A protac compound targeting degradation of lsd1 and preparation method and application thereof

Abstract

This invention discloses a PROTAC compound that targets and degrades LSD1, its preparation method, and its applications, belonging to the field of pharmaceutical chemistry. The preparation method includes two steps: first, 5-bromopentanoic acid is coupled with an LSD1 ligand precursor to obtain an intermediate, which is then subjected to a nucleophilic substitution reaction with cardamom to obtain the final product. Based on the PROTAC mechanism, this compound can degrade LSD1 protein in human non-small cell lung cancer NCI-H299 and HCC827 cells in a concentration- and time-dependent manner at the cellular level; at the animal level, it can significantly inhibit tumor growth in LA795 lung cancer-bearing mice, and has no significant toxicity to mouse body weight, blood routine tests, liver and kidney function, or major organs. This invention provides a new candidate compound for the development of novel LSD1-targeted anti-tumor drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemistry and biomedicine technology, specifically relating to the design, synthesis, and application of a protein degradation-targeting chimera (PROTAC) that targets the degradation of histone demethylase LSD1 in the treatment of lung cancer. Background Technology

[0002] Histone lysine-specific demethylase 1 (LSD1) is the first discovered histone demethylase, belonging to the flavin adenine dinucleotide (FAD)-dependent amine oxidase superfamily. It specifically removes monomethylation and dimethylation modifications at histone H3K4 and H3K9 sites, and also regulates the methylation status of non-histone substrates such as p53 and DNMT1, thereby participating in key biological processes such as gene expression regulation, tumor cell proliferation, invasion, and metastasis. Because LSD1 is abnormally highly expressed in various tumors, including acute myeloid leukemia, breast cancer, and lung cancer, and its expression level is positively correlated with tumor malignancy, LSD1 has become one of the core targets for tumor epigenetic therapy. The development of inhibitors or degraders targeting LSD1 has significant clinical application value.

[0003] Currently, drug development targeting LSD1 mainly focuses on two categories: small molecule inhibitors and protein degraders. Among them, LSD1 small molecule inhibitors are the earliest mature technology in this field and have been promoted in clinical research. Their core mechanism of action is to block the histone demethylation catalytic function of LSD1 by binding to the active site or cofactor of LSD1 through competitive binding or conformational inhibition, thereby inhibiting the abnormal transcription and expression of downstream oncogenes, and ultimately achieving the effects of inhibiting tumor cell proliferation and inducing tumor cell apoptosis. This type of inhibitor has the advantages of simple structure, low synthesis difficulty, and large space for drug optimization. LSD1 protein degraders have become a research hotspot in recent years, relying on novel targeted degradation technologies. Their core mechanism of action differs from the "inhibitory function" of traditional small molecule inhibitors. They specifically recognize and degrade LSD1 protein by mediating the intracellular ubiquitin-proteasome system, thereby fundamentally eliminating the presence of LSD1 protein. These drugs can not only block the catalytic activity of LSD1, but also eliminate the carcinogenic effects caused by its non-catalytic scaffold function. Compared with small molecule inhibitors, they are easier to overcome drug resistance problems, and often exhibit stronger anti-tumor activity at low doses. Therefore, they have become an important breakthrough direction for the development of LSD1 targeted drugs.

[0004] Reported LSD1 inhibitors fall into two categories: reversible inhibitors and irreversible inhibitors. Irreversible inhibitors present significant toxicity and high risk. Furthermore, some irreversible inhibitors exhibit unstable effects, making it difficult to determine their efficacy. Reversible inhibitors, on the other hand, have weak targeting specificity and insufficient structural diversity, mostly concentrated in specific core structures, limiting their optimization potential. Moreover, the development of reversible inhibitors is also susceptible to false-positive results, posing significant challenges to drug screening and activity validation.

[0005] Currently, developing novel PROTAC molecules that can efficiently degrade LSD1 and possess good drug-like properties remains a pressing technical problem in this field. This invention aims to design and synthesize a novel LSD1-targeting PROTAC compound to address the aforementioned problems in existing technologies. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing a PROTAC drug that targets and degrades LSD1 protein and to demonstrate its good inhibitory activity against lung cancer.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution: Firstly, a PROTAC compound targeting the degradation of LSD1, chemically named (2S,4R)-1-((S)-2-(5-(4-cinnamyl-3-hydroxy-5-methoxyphenoxy)pentanoylamino)-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide, has the following structural formula: .

[0008] Secondly, the present invention provides a method for preparing the PROTAC compound, comprising the following steps: Step S1: After activating 5-bromopentanoic acid, it is subjected to amidation reaction with (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide in the presence of a base to obtain intermediate compound 1: (2S,4R)-1-((S)-2-(4-bromobutamido)-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide; Step S2: The intermediate compound 1 obtained in step S1 is reacted with cardamom in the presence of a base and a catalyst to undergo a nucleophilic substitution reaction to obtain the compound described in claim 1.

[0009] Preferably, the activator is HATU, the base is DIPEA, and the reaction is carried out in a DCM solvent.

[0010] Preferably, the base is K2CO3, the catalyst is KI, and the reaction is carried out in DMF solvent under reflux conditions.

[0011] Preferably, in step S1, the equivalent ratio of 5-bromopentanoic acid, base, and (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide is 1.2:1.5:1; and in step S2, the equivalent ratio of cardamom, base, and intermediate compound 1 is 1:2.5:1.2.

[0012] Thirdly, the present invention provides a pharmaceutical composition comprising the said compound, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0013] Preferably, the dosage form of the pharmaceutical composition is an injection.

[0014] Preferably, the pharmaceutical composition is in the form of an injection. The daily injection concentration is approximately 0.8 mg / kg.

[0015] Fourthly, the present invention provides the use of the described PROTAC compound in the preparation of a medicament for degrading histone demethylase LSD1.

[0016] The use of the PROTAC compound in the preparation of medicaments for the prevention and / or treatment of lung cancer.

[0017] This invention specifically designed and synthesized a class of PROTAC (proteolytic targeting chimera) molecules with highly efficient targeted degradation function of LSD1 protein. These molecules achieve precise regulation of LSD1 protein through a "dual-function targeting" design. They mediate the selective ubiquitination and subsequent degradation of LSD1 protein through the ubiquitin-proteasome pathway, fundamentally reducing the homeostatic level of LSD1 protein in cells, rather than the "activity inhibition" mode of traditional inhibitors.

[0018] To systematically verify the targeting and biological function of the PROTAC molecule (compound 2), this invention, at the cellular level, used human non-small cell lung cancer NCI-H299 and HCC827 cell lines as models, and through Western blotting experiments, confirmed that the molecule can degrade LSD1 protein in a concentration- and time-dependent manner. At the animal level, a Balb / c mouse model of lung cancer LA795 cells was constructed. After tail vein injection, the tumor volume of the model mice was significantly reduced compared with the saline control group. Furthermore, through blood routine tests, liver and kidney function biochemical tests, and pathological observation of major organs, it was confirmed that within the effective therapeutic dose range, the drug had no significant effect on the weight gain of mice, and no pathological damage was observed in important organs such as the heart, liver, and kidneys, demonstrating excellent in vivo anti-lung cancer activity and good biosafety.

[0019] The present invention has the following beneficial effects: 1. A novel LSD1-targeting degrader with well-defined activity is provided. This molecule creatively couples an LSD1 inhibitor as the target ligand with a naturally derived E3 ligase ligand via an optimized linker chain, forming a completely new chemical entity (compound 2). Its structure was confirmed by mass spectrometry and nuclear magnetic resonance (see [link to article]). Figure 4 , 5 ).

[0020] 2. A fundamental breakthrough has been achieved, moving from "functional inhibition" to "protein clearance." In vitro experiments (Example 2) confirmed that compound 2 can significantly reduce the protein expression level of LSD1 in human lung cancer cells in a concentration- and time-dependent manner. Figure 6 This directly demonstrates its degradation function, rather than the traditional method of activity inhibition. This fundamentally avoids the inherent limitations of reversible / irreversible inhibitors.

[0021] 3. The potent antitumor efficacy and good safety profile were validated in disease models. In vivo pharmacodynamic experiments (Example 3) showed that compound 2, administered via tail vein injection in a mouse model of lung cancer, significantly inhibited tumor growth. Figure 7 A, B). More importantly, at effective doses, there was no significant decrease in animal body weight (A, B). Figure 7 C), blood routine and liver and kidney function indicators were normal. Figure 8 ), and no pathological damage was found in the major organs ( Figure 9 This demonstrates that the PROTAC molecule exhibits excellent anti-lung cancer activity while also possessing good in vivo biocompatibility, providing a new solution to the toxicity issues of traditional inhibitors (especially irreversible inhibitors).

[0022] 4. A clear and reproducible preparation route is provided. The target product is prepared efficiently and in high yield using well-defined starting materials, reagents, and reaction conditions. Attached Figure Description

[0023] Figure 1 Synthetic route diagram of compound 2; Figure 2 : Mass spectrum of compound 1; Figure 3 Compound 1 1 ¹H NMR (600 MHz, CDCl₃) plot (A) and 13 C NMR (151 MHz, CDCl3) plot (B); Figure 4 Liquid phase (A) and mass spectrum (B) of compound 2; Figure 5 Compound 2 1 H NMR (600 MHz, DMSO- d 6) Figure (A) and 13 C NMR (151 MHz, DMSO-) d 6) Figure (B); Figure 6 Compound 2 concentration (A) and time (B)-dependent degradation of LSD1 protein in different cells (all data are presented as mean ± standard deviation (mean ± SD) of at least three replicates. All statistical analyses were performed in GraphPadPrism 5.0 using one-way ANOVA and t-test). p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. Figure 7 Tumor volume (A), tumor image (B), and weight change (C) in tumor-bearing mice after drug treatment; Figure 8 : Blood routine indicators (A) and liver and kidney function biochemical indicators (B) of tumor-bearing mice after drug administration; Figure 9 H&E staining of heart, liver, spleen, lungs and kidneys in tumor-bearing mice after drug administration. Detailed Implementation

[0024] The technical solution of the present invention will be further described below with reference to the embodiments. The test materials used in the embodiments can all be obtained through conventional means.

[0025] Example 1: Preparation of PROTAC compounds targeting the histone demethylase LSD1 See the synthetic route of the compound. Figure 1 ; Synthesis of Intermediate Compound 1: (2S,4R)-1-((S)-2-(4-bromobutamido)-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide Accurately weigh 1.2 equivalents of 5-bromopentanoic acid and place it in a 50 mL dry three-necked round-bottom flask. Add 5 mL of ultra-dry DCM and stir magnetically until completely dissolved. Place the reaction apparatus in an ice bath (temperature controlled at 0±2℃) and activate for 10 min. Then, accurately add 1.5 equivalents of HATU and maintain a constant temperature of 0℃ with stirring for 30 min to complete the activation of the carboxylic acid (monitor the reaction system as it changes from turbid to clear and transparent). Maintaining ice bath conditions, a super-dry DCM solution (3 mL) containing (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide (1 equivalent) and DIPEA (2 equivalent) was slowly added dropwise through a constant pressure dropping funnel. The dropping rate was controlled at 1 drop / 2 s. After the addition was complete, the mixture was stirred in the ice bath for 30 min. Then the ice bath was removed, and the mixture was allowed to warm naturally to room temperature. The reaction was then magnetically stirred for 4 h (the reaction progress was monitored by TLC, with the developing solvent being dichloromethane and methanol at a ratio of 25:1, observed under a UV lamp at 254 nm. The reaction was considered complete when the starting material spot completely disappeared).

[0026] After the reaction was complete, 10 mL of deionized water was added to the reaction system to quench the reaction. The mixture was transferred to a 50 mL separatory funnel, allowed to stand for separation, and the organic phase was collected. The aqueous phase was extracted with DCM (10 mL × 3). The organic phases from the three extractions were combined, washed once with saturated brine (10 mL), dried for 30 min with anhydrous sodium sulfate (about 2 g), filtered to remove the drying agent, and the filtrate was concentrated by rotary evaporation until no liquid distilled off to obtain the crude product. The crude product was purified by silica gel column chromatography with dichloromethane:methanol = 30:1 (V / V) as the eluent, yielding 100 mg of a pale yellow solid powder. The purity was determined by HPLC to be ≥98%, yielding intermediate compound 1. Its mass spectrometry and... 1 H NMR, 13 C NMR (see) Figure 2 and Figure 3 The yield was 75% based on (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide.

[0027] Synthesis of final product compound 2: (2S,4R)-1-((S)-2-(5-(4-cinnamyl-3-hydroxy-5-methoxyphenoxy)pentanoylamino)-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide Accurately weigh cardamom (1 equivalent) into a 50 mL dry three-necked round-bottom flask, add 5 mL of ultra-dry DMF, and stir magnetically until completely dissolved. Add anhydrous K2CO3 (2.5 equivalents), and stir magnetically at room temperature for 30 min to completely deprotonate the phenolic hydroxyl groups of cardamom to form phenoxy anions (monitor the reaction system from pale yellow to light brown). Slowly add 2 mL of ultra-dry DMF solution of intermediate compound 1 (1.2 equivalents) through a constant-pressure dropping funnel at a dropping rate of 1 drop / 3 s. After the addition is complete, add 0.1 equivalents of KI as a catalyst, install a spherical condenser, raise the temperature to 80 °C, and reflux for 12 h (monitor the reaction progress by TLC, with the developing solvent: dichloromethane:methanol = 18:1, observed under a UV lamp at 254 nm; the reaction ends when the starting material spot completely disappears). After the reaction was completed, the reaction flask was placed in an ice-water bath to cool to room temperature. 15 mL of ice water was slowly added to quench the reaction, and the mixture was stirred for 10 min to allow the product to fully precipitate. The mixture was transferred to a 100 mL separatory funnel and extracted with EtOAc (20 mL × 3). The organic phases were combined. The organic phases were washed successively with deionized water (20 mL × 2) and saturated NaCl aqueous solution (20 mL). Anhydrous sodium sulfate (3 g) was added and dried for 40 min. The drying agent was removed by filtration, and the filtrate was concentrated to a viscous state using a rotary evaporator to obtain the crude product. The crude product was purified by silica gel column chromatography with dichloromethane:methanol = 20:1 (V / V) as the eluent to obtain target compound 2. Its HPLC chromatogram, mass spectrometry, and other parameters were obtained. 1 H NMR, 13 C NMR (see) Figure 4 and Figure 5 .

[0028] Example 2: Effect of compound 2 on LSD1 protein degradation in human lung cancer cell lines Human lung cancer cell lines NCI-H299 and HCC827 were revived and seeded into DMEM high-glucose medium containing 10% FBS and 1% penicillin-streptomycin antibiotics. The cells were then cultured in a CO2 incubator at 37°C, 5% CO2, and saturated humidity, with the medium changed every 2-3 days. Cells in the logarithmic growth phase were harvested and cultured at a rate of 5 × 10⁻⁶ cells / year. 4Cells were seeded at a density of 1 cell / well in 6-well cell culture plates, with 2 mL of culture medium added to each well. Cells were cultured for 24 h to allow adherence. Cells were divided into a blank control group (culture medium containing only 0.1% DMSO) and a drug treatment group (culture medium containing different concentrations of compound 2). Five concentration gradients were set in the drug treatment group: 0.5 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM, with three replicates per concentration; three treatment time gradients were also set: 0 h, 4 h, 8 h, and 12 h, corresponding to different culture time points. After drug administration, the culture plates were returned to a CO2 incubator for continued culture until the preset time. Total protein was extracted from both groups of cells. LSD1 protein expression levels were detected by Western blotting. The experimental results showed that compound 2 could target and degrade LSD1 protein in NCI-H299 and HCC827 cells in a time- and concentration-dependent manner. Figure 6 ).

[0029] Example 3: Study on the anti-lung cancer activity of compound 2 in mice A tumor-bearing model was established using a subcutaneous injection method: 0.2 mL of LA795 cell suspension (containing 2 × 10⁻⁶ cells) was subcutaneously injected into the right axilla of each Balb / c mouse. 6 (cells). After injection, mice were observed daily for their mental state, food and water intake, and tumor growth. The long diameter (a) and short diameter (b) of the tumor were measured using calipers, and the result was calculated using the formula V = 0.5 × a × b. 2 Calculate tumor volume when the tumor volume grows to 100-150 mm. 3 The tumor-bearing model was considered successfully established when the tumor was successfully established. Mice with successfully established tumor models were randomly divided into three groups of 10 mice each: a saline control group, a low-dose compound 2 group (2-L 10 mg / kg), and a high-dose compound 2 group (2-H, 20 mg / kg). Administration was via tail vein injection at a volume of 0.1 mL / 10 g body weight. The control group received an equal volume of saline containing 5% DMSO + 5% Tween 80. Mouse body weight was recorded daily during the administration period, and tumor volume was measured every 3 days. On day 7 after the end of administration, blood was collected from 5 randomly selected mice in each group for complete blood count and liver and kidney function biochemical tests. After blood collection, the mice were euthanized, and major organs such as the heart, liver, kidneys, and lungs were quickly dissected and stained using an HE staining kit. After staining, the morphology and structure of each organ tissue were observed under an optical microscope to determine if any pathological damage was present.

[0030] Experimental results showed that the tumor volume in the treatment group was significantly smaller than that in the saline control group (P<0.01); during and after the treatment period, the weight gain trend of the treatment group was not significantly different from that of the control group (P>0.05), indicating that compound 2 had no significant inhibitory effect on normal growth in mice. Figure 7 Blood routine and liver and kidney function tests showed that there were no significant differences in WBC, RBC, PLT, ALT, AST, CRE, and BUN levels between the drug-treated group and the control group (P>0.05). Figure 8 Pathological section observation results confirmed that, in both high-concentration and low-concentration groups, compound 2 did not cause pathological damage to vital organs such as the heart, liver, kidneys, and lungs in mice. Figure 9 This indicates that compound 2 possesses both excellent in vivo anti-lung cancer activity and good biocompatibility.

[0031] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. However, the above description is merely a specific embodiment of the present invention, and the technical features of the present invention are not limited thereto. Any other embodiments derived by those skilled in the art without departing from the technical solution of the present invention should be covered within the patent scope of the present invention.

Claims

1. A PROTAC compound for targeted degradation of LSD1, characterized in that, Its chemical name is (2S,4R)-1-((S)-2-(5-(4-cinnamyl-3-hydroxy-5-methoxyphenoxy)pentanoylamino)-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide, and its structural formula is as follows: 。 2. A method for preparing the PROTAC compound of claim 1, characterized in that, Includes the following steps: Step S1: After activating 5-bromopentanoic acid, it is subjected to amidation reaction with (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide in the presence of a base to obtain intermediate compound 1: (2S,4R)-1-((S)-2-(4-bromobutamido)-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide; Step S2: The intermediate compound 1 obtained in step S1 is reacted with cardamom in the presence of a base and a catalyst to undergo a nucleophilic substitution reaction to obtain the compound described in claim 1.

3. The method according to claim 2, characterized in that, In step S1, the activator is HATU, the base is DIPEA, and the reaction is carried out in DCM solvent.

4. The method according to claim 2, characterized in that, In step S2, the base is K2CO3, the catalyst is KI, and the reaction is carried out in DMF solvent under reflux conditions.

5. The method according to claim 2, characterized in that, In step S1, the equivalent ratio of 5-bromopentanoic acid, base, and (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide is 1.2:1.5:1; in step S2, the equivalent ratio of cardamom, base, and intermediate compound 1 is 1:2.5:1.

2.

6. A pharmaceutical composition, characterized in that, It comprises the compound of claim 1, or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

7. The pharmaceutical composition according to claim 6, characterized in that, The dosage form of the pharmaceutical composition is an injection.

8. The use of the PROTAC compound of claim 1 in the preparation of a medicament for degrading histone demethylase LSD1.

9. The use of the PROTAC compound of claim 1 in the preparation of a medicament for the prevention and / or treatment of lung cancer.

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