4-aminopyrimidine lrh-1 receptor antagonists and uses thereof

Abstract

This invention discloses 4-aminopyrimidine LRH-1 receptor antagonists and their applications, belonging to the field of pharmaceutical technology. This invention provides the application of 4-aminopyrimidine compounds and their derivatives, with the general structural formula shown in Formula (I), in the preparation of antitumor drugs. These compounds, as LRH-1 antagonists, exhibit significant downstream transcriptional repression activity against LRH-1 and have a high affinity for LRH-1 LBD protein, effectively inhibiting the proliferation of growth-dependent LRH-1 tumors such as breast cancer, colon cancer, pancreatic cancer, and prostate cancer cell lines. Therefore, these compounds can be applied to the treatment of diseases related to abnormal LRH-1 expression, including but not limited to the treatment of breast cancer, colon cancer, pancreatic cancer, and prostate cancer.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the pharmaceutical use of a 4-aminopyrimidine compound in the preparation of LRH-1 receptor antagonists, particularly in the preparation of drugs for treating breast cancer, colon cancer, pancreatic cancer, or prostate cancer. Background Technology

[0002] Liver receptor homolog 1 (LRH-1) is a nuclear receptor belonging to the NR5A subfamily, playing a crucial role in various physiological and pathological mechanisms. Similar to other nuclear receptors, the LRH-1 protein is mainly composed of four parts: an N-terminal domain, a DNA-binding domain, a ligand-binding domain (LBD), and a hinge region. LRH-1 can exert transcriptional activity in monomeric form and remains activated even without ligand binding. Its transcriptional activity is regulated by ligands, coactivators / inhibitors, and post-translational modifications such as phosphorylation and ubiquitination. LRH-1 is expressed at high levels in embryonic and adult endoderm-derived organs, such as the liver, pancreas, intestine, and gonads, and is widely involved in physiological processes such as embryonic development, cholesterol metabolism, and bile acid homeostasis regulation.

[0003] Studies have shown that LRH-1 expression is significantly upregulated in various tumor tissues and influences cancer progression. Specifically, LRH-1 plays an important role in the progression of intestinal inflammation, colon cancer, estrogen receptor-positive and estrogen receptor-negative breast cancer, androgen receptor-positive prostate cancer, and pancreatic cancer. Increased transcriptional levels promote the expression of downstream genes involved in steroid synthesis and cell cycle progression, thereby driving tumor proliferation and metastasis. LRH-1 participates in the occurrence and development of breast cancer by regulating the estrogen receptor signaling pathway, promoting the expression of genes such as aromatase Cyp19 and E-cadherin. In castration-resistant prostate cancer, LRH-1 expression is increased and upregulated in androgen synthases such as HSD3B1, Cyp17A1, and AKR1C3. Research has shown that the LRH-1 inverse agonist ML180 (which does not act on the LBD site) can inhibit the growth of androgen receptor-positive prostate cancer cells. LRH-1 can activate the transcriptional activity of Cyclin-D1 and E1 through synergistic action with β-catenin, promoting the growth and colony formation of pancreatic cancer cells. In colorectal cancer, LRH-1 can increase the expression of key steroid-producing enzymes Cyp11A1 and Cyp11B1, thereby increasing the level of immunomodulatory corticosteroids and promoting tumor escape. Silencing LRH-1 arrests cancer cell growth in the G0 / G1 phase, inhibiting the development of colorectal cancer. Therefore, LRH-1 holds promise as a key target for the treatment of various cancers.

[0004] Currently, research on small molecules targeting LRH-1 ligands is insufficient. In 2011, Whitby et al. discovered the small molecule agonists RJW100 and RJW101, which act on the LRH-1 LBD site. In 2012, Rey et al. discovered an LRH-1 antagonist with a benzothiophene core through virtual screening. In 2013, Benod et al. screened the ZINC database for the small molecule antagonist Cpd3, which acts on the LBD site, by homology modeling of the LRH-1 protein antagonistic conformation, and discovered Cpd3d2 through similarity search. Both antagonists have inhibitory activity against the proliferation of pancreatic cancer, colon cancer, and breast cancer cells. Furthermore, high-throughput screening revealed that ML179 and ML180 can act as inverse agonists of LRH-1, affecting the expression of proliferation-related genes in LRH-1-positive cancer cells. ML180 represents a novel molecular backbone with the potential to be developed into a therapeutic for LRH-1-driven malignancies. However, the number of antagonists discovered so far is still relatively small, and existing active molecules generally suffer from poor activity and drug-likeness, as well as unclear mechanisms of action.

[0005] Therefore, finding novel and more active LRH-1 antagonists for anticancer research has become an urgent issue for researchers in this field. Summary of the Invention

[0006] The purpose of this invention is to provide a compound with good antagonistic activity against liver receptor homolog 1 (LRH-1) and a clear mechanism of action, and to apply it to the development of therapeutic drugs for tumors associated with abnormal LRH-1 expression.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: This invention utilizes structure-based virtual screening technology to screen for compounds that can bind to the ligand-binding domain (LBD) of liver receptor homolog 1 (LRH-1) and antagonize its transcriptional activity, resulting in 4-aminopyrimidine compounds with the general structural formula shown in formula (I), as follows: (I); R1 is selected from: hydrogen, , , , ; R2 is selected from: hydrogen, , ; R3 is selected from: , , , , , .

[0008] This invention verifies, through transcriptional repression activity experiments, target binding verification experiments, and antitumor proliferation activity evaluation experiments, that the above-mentioned compounds have extremely high binding affinity to LRH-1 LBD protein; as LRH-1 LBD site antagonists, they exhibit significant downstream transcriptional repression activity; and they can inhibit the proliferation of LRH-1 growth-dependent tumor cells, thus exerting an anticancer effect.

[0009] Therefore, the present invention provides the use of 4-aminopyrimidine compounds, or pharmaceutically acceptable salts thereof, or solvates thereof, or stereoisomers thereof, or prodrug molecules thereof, or deuterated derivatives thereof, in the preparation of antitumor drugs, wherein the tumor is a tumor that abnormally expresses liver receptor homolog 1.

[0010] Furthermore, the structural formulas of the 4-aminopyrimidine compounds are shown in any of formulas (1) to (8). (1) (2) (3) (4) (5) (6) (7) (8).

[0011] Furthermore, the pharmaceutically acceptable salt is an organic acid salt, an inorganic acid salt, an organic base salt, or an inorganic base salt. The organic acid salt may be, but is not limited to, oxalate, lactate, p-toluenesulfonate, malate, citrate, fumarate, camphorsulfonate, or methanesulfonate. The inorganic acid salt may be, but is not limited to, nitrate, sulfate, hydrohalate, or phosphate. The organic base salt may be, but is not limited to, sodium methoxide, potassium methoxide, sodium ethoxide, potassium ethoxide, triethylamine, or tert-butylamine. The inorganic base salt may be, but is not limited to, sodium hydroxide, potassium hydroxide, or lithium hydroxide.

[0012] The solvate is a complex with variable stoichiometry formed by a solute (the compound shown in formula (Ⅰ) provided by this invention) and a solvent. The solvent can be, but is not limited to, pharmaceutically acceptable solvents such as water, methanol, ethanol, and acetic acid.

[0013] Mechanistic studies of this invention have shown that the 4-aminopyrimidine compounds and their derivatives exhibit significant antagonistic activity against LRH-1. As LRH-1 LBD antagonists, they inhibit the transcriptional activity of LRH-1, thereby suppressing tumor cell proliferation. Therefore, these compounds can be applied to the treatment of tumors associated with abnormal LRH-1 expression.

[0014] Furthermore, the tumor is LRH-1 positive breast cancer, colon cancer, pancreatic cancer, or prostate cancer. The occurrence and development of these tumors are associated with abnormal LRH-1 expression. In this invention, the pharmaceutical use of the compound is not limited to the preparation of drugs for the aforementioned tumors.

[0015] The therapeutic mechanism of the drug includes: its active ingredient, 4-aminopyrimidine compounds, inhibits tumor cell proliferation by antagonizing LRH-1 activity.

[0016] Furthermore, the drug comprises an effective dose of a 4-aminopyrimidine compound or a pharmaceutically acceptable salt thereof or a solvate thereof or a stereoisomer thereof or a prodrug molecule thereof or a deuterated thereof, and a pharmaceutically acceptable carrier.

[0017] The pharmaceutically acceptable carrier is any formulation or carrier medium capable of delivering an effective dose of the active substance of the present invention, without interfering with the biological activity of the active substance, and without toxic side effects on the host or subject.

[0018] Furthermore, the pharmaceutically acceptable carrier includes at least one of excipients, fillers, disintegrants, diluents, surfactants, absorption enhancers, adsorbents, binders, lubricants, humectants, flavorings, and sweeteners.

[0019] The compounds provided by this invention can be used in combination with existing antitumor drugs.

[0020] The pharmaceutical preparations of this invention can be prepared according to conventional methods in the pharmaceutical field, including but not limited to capsules, powders, tablets, granules, pills, injections, syrups, oral liquids, inhalers, ointments, suppositories, or patches.

[0021] The beneficial effects of this invention are as follows: This invention provides a series of novel 4-aminopyrimidine LRH-1 LBD receptor antagonists and their analogues. These compounds, as LRH-1 antagonists, exhibit significant downstream transcriptional repression activity against LRH-1 and possess high affinity for LRH-1 LBD proteins. Experimental results demonstrate that these compounds can effectively inhibit the proliferation of growth-dependent LRH-1 tumors such as breast cancer, colon cancer, pancreatic cancer, and prostate cancer cell lines. Therefore, 4-aminopyrimidine compounds can be used as LRH-1 LBD receptor antagonists in the treatment of diseases related to abnormal LRH-1 expression, including but not limited to breast cancer, colon cancer, pancreatic cancer, and prostate cancer. Attached Figure Description

[0022] Figure 1 The compound's downstream transcriptional repression activity against LRH-1 and its IC50 value. 50 value.

[0023] Figure 2 The results of TSA on the compound and LRH-1 LBD protein. Detailed Implementation

[0024] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0026] Example 1: Structure-based Virtual Filtering Experimental principle: This experiment uses structure-based virtual screening technology to predict the binding mode and binding free energy of compounds in commercial compound databases with receptor proteins, and performs structural similarity search to screen potential LRH-1 LBD receptor antagonists.

[0027] Experimental Methods: Based on the antagonistic conformational crystal model of the estrogen receptor (PDB ID: 3ERT), homology modeling of LRH-1 (PDB ID: 1YUC) was performed using SWISS-MODEL software to obtain the antagonistic conformational model of the LRH-1 LBD. Structure optimization was performed using the Protein preparation wizard module in the Schrodinger software package, followed by structure-based virtual screening of the ChemDiv small molecule database using the Glide molecular docking module. The RTMScore model was used for rescoring, and finally, structure clustering was performed using MOE software. Compounds were purchased for activity screening. For the selected compounds with transcriptional activity, molecular dynamics simulations were performed using Amber for 1 μs. Structural similarity and substructure search strategies were used to find analogs. Molecular simulation strategies were used to preliminarily predict their activity. A second batch of analogs was purchased, and finally, eight compounds were selected.

[0028] Experimental results: Eight potential LRH-1 LBD receptor antagonists were screened, and their chemical structures are shown in Table 1.

[0029] Table 1 Example 2: Downstream transcriptional repressive activity of compound LRH-1 Experimental Principle: This experiment uses a GA14 / UAS dual-luciferase reporter gene system to assess the repressive activity of LRH-1 downstream transcription. GA14 is a transcriptional regulator comprising two independent regions: a DNA-binding domain (BD) and an activation domain (AD). BD recognizes and binds to the upstream activating sequence (UAS) of GA14 effector genes, thereby initiating transcription of genes downstream of UAS. By constructing the expression vector Gal4-LRH-1-LBD plasmid containing the fusion of Gal4-BD and the nuclear receptor LBD, and the luciferase reporter gene vector 5×UAS-Luciferase plasmid containing UAS, the downstream transcriptional activity of LRH-1 LBD can be monitored. Due to the self-activating properties of LRH-1, the fluorescence signal decreases when a compound inhibits downstream transcription of LRH-1.

[0030] Experimental Methods: For the compounds with good activity obtained from the virtual screening above, downstream transcriptional repression activity was detected using a dual-luciferase reporter gene assay. The specific procedure was as follows: 293T cells in logarithmic growth phase were injected with 1×10⁻⁶ cells... 4 Seeds were planted at a density of 75 μL / well in 96-well plates. After 24 hours of culture, transfection was performed according to the transfection reagent instructions. 20 ng of Gal4-LRH-1-LBD plasmid (or an empty vector plasmid without the LRH-1 LBD sequence for the negative control), 75 ng of 5×UAS-Luciferase plasmid, and 5 ng of Renilla plasmid were added to each well, along with 0.25 μL of HG-Transgene transfection reagent and 25 μL of Opti-MEM medium. Six hours after transfection, the medium containing the transfection reagent was removed and replaced with DMEM medium containing 10% FBS, along with 10 μL of the test compound or DMSO (positive control). One day later, the Promega® Dual-Luciferase® Reporter Assay System was used. The medium in the wells was removed, the plates were washed with PBS, and 20 μL of 1×PLB lysis buffer was added. The plates were incubated at 37 °C for 15 min. After adding Luciferase AssayReagent II (LAR II) and detecting the fluorescence intensity of fireflies using a microplate reader, Stop & Glo® reagent was added to terminate the firefly luciferase reaction and initiate the renal luciferase reaction. The fluorescence intensity of the renal luciferase was then detected using a microplate reader. The firefly / renal luciferase ratio was calculated, and the transcriptional repressive activity (IC50) of the compound was calculated by examining the fluorescence values ​​of the positive and negative control wells. 50 ).

[0031] Experimental Results: The inhibitory activity curves of the eight compounds screened in Example 1 against LRH-1 LBD are as follows. Figure 1 As shown. The antagonistic activity IC of all compounds. 50 All were <10 μM, exhibiting good LRH-1 transcriptional repression activity, with compound LL08 showing the strongest antagonistic activity and an IC50 value of <10 μM. 50 = 0.86 μM.

[0032] Example 3: Compound Target Binding Verification Experiment Experimental Principle: This experiment uses the Thermal Shift Assay (TSA) to detect the interaction between proteins and ligands. The principle is that as proteins are gradually heated, their secondary structures open, exposing autofluorescent groups (tryptophan, tyrosine, etc.). By monitoring the changes in intrinsic fluorescence during protein denaturation, a melting curve can be plotted, thereby determining its melting temperature (T0). m When a protein binds to a ligand, it usually leads to a change in the protein's stability. By comparing changes in melting temperature, the binding status of the ligand can be inferred.

[0033] Experimental methods: Target validation experiments were performed on the above compounds. The pET50b-LRH-1 LBD-His tag plasmid was constructed and expressed in *E. coli* BL21, cultured to OD200. 600 When the nucleotide polymorphism (NPD) was 0.6–0.8, cells were induced for 4 hours at 30 °C, 160 rpm, and 0.5 mM IPTG. Cells were collected and lysed by sonication, and His-tagged fused LRH-1 LBD protein was purified using a nickel affinity chromatography column. TSA detection was performed using a NanoTemper / Prometheus Panta instrument. 0.4 mg / mL of LRH-1 LBD protein was incubated with 150 μM of the test compound or DMSO (final volume 20 μL) at room temperature for 30 min, followed by centrifugation at 12000 rpm. The test sample mixture was gradually heated from 25 °C to 95 °C at a rate of 1 °C / min, and fluorescence at 330 nm and 350 nm was recorded. The melting temperature (T0) of each sample was also obtained. m ), with DMSO group T m The filter condition is 3 times the SD value, T m Difference (ΔT) m A value greater than 3 times the SD value is considered to have a binding.

[0034] Experimental Results: The TSA experimental results of the 8 compounds screened in Example 1 are as follows: Figure 2As shown, all compounds exhibited significant temperature shifts, demonstrating their direct binding ability to the target LRH-1 LBD protein.

[0035] Example 4: Evaluation Experiment of the Anti-cell Proliferation Activity of Compounds Experimental Principle: Thiazol blue (MTT) can indicate cell survival, growth, proliferation, and toxicity by detecting cellular metabolic activity. Succinate dehydrogenase in the respiratory chain of living cells can reduce exogenous MTT to water-insoluble blue-violet formazan crystals, which then deposit in cells. Within a certain range, the amount of formazan crystals formed is directly proportional to the number of viable cells. These crystals can be dissolved using an SDS-HCl-PBS triple buffer, and the absorbance at 570 nm indirectly reflects the number of viable cells.

[0036] Experimental methods: Cell proliferation activity was evaluated using the MTT assay. HT29 colon cancer cells (3 × 10⁻⁶) were used. 3 (pieces / well), HCT116 (3×10) 3 (5 x 10⁶ cells / well), MCF-7 breast cancer cells (5 x 10⁶ cells / well) 3 (number / well), pancreatic cancer cells CFPAC-1 (3×10⁻⁶) 3 (number / well), prostate cancer cells LNCaP-FGC (5×10⁻⁶) 3 (pieces / hole), C4-2B (3×10) 3 Cells (90 μL / well) and 3T3 were seeded into 96-well plates, with 90 μL of cell suspension in each well. Cells were incubated in a cell culture incubator for 12 hours to allow adherence. Then, 10 μL of a concentration gradient compound or DMSO was added to each well, with ML180 as a positive control. After 72 hours of culture, 10 μL of MTT (5 mg / mL) was added to each well, and after incubation for 4 hours, 100 μL of SDS-HCl-PBS triple buffer was added overnight. Finally, the absorbance at 570 nm was measured using a microplate reader. After data processing, a dose-response curve of compound concentration versus cell viability was fitted to obtain a quantitative index (IC50) of the anti-cell proliferation activity of each compound. 50 ).

[0037] Experimental Results: The IC50 values ​​of the eight compounds screened in Example 1 showed that they exhibited inhibitory activity against the proliferation of the aforementioned cancer cells. 50 As shown in Table 2.

[0038] Table 2: IC50 of the compounds against the proliferation inhibition of six cancer cell types 50 (μM) and cytotoxicity Table 2 shows that most compounds exhibited some anti-proliferative activity against LRH-1 positive cancer cells, especially in prostate cancer (LNCaP, C4-2B). Among them, LL02 and LL08 showed excellent antagonistic activity against LNCaP, with IC50 values ​​of [missing information]. 50 The safety profile was significantly lower than that of the positive compound ML180. In normal 3T3 cells, all compounds, except LL08 which exhibited some toxicity, showed superior safety compared to ML180.

[0039] The above description is merely a specific embodiment of the present invention, intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and should not be construed as limiting the scope of protection of the present invention. All equivalent modifications or substitutions made based on the essence of the present invention should be covered within the scope of protection of the present invention.

Claims

The use of 1,4-aminopyrimidine compounds or their pharmaceutically acceptable salts or solvates or their stereoisomers or prodrug molecule or their deuterated derivatives in the preparation of antitumor drugs, characterized in that, The general structural formula of the 4-aminopyrimidine compound is shown in Formula (I), and the tumor is a tumor that abnormally expresses liver receptor homolog 1; （Ⅰ）； R1 is selected from: hydrogen, , , , ; R2 is selected from: hydrogen, , ; R3 is selected from: , , , , , .

2. The application as described in claim 1, characterized in that, The structural formulas of the 4-aminopyrimidine compounds are shown in any of formulas (1) to (8). （1）、 （2）、 （3）、 （4）、 （5）、 （6）、 （7）、 （8）。 3. The application as described in claim 1 or 2, characterized in that, The pharmaceutically acceptable salt is an organic acid salt, an inorganic acid salt, an organic base salt, or an inorganic base salt. The organic acid salt is oxalate, lactate, p-toluenesulfonate, malate, citrate, fumarate, camphorsulfonate, or methanesulfonate. The inorganic acid salt is nitrate, sulfate, hydrohalate, or phosphate. The organic base salt is sodium methoxide, potassium methoxide, sodium ethoxide, potassium ethoxide, triethylamine, or tert-butylamine. The inorganic base salt is sodium hydroxide, potassium hydroxide, or lithium hydroxide.

4. The application as described in claim 1 or 2, characterized in that, The compound acts as an antagonist of the ligand-binding domain of liver receptor homolog 1, inhibiting the transcriptional activity of liver receptor homolog 1 and thereby inhibiting tumor cell proliferation.

5. The application as described in claim 1 or 2, characterized in that, The tumor is breast cancer, colon cancer, pancreatic cancer, or prostate cancer.

6. The application as described in claim 5, characterized in that, The drug comprises an effective dose of a 4-aminopyrimidine compound or a pharmaceutically acceptable salt thereof or a solvate thereof or a stereoisomer thereof or a prodrug molecule thereof or a deuterated thereof, and a pharmaceutically acceptable carrier.

7. The application as described in claim 6, characterized in that, The pharmaceutically acceptable carriers include at least one of the following: excipients, fillers, disintegrants, diluents, surfactants, absorption enhancers, adsorbents, binders, lubricants, humectants, flavorings, and sweeteners.

8. The application as described in claim 6, characterized in that, The drug is formulated in the form of capsules, powders, tablets, granules, pills, injections, syrups, oral liquids, inhalers, ointments, suppositories, or patches.

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