NSD protein targeted inhibitor and preparation method thereof

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses an NSD protein targeted inhibitor and a preparation method thereof.The NSD protein targeted inhibitor is formed by assembling RK-0080552, rosmarinic acid, ferulic acid and TAT cell-penetrating peptide through non-covalent bonds, amino of the RK-0080552, phenolic hydroxyl of the rosmarinic acid and phenolic hydroxyl of the ferulic acid form a hydrogen bond, and the TAT cell-penetrating peptide forms a non-covalent bond. Arginine residues of the TAT cell-penetrating peptide are combined with carboxyl groups of rosmarinic acid and ferulic acid through electrostatic interaction to form a stable three-dimensional supramolecular structure; the preparation method comprises the following steps: respectively dissolving the components, mixing and crystallizing in proportion, washing, homogenizing at high pressure, and freeze-drying to obtain the NSD protein targeted inhibitor. According to the invention, drug activity enhancement and targeted delivery are realized through a supramolecular co-crystal technology, and the drug can be used for treating NSD2 high-expression tumors such as multiple myeloma and acute myelogenous leukemia, and has significant clinical application value.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and specifically to an NSD protein targeted inhibitor and a preparation method thereof. Background Art

[0002] The nuclear receptor-binding SET domain protein (NSD) family, as important histone methyltransferases, plays a key role in the development and progression of various malignancies. The NSD family includes three members, NSD1, NSD2, and NSD3, which regulate gene transcription and chromatin remodeling by catalyzing the dimethylation of lysine 36 at histone H3. Recent studies have found that abnormal activation of NSD family members is closely associated with various cancers: in hematological tumors, approximately 5-10% of acute myeloid leukemia patients have NSD1 gene mutations, and the resulting NUP98-NSD1 fusion protein has been shown to be highly oncogenic; in multiple myeloma, chromosomal translocations lead to NSD2 overexpression, which is significantly positively correlated with disease progression and clinical drug resistance; in the field of solid tumors, more than 60% of patients with squamous cell lung carcinoma have NSD3 gene amplification, which is an important driver of tumor growth.

[0003] Although NSD proteins have been identified as important therapeutic targets, the development of inhibitors targeting these proteins still faces numerous challenges. First, existing small-molecule inhibitors primarily target the PWWP1 domain of NSD, with limited inhibitory efficacy against methyltransferase activity. Second, most compound inhibitors struggle to distinguish between NSD1 / 2 / 3 isoforms, resulting in poor selectivity and off-target toxicity. Third, peptide inhibitors, such as H3K36me3 mimetic peptides, are susceptible to protease degradation and poor cell penetration. These limitations severely restrict the clinical application of NSD-targeted drugs.

[0004] Supramolecular cocrystal technology provides a new approach to solving the above problems. This technology assembles active molecules and functional modules through non-covalent bonding such as hydrogen bonding and π-π stacking, which not only enhances drug stability but also achieves multi-component synergistic enhancement and precise delivery. However, there are currently no reports on NSD inhibitors based on supramolecular cocrystals. In particular, in tumors with high NSD2 expression, such as multiple myeloma, there is still a lack of efficient targeted delivery solutions. Based on the above statements, the present application provides a NSD protein targeted inhibitor and a method for its preparation. Summary of the Invention

[0005] In order to solve the defects of existing NSD protein inhibitors such as poor targeting, poor selectivity, poor stability and low cell membrane penetration efficiency, the present application provides a NSD protein targeted inhibitor and a preparation method thereof.

[0006] In the first aspect, the present application provides an NSD protein targeting inhibitor, which is formed by non-covalent assembly of RK-0080552, rosmarinic acid-ferulic acid and TAT transmembrane peptide to form a supramolecular eutectic structure, wherein the molar ratio of RK-0080552, rosmarinic acid-ferulic acid and TAT transmembrane peptide is 1:0.5-1.5:0.8-1.2.

[0007] Preferably, the molar ratio of rosmarinic acid to ferulic acid in the rosmarinic acid-ferulic acid is 1:1.

[0008] Preferably, the X-ray diffraction pattern of the supramolecular eutectic structure has characteristic peaks at 2θ=7.5°±0.2°, 10.2°±0.2°, and 15.8°±0.2°.

[0009] In a second aspect, the present application provides a method for preparing a NSD protein targeted inhibitor, which adopts the following technical solution:

[0010] A method for preparing a NSD protein targeted inhibitor comprises the following steps:

[0011] S1. Weigh RK-0080552 and dissolve it in dimethyl sulfoxide to obtain an RK-0080552 solution; weigh rosmarinic acid and ferulic acid and dissolve them in ethanol to obtain a rosmarinic acid-ferulic acid solution; weigh TAT cell-penetrating peptide and dissolve it in deionized water to obtain a TAT cell-penetrating peptide solution;

[0012] S2. First, the RK-0080552 solution and the rosmarinic acid-ferulic acid solution were mixed according to the set molar ratio, and ultrasonically treated. Then, the TAT cell-penetrating peptide solution was added and stirred continuously to obtain a mixed solution. The mixed solution was transferred to a temperature-controlled reactor, the temperature was gradually decreased, and the mixture was allowed to stand for crystallization to obtain a supramolecular cocrystal.

[0013] S3, washing the supramolecular eutectic with deionized water to remove impurities, and then performing high-pressure homogenization to obtain a supramolecular eutectic suspension;

[0014] S4. Add a protective agent to the supramolecular eutectic suspension, and then freeze-dry to obtain an NSD protein targeted inhibitor.

[0015] Preferably, in step S1, the purity of RK-0080552, rosmarinic acid, ferulic acid and TAT cell-penetrating peptide is not less than 98%, and they are all prepared into 0.5-1.0 mol / L solutions.

[0016] Preferably, the specific operating steps of step S2 are as follows: according to the molar ratio of RK-0080552: rosmarinic acid - ferulic acid: TAT transmembrane peptide of 1: 0.5-1.5: 0.8-1.2, the RK-0080552 solution and the rosmarinic acid - ferulic acid solution are first mixed, stirred at 25-35 ° C and 200-300 rpm for 1-2 hours, then subjected to 35-45kHz ultrasonic treatment for 8-12 minutes, and then the TAT transmembrane peptide solution is added, and stirring is continued for 1-2 hours to obtain a mixed solution, and the mixed solution is transferred to a temperature-controlled reactor. Under nitrogen protection at a pressure of 0.5-1.5atm, a gradient cooling method is adopted, from 35 ° C to 20 ° C and then to 10 ° C, and each stage is maintained for 12-18 hours to form a supramolecular eutectic.

[0017] Preferably, the high-pressure homogenization treatment in step S3 uses a diamond alternating slit valve, and is cyclically treated at a pressure of 100-200 MPa for 3-5 times at 5-15°C.

[0018] Preferably, in step S4, the protective agent is trehalose with a final concentration of 10-20 g / L and poloxamer 188 with a final concentration of 10-20 g / L, and the freeze-drying parameters are as follows: pre-freezing at -20°C for 2-3 h, deep freezing at -80°C for 3-5 h, and freeze-drying at -50°C and 0.1-0.2 mBar for 20-28 h.

[0019] In a third aspect, the present application provides the use of NSD protein targeted inhibitors in the preparation of drugs for treating NSD2-overexpressing tumors.

[0020] Preferably, the NSD2-highly expressed tumor includes multiple myeloma, acute myeloid leukemia or diffuse large B-cell lymphoma.

[0021] In summary, this application has the following beneficial effects:

[0022] (1) Strong targeting: RK-0080552 specifically recognizes the key amino acid residues in the SET domain of the NSD2 protein, making the selectivity of NSD protein targeted inhibitors for NSD2 higher than that of existing technologies, effectively avoiding cross-inhibition of NSD1 / 3 isoforms.

[0023] (2) Excellent stability: The supramolecular eutectic structure is stably bound through hydrogen bonds and electrostatic interactions, making the inhibitor less likely to degrade in a physiological environment, extending the drug half-life and improving the drug's long-lasting efficacy.

[0024] (3) High delivery efficiency: The introduction of TAT transmembrane peptide increases the efficiency of drug uptake by tumor cells, solving the technical problem of poor cell membrane penetration of peptide inhibitors.

[0025] (4) Multiple synergistic effects: Rosmarinic acid-ferulic acid not only serves as an important component of the cocrystal structure to maintain structural stability, but its antioxidant properties can also effectively scavenge free radicals in the tumor microenvironment, achieving the dual therapeutic functions of "inhibition-antioxidation". Through complementary molecular forces and pharmacological properties, rosmarinic acid-ferulic acid enables the supramolecular cocrystal to surpass the single acid system in structural stability, targeted delivery, efficacy and process adaptability.

[0026] (5) Optimized preparation process: solvent crystallization combined with high-pressure homogenization technology ensures the uniformity and stability of supramolecular eutectics, with small batch-to-batch differences, making it suitable for large-scale production; ultrasound-assisted mixing combined with gradient cooling crystallization significantly shortens the crystallization time while improving the purity and yield of the eutectic; the composite protective agent works synergistically with the freeze-drying process to avoid destruction of the supramolecular structure and ensure the long-term stability of the preparation.

[0027] (6) Wide clinical application: This inhibitor shows significant therapeutic effects on malignant tumors such as multiple myeloma and acute myeloid leukemia with high expression of NSD2, providing a new strategy for clinical treatment.

[0028] (7) High safety: Natural ingredients such as rosmarinic acid and biocompatible materials such as TAT ​​peptide and trehalose are selected to reduce immunogenicity and improve safety. DETAILED DESCRIPTION

[0029] The present invention will be further described in detail below with reference to the following examples. It should be understood that the specific examples described herein are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0030] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0031] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0032] RK-0080552 was purchased from MedChemexpress Biotechnology Company, USA, CAS No.: 313527-11-6;

[0033] Rosmarinic acid was purchased from MedChemexpress Biotechnology Company, USA, CAS number: 20283-92-5;

[0034] Ferulic acid was purchased from MedChemexpress Biotechnology, USA, CAS No.: 537-98-4;

[0035] TAT transmembrane peptide was purchased from MedChemexpress Biotechnology Company, USA, CAS No.: HY-P0282A.

[0036] Example 1-3 provides a method for preparing a NSD protein targeting inhibitor.

[0037] Example 1

[0038] The preparation method of the NSD protein targeted inhibitor comprises the following steps:

[0039] S1. Weigh RK-0080552 and dissolve it in dimethyl sulfoxide to prepare a 0.5 mol / L RK-0080552 solution; weigh rosmarinic acid and ferulic acid at a molar ratio of 1:1 and dissolve them in ethanol to prepare a 0.5 mol / L rosmarinic acid-ferulic acid solution; weigh TAT cell-penetrating peptide and dissolve it in deionized water to prepare a 0.5 mol / L TAT cell-penetrating peptide solution;

[0040] S2. According to the molar ratio of RK-0080552:rosmarinic acid-ferulic acid:TAT transmembrane peptide of 1:0.5:0.8, the RK-0080552 solution and the rosmarinic acid-ferulic acid solution were first mixed, stirred at 25°C and 200 rpm for 1 hour, then subjected to 35 kHz ultrasonic treatment for 8 minutes, and then the TAT transmembrane peptide solution was added, and stirring was continued for 1 hour to obtain a mixed solution. The mixed solution was transferred to a temperature-controlled reactor, and the temperature was gradually decreased from 35°C to 20°C and then to 10°C under nitrogen protection at a pressure of 0.5 atm, with each stage maintained for 12 hours to form a supramolecular eutectic;

[0041] S3. Wash the supramolecular eutectic with deionized water to remove impurities, and then use a diamond alternating slit valve to perform high-pressure homogenization three times at a pressure of 100 MPa at 5°C to obtain a supramolecular eutectic suspension.

[0042] S4. Add trehalose with a final concentration of 10 g / L and poloxamer 188 with a final concentration of 10 g / L to the supramolecular eutectic suspension, then pre-freeze at -20°C for 2 h, deep freeze at -80°C for 3 h, and freeze-dry at -50°C, 0.1 mBar for 20 h to obtain an NSD protein targeted inhibitor.

[0043] Example 2

[0044] The preparation method of the NSD protein targeted inhibitor comprises the following steps:

[0045] S1. Weigh RK-0080552 and dissolve it in dimethyl sulfoxide to prepare a 0.75 mol / L RK-0080552 solution; weigh rosmarinic acid and ferulic acid at a molar ratio of 1:1 and dissolve them in ethanol to prepare a 0.75 mol / L rosmarinic acid-ferulic acid solution; weigh TAT cell-penetrating peptide and dissolve it in deionized water to prepare a 0.75 mol / L TAT cell-penetrating peptide solution;

[0046] S2. According to the molar ratio of RK-0080552: rosmarinic acid-ferulic acid: TAT transmembrane peptide of 1:1:1, the RK-0080552 solution and the rosmarinic acid-ferulic acid solution were first mixed, stirred at 30°C and 250 rpm for 1.5 hours, then subjected to 40 kHz ultrasonic treatment for 10 minutes, and then the TAT transmembrane peptide solution was added, and stirring was continued for 1.5 hours to obtain a mixed solution. The mixed solution was transferred to a temperature-controlled reactor, and the temperature was gradually decreased from 35°C to 20°C and then to 10°C under nitrogen protection at a pressure of 1 atm, with each stage maintained for 15 hours to form a supramolecular eutectic;

[0047] S3. Wash the supramolecular eutectic with deionized water to remove impurities, and then use a diamond alternating slit valve to perform high-pressure homogenization treatment four times at 150 MPa at 10°C to obtain a supramolecular eutectic suspension.

[0048] S4. Add trehalose with a final concentration of 15 g / L and poloxamer 188 with a final concentration of 15 g / L to the supramolecular eutectic suspension, then pre-freeze at -20°C for 2.5 h, deep freeze at -80°C for 4 h, and freeze-dry at -50°C and 0.15 mBar for 24 h to obtain an NSD protein targeted inhibitor.

[0049] Example 3

[0050] The preparation method of the NSD protein targeted inhibitor comprises the following steps:

[0051] S1. Weigh RK-0080552 and dissolve it in dimethyl sulfoxide to prepare a 1.0 mol / L RK-0080552 solution; weigh rosmarinic acid and ferulic acid at a molar ratio of 1:1 and dissolve them in ethanol to prepare a 1.0 mol / L rosmarinic acid-ferulic acid solution; weigh TAT cell-penetrating peptide and dissolve it in deionized water to prepare a 1.0 mol / L TAT cell-penetrating peptide solution;

[0052] S2. According to the molar ratio of RK-0080552:rosmarinic acid-ferulic acid:TAT transmembrane peptide of 1:1.5:1.2, the RK-0080552 solution and the rosmarinic acid-ferulic acid solution were first mixed, stirred at 35°C and 300 rpm for 2 hours, then subjected to 45 kHz ultrasonic treatment for 12 minutes, and then the TAT transmembrane peptide solution was added, and stirring was continued for 2 hours to obtain a mixed solution. The mixed solution was transferred to a temperature-controlled reactor, and the temperature was gradually decreased from 35°C to 20°C and then to 10°C under nitrogen protection at a pressure of 1.5 atm, with each stage maintained for 18 hours to form a supramolecular eutectic;

[0053] S3. Wash the supramolecular eutectic with deionized water to remove impurities, and then use a diamond alternating slit valve to perform high-pressure homogenization treatment at 200 MPa at 15°C for five cycles to obtain a supramolecular eutectic suspension.

[0054] S4. Add trehalose with a final concentration of 20 g / L and poloxamer 188 with a final concentration of 20 g / L to the supramolecular eutectic suspension, then pre-freeze at -20°C for 3 h, deep freeze at -80°C for 5 h, and freeze-dry at -50°C and 0.2 mBar for 28 h to obtain an NSD protein targeted inhibitor.

[0055] Comparative Example 1

[0056] The preparation method of the NSD protein targeted inhibitor comprises the following steps:

[0057] S1. Weigh RK-0080552 and dissolve it in dimethyl sulfoxide to prepare a 0.75 mol / L RK-0080552 solution; weigh rosmarinic acid and ferulic acid at a molar ratio of 1:1 and dissolve them in ethanol to prepare a 0.75 mol / L rosmarinic acid-ferulic acid solution;

[0058] S2. According to the molar ratio of RK-0080552 to rosmarinic acid-ferulic acid of 1:1, the RK-0080552 solution and the rosmarinic acid-ferulic acid solution were mixed, stirred at 30°C and 250 rpm for 3 hours, and then subjected to 40 kHz ultrasonic treatment for 10 minutes to obtain a mixed solution. The mixed solution was transferred to a temperature-controlled reactor and, under a nitrogen protection pressure of 1 atm, the temperature was gradually decreased from 35°C to 20°C and then to 10°C, with each stage maintained for 15 hours to form a supramolecular eutectic.

[0059] S3. Wash the supramolecular eutectic with deionized water to remove impurities, and then use a diamond alternating slit valve to perform high-pressure homogenization treatment four times at 150 MPa at 10°C to obtain a supramolecular eutectic suspension.

[0060] S4. Add trehalose with a final concentration of 15 g / L and poloxamer 188 with a final concentration of 15 g / L to the supramolecular eutectic suspension, then pre-freeze at -20°C for 2.5 h, deep freeze at -80°C for 4 h, and freeze-dry at -50°C and 0.15 mBar for 24 h to obtain an NSD protein targeted inhibitor.

[0061] Comparative Example 2

[0062] The preparation method of the NSD protein targeted inhibitor comprises the following steps:

[0063] S1. Weigh RK-0080552 and dissolve it in dimethyl sulfoxide to prepare a 0.75 mol / L RK-0080552 solution; weigh ferulic acid and dissolve it in ethanol to prepare a 0.75 mol / L ferulic acid solution; weigh TAT cell-penetrating peptide and dissolve it in deionized water to prepare a 0.75 mol / L TAT cell-penetrating peptide solution;

[0064] S2. According to the molar ratio of RK-0080552: ferulic acid: TAT transmembrane peptide of 1:1:1, the RK-0080552 solution and the ferulic acid solution were first mixed, stirred at 30°C and 250 rpm for 1.5 hours, then subjected to 40 kHz ultrasonic treatment for 10 minutes, and then the TAT transmembrane peptide solution was added, and stirring was continued for 1.5 hours to obtain a mixed solution. The mixed solution was transferred to a temperature-controlled reactor, and the temperature was gradually decreased from 35°C to 20°C and then to 10°C under nitrogen protection at a pressure of 1 atm, with each stage maintained for 15 hours to form a supramolecular eutectic;

[0065] S3. Wash the supramolecular eutectic with deionized water to remove impurities, and then use a diamond alternating slit valve to perform high-pressure homogenization treatment four times at 150 MPa at 10°C to obtain a supramolecular eutectic suspension.

[0066] S4. Add trehalose with a final concentration of 15 g / L and poloxamer 188 with a final concentration of 15 g / L to the supramolecular eutectic suspension, then pre-freeze at -20°C for 2.5 h, deep freeze at -80°C for 4 h, and freeze-dry at -50°C and 0.15 mBar for 24 h to obtain an NSD protein targeted inhibitor.

[0067] Comparative Example 3

[0068] The preparation method of the NSD protein targeted inhibitor comprises the following steps:

[0069] S1. Weigh RK-0080552 and dissolve it in dimethyl sulfoxide to prepare a 0.75 mol / L RK-0080552 solution; weigh rosmarinic acid and dissolve it in ethanol to prepare a 0.75 mol / L rosmarinic acid solution; weigh TAT cell-penetrating peptide and dissolve it in deionized water to prepare a 0.75 mol / L TAT cell-penetrating peptide solution;

[0070] S2. According to the molar ratio of RK-0080552:rosmarinic acid:TAT transmembrane peptide of 1:1:1, the RK-0080552 solution and the rosmarinic acid solution were first mixed, stirred at 30°C and 250 rpm for 1.5 hours, then subjected to 40 kHz ultrasonic treatment for 10 minutes, and then the TAT transmembrane peptide solution was added, and stirring was continued for 1.5 hours to obtain a mixed solution. The mixed solution was transferred to a temperature-controlled reactor, and the temperature was gradually decreased from 35°C to 20°C and then to 10°C under nitrogen protection at a pressure of 1 atm, with each stage maintained for 15 hours to form a supramolecular eutectic;

[0071] S3. Wash the supramolecular eutectic with deionized water to remove impurities, and then use a diamond alternating slit valve to perform high-pressure homogenization treatment four times at 150 MPa at 10°C to obtain a supramolecular eutectic suspension.

[0072] S4. Add trehalose with a final concentration of 15 g / L and poloxamer 188 with a final concentration of 15 g / L to the supramolecular eutectic suspension, then pre-freeze at -20°C for 2.5 h, deep freeze at -80°C for 4 h, and freeze-dry at -50°C and 0.15 mBar for 24 h to obtain an NSD protein targeted inhibitor.

[0073] Comparative Example 4

[0074] The preparation method of the NSD protein targeted inhibitor comprises the following steps:

[0075] S1. Weigh RK-0080552 and dissolve it in dimethyl sulfoxide to prepare a 0.75 mol / L RK-0080552 solution; weigh rosmarinic acid and ferulic acid at a molar ratio of 1:1 and dissolve them in ethanol to prepare a 0.75 mol / L rosmarinic acid-ferulic acid solution; weigh TAT cell-penetrating peptide and dissolve it in deionized water to prepare a 0.75 mol / L TAT cell-penetrating peptide solution;

[0076] S2. According to the molar ratio of RK-0080552:rosmarinic acid-ferulic acid:TAT cell-penetrating peptide of 1:1:1, the RK-0080552 solution and the rosmarinic acid-ferulic acid solution were first mixed, and stirred at 30°C and 250 rpm for 1.5 hours. Then, the TAT cell-penetrating peptide solution was added and stirred for 1.5 hours to obtain a mixed solution. The mixed solution was transferred to a reactor and allowed to stand at room temperature for 6 days under nitrogen protection at a pressure of 1 atm to form a supramolecular eutectic;

[0077] S3. Wash the supramolecular eutectic with deionized water to remove impurities, and then use a diamond alternating slit valve to perform high-pressure homogenization treatment four times at 150 MPa at 10°C to obtain a supramolecular eutectic suspension.

[0078] S4. Add trehalose with a final concentration of 15 g / L and poloxamer 188 with a final concentration of 15 g / L to the supramolecular eutectic suspension, then pre-freeze at -20°C for 2.5 h, deep freeze at -80°C for 4 h, and freeze-dry at -50°C and 0.15 mBar for 24 h to obtain an NSD protein targeted inhibitor.

[0079] Comparative Example 5

[0080] The preparation method of the NSD protein targeted inhibitor comprises the following steps:

[0081] S1. Weigh RK-0080552 and dissolve it in dimethyl sulfoxide to prepare a 0.75 mol / L RK-0080552 solution; weigh rosmarinic acid and ferulic acid at a molar ratio of 1:1 and dissolve them in ethanol to prepare a 0.75 mol / L rosmarinic acid-ferulic acid solution; weigh TAT cell-penetrating peptide and dissolve it in deionized water to prepare a 0.75 mol / L TAT cell-penetrating peptide solution;

[0082] S2. According to the molar ratio of RK-0080552: rosmarinic acid-ferulic acid: TAT transmembrane peptide of 1:1:1, the RK-0080552 solution and the rosmarinic acid-ferulic acid solution were first mixed, stirred at 30°C and 250 rpm for 1.5 hours, then subjected to 40 kHz ultrasonic treatment for 10 minutes, and then the TAT transmembrane peptide solution was added, and stirring was continued for 1.5 hours to obtain a mixed solution. The mixed solution was transferred to a temperature-controlled reactor, and the temperature was gradually decreased from 35°C to 20°C and then to 10°C under nitrogen protection at a pressure of 1 atm, with each stage maintained for 15 hours to form a supramolecular eutectic;

[0083] S3. Wash the supramolecular eutectic with deionized water to remove impurities, and then use a diamond alternating slit valve to perform high-pressure homogenization treatment four times at 150 MPa at 10°C to obtain a supramolecular eutectic suspension.

[0084] S4. Add trehalose with a final concentration of 15 g / L and poloxamer 407 with a final concentration of 15 g / L to the supramolecular eutectic suspension, then pre-freeze at -20°C for 2.5 h, deep freeze at -80°C for 4 h, and freeze-dry at -50°C and 0.15 mBar for 24 h to obtain an NSD protein targeted inhibitor.

[0085] Comparative Example 6

[0086] The preparation method of the NSD protein targeted inhibitor comprises the following steps:

[0087] S1. Weigh RK-0080552 and dissolve it in dimethyl sulfoxide to prepare a 0.75 mol / L RK-0080552 solution; weigh rosmarinic acid and ferulic acid at a molar ratio of 1:1 and dissolve them in ethanol to prepare a 0.75 mol / L rosmarinic acid-ferulic acid solution; weigh TAT cell-penetrating peptide and dissolve it in deionized water to prepare a 0.75 mol / L TAT cell-penetrating peptide solution;

[0088] S2. According to the molar ratio of RK-0080552:rosmarinic acid-ferulic acid:TAT transmembrane peptide of 1:2:1.5, the RK-0080552 solution and the rosmarinic acid-ferulic acid solution were first mixed, stirred at 30°C and 250 rpm for 1.5 hours, then subjected to 40 kHz ultrasonic treatment for 10 minutes, and then the TAT transmembrane peptide solution was added, and stirring was continued for 1.5 hours to obtain a mixed solution. The mixed solution was transferred to a temperature-controlled reactor, and the temperature was gradually decreased from 35°C to 20°C and then to 10°C under nitrogen protection at a pressure of 1 atm, with each stage maintained for 15 hours to form a supramolecular eutectic;

[0089] S3. Wash the supramolecular eutectic with deionized water to remove impurities, and then use a diamond alternating slit valve to perform high-pressure homogenization treatment four times at 150 MPa at 10°C to obtain a supramolecular eutectic suspension.

[0090] S4. Add trehalose with a final concentration of 15 g / L and poloxamer 188 with a final concentration of 15 g / L to the supramolecular eutectic suspension, then pre-freeze at -20°C for 2.5 h, deep freeze at -80°C for 4 h, and freeze-dry at -50°C and 0.15 mBar for 24 h to obtain an NSD protein targeted inhibitor.

[0091] In vitro antitumor activity assay (CCK-8 method)

[0092] Experimental method: Human multiple myeloma cells (MM.1S) with high expression of NSD2 were selected and the cells were seeded in 11 96-well plates (5×10 3 cells / well), and the following treatment groups were added to each 96-well plate:

[0093] Blank control group: equal volume of PBS;

[0094] Positive control group: 100 μM commercially available NSD2 inhibitor RK-0080552;

[0095] Example group: 100 μM NSD protein targeted inhibitor prepared in Examples 1-3;

[0096] Comparative Example Group: 100 μM NSD protein targeting inhibitors prepared in Comparative Examples 1-6.

[0097] After culturing for 48 h, 10 μL of CCK-8 reagent was added to each well and incubated for another 2 h. The absorbance (OD value) at 450 nm was measured using a microplate reader to calculate the cell survival rate. The specific data are shown in Table 1.

[0098] Table 1 Inhibitory effect of NSD protein targeted inhibitors on the proliferation of MM.1S cells

[0099] Experimental group (Cell survival rate %) Blank control group 100.0±3.2 Positive control group 52.3±4.1* Example 1 28.5±3.6** Example 2 22.7±2.9** Example 3 30.1±3.8** Comparative Example 1 50.4±4.7* Comparative Example 2 50.9±5.2* Comparative Example 3 38.6±4.3** Comparative Example 4 38.8±3.9** Comparative Example 5 34.8±3.1** Comparative Example 6 40.2±4.5*

[0100] Note: Data are expressed as mean ± standard deviation (Mean ± SD); significant marks: *p < 0.05, **p < 0.01 vs. blank control group.

[0101] This experiment systematically evaluated the proliferation inhibitory effect of NSD protein-targeted inhibitors on NSD2-overexpressing human multiple myeloma cells (MM.1S) using the CCK-8 method. Experimental data showed that the NSD protein-targeted inhibitors prepared in Examples 1-3 exhibited significant anti-tumor activity, and their cell survival rates were significantly lower than those in the positive control group. Among them, Example 2 showed the most excellent inhibitory effect, which fully demonstrated the advantages of supramolecular cocrystal technology: TAT transmembrane peptide significantly improved the cellular uptake efficiency of the drug, while rosmarinic acid-ferulic acid stabilized the cocrystal structure through hydrogen bond networks, and the synergistic effect of the two greatly enhanced the inhibitory effect of RK-0080552.

[0102] The comparative experimental results further verified the key role of each component and process. The cell survival rate of comparative example 1 increased, showing the decisive contribution of TAT membrane-penetrating peptide to drug internalization; the cell survival rate of comparative examples 2-3 increased, indicating that rosmarinic acid and ferulic acid are not only structural stabilizers, but their polyphenolic hydroxyl groups provide additional biological activity for the drug; in terms of process optimization, the inhibitory effect of comparative example 4 was significantly weaker than that of the embodiment group, confirming that ultrasound-assisted can effectively promote intermolecular interactions; and although comparative example 5 is still better than the positive control, the effect is slightly worse than that of the embodiment using poloxamer 188, suggesting that the latter has more advantages in maintaining particle stability; comparative example 6 changes the molar ratio of RK-0080552, rosmarinic acid-ferulic acid and TAT membrane-penetrating peptide, resulting in a cell survival rate significantly higher than that of Examples 1-3, further verifying the scientific nature of the molar ratio of the present application. These data confirm from multiple angles that the NSD protein-targeted inhibitor designed in this application achieves a significant improvement in efficacy.

[0103] In vivo tumor suppression assay

[0104] 1. Experimental animals and model establishment

[0105] Animal selection: 120 6-8 week old, male BALB / c nude mice weighing 18-22 g were used and housed in an SPF environment.

[0106] Tumor cell inoculation: Human diffuse large B-cell lymphoma cells (Raji) with high expression of NSD2 were selected and resuspended in PBS. 5×10 6 cells (volume 100 μL).

[0107] Tumor monitoring: Observe tumor growth daily until the tumor volume reaches 100-150mm 3 Dosage was started (approximately 10 days after inoculation).

[0108] 2. Experimental Grouping and Dosage Regimen

[0109] Ten BALB / c nude mice not inoculated with tumor cells served as a blank control group. The tumor-bearing mice were then randomly divided into 11 groups, with 10 mice in each group. The following dosing regimen was used:

[0110] Blank control group: healthy nude mice injected with equal volume of normal saline;

[0111] Model group: tumor-bearing BALB / c nude mice injected with equal volume of normal saline;

[0112] Positive control group: tumor-bearing BALB / c nude mice injected with 10 mg / kg of the commercially available NSD2 inhibitor RK-0080552;

[0113] Example 1 group: tumor-bearing BALB / c nude mice were injected with 10 mg / kg of the NSD protein targeted inhibitor prepared in Example 1;

[0114] Example 2 group: tumor-bearing BALB / c nude mice were injected with 10 mg / kg of the NSD protein targeted inhibitor prepared in Example 2;

[0115] Example 3 group: tumor-bearing BALB / c nude mice were injected with 10 mg / kg of the NSD protein targeted inhibitor prepared in Example 3;

[0116] Comparative Example 1: tumor-bearing BALB / c nude mice were injected with 10 mg / kg of the NSD protein targeted inhibitor prepared in Comparative Example 1;

[0117] Comparative Example 2 group: tumor-bearing BALB / c nude mice were injected with 10 mg / kg of the NSD protein targeted inhibitor prepared in Comparative Example 2;

[0118] Comparative Example 3 group: tumor-bearing BALB / c nude mice were injected with 10 mg / kg of the NSD protein targeted inhibitor prepared in Comparative Example 3;

[0119] Comparative Example 4 group: tumor-bearing BALB / c nude mice were injected with 10 mg / kg of the NSD protein targeted inhibitor prepared in Comparative Example 4;

[0120] Comparative Example 5 group: tumor-bearing BALB / c nude mice were injected with 10 mg / kg of the NSD protein targeted inhibitor prepared in Comparative Example 5;

[0121] Comparative Example 6 group: tumor-bearing BALB / c nude mice were injected with 10 mg / kg of the NSD protein targeting inhibitor prepared in Comparative Example 6.

[0122] According to the above experimental grouping and dosing schedule, mice were injected with tail vein once every 3 days for 4 weeks.

[0123] 3. Detection indicators and methods

[0124] Tumor volume monitoring: The long diameter (L) and short diameter (W) of the tumor were measured with a vernier caliper every 3 days according to the formula V = 0.5 × L × W 2 Calculate volume for 4 weeks;

[0125] End point analysis: 24 hours after the last administration, the mice were anesthetized and killed, the tumors were removed, and the volume and weight were accurately measured.

[0126] 4. Data processing

[0127] Tumor inhibition rate (%) = (1-average tumor weight of each group / average tumor weight of model group) × 100%;

[0128] The data were expressed as mean ± standard deviation, and the comparison among groups was performed using one-way analysis of variance (ANOVA). The specific data are shown in Table 2.

[0129] Table 2 Inhibitory effect of NSD protein targeted inhibitors on human diffuse large B-cell lymphoma cells

[0130]

[0131]

[0132] Note: Data are expressed as mean ± standard deviation (Mean ± SD); significant marks: *p < 0.05, **p < 0.001 vs. model group.

[0133] Analysis of the above results showed that the NSD protein-targeted inhibitor prepared in this experiment exhibited significant antitumor effects in vivo. Among them, Example 2 showed the most excellent tumor inhibition effect, with a tumor inhibition rate of 81.1%, significantly superior to the positive control group. Key component deletion experiments confirmed that TAT transmembrane peptide, rosmarinic acid, and ferulic acid played a decisive role in the efficacy. In terms of process optimization, ultrasound-assisted crystallization significantly improved the efficacy, and poloxamer 188 surpassed poloxamer 407 in terms of stability. Structural specificity analysis showed that the polyphenolic hydroxyl groups of rosmarinic acid and ferulic acid significantly contributed to the efficacy, with a 1:1:1 molar ratio being the optimal ratio. No significant toxicity was observed in any experimental group, confirming the good safety of the inhibitor. These results fully validate the advantages of supramolecular cocrystal design and other methods in improving the efficacy of NSD protein-targeted inhibitors.

[0134] The above specific embodiments are merely explanations of the present application and are not limitations of the present application. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A NSD protein targeted inhibitor, characterized in that The supramolecular eutectic structure is formed by assembling RK-0080552, rosmarinic acid-ferulic acid and TAT membrane-penetrating peptide through non-covalent bonds. The molar ratio of RK-0080552, rosmarinic acid-ferulic acid and TAT membrane-penetrating peptide is 1:0.5-1.5:0.8-1.

2.

2. The NSD protein targeted inhibitor according to claim 1, characterized in that The molar ratio of rosmarinic acid to ferulic acid in the rosmarinic acid-ferulic acid is 1:

1.

3. The NSD protein targeted inhibitor according to claim 1, characterized in that The X-ray diffraction spectrum of the supramolecular eutectic structure has characteristic peaks at 2θ=7.5°±0.2°, 10.2°±0.2°, and 15.8°±0.2°.

4. A method for preparing the NSD protein targeted inhibitor according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1. Weigh RK-0080552 and dissolve it in dimethyl sulfoxide to obtain an RK-0080552 solution; weigh rosmarinic acid and ferulic acid and dissolve them in ethanol to obtain a rosmarinic acid-ferulic acid solution; weigh TAT cell-penetrating peptide and dissolve it in deionized water to obtain a TAT cell-penetrating peptide solution; S2. First, the RK-0080552 solution and the rosmarinic acid-ferulic acid solution were mixed according to the set molar ratio, and ultrasonically treated. Then, the TAT cell-penetrating peptide solution was added and stirred continuously to obtain a mixed solution. The mixed solution was transferred to a temperature-controlled reactor, the temperature was gradually decreased, and the mixture was allowed to stand for crystallization to obtain a supramolecular cocrystal. S3, washing the supramolecular eutectic with deionized water to remove impurities, and then performing high-pressure homogenization to obtain a supramolecular eutectic suspension; S4. Add a protective agent to the supramolecular eutectic suspension, and then freeze-dry to obtain an NSD protein targeted inhibitor.

5. The method for preparing the NSD protein targeted inhibitor according to claim 4, characterized in that: In step S1, the purity of RK-0080552, rosmarinic acid, ferulic acid and TAT cell-penetrating peptide is not less than 98%, and they are all prepared into 0.5-1.0 mol / L solutions.

6. The method for preparing the NSD protein targeted inhibitor according to claim 4, characterized in that: The specific operating steps of step S2 are as follows: according to the molar ratio of RK-0080552: rosmarinic acid-ferulic acid: TAT transmembrane peptide of 1:0.5-1.5:0.8-1.2, the RK-0080552 solution and the rosmarinic acid-ferulic acid solution are first mixed, stirred at 25-35°C and 200-300 rpm for 1-2 hours, then subjected to 35-45kHz ultrasonic treatment for 8-12 minutes, and then the TAT transmembrane peptide solution is added, and stirring is continued for 1-2 hours to obtain a mixed solution, and the mixed solution is transferred to a temperature-controlled reactor. Under nitrogen protection at a pressure of 0.5-1.5atm, a gradient cooling method is used from 35°C to 20°C and then to 10°C, with each stage maintained for 12-18 hours to form a supramolecular eutectic.

7. The method for preparing the NSD protein targeted inhibitor according to claim 4, characterized in that: In step S3, the high-pressure homogenization treatment uses a diamond alternating slit valve and is cycled at a pressure of 100-200 MPa for 3-5 times at 5-15°C.

8. The method for preparing the NSD protein targeted inhibitor according to claim 4, characterized in that: In step S4, the protective agents are trehalose with a final concentration of 10-20 g / L and poloxamer 188 with a final concentration of 10-20 g / L, and the freeze-drying parameters are as follows: pre-freezing at -20°C for 2-3 hours, deep freezing at -80°C for 3-5 hours, and freeze-drying at -50°C and 0.1-0.2 mBar for 20-28 hours.

9. Use of the NSD protein targeted inhibitor according to any one of claims 1 to 3 in the preparation of a drug for treating tumors with high NSD2 expression.

10. Use of the NSD protein targeted inhibitor according to claim 9 in the preparation of a drug for treating NSD2-overexpressing tumors, characterized in that: The NSD2-highly expressed tumors include multiple myeloma, acute myeloid leukemia or diffuse large B-cell lymphoma.

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