Use of amide alkaloids for the preparation of inhibitors of osteoclast differentiation

Abstract

The application relates to application of an amide alkaloid in preparation of an osteoclast differentiation inhibitor, wherein the amide alkaloid has a structure as shown in formula (I) or formula (II). The specific amide alkaloid of the application has a significant inhibiting effect on RANKL-induced osteoclast differentiation, has small cytotoxicity, has good anti-osteoclast differentiation activity, and can be used for preparing the osteoclast differentiation inhibitor.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically, to the application of amide alkaloids in the preparation of osteoclast differentiation inhibitors. Background Technology

[0002] Osteoclasts are bone-degrading cells that initiate normal bone remodeling by increasing their bone resorption activity. Osteoclasts originate from precursors in the bone marrow or monocyte lineage. These precursor cells differentiate into osteoclasts upon induction by receptor activator of nuclear factor-κB ligand (RANKL). Osteoclasts are currently the only known cells with bone resorption function and play a crucial role in bone formation, development, remodeling, and bone mass regulation. The body's skeletal health is determined by the dynamic balance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. Abnormal osteoclast differentiation can lead to various skeletal diseases, such as osteoporosis, osteoarthritis, and bone metastases.

[0003] Currently, bone resorption inhibitors that inhibit osteoclast differentiation are mainly used as the main means of long-term treatment for osteoporosis in clinical practice. The bone resorption inhibitors currently in use in clinical practice mainly include: (1) bisphosphonates such as glycolate phosphonate, clodronate, pamidronate, halogenated phosphates, Allen phosphate, risedronate sodium, zoledronic phosphate, ibandronate sodium, etc.; (2) estrogen receptor modulators such as tamoxifen, toremifene, droloxifen, raloxifene, azoxifen, bardoxifene, epsilonide, etc.; (3) the novel bone resorption inhibitor RANKL monoclonal antibody drug denosumab (trade name Prolia). While the aforementioned drugs can prevent bone density loss to some extent, they also have varying degrees of side effects. For example, bisphosphonates, first-line anti-osteoporosis drugs, can cause increased bone fragility and mandibular osteonecrosis; raloxifene can cause venous thrombosis; and RANKL monoclonal antibodies are expensive and carry serious side effects such as high risk of infection, hypercalcemia, mandibular osteonecrosis, and accelerated bone loss after discontinuation. Therefore, there is an urgent need to develop safer and more effective small molecule drugs to inhibit osteoclast differentiation. Summary of the Invention

[0004] The primary objective of this invention is to overcome the problems of significant side effects of bone resorption inhibitors that inhibit osteoclast differentiation and the limited availability of small molecule drugs that inhibit RANKL-induced osteoclast differentiation in the prior art. This invention provides the application of amide alkaloids in the preparation of osteoclast differentiation inhibitors. The specific amide alkaloids of this invention exhibit significant inhibitory effects on RANKL-induced osteoclast differentiation, with low cytotoxicity and good osteoclast differentiation inhibitory activity, and can be used to prepare osteoclast differentiation inhibitors.

[0005] The above-mentioned objective of the present invention is achieved through the following technical solution: Application of amide alkaloids in the preparation of osteoclast differentiation inhibitors, wherein the amide alkaloids have the structure shown in formula (I) or formula (II): , ; Wherein, R1 is H or OH; R2, R3, and R4 are independently H, OH, or C. 1~6 alkoxy group, or R2 is H, OH or C 1~6 The alkoxy groups, R3 and R4, together with the carbon atoms attached to them, form the following segment structure: ; In formula (Ⅰ), It is either a carbon-carbon double bond or a carbon-carbon single bond.

[0006] The amide alkaloids of this invention can be extracted from Lycium bark, see the literature "Research Progress on Chemical Composition of Lycium Bark", Lu Lihe et al.

[0007] The inventors of this invention have discovered that specific amide alkaloids have a significant inhibitory effect on RANKL-induced osteoclast differentiation, and have low cytotoxicity, exhibiting good anti-osteoclast differentiation activity, and can be used to prepare osteoclast differentiation inhibitors.

[0008] Preferably, the C 1~6 The alkoxy group is a methoxy group.

[0009] Preferably, the amide alkaloid is any one of compounds numbered 1 to 6: , , , , , .

[0010] The inventors of this invention further discovered that the amide alkaloids numbered 1, 4, and 6 had lower half-maximal inhibitory concentrations (WMCs) for inhibiting osteoclast differentiation and better anti-osteoclast differentiation activity; the amide alkaloids numbered 1, 2, and 6 had higher selective inhibition constants (SI) for inhibiting osteoclast differentiation and were more less toxic and effective.

[0011] Preferably, the osteoclast differentiation inhibitor is a drug for treating and / or preventing bone diseases caused by osteoclast differentiation.

[0012] More preferably, the drug further includes pharmaceutically acceptable excipients.

[0013] More preferably, the excipient is at least one of a lubricant, filler, binder, disintegrant, surfactant, antioxidant, or pH adjuster.

[0014] More preferably, the dosage form of the drug is an injection, tablet, oral liquid, granules, or capsule.

[0015] More preferably, the bone disease is osteoporosis, osteoarthritis, or bone metastasis.

[0016] Preferably, the osteoclast differentiation inhibitor is an inhibitor of RANKL-induced osteoclast differentiation.

[0017] Preferably, the amide alkaloid is a deuterium isotope variant of the amide alkaloid.

[0018] Compared with the prior art, the beneficial effects of the present invention are: The specific amide alkaloids of this invention have a significant inhibitory effect on RANKL-induced osteoclast differentiation, and have low cytotoxicity. They have good anti-osteoclast differentiation activity and can be used to prepare osteoclast differentiation inhibitors. Attached Figure Description

[0019] Figure 1 The figure shows the results of the osteoclast differentiation inhibition and cytotoxicity experiments of amide alkaloids in Example 1, where: A is the IC50 value of the osteoclast differentiation inhibition experiment of amide alkaloids in Example 1. 50 Curve B; B represents the CC curve from the cytotoxicity test of the amide alkaloids in Example 1. 50 Curve; C is a representative image of the experimental results of the osteoclast differentiation inhibition experiment of amide alkaloids in Example 1.

[0020] Figure 2 Figure 1 shows the results of the osteoclast differentiation inhibition and cytotoxicity experiments of amide alkaloids in Example 2, where: A is the IC50 value of the osteoclast differentiation inhibition experiment of amide alkaloids in Example 2. 50 Curve B; B represents the CC curve from the cytotoxicity test of amide alkaloids in Example 2. 50 Curve; C is a representative image of the experimental results of the osteoclast differentiation inhibition experiment of amide alkaloids in Example 2.

[0021] Figure 3 Figure 1 shows the results of the osteoclast differentiation inhibition and cytotoxicity experiments of amide alkaloids in Example 3, where: A is the IC50 value of the osteoclast differentiation inhibition experiment of amide alkaloids in Example 3. 50 Curve B; B represents the CC curve from the cytotoxicity test of the amide alkaloids in Example 3. 50 Curve; C is a representative image of the experimental results of the osteoclast differentiation inhibition experiment of amide alkaloids in Example 3.

[0022] Figure 4Figure 1 shows the results of the osteoclast differentiation inhibition and cytotoxicity experiments of amide alkaloids in Example 4, where: A is the IC50 value of the osteoclast differentiation inhibition experiment of amide alkaloids in Example 4. 50 Curve B represents the CC curve from the cytotoxicity test of amide alkaloids in Example 4. 50 Curve; C is a representative image of the experimental results of the osteoclast differentiation inhibition experiment of amide alkaloids in Example 4.

[0023] Figure 5 Figure 1 shows the results of the osteoclast differentiation inhibition and cytotoxicity experiments of amide alkaloids in Example 5, where: A is the IC50 value of the osteoclast differentiation inhibition experiment of amide alkaloids in Example 5. 50 Curve B; B represents the CC curve from the cytotoxicity test of the amide alkaloids in Example 5. 50 Curve; C is a representative image of the experimental results of the osteoclast differentiation inhibition experiment of amide alkaloids in Example 5.

[0024] Figure 6 Figure 1 shows the results of the osteoclast differentiation inhibition and cytotoxicity experiments of amide alkaloids in Example 6, where: A is the IC50 value of the osteoclast differentiation inhibition experiment of amide alkaloids in Example 6. 50 Curve B; B represents the CC curve from the cytotoxicity test of the amide alkaloids in Example 6. 50 Curve; C is a representative image of the experimental results of the osteoclast differentiation inhibition experiment of amide alkaloids in Example 6. Detailed Implementation

[0025] To more clearly and completely describe the technical solution of the present invention, the present invention will be further described in detail below through specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention. Various changes can be made within the scope of the claims of the present invention.

[0026] The present invention will be further described below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. It should be understood that these embodiments and examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. The purpose of providing these embodiments and examples is to make the disclosure of the present invention more thorough and complete. It should also be understood that the present invention can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present invention, and the equivalent forms obtained also fall within the protection scope of the present invention. For example, features described or described as part of one embodiment can be combined in a suitable manner in another embodiment to produce new embodiments. In addition, numerous specific details are set forth in the following description to provide a more complete understanding of the present invention. It should be understood that the present invention can be implemented without one or more of these details. Unless otherwise stated, the raw materials and reagents used in the embodiments of the present invention are conventionally purchased raw materials and reagents. The mass or weight of the relevant components mentioned in the specification of the embodiments of the present invention can refer not only to the specific content of each component, but also to the proportional relationship of mass or weight between the components. Therefore, as long as the content of the relevant components is scaled up or down proportionally according to the specification of the embodiments of the present invention, it is within the scope disclosed in the specification of the embodiments of the present invention. Specifically, the mass or weight described in the specification of the embodiments of the present invention can be units known in the chemical field such as μg, mg, g, kg.

[0027] Example 1 This embodiment provides the application of amide alkaloids in the preparation of osteoclast differentiation inhibitors. The amide alkaloid in this embodiment is compound number 1, with the chemical name trans-N-p-hydroxyphenylethyl caffeamide, and its structure is as follows: .

[0028] 1. Extraction and culture of mouse BMM cells Tibial bone marrow cells from 3-6 month old C57BL / 6 mice were cultured in MEM-α complete medium (containing 10% FBS and 1% penicillin antibiotics) with 30 ng / mL M-CSF. After 24 h, the cells adhered to the culture vessel; these adherent cells were bone marrow-derived macrophages (BMMs). After successful culture, the adherent cells were detached from the culture vessel using the complete medium and prepared into a cell suspension for subsequent experiments.

[0029] 2. Cytotoxicity test (1) Cell plating and grouping BMMs cells were diluted to a density of 1×10⁻⁶ cells using MEM-α complete medium containing 30 ng / mL M-CSF. 5A suspension of cells / mL was added to 100 μL per well in a 96-well cell culture plate and cultured in a CO2 incubator for 24 h. Cells were divided into a control group and a treatment group, with three replicates in each group.

[0030] (2) Drug intervention In the treatment group, amide alkaloids were prepared into a 10 mM stock solution using DMSO. Different concentrations of the stock solution (0.03 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, 33 μM) were prepared using MEM-α complete medium containing 30 ng / mL M-CSF. The samples were cultured for 2 consecutive days, and cytotoxicity was detected on the 3rd day.

[0031] Control group: The treatment method for the control group was basically the same as that for the drug treatment group, except that the test drug was not added.

[0032] (3) Test methods and result processing After drug intervention, the culture medium was discarded. The MTT assay [3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide] was used to determine the cell growth inhibition rate. Four hours after adding the MTT reagent, the precipitate was dissolved in DMSO, and the absorbance (OD value) was measured at 490 nm using a microplate reader. The growth inhibition rate of the drug on cells was calculated using the following formula:

[0033] The drug concentration and its corresponding cell growth inhibition rate were input into GraphPad Prism 8 software. The Log value of the compound concentration was plotted on the x-axis, and the inhibition rate on the y-axis. The CC value was calculated using the software's built-in nonlinear fitting curve method. 50 value.

[0034] 3. Osteoclast differentiation inhibition experiment (1) Cell plating and grouping BMMs cells were diluted to a density of 1×10⁻⁶ cells using MEM-α complete medium containing 30 ng / mL M-CSF. 5 A suspension of cells / mL was added at a rate of 100 μL to each well of a 96-well cell culture plate and cultured in a CO2 incubator for 24 h. The culture medium was then discarded. Cells were divided into a control group and a treatment group, with three replicates in each group.

[0035] (2) Osteoclast differentiation and drug intervention methods Drug-treated groups: RANKL and M-CSF were added to complete culture medium at final concentrations of 100 ng / mL and 30 ng / mL, respectively. Simultaneously, different concentrations of amide alkaloids (0.03 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 33 μM) were added for culturing. The culture was continued for 5 days, with the medium containing the same concentrations of RANKL, M-CSF, and amide alkaloids replaced every 2 days. The experiment ended on day 5. Cells were fixed with 4% paraformaldehyde. Three replicates were performed for each concentration.

[0036] Control group: The treatment method for the control group was basically the same as that for the drug administration group, except that no amide alkaloids were added.

[0037] (3) Methods for detecting osteoclast differentiation activity.

[0038] Osteoclasts were washed with 4% paraformaldehyde using PBS buffer and then stained with a TRAP staining kit. After staining, osteoclasts were observed and counted under an inverted microscope. The inhibition rate of RANKL-induced osteoclast differentiation by amide alkaloids was calculated using the following formula:

[0039] The drug concentrations and their corresponding osteoclast inhibition rates were input into GraphPad Prism 8 software. Based on the inhibition rates of each compound on osteoclast differentiation, the IC50 was calculated using the software's built-in nonlinear fitting curve method. 50 value.

[0040] 4. Calculation of the suppression constant The selected inhibition constant (SI) can be used to evaluate the safety of a drug. The formula for calculating the selected inhibition constant is: SI = CC 50 / IC 50 SI < 1.0 indicates that the compound is toxic and ineffective, while SI ≥ 1.0 indicates that the compound is low in toxicity and effective.

[0041] Example 2 This embodiment provides the application of amide alkaloids in the preparation of osteoclast differentiation inhibitors. The amide alkaloid in this embodiment is compound number 2, with the chemical name dihydrocaffeoyltyramine, and its structure is as follows: .

[0042] The cytotoxicity and osteoclast differentiation inhibition activities of the amide alkaloids in this embodiment were determined according to the experimental method of Example 1.

[0043] Example 3 This embodiment provides the application of amide alkaloids in the preparation of osteoclast differentiation inhibitors. The amide alkaloid in this embodiment is compound number 3, with the chemical name N-trans-coumaroyloctopamine, and its structure is as follows: .

[0044] The cytotoxicity and osteoclast differentiation inhibition activities of the amide alkaloids in this embodiment were determined according to the experimental method of Example 1.

[0045] Example 4 This embodiment provides the application of amide alkaloids in the preparation of osteoclast differentiation inhibitors. The amide alkaloid in this embodiment is compound number 4, with the chemical name trans-N-feruloylnorsynephrine, and its structure is as follows: .

[0046] The cytotoxicity and osteoclast differentiation inhibition activities of the amide alkaloids in this embodiment were determined according to the experimental method of Example 1.

[0047] Example 5 This embodiment provides the application of amide alkaloids in the preparation of osteoclast differentiation inhibitors. The amide alkaloid in this embodiment is compound number 5, with the chemical name grossamide, and its structure is as follows: .

[0048] The cytotoxicity and osteoclast differentiation inhibition activities of the amide alkaloids in this embodiment were determined according to the experimental method of Example 1.

[0049] Example 6 This embodiment provides the application of amide alkaloids in the preparation of osteoclast differentiation inhibitors. The amide alkaloid in this embodiment is compound number 6, with the chemical name 1,2-dihydro-6,8-dimethoxy-7-hydroxy-1-(3,4-dihydroxyphenylethyl)-aza-1-aza-2-di-[2-(4-hydroxyphenyl)ethyl]-2,3-naphthyldimethylamide, and its structure is as follows: .

[0050] The cytotoxicity and osteoclast differentiation inhibition activities of the amide alkaloids in this embodiment were determined according to the experimental method of Example 1.

[0051] Test Results Figures 1-6 A represents the IC50 values ​​of the osteoclast differentiation inhibition experiments of amide alkaloids in Examples 1-6. 50 curve; Figures 1-6B represents the CC values ​​from the cytotoxicity experiments of the amide alkaloids in Examples 1-6. 50 curve; Figures 1-6 C represents representative images of the osteoclast differentiation inhibition experiments of amide alkaloids in Examples 1-6. From Figures 1-6 As can be seen from C, the amide alkaloids of the present invention can inhibit RANKL-induced osteoclast differentiation in a dose-dependent manner.

[0052] Table 1 shows the test data of cytotoxicity and osteoclast differentiation inhibition activity of the amide alkaloids in Examples 1-6, as well as the calculated selection inhibition constants.

[0053] Table 1. Experimental results of Examples 1-6

[0054] Table 1 shows the cytotoxicity (CC) of the amide alkaloids in Examples 1-6. 50 All values ​​were above 204.30±96.40 µM, with an osteoclast inhibition IC50 value. 50 All values ​​were below 14.34 ± 6.22 µM, and the selection inhibition constant (SI) was above 8.29, indicating that the specific amide alkaloids of this invention have a significant inhibitory effect on RANKL-induced osteoclast differentiation, and have low cytotoxicity and good anti-osteoclast differentiation activity, and can be used to prepare osteoclast differentiation inhibitors.

[0055] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. The application of amide alkaloids as the sole active ingredient in the preparation of drugs for treating and / or preventing osteoporosis caused by osteoclast differentiation, characterized in that, The amide alkaloids have the structure shown in number 1: .

2. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

3. The application according to claim 2, characterized in that, The excipients are at least one of lubricants, fillers, binders, disintegrants, surfactants, antioxidants, or pH adjusters.

4. The application according to claim 1, characterized in that, The dosage form of the drug is injection, tablet, oral liquid, granules or capsule.

5. The application according to claim 1, characterized in that, The drug is an inhibitor of RANKL-induced osteoclast differentiation.

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