A compound, uses and inhibitors and medicaments

Abstract

The present application relates to a kind of human 17 hydroxysteroid dehydrogenase type 1 inhibitor, to a kind of compound and application and inhibitor and drug. Human 17 hydroxysteroid dehydrogenase type 1 is a key enzyme in human body catalyzing androgen to estrogen conversion, and it is proved to be an important reason of aromatase inhibitor drug resistance. By inhibiting 17 hydroxysteroid dehydrogenase type 1 activity, estrogen level in vivo can be reduced, androgen / estrogen ratio level in vivo can be improved, and various diseases caused by androgen / estrogen ratio imbalance and excessive estrogen level can be alleviated. The present application obtains a kind of high selectivity, high efficiency 17 hydroxysteroid dehydrogenase type 1 inhibitor by structure-activity relationship design under the guidance of protein structure, can significantly inhibit the proliferation of estrogen receptor positive breast cancer cell line, ovarian cancer cell line and endometrial adenocarcinoma cell line.

Description

Technical Field

[0001] This invention relates to a novel human 17-hydroxysteroid dehydrogenase type 1 inhibitor and its application in the treatment of estrogen-dependent cancers, specifically the application of human 17-hydroxysteroid dehydrogenase type 1 inhibitor (PBRR) in inhibiting the proliferation of estrogen receptor-positive breast cancer cells, ovarian cancer cells, and endometrial adenocarcinoma cells. Background Technology

[0002] Numerous studies have shown that estradiol plays a crucial role in the course of estrogen-dependent diseases in women, such as breast cancer, endometrial cancer, endometriosis, and uterine cancer. In all breast cancer cases, 60% of premenopausal women and 75% of postmenopausal women are estrogen-dependent (ER+), and up to 80% of endometrial breast cancer patients are also ER+. Therefore, estrogen-dependent cancers are the most common malignant tumors among women worldwide, seriously threatening women's health and lives.

[0003] In recent years, molecular therapy for ER+ breast cancer has yielded a landmark treatment: aromatase inhibitors. These inhibitors target and inhibit aromatase, a key enzyme in estrogen synthesis, thereby blocking estradiol synthesis. Studies have found that aromatase inhibitors achieve better therapeutic effects than estrogen receptor modulators like tamoxifen in postmenopausal estrogen receptor-positive breast cancer patients, with relatively fewer side effects. Although the clinical use of aromatase inhibitors has greatly improved cancer-free survival in estrogen receptor-positive breast cancer patients, recent studies have shown that up to 37% of patients develop resistance during aromatase inhibitor treatment. Research has identified the synthesis of aromatase-independent estrogens androst-5-ene-3β,17β-diol (5-diol) and 5α-androstane-3β,17β-diol (3β-diol) as one of the main mechanisms of this resistance.

[0004] Human 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) is one of the most important enzymes for estrogen activation, possessing a dual function of activating estrogen and degrading androgens. It catalyzes the biosynthesis of estrone (E1) to estradiol (E2) with high activity and can convert dehydroepiandrosterone (DHEA) to 5-diol and dehydrotestosterone (DHT) to 3β-diol, stimulating the growth of ER+ breast cancer cells. Therefore, it is an important target for estrogen-dependent diseases and has received considerable attention in decades of breast cancer treatment efforts. Thus, the development of 17β-HSD1 inhibitors is of great therapeutic significance. International research on 17β-HSD1 inhibitors has a history of about fifty years. However, due to the use of analogs of natural substrates as the starting point for inhibitor design, and the extremely similarity of substrates recognized by members of this enzyme family (especially 17β-HSD2, which catalyzes the E2 to E1 reaction), inhibitors of this enzyme often exhibit estrogenicity or lack sufficient specificity. Building upon the previously reported high-affinity 17β-HSD1 inhibitor CC-156 (16β-(m-carbamoylbenzyl)E2), researchers developed a new generation of highly effective 17β-HSD1 inhibitor, PBRM (3-{[(16b,17b)-3-(2-iodoethyl)-17-hydroxyestra-1,3,5(10)-trien-16-yl]methyl}benzamide), by adding side-chain groups at the C2, C3, and C7 positions of the steroid core to reduce its estrogenicity. This inhibitor is an irreversible competitive inhibitor; its low-reactivity brominated side-chain group at C3 specifically undergoes a nucleophilic substitution reaction with the nitrogen atom on the histidine side chain at position 221 of 17β-HSD1, thus exhibiting extremely high specificity. This inhibitor demonstrates high inhibitory activity (IC50) against the E1 to E2 response of 17β-HSD1. 50 The enzyme has a molecular weight of 68 nM and has shown good tumor-suppressive effects in cell and animal experiments. Furthermore, in studying the substrate inhibitory effect of 17β-HSD1, the applicant discovered that during the binding of the enzyme to substrate E1, E1 exhibits a reverse binding phenomenon, forming a dead-end complex. More importantly, the 17β-HSD1 enzyme molecule shows a higher affinity for the reverse-bound E1 than for the forward-bound E1. Therefore, inhibitors designed based on this binding mechanism are expected to achieve better inhibitory effects, especially further improving inhibitor specificity. Currently, there are no reports on inhibitors based on the structural feature of the 17β-HSD1 enzyme molecule's reverse binding to E1.

[0005] Based on the previously elucidated structural characteristics of 17β-HSD1's reverse binding to substrates, and the characteristic that the nitrogen atom on the side chain of the histidine residue at position 221 of the enzyme can participate in affinity substitution reactions, this invention employs computer-aided drug design methods to design a novel, highly specific, and highly efficient 17β-HSD1 inhibitor. Summary of the Invention

[0006] The purpose of this invention is to provide an inhibitor of human 17-hydroxysteroid dehydrogenase type 1.

[0007] 1) Its molecular structure is shown in Formula 1;

[0008]

[0009] Formula 1: Molecular structure of PBRR.

[0010] 2) Its molecular weight is 498 Daltons.

[0011] 3) Its molecular formula is C 27 H 34 BrN2O2

[0012] Human 17-hydroxysteroid dehydrogenase type 1 (HHDDE1) is one of the most important enzymes for estrogen activation in the human body. This enzyme has a dual function of activating estrogen and degrading androgens. Inhibiting HHDDE1 activity can lower estrogen levels in the body while increasing androgen levels, thereby inhibiting the proliferation of estrogen-dependent tumor cells.

[0013] This invention, through analysis of the specific conformation of estrone in the substrate binding pocket of human 17-hydroxysteroid dehydrogenase type 1, rationally designed a novel specific inhibitor (PBRR) using computational and analytical methods. Its molecular formula is C1. 27 H 34 BrN2O2, with a molecular structure as shown in Formula 1, is similar to the best existing inhibitor PBRM (IC). 50 Compared to 83 nM, PBRR has a stronger binding affinity to 17β-HSD1, reaching 30 nM, which will help reduce the dosage used.

[0014] This invention uses cell biology analysis to verify the inhibitory effect of PBRR on the proliferation of estrogen receptor-positive breast cancer cell lines (T47D, MCF-7), endometrial adenocarcinoma cell lines (Ishikawa), and ovarian cancer cell lines (OVCAR-3).

[0015] This invention provides a highly selective and efficient 17-hydroxysteroid dehydrogenase type 1 inhibitor through structure-activity design guided by protein structure. This inhibitor can significantly inhibit the proliferation of estrogen receptor-positive breast cancer cell lines, ovarian cancer cell lines, and endometrial adenocarcinoma cell lines. Attached Figure Description

[0016] Figure 1 The binding of PBRR to 17β-HSD1.

[0017] Figure 2 :PBRR 13 C NMR and 2D COSY NMR analysis spectra ( 13 In a C NMR spectrum, the chemical shift values ​​corresponding to atoms are marked near the corresponding atoms.

[0018] Figure 3 PBRR inhibits the proliferation of breast cancer T47D and MCF-7 cell lines.

[0019] Figure 4 PBRR inhibits the proliferation of Ishikawa cell line, an endometrial adenocarcinoma.

[0020] Figure 5 PBRR inhibits the proliferation of ovarian cancer OVCAR-3 cell line. Detailed Implementation

[0021] Example 1: Rational Design of Human 17-Hydroxysteroid Dehydrogenase Type 1 Inhibitors

[0022] The development of 17β-HSD1 inhibitors has long been hampered by bottlenecks. Most inhibitors developed by previous researchers failed to enter clinical trials due to insufficient endogenous estrogenicity and specificity. Previous inhibitors designed based on the steroid core have referenced the crystal structure of the complex between estradiol (E2) and the enzyme. Since the D ring of the steroid core is located at the catalytic center, it is often used as a design site for substituents. The applicant, through studying the mechanism of mutual recognition and binding between 17β-HSD1 and E1, demonstrated that the natural substrate E1 of this enzyme binds more readily to the enzyme in a reverse manner. Based on this, the applicant used SEESAR software to start with E1, adding a benzamide group at position 3 to enhance the binding of the inhibitor to the amino acid residue at the catalytic center. Simultaneously, a bromoethyl group was designed at position 17 to endow the inhibitor with the ability to covalently react with the His221 side chain, and an amino group was added at position 16 to promote the electrostatic interaction between the inhibitor and the Glu282 side chain. This inhibitor exhibits extremely high specificity for 17β-HSD1, with an affinity reaching 30 nM. The inhibitor is named PBRR(3-(((8S,9S,13S,14S,16R,17R)-16-amino-17-(2-bromoethyl)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-3-yl)oxy)benzamide), and its molecular formula is C 27 H 34 BrN2O2, molecular structure is shown in Formula 1.

[0023]

[0024] Formula 1: Molecular structure of PBRR

[0025] Molecular docking calculations revealed that PBRR can covalently bind to the side chain of His221 residue of 17β-HSD1 via a bromine substitution reaction, similar to PBRM. A salt bridge is formed between the amino group attached to the D ring and the carboxyl group of the Glu282 side chain. A π bond is formed between the benzamide group attached to the A ring and the benzene ring of the Tyr155 side chain. Furthermore, its amide group can form hydrogen bonds with Asn152 and Leu95. Figure 1 The close interaction is consistent with the high inhibitory activity of this inhibitor. Furthermore, molecular docking analysis indicates that PBRR does not bind to estrogen receptors.

[0026] The synthetic route of PBRR is shown in Formula 2. Raw material a (CAS: 74536-67-7) was purchased from Hong Kong Chemhere Ltd. A (1 g), imidazole (0.64 g), and tert-butyldimethylchlorosilane (0.67 g) were dissolved in 13 mL of dimethylformamide. The mixture was stirred overnight at room temperature under nitrogen protection. Then, an equal volume of water was added to the reaction mixture, and the mixture was extracted with dichloromethane (three times, 10 mL each time). The solvent was then evaporated to obtain b. First, 0.052 mg of NaH was dissolved in 1.5 mL of tetrahydrofuran at room temperature under nitrogen protection. Then, 0.32 g of triethyl phosphonate and 0.2 g of b were added, and the mixture was heated (60 °C) and rotary evaporated overnight. Then, an equal volume of water was added to the reaction mixture, and the mixture was extracted with ethyl acetate (three times, 10 mL each time). The solvent was then evaporated to obtain c. c (0.0498 g) was dissolved in 3.0 mL of dimethylformamide. In N,N-dimethylformamide (DMF), 0.0561 g of 3-bromobenzamide and 0.0342 g of anhydrous potassium carbonate were added, and the mixture was stirred overnight at room temperature. Then, an equal volume of water was added, and the mixture was extracted with dichloromethane (10 mL three times). The organic solvent was removed by rotary evaporation to obtain d. d (0.0356 g) was dissolved in 3.0 mL of lithium aluminum hydride (1.0 M)-tetrahydrofuran solution, and stirred overnight at room temperature. Then, a 10% (w / w) aqueous solution of ammonium chloride (equal molar amounts of ammonium chloride and lithium aluminum hydride) was added, and the mixture was stirred with ethyl acetate. The ester was extracted three times (50 mL each time), the organic phases were combined, and the organic solvent was removed by rotary evaporation to obtain intermediate e; e (0.0471 g) was dissolved in 3.0 mL of carbon tetrachloride, and N-bromosuccinimide (NBS) (0.0267 g) was added. The mixture was stirred at room temperature and irradiated with 350 nm light for 21 hours. An equimolar amount of sodium bicarbonate was added and stirred until homogeneous. The mixture was then extracted with a 1:1 (v / v) dichloromethane aqueous solution, and the organic solvent was removed by rotary evaporation to obtain intermediate f; f (0.0534 g) was dissolved in 3.0 mL of carbon tetrachloride. DMSO was added, and lithium azide (0.0075 g) was added and stirred for 6 hours. Then, 0.5 μL of lithium aluminum hydride (1.0 M)-tetrahydrofuran solution was added, and the mixture was stirred overnight at room temperature. The product was extracted with diethyl ether and water (1:1 v / v), and the organic solvent was removed by rotary evaporation. Finally, the product was separated by an Agilent Poroshell 120 Chiral-CF chiral column (mobile phase 100 / 0.2 / 0.05: Methanol / Acetic Acid / Ammonium Hydroxide) to obtain the final product PBRR, with a yield of approximately 70%. PBRR... 13 The C NMR and 2D COSYNMR spectra are shown below. Figure 2 .

[0027]

[0028] Equation 2: Synthesis path of PBRR

[0029] Example 2: Inhibition of the proliferation of estrogen receptor-positive breast cancer cell lines (T47D, MCF-7) (purchased from ATCC, USA) by PBRR. Estrogen-dependent human breast cancer cells MCF7 and T47D were seeded into the wells of different 12-well plates, with an seeding density of approximately 1 × 10⁻⁶ cells per well. 5 Cells were grown at a constant temperature and humidity of 37°C under 5% CO2 atmospheric conditions for 24 hours. The culture medium for MCF7 was DMEM low-glucose (Gibco, catalog number: 11885084) hormone-free medium containing 10% fetal bovine serum (incubated overnight at 4°C with 2% activated charcoal, centrifuged first, and then the supernatant was filtered through a 0.2-micron sterile filter for sterilization).

[0030] The culture medium for T47D was DMEM high glucose (Gibco, catalog number: 11965092) Hormone-free medium containing 10% fetal bovine serum (incubated overnight at 4°C with 2% activated carbon by volume, centrifuged first, and then the supernatant was filtered through a 0.2-micron sterile filter).

[0031] Divide each of the 12 wells in the two 12-well plates into three groups: a Hormone-Free group, a Control group, and a PBRR treatment group. Replace the medium with the same fresh culture medium as the cell culture medium described above. The Hormone-Free group received no estrone (E1) substrate and no PBRR inhibitor. The Control group received E1 at a final concentration of 0.1 nM. The PBRR treatment group received E1 at a final concentration of 0.1 nM and PBRR at a final concentration of 400 nM. After incubation for 2 days, replace the medium with the same fresh culture medium and add the corresponding concentrations of substrate and inhibitor. After 4 days, end the culture and remove the medium. Quantify the total DNA content of the cell plate samples according to the CyQUANT (Invitrogen) cell proliferation kit instructions.

[0032] Experimental results:

[0033] like Figure 3As shown, by quantifying the total DNA content of cells in different treatment groups, it was found that under the condition of 400 nM PBRR, the total DNA growth rate (cell proliferation rate) of T47D and MCF7 cells decreased to 85.67±4.04% and 84.33±4.04% of the hormone-free group, respectively, while the growth rate of the hormone control group reached 146.33±3.22% and 165.00±5.00% of the hormone-free group, respectively. This indicates that 400 nM PBRR can completely inhibit the proliferation of T47D and MCF7 cells induced by 0.1 nM E1.

[0034] Example 3: Inhibition of proliferation of estrogen receptor-positive endometrial adenocarcinoma cell line (Ishikawa) (purchased from ATCC, USA) by PBRR

[0035] Estrogen-dependent endometrial adenocarcinoma cells Ishikawa (purchased from Merck, catalog number: 99040201) were seeded into 12-well plates, with approximately 1 × 10⁶ cells per well. 5 Cells were cultured at 37°C and constant humidity under 5% CO2 concentration for 24 hours. The culture medium was MEM (Merck, catalog number: M8042) Hormone-Free medium containing 5% fetal bovine serum (incubated overnight at 4°C with 2% activated charcoal, centrifuged, and the supernatant filtered through a 0.2-micron sterile filter). The 12 wells were divided into three groups: Hormone-Free, Control, and PBRR-treated. The culture medium was replaced with the same fresh medium as the previous cell culture. The Hormone-Free group did not contain the substrate estrone (E1) or the inhibitor PBRR; the Control group contained 0.1 nM E1; and the PBRR-treated group contained 0.1 nM E1 and 550 nM PBRR. After incubation for 2 days, the culture medium was replaced with the same fresh medium as the cell culture above, and the corresponding concentrations of substrate and inhibitor were added. After 4 days, the culture was ended and the culture medium was removed. The total amount of DNA in the cell plate sample was quantitatively detected according to the instructions of the CyQUANT (Invitrogen) cell proliferation kit.

[0036] Experimental results:

[0037] like Figure 4 As shown, by quantifying the total DNA content of cells in different treatment groups, it was found that the proliferation rate of Ishikawa cells under the condition of 550 nM PBRR decreased to 89.33 ± 4.04% of the hormone-free group, while the growth rate of the hormone control group reached 144.00 ± 4.00% of the hormone-free group. This indicates that 550 nM PBRR can completely inhibit the proliferation of Ishikawa cells induced by 0.1 nM E1.

[0038] Example 4: Inhibition of proliferation of estrogen receptor-positive ovarian cancer cell line (OVCAR-3) (purchased from ATCC, USA) by PBRR

[0039] Estrogen-dependent human ovarian cancer cells (OVCAR-3) were seeded into 12-well plates, with approximately 1 × 10⁻⁶ cells per well. 5 Cells were cultured at 37°C and constant humidity under 5% CO2 concentration for 24 hours. The culture medium was RPMI-1640 (Gibco, catalog number: 11875135) Hormone-Free medium containing 20% ​​fetal bovine serum (incubated overnight at 4°C with 2% activated charcoal, centrifuged, and the supernatant filtered through a 0.2-micron sterile filter). The 12 wells were divided into three groups: Hormone-Free, Control, and PBRR-treated. The culture medium was replaced with the same fresh medium as the previous cell culture. The Hormone-Free group did not contain the substrate estrone (E1) or the inhibitor PBRR; the Control group contained 0.1 nM E1; and the PBRR-treated group contained 0.1 nM E1 and 500 nM PBRR. After incubation for 2 days, the culture medium was replaced with fresh medium and the corresponding concentrations of substrate and inhibitor were added. After 4 days, the culture was terminated and the culture medium was removed. The total DNA content of the cell plate samples was quantitatively detected according to the instructions of the CyQUANT (Invitrogen) cell proliferation kit.

[0040] Experimental results:

[0041] like Figure 5 As shown, by quantifying the total DNA content of cells in different treatment groups, it was found that under the condition of 500 nM PBRR, the proliferation rate of T47D and MCF7 cells decreased to 91.00 ± 3.61% of the hormone-free group, while the growth rate of the hormone control group reached 165.00 ± 5.00% of the hormone-free group. This indicates that 500 nM PBRR can completely inhibit the proliferation of OVCAR-3 cells induced by 0.1 nM E1.

Claims

1. A compound, characterized in that: Its molecular structure is shown in Formula 1; ； Formula 1: Molecular structure of PBRR.

2. The use of the compound of claim 1 in the preparation of a drug for inhibiting the proliferation of estrogen receptor-positive tumor cell lines.

3. The application according to claim 2, characterized in that: Including one, two, or three of the following applications: 1) Application in the preparation of drugs that inhibit the proliferation of estrogen receptor-positive breast cancer cell lines; 2) Application in the preparation of drugs that inhibit the proliferation of estrogen receptor-positive ovarian cancer cell lines; 3) Application in the preparation of drugs that inhibit the proliferation of estrogen receptor-positive endometrial adenocarcinoma cell lines.

4. The application according to claim 3, characterized in that: The concentration of the compound used in cellular environments is 30 nM to 600 nM.

5. The use of the compound of claim 1 in the preparation of one, two, or three of the therapeutic agents for estrogen receptor-positive breast cancer, ovarian cancer, and endometrial adenocarcinoma, or as a human 17-hydroxysteroid dehydrogenase type 1 inhibitor.

6. A cancer treatment drug for human 17-hydroxysteroid dehydrogenase type 1 inhibitors or estrogen receptor-positive cells, characterized in that: It uses the compound described in claim 1 as its active ingredient.

7. The inhibitor or cancer treatment drug according to claim 6, characterized in that: It contains a pharmaceutically or pharmacologically acceptable carrier.

8. The inhibitor or cancer treatment drug according to claim 6, characterized in that: Cancers with estrogen receptor-positive cells include one, two, or three of the following: estrogen receptor-positive breast cancer, ovarian cancer, and endometrial adenocarcinoma.

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