CN106943397B

CN106943397B - Androgen receptor antagonists and uses thereof

Info

Publication number: CN106943397B
Application number: CN201710117698.9A
Authority: CN
Inventors: 李丹; 周文方; 侯廷军
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2017-03-01
Filing date: 2017-03-01
Publication date: 2020-08-04
Anticipated expiration: 2037-03-01
Also published as: CN109846879A; CN109700811B; CN109700794A; CN106943397A; CN109700811A; CN109730996B; CN109700798A; CN109700804A; CN109730996A; CN109793730B; CN109700794B; CN109846879B; CN109793730A; CN109793737B; CN109793737A; CN109700804B

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 antagonists and uses thereof

Technical Field

The invention relates to the technical field of biochemistry, in particular to an androgen receptor antagonist and application thereof.

Background

Prostate cancer is the second most lethal tumor of men in western countries, and in recent years, the incidence rate of prostate cancer in China is on the trend of obvious increase along with the improvement of living standard and the change of dietary structure of people in China. For patients with limited stage (early stage) prostate cancer, radical operation and radiotherapy of prostate cancer can achieve better curative effect. However, because early clinical symptoms of the prostate cancer are not obvious, the disease condition is hidden, and the screening is not popular, many patients are in the late stage of the metastatic tumor when seeing a doctor, and at the moment, the radical treatment and the chemotherapy of the prostate cancer hardly achieve the ideal effect, and endocrine treatment is required to be additionally used. For patients with hormone-sensitive advanced prostate cancer, endocrine therapy is the main treatment means, including surgical castration, drug castration, and the use of antiandrogen drugs, mainly Androgen Receptor (AR) antagonists; for Prostate Cancer (CRPC) patients who have progressed to be refractory, AR antagonists are one of the important therapeutic approaches.

The use of AR antagonists (antiandrogens) in the treatment of prostate cancer has been long-lived, initially in combination with the chemocastration drug gonadotropin Releasing Hormone (L lutenizing Hormone Releasing Hormone, L HRH) analogues, as a complementary means in Androgen Deprivation Therapy (ADT), primarily to block the exacerbation of disease symptoms caused by short-term increases in testosterone levels in patients during the initial period of drug castration.

AR antagonists can be classified into steroids and non-steroids according to structural type; steroid antagonists have limited clinical use due to defects such as hepatotoxicity, interfering libido, cardiovascular side effects, inefficiency and the like; since the eighties of the last century, the clinical use of non-steroidal antagonists has emerged as the first generation antagonists flutamide, hydroxyflutamide, bicalutamide, nilutamide, and the second generation antagonist enzalutamide. Studies have shown that AR and its dominant signaling pathways play a critical role in the progression of prostate cancer, and that endocrine treatment of advanced prostate cancer is also primarily aimed at. Castration treatment may cut off as much of the major source of androgen in the patient as possible, leaving AR devoid of natural ligand binding to inhibit this pathway; AR antagonists can reduce the pathway activation of androgens from other sources in the patient by competitively binding AR with the androgen, thereby achieving a complete hormone blocking effect. As such, AR antagonists were later approved for use in ADT as monotherapy as well.

The first generation of non-steroidal antagonists were all derived from flutamide and therefore have a similar structural backbone; bicalutamide, being the best, most stable and most widely used among them, exerts a cancer suppressing effect by decreasing the stability of AR, in addition to competitively binding AR to be antagonistic and thus difficult to aggregate coactivators, bind DNA. If the first generation of AR antagonists were said to have only limited adjuvant utility in the treatment of prostate cancer, the emergence of the second generation antagonist enzalutamide pushed AR antagonists to a new significant position for CRPC standard therapy. Compared with bicalutamide, enzalutamide has higher affinity to AR, thereby bringing about stronger drug effect; besides the characteristics of the first-generation non-steroidal antagonist, the action mechanism can also inhibit the nuclear transfer of AR, so that the AR cannot enter the nucleus to play the role of a transcription factor. Enzalutamide was initially approved in 2012 for the treatment of CRPC patients with spread of cancer after endocrine therapy and chemotherapy, and was further approved in 2014 for the treatment of asymptomatic or mildly symptomatic metastatic CRPC with failed ADT treatment but not receiving chemotherapy; therefore, for the late-stage metastatic CRPC with few available medicines, the new generation AR antagonist enzalutamide which can be used independently has a heavy-drug position; and with further clinical studies, its therapeutic range will continue to expand in prostate cancer.

However, after each prostate cancer drug is used, resistance always occurs along with the progress of the disease, the specific reason for the resistance of the AR antagonist is not fully elucidated, and a great deal of research observes that the mutation of the AR protein is a very critical point. Point mutations in the AR protein not only cause antagonist failure, but also result in reversal of the small molecule function once antagonized to produce agonist effects, even though second generation antagonists with unique therapeutic advantages inevitably undergo reversal over time. Therefore, a new generation of novel antagonist molecules with high affinity for AR and with framework structure different from that of the existing AR antagonists is still the focus of the research on prostate cancer drugs, and there is an urgent clinical need.

The new generation of AR antagonist drugs in clinical research mainly comprises ARN-509, ODM-201 and AZD3514, wherein the two drugs respectively aim at prostate patients who have received different treatments and are in different stages, and currently, the prostate patients respectively progress to the three-stage clinical test stage, and the treatment line is very expected to be added for approval in the near future.

Summarizing the research conditions of AR antagonists at home and abroad, it can be found that the development of a new generation of antagonist which targets HBP sites and has a novel framework structure, high affinity and high selectivity is still the focus of research, and the new generation of antagonist has great clinical requirements along with the aggravation of the aging problem of the population. non-HBP antagonist targeting other areas of AR protein can overcome the defect of drug resistance of traditional antagonist, and the research still has great clinical blank; the development of new AR antagonist drugs is of great importance.

Disclosure of Invention

The invention aims to provide a compound with androgen receptor antagonistic activity, which is applied to the preparation of androgen receptor antagonists and anti-prostate tumor drugs.

The invention realizes the purpose through the following technical scheme:

the invention adopts a means of computer-aided drug molecule design to discover a lead compound of a targeted androgen receptor, and then carries out molecule-based docking on a plurality of small molecule compound three-dimensional structure databasesVirtual screening, to obtain the top 1000 compounds (lower energy, higher score), then through prostate cancer classical cell line L NCaP MTT cell proliferation experiment, AR transcription factor activity inhibition experiment and using kit PolarScreen^TMAR completioassay, green (thermo Fisher scientific) examined the binding of compounds to the ligand binding domain L BP of AR, and finally 2 representative active compounds were screened, each:

the structural formula of the 5-amino-2- (naphthalene-1-yl) isoindole-1, 3-dione is shown as the formula (1);

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

the invention further tests the biological activity of the screened compounds, and finds that the compounds have obvious antagonistic activity on androgen receptor, so the invention provides the application of any one of the compounds or pharmaceutically acceptable salts thereof in preparing androgen receptor antagonists.

The research of the invention finds that: the two compounds have good effects in the anti-prostate tumor experiments at the protein level and the cell level, so the invention provides the application of any compound or the medicinal salt thereof in preparing the prostate cancer cell proliferation inhibitor.

The invention also provides application of any compound or medicinal salt thereof in preparing an anti-prostate tumor medicament.

The invention also provides a pharmaceutical composition comprising any one of the compounds or a pharmaceutically acceptable salt thereof as an active ingredient.

The compound as the effective component is an androgen receptor antagonist, so that the pharmaceutical composition of the invention can be used as a therapeutic drug for diseases related to androgen receptor.

The medicinal salt is hydrochloride, phosphate, sulfate, acetate, maleate, citrate, benzene sulfonate, methyl benzene sulfonate, fumarate or tartrate.

The pharmaceutical composition further comprises a pharmaceutically acceptable excipient, diluent or carrier. Specifically, syrup, gum arabic, starch, etc. can be used. The pharmaceutical composition can be administered by intravenous, oral, sublingual, intramuscular or subcutaneous routes, or by the skin mucosa route.

The pharmaceutical composition is prepared in a liquid preparation or a solid preparation. Such as tablet, capsule and injection. The preparation can be prepared by a conventional pharmaceutical method.

The invention has the following beneficial effects:

the invention discovers that 2 compounds have obvious antagonistic activity on androgen receptor based on a virtual screening method of molecular docking and biological activity measurement, can be used as androgen receptor antagonist to be applied to the treatment of diseases related to androgen receptor, and provides a new choice for the research of the current drugs for treating prostate cancer.

Drawings

FIG. 1 shows the results of the AR binding assay of 13 compounds of the present invention at a concentration of 10. mu.M.

FIG. 2 shows the results of the AR binding assay of Compound 1 over a series of concentration gradients.

FIG. 3 shows the results of the AR binding assay of Compound 3 at a series of concentration gradients.

FIG. 4 shows the results of the AR binding assay of Compound 4 at a series of concentration gradients.

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

FIG. 6 shows the results of experiments on the antagonistic activity against AR of 13 compounds at a concentration of 10. mu.M.

FIG. 7 shows the results of compounds 5-7 tested for AR antagonist activity over a series of concentration gradients.

FIG. 8 shows the results of compounds 8-10 tested for AR antagonist activity over a series of concentration gradients.

FIG. 9 shows the results of compounds 11-13 tested for AR antagonist activity over a series of concentration gradients.

FIG. 10 shows the results of the inhibitory activity of compounds 1 to 8 on the proliferation of prostate cancer cells at a concentration of 10. mu.M.

FIG. 11 shows the results of the inhibitory activity of compounds 9-13 on the proliferation of prostate cancer cells.

Detailed Description

The present invention will be further described with reference to the following specific examples.

Example 1

(1) Molecular docking-based virtual screening

The experimental principle is as follows: the interactions between compounds in the compound database and AR are predicted, analyzed and evaluated using molecular docking methods to identify antagonist molecules capable of binding to AR.

The experimental method comprises the following steps: based on the crystal structure of complexes formed by AR and androgen (PDB numbers: 2PNU, 2Q7I and 3V49), a virtual screening study based on molecular docking was performed using the Glide module in Schrodinger molecular simulation software. The compound library adopted by the virtual screening comprises the latest version of Chembridge, ChemDiv and a Chinese herbal medicine effective component three-dimensional structure database which is developed by a project group of the applicant and contains more than 6 ten thousand compounds. We evaluated the 2000 compounds best scored by virtual screening using the Reos rule, rejecting molecules containing reactive groups.

The experimental results are as follows: molecular docking can more accurately determine small organic 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 performed subsequent molecular-level-based binding experiments (PolarScreen)^TMAR completistor Assay, Green, Thermo FisherScientific) from which a panel of small molecule compounds with significant AR antagonistic activity was discovered, see table 1 for details.

TABLE 1

The structural formula of the compound is as follows:

(2) competitive binding assay for AR

Experimental principle measurement of AR binding capacity of compound using fluorescence correction experiment by Invitrogen (Thermo fisher scientific) androgen receptor { AR-L BD (His-GST) } binds to an androgen ligand with fluorescence (fluoromonetma L Green) to form a binary complex (AR-L BD (His-GST)/fluoromonetma L Green) having a high fluorescence correction value.

The experimental method comprises the steps of mixing AR L BD protein and high-affinity fluorescent ligand in a buffer solution, adding test compounds (virtual screening compounds) with different concentrations, and taking androgen Dihydrotestosterone (DHT) as a positive control), if the test compounds have higher affinity to AR L BD, the test compounds can replace the fluorescent ligand in a binary compound as a competitive ligand, so that the fluorescence bias value of the system is reduced, if the added test compounds basically have no binding capacity to AR L BD, the fluorescence bias value of the system can still be maintained at a higher value, and the binding capacity (binding affinity) of the virtual screening compounds to AR can be quantitatively measured by measuring the change of the fluorescence polarization value of the system by using a multifunctional microplate reader.

The experimental results are as follows: as shown in FIG. 1, compounds Nos. 1 to 13 all showed AR binding rates exceeding 30%. We test the binding capacity of compounds with different concentrations and find that the series of compounds have good binding capacity and half-inhibitory concentration IC for inhibiting the fluorescent ligand binding of AR₅₀All on the micromolar scale, as shown in FIGS. 2, 3 and 4, wherein the IC of the compound No. 1,3 and 4₅₀The values were 33. mu.M, 50-100. mu.M and 2.6. mu.M, respectively.

(3) Evaluation of interaction Pattern between antagonist and AR

The experimental principle is as follows: based on molecular docking and molecular dynamics simulations, the interaction pattern between AR antagonist and AR is predicted from an atomic scale.

The experimental steps are as follows: based on the results of molecular docking predictions, a 50ns molecular dynamics simulation was performed on the antagonist/AR using AMBER 14.

The experimental results are as follows: the interaction between the antagonist and AR by molecular docking prediction and molecular dynamics simulation is shown in figure 5. The predicted structure suggests that molecular recognition between the antagonist and AR is primarily through van der waals and hydrogen bonding interactions. The hydroxyl on the antagonist can form a stable hydrogen bond with the Ser 110; two benzene rings produce strong van der waals interactions with surrounding hydrophobic residues.

(4) Evaluation experiment of AR antagonistic ability

The experimental principle is that AR is used as a transcription factor and needs to be combined with a specific sequence, namely an ARE reaction element, so as to exert the transcription activity, therefore, reporter gene enhanced green fluorescent protein EGFP controlled by an ARR2PB promoter is introduced into L NCaP of prostate cancer cells with positive AR, and after the cells ARE treated by administration of test compounds with different concentrations, the expression level of EGPF in the cells is measured, so that the strength of the compounds on the AR antagonistic capability can be obtained.

The experimental steps are as follows: we use a previously constructed ARR containing a strong response to AR₂EGFP (enhanced green fluorescent protein) reporter gene plasmid controlled by PB promoter, and stable expression EGFP prostate cancer cell line regulated and controlled by AR (augmented reality) obtained by adopting method of stably transfecting L NCaP cells by using lentivirus(LN-ARR₂PB-EGFP)。LN-ARR₂PB-EGFP cells are cultured in a complete culture medium without androgen for several days to reduce the background fluorescence value to a lower level, then the PB-EGFP cells are inoculated into a 96-well plate with black bottom penetration at the density of 40000 cells/well, after the cells are stably attached to the wall, androgen and test compounds (virtually screened compounds and marketed antagonist drug enzalutamide) with different concentrations are simultaneously given, after incubation is carried out for 24-48h, the fluorescence intensity value near the wavelength of 530nm is detected by a multifunctional enzyme-labeling instrument under excitation light with the wavelength of 485nm, and the inhibition rate of the test compounds on AR protein can be quantitatively calculated.

The experimental results are as follows:

as shown in FIG. 6, the inhibition rate of compounds No. 1-13 was 30% or more.

As shown in FIGS. 7-9, after L N-ARR2PB-EGFP cells are treated by different concentrations of compounds for 36h, the compounds have obvious down-regulation effect on the expression of reporter gene EGFP and show a dose-dependent relationship, which indicates that all the compounds listed by us are potential AR antagonists with good activity.

(5) MTT method for detecting prostate tumor cell proliferation resisting activity of compound

The experimental principle is as follows: succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTT ((3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazole bromide)) into water-insoluble blue-purple crystalline Formazan (Formazan) and deposit in the cells, while dead cells do not have the function, buffer is added to dissolve Formazan formed in the cells, and the light absorption value is measured by an enzyme linked immunosorbent detector at 490nm wavelength, thereby indirectly reflecting the number of the living cells.

The experimental steps comprise inoculating and culturing cancer cells in a 96-well plate by using a complete androgen-free culture medium at a density of 3000 per well, simultaneously administering 1nM DHT and test compounds (virtual screening compounds, marketed antagonist drugs or DMSO) with different concentrations after the cells are stably attached to the wall, adding 10 mu L5 mg/ml MTT into each well after incubating for 4days, continuously incubating for 3hours in an incubator, adding 100 mu L SDS-HCl-PBS triple buffer into each well, incubating at 37 ℃ overnight, detecting the absorbance value of each well at 570nM under a microplate reader, and converting the absorbance value into the survival rate to obtain the I of the administered compoundsC₅₀The value is obtained.

As shown in FIGS. 10 and 11, the compounds of the present invention have obvious proliferation inhibiting ability and IC inhibiting ability on prostate cancer cell L NCaP₅₀Can reach the level similar to that of the marketed medicine enzalutamide.

Claims

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,