Thiophene-containing cyclic derivatives, their preparation methods and their pharmaceutical applications

Abstract

This invention provides thiophene-containing cyclic derivatives, their preparation methods, and their pharmaceutical applications. Specifically, this invention relates to a compound of general formula (I) and its pharmaceutically acceptable salt, their preparation methods, and their use as gonadotropin-releasing hormone (GnRh) receptor antagonists, wherein the definitions of each substituent in general formula (I) are the same as those in the specification.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field and relates to a thiophene ring derivative and its pharmaceutically usable salt, its preparation method, a pharmaceutical composition containing the derivative, and its use as a gonadotropin-releasing hormone (GnRh) receptor antagonist. Background Technology

[0002] Gonadoliberin (GnRH), also known as luteinizing hormone-releasing hormone (LHRH), is a decapeptide hormone (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) synthesized by hypothalamic neuroendocrine cells. It is a central regulatory factor in the endocrine-reproductive system. Transported to the pituitary gland via the hypothalamic-pituitary portal circulation system, it binds to GnRH receptor cells in the anterior pituitary gland. This triggers the secretion and release of gonadotropins such as luteinizing hormone (LH) and follicle-stimulating hormone (FSH), regulating the normal development of the ovary and corpus luteum, and playing a crucial role in the hypothalamic-pituitary-gonadal axis. GnRH receptors exert their regulatory function by coupling with G proteins that can activate the phosphatidylinositol calcium second messenger system, while LH regulates the production of sex steroids, and FSH regulates spermatogenesis in men and follicle development in women.

[0003] LH and FSH are released into the circulation and bind to specific receptors on cells of the ovaries or testes, stimulating steroid production. In the presence of sex steroids, conditions such as endometriosis, uterine fibroids, and prostate cancer are exacerbated, requiring treatment with long-acting peptide GnRH receptor agonists and antagonists for control.

[0004] Peptide GnRH receptor antagonists include linear GnRH-derived peptides (US 5,171,835), cyclic hexapeptide derivatives (US 2002 / 0065309), and bicyclic peptide derivatives (Journal of Medicinal Chemistry, 1993; 36: 3265-73); while peptide GnRH receptor agonists include leuprorelin (pGlu-His-Trp-Ser-Tyr-d-Leu-Leu-Arg-Pro-NHEt). However, peptide compounds have many unresolved issues, including oral absorption, dosage form, dose-volume relationship, drug stability, duration of action, and metabolic stability. The main reason why small molecule GnRH receptor antagonists are superior to existing peptide-based therapies is that they can be administered orally, which is convenient and rapid.

[0005] Currently, a series of patents for small molecule GnRH receptor antagonists have been disclosed, including WO2006096785, WO2010026993, WO2011076687, and WO2012175514.

[0006] Despite the significant amount of research already conducted in this field, further research is needed to develop more effective small-molecule GnRH receptor antagonists. This invention provides a novel GnRH receptor antagonist structure and has found that compounds with this structure exhibit good activity and can effectively treat endocrine and reproductive system diseases. Summary of the Invention

[0007] This invention provides a compound of general formula (I) and a pharmaceutically acceptable salt thereof:

[0008]

[0009] in:

[0010] X1 and X2 are each independently selected from halogen or C. 1-3 Halogenated alkyl; preferably fluorine, chloro, or trifluoromethyl;

[0011] n is selected from 1, 2, or 3.

[0012] In a preferred embodiment of the present invention, the compound is selected from:

[0013]

[0014] The present invention also provides a method for preparing the compound of general formula (I) and its pharmaceutically acceptable salt, comprising:

[0015]

[0016] The compound of general formula (II) undergoes an intramolecular amidation reaction in a solvent in the presence of a condensing agent to give the compound of general formula (I);

[0017] X1 and X2 are each independently selected from halogen or C. 1-3 Halogenated alkyl; preferably fluorine, chloro, or trifluoromethyl;

[0018] n is selected from 1, 2, or 3;

[0019] The condensing agent is selected from carbodiimide condensing agents; preferably DCC, DIC, or EDCI.

[0020] In a preferred embodiment of the present invention, the condensing agent in the above preparation method is selected from DCC or EDCI.

[0021] In a further preferred embodiment of the present invention, the solvent used in the above preparation method is selected from DMF or DMA.

[0022] In a further preferred embodiment of the present invention, the molar ratio of the condensing agent to the compound of general formula (II) in the above preparation method is 1:1 to 3:1.

[0023] In a further preferred embodiment of the present invention, the reaction temperature of the above preparation method is 40–100°C; preferably 60–80°C.

[0024] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the above-mentioned compound and its pharmaceutically acceptable salt, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0025] The present invention also relates to the use of the above-described compounds and their pharmaceutically usable salts or the above-described pharmaceutical compositions in the preparation of medicaments for GnRH receptor antagonists. Invention Details

[0027] Unless otherwise stated, the terms used in the specification and claims shall have the following meanings.

[0028] "alkyl" refers to a saturated aliphatic hydrocarbon group, including straight-chain and branched groups with 1 to 20 carbon atoms. Preferably, it is an alkyl group containing 1 to 10 carbon atoms, more preferably an alkyl group containing 1 to 6 carbon atoms, most preferably an alkyl group containing 1 to 3 carbon atoms, and most preferably methyl. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, and n-pentyl.

[0029] "Halogenated alkyl" refers to an alkyl group that has been substituted with one or more halogens, where the definition of alkyl is as described above.

[0030] "Halogen" refers to fluorine, chlorine, bromine, or iodine.

[0031] "Pharmaceutical composition" means a mixture containing one or more of the compounds described herein or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity.

[0032] "DCC" refers to dicyclohexylcarbodiimide;

[0033] "DIC" refers to diisopropylcarbodiimide;

[0034] “EDCI” refers to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide;

[0035] "DMF" refers to N,N-dimethylformamide;

[0036] “DMA” refers to N,N-dimethylacetamide;

[0037] "DMAP" refers to 4-dimethylaminopyridine. Detailed Implementation

[0038] The present invention will be explained in more detail below with reference to the embodiments. The embodiments of the present invention are only used to illustrate the technical solutions of the present invention and are not intended to limit the nature and scope of the present invention.

[0039] Experimental methods in this invention, where specific conditions are not specified, are generally performed under conventional conditions or as recommended by the raw material or product manufacturer. Reagents whose specific source is not specified are commercially available, conventional reagents.

[0040] Compound 1-1 of Example 1 of this invention was prepared according to the method disclosed in CN104703992B.

[0041] Example

[0042] The structures of the compounds were determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR chemical shifts (6) are given in units of 10⁻⁶ (ppm). NMR measurements were performed using a Varian INOVA-400 NMR spectrometer with deuterated chloroform (CDCl₃) as the solvent and tetramethylsilane (TMS) as the internal standard.

[0043] MS measurements were performed using an ACQUITY UPLC (ESI) mass spectrometer under the following conditions: ESI source, negative ion mode.

[0044] HPLC determinations were performed using an Agilent 1200DAD high-performance liquid chromatograph (Sunfire C18 150×4.6mm column) and a Waters 2695-2996 high-performance liquid chromatograph (Gimini C18 150×4.6mm column).

[0045] The average inhibition rate and IC50 value of the kinase were determined using a NovoStar microplate reader (BMG GmbH, Germany).

[0046] Thin-layer chromatography silica gel plates are Yantai Huanghai HSGF254 or Qingdao GF254. The silica gel plates used in thin-layer chromatography (TLC) have a diameter of 0.15 mm to 0.2 mm, and the diameter of the silica gel plates used for thin-layer chromatography separation and purification products is 0.4 mm to 0.5 mm.

[0047] The known starting materials of this invention can be synthesized using or according to methods known in the art, or can be purchased from companies such as ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, AccelaChemBio Inc, and Darui Chemicals.

[0048] Example 1

[0049] Preparation of Compound 1

[0050]

[0051] 40 mg DMAP, 440 mg EDCI (2.312 mmol), and compound 1-1 (1 g, 1.926 mmol) were added to a 100 mL three-necked flask. After purging with nitrogen, the mixture was dissolved in 25 mL DMF. The mixture was heated to 60-80 °C and stirred for 6-8 h. The color of the reaction solution gradually darkened. The reaction was monitored by HPLC until it ended. If there was any remaining reactant, EDCI could be added until the reactant was completely reacted. The temperature was lowered to 0-15 °C, and 50 mL of water was slowly added to the reaction solution while stirring. A large amount of solid precipitated. The temperature was further lowered to -5-0 °C and stirred for 1-2 h. The target product was collected by filtration, the filter cake was washed with water, and dried to obtain 0.86 g of the compound from Example 1. Yield: 91%, purity: 97.4% (HPLC).

[0052] 1H NMR (400MHz, CDCl3) δ7.21-7.23 (m, 2H), 7.27-7.4923 (m, 2H), 7.17-7.21 (m, 1H), 7.21-7. 23(t,2H),5.68(br,2H),4.47(s,2H),4.21-4.26(q,2H),4.47(s,3H),1.23-1.27(t,3H).

[0053] MS m / s (ESI): 488.2 [M+1] +

[0054] Examples 2-6 were synthesized using the same method as in Example 1. The data are as follows:

[0055]

[0056]

[0057] Biological evaluation

[0058] Test Example 1: Determination of the in vitro protein activity of the compounds of the present invention against human GnRHr (GnRH receptor)

[0059] Experimental objective: This method is used to determine the inhibitory effect of the compounds in this invention on the activity of human GnRHr protein expressed in human GnRHr / CHO stable cell line cells.

[0060] Experimental materials and instruments:

[0061] 1. Fluo-4NW Calcium Assay Kits (F36206, invitrogen);

[0062] 2. DMEM / F12 (SH30023.01B, thermo);

[0063] 3. G418 (11811-031, invitrogen);

[0064] 4. FlexStation3 microplate reader.

[0065] Experimental plan:

[0066] The mammalian expression vector containing the human GnRHr gene was transformed into CHO cells using LTX reagent containing Plus™; antibiotics were added the following day for screening, and single-clonal cell lines were selected.

[0067] Human GnRHr / CHO stable transgenic cells were seeded at a density of 25,000 cells / well in 96-well plates. The next day, the culture medium was removed, and 100 μL of loading buffer containing Fluo-4 dye was added to each well. The plates were incubated at 37°C for 30 minutes. After 30 minutes, the plates were allowed to equilibrate to room temperature for 10 minutes. Seven concentrations of each compound were prepared using DMSO: 100 μM, 10 μM, 1 μM, 0.1 μM, 0.01 μM, 0.001 μM, and 0.0001 μM. 1 μL of each compound was added to each well and incubated at room temperature for 10 minutes. Detection was performed using a FlexStation 3 microplate reader. The reader automatically added 50 μL of GnRH peptide stimulation solution and immediately read the values ​​at 494 / 516 nM. The IC50 values ​​of the compounds were... 50 The value can be obtained by software calculation using fluorescence values ​​corresponding to different concentrations.

[0068] The inhibitory activity of the compound against human GnRHr in this invention was determined by the above experiments, and the measured IC50 values ​​were... 50 The values ​​are shown in Table 1.

[0069] Table 1

[0070] Example number <![CDATA[IC 50 (nM)]]> 1 0.9 6 10.2

[0071] in conclusion:

[0072] The compounds of this invention have a significant inhibitory effect on human GnRHr activity.

Claims

1. A compound of general formula (I) and a pharmaceutically acceptable salt thereof: in: X1 and X2 are each independently selected from halogen or C. 1-3 Halogenated alkyl groups; n is selected from 1, 2, or 3.

2. The compound of general formula (I) according to claim 1 and its pharmaceutically acceptable salt, characterized in that, X1 and X2 are each independently selected from fluorine, chlorine, or trifluoromethyl.

3. The compound of general formula (I) according to claim 1 and its pharmaceutically acceptable salt, characterized in that, The compound is selected from:

4. A method for preparing the compound of general formula (I) as described in claim 1 and its pharmaceutically acceptable salt, comprising: The compound of general formula (II) undergoes an intramolecular amidation reaction in a solvent in the presence of a condensing agent to give the compound of general formula (I); X1 and X2 are each independently selected from halogen or C. 1-3 Halogenated alkyl groups; n is selected from 1, 2, or 3; The condensing agent is selected from carbodiimide condensing agents.

5. The preparation method according to claim 4, characterized in that, X1 and X2 are each independently selected from fluorine, chlorine, or trifluoromethyl.

6. The preparation method according to claim 4, characterized in that, The condensing agent is selected from DCC, DIC, or EDCI.

7. The preparation method according to claim 4, characterized in that, The condensing agent is selected from DCC or EDCI.

8. The preparation method according to claim 4, characterized in that, The solvent is selected from DMF or DMA.

9. The preparation method according to claim 4, characterized in that, The molar ratio of the condensing agent to the compound of general formula (II) is 1:1 to 3:

1.

10. The preparation method according to claim 4, characterized in that, The reaction temperature is 40–100°C.

11. The preparation method according to claim 10, characterized in that, The reaction temperature is 60–80°C.

12. A pharmaceutical composition comprising a therapeutically effective amount of the compound according to claim 1 or 3 and its pharmaceutically acceptable salt, and one or more pharmaceutically acceptable carriers, diluents or excipients.

13. Use of the compound according to claim 1 or 3 and its pharmaceutically usable salt in the preparation of a medicament for a GnRH receptor antagonist.

14. Use of the pharmaceutical composition according to claim 12 in the preparation of a medicament for a GnRH receptor antagonist.

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