Application of amide alkaloid in preparation of osteoclast differentiation inhibitor

Abstract

The invention relates to application of amide alkaloid in preparation of an osteoclast differentiation inhibitor, the amide alkaloid has a structure as shown in a formula (I) or a formula (II): # imgabs0 # imgabs1 #. The specific amide alkaloid has a remarkable inhibiting effect on RANKL-induced osteoclast differentiation, is low in cytotoxicity, has good osteoclast differentiation resisting activity, and can be used for preparing an osteoclast differentiation inhibitor. The compound can be used for preparing an osteoclast differentiation inhibitor.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and more specifically, to the application of amide alkaloids in the preparation of osteoclast differentiation inhibitors. Background Art

[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 monocytic lineage. The precursor cells are induced by receptor activator of nuclear factor-κB ligand (RANKL) to differentiate into osteoclasts. Osteoclasts are the only known cells with bone resorption function and play a key role in the formation, development, reconstruction and regulation of bone mass. The body's bone health is determined by the dynamic balance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. Abnormal differentiation of osteoclasts may lead to a variety of bone diseases, such as osteoporosis, osteoarthritis and bone metastasis.

[0003] At present, bone resorption inhibitors that inhibit osteoclast differentiation are mainly used as the main means of long-term treatment for osteoporosis in clinical practice. The main bone resorption inhibitors currently in use in clinical practice include: (1) bisphosphonates such as glycolic acid phosphonates, clodronate, pamidronate, halogenated phosphates, eldronate phosphate, risedronate sodium, zoledronic acid phosphate, ibandronate sodium, etc.; (2) estrogen receptor modulators such as tamoxifen, toremifene, droloxifene, raloxifene, arzoxifene, bazedoxifene, ipriflavone, etc.; (3) a new type of bone resorption inhibitor RANKL monoclonal antibody drug Denosumab (trade name Prolia). Although the above drugs can prevent the decrease of bone density to a certain extent, they have side effects of varying degrees. For example, bisphosphonates, the first-line anti-osteoporosis drugs in clinical practice, can cause increased bone fragility and mandibular osteonecrosis; raloxifene can cause venous thrombosis; RANKL monoclonal antibodies are expensive and have serious side effects such as high infection risk, hypercalcemia, mandibular osteonecrosis, and increased bone loss after drug withdrawal. Therefore, it is urgent to develop safer and more effective small molecule drugs that inhibit osteoclast differentiation. Summary of the invention

[0004] The primary purpose of the present invention is to overcome the problems of large side effects of bone resorption inhibitors that inhibit osteoclast differentiation in the above-mentioned prior art and the small number of small molecule drugs that inhibit RANKL-induced osteoclast differentiation, and to provide the use of amide alkaloids in the preparation of osteoclast differentiation inhibitors. The specific amide alkaloids of the present invention have a significant inhibitory effect on RANKL-induced osteoclast differentiation, low cytotoxicity, good osteoclast differentiation inhibitory activity, and can be used to prepare osteoclast differentiation inhibitors.

[0005] The above-mentioned object of the present invention is achieved by the following technical solutions:

[0006] The use of amide alkaloids in the preparation of osteoclast differentiation inhibitors, wherein the amide alkaloids have a structure as shown in formula (I) or formula (II):

[0007]

[0008] Wherein, R1 is H or OH;

[0009] R2, R3, R4 are independently H, OH or C 1～6 Alkoxy, or R2 is H, OH or C 1～6 Alkoxy, R3, R4 and the carbon atoms to which they are connected together form the following fragment structure:

[0010]

[0011] In formula (I), It is a carbon-carbon double bond or a carbon-carbon single bond.

[0012] The amide alkaloids of the present invention can be extracted from the cortex lycopersicum, as shown in the document "Progress in Research on Chemical Components of Cortex lycopersicum", Lu Lihe et al.

[0013] The inventors of the present invention have found that specific amide alkaloids have a significant inhibitory effect on RANKL-induced osteoclast differentiation, have low cytotoxicity, have good anti-osteoclast differentiation activity, and can be used to prepare osteoclast differentiation inhibitors.

[0014] Preferably, the C 1～6 Alkoxy is methoxy.

[0015] Preferably, the amide alkaloid is any one of the compounds numbered 1 to 6:

[0016]

[0017] The inventors of the present invention further studied and found that the amide alkaloids numbered 1, 4, and 6 have lower half-inhibitory concentrations for inhibiting osteoclast differentiation and better anti-osteoclast differentiation activity; the amide alkaloids numbered 1, 2, and 6 have higher selective inhibition constants (SI) for inhibiting osteoclast differentiation and are less toxic and effective.

[0018] Preferably, the osteoclast differentiation inhibitor is a drug for treating and / or preventing bone diseases caused by osteoclast differentiation.

[0019] More preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0020] More preferably, the auxiliary material is at least one of a lubricant, a filler, a binder, a disintegrant, a surfactant, an antioxidant or a pH adjuster.

[0021] More preferably, the drug is in the form of injection, tablet, oral solution, granule or capsule.

[0022] More preferably, the bone disease is osteoporosis, osteoarthritis or bone metastasis.

[0023] Preferably, the osteoclast differentiation inhibitor is an inhibitor of RANKL-induced osteoclast differentiation.

[0024] Preferably, the amide alkaloid is a deuterated isotope variant of the amide alkaloid.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The specific amide alkaloids of the present invention have a significant inhibitory effect on RANKL-induced osteoclast differentiation, have low cytotoxicity, have good anti-osteoclast differentiation activity, and can be used to prepare osteoclast differentiation inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 A is the IC of the osteoclast differentiation inhibition experiment of amide alkaloids in Example 1 50 curve; Figure 1 B is the CC of the cytotoxicity test of amide alkaloids in Example 1 50 curve; Figure 1 C is a representative picture of the experimental results of the osteoclast differentiation inhibition experiment of amide alkaloids in Example 1.

[0028] Figure 2 A is the IC of the osteoclast differentiation inhibition experiment of amide alkaloids in Example 2 50 curve; Figure 2 B is the CC of the cytotoxicity test of amide alkaloids in Example 2 50 curve; Figure 2 C is a representative picture of the experimental results of the osteoclast differentiation inhibition experiment of amide alkaloids in Example 2.

[0029] Figure 3 A is the IC of the osteoclast differentiation inhibition experiment of amide alkaloids in Example 3 50 curve; Figure 3 B is the CC of the cytotoxicity test of amide alkaloids in Example 3 50 curve; Figure 3 C is a representative picture of the experimental results of the osteoclast differentiation inhibition experiment of amide alkaloids in Example 3.

[0030] Figure 4 A is the IC of the osteoclast differentiation inhibition experiment of amide alkaloids in Example 450 curve; Figure 4 B is the CC of the cytotoxicity test of amide alkaloids in Example 4 50 curve; Figure 4 C is a representative picture of the experimental results of the osteoclast differentiation inhibition experiment of amide alkaloids in Example 4.

[0031] Figure 5 A is the IC of the osteoclast differentiation inhibition experiment of amide alkaloids in Example 5 50 curve; Figure 5 B is the CC of the cytotoxicity test of amide alkaloids in Example 5 50 curve; Figure 5 C is a representative picture of the experimental results of the osteoclast differentiation inhibition experiment of amide alkaloids in Example 5.

[0032] Figure 6 A is the IC of the osteoclast differentiation inhibition experiment of amide alkaloids in Example 6 50 curve; Figure 6 B is the CC of the cytotoxicity test of amide alkaloids in Example 6 50 curve; Figure 6 C is a representative picture of the experimental results of the osteoclast differentiation inhibition experiment of amide alkaloids in Example 6. DETAILED DESCRIPTION

[0033] In order to more clearly and completely describe the technical solution of the present invention, the present invention is further described in detail through specific embodiments below. It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention. Various changes can be made within the scope of the rights of the present invention.

[0034] The present invention is further described below in conjunction with specific embodiments, but the embodiments do not limit the present invention in any form. It should be understood that these embodiments and embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. The purpose of providing these embodiments and embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. It should also be understood that the present invention can be implemented in many different forms, and is not limited to the embodiments and embodiments described herein. Those skilled in the art can make various changes or modifications without violating the connotation of the present invention, and the equivalent forms obtained also fall within the scope of protection of the present invention. For example, the features described or described as part of an embodiment can be combined in another embodiment in a suitable manner to produce a new embodiment. In addition, in the following description, a large number of specific details are given in order to provide a more comprehensive 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 specified, the raw material reagents used in the embodiments of the present invention are conventionally purchased raw material reagents. The mass or weight of the relevant components mentioned in the description of the embodiments of the present invention can not only refer to the specific content of each component, but also represent the proportional relationship of the mass or weight between the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the description of the embodiments of the present invention, it is within the scope disclosed in the description of the embodiments of the present invention. Specifically, the mass or weight described in the description of the embodiments of the present invention may be units known in the chemical field such as μg, mg, g, kg, etc.

[0035] Example 1

[0036] This example provides the use of amide alkaloids in the preparation of osteoclast differentiation inhibitors. The amide alkaloids in this example are compounds numbered 1, whose chemical name is trans-N-p-hydroxyphenylethyl caffeamide, and whose structure is as follows:

[0037]

[0038] 1. Extraction and culture of mouse BMMs cells

[0039] Tibia bone marrow cells from 3-6 month old C57BL / 6 mice were cultured in MEM-α complete medium (containing 10% FBS and 1% double antibody) containing 30ng / mL M-CSF. After 24 hours, the cells adhered to the wall, and the adherent cells were bone marrow-derived macrophages (BMMs). After successful culture, the adherent cells were blown off with complete medium and prepared into cell suspension for subsequent experiments.

[0040] 2. Cytotoxicity assay

[0041] (1) Cell plating and grouping

[0042] The BMMs cells were diluted to a density of 1×10 in MEM-α complete medium containing 30 ng / mL M-CSF. 5 The suspension of 100 μL / mL was added to each well of a 96-well cell culture plate and cultured in a carbon dioxide incubator for 24 hours. The cells were divided into a control group and a drug-treated group, with 3 replicates in each group.

[0043] (2) Drug intervention

[0044] Drug administration group: 10 mM stock solution of amide alkaloids was prepared using DMSO, and different concentrations of test samples (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 continuously for 2 days, and the cytotoxicity was detected on the 3rd day.

[0045] Control group: The treatment method of the control group is basically the same as that of the drug-treated group, except that no test drug is added.

[0046] (3) Test methods and result processing

[0047] After the drug intervention, the culture medium was discarded. The MTT [3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide] method was used to determine the drug-treated cells. After 4 hours of adding the MTT reagent, the precipitate was dissolved with DMSO, and finally the absorbance value (OD value) was measured at a wavelength of 490nm using an enzyme marker. The growth inhibition rate of the drug on the cells was calculated according to the following formula:

[0048]

[0049] The drug concentration and its corresponding cell growth inhibition rate were input into GraphPad Prism 8 software, with the Log value of the compound concentration as the horizontal axis and the inhibition rate as the vertical axis. The nonlinear fitting curve method provided by the software was used to calculate CC. 50 value.

[0050] 3. Osteoclast differentiation inhibition experiment

[0051] (1) Cell plating and grouping

[0052] The BMMs cells were diluted to a density of 1×10 in MEM-α complete medium containing 30 ng / mL M-CSF. 5 The suspension of cells / mL was added to each well of a 96-well cell culture plate at a rate of 100 μL, and the culture medium was discarded after culturing in a carbon dioxide incubator for 24 hours. The cells were divided into a control group and a drug-treated group, with 3 replicates in each group.

[0053] (2) Osteoclast differentiation and drug intervention methods

[0054] Drug administration group: RANKL and M-CSF were added to the complete culture medium, and the final concentrations of RANKL and M-CSF were 100ng / mL and 30ng / mL, respectively. At the same time, different concentrations of amide alkaloids (0.03μM, 0.1μM, 0.3μM, 1μM, 3μM, 10μM, 33μM) were added for culture. The culture was continued for 5 days, and the culture medium with the same concentration of RANKL, M-CSF and amide alkaloids was replaced every 2 days. The experiment was terminated on the 5th day. The cells were fixed with 4% paraformaldehyde. Three parallel wells were set for each concentration.

[0055] Control group: The treatment method of the control group was basically the same as that of the drug-treated group, except that no amide alkaloids were added.

[0056] (3) Osteoclast differentiation activity detection method.

[0057] 4% paraformaldehyde was washed with PBS buffer, and then osteoclasts were stained with TRAP staining kit. After staining, osteoclasts were observed and counted under an inverted microscope. The inhibition rate of amide alkaloids on RANKL-induced osteoclast differentiation was calculated according to the following formula:

[0058]

[0059] The drug concentration and its corresponding osteoclast inhibition rate were input into GraphPad Prism 8 software. According to the inhibition rate of osteoclast differentiation of each compound, the nonlinear fitting curve method provided by the software was used to calculate the IC 50 value.

[0060] 4. Calculation of the selection inhibition constant

[0061] The selective inhibition constant (SI) can be used to evaluate the safety of drug use. The formula for calculating the selective inhibition constant is: SI = CC 50 / IC 50 ; SI < 1.0 indicates that the compound is toxic and ineffective, and SI ≥ 1.0 indicates that the compound is less toxic and effective.

[0062] Example 2

[0063] This embodiment provides the use of amide alkaloids in the preparation of osteoclast differentiation inhibitors. The amide alkaloids in this embodiment are compounds numbered 2, the chemical name of which is dihydrocaffeoyltyramide, and the structure thereof is as follows:

[0064]

[0065] The cytotoxicity and osteoclast differentiation inhibitory activity of the amide alkaloids of this example were determined according to the experimental method of Example 1.

[0066] Example 3

[0067] This example provides the use of an amide alkaloid in the preparation of an osteoclast differentiation inhibitor. The amide alkaloid in this example is a compound numbered 3, with a chemical name of N-trans-coumaroyloctopamine, and its structure is as follows:

[0068]

[0069] The cytotoxicity and osteoclast differentiation inhibitory activity of the amide alkaloids of this example were determined according to the experimental method of Example 1.

[0070] Example 4

[0071] This example provides the use of an amide alkaloid in the preparation of an osteoclast differentiation inhibitor. The amide alkaloid in this example is a compound numbered 4, whose chemical name is trans-N-feruloylnorsynephrine, and whose structure is as follows:

[0072]

[0073] The cytotoxicity and osteoclast differentiation inhibitory activity of the amide alkaloids of this example were determined according to the experimental method of Example 1.

[0074] Example 5

[0075] This example provides the use of amide alkaloids in the preparation of osteoclast differentiation inhibitors. The amide alkaloids in this example are compounds numbered 5, with a chemical name of grossamide, and a structure as follows:

[0076]

[0077] The cytotoxicity and osteoclast differentiation inhibitory activity of the amide alkaloids of this example were determined according to the experimental method of Example 1.

[0078] Example 6

[0079] This embodiment provides the use of amide alkaloids in the preparation of osteoclast differentiation inhibitors. The amide alkaloids in this embodiment are compounds numbered 6, and their chemical name is 1,2-dihydro-6,8-dimethoxy-7-hydroxy-1-(3,4-dihydroxyphenylethyl)-nitrogen 1 nitrogen 2-di-[2-(4-hydroxyphenyl)ethyl]-2,3-naphthalene dimethylamide, and their structure is as follows:

[0080]

[0081] The cytotoxicity and osteoclast differentiation inhibitory activity of the amide alkaloids of this example were determined according to the experimental method of Example 1.

[0082] Test Results

[0083] Figures 1 to 6 A and B are respectively the IC values ​​of the osteoclast differentiation inhibition experiments of the amide alkaloids of Examples 1 to 6. 50 curve; Figures 1 to 6 B are CC of the cytotoxicity experiments of amide alkaloids in Examples 1 to 6. 50 curve; Figures 1 to 6 C are representative pictures of the experimental results of the osteoclast differentiation inhibition experiment of amide alkaloids in Examples 1 to 6. Figures 1 to 6 It can be seen from C that the amide alkaloids of the present invention can inhibit RANKL-induced osteoclast differentiation in a dose-dependent manner.

[0084] Table 1 shows the test data of the cytotoxicity and osteoclast differentiation inhibition activity of the amide alkaloids of Examples 1 to 6, and the calculated selective inhibition constants.

[0085] Table 1 Experimental results of Examples 1 to 6

[0086]

[0087] As can be seen from Table 1, the cytotoxic CC of the amide alkaloids of Examples 1 to 6 50 All were above 204.30±96.40μM, and the IC 50 All of them were below 14.34±6.22 μM, and the selected inhibition constants (SI) were all above 8.29, indicating that the specific amide alkaloids of the present invention have a significant inhibitory effect on RANKL-induced osteoclast differentiation, have low cytotoxicity, have good anti-osteoclast differentiation activity, and can be used to prepare osteoclast differentiation inhibitors.

[0088] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. The use of amide alkaloids in the preparation of osteoclast differentiation inhibitors, characterized in that: The amide alkaloid has a structure as shown in formula (I) or formula (II): Wherein, R1 is H or OH; R2, R3, R4 are independently H, OH or C 1～6 Alkoxy; or R2 is H, OH or C 1～6 Alkoxy, R3, R4 and the carbon atoms to which they are connected together form the following fragment structure: In formula (I), It is a carbon-carbon double bond or a carbon-carbon single bond.

2. The application according to claim 1, characterized in that: The C 1～6 Alkoxy is methoxy.

3. The application according to claim 1, characterized in that: The amide alkaloid is any one of the compounds numbered 1 to 6:

4. The use according to claim 1, characterized in that: The osteoclast differentiation inhibitor is a drug for treating and / or preventing bone diseases caused by osteoclast differentiation.

5. The use according to claim 4, characterized in that: The drug also includes pharmaceutically acceptable excipients.

6. The use according to claim 5, characterized in that: The auxiliary material is at least one of a lubricant, a filler, a binder, a disintegrant, a surfactant, an antioxidant or a pH regulator.

7. The use according to claim 4, characterized in that: The dosage form of the medicine is injection, tablet, oral solution, granule or capsule.

8. The use according to claim 4, characterized in that: The bone disease is osteoporosis, osteoarthritis or bone metastasis.

9. The use according to claim 1, characterized in that: The osteoclast differentiation inhibitor is an inhibitor of RANKL-induced osteoclast differentiation.

10. The use according to claim 1, characterized in that: The amide alkaloids are deuterium isotope variants of amide alkaloids.

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