Application of compound with benzo-aza structure in preparation of medicine for inhibiting osteoclast formation

Abstract

The invention discloses application of a compound with a benzo-aza structure in preparation of a medicine for inhibiting osteoclast formation, the structure of the compound is shown as a formula (1), and R substituent is one of H, OCH3, Br, F and i-Pr. Cell experiments prove that the compound with the benzo-aza structure has a remarkable inhibiting effect on RANKL-induced osteoclast differentiation and marker gene expression, can be used as a candidate medicine which is novel in structure and is used for inhibiting osteoclast formation, and is used for targeting osteoclast to treat osteoporosis related diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of osteoclast differentiation inhibitors, and particularly relates to the use of a compound with a benzazepine structure in the preparation of a drug for inhibiting osteoclast formation, and the preparation of a drug for treating osteoporosis, tumor bone metastasis or inflammatory bone destruction. BACKGROUND TECHNICAL FIELD

[0002] Osteoclasts (OCs) are multinucleated cells derived from myeloid hematopoietic precursor cells and are mainly involved in the process of bone resorption. They play an important role in bone-related diseases such as osteoporosis, rheumatoid arthritis, ankylosing spondylitis, etc., because the excessive differentiation of osteoclasts affects bone tissue resorption in these diseases. In osteoporosis, increased osteoclast activity and / or decreased bone formation are the main causes of bone loss, and the dysfunction of osteoclasts is an important pathogenic factor. Osteoclasts also play a key role in the process of bone fracture repair, they are recruited to the bone fragments for resorption after the inflammatory stage of the fracture, and remodel the hard callus at the end of the healing process.

[0003] Currently, the therapeutic drugs for osteoporosis mainly include anti-resorption drugs and anabolic drugs. Anti-resorption drugs such as bisphosphonates and selective estrogen receptor modulators treat osteoporosis by inhibiting bone resorption. Anabolic drugs, including parathyroid hormone (PTH) analogs and sclerostin inhibitors, treat osteoporosis by stimulating bone formation. However, these drugs have some problems, such as severe side effects and loss of efficacy, which can limit the long-term use of a single drug. For example, long-term use of bisphosphonates can increase the risk of atypical femoral fractures, and the use of PTH analogs requires strict control of dosage and time to avoid potential side effects.

[0004] In view of the limitations of existing therapeutic drugs, the development of new drugs has become a top priority. The development of new drugs aims to find a treatment strategy with fewer side effects and higher efficiency. The successful development of such new drugs will help to improve the treatment effect of osteoporosis patients, reduce side effects, and improve the quality of life of patients. SUMMARY

[0005] The purpose of the present application is to overcome the above-mentioned deficiencies in the prior art, and to provide a compound with a benzazepine structure for the preparation of a drug for inhibiting osteoclast formation, and a drug for treating osteoporosis, tumor bone metastasis or inflammatory bone destruction. BACKGROUND

[0006] The present application provides a compound with a benzazepine structure for the preparation of a drug for inhibiting osteoclast formation, and a drug for treating osteoporosis, tumor bone metastasis or inflammatory bone destruction. The compound with the structure can significantly inhibit the expression of Trap and Ctsk genes in cells, and can significantly inhibit the osteoclast differentiation induced by RANKL, and can be used as a candidate drug for inhibiting osteoclast formation.

[0007] To achieve the above-mentioned purpose, the technical scheme adopted by the present application to solve its technical problems is:

[0008] In a first aspect, the present application provides a compound with a benzazepine structure in the preparation of an osteoclast differentiation inhibitor, and the compound has the structural formula:

[0009]

[0010] In the formula, R is one of H, OCH3, Br, F and i-Pr.

[0011] More specifically, the compound is selected from the following structure:

[0012]

[0013] In a second aspect, the present application provides an application of an osteoclast formation inhibitor in the preparation of a drug for treating postmenopausal osteoporosis and bone destruction diseases.

[0014] Compared with the prior art, the present application has the following advantages:

[0015] The compound with the benzazepine structure provided by the present application can significantly inhibit the expression of Trap and Ctsk genes in cells, and can significantly inhibit the osteoclast formation induced by RANKL. BRIEF DESCRIPTION OF DRAWINGS

[0016] The accompanying drawings, which are incorporated into and constitute a part of the specification, illustrate embodiments consistent with the present application and, together with the specification, serve to explain the principles of the present application.

[0017] Figure 1 is a 2-benzoyl-1,5-dihydro-4H-benzo[b]azepine-4-one nuclear magnetic hydrogen spectrum diagram given by example compound A1. -4-ketone.

[0018] Figure 2 is a 2-benzoyl-7-methoxy-1,5-dihydro-4H-benzo[b]azepine-4-one nuclear magnetic hydrogen spectrum diagram given by example compound A2. -4-ketone.

[0019] Figure 32-benzoyl-7-bromo-1,5-dihydro-4H-benzo[ ]azepin-4-one -4-ketone proton NMR spectrum.

[0020] Figure 4 2-benzoyl-7-fluoro-1,5-dihydro-4H-benzo[ ]azepin-4-one -4-ketone proton NMR spectrum.

[0021] Figure 5 2-benzoyl-7-isopropyl-1,5-dihydro-4H-benzo[ ]azepin-4-one -4-ketone proton NMR spectrum.

[0022] Figure 6 2-benzoyl-1,5-dihydro-4H-benzo[ ]azepin-4-one -4-ketone carbon NMR spectrum.

[0023] Figure 7 2-benzoyl-7-methoxy-1,5-dihydro-4H-benzo[ ]azepin-4-one -4-ketone carbon NMR spectrum.

[0024] Figure 8 2-benzoyl-7-bromo-1,5-dihydro-4H-benzo[ ]azepin-4-one -4-ketone carbon NMR spectrum.

[0025] Figure 9 2-benzoyl-7-fluoro-1,5-dihydro-4H-benzo[ ]azepin-4-one -4-ketone carbon NMR spectrum.

[0026] Figure 10 2-benzoyl-7-isopropyl-1,5-dihydro-4H-benzo[ ]azepin-4-one -4-ketone carbon NMR spectrum.

[0027] Figure 11 Results of the experiment to detect the effect of Example Compound A1 on the cell viability of mouse bone marrow-derived macrophages (BMMs) by CCK-8 method. Results of the experiment to detect the effect of Example Compound A1 on the cell viability of mouse bone marrow-derived macrophages (BMMs) by CCK-8 method.

[0028] Figure 12 Results of the experiment to detect the effect of Example Compound A1 on the formation of osteoclasts induced by RANKL by TRAP staining. Results of the experiment to detect the effect of Example Compound A1 on the formation of osteoclasts induced by RANKL by TRAP staining. Scale bar: 200 pm.

[0029] Figure 13 TRAP staining to detect the presence of benzoazepine The experimental results of the effect of structured compounds A2-A5 on RANKL-induced osteoclast formation, scale: 200 μm.

[0030] Figure 14 qRT-PCR to detect the presence of benzoazepine The experimental results of the effect of structured compound A1 on the expression of osteoclast formation marker genes, data expressed as mean ± standard deviation. ## P<0.01 vs Ctrl / 0 μM group, *P<0.05, **P<0.01 vs RANKL / 0 μM group. DETAILED DESCRIPTION

[0031] The present application will be further described below in conjunction with examples and drawings. The following examples are only illustrative of the present application, and are not intended to limit the present application in any way.

[0032] Example 1 2-benzoyl-1,5-dihydro-4H-benzo[b]azepine -4-ketone, labeled as A1, with the following structure:

[0033]

[0034] Compound A1 was prepared using the method of Example 1 of the invention patent application with publication number CN 118271243 A.

[0035] A1 was subjected to nuclear magnetic detection, and the results are as follows: 1 H NMR (400 MxHz, CDCl3) δ 8.13 (s, 1H), 7.65 (d, J = 8.0 Hz, 2H), 7.51 (t, J = 7.6 Hz, 1H), 7.38 (t, J = 7.6 Hz, 2H), 7.21 (dd, J = 8.0, 4.0 Hz, 1H), 7.16 (d, J = 4.0 Hz, 2H), 7.07 (d, J = 7.6 Hz, 1H), 5.95 (s, 1H), 3.63 (s, 2H). 13 C NMR (100 MHz, CDCl3) δ 194.4, 189.8, 144.7, 139.0, 135.2, 133.2, 130.5, 129.7, 128.5, 127.9, 126.7, 123.2, 119.9, 114.4, 50.0. HRMS (ESI): m / z calcd for C 17 H 13 NO2[M+H] + : 264.0980, found: 264.0958.

[0036] Example 2 2-Benzoyl-7-methoxy-1,5-dihydro-4H-benzo[b]aza -4-Ketone, labeled A2, has the following structural formula:

[0037]

[0038] Compound A2 was prepared using the method described in Example 13 of the invention patent application with publication number CN 118271243 A.

[0039] A2 was subjected to NMR analysis, and the results are as follows: 1 H NMR (400MHz, CDCl3) δ8.17(s,1H),7.75(d,J=7.2Hz,2H),7.62(t,J=7.6Hz,1H),7.48(t,J=7.6Hz,2H),7.11 (d,J=8.8Hz,1H),6.87(dd,J=8.8,2.8Hz,1H),6.76(d,J=2.8Hz,1H),5.99(s,1H),3.82(s,3H),3.69(s,2H). 13 CNMR (100MHz, CDCl3) δ194.6,190.1,189.7,145.1,144.6,139.9,139.0,138.1,137.9,135.4,133.3, 130.4,129.8,128.6,128.2,127.8,127.4,122.5,120.3,117.9,115.5,114.4,49.7,45.3,21.1,20.2. HRMS(ESI):m / z calcd for C 18 H 15 NO3[M+H] + :294.1085, found:294.1083.

[0040] Example 3 2-Benzoyl-7-bromo-1,5-dihydro-4H-benzo[b]aza -4-keto, labeled A3, has the following structural formula:

[0041]

[0042] Compound A3 was prepared using the method described in Example 20 of the invention patent application with publication number CN 118271243 A.

[0043] A3 was subjected to MRI, and the results are as follows: 1H NMR (400MHz, CDCl3) δ8.31 (s, 1H), 7.86 (d, J = 7.6Hz, 2H), 7.72 (d, J = 7.2Hz, 1H), 7.59 (t, J = 7. 6Hz, 2H), 7.25 (dd, J = 8.4, 4.4Hz, 1H), 7.11 (dd, J = 21.2, 8.4Hz, 2H), 6.14 (s, 1H), 3.81 (s, 2H). 13 C NMR (100MHz, CDCl3) δ194.4,189.0,162.6,160.1,144.7,135.4,135.1,133.4,12 9.8,128.7,125.5,125.4,121.5,121.4,116.7,116.5,115.2,115.0,114.3,49.8. HRMS(ESI):m / z calcd for C 17 H 12 FNO2[M+H] + :282.0852, found:282.0858.

[0044] Example 4 2-Benzoyl-7-fluoro-1,5-dihydro-4H-benzo[b]aza -4-keto, labeled A4, has the following structural formula:

[0045]

[0046] Compound A4 was prepared using the method described in Example 21 of the invention patent application with publication number CN 118271243 A.

[0047] An A4 sample was taken for MRI analysis, and the results are as follows: 1 H NMR (400MHz, CDCl3) δ8.20(s,1H),7.74(d,J=7.6Hz,2H),7.63(t,J=7.6Hz,1H),7.49(t ,J=7.6Hz,2H),7.41(d,J=10.0Hz,2H),7.06(d,J=8.4Hz,1H),6.07(s,1H),3.70(s,2H). 13 C NMR (100MHz, CDCl3) δ194.2,189.1,144.5,138.3,135.1,133.5,133.1,131.0,129.8,128.7,125.2,121.6,119.4,114.9,49.7. HRMS(ESI):m / z calcd for C 17 H 12 BrNO2[M+H]+ :342.0051, found:342.0052.

[0048] Example 5 2-Benzoyl-7-isopropyl-1,5-dihydro-4H-benzo[b]aza -4-Ketone, labeled A5, has the following structural formula:

[0049]

[0050] Compound A5 was prepared using the method described in Example 17 of the invention patent application with publication number CN 118271243 A.

[0051] An A5 sample was taken for NMR analysis, and the results are as follows: 1 H NMR (400MHz, CDCl3) δ8.21(s,1H),7.75(d,J=7.6Hz,2H),7.61(t,J=7.2Hz,1H),7.48(t,J=7.6Hz,2H),7.18(d,J= 8.0Hz, 1H), 7.11 (d, J = 6.0Hz, 2H), 6.02 (s, 1H), 3.71 (s, 2H), 2.92 (dt, J = 13.6, 6.8Hz, 1H), 1.25 (d, J = 6.8Hz, 6H). 13 C NMR (100MHz, CDCl3) δ194.6,190.0,147.9,144.7,136.9,135.4,133.3,129.8,128.6,128.2,126.2,123.3,119.9,114.1,50.3,33.8,24.1. HRMS(ESI):m / z calcd forC 20 H 19 NO2[M+H] + :306.1416, found:306.1413.

[0052] Example 6 possesses benzo[a]aza Cytotoxicity of structural compounds and their inhibitory effect on RANKL-induced osteoclast differentiation

[0053] The specific experiment is as follows:

[0054] Experiment 1: Detection of benzo[a]azine-containing organisms using the CCK-8 method The cytotoxicity of structural compounds to BMMs (using 2-benzoyl-1,5-dihydro-4H-benzo[b]aza) (Taking -4-ketoA1 as an example)

[0055] (1) Isolation and culture of mouse BMMs

[0056] Isolation of C57BL / 6 mouse femur, flush the bone marrow cavity with α-MEM complete medium (10% fetal bovine serum, 1% penicillin-streptomycin double antibiotic, α-MEM medium), centrifuge the flush liquid at 1000 rpm for 5 min to obtain primary bone marrow cells, discard the supernatant and add BMMs complete medium (40 ng / ml M-CSF, 10% fetal bovine serum, 1% penicillin-streptomycin double antibiotic, α-MEM medium, hereinafter referred to as BMMs complete medium), and culture in a 37°C, 5% CO2 incubator. Change the medium on the second day to remove unattached cells, and continue to culture until the bone marrow-derived mononuclear macrophages grow to more than 80% density, which are BMMs.

[0057] (2) CCK-8 method for detecting the cytotoxicity of benzazepine compound on BMMs (taking A1 compound as an example)

[0058] BMMs were seeded into 96-well plates at a cell density of 5×10 3 cells / well, and 0, 0.03125, 0.0625, 0.125, 0.25, 0.5 and 1 μM A1 compound groups were set up, with 6 replicate wells in each group. The culture medium was BMMs complete medium, and the cells were cultured for 24, 48 and 72 hours, with medium change every other day. At the corresponding time points, 10 μl of CCK-8 reagent was added to each well, and after 2 hours of incubation in the incubator, the plates were removed, shaken well on the microplate reader and the absorbance value at 450 nm was measured. The experimental results showed that A1 compound at a concentration of 1 μM and below had no significant inhibitory effect on BMMs. Figure 11

[0059] Experiment 2: Evaluation of the effect of benzazepine compound on osteoclast differentiation by tartrate-resistant acid phosphatase (TRAP) staining (taking A1 compound as an example)

[0060] BMMs were seeded into 96-well plates at a cell density of 5×10 3 ​Cells / well, set 0, 0.03125, 0.0625, 0.125, 0.25, 0.5 and 1 μM of A1 compound groups, 6 replicates in each group. The next day, the medium was replaced with osteoclast induction medium (50 ng / ml RANKL, 40 ng / ml M-CSF, 10% fetal bovine serum, 1% penicillin-streptomycin double antibody, α-MEM medium, hereinafter referred to as osteoclast induction medium), and 0, 0.03125, 0.0625, 0.125, 0.25, 0.5 and 1 μM of A1 compound were added, respectively. The medium was changed every 48 h, and the culture was terminated after about 6 days when the 0 μM control group had obvious mature and hypertrophic osteoclasts. After the medium was aspirated, phosphate buffer was washed once, 500 μl of 4% paraformaldehyde was added per well to fix the cells for 5 min, and the anti-tartaric acid phosphatase staining was performed using a kit. The effect of A1 compound on osteoclast formation was evaluated under an inverted microscope. The TRAP staining results showed that the number of osteoclasts in the 0.03125 μM A1 compound treatment group had no significant difference compared with the 0 μM control group, however, the number of osteoclasts in the 0.0625, 0.125 μM, 0.25 μM, 0.5 μM and 1 μM A1 compound treatment groups were significantly less than the control group + The size and number of osteoclasts were significantly less than the control group Figure 12 ); and A2-A5 compounds had similar inhibitory effect on osteoclast differentiation Figure 13

[0061] Experiment 3: qRT-PCR method was used to detect the effect of compounds with benzazepine structure on the expression of osteoclast differentiation marker genes (taking A1 compound as an example)

[0062] 6 different concentrations of A1 compound groups, RANKL induction group, and different concentrations of drug treatment group were set, 3 replicates in each group. BMMs were plated at 2 x 10 5 ​The cells were seeded in 24-well plates at a density of 1 cell per well, and the next day the medium was changed to one containing different concentrations of the A1 compound. The cells were divided into the following groups: a control group containing 0, 0.03125, 0.0625, 0.125, 0.25, 0.5 and 1 μM concentrations of the A1 compound, and the medium was BMM complete medium; an induction group containing the corresponding concentrations of the A1 compound, and the medium was osteoclast induction medium. After 3 days of culture, the RNA was extracted and qRT-PCR was performed to detect the expression of the osteoclast differentiation marker genes Trap, NFATc1, DC-Stamp and Ctsk. The qRT-PCR results showed that RANKL significantly induced the expression of Trap, NFATc1, DC-Stamp and Ctsk; and the A1 compound inhibited the RANKL-induced expression of Trap and Ctsk at 0.0625 μM, and significantly inhibited the expression of Trap, NFATc1, DC-Stamp and Ctsk at 0.125 μM, showing an obvious dose-dependent effect. Figure 14

[0063] The raw materials and equipment used in the present application are conventional raw materials and equipment in the art, unless otherwise specified; the methods used in the present application are conventional methods in the art, unless otherwise specified.

[0064] The above only describes some preferred embodiments of the present application, and is used to help explain the present application. The protection scope of the present application is not limited to the above-described embodiments, but any technical solution falling within the concept of the present application falls within the protection scope of the present application.​

Claims

1. Possesses benzo[a]aza The compound with the structure is used in the preparation of drugs that inhibit osteoclast formation, characterized by, The aforementioned benzo[a]aza The structural formula of the compound is shown in formula (1): R is one of H, OCH3, Br, F, or i-Pr.

2. Possesses benzo[a]aza The application of compounds with a specific structure in the preparation of drugs for treating osteoporosis, characterized in that... The aforementioned benzo[a]aza The structural formula of the compound is shown in formula (1): R is one of H, OCH3, Br, F, or i-Pr.

3. Possesses benzo[a]aza The application of compounds with a specific structure in the preparation of drugs for treating bone metastases of tumors, characterized in that, The aforementioned benzo[a]aza The structural formula of the compound is shown in formula (1): R is one of H, OCH3, Br, F, or i-Pr.

4. Possesses benzo[a]aza The application of compounds with a specific structure in the preparation of medicaments for treating inflammatory bone destruction, characterized in that... The aforementioned benzo[a]aza The structural formula of the compound is shown in formula (1): R is one of H, OCH3, Br, F, or i-Pr.

5. The application as described in any one of claims 1 to 4, characterized in that, The aforementioned benzo[a]aza The compounds with the following structure are:

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