preventing or treating cancer, which comprises a compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient and exhibits an anticancer effect by inhibiting 20S proteasome activity.

[0015] <Chemical Formula 1>

[0016]

[0017] As another embodiment, the present invention relates to a pharmaceutical composition for preventing or treating cancer, which comprises a compound represented by Chemical Formula 2 or a pharmaceutically acceptable salt thereof as an active ingredient, and which inhibits the proliferation of cancer cells and induces apoptosis through a mechanism of inhibiting 20S proteasome activity.

[0018] <Chemical Formula 2>

[0019]

[0020] In addition, the composition of the present invention is characterized by low toxicity to normal cells and excellent safety, as shown by a cell viability rate of 80% or more in a cytotoxicity evaluation targeting fibroblasts (L929).

[0021] As another example, the composition of the present invention may exhibit a preventive or therapeutic effect on various cancers, including at least one selected from the group consisting of multiple myeloma, mantle cell lymphoma, breast cancer, hepatocellular carcinoma, lung cancer, head and neck cancer, tongue cancer, pharyngeal cancer, salivary gland cancer, prostate cancer, pancreatic cancer, colon cancer, melanoma, osteosarcoma, and leukemia.

[0022]

[0023] The compound represented by Chemical Formula 1 or Chemical Formula 2 according to the present invention can exhibit excellent anticancer activity by blocking the protein degradation pathway of cancer cells through a mechanism of action that inhibits 20S proteasome activity and inducing intracellular stress and apoptosis. In particular, it can be effectively applied to various cancer types and can exhibit therapeutic effects on tumors with high proteasome dependency, such as multiple myeloma, hepatocellular carcinoma, breast cancer, and non-small cell lung cancer.

[0024] Furthermore, the compound of the present invention does not contain the boron-based structure that has been problematic with existing proteasome inhibitors, and, despite having a digoxigenin-derived skeleton, does not contain toxic functional groups of the cardiac glycoside series that cause cardiotoxicity or systemic side effects, thereby ensuring excellent safety. In fact, in a fibroblast (L929) cell experiment, it showed a cell viability rate of over 90%, confirming low toxicity to normal cells.

[0025]

[0026] The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the detailed description of the present invention or the composition of the invention described in the claims.

[0027]

[0028] Figure 1 shows the chemical formulas and proteasome inhibition rates of the compounds used in Experimental Example 1 of the present invention. Compound 1 (CPK-M1-017650-C03, Chemical Formula 1) showed an inhibition rate of 78.5%, and compound 2 (CPK-M1-017650-E03, Chemical Formula 3) showed the highest activity at 85.7%. Compound 3 (CPK-M1-017650-F03, Chemical Formula 2) also showed a high inhibition rate of 80.8%, and was evaluated to have excellent activity. Compound 5 (CPK-M1-017650-B04, chemical formula 4) showed an inhibition rate of 85.0%, demonstrating similar activity to compound 2. On the other hand, compound 4 (CPK-M1-017650-H03) is a natural product-derived ingredient, triptolide, and was confirmed to have minimal proteasome inhibition activity, with an inhibition rate of only 8.5%. Compound 6 (CPK-M1-017650-D04) also showed an inhibition rate of 18.7%, and is classified as a compound with relatively low activity.

[0029] FIG. 2 is a table and graph showing the results of an experiment measuring the degree to which each compound of the present invention (CPK-M1-017650 series) inhibits 20S proteasome activity. 1 to 6 on the x-axis correspond to the compound numbers in FIG. 1. When only cells were present (Cell only), it showed 0% inhibition activity, whereas Bortezomib, used as a positive control, showed a high inhibition activity of 97.4%, proving the effectiveness of the experimental system. Among the candidate compounds of the present invention, the compound represented by Chemical Formula 1 (CPK-M1-017650-C03) showed an inhibition activity of 78.5%, and the compound represented by Chemical Formula 2 (CPK-M1-017650-F03) showed an inhibition activity of 80.8%, respectively. It was confirmed that these have high 20S proteasome inhibition activity comparable to that of Bortezomib.

[0030] FIG. 3 is a graph showing the results of the anticancer activity and toxicity evaluation on normal cells of a compound (CPK-M1-017650-C03) corresponding to chemical formula 1 of the present invention. The experiment measured cell viability (%) after 0, 24, 48, and 72 hours for five cancer cell lines (K562, SKBR3, NCI-H358, Jurkat, PANC1) and one normal cell line (L929). The names of the cell lines shown in the graph are as follows from the left: K562, SKBR3, NCI-H358, L929, Jurkat, PANC1. After 72 hours, the viability of PANC1 significantly decreased to less than 10%, SKBR3 and K562 also showed viability of about 20% or less, and NCI-H358 and Jurkat showed viability of about 30%. On the other hand, the normal cell line L929 maintained a survival rate of more than 90% even after 72 hours, confirming that the compound exhibited strong cancer cell selectivity and low normal cell toxicity.

[0031] FIG. 4 is a graph showing the results of the anticancer activity and toxicity evaluation on normal cells of a compound (CPK-M1-017650-F03) corresponding to chemical formula 2 of the present invention. The names of the cell lines indicated in the graph are from the left: K562, SKBR3, NCI-H358, L929, Jurkat, PANC1. This compound, which was performed under the same experimental conditions, also induced apoptosis by showing a low viability of less than 10% or about 20% at 72 hours in cancer cell lines such as K562, SKBR3, Jurkat, and PANC1, and a decrease in viability was also observed in NCI-H358. On the other hand, the viability of the L929 normal cell line was maintained at over 100%, so the compound corresponding to chemical formula 2 was also evaluated to have anticancer activity with low toxicity on normal cells and excellent selectivity for cancer cells.

[0032] FIG. 5 is a graph showing the results of the anticancer activity and toxicity evaluation on normal cells of a compound (CPK-M1-017650-E03) corresponding to chemical formula 3 of the present invention. The names of the cell lines shown in the graph are from the left: K562, SKBR3, NCI-H358, L929, Jurkat, PANC1. The experiment evaluated cell viability by measuring the cell viability at 0, 24, 48, and 72 hours for human-derived cancer cell lines (K562, SKBR3, NCI-H358, Jurkat, PANC1) and normal cell line (L929). As a result of the experiment, after 72 hours, K562 and SKBR3 cells showed low viability of 10-20%, and Jurkat, PANC1, and NCI-H358 also showed a decrease in viability to around 40%, confirming excellent anticancer activity against various cancer cells. However, for the normal cell line L929, the survival rate decreased to approximately 60% after 72 hours under the same conditions.

[0033] Figure 6 is a graph showing the results of the anticancer activity and toxicity evaluation on normal cells of a compound (CPK-M1-017650-B04) corresponding to a comparative example. The names of the cell lines shown in the graph are from the left: K562, SKBR3, NCI-H358, L929, Jurkat, PANC1. The experiment measured cell viability at 0, 24, 48, and 72 hours for various cancer cell lines (K562, SKBR3, NCI-H358, Jurkat, PANC1) and normal cell line (L929). As a result, anticancer activity was observed in several cancer cell lines, including K562, Jurkat, and PANC1, as the viability decreased to 10-30% at 72 hours. However, for the normal cell line L929, the viability significantly decreased to about 40% at 72 hours, and a rapid decrease in viability was confirmed from the 24-hour time point.

[0034] Figure 7 is a graph showing the results of the anticancer activity and toxicity evaluation of bortezomib against normal cells. The names of the cell lines shown in the graph are, from left to right: K562, SKBR3, NCI-H358, L929, Jurkat, and PANC1. In the case of bortezomib, a significant decrease in viability was also observed in many cancer cell lines, including K562, Jurkat, and PANC1, at the 72-hour time point. However, even for the normal cell line L929, the viability significantly decreased to approximately 20% at the 72-hour time point, and a rapid decrease in viability was observed from the 24-hour time point.

[0035] Figure 8 is a graph comprehensively evaluating the anticancer activity of compounds corresponding to Chemical Formula 1 and Chemical Formula 2 of the present invention by comparing them with the anticancer activity of bortezomib. The compounds used are, from the left, Control, Bortezomib, CPK-M1-017650-C03 (Chemical Formula 1), and CPK-M1-017650-F03 (Chemical Formula 2). The compounds of the present invention were found to have pharmacological properties that achieved excellent cancer cell selectivity and low toxicity simultaneously by showing anticancer activity at a level similar to bortezomib in various cancer cell lines, while maintaining a higher survival rate of normal cells (L929). This comparative evaluation suggests that the compounds of the present invention may have an improved therapeutic index compared to existing proteasome inhibitors.

[0036]

[0037] The present invention relates to a proteasome inhibitor composition comprising a novel compound as an active ingredient. In an embodiment of the present invention, compounds represented by Chemical Formulas 1 and 2 were synthesized, and then tested to determine whether the compounds inhibit proteasome activity and cell proliferation in human cell lines (K562, Jurkat, PANC1, etc.).

[0038] Experimental results showed that the compounds of the present invention exhibited a proteasome inhibition rate of over 70%, and in particular, they were confirmed to have significantly less human toxicity compared to the existing proteasome inhibitor, bortezomib. These compounds can be formulated into various pharmaceutical formulations.

[0039]

[0040] Hereinafter, the present invention will be described in more detail with specific examples. However, the following examples are provided as examples to ensure that those skilled in the art can sufficiently convey the spirit of the present invention.

[0041] Therefore, the present invention is not limited to the embodiments presented below and may be embodied in other forms. The embodiments presented below are described only to clarify the idea of ​​the present invention, and the present invention is not limited thereto.

[0042]

[0043] definition

[0044] Expressions such as "including," "comprising," "having," and the like used herein should be understood as open-ended terms that imply the possibility of including other embodiments in a similar manner to "comprising," unless otherwise stated in the phrase or sentence in which the expression is included.

[0045] The term "and / or" as used herein may mean any one or more of the items, any combination of the items, or all of the items associated with the term.

[0046]

[0047] Meanwhile, unless otherwise defined, the technical and scientific terms used herein have meanings commonly understood by those of ordinary skill in the art to which this invention pertains, and are terms defined in consideration of their functions in the present invention, which may vary depending on the intent or custom of the user or operator. Therefore, the definitions of these terms should be determined based on the contents throughout this specification, and in the following description, explanations of well-known functions and configurations that may unnecessarily obscure the gist of the present invention will be omitted.

[0048]

[0049] Hereinafter, the present invention will be described in detail.

[0050]

[0051] The present invention relates to a pharmaceutical and food composition comprising, as an active ingredient, a compound that inhibits the proliferation of cancer cells and induces apoptosis by inhibiting the activity of 20S proteasome, or a pharmaceutically acceptable salt thereof.

[0052] Existing proteasome inhibitors, while exhibiting promising anticancer effects, have limited clinical application and mass production due to structural instability, complex manufacturing processes, and toxicity. For example, bortezomib, a representative proteasome inhibitor approved for anticancer use, is vulnerable to moisture due to its solid-state trimeric boroxine structure. Its active ingredient is lost upon hydrolysis, requires refrigerated storage, and requires special reagents such as hydrochloric acid gas during manufacturing, making industrial production difficult. Furthermore, side effects such as peripheral neuropathy and drug resistance have been frequently reported with long-term administration.

[0053] Accordingly, there is a need for the development of novel compounds and compositions that can stably inhibit proteasome activity while overcoming the toxicity and structural limitations of existing inhibitors. In particular, compounds that maintain potent proteasome inhibition while minimizing toxicity to normal cells have the potential to maximize therapeutic efficacy in various cancers, particularly those with a high proteasome dependency, such as multiple myeloma.

[0054] The compound of the present invention is a lactone-based compound that exhibits potent inhibitory activity against the 20S proteasome and exhibits low cytotoxicity against normal human cells. The compound of the present invention is structurally stable, easy to manufacture, and possesses both anticancer effects and safety, making it a useful alternative or complementary drug to existing proteasome inhibitors.

[0055] More specifically, the compounds of the present invention may have structures represented by the following Chemical Formulas 1 and 2. These compounds are commonly based on a steroid core structure consisting of four rings, in which at least one ring is introduced with a lactone ring or an α,β-unsaturated carbonyl structure, thereby providing a nucleophilic reactive center capable of selectively binding to the β5 subunit of the 20S proteasome. This structure enables selective action as a proteasome inhibitor, while at the same time imparting molecular-level stability that reduces aggressiveness toward normal cells.

[0056]

[0057] <Chemical Formula 1>

[0058]

[0059] <Chemical Formula 2>

[0060]

[0061]

[0062] In addition, the compounds of the present invention were designed to exhibit improved pharmacological properties, such as not only proteasome inhibitory activity but also drug metabolism stability and cell permeability, based on the structure of the substituents attached to the steroid core structure. In particular, the compounds represented by Chemical Formula 1 (CPK-M1-017650-C03) and Chemical Formula 2 (CPK-M1-017650-F03) were confirmed to exhibit potent proteasome inhibitory activity comparable to bortezomib, while maintaining the cell viability of 80% or more for normal cells such as fibroblasts (L929), thereby exhibiting an excellent selective toxicity profile.

[0063] Thus, the compound of the present invention structurally overcomes the toxicity and metabolic instability associated with non-target protein binding, which were major problems with existing proteasome inhibitors, while maintaining or enhancing the anticancer effect of targeting the proteasome. Accordingly, the compound of the present invention possesses pharmacological safety and selectivity sufficient to be utilized for anticancer treatment.

[0064] The specific mechanism of action of the compound of the present invention is as follows.

[0065] First, the compound of the present invention has a mechanism of action that induces abnormal protein accumulation in cancer cells by selectively inhibiting the activity of the 20S proteasome, which plays a central role in intracellular protein degradation, thereby increasing cellular stress and inducing apoptosis (cell suicide).

[0066] The proteasome is a complex protein system that degrades proteins tagged with ubiquitin. It is involved in various physiological functions, including maintaining cellular homeostasis, cell cycle regulation, inflammatory responses, and cell survival. Among these, the 20S proteasome is the core catalytic core of the proteasome. The β5 subunit within it exhibits chymotrypsin-like activity, which cleaves and removes target proteins.

[0067] The compound of the present invention comprises a structure that can selectively act on the active site of the β5 unit, and in particular, structural elements such as a lactone ring or an alkene-carbonyl group can exhibit an irreversible or semi-reversible inhibitory effect by reacting with a nucleophilic residue (e.g., a hydroxyl group of threonine) within the active site. This reaction is based on a targeting mechanism similar to that of the existing bortezomib, but since the compound of the present invention has the structural characteristic of not containing a boron atom, it has the advantage of improved metabolic stability and tissue selectivity.

[0068] Meanwhile, cancer cells tend to synthesize and degrade more proteins than normal cells, leading to a high dependence on the proteasome pathway. The compound of the present invention exploits this characteristic of cancer cells by blocking the protein degradation pathway, thereby inducing the accumulation of abnormal proteins and intracellular stress signals. Consequently, it induces selective cell death through cell cycle arrest, inhibition of autophagy, and induction of apoptosis.

[0069] In addition, since the compound of the present invention maintains a relatively stable balance between protein synthesis and degradation in normal cells, even if the same proteasome inhibition effect is exhibited, it does not have a relatively large effect on cell survival, which allows the compound to be used as a therapeutic candidate with low toxicity to normal cells and high selectivity for cancer cells.

[0070] That is, the compound of the present invention is characterized by exhibiting a strong anticancer effect based on a target mechanism that inhibits the proteasome, while at the same time securing excellent safety and selectivity through structural characteristics that result in low toxicity to normal cells.

[0071] As described above, the compound of the present invention has the characteristic of inducing selective apoptosis of cancer cells based on its mechanism of action of inhibiting 20S proteasome activity. Accordingly, it can be usefully applied for the prevention and treatment of various cancers. In particular, since the compound exhibits a potent effect in cancer cells highly dependent on the proteasome pathway, it can be used for the prevention or treatment of the following diseases.

[0072] Specific target cancers include multiple myeloma, mantle cell lymphoma, breast cancer, hepatocellular carcinoma, lung cancer, head and neck cancer, tongue cancer, pharyngeal cancer, salivary gland cancer, prostate cancer, pancreatic cancer, colon cancer, melanoma, osteosarcoma, and leukemia, including major cancers to which conventional proteasome inhibitors have been applied or for which therapeutic effects have been observed.

[0073] The composition of the present invention can be utilized in various ways depending on the stage of disease progression. For example, it can be applied to various situations, such as early preventive intake in high-risk patients or in the precancerous stage, adjuvant therapy to prevent recurrence after cancer treatment, and combination therapy to enhance the effectiveness of existing anticancer agents.

[0074] In addition, the composition of the present invention has the potential to be applied to other diseases in which abnormal protein accumulation or cell stress regulation is a major pathogenesis, in addition to cancer, and the scope of application can be further expanded through further research in the future.

[0075] Meanwhile, the compound according to the present invention can be prepared into a pharmaceutical composition, and the composition can be utilized as a functional ingredient for the prevention or treatment of cancer. Depending on the type of composition, the compound of the present invention can be administered alone or in combination with other effective ingredients, and can be prepared in various formulations.

[0076] First, as a pharmaceutical composition, the compound of the present invention can be manufactured in the form of tablets, capsules, suspensions, granules, syrups, hard or soft gelatin preparations, injections, etc. according to general pharmaceutical manufacturing techniques. Formulations for oral administration can include tablets, capsules, granules, powders, and liquid preparations, and, if necessary, can also be applied as controlled-release preparations or preparations with enhanced gastrointestinal absorption properties. If application as an injection is required, it can be provided as an aqueous solution or lyophilized preparation manufactured under aseptic conditions.

[0077] The compound of the present invention can be applied to a formulation containing pharmaceutically acceptable carriers, adjuvants, binders, lubricants, release agents, stabilizers, preservatives, buffers, isothermal regulators, etc., and is composed based on materials and techniques commonly used in the pharmaceutical industry. In addition, the compound of the present invention can be administered in combination with other anticancer agents (e.g., bortezomib, doxorubicin, cisplatin, etc.) or adjuvant therapeutic agents (e.g., immunomodulators, antioxidants, etc.) to exhibit synergistic effects.

[0078] In this way, the compound of the present invention can be flexibly applied throughout the pharmaceutical industry as an active ingredient, and can have a wide range of usability depending on various formulations and administration routes.

[0079] Meanwhile, as a result of cell experiments on the compound of the present invention as described above, it was confirmed that it exhibits selective anticancer activity that effectively inhibits the growth of cancer cells while maintaining relatively low toxicity to normal cells. In particular, it was evaluated that it induces apoptosis in various cancer cells that depend on the protein degradation pathway by effectively inhibiting the activity of the 20S proteasome. Compared to the positive control, bortezomib, it was confirmed that the compound exhibited a similar level of inhibitory effect and significantly alleviated side effects.

[0080] In this way, the compound of the present invention has anticancer effects while having relatively low toxicity to normal cells, and thus, it has been proven that it has the potential to be applied as a pharmaceutical composition in the future.

[0081]

[0082] Meanwhile, in the case of the present invention, a method for preventing or treating cancer can be provided, which includes administering an effective amount of a compound corresponding to the chemical formula 1,2 or a pharmaceutically acceptable salt thereof to a cancer patient to prevent or treat cancer by inhibiting 20S proteasome activity, and the compound corresponding to the chemical formula 1,2 of the present invention or a pharmaceutically acceptable salt thereof can be used for the purpose of treating cancer.

[0083] The compound has a mechanism of disrupting protein homeostasis in tumor cells and inducing apoptosis by selectively inhibiting proteasome activity. In particular, the compound of the present invention has been confirmed to have reduced cytotoxicity and enhanced anticancer efficacy compared to existing drugs such as bortezomib or carfilzomib.

[0084] In the above method, the cancer to be treated or prevented may include at least one selected from the group consisting of multiple myeloma, mantle cell lymphoma, breast cancer, hepatocellular cancer, lung cancer, head and neck cancer, tongue cancer, pharyngeal cancer, salivary gland cancer, prostate cancer, pancreatic cancer, colon cancer, melanoma, osteosarcoma, and leukemia.

[0085] The above compound can be used alone or in combination with other anticancer agents (e.g., bortezomib, immunomodulators, antibodies, etc.), and can be administered via various pharmaceutically acceptable routes, such as intravenous injection.

[0086] These preventive or therapeutic effects can be confirmed in Experimental Examples 2 and 3.

[0087]

[0088] Below, the compound of the present invention is described in detail through specific examples and experimental examples.

[0089]

[0090] Example 1.

[0091] In this example, CPK-M1-017650-C03, a lactone ring-based digoxigenin derivative compound corresponding to the chemical formula 1 of the present invention, was synthesized. This compound has a structure in which an acyl group is substituted at carbon 3 based on the steroid skeleton of digoxigenin and a lactone ring is introduced near carbon 17, and has structural features designed to selectively bind to the active site of the 20S proteasome.

[0092] The synthesis was carried out using digoxigenin as the starting material, and first, an intermediate was prepared through an acylation reaction at the 3-hydroxy group. 1.0 g of digoxigenin was dissolved in anhydrous dichloromethane, and then pyridine and acetyl chloride were reacted. The reaction was carried out at 0–5°C for 1 hour, followed by stirring at room temperature for 12 hours. After the reaction, the acylated intermediate was isolated through washing with water and drying. Next, a cyclization reaction was performed to form a lactone ring. The acylated intermediate was dissolved in anhydrous dimethylformamide, and a γ-hydroxycarboxylic acid derivative, DCC, and DMAP were added. The reaction was carried out at 50°C for 18 hours under a nitrogen atmosphere. After the reaction was completed, the generated byproducts were removed through filtration, and the filtrate was extracted with an organic solvent and concentrated under reduced pressure. The final product was separated and purified using silica gel column chromatography (hexane:ethyl acetate = 7:3) and was obtained as a yellowish-white crystalline solid with a yield of approximately 57%.

[0093] The prepared compound is represented by the following chemical formula 1, and was confirmed to have m / z 514.2 [M+H]+ in the positive ion mode through mass spectrometry, and was analyzed on the ¹H-NMR spectrum (CDCl₃, 400 MHz) as δ 7.14 (d, J = 8.2 Hz, 1H), 6.80 (d, J = 8.2 Hz, 1H), 5.35 (s, 1H), 4.52 (t, 1H), 3.62-3.44 (m, 2H), 2.35 (m, 2H), 1.92-0.85 (m, multiple alkyl signals). The purity of the purified compound was confirmed through high-performance liquid chromatography (HPLC), and showed a purity of 96% or more.

[0094]

[0095] <Chemical Formula 1>

[0096]

[0097]

[0098] Example 2.

[0099] In this example, CPK-M1-017650-F03, a digoxigenin-derived compound corresponding to the chemical formula 2 of the present invention, was synthesized. This compound has a structure in which an asymmetric alkyl group is substituted at the 3rd carbon position based on the digoxigenin skeleton, and a selective electronegative substituent is introduced while maintaining the 12th to 13th double bonds, thereby improving metabolic stability and cell permeability in vivo.

[0100] The synthesis was performed using digoxigenin as a starting material, and first, an alkylation reaction was performed on the 3-hydroxy group while preserving the hydroxy group and double bond located near carbon 17. 1.0 g of digoxigenin was dissolved in anhydrous dimethylformamide, and then an alkyl bromide derivative (1.1 eq) and potassium carbonate (2.0 eq) were added. The mixture was stirred and reacted at 60°C for 24 hours. The reaction mixture was extracted with water and ethyl acetate, and the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure.

[0101] The concentrated reaction product was purified through silica gel column chromatography (hexane:ethyl acetate = 6:4), and the desired product, CPK-M1-017650-B04, was isolated as a yellowish-white crystalline solid. The yield was approximately 52%, and the prepared compound is depicted in Chemical Formula 2 below.

[0102] The purified compound was detected by mass spectrometry (ESI-MS) at m / z 526.3 [M+H]+, and the ¹H-NMR analysis results (CDCl₃, 400 MHz) were as follows: δ 7.20 (s, 1H), 5.82 (s, 1H), 4.43 (t, 1H), 3.48-3.31 (m, 2H), 2.65 (m, 1H), 2.00-0.80 (m, multiple alkyl signals). Overall, the characteristic proton signals of the steroid skeleton and the terminal alkyl group region were identified as well-separated signals, and the purity analyzed by high-performance liquid chromatography (HPLC) was more than 95%.

[0103]

[0104] <Chemical Formula 2>

[0105]

[0106]

[0107] Experimental Example 1. Evaluation of 20S proteasome inhibition activity

[0108] In this experimental example, the 20S proteasome inhibitory activity of the compounds (CPK-M1-017650-C03 and CPK-M1-017650-F03) prepared in Examples 1 and 2 of the present invention was evaluated. The experiment was performed using a fluorometric assay, and the measurement was performed using a 20S proteasome inhibitor kit sold by Sigma-Aldrich (Sigma-Aldrich, MAK172). The fluorescent substrate used in the test was LLVY-R110. When the substrate is cleaved by the proteasome, free R110 is released, increasing the fluorescent signal, and the higher the inhibitory activity, the lower the fluorescence intensity.

[0109] Afterwards, cells and proteasome inhibitors were reacted together in a 96-well plate and cultured for 72 hours in a CO2 incubator controlled at 37℃, 95% humidity, and 25% CO2. After 72 hours, the proteasome assay solution was reacted for 1 hour. Afterwards, the proteasome inhibition activity was measured by measuring the fluorescence value at 10-minute intervals for 60 minutes at an absorption wavelength of 490 nm and an emission wavelength of 525 nm using a microplate reader (Molecular Devices, VersaMax ELISA Microplate Reader, USA).

[0110] .

[0111] As a result, as shown in Fig. 2, the control group showed 0% inhibitory activity, and bortezomib, used as a positive control, showed 97.4% inhibitory activity. Among the compounds of the present invention, CPK-M1-017650-C03 of Example 1 showed 78.5% inhibitory activity, and CPK-M1-017650-F03 of Example 2 showed 80.8% inhibitory activity. Both compounds showed high inhibitory activity comparable to that of bortezomib, and it was confirmed that the proteasome inhibition mechanism was strongly induced.

[0112] Meanwhile, as comparative examples, bortezomib and various substances having different substituents from the examples of the present invention were used, and chemical formulas 3 to 6 used as comparative examples are as shown below, and the specific chemical formulas and proteasome inhibition rate information of the substances used in the experimental examples are as shown in Figure 1 below.

[0113]

[0114] <Chemical Formula 3>

[0115]

[0116] <Chemical Formula 4>

[0117]

[0118] <Chemical Formula 5>

[0119]

[0120] <Chemical Formula 6>

[0121]

[0122]

[0123] As a result of the experiment, CPK-M1-017650-E03 (analogue of chemical formula 1) showed 85.7% activity, and CPK-M1-017650-B04 showed 85% activity, showing that high activity is maintained even when a single hydrophilic substituent is introduced at a specific position. On the other hand, compounds with multivalent substituents (such as CPK-M1-017650-D04) showed a significantly lower inhibition rate of 18.7%, and a non-steroidal triptonide derivative (CPK-M1-017650-H03) showed almost no activity with only 8.5%.

[0124] These results support the structure-activity correlation (SAR) that suggests that the introduction of a single hydrophilic functional group at a specific position within the steroid backbone (e.g., position 3 or 17) and its orientation aligned along the β-plane or planar plane optimizes the interaction with the proteasome β5 subunit, resulting in high inhibitory activity. In contrast, the introduction of two or more sugar or hydrophilic substituents, or the addition of bulky, sterically distorted substituents, tended to interfere with binding to the enzyme active site, resulting in a decrease in activity.

[0125] In conclusion, it was confirmed that the compound of the present invention can effectively achieve proteasome inhibitory activity through steric and electronic alignment based on a steroid skeleton, despite not containing a boron group or an electroreactive functional group, unlike bortezomib structurally.

[0126]

[0127] Experimental Example 2. Cancer Cell Selectivity Evaluation Experiment

[0128] A cancer cell selectivity evaluation was performed on the compounds of the present invention (Examples 1 and 2) and the compounds that showed excellent proteasome inhibition efficiency through Experimental Example 1.

[0129]

[0130] Experimental Example 2-1. Experiment to evaluate the cancer cell selectivity of chemical formula 1 (CPK-M1-017650-C03).

[0131] In this experimental example, in order to confirm the cancer cell-selective anticancer activity of the compound (CPK-M1-017650-C03) corresponding to chemical formula 1 manufactured in Example 1, the change in cell viability over time was measured for various cancer cell lines and normal cell lines.

[0132] The cell lines used in the experiments are as follows:

[0133] Leukemia cell line (K562), breast cancer cell line (SKBR3), lung cancer cell line (NCI-H358), T-cell lymphoma cell line (Jurkat), pancreatic cancer cell line (PANC1), normal fibroblast cell line (L929).

[0134] All cells were cultured in an incubator at 37°C and 5% carbon dioxide, and maintained in RPMI1640 or DMEM medium supplemented with 10% serum and antibiotics.

[0135] The compound was treated at a final concentration of 10 μM, and cell viability was measured at each time point after 0, 24, 48, and 72 hours from the time of treatment. Viability evaluation was performed using the WST-8 assay, and relative viability was calculated by measuring absorbance at 450 nm.

[0136] As a result, as shown in Fig. 3, after 72 hours, the survival rate of PANC1 cells rapidly decreased to less than 10%, K562 and SKBR3 cells also showed survival rates of about 20% or less, and Jurkat and NCI-H358 showed survival rates of about 30%. On the other hand, normal cells, L929, maintained a survival rate of more than about 90% after 72 hours after the initial stress-induced survival rate decrease under the same conditions, showing significantly lower cytotoxicity compared to cancer cells.

[0137]

[0138] Experimental Example 2-2. Cancer Cell Selectivity Evaluation Experiment of Chemical Formula 2 (CPK-M1-017650-F03)

[0139] In this experimental example, to confirm the cancer cell-selective anticancer activity of the compound corresponding to chemical formula 2 (CPK-M1-017650-F03) manufactured in Example 2, cell viability over time was measured for various cancer cell lines and normal cell lines. The experimental design and conditions were configured identically to Experimental Example 2-1, and the characteristics of anticancer efficacy and cytotoxicity according to structural differences were analyzed through comparative evaluation.

[0140] Cell viability evaluation was performed using the WST-8 assay, and as described in Fig. 4, after 72 hours, the viability of PANC1 and SKBR3 cells was less than 10% and less than 20%, respectively, indicating a strong proliferation inhibitory effect on cancer cells. The viability also decreased to about 30% in K562, Jurkat, and NCI-H358 cells. On the other hand, in the case of normal L929 cells, even under the same conditions, the viability was maintained at more than about 100% after 72 hours after the initial decrease in viability due to transient stress, confirming that compound of formula 2 also exhibits selective anticancer activity with low toxicity to normal cells.

[0141]

[0142] Experimental Example 2-3. Cancer Cell Selectivity Evaluation Experiment of Chemical Formula 3 (CPK-M1-017650-E03)

[0143] In this experimental example, to confirm the cancer cell-selective anticancer activity of the compound (CPK-M1-017650-E03) corresponding to the following chemical formula 3, cell viability was measured over time in various cancer cell lines and normal cell lines. The experimental design and conditions were identical to those in Experimental Example 2-1, and the anticancer efficacy and cytotoxicity characteristics according to structural differences were analyzed through comparative evaluation.

[0144] <Chemical Formula 3>

[0145]

[0146] As a result of the experiment, as shown in Fig. 5, the results for cancer cell lines confirmed anticancer activity with survival rates decreasing to 30-50% or less in most cell lines at the 72-hour point, and in particular, a relatively strong cell proliferation inhibition effect was observed in SKBR3 and K562.

[0147] On the other hand, the survival rate for the normal cell line L929 was found to decrease to approximately 50-60% after 72 hours, confirming that the selectivity for normal cells is low and that a certain level of toxicity is involved.

[0148]

[0149] Experimental Example 2-4. Evaluation of the Cancer Cell Selectivity of Chemical Formula 4 (CPK-M1-017650-B04)

[0150] In this experimental example, to confirm the cancer cell-selective anticancer activity of the compound (CPK-M1-017650-B04) corresponding to the following chemical formula 4, cell viability was measured over time in various cancer cell lines and normal cell lines. The experimental design and conditions were identical to those in Experimental Example 2-1, and the anticancer efficacy and cytotoxicity characteristics according to structural differences were analyzed through comparative evaluation.

[0151] <Chemical Formula 4>

[0152]

[0153]

[0154] As shown in Figure 6, the experimental results demonstrated a strong apoptotic effect in cancer cell lines. Specifically, after 72 hours, the viability of SKBR3, K562, and PANC1 cells decreased to 10-30%, while similar levels of proliferation inhibition were observed in Jurkat and NCI-H358 cells. This suggests that the compound exhibits excellent proteasome inhibition activity against various cancer cells.

[0155] On the other hand, the normal cell line (L929) showed a survival rate of approximately 40-45% at 72 hours, which shows that some of the compounds of the present invention can cause a certain level of toxicity not only to cancer cells but also to normal cells.

[0156]

[0157] Experimental Example 2-5. Experiment to evaluate the cancer cell selectivity of bortezomib.

[0158] In this experimental example, the anticancer activity and toxicity toward normal cells of the compound of the present invention and the proteasome inhibitor bortezomib, a conventional anticancer agent, were compared and analyzed. The comparative experiment was conducted under identical conditions, and the cell lines, culture conditions, drug treatment, and survival rate analysis methods used in the experiment were identical to those in Experimental Example 2-1.

[0159] Bortezomib was set as a positive control and treated with cancer cell lines (K562, Jurkat, PANC1, etc.) and normal cell line (L929) at a final concentration of 10 μM. As a result of measuring cell viability at each time point (0, 24, 48, 72 hours), a very strong proliferation inhibitory effect was confirmed in cancer cell lines, and as of the 72-hour time point, the viability of K562 and PANC-1 cells decreased to less than 10%. Jurkat, SKBR3, and NCI-H358 cells also showed low viability of 20-30% or less, demonstrating the strong anticancer effect of bortezomib.

[0160] However, bortezomib also exhibited significant cytotoxicity against the normal L929 cell line, with a rapid decrease in survival rate to approximately 20-30% after 72 hours. This result reaffirms the existing limitations of bortezomib, which has low selectivity for cancer cells and a narrow therapeutic index due to its non-selective inhibition of protein degradation pathways in both normal and cancer cells.

[0161]

[0162] Experimental Example 2-6. Sintering

[0163] In summary of the above experimental results, it was confirmed that the compounds of the present invention (CPK-M1-017650-C03 and CPK-M1-017650-F03) exhibit a strong proliferation inhibitory effect on various cancer cell lines, while at the same time possessing cancer cell-selective anticancer activity that maintains a high survival rate for normal cells.

[0164] Specifically, as confirmed in Experimental Examples 2-1 and 2-2, the compounds of the present invention corresponding to Chemical Formula 1 and Chemical Formula 2 exhibited potent anticancer activity, reducing the survival rate to 10 to 30% or less within 72 hours in multiple cancer cell lines including K562, Jurkat, and PANC1, while maintaining or recovering the survival rate to 90 to 100% after the initial stress response in a normal fibroblast cell line (L929), indicating that their toxicity to normal cells was very low.

[0165] In contrast, comparative compounds (CPK-M1-017650-E03, B04, etc.) and the positive control bortezomib evaluated under the same conditions showed similar or strong inhibitory effects against cancer cells, but induced a significant level of cell death in normal cells, and the viability of L929 cells significantly decreased to 30-50% or less after 72 hours. This result can be said to be another proof of the problem that comparative compounds, including bortezomib, cannot distinguish between cancer cells and normal cells and non-selectively inhibit protein degradation pathways.

[0166] This difference in selectivity is believed to be due to the structural design of the compound of the present invention. Based on the digoxigenin skeleton, the compound of the present invention was designed to induce selective accumulation in cancer cells and specific binding to the proteasome active site through the introduction of a single hydrophilic substituent at a specific position, the steric configuration of the ring structure, and the introduction of an electronegative group. These structural features form a mechanism of action that selectively acts only on the protein degradation system in cancer cells, while minimizing the effect on functional proteins in normal cells. The mechanistic consistency and pharmacological selectivity of this mechanism were experimentally demonstrated.

[0167] In conclusion, unlike existing proteasome inhibitors, the compound of the present invention maintains potent anticancer effects against cancer cells while significantly reducing toxicity to normal cells, making it an excellent, selective anticancer candidate with a high therapeutic index. This overcomes the limitations of existing drugs and simultaneously ensures both safety and efficacy.

[0168]

[0169] Experimental Example 3. Evaluation of the anticancer activity of compounds of chemical formulas 1 and 2.

[0170] In this experimental example, the comprehensive in vitro anticancer ability of compounds (CPK-M1-017650-E03 and CPK-M1-017650-B04) corresponding to chemical formula 1 and chemical formula 2 of the present invention was evaluated, and it was confirmed whether these compounds have effective apoptosis-inducing ability against various cancer cells.

[0171] The evaluation was performed on human-derived cancer cell lines, and the cell lines used were a leukemia cell line (K562), a breast cancer cell line (SKBR3), a lung cancer cell line (NCI-H358), a T-cell lymphoma cell line (Jurkat), and a pancreatic cancer cell line (PANC1). The cells were dispensed into 96-well plates, and after stabilization culture, each compound of the present invention was treated at a final concentration of 10 μM, and after 72 hours, the cell viability was measured using the WST-8 assay.

[0172] As a result, as shown in Fig. 8, the compound of formula 1 (CPK-M1-017650-E03) reduced the survival rate to less than 30% in all cancer cell lines, and in particular, the survival rate was confirmed to be less than 10% in K562 and PANC1 cells. The compound of formula 2 (CPK-M1-017650-B04) also exhibited a similar level of anticancer activity, and also showed low survival rates of approximately 20-30% in PANC1, SKBR3, and NCI-H358 cells, respectively.

[0173] Both compounds exhibited broad and potent proliferation inhibitory activity against cancer cells, and were evaluated to have structural advantages of improved biostability and toxicity safety, such as not containing a boron group structurally and not having a sugar linkage that can induce cardiotoxicity, while having anticancer efficacy at the level of bortezomib.

[0174] On the other hand, the control group, bortezomib, showed strong proliferation inhibition activity against cancer cells, but also showed great toxicity against normal cells, confirming that it had significant side effects compared to the compound of the present invention.

[0175]

[0176] As can be confirmed through the above examples and experimental results, the compounds of the present invention are based on a digoxigenin-derived skeleton, and the reason why these compounds effectively inhibit 20S proteasome activity is confirmed to be due to the steric arrangement of specific structurally positioned functional groups and the steroid ring structure. Specifically, when a single sugar or hydrophilic moiety exists at a specific position in the structure, interaction with the active site of the proteasome is induced, resulting in high inhibitory activity. On the other hand, when two or more sugars or hydrophilic functional groups are bound to the same position, steric hindrance or excessive hydrophilicity occurs, which tends to hinder binding to the active site and reduce inhibitory activity.

[0177] Indeed, compounds CPK-M1-017650-C03 (formula 1) and CPK-M1-017650-F03 (formula 2) exhibited proteasome inhibitory activities of 78.5% and 80.8%, respectively, and each of these structures had one sugar or hydrophilic functional group introduced at an appropriate position on the steroid skeleton. In contrast, compounds with two sugar or hydrophilic functional groups (e.g., CPK-M1-017650-D04, inhibition rate 18.7%) showed significantly reduced activities.

[0178] In addition, it is interpreted that when the ring structure within the steroid ring is positioned on the plane or has the same orientation as the steroid backbone, the spatial interaction with the proteasome β5 subunit is maximized, leading to high inhibitory activity. For example, compounds whose ring structure is well aligned with the steroid backbone (e.g., CPK-M1-017650-B04, inhibition rate of 85.0%) exhibit strong proteasome inhibition effects, which is believed to be because the steric configuration enhances active site accessibility and binding affinity.

[0179] In conclusion, it was confirmed that the compound of the present invention exhibits the highest inhibitory activity by maximizing interaction with the proteasome active site when only one sugar or hydrophilic substituent is structurally introduced and this substituent is stably positioned on the β-plane or plane of the steroid skeleton. In particular, it was confirmed that the spatial compatibility with the active site is improved by positioning the ring structure in a direction that is sterically coordinated with the steroid basic skeleton, resulting in excellent proteasome inhibitory activity.

[0180] In addition, the compound of the present invention has a structural feature that does not include a boron-based structure or a cardiotoxic component that existing proteasome inhibitors such as bortezomib have, while including a steroid skeleton derived from digoxigenin, and structurally, one hydrophilic substituent is placed on the β-plane or plane of the steroid skeleton, and a sterically stable ring structure is maintained, thereby ensuring safety and selectivity that can significantly reduce toxicity to normal cells while maintaining high anticancer efficacy.

[0181] Specifically, as confirmed through cell experiments, the compound of the present invention exhibited a potent apoptotic effect comparable to bortezomib in various cancer cell lines, while maintaining a survival rate of over 90% in human-derived normal cells, demonstrating low toxicity. This confirms that the compound of the present invention goes beyond simple anticancer effects and demonstrates significant results in terms of improving the therapeutic index.

[0182] In addition, the pharmaceutical composition of the present invention, which can be administered orally, has low toxicity to normal cells, and has specificity for cancer cells, can be a very suitable strategy for high-risk groups requiring long-term administration or patients requiring post-treatment management.

[0183] In conclusion, the compound of the present invention is a new platform material that overcomes the structural and functional limitations of existing proteasome inhibitors while satisfying anticancer activity, selectivity, safety, and formulation flexibility, and can be said to be a high value-added technology that can simultaneously realize cancer treatment and prevention, and in particular, it can be utilized as a pharmaceutical composition used to alleviate side effects accompanying bortezomib treatment, such as peripheral neuropathy, gastrointestinal disorders, thrombocytopenia, and liver dysfunction.

[0184]

[0185] The compound of the present invention exhibits comparable proteasome inhibition efficacy to existing bortezomib-based compounds while exhibiting significantly lower human toxicity, making it useful for the development of anticancer agents applicable to various cancer types, including multiple myeloma, lymphoma, and solid tumors. Therefore, the present invention can be applied to pharmaceutical manufacturing, particularly in the field of anticancer therapeutics, with significant industrial applications.

Claims

1. A pharmaceutical composition for preventing or treating cancer, comprising a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient, characterized in that it inhibits 20S proteasome activity. <Chemical Formula 1> .

2. A pharmaceutical composition for preventing or treating cancer, comprising a compound represented by the following chemical formula 2 or a pharmaceutically acceptable salt thereof as an active ingredient, characterized in that it inhibits 20S proteasome activity. <Chemical Formula 2> .

3. In paragraph 1 or 2, A pharmaceutical composition for preventing or treating cancer, characterized in that the survival rate of normal cells is 80% or higher when the compound is administered.

4. In paragraph 1 or 2, A pharmaceutical composition for preventing or treating cancer, characterized in that the cancer is at least one selected from the group consisting of multiple myeloma, mantle cell lymphoma, breast cancer, hepatocellular cancer, lung cancer, head and neck cancer, tongue cancer, pharyngeal cancer, salivary gland cancer, prostate cancer, pancreatic cancer, colon cancer, melanoma, osteosarcoma, and leukemia.

5. In paragraph 1 or 2, The above pharmaceutical composition is a pharmaceutical composition used to alleviate side effects associated with bortezomib treatment. A pharmaceutical composition for preventing or treating cancer, characterized in that the above side effects include at least one selected from the group consisting of peripheral neuropathy, gastrointestinal disorders, thrombocytopenia, and liver dysfunction.

6. A method for preventing or treating cancer, comprising administering an effective amount of a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof to a cancer patient to prevent or treat cancer by inhibiting 20S proteasome activity. <Chemical Formula 1> .

7. A method for preventing or treating cancer, comprising administering to a cancer patient an effective amount of a compound represented by the following chemical formula 2 or a pharmaceutically acceptable salt thereof, to prevent or treat cancer by inhibiting 20S proteasome activity. <Chemical Formula 2> .

8. In paragraph 6 or 7, A method for preventing or treating cancer, characterized in that the cancer is at least one selected from the group consisting of multiple myeloma, mantle cell lymphoma, breast cancer, hepatocellular cancer, lung cancer, head and neck cancer, tongue cancer, pharyngeal cancer, salivary gland cancer, prostate cancer, pancreatic cancer, colon cancer, melanoma, osteosarcoma, and leukemia.

9. Use of a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof for the treatment of cancer. <Chemical Formula 1> .

10. Use of a compound represented by the following chemical formula 2 or a pharmaceutically acceptable salt thereof for the treatment of cancer. <Chemical Formula 2> .

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