CN106943397B - Androgen receptor antagonist and its application - Google Patents

Abstract

The invention discloses an androgen receptor antagonist and application thereof, belonging to the technical field of biochemistry. The 13 compounds provided by the invention have obvious antagonistic activity on androgen receptor, so that the compounds can be applied to preparation of androgen receptor antagonists, prostate cancer cell proliferation inhibitors and prostate tumor resisting medicines. The invention also provides a pharmaceutical composition with the compound as an active ingredient, and provides a new choice for the research of the current drugs for treating prostate cancer.

Description

Androgen receptor antagonist and its application

technical field

The invention relates to the technical field of biochemistry, in particular to androgen receptor antagonists and applications thereof.

Background technique

Prostate cancer is the second leading cause of death in men in Western countries. In recent years, with the improvement of people's living standards and changes in dietary structure, the incidence of prostate cancer in China has shown a trend of increasing significantly. For patients with limited stage (early stage) prostate cancer, radical prostatectomy and radiotherapy can achieve better curative effect. However, due to the lack of obvious clinical symptoms and hidden disease in the early stage of prostate cancer, and the lack of widespread screening, many patients are already in the advanced stage when the tumor has metastasized. Endocrine therapy. For patients with hormone-sensitive advanced prostate cancer, endocrine therapy is the main treatment method, including surgical castration, medical castration and the use of antiandrogen drugs based on androgen receptor (AR) antagonists; AR antagonists are one of the important treatments for patients with castration-resistant Prostate Cancer (CRPC).

AR antagonists (anti-androgens) have been used for a long time in the treatment of prostate cancer, initially in combination with analogs of the chemical castration drug Luteinizing Hormone Releasing Hormone (LHRH) as an androgen A supplementary method in Androgen Deprivation Therapy (ADT), its main role is to block the aggravation of disease symptoms caused by the short-term increase in testosterone levels in the initial stage of drug castration.

AR antagonists can be divided into steroids and non-steroids according to their structural types; steroid antagonists are limited in clinical use due to defects such as hepatotoxicity, interference with libido, cardiovascular side effects and low efficacy; non-steroids Clinical Use of Antagonists Since the 1980s, the first-generation antagonists flutamide, hydroxyflutamide, bicalutamide, nilutamide, and the second-generation antagonist enzalutamide have appeared. Studies have shown that AR and its dominant signaling pathways play a key role in the progression of prostate cancer, and endocrine therapy for advanced prostate cancer is also mainly aimed at this. Castration therapy can cut off the main source of androgens in patients as much as possible, so that AR lacks the binding of natural ligands to inhibit this pathway; AR antagonists can compete with androgens to bind AR to reduce other sources of AR in patients. Androgen pathway activation, so as to achieve complete hormone blocking effect. Because of this, AR antagonists were also later approved as monotherapy in ADT.

The first generation of non-steroidal antagonists are all derived from flutamide, so they have a similar structural skeleton; bicalutamide, as the best tolerated, most stable and most widely used leader, in addition to being able to compete In addition to binding AR to make it in an antagonistic state, it is difficult to aggregate coactivators and bind to DNA, and it can also reduce the stability of AR to exert a tumor suppressor effect. If the first-generation AR antagonists have only limited adjuvant effect in the treatment of prostate cancer, the emergence of the second-generation antagonist enzalutamide has pushed AR antagonists to a new important position in the standard treatment of CRPC. Compared with bicalutamide, enzalutamide has a higher affinity for AR, resulting in stronger efficacy; in addition to the first-generation non-steroidal antagonist properties, it can inhibit the mechanism of action. The nuclear transfer of AR prevents it from entering the nucleus to function as a transcription factor. Enzalutamide was originally approved in 2012 for the treatment of CRPC patients whose cancer has spread after endocrine therapy and chemotherapy, and was further approved in 2014 for asymptomatic or mildly symptomatic metastases who failed ADT but did not receive chemotherapy It can be seen that enzalutamide, a new-generation AR antagonist that can be used alone, plays an important role as a blockbuster drug for advanced metastatic CRPC for which few drugs are available; and with further clinical research, its role in The range of treatments in prostate cancer will continue to expand.

However, after each prostate cancer drug is used, drug resistance will always develop as the disease progresses. The specific reasons for AR antagonist resistance have not yet been fully elucidated. A large number of studies have observed that AR protein mutations are very important among them. Crucial point. The point mutation of AR protein will not only make the antagonist ineffective, but also lead to the reversal of the function of the small molecule that once played an antagonistic role to produce an agonist effect, even if the second-generation antagonists with unique advantages in efficacy are used for a period of time. A reversal effect occurs. Therefore, a new generation of novel antagonist molecules with high affinity for AR and a skeleton structure different from previous AR antagonists is still the key direction of prostate cancer drug research, and there is also an urgent clinical need.

The next-generation AR antagonist drugs under clinical research mainly include ARN-509, ODM-201 and AZD3514. The former two have respectively progressed to phase III clinical trials for prostate patients who have received different therapies and are in different stages. It is expected to be approved to join the therapeutic ranks in the near future. ARN-509 has a very similar structure to enzalutamide. The current research results show that compared with enzalutamide, it has stronger receptor binding ability, requires a lower dose, and has a lower central Nervous system penetration and epileptogenic side effects, but because of the excessive structural similarity, the F876L mutation that is resistant to enzalutamide also produces resistance to ARN-509. ODM-201 and its in vivo metabolite ORM-15341 have a more novel chemical structure, and its mechanism of action is similar to that of second-generation antagonists, but its affinity for AR even exceeds that of ARN-509 and enzalutamide.

Summarizing the research status of AR antagonists at home and abroad, it can be found that the development of a new generation of antagonists targeting HBP sites with novel skeleton structure, high affinity and high selectivity is still the focus of research. There is a huge clinical need. Non-HBP antagonists targeting other regions of AR protein can overcome the defects of traditional antagonist resistance, and there is still a very large clinical gap in this aspect; drug development of new AR antagonists is a long way to go.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a compound with androgen receptor antagonistic activity, which can be used in the preparation of androgen receptor antagonists and anti-prostate tumor drugs.

The present invention realizes above-mentioned purpose through following technical scheme:

In the present invention, the method of computer-aided drug molecular design is used to discover leading compounds targeting androgen receptors, and then virtual screening based on molecular docking is performed on the three-dimensional structure database of multiple small molecular compounds to obtain 1000 compounds with high scores (energy The lower the score, the higher the score). Then, the MTT cell proliferation experiment of the classic prostate cancer cell line LNCaP, the inhibition experiment of AR transcription factor activity and the use of the kit PolarScreen ^TM AR CompetitorAssay, Green (Thermo Fisher Scientific) were used to investigate the binding of the compound to the ligand binding domain of AR LBP. Two representative active compounds were finally screened, which are:

5-amino-2-(naphthalen-1-yl)isoindole-1,3-dione, the structural formula is shown in formula (1);

2-(1,2-Dihydroacenaphthylene-5-yl)isoindole-1,3-dione, the structural formula is shown in formula (2);

The present invention further tests the biological activity of the above screened compounds, and it is found that the above compounds have obvious antagonistic activity to androgen receptors. Therefore, the present invention provides any of the above compounds or their pharmaceutically acceptable salts in the preparation of androgen receptors use of antagonists.

The research of the present invention finds that the above two compounds show good effects in the anti-prostate tumor experiments at the protein level and the cell level. Therefore, the present invention provides any one of the above compounds or a pharmaceutically acceptable salt thereof in the preparation of prostate cancer cell proliferation inhibitors applications in .

The present invention also provides the application of any of the above compounds or their pharmaceutically acceptable salts in the preparation of anti-prostate tumor medicaments.

The present invention also provides a pharmaceutical composition comprising any compound or a pharmaceutically acceptable salt thereof as an active ingredient.

The compound as an active ingredient is an androgen receptor antagonist, therefore, the pharmaceutical composition of the present invention can be used as a therapeutic drug for diseases related to androgen receptor.

The pharmaceutically acceptable salt is hydrochloride, phosphate, sulfate, acetate, maleate, citrate, benzenesulfonate, toluenesulfonate, fumarate or tartrate .

The pharmaceutical composition also includes a pharmaceutically acceptable excipient, diluent or carrier. Specifically, such as syrup, gum arabic and starch. The pharmaceutical composition can be administered by intravenous, oral, sublingual, intramuscular or subcutaneous, mucocutaneous route.

The preparation form of the pharmaceutical composition is a liquid preparation or a solid preparation. Specifically, such as tablets, capsules and injections. Each dosage form can be prepared by conventional methods of pharmacy.

The beneficial effects that the present invention has:

The virtual screening method based on molecular docking and the biological activity measurement of the present invention finds that two compounds have obvious antagonistic activity to androgen receptor, and can be used as androgen receptor antagonist in the treatment of diseases related to androgen receptor Among them, it provides new options for the current drug research for the treatment of prostate cancer.

Description of drawings

Figure 1 shows the experimental results of AR binding ability of 13 compounds of the present invention at a concentration of 10 μM.

Figure 2 shows the experimental results of compound 1 on AR binding ability under a series of concentration gradients.

Figure 3 shows the experimental results of compound 3 on AR binding ability under a series of concentration gradients.

Figure 4 shows the experimental results of compound 4 on AR binding ability under a series of concentration gradients.

Figure 5(a) is the binding conformation of the antagonist in the AR active pocket (the protein is in the shape of a strip, and the antagonist is shown in the stick model); (b) is the interaction mode between the antagonist and the AR active pocket residues .

Figure 6 shows the experimental results of the antagonistic activity of 13 compounds against AR at a concentration of 10 μM.

Figure 7 shows the experimental results of compounds 5-7 on AR antagonism under a series of concentration gradients.

Figure 8 shows the experimental results of compounds 8-10 on AR antagonism under a series of concentration gradients.

Figure 9 shows the experimental results of compounds 11-13 on AR antagonism under a series of concentration gradients.

Figure 10 shows the results of the inhibitory ability of compounds 1-8 on the proliferation of prostate cancer cells at a concentration of 10 μM.

Figure 11 shows the results of the inhibitory ability of compounds 9-13 on prostate cancer cell proliferation.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments.

Example 1

(1) Virtual screening based on molecular docking

Experimental principle: Use molecular docking method to predict, analyze and evaluate the interaction between the compounds in the compound database and AR, so as to determine the antagonist molecules that can bind to AR.

Experimental methods: Based on the crystal structures of AR and androgen complexes (PDB numbers: 2PNU, 2Q7I and 3V49), a molecular docking-based virtual screening study was performed using the Glide module in Schrodinger molecular simulation software. The compound libraries used in the virtual screening include the latest versions of Chembridge, ChemDiv and the three-dimensional structure database of active ingredients of Chinese herbal medicine containing more than 60,000 compounds developed by the applicant's research group. We evaluated the 2000 compounds with the best scores in the virtual screening using the Reos rule, excluding molecules containing reactive groups.

Experimental results: Molecular docking can more accurately determine the organic small molecules that can form strong interactions with AR. Based on the predicted results of molecular docking, we purchased more than 200 compounds from commercial compound libraries, and conducted subsequent activity tests based on molecular-level binding experiments (PolarScreen ^TM AR Competitor Assay, Green, Thermo Fisher Scientific), and found a A batch of small molecule compounds with obvious AR antagonistic activity is shown in Table 1.

Table 1

The structural formula of the above compound is as follows:

(2) AR Competitive Binding Experiment

Experimental principle: The measurement of the AR binding ability of the compound adopts the fluorescence bias experiment of Invitrogen (under Thermo Fisher Scientific). Androgen receptor {AR-LBD(His-GST)} binds to a fluorescent androgen ligand (FluormoneTMAL Green) to form a binary complex (AR-LBD(His-GST)/FluormoneTMAL Green), which with high fluorescence bias. This complex is added to the microplate containing the test compound, and the test compound will replace the fluorescent ligand (FluormoneTMALGreen) in the binary complex as a competitive ligand, so that the fluorescence deviation decreases. If a non-competing ligand that does not have the ability to replace the fluorescent compound is added, its bias value will remain high. Therefore, after adding the test compound, the change of the fluorescence bias value can be used to quantitatively measure the relative affinity of the test compound to AR-LBD (His-GST).

Experimental method: After mixing AR LBD protein and high-affinity fluorescent ligand in buffer, different concentrations of test compounds (virtual screening compounds) were added, and the androgen dihydrotestosterone (DHT) was used as a positive control). If the test compound has a high affinity for AR LBD, it will replace the fluorescent ligand in the binary complex as a competitive ligand, so that the fluorescence bias value of the system will decrease; if the added test compound has no binding to AR LBD The fluorescence polarization value of the system will remain at a high value. We can quantitatively measure the binding affinity of the virtual screening compound to AR by measuring the change of the fluorescence polarization value of the system with a multi-function microplate reader.

Experimental results: As shown in Figure 1, the AR binding rates of compounds 1-13 all exceeded 30%. We tested the binding ability of different concentrations of compounds to AR, and found that a series of compounds have good binding ability, and their half-inhibitory concentration _IC50 for inhibiting the binding of AR fluorescent ligands is all micromolar, as shown in Figures 2, 3, and 4 As shown, the _IC50 values of compounds 1, 3 and 4 were 33 μM, 50-100 μM and 2.6 μM, respectively.

(3) Evaluation of the interaction mode between antagonists and AR

Experimental principle: Predict the interaction mode between AR antagonists and AR at the atomic scale based on molecular docking and molecular dynamics simulations.

Experimental steps: Based on the results obtained by molecular docking prediction, a molecular dynamics simulation of 50 ns was performed on the antagonist/AR, and AMBER14 was used for the simulation.

Experimental results: The interaction between antagonist and AR obtained by molecular docking prediction and molecular dynamics simulation is shown in Fig. 5. The predicted structures suggest that the molecular recognition between the antagonist and AR is mainly through van der Waals and hydrogen bonding interactions. The hydroxyl group on the antagonist forms stable hydrogen bonds with Ser110; the two benzene rings produce strong van der Waals interactions with multiple surrounding hydrophobic residues.

(4) AR antagonistic ability evaluation experiment

Experimental principle: AR, as a transcription factor, needs to bind to a specific sequence, namely the ARE response element, to exert its transcriptional activity; therefore, the reporter gene-enhanced green fluorescent protein EGFP, controlled by the ARR2PB promoter, was introduced into AR-positive prostate cancer cells LNCaP. After different concentrations of test compounds are administered and treated, the level of EGPF expression in cells can be measured to obtain the strength of the compound's ability to antagonize AR.

Experimental steps: We used a pre-constructed EGFP (enhanced green fluorescent protein) reporter gene plasmid controlled by the ARR ₂ PB promoter that has a strong response to AR, and stably transfected LNCaP cells with lentivirus to obtain AR-regulated cells. of stably expressing EGFP prostate cancer cell line (LN-ARR ₂ PB-EGFP). LN-ARR ₂ PB-EGFP cells were first cultured in androgen-free complete medium for several days to reduce the background fluorescence value to a low level, and then seeded at a density of 40,000 cells/well into black bottom transparent 96 cells. In the well plate, after the cells are stably attached to the wall, androgens and test compounds at different concentrations (virtual screening compounds, the listed antagonist drug enzalutamide) are administered at the same time, and after incubation for 24-48h, the cells are incubated with a multi-function microplate reader at the wavelength of The inhibition rate of the test compound antagonizing AR protein can be quantitatively calculated by detecting the fluorescence intensity value near the wavelength of 530nm under the excitation light of 485nm.

Experimental results:

As shown in Figure 6, compounds Nos. 1-13 can achieve an inhibition rate of more than 30%.

As shown in Figures 7-9, after different concentrations of compounds were treated in LN-ARR2PB-EGFP cells for 36h, the compounds significantly down-regulated the expression of the reporter gene EGFP, and showed a dose-dependent relationship, which indicated that the compounds we listed were all Potential AR antagonist with good activity.

(5) MTT assay to detect compound anti-proliferative activity of prostate tumor cells

Experimental principle: succinate dehydrogenase in the mitochondria of living cells can make exogenous MTT ((3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide)) It is reduced to water-insoluble blue-purple crystalline formazan (Formazan) and deposited in cells, while dead cells do not have this function. Add buffer to dissolve the formazan formed in cells, and measure it with an enzyme-linked immunosorbent assay at a wavelength of 490 nm. The light absorption value can indirectly reflect the number of living cells.

Experimental steps: Inoculate and culture cancer cells in a 96-well plate at a density of 3000 cells/well in androgen-free complete medium. After the cells stably adhere to the wall, 1nM DHT and different concentrations of test compounds (virtual screening compounds, marketed compounds) were administered at the same time. Antagonist drugs or DMSO), after 4 days of incubation, add 10 μL 5mg/ml MTT to each well, continue to incubate for 3 hours in the incubator, and then add 100 μL SDS-HCl-PBS triple buffer to each well, after overnight incubation at 37 degrees, in the microplate reader Detect the absorbance value of each well at 570nM at the next step, and convert it into the survival rate to obtain the IC ₅₀ value of the administered compound.

Experiment results: As shown in Figures 10 and 11, the compounds of the present invention have obvious ability to inhibit the proliferation of prostate cancer cells LNCaP, and the inhibition IC ₅₀ can reach a level similar to that of the marketed drug enzalutamide.

Claims (1)

1. The application of any compound with a structural formula shown in formulas (1) to (2) or pharmaceutically acceptable salt thereof in preparing the anti-prostate tumor medicament is characterized in that the anti-prostate tumor medicament is an androgen receptor antagonist,

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