Aromatic hydrocarbon receptor agonist compound and use thereof

Abstract

Disclosed is an aromatic hydrocarbon receptor agonist compound represented by general formula (I), where Y is N or CR4; Z is O or NR8; R2 and R3 together with the atoms to which they are linked form an unsubstituted or 1-2 Q'-substituted 3-8 membered ring, where Q' is Q, or the C on Q' together with R1 and the atoms to which they are linked form an unsubstituted or 1-2 Q-substituted 3-8 membered ring; and / or, R5 and R6 together with the atoms to which they are linked form an unsubstituted or 1-2 Q-substituted 3-8 membered ring. R8 and R1 together with the atoms to which they are linked form an unsubstituted or 1-2 Q-substituted 3-8 membered ring. The aromatic hydrocarbon receptor agonist compound represented by general formula (I) of the present invention has excellent AhR agonist activity.

Description

Aromatic hydrocarbon receptor agonist compounds and their applications Technical Field

[0001] This invention belongs to the field of compound synthesis technology, specifically relating to an aromatic hydrocarbon receptor agonist compound and its applications. Background Technology

[0002] Aromatic hydrocarbon receptors (AhRs) are transcription factors that regulate multiple aspects of immune function, most notably by suppressing adaptive immune responses (Ehrlich et al., Curr. Opin. Toxicol., 2, 72-78 (2017)). AhRs also regulate the expression of CYP1A1, CYP1A2, and CYP1B1, which catalyze the metabolism of polycyclic aromatic hydrocarbons (PAHs) and other aromatic compounds, such as estrogens. While this metabolism can lead to the formation of reactive substances in some cases (e.g., with benzo[a]pyrene), CYP induction is also considered crucial for the detoxification and metabolic clearance of PAHs, reducing the likelihood of bioactivation and DNA adduct formation. Following FDA approval, some marketed drugs were found to activate AhR (thereby upregulating CYP1A1, CYP1A2, and CYP1B1), but their long-term use was not associated with dioxin-like toxicity (Ehrlich et al., Curr. Opin. Toxicol., 2, 72-78 (2017)). Therefore, CYP induction is no longer considered an obstacle to the use of AhR agonists in treatment (Ehrlich et al., Curr. Opin. Toxicol., 2, 72-78 (2017)).

[0003] AhR signaling plays a crucial role in maintaining skin homeostasis by regulating the skin's immune network, keratinocyte differentiation, skin barrier function, pigmentation, and response to oxidative stress. Cells found in the skin, including keratinocytes, sebaceous gland cells, fibroblasts, melanocytes, endothelial cells, Langerhans cells, and other immune cells, possess AhR. Therefore, the regulation of the AhR signaling pathway is related to the pathology of various skin diseases and conditions. On the other hand, AhR regulates osteoclast differentiation and function, playing a key role in osteoporosis. AhR also plays an important role in regulating hormone levels and metabolism through interactions with the endocrine system.

[0004] Endogenous AhR agonists such as kynurenine and FICZ have been shown to promote the transformation of macrophages, dendritic cells, and NK cells into immunosuppressive phenotypes, affect the differentiation of Th17 cells and immunosuppressive Treg and Tr1 cells, and inhibit the release of a series of inflammatory factors such as IL-2, IL-6, IL-22, TNF-α, and IFN-γ. Therefore, by regulating AhR activity, the progression of neurodegenerative diseases can be intervened.

[0005] Subcellular localization of nuclear factor E2-related factor 2 (Nrf2) is an important factor in cellular defense against oxidative stress and electrophilic agent attack. It participates in the composition and control of oxidative stress defense pathways and is associated with oxidative stress-related diseases (including cancer, neurodegenerative diseases, cardiovascular diseases, and aging). It has been shown to be a potential target for the treatment of many diseases.

[0006] The NMPA has approved icariin, an AhR agonist, for the treatment of liver cancer. The FDA and NMPA have approved Benvimod, a topical cream containing 1% tapinarof (an AhR agonist). It is used for the treatment of adult plaque psoriasis. Nevertheless, new oral, selective, and potent AhR agonists are still needed to treat immune-modulating diseases. Summary of the Invention

[0007] The purpose of this invention is to provide an aromatic hydrocarbon receptor agonist compound having the structure shown in general formula (I):

[0008] Where Y is N or CR4;

[0009] R1 and R7 are selected from hydrogen, unsubstituted or independently selected from those substituted by D, halogens, OR, SR, N(R)2, -NO2, -CN, -C(O)OR, -C(O)N(R)2, RC(O)O-, RC(O)N(R)-, and -S(O). 1～2 R, -S(O) 1～2 N(R)2 and RS(O) 1～2 N(R)-substituted C1-C8 alkyl, C3-C8 cycloalkyl;

[0010] Z is O or NR8; R8 and R1 together with the linked atoms form an unsubstituted or substituted 1-2 Q-shaped 4- to 8-membered heterocycle containing 2-3 N atoms and 0-2 O and S atoms;

[0011] R2, R3, R4, R5, and R6 are independently selected from H or Q; or, R2 and R1 together with the linked atoms form an unsubstituted or substituted 3- to 8-membered heterocycle containing 1- to 2 N atoms and 0- to 2 O and S atoms.

[0012] Alternatively; R2 and R3 together with the linked atoms form unsubstituted or substituted 1-2 Q' rings of 3-8 members, where Q' is Q, or Q' together with R1 and the linked atoms form unsubstituted or substituted 1-2 Q' rings of 3-8 members containing 1-2 N atoms and 0-2 O and S atoms; and / or, R5 and R6 together with the linked atoms form unsubstituted or substituted 1-2 Q' rings of 3-8 members.

[0013] Each Q is independently selected from T' or from T substituted with 1 to 3 T', where T' is D, halogen, -NO2, -CN, oxo, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C3-C8 cycloalkyl, OR, SR, N(R)2, -COOR, RCOO-, -CON(R)2, ROCON(R)-, -S(O). 1～2 R, -S(O) 1～2 N(R)2, RC(=O)N(R)-, RS(O) 1～2 N(R)-, RN(R)COO-, or heteroalkyl rings with 1 to 3 heteroatoms selected from N, O, or S (3 to 8-membered heteroalkyl rings); T is selected from C1 to C8 alkyl, C2 to C8 alkenyl, C2 to C8 ynyl, C3 to C8 cycloalkyl, OR, SR, N(R)2, -COOR, RCOO-, -CON(R)2, ROCON(R)-, -S(O). 1～2 R, -S(O) 1～2 N(R)2, RC(=O)N(R)-, RS(O) 1～2 N(R)-, RN(R)COO- or heteroalkyl rings consisting of 1 to 3 3 to 8-membered heteroalkyl rings selected from N, O or S heteroatoms;

[0014] R is independently selected from H, D, -CH2CH2N(R')2, C1-C8 alkyl and C3-C8 cycloalkyl, wherein R' is C1-C8 alkyl, C2-C8 alkenyl or C2-C8 ynyl.

[0015] In a preferred embodiment,

[0016] The 3-8 membered ring is a 3-8 membered alkane ring, a 3-8 membered alkene ring, a 6-8 membered aromatic ring, a 3-8 membered heteroalkane ring with 1-3 preferred heteroatoms selected from N, O or S heteroatoms, a 3-8 membered heteroalkene ring with 1-3 preferred heteroatoms selected from N, O or S heteroatoms, or a 3-8 membered heteroaromatic ring with 1-3 preferred heteroatoms selected from N, O or S heteroatoms.

[0017] In a further preferred embodiment,

[0018] The 3-8 membered ring is preferably a 3-7 membered ring, more preferably a 3-7 membered alkane ring, a 3-7 membered alkene ring, a 6-8 membered aromatic ring, a heteroalkyl ring with 1-3 preferred heteroalkyl atoms with 1-2 heteroalkyl atoms selected from N, O or S heteroatoms, a heteroalkyl ring with 1-3 preferred heteroalkyl atoms with 1-2 heteroalkyl atoms selected from N, O or S heteroatoms, or a heteroaromatic ring with 1-3 preferred heteroalkyl atoms with 1-2 heteroalkyl atoms selected from N, O or S heteroatoms.

[0019] In a further preferred embodiment,

[0020] The 3-8 membered ring is preferably a 5-6 membered ring, more preferably a 5-6 membered alkane ring, a 5-6 membered alkene ring, a 6 membered aromatic ring, a heteroalkyl ring with 1-3 (preferably 1-2) heteroalkyl atoms selected from N, O or S heteroatoms, a heteroalkene ring with 1-3 (preferably 1-2) heteroalkyl atoms selected from N, O or S heteroatoms, or a heteroaromatic ring with 1-3 (preferably 1-2) heteroalkyl atoms selected from N, O or S heteroatoms.

[0021] In a preferred embodiment,

[0022] R2 and R3 together with the linked atoms form unsubstituted or substituted 5-6 membered rings, while R5 and R6 together with the linked atoms form unsubstituted or substituted 5-6 membered aromatic rings.

[0023] In the first preferred embodiment, R2 and R3 together with the linked atoms form an unsubstituted or substituted 6-membered aromatic ring with 1 to 2 Q's, and R5 and R6 together with the linked atoms form an unsubstituted or substituted 6-membered aromatic ring with 1 to 2 Q's; then general formula (Ⅰ) becomes general formula (Ⅰ1) as follows.

[0024] Among them, A1, A2, A3, and A4 are independently selected from CH, CQ, or N;

[0025] A5, A6, A7, and A8 are independently selected from CH, CQ', or N;

[0026] Preferably, when Y is CR4, general formula (Ⅰ1) becomes the following general formula (Ⅰ1a).

[0027] Preferably, when Y is N, the general formula (Ⅰ1) becomes the following general formula (Ⅰ1b).

[0028] The second preferred case is that Y is CR4, R2 and R3 together with the linked atoms form an unsubstituted or substituted 5-membered aromatic ring, and R5 and R6 together with the linked atoms form an unsubstituted or substituted 6-membered aromatic ring; then general formula (Ⅰ) becomes general formula (Ⅰ2).

[0029] Among them, A1, A2, A3, and A4 are independently selected from CH, CQ, or N;

[0030] A5, A6, and A7 are selected from O and S, while the other two are independently selected from CH, CQ', or N.

[0031] The third preferred scenario is,

[0032] If Y is CR4, R2 and R3 together with the linked atoms form an unsubstituted or substituted 5-membered ring with 1 to 2 Q's, and R5 and R6 together with the linked atoms form an unsubstituted or substituted 6-membered aromatic ring with 1 to 2 Q's, then general formula (Ⅰ) becomes general formula (Ⅰ3).

[0033] Among them, A1, A2, A3, and A4 are independently selected from CH, CQ, or N;

[0034] A5, A6, and A7 are independently selected from CH2, CHQ', CQ'2, NH, NQ', O, or S.

[0035] The fourth preferred case is that Y is CR4, R2 and R3 together with the linked atoms form an unsubstituted or substituted 6-membered ring with 1 to 2 Q's, and R5 and R6 together with the linked atoms form an unsubstituted or substituted 6-membered aromatic ring with 1 to 2 Q's. Then general formula (I) becomes general formula (I4).

[0036] Among them, A1, A2, A3, and A4 are independently selected from CH, CQ, or N;

[0037] A5, A6, A7, and A8 are independently selected from CH2, CHQ', CQ'2, NH, NQ', O, or S.

[0038] The fifth preferred case is that Y is CR4, R2 and R3 together with the linked atoms form an unsubstituted or substituted 6-membered aromatic ring, and R5 and R6 together with the linked atoms form an unsubstituted or substituted 5-membered aromatic ring, then general formula (Ⅰ) becomes general formula (Ⅰ5).

[0039] Among them, one of A1, A2, and A3 is selected from O and S, and the other two are independently selected from CH, CQ, or N;

[0040] A5, A6, A7, and A8 are independently selected from CH, CQ', or N.

[0041] In the example above, Z is preferably 0.

[0042] In a preferred example,

[0043] Z is NR8;

[0044] R8 and R1 together with the linked atoms form an unsubstituted or substituted 1 to 2 Q-substituted 4 to 8-membered heterocycle containing 2 to 4 N atoms and 0 to 2 O and S atoms, preferably a 4 to 7-membered heterocycle containing 2 to 3 N atoms and 0 to 1 O and S atoms, more preferably a 5 to 6-membered heteroaromatic ring containing 2 to 3 N atoms.

[0045] Furthermore, R8 and R1 together form unsubstituted or substituted with 1 to 2 Q-, -NCH-, -NHCO-, -CH=CH-, or -CH2CH2-.

[0046] In another preferred example,

[0047] Z is O;

[0048] Q' and R1 together with the linked atoms form an unsubstituted or substituted 3- to 8-membered heterocycle containing 1 to 2 N atoms and 0 to 2 O and S atoms, preferably a 4- to 7-membered heterocycle containing 1 to 2 N atoms and 0 to 1 O and S atoms, more preferably a 5- to 6-membered heterocycle containing 1 to 2 N atoms and 0 to 1 O and S atoms, and most preferably a 5- to 6-membered heterocycle containing 1 N atom.

[0049] Furthermore, Q' and R1, together with the already linked atoms, form the following general formula (Ⅰ1c).

[0050] In another example, R2 and R1 together with the linked atoms form an unsubstituted or substituted 3- to 8-membered heterocycle containing 1 to 2 N atoms and 0 to 2 O and S atoms, preferably a 4- to 7-membered heterocycle containing 1 to 2 N atoms and 0 to 1 O and S atoms, more preferably a 5- to 6-membered heterocycle containing 1 to 2 N atoms and 0 to 1 O and S atoms, and most preferably a 5- to 6-membered heterocycle containing 1 N atom.

[0051] In a preferred embodiment of the present invention,

[0052] R1 and R7 are selected from hydrogen, unsubstituted or independently selected from those substituted by D, halogens, OR, SR, N(R)2, -NO2, -CN, -C(O)OR, -C(O)N(R)2, RC(O)O-, RC(O)N(R)-, and -S(O).1～2 R, -S(O) 1～2 N(R)2 and RS(O) 1～2 N(R)-substituted C1-C6 alkyl, C3-C7 cycloalkyl;

[0053] Preferably, R1 and R7 are selected from hydrogen, unsubstituted or independently selected from D, halogens, OR, SR, N(R)2, -NO2, -CN, -C(O)OR, -C(O)N(R)2, RC(O)O-, RC(O)N(R)-, and -S(O). 1～2 R, -S(O) 1～2 N(R)2 and RS(O) 1～2 N(R)-substituted C1-C4 alkyl, C5-C6 cycloalkyl;

[0054] More preferably, R1 is selected from hydrogen, unsubstituted or independently selected from D, halogens, OR, SR, N(R)2, -NO2, -CN, -C(O)OR, -C(O)N(R)2, RC(O)O-, RC(O)N(R)-, -S(O). 1～2 R, -S(O) 1～2 N(R)2 and RS(O) 1～2 N(R)-substituted C1-C3 alkyl, C5-C6 cycloalkyl; R7 is selected from hydrogen, methyl or hydroxymethyl.

[0055] In a preferred embodiment of the present invention,

[0056] Each Q is independently selected from T' or from T substituted with 1 to 2 T', wherein T' is D, halogen, -NO2, -CN, oxo, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 cycloalkyl, OR, SR, N(R)2, -COOR, RCOO-, -CON(R)2, ROCON(R)-, -S(O). 1～2 R, -S(O) 1～2 N(R)2, RC(=O)N(R)-, RS(O) 1～2 N(R)-, RN(R)COO-, or a heteroalkyl ring consisting of 1 to 2 3- to 7-membered heteroatoms selected from N, O, or S; T is selected from C1 to C6 alkyl, C2 to C6 alkenyl, C2 to C6 ynyl, C3 to C7 cycloalkyl, OR, SR, N(R)2, -COOR, RCOO-, -CON(R)2, ROCON(R)-, -S(O). 1～2 R, -S(O) 1～2 N(R)2, RC(=O)N(R)-, RS(O) 1～2 N(R)-, RN(R)COO- or heteroalkyl rings consisting of 1 to 2 3 to 7-membered heteroalkyl rings selected from N, O or S heteroatoms;

[0057] Preferably, each Q is independently selected from T' or from T substituted with 1 to 2 T', wherein T' is D, halogen, -NO2, -CN, oxo, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C5-C6 cycloalkyl, OR, SR, N(R)2, -COOR, RCOO-, -CON(R)2, ROCON(R)-, -S(O). 1～2 R, -S(O) 1～2 N(R)2, RC(=O)N(R)-, RS(O) 1～2 N(R)-, RN(R)COO-, or a heteroalkyl ring consisting of 1 to 2 5- or 6-membered heteroatoms selected from N, O, or S; T is selected from C1-C4 alkyl, C2-C4 alkenyl, C2-C4 ynyl, C5-C6 cycloalkyl, OR, SR, N(R)2, -COOR, RCOO-, -CON(R)2, ROCON(R)-, -S(O). 1～2 R, -S(O) 1～2 N(R)2, RC(=O)N(R)-, RS(O) 1～2 N(R)-, RN(R)COO- or heteroalkyl rings consisting of 1 to 2 5- or 6-membered heteroalkyl rings selected from N, O or S heteroatoms;

[0058] More preferably, each Q is independently selected from T' or from T substituted with one T', wherein T' is D, halogen, -NO2, -CN, oxo, C1-C3 alkyl, C2-C3 alkenyl, C2-C3 alkynyl, C5-C6 cycloalkyl, OR, SR, N(R)2, -COOR, RCOO-, -CON(R)2, ROCON(R)-, -S(O). 1～2 R, -S(O) 1～2 N(R)2, RC(=O)N(R)-, RS(O) 1～2 N(R)-, RN(R)COO-, or a heteroalkyl ring consisting of 1 to 2 5- or 6-membered heteroatoms selected from N, O, or S; T is selected from C1- or C3 alkyl, C2- or C3 alkenyl, C2- or C3 alkynyl, C5- or C6 cycloalkyl, OR, SR, N(R)2, -COOR, RCOO-, -CON(R)2, ROCON(R)-, -S(O). 1～2 R, -S(O) 1～2 N(R)2, RC(=O)N(R)-, RS(O) 1～2 N(R)-, RN(R)COO-, or preferably a 5- or 6-membered heteroalkyl ring selected from N, O, or S heteroatoms.

[0059] In a preferred embodiment of the present invention,

[0060] R is independently selected from H, D, -CH2CH2N(R')2, C1-C6 alkyl and C3-C7 cycloalkyl, wherein R' is C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl;

[0061] Preferably,

[0062] R is independently selected from H, D, -CH2CH2N(R')2, C1-C4 alkyl and C5-C6 cycloalkyl, wherein R' is C1-C4 alkyl, C2-C4 alkenyl or C2-C4 alkynyl;

[0063] More preferably,

[0064] R is independently selected from H, D, -CH2CH2N(R')2, C1-C3 alkyl and C5-C6 cycloalkyl, wherein R' is C1-C3 alkyl, C2-C3 alkenyl or C2-C3 alkynyl.

[0065] The aromatic hydrocarbon receptor agonist compound of the present invention has the following compound structure:

[0066] Another object of the present invention is to provide pharmaceutically acceptable salts, deuterated derivatives, solvates, hydrates, and prodrugs of aromatic hydrocarbon receptor agonist compounds of general formula (I).

[0067] Another objective of this invention is to provide the application of aryl hydrocarbon receptor agonist compounds of general formula (I) in the preparation of drugs for treating diseases related to aryl hydrocarbon receptor imbalance and disorder, including but not limited to drugs for treating central nervous system diseases, endocrine system-related cancers, obesity, or drugs for immune regulation, hematopoiesis, cell cycle or intestinal barrier regulation, regulation of osteoclast differentiation and function, and antioxidant effects; preferably, the central nervous system diseases are selected from Alzheimer's disease and Parkinson's disease; the immune regulation is selected from the immune regulation of psoriasis, atopic dermatitis, lupus erythematosus, multiple sclerosis, vitiligo, and acne; the intestinal barrier is selected from inflammatory bowel diseases such as ulcerative colitis and Crohn's disease; the endocrine system-related cancers are selected from prostate cancer, liver cancer, breast cancer, cervical cancer, and lung cancer; and the regulation of osteoclast differentiation and function refers to the regulation of osteoclast differentiation and function in osteoporosis.

[0068] Another object of this invention is to provide the application of aryl hydrocarbon receptor agonist compounds of general formula (I) in drugs for treating diseases related to aryl hydrocarbon receptor imbalance and disorder, including but not limited to drugs for treating central nervous system diseases, endocrine system-related cancers, obesity, or drugs for immune regulation, hematopoiesis, cell cycle or intestinal barrier regulation, regulation of osteoclast differentiation and function, and antioxidant effects; preferably, the central nervous system diseases are selected from Alzheimer's disease and Parkinson's disease; the immune regulation is selected from the immune regulation of psoriasis, atopic dermatitis, lupus erythematosus, multiple sclerosis, vitiligo, and acne; the intestinal barrier is selected from inflammatory bowel diseases such as ulcerative colitis and Crohn's disease; the endocrine system-related cancers are selected from prostate cancer, liver cancer, breast cancer, cervical cancer, and lung cancer; and the regulation of osteoclast differentiation and function refers to the regulation of osteoclast differentiation and function in osteoporosis.

[0069] The positive and progressive effects of the present invention are as follows: the aromatic hydrocarbon receptor agonist compound of general formula (I) of the present invention has excellent AhR agonist activity. Detailed Implementation

[0070] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0071] Example 1

[0072] first step

[0073] 1-Methyl-2-oxoquinoline-3-carboxaldehyde (100 mg, 0.53 mmol) and o-phenylenediamine (69 mg, 0.64 mmol) were dissolved in acetic acid (2 mL) and reacted at 80 °C for 18 hours. The reactants were cooled and concentrated under reduced pressure. The residue was purified by preparative chromatography to give compound 1 (45 mg, 0.16 mmol, yield 30.6%) as a yellow solid. (Synthetic Method A1)

[0074] Compound 1 can also be synthesized using synthetic method A2:

[0075] 1-Methyl-2-oxoquinoline-3-carboxaldehyde (100 mg, 0.53 mmol) and o-phenylenediamine (63 mg, 0.58 mmol) were dissolved in ethanol (10 mL) and water (0.5 mL). Sodium metabisulfite (0.7 mg, 0.37 mmol) was added, and the mixture was heated to 70 °C for 4 hours. The reaction mixture was quenched with water, and saturated potassium carbonate solution (5 mL) was added. The mixture was extracted three times with ethyl acetate. The organic phases were combined, dried, and concentrated. The residue was purified by silica gel column chromatography to give compound 1 (75 mg, 0.27 mmol, yield 50.9%) as a yellow solid. (Synthetic method A2)

[0076] LCMS(ESI):[M+H] + =276.2; 1 H NMR (400MHz, DMSO-d6) δ12.71(s,1H),9.13(s,1H),8.06-8.01(m,1H),7.78-7.63(m,4H),7.42-7.36(m,1H),7.24-7.18(m,2H),3.84(s,3H).

[0077] The series of compounds 1-1 to 1-16 in Table 1 were synthesized using the synthetic method of compound 1 (method A1 or A2).

[0078] Table 1

[0079] Example 2

[0080] first step

[0081] Phosphorus oxychloride (1.96 mL, 21.1 mmol) was added to the reaction flask and cooled to 5-15 °C. N,N-dimethylformamide (0.81 mL, 10.5 mmol) was slowly added dropwise over 30 minutes. After the addition was complete, the mixture was stirred for 15 minutes, and then 1-acetylindoline (1.7 g, 10.5 mmol) was added. The temperature was slowly increased to 75-80 °C over 2 hours, and the reaction was stirred at 80 °C for 12 hours. The reaction mixture was cooled and poured into ice water (400 mL), and extracted three times with ethyl acetate. The organic phases were combined, dried, and concentrated. The residue was purified by silica gel column chromatography to give intermediate 2-A (300 mg, 1.51 mmol, yield 14.3%), a pale yellow solid.

[0082] LCMS(ESI):[M+H] + =200.2; 1H NMR (400MHz, DMSO-d6) δ10.29(s,1H),8.51(s,1H),7.74-7.71(d,J=8.0Hz,1H),7.58-7.55( d,J=6.8Hz,1H),7.25-7.21(m,1H),4.40-4.35(t,J=8.0Hz,2H),3.44-3.40(t,J=8.0Hz,2H).

[0083] Step 2

[0084] 2-A (100 mg, 0.5 mmol) was dissolved in acetic acid (1 mL), and o-phenylenediamine (65 mg, 0.6 mmol) was added. The mixture was reacted at 100 °C for 18 hours. The reactants were alkalized with saturated potassium carbonate solution and extracted three times with ethyl acetate. The organic phases were combined and concentrated. The residue was purified by reverse-phase column chromatography and further purified by slurrying with N,N-dimethylformamide to give compound 2 (11 mg, 0.038 mmol, yield 7.63%) as a pale yellow solid.

[0085] LCMS(ESI):[M+H] + =288.2; 1 H NMR (400MHz, DMSO-d6) δ12.70(s,1H),9.13(s,1H),7.77-7.73(m,1H),7.73-7.69(m,1H),7.67-7.62(m,1H),7. 54-7.50(m,1H),7.30-7.24(m,1H),7.23-7.18(m,1H),4.55-4.47(t,J=8.0Hz,2H),3.51-3.44(t,J=8.0Hz,2H).

[0086] The series of compounds 2-1 to 2-9 in Table 2 were synthesized using the synthetic method of compound 2 (method B).

[0087] Table 2

[0088] Example 3

[0089] first step

[0090] 500 mg of benzimidazole-2-ethyl acetate (2.448 mmol) was dissolved in 5 mL of anhydrous tetrahydrofuran. 1 M lithium bis(trimethylsilyl)amino (3.67 mL, 3.67 mmol) was added dropwise at 0 °C. After the addition was complete, the mixture was stirred at 0 °C for 1 hour. Then, a solution of 433 mg of 1-methyl-2,4-dihydro-1-hydrobenzo[d][1,3]oxazine-2,4-dione (2.45 mmol) dissolved in 5 mL of anhydrous tetrahydrofuran was added dropwise, and the reaction was stirred for 6 hours. The reaction mixture was quenched with saturated ammonium chloride and extracted with ethyl acetate. The organic phase was dried and concentrated. The residue was purified by preparative chromatography to give 3-A (50 mg, 0.17 mmol, yield 7.01%), an off-white solid.

[0091] LCMS(ESI):[M+H] + =292.2; 1 H NMR(400MHz,DMSO-d6)δ13.67(s,2H),8.24-8.18(m,1H),7.82-7.74(m,2H),7.67- 7.61(m,1H),7.48-7.42(m,1H),7.36-7.30(m,2H),7.27-7.20(m,1H),3.62(s,3H).

[0092] Step 2

[0093] 3-A (30 mg, 0.1 mmol) was mixed with phosphorus oxychloride (1 mL) and heated to 100 °C for 24 hours. The reaction mixture was quenched with 0.5 M sodium hydroxide and extracted with ethyl acetate. The organic phase was dried and concentrated. The residue was purified by silica gel column chromatography to give 3-B (5 mg, 0.016 mmol, yield 15.7%), an off-white solid.

[0094] LCMS(ESI):[M+H] + =310.2; 312.2; 1 H NMR(400MHz,DMSO-d6)δ12.70(s,1H),8.17-8.13(m,1H),7.89-7.82(m,1H),7.77- 7.73(m,1H),7.70-7.59(m,2H),7.52-7.45(m,1H),7.27-7.22(m,2H),3.75(s,3H).

[0095] Step 3

[0096] 3-B (50 mg, 0.16 mmol) was dissolved in 7 M ammonia / ethanol solution (7 mL, 49 mmol), and the mixture was reacted in a sealed tube at 100 °C for 12 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by preparative chromatography to give compound 3 (15 mg, 0.052 mmol, yield 32.3%), an off-white solid.

[0097] Compound 3 can also be synthesized using the following method:

[0098] 2-Methylaminobenzonitrile (0.5 g, 3.8 mmol) was dissolved in toluene (25 mL), and benzimidazole-2-ethyl acetate (775 mg, 3.8 mmol) and tin tetrachloride (1.98 g, 7.6 mmol) were added. The mixture was heated to 110 °C and reacted for 18 hours. A saturated potassium carbonate solution (20 mL) of water (25 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic phases were combined, dried, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give compound 3 (0.3 g, 1.03 mmol, yield 27.1%), an off-white solid.

[0099] LCMS(ESI):[M+H] + =291.4; 1 H NMR(400MHz,DMSO-d6)δ12.99(br,1H),11.17(br,1H),8.41-8.15(m,2H),7.77-7 .61(m,3H),7.61-7.54(m,1H),7.40-7.32(m,1H),7.23-7.13(m,2H),3.71(s,3H).

[0100] Example 4

[0101] first step

[0102] 2-O-1-H-quinoline-3-carboxaldehyde (100 mg, 0.58 mmol) was dissolved in N,N-dimethylformamide (4 mL), and potassium carbonate (120 mg, 0.87 mmol) and iodoethane (100 mg, 0.64 mmol) were added. The mixture was stirred at room temperature for 18 hours. The reaction mixture was extracted with ethyl acetate after adding saturated ammonium chloride solution. The organic phases were combined, dried, and concentrated. The residue was purified by silica gel column chromatography to give 4-A (90 mg, 0.48 mmol, 77.5% yield), an off-white solid.

[0103] LCMS(ESI):[M+H] + =202.2; 1H NMR(400MHz,DMSO-d6)δ10.29(s,1H),8.51(s,1H),8.03-8.00(m,1H),7.82-7.76( m,2H),7.69-7.66(m,1H),7.37-7.33(m,1H),4.37-4.32(m,2H),1.27-1.24(m,3H).

[0104] Step 2

[0105] 4-A (90 mg, 0.48 mmol) was dissolved in acetic acid (2 mL), and o-phenylenediamine (97 mg, 0.9 mmol) was added. The mixture was heated to 80 °C and reacted for 18 hours. A saturated potassium carbonate solution (15 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic phases were combined, dried, and concentrated. The residue was purified by reverse-phase column chromatography to give compound 4 (13 mg, 0.045 mmol, yield 10.1%) as a yellow solid.

[0106] LCMS(ESI):[M+H] + =294.2; 1 H NMR(400MHz,DMSO-d6)δ12.73(s,1H),9.12(s,1H),8.09-8.00(m,1H),7.79-7.61(m,4H ),7.43-7.33(m,1H),7.26-7.15(m,2H),4.55-4.43(m,2H),1.40-1.27(t,J=6.8Hz,3H).

[0107] Compounds 4-1 to 4-3 in Table 3 were synthesized using the synthetic method of compound 4 (method D).

[0108] Table 3

[0109] Example 5

[0110] first step

[0111] Ethyl 3-oxo-4-hydro-quinoxaloline-2-carboxylate (1 g, 4.58 mmol) was dissolved in N,N-dimethylformamide (20 mL), potassium carbonate (0.63 g, 4.58 mmol) was added, and iodomethane (1 mL, 16 mmol) was added dropwise. The mixture was stirred at room temperature for 18 hours. The reaction mixture was filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give 5-A (900 mg, 3.88 mmol, yield 84.9%), a pale yellow solid.

[0112] LCMS(ESI):[M+H] + =233.2; 1H NMR (400MHz, CDCl3) δ7.97-7.94(m,1H),7.68-7.62(m,1H),7.42-7.34(m,2H),4.54-4.48(m,2H),3.73(s,3H),1.46-1.42(m,3H).

[0113] Step 2

[0114] 5-A (200 mg, 0.86 mmol) was dissolved in 1,4-dioxane (10 mL), o-phenylenediamine (112 mg, 1.03 mmol) was added, followed by potassium tert-butoxide (326 mg, 2.58 mmol). The mixture was heated to 100 °C and reacted for 8 hours. After the reaction was complete, a saturated ammonium chloride solution (10 mL) was added to quench the reaction. The mixture was extracted with ethyl acetate, and the organic phases were combined and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give compound 5 (7.2 mg, 0.025 mmol, yield 2.94%), a pale yellow solid.

[0115] LCMS(ESI):[M+H] + =276.4; 1 H NMR (400MHz, DMSO-d6) δ13.01(s,1H),8.04-7.97(m,1H),7.79-7.72(m,3H),7.72-7.67(m,1H),7.54-7.48(m,1H),7.33-7.24(m,2H),3.80(s,3H).

[0116] Example 6

[0117] first step

[0118] 3-Methylcyclopentanone (5 g, 51 mmol) was dissolved in N,N-dimethylformamide (100 mL), and N,N-dimethylformamide dimethyl acetal (8.2 mL, 61.7 mmol) was added. The mixture was heated to 100 °C for 24 hours under nitrogen protection. The reaction system was concentrated under reduced pressure to obtain a mixture of 6-1A and 6-2A (8 g, 52.2 mmol, yield 102.4%), a pink oily substance, which was used directly in the next step of the reaction.

[0119] LCMS(ESI):[M+H] + =154.2;

[0120] Step 2

[0121] A mixture of 6-1A and 6-2A (8 g, 52.2 mmol) was dissolved in methanol (60 mL), and methyl cyanoacetate (10 mL, 113.3 mmol) and piperidine (5 mL, 54.6 mmol) were added. The mixture was heated to 80 °C for 18 hours under nitrogen protection. Saturated brine (100 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The combined organic phases were dried, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give a mixture of 6-1B and 6-2B (5.5 g, 26.5 mmol, 50.8%) as a pink solid.

[0122] LCMS(ESI):[M+H] + =208.2;

[0123] Step 3

[0124] A mixture of 6-1B and 6-2B (2 g, 9.65 mmol) was dissolved in acetone (120 mL), and potassium carbonate (2.67 g, 19.3 mmol) and methyl iodide (2.4 mL, 38.6 mmol) were added. The mixture was stirred at room temperature for 12 hours. Saturated brine (100 mL) was added to the reaction mixture, and the solution was extracted with ethyl acetate. The organic phase was dried, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give a mixture of 6-1C and 6-2C (1 g, 4.52 mmol, yield 46.7%) as a pink solid.

[0125] LCMS(ESI):[M+H] + =222.2;

[0126] Step 4

[0127] A mixture of 6-1C and 6-2C (0.4 g, 1.8 mmol) was dissolved in dichloromethane. 1 N of bis(2-isobutyl)aluminum hydride (2.04 mL, 2.04 mmol) was added at -78 °C, and the mixture was stirred for 30 minutes. Sodium sulfate decahydrate (200 mg) was added to the reaction mixture, and stirring was continued for another 30 minutes. The reaction mixture was filtered and washed with ethyl acetate. The organic phase was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give a mixture of 6-1D and 6-2D (115 mg, 0.6 mmol, yield 35.4%) as a pink oil.

[0128] LCMS(ESI):[M+H] + =192.2;

[0129] Step 5

[0130] A mixture of 6-1D and 6-2D (115 mg, 0.6 mmol) was dissolved in ethanol (20 mL), and o-phenylenediamine (80 mg, 0.74 mmol), sodium metabisulfite (72 mg, 0.38 mmol), and water (1.2 mL) were added. The reaction mixture was heated to 70 °C and stirred for 4 hours. The reactants were concentrated under reduced pressure, and the residue was extracted with ethyl acetate after adding water (5 mL) and saturated potassium carbonate solution (5 mL). The organic phase was dried, concentrated under reduced pressure, and the residue was purified by preparative chromatography and SFC to give compound 6-1 (32 mg, 0.11 mmol, yield 18.3%) and compound 6-2 (10.3 mg, 0.037 mmol, yield 6.2%) as pale yellow solids.

[0131] Compound 6-1: LCMS (ESI): [M+H] + =280.2; 1 H NMR(400MHz,DMSO-d6)δ8.53(s,1H),7.88-7.81(m,2H),7.52-7.47(m,2H),3.63(s,3H),3.1 6-3.07(m,2H),2.81-2.68(m,1H),2.46-2.39(m,1H),1.77-1.65(m,1H),1.36-1.24(m,3H).

[0132] Compound 6-2: LCMS (ESI): [M+H] + =280.2; 1 H NMR(400MHz,DMSO-d6)δ8.49(s,1H),7.84-7.78(m,2H),7.48-7.41(m,2H),3.63(s,3H),3.1 1-3.01(m,2H),2.76-2.64(m,1H),2.43-2.36(m,1H),1.68-1.61(m,1H),1.21-1.13(m,3H).

[0133] The series of compounds 6-1 to 6-4 in Table 4 were synthesized using the fourth step of the method for compound 6.

[0134] Table 4

[0135] Example 7

[0136] first step

[0137] 2-Chloroquinoline-3-carboxaldehyde (0.5 g, 2.61 mmol) and o-phenylenediamine (0.21 g, 1.94 mmol) were dissolved in chloroform (10 mL), and acetic acid (0.5 mL, 8.88 mmol) was added. The mixture was stirred at 30 °C for 18 hours. The reaction system was alkalized with saturated potassium carbonate solution and extracted three times with ethyl acetate. The organic phases were combined and concentrated, and the residue was purified by silica gel column chromatography to give 7-A (160 mg, 0.57 mmol, yield 29.5%), an off-white solid.

[0138] LCMS(ESI):[M+H] + =280.2,282.2; 1 H NMR(400MHz,DMSO-d6)δ12.94(s,1H),9.00(s,1H),8.22-8.18(m,1H),8.09-8.05( m,1H),7.96-7.91(m,1H),7.79-7.72(m,2H),7.66-7.60(m,1H),7.33-7.24(m,2H).

[0139] Step 2

[0140] 7-A (150 mg, 0.54 mmol) was dissolved in pyridine (2 mL), and formyl hydrazine (50 mg, 0.83 mmol) was added under nitrogen protection. The mixture was heated to 120 °C and reacted for 18 hours. A saturated sodium chloride solution (10 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic phases were combined and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give compound 7 (58 mg, 0.2 mmol, yield 37.9%), an off-white solid.

[0141] LCMS(ESI):[M+H] + =286.4; 1 H NMR (400MHz, CD3OD) δ9.98(s,1H),8.75(s,1H),8.44-8.39(m,1H),8.19-8.14(m,1H),7.91-7.85(m,1H),7.79-7.69(m,3H),7.38-7.33(m,2H).

[0142] The series of compounds 7-1 to 7-3 in Table 4 were synthesized using the method of compound 7.

[0143] Table 5

[0144] Example 8

[0145] first step

[0146] Phosphorus oxychloride (1.96 mL, 21.1 mmol) was added to the reaction flask and cooled to 5-15 °C. N,N-dimethylformamide (0.81 mL, 10.5 mmol) was slowly added dropwise over 30 minutes. After the addition was complete, the mixture was stirred for 15 minutes, followed by the addition of N-(4-bromophenyl)-N-methylacetamide (2.38 g, 10.5 mmol). The temperature was slowly increased to 120 °C over 2 hours, and the reaction mixture was stirred at 120 °C for 12 hours. The reaction mixture was cooled and poured into ice water (400 mL), and extracted three times with ethyl acetate. The organic phases were combined, dried, and concentrated. The residue was purified by silica gel column chromatography to give 8-A (580 mg, 2.17 mmol, yield 20.7%), a pale yellow solid.

[0147] LCMS(ESI):[M+H] + =266.2,268.2; 1 H NMR (400MHz, DMSO-d6) δ10.27(s,1H),8.49(s,1H),8.29-8.27(m,1H),7.94-7.90(m,H),7.60-7.57(m,1H),3.66(s,3H).

[0148] Step 2

[0149] 8-A (80 mg, 0.3 mmol) was dissolved in ethanol (10 mL), and o-phenylenediamine (40 mg, 0.37 mmol), sodium metabisulfite (40 mg, 0.21 mmol), and water (0.5 mL) were added under nitrogen protection. The mixture was heated to 70 °C and reacted for 3 hours. The reaction mixture was quenched with water, and saturated potassium carbonate solution (5 mL) was added. The mixture was extracted three times with ethyl acetate. The organic phases were combined, dried, and concentrated. The residue was purified by silica gel column chromatography to give compound 8-B (35 mg, 0.099 mmol, yield 32.9%) as a yellow solid.

[0150] LCMS(ESI):[M+H] + =354.4,356.4; 1 H NMR (400MHz, DMSO-d6) δ12.73 (s, 1H), 9.11 (s, 1H), 8.33-8.29 (d, J = 2.4Hz, 1H), 7.89-7.84 (m, 1H),7.73-7.69(m,1H),7.69-7.65(m,1H),7.65-7.61(m,1H),7.25-7.19(m,2H),3.81(s,3H).

[0151] Step 3

[0152] 8-B (30 mg, 0.085 mmol) was dissolved in N,N-dimethylformamide (3 mL), and 2-dimethylaminoethylamine (0.1 mL, 0.91 mmol), methanesulfonic acid (2-dicyclohexylphosphine-3,6-dimethoxy-2',4',6'-triisopropyl-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl)palladium (25 mg, 0.028 mmol), sodium tert-butoxide (40 mg, 0.42 mmol), and tris(dibenzylacetone)dipalladium (25 mg, 0.027 mmol) were added. The mixture was heated to 130 °C for 18 hours under nitrogen protection. The reaction mixture was diluted with saturated brine, extracted with ethyl acetate, and the organic phases were combined and concentrated under reduced pressure. The residue was purified by preparative chromatography to give compound 8 (10.8 mg, 0.03 mmol, yield 35.3%) as a yellow solid.

[0153] LCMS(ESI):[M+H] + =362.2; 1 H NMR (400MHz, DMSO-d6) δ12.70(s,1H),8.95(s,1H),7.72-7.66(m,1H),7.66-7.60(m,1H),7.48-7.42(d,J=9.2Hz,1H),7.22- 7.15(m,3H),7.07-7.05(d,J=2.4Hz,1H),5.62-5.55(m,1H),3.77(s,3H),3.21-3.13(m,2H),2.53-2.47(m,2H),2.22(s,6H).

[0154] The series of compounds 8-1 to 8-16B listed in Table 6 were synthesized using the method described for compound 8. Compounds 8-2B and 8-2 were synthesized from compound 8-1B using the following derivatization method:

[0155] 8-1B (50 mg, 0.16 mmol) was dissolved in dichloromethane (1 mL), and 1 N boron tribromide solution (1 mL, 1 mmol) was added at -20 °C. The mixture was stirred at 0 °C for 6 hours. The reaction mixture was quenched with ice water and extracted with ethyl acetate. The organic phases were combined, dried, and concentrated under reduced pressure. The residue was crystallized from dichloromethane to give compound 8-2B (25 mg, 0.086 mmol, yield 53.8%) as a yellow solid.

[0156] Compound 8-2B (70 mg, 0.24 mmol) and N,N-dimethylethanolamine (32 mg, 0.36 mmol) were dissolved in anhydrous tetrahydrofuran (6 mL). Triphenylphosphine (94 mg, 0.36 mmol) was added, followed by the dropwise addition of diisopropyl azodicarbonate (73 mg, 0.36 mmol). The mixture was stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography and preparative chromatography to give compound 8-2 (35 mg, 0.097 mmol, yield 40.4%).

[0157] Table 6

[0158] Example 9

[0159] first step

[0160] 4-Aminopyridine-3-carboxaldehyde (1 g, 8.2 mmol) and benzimidazole-2-ethyl acetate (1.67 g, 8.2 mmol) were dissolved in ethanol (25 mL), and piperidine (0.75 mL, 8.2 mmol) was added. The mixture was heated to 80 °C and reacted for 18 hours. The reaction system was cooled, the solid was collected by filtration, washed with ethanol (10 mL), and dried to give 9-A (1.48 g, 5.64 mmol, yield 68.8%), a yellow solid.

[0161] LCMS(ESI):[M+H] + =263.2; 1 H NMR (400MHz, DMSO-d6) δ12.69(s,1H),9.19(s,1H),9.14(s,1H),8.56-8.54(m,1H),7.74-7.64(m,2H),7.33-7.31(m,1H),7.24-7.19(m,2H).

[0162] Step 2

[0163] 9-A (0.2 g, 0.76 mmol) was dissolved in N,N-dimethylformamide (5 mL), and potassium carbonate (0.21 g, 1.52 mmol) and methyl iodoforme (0.04 mL, 0.64 mmol) were added. The mixture was stirred at room temperature for 4 hours. A saturated sodium chloride solution (20 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic phases were combined, dried, and concentrated under reduced pressure. The residue was purified by preparative chromatography to give compound 9 (0.02 g, 0.072 mmol, yield 9.5%) as a yellow solid.

[0164] LCMS(ESI):[M+H] + =277.2;1 H NMR(400MHz,DMSO-d6)δ12.67(s,1H),9.20(s,1H),9.18(s,1H),8.68-8.66( m,1H),7.70-7.67(m,2H),7.62-7.60(m,1H),7.23-7.20(m,2H),3.78(s,3H).

[0165] Test Example 1: Human Aromatic Hydrocarbon Receptor (AhR) Agonist Luciferase Assay

[0166] Cytochrome P450 family 1 member A1 (CYP1A1) is one of the hallmark genes regulated by the AhR signaling pathway. The upstream promoter of the CYP1A1 gene contains a specific sequence called the dioxin responsive element (DRE), which binds to activated AhR to initiate CYP1A1 expression. To detect AhR activation, the gene sequence from -1200 bp to 0 bp upstream of CYP1A1 was placed before the Nano-luciferase reporter gene to construct a reporter gene plasmid. The reporter gene plasmid was introduced into human hepatocellular carcinoma cells (HepG2) via liposome transfection, and cells stably expressing the gene were selected using puromycin. The selected cells were further screened to identify single clones, and one morphologically normal, growing clone with the highest induction signal was selected for human AhR agonist luciferase assays.

[0167] HepG2 monoclonal cell lines stably expressing reporter genes were counted on day 1 at a rate of 1-8 × 10⁻⁸. 4 / The density of each well was determined by inoculating 100 μL of complete medium (MEM medium without tryptophan and phenol red, 10% fetal bovine serum, 1× non-essential amino acids, 1× sodium pyruvate and 1× GlutaMax) into white opaque 96-well plates and incubating at 37°C and 5% CO2 for 24 hours.

[0168] After synthesis, the test compounds were prepared into a 10 mM stock solution using dimethyl sulfoxide (DMSO) and stored at 4°C in the dark. Before treatment, the stock solutions, positive control compounds (such as dioxins, ITE, kynurenine, etc.), and negative control (DMSO) were initially diluted (determined based on preliminary experiments) using complete medium without tryptophan and phenol red, and then serially diluted 3-20 times to obtain 11 concentration points, yielding 2× dilutions. When treating cells, 50 μL of medium was aspirated from the 96-well plate seeded with cells, and then 50 μL of the serially diluted 2× dilution was added. Two replicates were set up for each compound, and each replicate included one well with a concentration of 0 (i.e., containing only medium) to determine the baseline. Report cells were incubated in an incubator for 4-24 hours after treatment.

[0169] After processing, 100 μL / well of Nano-Glo luciferase assay reagent was added to each well of a 96-well plate, and the RLU (relative fluorescence intensity) of each well was measured. The baseline Ave RLU was calculated by averaging the RLU values ​​at a concentration of 0 for each 96-well plate. Vehicle The RLU of the test compound at different concentrations in the experimental group. Test Cmpd and baseline Ave RLU Vehicle The ratio of the two compounds is used to calculate the activity of AhR under different concentrations of the test compound according to formula (1) to determine the activation factor.

[0170] Formula (1)

[0171] The activation fold and corresponding compound concentration were fitted using Graphpad Prism 9 with an Agonist vs. response-variable slope (four parameters) to calculate the EC50 of compound activation of AhR. 50 EC values ​​of each compound 50 The values ​​are shown in Table 7, A:EC 50 ≤1nM, B:1nM<EC 50 ≤100nM, C:100nM<EC 50 ≤1000nM, D:≥1000uM.

[0172] Test Example 2: Mouse-derived aryl hydrocarbon receptor (AhR) agonist luciferase assay

[0173] To detect AhR activation, six tandem xenobiotic responsive elements (XREs, see Buckley, SMK et al., Sci. Rep. 2015; 5:11842.) were placed before the Nano-luciferase reporter gene to construct a reporter gene plasmid. The reporter gene plasmid was introduced into mouse hepatocellular carcinoma cells Hepa1-6 via liposome transfection, and stably expressing cells were selected using puromycin for mouse AhR agonist luciferase assays.

[0174] The Hepa1-6 cell line, which stably expresses the reporter gene, was counted on day 1 at a rate of 1-8 × 10⁻⁶ cells / year. 4 The well density was determined by inoculating 100 μL of phenol red-free DMEM medium (phenol red-free DMEM, 10% fetal bovine serum) into white opaque 96-well plates and incubating at 37°C with 5% CO2 for 24 hours.

[0175] After synthesis, the test compounds were prepared into a 10 mM stock solution using dimethyl sulfoxide (DMSO) and stored at 4°C in the dark. Before treatment, the stock solutions, positive control compounds (such as dioxins, ITE, kynurenine, etc.), and negative control (DMSO) were initially diluted (determined based on preliminary experiments) using complete culture medium without tryptophan and phenol red, resulting in 11 serial dilutions ranging from 3 to 20 times to obtain a 10× dilution. When treating cells, 10 μL of culture medium was aspirated from the 96-well plate from which cells were seeded, followed by the addition of 10 μL of the serially diluted 10× dilution. Two replicates were performed for each compound, with one well in each replicate containing only 0% concentration (i.e., culture medium only) to determine the baseline. Report cells were incubated in an incubator for 4–24 hours after treatment.

[0176] After processing, 100 μL / well of Nano-Glo luciferase assay reagent was added to each well of a 96-well plate, and the RLU (relative fluorescence intensity) of each well was measured. The baseline Ave RLU was calculated by averaging the RLU values ​​at a concentration of 0 for each 96-well plate. Vehicle The RLU of the test compound at different concentrations in the experimental group. Test Cmpd and baseline Ave RLU Vehicle The ratio of the two compounds is used to calculate the activity of AhR under different concentrations of the test compound according to formula (1) to determine the activation factor.

[0177] Formula (1)

[0178] The activation fold and corresponding compound concentration were fitted using Graphpad Prism 9 with an Agonist vs. response-variable slope (four parameters) to calculate the EC50 of compound activation of AhR. 50 EC values ​​of each compound 50 The values ​​are shown in Table 7, where A: EC 50 ≤1nM, B:1nM<EC 50 ≤100nM, C:100nM<EC 50 ≤1000nM, D:≥1000uM.

[0179] Test Example 3: Human Nrf2 Agonist Luciferase Assay

[0180] Nrf2 (Nuclear factor erythroid 2-related factor 2) is a key transcription factor for oxidative stress resistance in cells. In its resting state, Nrf2 forms a complex with Keap1 (Kelch-like ECH-associated protein 1). Keap1 can persistently ubiquitinate Nrf2 by recruiting the ubiquitin ligase Cullin3 and related ubiquitination complexes, leading to Nrf2 degradation. Under oxidative stress conditions such as those caused by reactive oxygen species (ROS) and electrophilic substances, the cysteine ​​sulfhydryl groups in Keap1 are modified, triggering a conformational change in Keap1 and releasing Nrf2. This inhibits Nrf2 degradation, allowing it to accumulate in the cell and enter the nucleus. In the cell nucleus, Nrf2 forms a dimer with sMaf protein, which binds to the antioxidant response element (ARE), thereby activating the expression of downstream key antioxidant genes such as NAD(P)H quinone oxidoreductase 1 and glutathione S-transferase.

[0181] To detect Nrf2 activation, four tandem antioxidant response elements (see Wu, P. et al., J. Biol. Chem., 2016; 291:22288-22301.) were placed before the Nano-luciferase reporter gene to construct a reporter gene plasmid. The reporter gene plasmid was introduced into human hepatocellular carcinoma cells (HepG2) via liposome transfection, and stably expressing cells were selected using puromycin. The selected cells were further screened to identify single clones, and one morphologically normal, growing cell with the highest induction signal was selected for human Nrf2 agonist luciferase assays.

[0182] HepG2 monoclonal cell lines stably expressing reporter genes were counted on day 1 at a rate of 1-8 × 10⁻⁸. 4 / The wells were inoculated with 100 μL of complete medium (MEM basal medium, 10% fetal bovine serum, 1× non-essential amino acids, 1× sodium pyruvate and 1× GlutaMax) into white opaque 96-well plates and incubated at 37°C with 5% CO2 for 24 hours.

[0183] After synthesis, the test compounds were prepared into a 10 mM stock solution using dimethyl sulfoxide (DMSO) and stored at 4°C in the dark. Before treatment, the stock solutions, positive control compounds (such as DL-sulforaphane, CDDO-Me, etc.), and negative control (DMSO) were initially diluted with complete culture medium (determined based on preliminary experiments), and then serially diluted 3-20 times to obtain three 2× dilutions. When treating cells, 50 μL of culture medium was aspirated from the 96-well plate from which cells were seeded, and then 50 μL of the serially diluted 2× dilution was added. Two replicates were set up for each compound. In each experiment, 6 wells were set up with a concentration of 0 (i.e., containing only complete culture medium) to determine the baseline. After treatment, the cells were returned to the incubator and incubated for 4-24 hours.

[0184] After processing, 100 μL / well of Nano-Glo luciferase assay reagent was added to each well of a 96-well plate, and the RLU (relative fluorescence intensity) of each well was measured. The baseline Ave RLU was calculated by averaging the RLU values ​​at a concentration of 0 for each 96-well plate. Vehicle The RLU of the test compound at different concentrations in the experimental group. Test Cmpd and baseline Ave RLU Vehicle The ratio of Nrf2 to Nrf2 is used to calculate the activity of Nrf2 under different concentrations of the test compound according to formula (1) to determine the activation factor.

[0185] Formula (1)

[0186] The activation factors of each compound for Nrf2 are shown in Table 7, where A: ≥5 times and B: <5 times.

[0187] Table 7 shows the effects of compounds on AhR (EC). 50 Activation activity of Nrf2 (@20uM concentration) and Nrf2 (value)

[0188] The compound's effect on AhR (EC) 50 Activation activity of Nrf2 (@20uM concentration) and Nrf2 (value)

[0189] The following control standards were used in the above tests:

[0190] AhR agonist: 1-Methyl-2-oxo-N-(pyrimidin-2-yl)-1,2-dihydroquinoline-3-carboxamide

[0191] Nrf2 agonist: Methylbardoxolone (CDDO-Me)

[0192] The present invention has been described in detail above with reference to the accompanying drawings and embodiments. Those skilled in the art can make various modifications to the present invention based on the above description. Therefore, certain details in the embodiments should not be construed as limiting the present invention, and the scope of protection of the present invention shall be defined by the appended claims.

Claims

1. An aromatic hydrocarbon receptor agonist compound, characterized in that... The aromatic hydrocarbon receptor agonist has the structure shown in general formula (Ⅰ): Where Y is N or CR4; R1 and R7 are selected from hydrogen, unsubstituted or independently selected from those substituted by D, halogens, OR, SR, N(R)2, -NO2, -CN, -C(O)OR, -C(O)N(R)2, RC(O)O-, RC(O)N(R)-, and -S(O). 1～2 R, -S(O) 1～2 N(R)2 and RS(O) 1～2 N(R)-substituted C1-C8 alkyl, C3-C8 cycloalkyl; Z is O or NR8; R8 and R1 together with the linked atoms form an unsubstituted or substituted 1-2 Q-substituted 4-8 membered heterocycles containing 2-4 N atoms and 0-2 O and S atoms; R2, R3, R4, R5, and R6 are independently selected from H or Q; or, R2 and R1 together with the linked atoms form an unsubstituted or substituted 3- to 8-membered heterocycle containing 1- to 2 N atoms and 0- to 2 O and S atoms. Alternatively; R2 and R3 together with the linked atoms form unsubstituted or substituted 1-2 Q' rings of 3-8 members, where Q' is Q, or Q' together with R1 and the linked atoms form unsubstituted or substituted 1-2 Q' rings of 3-8 members containing 1-2 N atoms and 0-2 O and S atoms; and / or, R5 and R6 together with the linked atoms form unsubstituted or substituted 1-2 Q' rings of 3-8 members. Each Q is independently selected from T' or from T substituted with 1 to 3 T', where T' is D, halogen, -NO2, -CN, oxo, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C3-C8 cycloalkyl, OR, SR, N(R)2, -COOR, RCOO-, -CON(R)2, ROCON(R)-, -S(O). 1～2 R, -S(O) 1～2 N(R)2, RC(=O)N(R)-, RS(O) 1～2 N(R)-, RN(R)COO-, or heteroalkyl rings with 1 to 3 heteroatoms selected from N, O, or S (3 to 8-membered heteroalkyl rings); T is selected from C1 to C8 alkyl, C2 to C8 alkenyl, C2 to C8 ynyl, C3 to C8 cycloalkyl, OR, SR, N(R)2, -COOR, RCOO-, -CON(R)2, ROCON(R)-, -S(O). 1～2 R, -S(O) 1～2 N(R)2, RC(=O)N(R)-, RS(O) 1～2 N(R)-, RN(R)COO- or heteroalkyl rings consisting of 1 to 3 3 to 8-membered heteroalkyl rings selected from N, O or S heteroatoms; R is independently selected from H, D, -CH2CH2N(R')2, C1-C8 alkyl and C3-C8 cycloalkyl, wherein R' is C1-C8 alkyl, C2-C8 alkenyl or C2-C8 ynyl.

2. The aromatic hydrocarbon receptor agonist compound according to claim 1, characterized in that... The 3-8 membered ring is a 3-8 membered alkane ring, a 3-8 membered alkene ring, a 6-8 membered aromatic ring, a 3-8 membered heteroalkane ring with 1-3 preferred heteroatoms selected from N, O or S heteroatoms, a 3-8 membered heteroalkene ring with 1-3 preferred heteroatoms selected from N, O or S heteroatoms, or a 3-8 membered heteroaromatic ring with 1-3 preferred heteroatoms selected from N, O or S heteroatoms.

3. The aromatic hydrocarbon receptor agonist compound as described in claim 2, characterized in that... The 3-8 membered ring is preferably a 3-7 membered ring, more preferably a 3-7 membered alkane ring, a 3-7 membered alkene ring, a 6-8 membered aromatic ring, a heteroalkyl ring with 1-3 preferred heteroalkyl atoms with 1-2 heteroalkyl atoms selected from N, O or S heteroatoms, a heteroalkyl ring with 1-3 preferred heteroalkyl atoms with 1-2 heteroalkyl atoms selected from N, O or S heteroatoms, or a heteroaromatic ring with 1-3 preferred heteroalkyl atoms with 1-2 heteroalkyl atoms selected from N, O or S heteroatoms.

4. The aromatic hydrocarbon receptor agonist compound as described in claim 3, characterized in that... The 3-8 membered ring is preferably a 5-6 membered ring, more preferably a 5-6 membered alkane ring, a 5-6 membered alkene ring, a 6 membered aromatic ring, a heteroalkyl ring with 1-3 (preferably 1-2) heteroalkyl atoms selected from N, O or S heteroatoms, a heteroalkene ring with 1-3 (preferably 1-2) heteroalkyl atoms selected from N, O or S heteroatoms, or a heteroaromatic ring with 1-3 (preferably 1-2) heteroalkyl atoms selected from N, O or S heteroatoms.

5. The aromatic hydrocarbon receptor agonist compound as described in claim 6, characterized in that... R2 and R3 together with the linked atoms form unsubstituted or substituted 5-6 membered rings, while R5 and R6 together with the linked atoms form unsubstituted or substituted 5-6 membered aromatic rings.

6. The aromatic hydrocarbon receptor agonist compound as described in claim 5, characterized in that... R2 and R3 together with the linked atoms form an unsubstituted or substituted 6-membered aromatic ring with 1 to 2 Q's, and R5 and R6 together with the linked atoms form an unsubstituted or substituted 6-membered aromatic ring with 1 to 2 Q's; then general formula (Ⅰ) becomes general formula (Ⅰ1) as follows. Among them, A1, A2, A3, and A4 are independently selected from CH, CQ, or N; A5, A6, A7, and A8 are independently selected from CH, CQ', or N; Preferably, when Y is CR4, general formula (Ⅰ1) becomes the following general formula (Ⅰ1a). Preferably, when Y is N, the general formula (Ⅰ1) becomes the following general formula (Ⅰ1b).

7. The aromatic hydrocarbon receptor agonist compound as described in claim 5, characterized in that... Y is CR4, R2 and R3 together with the linked atoms form an unsubstituted or substituted 5-membered aromatic ring with 1 to 2 Q's, and R5 and R6 together with the linked atoms form an unsubstituted or substituted 6-membered aromatic ring with 1 to 2 Q's; then general formula (Ⅰ) becomes general formula (Ⅰ2). Among them, A1, A2, A3, and A4 are independently selected from CH, CQ, or N; A5, A6, and A7 are selected from O and S, while the other two are independently selected from CH, CQ', or N.

8. The aromatic hydrocarbon receptor agonist compound as described in claim 5, characterized in that... If Y is CR4, R2 and R3 together with the linked atoms form an unsubstituted or substituted 5-membered ring with 1 to 2 Q's, and R5 and R6 together with the linked atoms form an unsubstituted or substituted 6-membered aromatic ring with 1 to 2 Q's, then general formula (Ⅰ) becomes general formula (Ⅰ3). Among them, A1, A2, A3, and A4 are independently selected from CH, CQ, or N; A5, A6, and A7 are independently selected from CH2, CHQ', CQ'2, NH, NQ', O, or S.

9. The aromatic hydrocarbon receptor agonist compound as described in claim 5, characterized in that... If Y is CR4, R2 and R3 together with the linked atoms form an unsubstituted or substituted 6-membered ring with 1 to 2 Q's, and R5 and R6 together with the linked atoms form an unsubstituted or substituted 6-membered aromatic ring with 1 to 2 Q's, then general formula (Ⅰ) becomes general formula (Ⅰ4). Among them, A1, A2, A3, and A4 are independently selected from CH, CQ, or N; A5, A6, A7, and A8 are independently selected from CH2, CHQ', CQ'2, NH, NQ', O, or S.

10. The aromatic hydrocarbon receptor agonist compound as described in claim 5, characterized in that... If Y is CR4, R2 and R3 together with the linked atoms form an unsubstituted or substituted 6-membered aromatic ring with 1 to 2 Q's, and R5 and R6 together with the linked atoms form an unsubstituted or substituted 5-membered aromatic ring with 1 to 2 Q's, then general formula (Ⅰ) becomes general formula (Ⅰ5). Among them, one of A1, A2, and A3 is selected from O and S, and the other two are independently selected from CH, CQ, or N; A5, A6, A7, and A8 are independently selected from CH, CQ', or N.

11. The aromatic hydrocarbon receptor agonist compound according to any one of claims 1, 5 to 10, characterized in that... Z is O.

12. The aromatic hydrocarbon receptor agonist compound as described in claim 1 or 6, characterized in that... Z is NR8; R8 and R1 together with the linked atoms form an unsubstituted or substituted 1 to 2 Q-substituted 4 to 8-membered heterocycle containing 2 to 4 N atoms and 0 to 2 O and S atoms, preferably a 4 to 7-membered heterocycle containing 2 to 3 N atoms and 0 to 1 O and S atoms, more preferably a 5 to 6-membered heteroaromatic ring containing 2 to 3 N atoms.

13. The aromatic hydrocarbon receptor agonist compound as described in claim 1 or 6, characterized in that... Z is NR8; R8 and R1 together form unsubstituted or substituted with 1 to 2 Qs -NCH-, -NHCO-, -CH=CH-, or -CH2CH2-.

14. The aromatic hydrocarbon receptor agonist compound as described in claim 6, characterized in that... Z is O; Q' and R1 together with the linked atoms form an unsubstituted or substituted 3- to 8-membered heterocycle containing 1 to 2 N atoms and 0 to 2 O and S atoms, preferably a 4- to 7-membered heterocycle containing 1 to 2 N atoms and 0 to 1 O and S atoms, more preferably a 5- to 6-membered heterocycle containing 1 to 2 N atoms and 0 to 1 O and S atoms, and most preferably a 5- to 6-membered heterocycle containing 1 N atom.

15. The aromatic hydrocarbon receptor agonist compound of claim 14, characterized in that... Q' and R1, together with the already linked atoms, form the following general formula (Ⅰ1c).

16. The aromatic hydrocarbon receptor agonist compound as claimed in claim 1, characterized in that... R2 and R1, together with the linked atoms, form an unsubstituted or substituted 3- to 8-membered heterocycle containing 1 to 2 N atoms and 0 to 2 O and S atoms, preferably a 4- to 7-membered heterocycle containing 1 to 2 N atoms and 0 to 1 O and S atoms, more preferably a 5- to 6-membered heterocycle containing 1 to 2 N atoms and 0 to 1 O and S atoms, and most preferably a 5- to 6-membered heterocycle containing 1 N atom.

17. The aromatic hydrocarbon receptor agonist compound according to claim 1, characterized in that... R1 and R7 are selected from hydrogen, unsubstituted or independently selected from those substituted by D, halogens, OR, SR, N(R)2, -NO2, -CN, -C(O)OR, -C(O)N(R)2, RC(O)O-, RC(O)N(R)-, and -S(O). 1～2 R, -S(O) 1～2 N(R)2 and RS(O) 1～2 N(R)-substituted C1-C6 alkyl, C3-C7 cycloalkyl; Preferably, R1 and R7 are selected from hydrogen, unsubstituted or independently selected from D, halogens, OR, SR, N(R)2, -NO2, -CN, -C(O)OR, -C(O)N(R)2, RC(O)O-, RC(O)N(R)-, and -S(O). 1～2 R, -S(O) 1～2 N(R)2 and RS(O) 1～2 N(R)-substituted C1-C4 alkyl, C5-C6 cycloalkyl; More preferably, R1 is selected from hydrogen, unsubstituted or independently selected from D, halogens, OR, SR, N(R)2, -NO2, -CN, -C(O)OR, -C(O)N(R)2, RC(O)O-, RC(O)N(R)-, -S(O). 1～2 R, -S(O) 1～2 N(R)2 and RS(O) 1～2 N(R)-substituted C1-C3 alkyl, C5-C6 cycloalkyl; R7 is selected from hydrogen, methyl or hydroxymethyl.

18. The aromatic hydrocarbon receptor agonist compound as claimed in claim 1, characterized in that... Each Q is independently selected from T' or from T substituted with 1 to 2 T', wherein T' is D, halogen, -NO2, -CN, oxo, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 cycloalkyl, OR, SR, N(R)2, -COOR, RCOO-, -CON(R)2, ROCON(R)-, -S(O). 1～2 R, -S(O) 1～2 N(R)2, RC(=O)N(R)-, RS(O) 1～2 N(R)-, RN(R)COO-, or a heteroalkyl ring consisting of 1 to 2 3- to 7-membered heteroatoms selected from N, O, or S; T is selected from C1 to C6 alkyl, C2 to C6 alkenyl, C2 to C6 ynyl, C3 to C7 cycloalkyl, OR, SR, N(R)2, -COOR, RCOO-, -CON(R)2, ROCON(R)-, -S(O). 1～2 R, -S(O) 1～2 N(R)2, RC(=O)N(R)-, RS(O) 1～2 N(R)-, RN(R)COO- or heteroalkyl rings consisting of 1 to 2 3 to 7-membered heteroalkyl rings selected from N, O or S heteroatoms; Preferably, each Q is independently selected from T' or from T substituted with 1 to 2 T', wherein T' is D, halogen, -NO2, -CN, oxo, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C5-C6 cycloalkyl, OR, SR, N(R)2, -COOR, RCOO-, -CON(R)2, ROCON(R)-, -S(O). 1～2 R, -S(O) 1～2 N(R)2, RC(=O)N(R)-, RS(O) 1～2 N(R)-, RN(R)COO-, or a heteroalkyl ring consisting of 1 to 2 5- or 6-membered heteroatoms selected from N, O, or S; T is selected from C1-C4 alkyl, C2-C4 alkenyl, C2-C4 ynyl, C5-C6 cycloalkyl, OR, SR, N(R)2, -COOR, RCOO-, -CON(R)2, ROCON(R)-, -S(O). 1～2 R, -S(O) 1～2 N(R)2, RC(=O)N(R)-, RS(O) 1～2 N(R)-, RN(R)COO- or heteroalkyl rings consisting of 1 to 2 5- or 6-membered heteroalkyl rings selected from N, O or S heteroatoms; More preferably, each Q is independently selected from T' or from T substituted with one T', wherein T' is D, halogen, -NO2, -CN, oxo, C1-C3 alkyl, C2-C3 alkenyl, C2-C3 alkynyl, C5-C6 cycloalkyl, OR, SR, N(R)2, -COOR, RCOO-, -CON(R)2, ROCON(R)-, -S(O). 1～2 R, -S(O) 1～2 N(R)2, RC(=O)N(R)-, RS(O) 1～2 N(R)-, RN(R)COO-, or a heteroalkyl ring consisting of 1 to 2 5- or 6-membered heteroatoms selected from N, O, or S; T is selected from C1- or C3 alkyl, C2- or C3 alkenyl, C2- or C3 alkynyl, C5- or C6 cycloalkyl, OR, SR, N(R)2, -COOR, RCOO-, -CON(R)2, ROCON(R)-, -S(O). 1～2 R, -S(O) 1～2 N(R)2, RC(=O)N(R)-, RS(O) 1～2 N(R)-, RN(R)COO-, or one to two 5- to 6-membered heteroalkyl rings selected from N, O, or S heteroatoms.

19. The aromatic hydrocarbon receptor agonist compound as described in claim 17 or 18, characterized in that... R is independently selected from H, D, -CH2CH2N(R')2, C1-C6 alkyl and C3-C7 cycloalkyl, wherein R' is C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl; Preferably, R is independently selected from H, D, -CH2CH2N(R')2, C1-C4 alkyl and C5-C6 cycloalkyl, wherein R' is C1-C4 alkyl, C2-C4 alkenyl or C2-C4 alkynyl; More preferably, R is independently selected from H, D, -CH2CH2N(R')2, C1-C3 alkyl and C5-C6 cycloalkyl, wherein R' is C1-C3 alkyl, C2-C3 alkenyl or C2-C3 alkynyl.

20. The aromatic hydrocarbon receptor agonist compound according to claim 1, characterized in that... It has the following structure:

21. A pharmaceutically acceptable salt, deuterated product, solvate, hydrate, or prodrug of an aromatic hydrocarbon receptor agonist compound of general formula (I) as claimed in claim 1.

22. The use of an aryl hydrocarbon receptor agonist compound of general formula (I) according to claim 1 in the preparation of a medicament for treating diseases related to aryl hydrocarbon receptor imbalance and disorder, including but not limited to the use in medicaments for treating central nervous system diseases, endocrine system-related cancers, obesity, or in medicaments for immune regulation, hematopoiesis, cell cycle or intestinal barrier, regulation of osteoclast differentiation and function, and antioxidant effects; preferably, the neurodegenerative disease is selected from Alzheimer's disease and Parkinson's disease; the immune regulation is selected from the immune regulation of psoriasis, atopic dermatitis, lupus erythematosus, multiple sclerosis, vitiligo, and acne; the intestinal barrier is selected from inflammatory bowel diseases such as ulcerative colitis and Crohn's disease; the endocrine system-related cancers are selected from prostate cancer, liver cancer, breast cancer, cervical cancer, and lung cancer; and the regulation of osteoclast differentiation and function refers to the regulation of osteoclast differentiation and function in osteoporosis.

23. The use of an aryl hydrocarbon receptor agonist compound of general formula (I) according to claim 1 in a medicament for treating diseases related to aryl hydrocarbon receptor imbalance and disorder, including but not limited to the use in treating central nervous system diseases, endocrine system-related cancers, obesity, or in immune regulation, hematopoiesis, cell cycle or intestinal barrier, regulation of osteoclast differentiation and function, and antioxidant effects; preferably, the central nervous system diseases are selected from Alzheimer's disease and Parkinson's disease; the immune regulation is selected from the immune regulation of psoriasis, atopic dermatitis, lupus erythematosus, multiple sclerosis, vitiligo, and acne; the intestinal barrier is selected from inflammatory bowel diseases such as ulcerative colitis and Crohn's disease; the endocrine system-related cancers are selected from prostate cancer, liver cancer, breast cancer, cervical cancer, and lung cancer; and the regulation of osteoclast differentiation and function refers to the regulation of osteoclast differentiation and function in osteoporosis.

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