CN116650481B - Aromatic compounds as activators of plexin protein-B2 and their use in preparing drugs for treating osteoporosis - Google Patents

Abstract

The present invention discloses an aromatic compound as an activator of plexin protein-B2 and its application in the preparation of a drug for treating osteoporosis. The aromatic compound is a compound as shown in formula I, and the compound as shown in formula I can inhibit the expression level of osteoclast gene CTSK-mRNA, provide a research basis and theoretical basis for the development of drugs for treating OP, and can be used to prepare drugs for preventing and/or treating osteoporosis.

Description

Aromatic compounds as activators of plexin protein-B2 and their use in the preparation of drugs for the treatment of osteoporosis

This application is a divisional application of Chinese patent application 202111502631.X with a filing date of 2021/12/10.

Technical Field

The invention belongs to the technical field of medicine and biochemistry, and specifically relates to an aromatic compound as an activator of plexin protein-B2 (Plexin-B2) and the application of the aromatic compound in the preparation of a drug for treating osteoporosis.

Background technique

Osteoporosis (OP) is the most common bone metabolic disease, characterized by decreased bone mass and destruction of bone microstructure, which leads to increased bone fragility and increased risk of fractures. OP mainly includes senile osteoporosis and postmenopausal osteoporosis, and the high-risk groups are the elderly and postmenopausal women.

At present, clinical anti-osteoporosis drugs mainly include basic supplements (such as calcium, vitamin D), bone resorption inhibitors (such as bisphosphonates) and bone formation promoting drugs (such as parathyroid hormone). From the current treatment situation, they all have problems such as general therapeutic effect, high cost of long-term treatment, certain side effects, and poor patient compliance. Therefore, the development of effective anti-osteoporosis drugs is urgently needed.

Plexins are a family of widely distributed transmembrane receptor proteins. Plexin-B2 is a member of the Plexin-B subfamily. So far, there have been no reports on the use of Plexin-B2 activators in the preparation of drugs for the treatment of osteoporosis.

Summary of the invention

The technical problem to be solved by the present invention is to overcome the problem of unsatisfactory therapeutic effect of existing osteoporosis treatment drugs, and to provide an aromatic compound as an activator of plexin protein-B2 and its use in the preparation of osteoporosis treatment drugs. The present invention can provide a research basis and theoretical basis for the development of a new drug for the treatment of osteoporosis.

The specific technical solutions provided by the present invention are as follows.

The present invention provides an application of an aromatic compound as an activator of plexin protein-B2, wherein the aromatic compound is one or more of a compound as shown in formula I, a compound as shown in formula II, and a compound as shown in formula III;

The present invention also provides an application of an aromatic compound in the preparation of a drug for preventing and/or treating a disease related to plexin protein-B2, wherein the aromatic compound is one or more of the compound shown in formula I, the compound shown in formula II, and the compound shown in formula III.

The present invention also provides an application of an aromatic compound in the preparation of a drug for preventing and/or treating osteoporosis, wherein the aromatic compound activator is one or more of a compound as shown in formula I, a compound as shown in formula II, and a compound as shown in formula III;

In the present invention, the aromatic compound is preferably the compound shown in Formula I, the compound shown in Formula II or the compound shown in Formula III, for example, the compound shown in Formula II.

In the present invention, K784-9621 (the compound shown in Formula II) is one of the small molecule activators of Plexin-B2, which can significantly increase the expression levels of Plexin-B2 protein and gene and activate its downstream signaling pathway.

In the present invention, L087-0246 (the compound shown in formula I), K784-9621 (the compound shown in formula II) and G856-6766 (the compound shown in formula III) can inhibit the expression level of osteoclast gene CTSK-mRNA.

The present invention also provides an application of an aromatic compound as an expression promoter for genes Runx2, Sp7, and Alp, wherein the aromatic compound is one or more of the compound shown in formula I, the compound shown in formula II, and the compound shown in formula III.

In the present invention, the aromatic compound is preferably the compound shown in Formula I, the compound shown in Formula II or the compound shown in Formula III, for example, the compound shown in Formula II.

The present invention also provides an application of an aromatic compound as an inhibitor of gene NFATc1, Rank, and CTSK expression, wherein the aromatic compound is one or more of the compound shown in formula I, the compound shown in formula II, and the compound shown in formula III.

In the present invention, the aromatic compound is preferably the compound shown in Formula I, the compound shown in Formula II or the compound shown in Formula III, for example, the compound shown in Formula II.

The present invention also provides an application of an aromatic compound in the preparation of a drug for preventing and/or treating postmenopausal osteoporosis, wherein the aromatic compound is the compound shown in Formula II.

The reagents and raw materials used in the present invention are commercially available.

The positive and progressive effects of the present invention are:

In the present invention, L087-0246 (a compound as shown in formula I), K784-9621 (a compound as shown in formula II) and G856-6766 (a compound as shown in formula III) can inhibit the expression level of the osteoclast gene CTSK-mRNA. In particular, K784-9621 in the present invention can not only inhibit osteoclastogenesis, but also significantly promote osteoblastogenesis, thereby significantly increasing bone mass and delaying the development of osteoporosis. Specifically:

(1) K784-9621 can significantly promote osteoblastogenesis in vitro. The expression of osteoblast-related genes Runx2, Sp7, and Alp after K784-9621 intervention was significantly increased compared with the control group;

(2) K784-9621 can significantly inhibit osteoclastogenesis in vitro. The expression of osteoclast-related genes NFATc1, Rank, and CTSK after K784-9621 intervention was significantly reduced compared with the control group;

(3) K784-9621 can improve bone mass, increase bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and bone formation rate, and reduce trabecular separation (Tb.Sp);

(4) K784-9621 can increase OCN ⁺ osteoblasts and reduce Trap ⁺ osteoclasts.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1A is a structural diagram of the small molecule compound in Example 1;

FIG1B is a schematic diagram of the qPCR results of the effects of various small molecule compounds on the expression level of the osteoclast gene CTSK;

FIG. 2 is a schematic diagram showing the effect of K784-9621 on Plexin-B2 protein; FIG. 2A is a schematic diagram showing the result of K784-9621 promoting the expression of Plexin-B2 protein; FIG. 2B is a statistical analysis diagram showing the effect of K784-9621 promoting the expression of Plexin-B2 protein;

FIG3 is a schematic diagram of the qPCR results of K784-9621 promoting Plexin-B2 gene expression;

FIG4 is a schematic diagram of the qPCR results of K784-9621 promoting the expression of osteogenesis-related genes;

FIG. 5 is a schematic diagram of the effect of K784-9621 on osteoclasts; FIG. 5A is a schematic diagram of tartrate-resistant acid phosphatase (Trap) staining of K784-9621 inhibiting osteoclastogenesis; FIG. 5B is a statistical analysis of the number of Trap-positive (Trap ⁺ ) osteoclasts; and FIG. 5C is a schematic diagram of qPCR results of K784-9621 inhibiting osteoclast-related gene expression;

FIG6 is a schematic diagram of the effect of K784-9621 on bone mass and bone morphological parameters of osteoporotic mice; FIG6A is a three-dimensional reconstruction of bone scans showing the increase in bone mass of postmenopausal osteoporotic mice after ovariectomy and intraperitoneal injection of K784-9621; FIG6B is a schematic diagram of micro-CT bone parameter analysis of mice under different treatment conditions;

FIG7 is a schematic diagram showing the effect of K784-9621 on the number of OCN-positive osteoblasts in osteoporotic mice; FIG7A is a schematic diagram showing osteocalcin (OCN) staining in mice under different treatment conditions, with arrows indicating OCN-positive (OCN ⁺ ) cells; FIG7B is a statistical analysis of the number of OCN ⁺ cells in mice under different treatment conditions;

Figure 8 is a schematic diagram of the effect of K784-9621 on the number of Trap-positive osteoclasts in osteoporotic mice; wherein, Figure 8A is a schematic diagram of Trap staining of mice under different treatment conditions, and arrows indicate Trap-positive (Trap ⁺ ) cells; Figure 8B is a statistical analysis of the number of Trap-positive (Trap ⁺ ) cells in mice under different treatment conditions.

Detailed ways

The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

Example 1: Small molecule compounds that can bind to Plexin-B2 are obtained by computer drug-aided design screening, and small molecule compounds that affect the expression level of osteoclast gene CTSK are screened;

1. Prepare 10 mg/ml storage solution: ① The small molecule compounds (as shown in Table 1) were obtained through computer drug-aided design screening and purchased from Chemdiv. Centrifuge at 1000 r/min for 5 mins; ② In an ultra-clean workbench, pipette 100 ul DMSO into 1 mg powder and mix by repeatedly pipetting; ③ Divide the mixed liquid into aliquots and store at -20 degrees.

2. Prepare 10ug/ml working solution: ① Take the aliquoted storage solution and place it at room temperature to rewarm; ② Add 1ul of storage solution to every 1ml of culture medium to prepare the working solution for subsequent cell experiments.

3. Mouse bone marrow mononuclear cells were inoculated at a density of 1×10^ ⁵ cells/well in α-MEM medium containing macrophage colony stimulating factor (M-CSF, 30 ng/ml), nuclear factor κB receptor activator ligand (Rankl, 100 ng/ml), 10% fetal bovine serum, and 1% double antibody, and divided into a control group (Control) (untreated group) and various small molecule compound treatment groups (the structures of each compound are shown in Figure 1A). The medium was changed every 3 days. After 9 days of differentiation culture, Trap staining was performed to detect the number of osteoclasts; after 7-8 days of differentiation culture, cell RNA was extracted, and the expression level of osteoclast gene CTSK-mRNA was detected by qPCR (the specific experimental method of qPCR is referenced: Chang-Jun Li, et al. Long noncoding RNABmncr regulatesmesenchymal stem cell fate during skeletal aging. J Clin Invest, 2018, 128(12): 5251-5266.).

See Figure 1B and Table 1. Each value is the mean ± SD of three tests. Compared with the Control group, *P < 0.05, **P < 0.01, ***p < 0.001.

Table 1

Group Relative expression level of CTSK Control 20.000±2.843 L087-0246 11.200±5.432* G639-1239 23.981±7.250 1109-0033 14.520±4.744 K784-9621 3.943±1.347** K781-0814 14.406±0.379 G717-1369 18.382±2.935 D678-0039 16.458±4.076 T630-1975 17.133±5.219 G856-6766 12.559±0.254*

The effects of the 9 compounds shown in Figure 1A on the expression level of the osteoclast gene CTSK are shown in Table 1 and Figure 1B. This result shows that K784-9621 has the most significant inhibitory effect on the osteoclast gene CTSK.

Example 2 K784-9621 can significantly promote the expression of Plexin-B2, promote osteoblastogenesis and inhibit osteoclastogenesis

(1) The specific experimental steps for detecting Plexin-B2 protein and gene expression are as follows:

Mouse bone marrow mononuclear cells were inoculated at a density of 1×10^ ⁵ cells/well in α-MEM medium containing macrophage colony stimulating factor (M-CSF, 30ng/ml), 10% fetal bovine serum, and 1% double antibody, and divided into a control group (Control) and a K784-9621 treatment group (K784-9621). After 48 hours of K784-9621 intervention, cell protein and RNA were extracted, and the expression levels of Plexin-B2 protein and gene were detected by WB and q-PCR, respectively. (Specific experimental method of WB reference: Chang-JunLi, et al. Long noncoding RNA Bmncr regulates mesenchymal stem cell fate during skeletal aging. J Clin Invest, 2018, 128 (12): 5251-5266.).

For specific results, please refer to Figures 2 and 3, Tables 2 and 3. Each value is expressed as the mean ± SD of three tests. Compared with the Control group, *P < 0.05, **P < 0.01, ***p < 0.001.

Table 2

Group Relative expression level of Plexin-B2 Control 0.442±0.051 K784-9621 0.990±0.175*

As can be seen from Figures 2A and 2B, K784-9621 can significantly increase the expression level of Plexin-B2 protein compared with the DMSO-treated Control group.

table 3

Group Relative expression level of Plexin-B2 Control 1.000±0.095 K784-9621 1.534±0.057**

As can be seen from Figure 3, compared with the Control group, K784-9621 can significantly increase the gene expression level of Plexin-B2.

(2) The specific experimental steps for detecting the expression level of osteogenesis-related genes are as follows:

Bone marrow mesenchymal stem cells (BMSCs) were ^seeded in α-MEM medium containing 0.1 mM dexamethasone, 10 mM β-glycerophosphate, 50 mM ascorbic acid-2-phosphate, and 10% fetal bovine serum at a cell density of 5×10 ³ /cm 2 , and were divided into a control group (Control) and a K784-9621 treatment group (K784-9621). The medium was changed every 3 days. After 6 days of differentiation culture, cell RNA was extracted, and the expression levels of osteogenic-related genes were detected by qPCR.

For specific results, see Figure 4 and Table 4. Each value is expressed as the mean ± SD of three tests. Compared with the Control group, *P < 0.05, **P < 0.01, ***p < 0.001.

Table 4

Group Runx2 relative expression level Relative expression level of Sp7 Alp relative expression level Control 1.059±0.073 1.039±0.101 1.000±0.067 K784-9621 1.722±0.085** 1.880±0.020** 1.898±0.042***

As can be seen from Figure 4 and Table 4, compared with the control group, the expression levels of mRNA of osteogenesis-related genes Runx2, Sp7, and Alp were significantly increased after treatment with K784-9621. This result shows that K784-9621 can promote the differentiation of BMSCs into osteoblasts.

(3) The specific experimental steps for detecting the expression level of osteoclast-related genes are as follows:

Mouse bone marrow mononuclear cells were inoculated at a density of 1×10^ ⁵ cells/well in α-MEM culture medium containing macrophage colony stimulating factor (M-CSF, 30ng/ml), nuclear factor κB receptor activator ligand (Rankl, 100ng/ml), 10% fetal bovine serum, and 1% double antibody. They were divided into a control group (Control) and a K784-9621 treatment group (K784-9621). The medium was changed every 3 days. After 9 days of differentiation culture, Trap staining was performed to detect the number of osteoclasts; after 7-8 days of differentiation culture, cell RNA was extracted, and the expression levels of osteoclast-related genes were detected by qPCR.

See Figure 5 and Table 5. Each value is expressed as the mean ± SD of three tests. Compared with the Control group, *P < 0.05, **P < 0.01, ***p < 0.001.

table 5

As can be seen from Figures 5A and 5B and Table 5 , the number of osteoclasts was significantly reduced after K784-9621 intervention compared with the control group.

Table 6

Group NFATc1 relative expression level Rank relative expression level CTSK relative expression level Control 1.000±0.133 1.000±0.124 1.000±0.113 K784-9621 0.517±0.032** 0.444±0.054** 0.143±0.054**

As can be seen from Figure 5C and Table 6, compared with the control group, the mRNA expression levels of osteoclast-related genes NFATc1, Rank, and CTSK were significantly reduced after K784-9621 treatment. This result shows that K784-9621 can inhibit osteoclastogenesis.

Example 3 K784-9621 can increase bone mass in postmenopausal osteoporotic mice by promoting bone formation and inhibiting bone resorption

Postmenopausal osteoporosis (PMO) is a metabolic bone disease caused by a decrease in estrogen, which leads to significant bone loss. It is a high-conversion osteoporosis, characterized by a significant increase in osteoclastogenesis and a slight increase in osteoblasts. Ultimately, bone absorption is greater than bone formation, leading to a significant decrease in bone mass. Since we previously observed that K784-9621 can significantly inhibit osteoclastogenesis and promote osteoblastogenesis in vitro, we further speculated whether it has an improvement effect on the bone loss of postmenopausal osteoporosis. After 1 week of adaptive feeding, C57BL/C mice were divided into sham operation + vehicle group (sham), ovariectomy + vehicle group (OVX) and ovariectomy + K784-9621 group (K784-9621). One week after the operation, vehicle or K784-9621 (70 mg/kg, once a day) was injected intraperitoneally. Seven weeks after the injection, the mice in each group were weighed, blood was collected from the apex of the heart, and bilateral femurs were collected. One side of the femur was fixed with formaldehyde and placed in a sample tube for CT scanning, and then micro-CT analysis and three-dimensional reconstruction were performed to observe the bone mass; the other side of the femur was decalcified in 10% EDTA decalcification solution for 3 weeks, embedded in paraffin, and sectioned, and then OCN and Trap staining were performed respectively.

For specific results, see Figures 6-8 and Tables 7-9. Each value is expressed as the mean ± SD of three tests. Compared with the sham group, *P<0.05, **P<0.01, ***p<0.001; compared with the OVX group, #P<0.05, ##P<0.01, ###p<0.001.

Table 7

From the three-dimensional reconstruction of bone scan in Figure 6A, it can be seen that compared with the sham group, the bone mass of the OVX group after ovariectomy was significantly reduced, while the bone mass of mice treated with K784-9621 was significantly increased compared with the OVX group. From the mirco-CT analysis parameters in Figure 6B, it can be seen that compared with the sham group, the bone volume fraction (BV/TV), trabecular number (Tb.N) and trabecular thickness (Tb.Th) of the OVX group mice were significantly reduced, while the trabecular separation (Tb.Sp) was significantly increased, and K784-9621 can improve the above bone mass parameters of mice after ovariectomy. This result shows that K784-9621 can improve the bone loss of postmenopausal osteoporosis mice.

Table 8

Group OCN ⁺ osteoblasts (N/mm) sham 8.723±0.861 OVX 8.690±0.813 K784-9621 10.616±0.556##

As can be seen from Figure 7A and Figure 7B, compared with the sham group, the OCN ⁺ osteoblasts in the OVX group did not change significantly; compared with the OVX group, K784-9621 can significantly increase the number of osteoblasts. This result shows that K784-9621 can promote osteoblastogenesis in vivo.

Table 9

Group Trap ⁺ osteoclasts (N/mm) sham 3.682±0.189 OVX 8.392±0.587*** K784-9621 6.226±0.731###

As can be seen from Figure 8A and Figure 8B, compared with the sham group, the number of Trap ⁺ osteoclasts in the OVX group increased significantly; compared with the OVX group, K784-9621 could significantly reduce the number of osteoclasts. This result shows that K784-9621 can inhibit osteoclastogenesis in vivo.

The above results collectively indicate that K784-9621 can increase the bone mass of postmenopausal osteoporosis mice by inhibiting osteoclastogenesis and promoting osteoblastogenesis.

The above is only a preferred specific implementation manner of the present invention, and the protection scope of the present invention is not limited thereto. Any simple change or equivalent replacement of the technical solution that can be obviously obtained by any technician familiar with the technical field within the technical scope disclosed in the present invention shall fall under the protection of the present invention.

Claims (1)

1. Use of an aromatic compound in the preparation of a drug for preventing and/or treating osteoporosis, wherein the aromatic compound is a compound as shown in formula I;