CN113072467B - Substituted guanidine group-containing derivative and its application in the preparation of osteoclast differentiation inhibitor - Google Patents

Abstract

The invention discloses an application of a derivative containing substituted guanidino in preparing an osteoclast differentiation inhibitor, wherein the derivative containing substituted guanidino has a direct and obvious inhibiting effect on the activity of inducing osteoclast differentiation and bone resorption by RANKL, so that the derivative can be used as an osteoclast differentiation inhibitor drug and used for treating diseases treated by the osteoclast differentiation inhibitor.

Description

Substituted guanidine group-containing derivatives and their application in the preparation of osteoclast differentiation inhibitors

technical field

The invention relates to the field of medicine, in particular to a substituted guanidine group derivative and a new use thereof.

Background technique

Osteoclasts (osteoclasts, OCs) are the only cells in the body responsible for bone resorption. Factors such as estrogen deficiency and elevated inflammatory factors can lead to increased RANKL levels in the body, resulting in high activation of osteoclasts. ) interacts with extracellular matrix proteins and is tightly attached to the bone surface. Then, the proton pump at the fold boundary of osteoclasts will generate an acidic environment, and under the action of matrix enzymes synthesized by osteoclasts, calcium is easily dissolved from the bone. Therefore, when the bone resorption function of osteoclasts is dominant, it is easy to cause an increase in the resorption of organic bone matrix, which in turn leads to the occurrence and development of osteolytic diseases. Osteolytic bone destruction diseases mainly include osteoporosis (OP), inflammatory disease Femoral destruction and tumor metastases bone destruction.

Denosumab, a RANKL monoclonal antibody approved by the US FDA in June 2010, plays an important role in the proliferation, differentiation and survival of osteoclasts. Denosumab can compete with RANK on the surface of osteoclasts and osteoclast precursor cells to bind to the DE ring structure on human RANKL protein, block the effective binding between receptors and ligands, and inhibit the formation and differentiation of osteoclasts. and survival, reduce osteoclast bone resorption activity, inhibit the process of osteolysis, improve bone strength and bone density, clinical results show that denosumab treatment can reduce vertebral fractures by 68% and hip fractures by 40%. Although denosumab is considered to be a good example of targeted osteoclast therapy, it requires subcutaneous injection and is expensive, which limits its use.

Therefore, continuing to study convenient, safe and effective targeted osteoclast-related drugs can not only promote the research of bone loss-related diseases, but also further promote its treatment, promote the development of such drugs, improve the suffering of patients, and improve the quality of life. .

SUMMARY OF THE INVENTION

The object of the present invention is to provide a substituted guanidine group-containing derivative which can inhibit osteoclast differentiation.

Another object of the present invention is to provide an application of a substituted guanidine group-containing derivative in the preparation of an osteoclast differentiation inhibitor.

The object of the present invention is achieved in this way: an application of a substituted guanidine-containing derivative in the preparation of an osteoclast differentiation inhibitor, the general structural formula of the substituted guanidine-containing derivative is:

wherein R ¹ is selected from the following groups: 1H-pyrro[2,3-b]pyridyl, N-methylindolyl, N-ethylindolyl, N-acetylindolyl, 5-chlorobenzene furanyl, 5-chlorobenzothienyl, 5-chloro-1-methyl-1H-indolyl, 4-chlorophenylmethylselenyl or chlorophenyl.

The osteoclast differentiation inhibitor is a drug for treating osteolytic diseases caused by excessive activation of osteoclasts.

The osteoclast differentiation inhibitor is a drug for treating osteoporosis, rheumatoid joints, and tumor metastases and bone destruction.

The osteoclast differentiation inhibitor is in oral, injection or external dosage form.

The osteoclast differentiation inhibitor includes substituted guanidino derivatives as active ingredients and medically acceptable pharmaceutical excipients.

The substituted guanidine group-containing derivative of the present invention has a direct and significant inhibitory effect on the osteoclast differentiation and bone resorption activities induced by RANKL, so that it can be used as an osteoclast differentiation inhibitor drug for an osteoclast differentiation inhibitor The disease to be treated is convenient to take, safe and effective, and the treatment cost is low.

Description of drawings

Figures 1 to 4 show that Sephin1, Sen-01, Sen-04, and Sen-06, substituted guanidino derivatives of the present invention, inhibit RANKL-induced osteoclastogenesis in mice, and Sephin1 (0.78-12.5 μm) inhibits bone resorption;

Fig. 5-Fig. 6 is that the substituted guanidino derivative Sephin1 of the present invention improves the bone mineral density of the ovariectomized (OVX) mouse osteoporosis model in vivo;

Fig. 7-Fig. 10 respectively show that the substituted guanidino derivative Sephin1 of the present invention inhibits osteoclast differentiation and bone activity in patients with osteoporosis;

Among them: RANKL is the ligand that activates nuclear factor NF-κB receptor (Note: compared with RANKL group, *P<0.05, **P<0.01, ***P<0.001).

Detailed ways

The present invention relates to the application of a substituted guanidino-containing derivative in the preparation of an osteoclast differentiation inhibitor, and the general structural formula of the substituted guanidine-containing derivative is:

wherein R ¹ is selected from the following groups: 1H-pyrro[2,3-b]pyridyl, N-methylindolyl, N-ethylindolyl, N-acetylindolyl, 5-chloro- 1-methyl-1H-indolyl, 5-chlorobenzothienyl, 5-chlorobenzofuranyl, 4-chlorophenylmethylselenyl or chlorophenyl.

Preferably, the osteoclast differentiation inhibitor is a drug for treating osteolytic diseases caused by excessive activation of osteoclasts. More preferably, the osteoclast differentiation inhibitor is a drug for the treatment of osteoporosis, rheumatoid joints, and tumor metastases and bone destruction.

The osteoclast differentiation inhibitor can be in oral, injection or external dosage forms. The osteoclast differentiation inhibitor includes substituted guanidino derivatives as active ingredients and medically acceptable pharmaceutical excipients.

When the substituted guanidine group-containing derivative is prepared, compound III can be produced by reacting the raw material shown in chemical formula I with aminoguanidine hydrochloride.

R ¹ is selected from the aforementioned groups, and the corresponding compound III obtained is one of the following compounds:

As a safe neuroprotective drug, Sephin1 has a good clinical effect in the treatment of patients with multiple sclerosis, but whether it can improve osteoclast-related bone destruction diseases by inhibiting osteoclasts has not been reported. And Sen-01 to Sen-08 are newly synthesized substituted guanidino derivatives, which have not been reported before.

Compound preparation and identification

(1) The synthetic steps of the substituted guanidino derivative Sephin1 are as follows:

The starting aldehyde compound (1 equiv.), aminoguanidine hydrochloride (1.2 equiv.), potassium hydroxide (1.2 equiv.) were put into a methanol solution. React at 80 degrees for 2 hours. After the completion of the reaction, the reaction mixture was poured into water to obtain a yellow solid product, which was slurried with methyl tert-butyl ether to obtain the target product Sephin1. The identification results of the target product Sephin1 are as follows:

(E)-2-(2-chlorobenzylidene)hydrazine-1-carboximidamide(Sephin1) ¹ H NMR(400MHz,DMSO)δ7.95(s,1H),7.84(s,1H),7.78(s,2H), 7.64(s, 1H), 7.46(d, J=15.6Hz, 2H), 7.35(s, 1H), 6.49(s, 1H). MS: m/z=197.2[M+H ⁺ ].

(2) The preparation methods of the substituted guanidino-containing derivatives Sen-01 to Sen-08 are the same as the above-mentioned preparation method of the substituted guanidino-containing derivative Sephin1. The identification results of the target products Sen-01 to Sen-08 are as follows:

(E)-2-((1H-pyrrolo[2,3-b]pyridin-4-yl)methylene)hydrazine-1-carboximidamide(Sen-01). ¹ H NMR(400MHz,DMSO)δ11.83(s ,1H),8.46(s,1H),8.26(d,J=4.8Hz,1H),7.57(s,1H),7.45(d,J=4.9Hz,1H),7.29(s,4H),6.93 (s, 1H). MS: m/z=203.2 [M+H ⁺ ].

(E)-2-((1-methyl-1H-indol-4-yl)methylene)hydrazine-1-carboximidamide(Sen-02). ¹ H NMR(400MHz,DMSO)δ7.90(s,1H), 7.84(s,1H),7.78(s,2H),7.66(s,1H),7.50(s,1H),7.46(s,1H),7.24(s,2H),6.91(s,1H),3.82 (s, 3H). MS: m/z=216.2 [M+H ⁺ ].

(E)-2-((1-ethyl-1H-indol-4-yl)methylene)hydrazine-1-carboximidamide(Sen-03). ¹ H NMR(400MHz,DMSO)δ7.90(s,1H), 7.84(s,1H),7.78(s,2H),7.66(s,1H),7.51(s,1H),7.46(s,1H),7.28(s,1H),7.21(s,1H),6.92 (s, 1H), 4.27 (d, J=24.1Hz, 2H), 1.16 (s, 3H). MS: m/z=230.3 [M+H ⁺ ].

(E)-2-((1-acetyl-1H-indol-4-yl)methylene)hydrazine-1-carboximidamide(Sen-04). ¹ H NMR(400MHz,DMSO)δ8.39–8.24(m,2H ), 7.90(s, 1H), 7.84(s, 1H), 7.78(s, 2H), 7.55(s, 1H), 7.42(s, 1H), 7.31(d, J＝3.8Hz, 2H), 2.70 (s, 3H). MS: m/z=244.3 [M+H ⁺ ].

(E)-2-((5-chlorobenzofuran-4-yl)methylene)hydrazine-1-carboximidamide(Sen-05). ¹ H NMR (400MHz, DMSO) δ7.90(s,1H), 7.86–7.76( m, 4H), 7.61(s, 1H), 7.40(s, 1H), 7.28(s, 1H), 7.12(s, 1H). MS: m/z=237.1[M+H ⁺ ].

(E)-2-((5-chlorobenzo[b]thiophen-4-yl)methylene)hydrazine-1-carboximidamide(Sen-06). ¹ H NMR(400MHz, DMSO)δ8.08(s,1H), 7.92–7.82(m, 3H), 7.78(s, 2H), 7.65(s, 1H), 7.60(s, 1H), 7.28(s, 1H). MS: m/z=253.1 [M+H ⁺ ] .

(E)-2-((5-chloro-1-methyl-1H-indol-4-yl)methylene)hydrazine-1-carboximidamid e(Sen-07). ¹ H NMR(400MHz,DMSO)δ7.90( s,1H),7.84(s,1H),7.78(s,2H),7.57(s,1H),7.37(s,1H),7.28(s,1H),7.19(s,1H),6.93(s ,1H),3.81(s,3H).MS: m/z=250.2[M+H ⁺ ].

(E)-2-(3-(methylselanyl)benzylidene)hydrazine-1-carboximidamide(Sen-08) ^.1H NMR(500MHz,Chloroform)δ7.97(s,1H),7.84(s,1H),7.78 (s, 2H), 7.62(s, 1H), 7.48(t, J=15.3Hz, 3H), 7.23(s, 1H), 2.29(s, 3H). MS: m/z=257.2[M+H ⁺ ].

Experimental Example 1 Substituted guanidino derivatives Sephin1, Sen-01, Sen-04, and Sen-06 inhibit RANKL-induced osteoclastogenesis or bone resorption in mice

Take the femur and tibia of C57BL/6 mice, peel off the muscle and cut off both ends, use a syringe to suck α-MEM to wash the bone marrow cavity of the femur and tibia repeatedly, flush out the bone marrow until the bone marrow cavity turns white, and culture at 37°C, 5% CO ₂ . After 48 hours in the box, adherent cells were taken, and the cells were seeded in 96-well plates. After that, drugs of different concentrations were added to the drug group, and the stimulator RANKL was added after 2 hours (the final concentration of RANKL was 50 ng/ml). TRAP staining was performed 3-4 days after induction, observed and photographed under a microscope, and osteoclasts were counted as TRAP positive and more than 3 nuclei. The bone resorption plate was washed off the cells on the 7th day of induction, observed and photographed under a 40×Nikon inverted optical microscope, and the percentage of bone resorption area in each well was calculated by Image-Pro Plus software.

The results showed that the substituted guanidino derivatives Sephin1, Sen-01, Sen-04, and Sen-06 could significantly inhibit RANKL-induced osteoclastogenesis in vitro, as shown in Figure 1 and Figure 2. Sephin1 (0.78-12.5 μm) significantly inhibited bone resorption activity, and the results are shown in Figure 3 and Figure 4 .

Experimental Example 2 Substituted guanidine derivative Sephin1 improves bone mineral density in ovariectomized (OVX) mouse osteoporosis model in vivo

15 C57BL/6 female mice were 8 weeks old, the negative control group (sham) was 5 mice in the sham operation group, and the remaining 10 OVX model mice were randomly divided into the positive control group (ovx), the drug group Sephin1 group (ovx). +Sephin1 (4mg/kg)), 5 in each group. The negative control group and the positive control group were injected with normal saline; in the drug intervention group, 4 mg/kg of Sephin1 was injected intraperitoneally, and the drug was injected intraperitoneally every other day. Reserve in formalin.

Micro-CT scans showed that compared with the negative control group, the bone mass of the mice in the OVX model group was significantly reduced, while the Sephin1 intervention group significantly increased the bone mineral density (BMD) of the mice with bone destruction, suggesting that Sephin1 can pass Inhibition of osteopenia in OVX mice in vivo has an improving effect on OVX-induced osteoporosis, as shown in Figure 5 and Figure 6.

Based on the above results, Sephin1 can significantly inhibit the RANKL-induced osteoclastogenesis and bone resorption activities of mouse osteoclasts in vitro and osteoporosis patients, and Sephin1 can improve OVX-induced osteoporosis in vivo.

Experimental Example 3 Substituted guanidine derivative Sephin1 inhibits osteoclast differentiation and bone activity in patients with osteoporosis

Osteo Assay 96孔板培养，药物组加入不同浓度的药物，2h后均加刺激剂RANKL(RANKL终浓度为5ng/ml)，每三天换一次液，在诱导6天后进行TRAP染色，在显微镜下观察拍照，并按TRAP阳性并且大于3个核的为破骨细胞进行计数。骨吸收板每三天换一次液，在诱导7天洗去细胞，在40×Nikon倒置光学显微镜光镜观察拍照，通过Image-Pro Plus软件计算每孔中骨吸收面积百分比。Lymphocytes were isolated from the peripheral blood of patients with osteoporosis, and the lymphocytes were sorted using CD14 magnetic beads. The obtained cells were seeded on artificial bone chips coated with

The Osteo Assay was cultured in 96-well plates. The drug group was added with different concentrations of drugs, and the stimulator RANKL was added after 2 hours (the final concentration of RANKL was 5ng/ml). The medium was changed every three days. Observe and photograph, and count osteoclasts that are TRAP-positive and more than 3 nuclei. The liquid of the bone resorption plate was changed every three days, and the cells were washed away on the 7th day of induction, observed and photographed under a 40×Nikon inverted optical microscope, and the percentage of bone resorption area in each well was calculated by Image-Pro Plus software.

The results showed that Sephin1 at concentrations of 3.125 μM and 12.5 μM could significantly reduce the area of the bone depression formed by osteoclasts in osteoporosis patients, and had an inhibitory effect on the bone resorption activity of osteoclasts in patients with osteoporosis, as shown in Figure 7-figure 10 shown.

Claims (7)

1. an application of a substituted guanidino derivative in the preparation of an osteoclast differentiation inhibitor, the general structural formula of the substituted guanidino derivative is:

wherein R ¹ is selected from the following groups: 1H-pyrro[2,3-b]pyridyl, N-methylindolyl, N-ethylindolyl, N-acetylindolyl, 5-chloro- 1-Methyl-1H-indolyl, 5-chlorobenzothienyl or 5-chlorobenzofuranyl. 2 . The application of the substituted guanidine group-containing derivative according to claim 1 in the preparation of an osteoclast differentiation inhibitor, characterized in that: the osteoclast differentiation inhibitor is for the treatment of lysis caused by excessive activation of osteoclasts. Medications for bone diseases. 3 . The application of the substituted guanidine group-containing derivative according to claim 1 in the preparation of an osteoclast differentiation inhibitor, wherein the osteoclast differentiation inhibitor is for the treatment of osteoporosis and rheumatoid joints. 4 . , Tumor metastases and bone destruction drugs. 4 . The application of the substituted guanidine group-containing derivative according to claim 1 in the preparation of an osteoclast differentiation inhibitor, wherein the osteoclast differentiation inhibitor is an oral, injection or external dosage form. 5 . 5 . The application of the substituted guanidine-containing derivative according to claim 1 in the preparation of an osteoclast differentiation inhibitor, characterized in that: the osteoclast differentiation inhibitor comprises the active ingredients substituted guanidine-containing derivative and Medically acceptable pharmaceutical excipients. 6. The application of a substituted guanidine-containing derivative in the preparation of a medicine for the treatment of osteoporosis, wherein the general structural formula of the substituted guanidine-containing derivative is:

wherein R ¹ is selected from the following groups: chlorophenyl. 7. a kind of substituted guanidine derivatives, its general structural formula is:

wherein R ¹ is selected from the following groups: 1H-pyrro[2,3-b]pyridyl, N-methylindolyl, N-ethylindolyl, N-acetylindolyl, 5-chloro- 1-Methyl-1H-indolyl, 5-chlorobenzothienyl or 5-chlorobenzofuranyl.

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