CN109730996B

CN109730996B - Quinoline structure type androgen receptor antagonist and application thereof

Info

Publication number: CN109730996B
Application number: CN201910136005.XA
Authority: CN
Inventors: 侯廷军; 李丹; 周文方
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2017-03-01
Filing date: 2017-03-01
Publication date: 2021-08-24
Anticipated expiration: 2037-03-01
Also published as: CN109700804B; CN109793730B; CN109846879A; CN109700811B; CN109793737A; CN106943397B; CN109793737B; CN106943397A; CN109700798A; CN109700804A; CN109700794A; CN109793730A; CN109730996A; CN109700811A; CN109846879B; CN109700794B

Abstract

The divisional application discloses a quinoline structure type androgen receptor antagonist and application thereof, belonging to the technical field of biochemistry. The compound provided by the invention has obvious antagonistic activity on androgen receptor, so that the compound 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

Quinoline structure type androgen receptor antagonist and application thereof

The application is divisional application with application number 201710117698.9, application date 2017, 3/1, invention title "androgen receptor antagonist and application 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. Endocrine therapy is the main treatment means for hormone-sensitive advanced prostate cancer patients, 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 (LHRH) analogues, as a supplementary means in Androgen Deprivation Therapy (ADT), primarily to arrest 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 are mainly ARN-509, ODM-201 and AZD3514, which have been currently progressing to the three-phase clinical trial stage separately for prostate patients who have received different therapies and are in different stages, and are very expected to be approved for the addition of treatment lines in the near future. ARN-509 has a structure very similar to that of enzalutamide, and the current research results show that compared with enzalutamide, the novel compound has stronger receptor binding capacity, lower dosage required to be taken and lower central nervous system infiltration and epileptogenic side effects, but due to the excessive similarity of the structure, the F876L mutation which has the resistance to enzalutamide can also generate the resistance to ARN-509. ODM-201 and its in vivo metabolite ORM-15341 have a more novel chemical structure, acting in a mechanism similar to the second generation antagonists, but with affinity for AR even exceeding ARN-509 and enzalutamide.

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 computer-aided drug molecule design means to find a lead compound of a targeted androgen receptor, and then virtual screening based on molecule docking is carried out on a plurality of small molecule compound three-dimensional structure databases to obtain 1000 compounds with front scores (the lower the energy, the more the front the score). Then MTT cell proliferation experiment of prostate cancer classical cell line LNCaP, AR transcription factor activity inhibition experiment and the use of kit PolarScreen^TMAR completor Assay, Green (thermo Fisher scientific) investigated the binding of compounds to the ligand binding domain LBP of AR, and finally screened 1 representative active compound: ethyl 7-chloro-4- ((3-hydroxyphenyl) amino) -8-methylquinoline-3-carboxylic acid, the structural formula is shown as formula (1);

the invention further tests the biological activity of the screened compound, and finds that the compound has obvious antagonistic activity on androgen receptor, so the invention provides the application of the compound or the medicinal salt thereof in preparing androgen receptor antagonist.

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

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

The present invention also provides a pharmaceutical composition comprising the compound 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 for the first time that the compound ethyl 7-chloro-4- ((3-hydroxyphenyl) amino) -8-methylquinoline-3-carboxylic acid has obvious antagonistic activity to androgen receptor based on a virtual screening method of molecular docking and biological activity measurement, can be used as an 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 medicaments 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 is a binding conformation of the antagonist in the AR active pocket and the interaction pattern between the antagonist and AR active pocket residues, wherein (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 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 Fisher Scientific) 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

The experimental principle is as follows: the binding capacity of compound AR was measured using a fluorescence bias experiment from Invitrogen (Thermo Fisher Scientific). The 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) having a higher fluorescence bias value. When the complex is added into a micropore plate containing a test compound, the test compound serving as a competitive ligand replaces a fluorescent ligand (fluoromone ML Green) in a binary complex, so that the fluorescence bias positive value is reduced. If a noncompetitive ligand is added that does not have the ability to replace the fluorescent compound, the bias value will remain high. Thus, the change in fluorescence bias upon addition of the test compound can be used to quantitatively measure the relative affinity of the test compound for AR-LBD (His-GST).

The experimental method comprises the following steps: after mixing the AR LBD protein and the high affinity fluorescent ligand in buffer, different concentrations of test compound (virtual screening compound) were added with the androgen Dihydrotestosterone (DHT) as positive control). If the test compound has higher affinity to AR LBD, the fluorescent ligand in the binary compound can be replaced by the competitive ligand, so that the fluorescence bias value of the system is reduced; if the added test compound has no binding capacity to AR LBD basically, the fluorescence bias value of the system can be maintained at a higher value, and the binding capacity (binding affinity) of the virtual screening compound 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 as follows: AR, as a transcription factor, needs to bind to a specific sequence, an ARE response element, to exert transcription activity; therefore, reporter gene enhanced green fluorescent protein EGFP controlled by an ARR2PB promoter is introduced into AR positive prostate cancer cells LNCaP, and after the treatment of 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 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 (LN-ARR) regulated and controlled by AR (endothelial growth factor receptor) obtained by adopting method of stably transfecting LNCaP cell by using lentivirus₂PB-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 LN-ARR2PB-EGFP cells were treated with different concentrations of the compounds for 36h, the compounds produced significant down-regulation of reporter EGFP gene expression and exhibited dose-dependent relationship, indicating that all of the compounds listed 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 are as follows: inoculating and culturing cancer cells on a 96-well plate by using a complete medium without androgen at a density of 3000 per well, after the cells are stably attached to the wall, simultaneously administering 1nM DHT and test compounds (virtual screening compounds, marketed antagonist drugs or DMSO) with different concentrations, adding 10 mu L5 mg/ml MTT into each well after incubating for 4days, continuously incubating for 3hours in an incubator, then adding 100 mu L SDS-HCl-PBS triple buffer into each well, after incubating at 37 ℃ overnight, detecting the absorbance value of each well at 570nM position under an enzyme labeling instrument, converting the absorbance value into the survival rate, and obtaining the IC of the administered compounds₅₀The value is obtained.

The experimental results are as follows: as shown in figures 10 and 11, the compound of the invention has obvious proliferation inhibition capacity and IC inhibition effect on prostate cancer cells LNCaP₅₀Can reach the level similar to that of the marketed medicine enzalutamide.

Claims

1. The application of the compound with the structural formula shown as the formula (1) or the medicinal salt thereof in preparing the anti-prostate tumor medicament is characterized in that the compound or the medicinal salt thereof has androgen receptor antagonistic activity and inhibits the proliferation of prostate cancer cells, the prostate cancer cells are androgen receptor positive prostate cancer cells,

(1)。

2. the use of claim 1, wherein the pharmaceutically acceptable salt is a hydrochloride, phosphate, sulfate, acetate, maleate, citrate, benzenesulfonate, methylbenzenesulfonate, fumarate, or tartrate salt.

3. The use of claim 1, wherein the medicament further comprises a pharmaceutically acceptable excipient or carrier.

4. The use according to claim 3, wherein the medicament is formulated as a liquid formulation or a solid formulation.