Bone-targeted ald / no prodrug

Abstract

The present application relates to the field of orthopedics, and more particularly to a bone-targeting ALD / NO prodrug, and provides a compound having the structure of formula I or a pharmaceutically acceptable salt thereof. The compound or the pharmaceutically acceptable salt thereof can improve the activity of osteoblasts, promote calcium deposition, and does not inhibit the function of osteoclasts. The compound has great significance for the regeneration and repair of damaged bone tissue, realizes the accurate delivery of NO gas molecules in the local tissue at the interface between the implant and the bone tissue, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of orthopedics, in particular to a bone-targeting ALD / NO prodrug. BACKGROUND

[0002] NO is an important gaseous signaling molecule, not only a key factor for maintaining the health of the vascular system, but also an important signaling molecule affecting the physiological process of bone metabolism 1 . A large number of studies have shown that NO can not only promote the proliferation and migration of endothelial cells, thereby promoting the sprouting of new blood vessels from existing blood vessels 2 ; but also regulate the expansion of vascular smooth muscle cells through the cGMP signaling pathway to provide sufficient blood supply for the new blood vessels in the granulation tissue and promote the growth of new blood vessels 3 . More importantly, recent studies have also found that NO can activate the glycolysis pathway of osteoblasts to promote osteoblast proliferation and differentiation 4 . Current research evidence shows that NO produced by H-type vascular endothelial cells in bone tissue can activate the NOTCH signaling pathway in multiple ways 5,6 , regulate bone tissue angiogenesis and osteogenesis. However, with age, H-type blood vessels in bone tissue degenerate and decrease, leading to a decrease in NO production by vascular endothelial cells in bone tissue, which not only affects the neovascularization during bone injury repair, but also affects bone metabolism, which is an important reason for bone loss 7 , while exogenous NO can effectively promote bone tissue regeneration and improve or even reverse osteoporosis 8 .

[0003] However, NO is chemically active and can be easily oxidized to nitrite (NO2 - ) by oxygen and free radicals under physiological conditions, thereby losing activity. Its effective diffusion distance is limited to about 100 μm, and its half-life is 1-3 seconds. Therefore, there are challenges in achieving effective delivery of exogenous NO in bone tissue. Therefore, the core problem of pushing NO gas therapy to the clinical application of implants is how to design NO donor prodrugs with bone tissue targeting function and achieve precise delivery of NO gas molecules in the local tissue at the implant-bone tissue interface.

[0004] Although many clinical studies on the local application of bisphosphonates in periodontal and implant areas have shown that it is an important treatment strategy for improving implant bone integration, there are still challenges in clinical application due to the inhibition of bisphosphonate drugs on osteoclast activity while inhibiting osteoblast function. Therefore, those skilled in the art are committed to developing a new type of ALD drug molecule that can target bone tissue to release NO gas.

[0005] 1. Gao, P.; Qiu, H.; Xiong, K.; et al., Metal-catechol-(amine) networks for surface synergistic catalytic modification: Therapeutic gas generation and biomolecule grafting. Biomaterials 2020, 248, 119981. https: / / doi.org / 10.1016 / j.biomaterials.2020.119981.

[0006] 2. Smith, T. L.; Oubaha, M.; Cagnone, G.; et al., eNOS controls angiogenic sprouting and retinal neovascularization through the regulation of endothelial cell polarity. Cellular and Molecular Life Sciences 2022, 79(1), 37. https: / / pubmed.ncbi.nlm.nih.gov / 34971428 / .

[0007] 3. Chen, Y.; Gao, P.; Huang, L.; et al., A tough nitric oxide-eluting hydrogel coating suppresses neointimal hyperplasia on vascular stent. Nature communications 2021, 12(1), 7079. https: / / www.nature.com / articles / s41467-021-27368-4

[0008] 4. Jin, Z.; Kho, J.; Dawson, B.; Jiang, M. M.; et al., Nitric oxide modulates bone anabolism through regulation of osteoblast glycolysis and differentiation. J Clin Invest 2021, 131(5). https: / / www.jci.org / articles / view / 138935.

[0009] 5. Charles, N.; Ozawa, T.; Squatrito, M.; et al., Perivascular nitric oxide activates notch signaling and promotes stem-like character in PDGF-induced glioma cells. Cell stem cell 2010, 6 (2), 141-152.

[0010] https: / / pubmed.ncbi.nlm.nih.gov / 20144787 / .

[0011] 6. Majumdar, U.; Manivannan, S.; Basu, M.; et al., Nitric oxide prevents aortic valve calcification by S-nitrosylation of USP9X to activate NOTCH signaling. Science Advances 2021, 7 (6), eabe3706. https: / / pubmed.ncbi.nlm.nih.gov / 33547080 / .

[0012] 7. Kusumbe, A. P.; Ramasamy, S. K.; Adams, R. H., Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone. Nature 2014, 507 (7492), 323-328. https: / / pubmed.ncbi.nlm.nih.gov / 24646994 / .

[0013] 8. Lin, Y. J.; Chen, C. C.; Chi, N. W.; et al., In Situ Self-Assembling Micellar Depots that Can Actively Trap and Passively Release NO with Long-Lasting Activity to Reverse Osteoporosis. Adv Mater 2018, 30 (22), e1705605. https: / / pubmed.ncbi.nlm.nih.gov / 29665153 / . SUMMARY

[0014] In view of the above defects of the prior art, the technical problem to be solved by the present application is how to achieve precise delivery of NO molecules to improve the insufficient bone regeneration ability in bone injury repair and enhance the osteogenic ability.

[0015] To achieve the above-mentioned purpose, the present application provides a compound with the structure of formula I or a pharmaceutically acceptable salt thereof, as follows:

[0016]

[0017] In a preferred embodiment of the present application, the present application provides a preparation method of a compound with the structure of formula I, comprising the following steps:

[0018] Step 1: 2-amino-3-methyl-3-mercaptobutyric acid is dissolved in pyridine, then acetic anhydride is added dropwise at 0°C, the reaction mixture is stirred at 25°C overnight, after the reaction is completed, the mixture is adjusted to pH < 7 with 1M hydrochloric acid, and extracted with dichloromethane three times, the organic phase is combined, washed with saturated brine, dried with sodium sulfate hydrate, filtered and concentrated to dryness under reduced pressure, to obtain a crude product, the crude product is stirred with a mixture of petroleum ether and methanol, the suspension is separated by filtration, and the filter cake is washed with petroleum ether and dried under reduced pressure to obtain white solid N-(2,2-dimethyl-4-oxothiazolidin-3-yl)acetamide;

[0019] Step 2: 1M aqueous NaOH solution is added to a suspension of (4-amino-1-hydroxy-1- phosphonobutyl)phosphonic acid until a clear solution is formed, then N-(2,2-dimethyl-4-oxothiazolidin-3-yl)acetamide is dissolved in acetonitrile, and added in four portions each for 15 minutes, the reaction mixture is stirred at 25°C overnight, the crude product is purified by reverse phase to obtain white solid [10-hydroxy-2,5-dioxo-10-phosphonoyl-4-(2-mercaptopropan-2-yl)-3,6-diaza-10-yl] phosphonic acid;

[0020] Step 3: Dissolve [10-hydroxy-2,5-dioxo-10-phosphono-4-(2-mercaptopropan-2-yl)- 3,6-diaza-10-yl] phosphonic acid in water, then add 4M aqueous sodium nitrite dropwise at 0°C, then add 1M hydrochloric acid dropwise at 0°C, stir the mixture at 0°C for 30 minutes, purify the product by filtration, recrystallization and drying to obtain green solid ALD-SNAP.

[0021] In another preferred embodiment of the present application, the present application provides a pharmaceutical composition comprising a compound of the present application or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0022] Preferably, the pharmaceutical composition can be formulated as an injectable fluid, an aerosol, a cream, a gel, a pill, a capsule, a syrup, a transdermal patch or an excipient.

[0023] In another preferred embodiment of the present application, the present application provides the use of a compound of the present application or a pharmaceutically acceptable salt thereof and a pharmaceutical composition thereof in the preparation of a medicament for treating a bone disease.

[0024] Preferably, the bone disease comprises osteosclerosis, osteoporosis, bone fracture, bone defect, femoral head necrosis.

[0025] Preferably, the bone disease is osteoporosis.

[0026] Preferably, the bone disease is induced by diabetes.

[0027] In a preferred embodiment of the present application, the present application provides a compound of the present application or a pharmaceutically acceptable salt thereof and a pharmaceutical composition thereof, characterized in that it is used for treating a bone disease or atherosclerosis.

[0028] Preferably, the bone disease comprises osteosclerosis, osteoporosis, bone fracture, bone defect, femoral head necrosis, preferably the bone disease is osteoporosis; optionally, the treatment of the bone disease is to promote the proliferation and differentiation of bone cells, bone tissue regeneration; the treatment of atherosclerosis is to promote the proliferation and migration of endothelial cells, to promote angiogenesis.

[0029] Preferably, the bone disease is induced by diabetes.

[0030] In another preferred embodiment of the present application, the present application provides the use of a compound of the present application or a pharmaceutically acceptable salt thereof and a pharmaceutical composition thereof in the preparation of a medicament for promoting osteogenesis and / or reducing bone resorption.

[0031] Preferably, the medicament or preparation is administered orally or by injection at a dose of once a day.

[0032] Preferably, the drug or preparation is used alone or in combination with other drugs.

[0033] The prominent features and benefits of the present application are:

[0034] The compound can improve osteoblast activity and promote calcium deposition without inhibiting the function of osteoclasts, and is of great significance for the regeneration and repair of damaged bone tissue, and realizes the accurate delivery of NO gas molecules in the local tissue at the interface between the implant and the bone tissue, and has a wide application prospect.

[0035] The concept, specific structure and technical effects of the present application will be further described below in combination with the drawings, so as to fully understand the purpose, features and effects of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 is a schematic diagram of ALD / NO twin drug synthesis.

[0037] Figure 2 is the structure formula of ALD / NO twin drug.

[0038] Figure 3 is a qualitative result diagram of alkaline phosphatase (ALP) staining.

[0039] Figure 4 is a quantitative result diagram of alkaline phosphatase (ALP) staining.

[0040] Figure 5 is a qualitative result diagram of alizarin red staining.

[0041] Figure 6 is a quantitative result diagram of alizarin red staining.

[0042] Figure 7 is a qualitative result diagram of TRAP staining.

[0043] Figure 8 is a quantitative result diagram of TRAP staining.

[0044] Figure 9 is a result diagram of color reaction after ALD, NAP, SNAP and SNAP-ALD are added to griess reagent.

[0045] Figure 10 is a result diagram of NO catalytic release rate after ALD, NAP, SNAP and SNAP-ALD are targeted to hydroxyapatite. DETAILED DESCRIPTION

[0046] The technical content of the present application will be more clearly and conveniently understood through the following description of the preferred embodiments of the present application with reference to the accompanying drawings. The present application can be embodied in many different forms and the scope of the present application is not limited to the embodiments mentioned herein.

[0047] Example 1 Preparation and characterization of ALD / NO prodrug

[0048] Step 1: 2-amino-3-methyl-3-mercaptobutyric acid (20 g, 134.04 mmol) was dissolved in pyridine (80 mL) and acetic anhydride (35.48 mL, 375.31 mmol) was added dropwise at 0 °C. The reaction mixture was stirred at 25 °C overnight. After the reaction was completed, the mixture was adjusted to pH < 7 with 1 M hydrochloric acid and extracted with dichloromethane (3 times). The organic phase was combined, washed with saturated brine, dried over sodium sulfate, filtered and concentrated to dryness under reduced pressure to obtain the crude product. The crude product was stirred with a mixture of petroleum ether and methanol, and the suspension was separated by filtration. The filter cake was washed with petroleum ether and dried under reduced pressure to obtain N-(2,2-dimethyl-4-oxothiazolidin-3-yl)acetamide (6000 mg, 34.64 mmol, 25.84%) as a white solid.

[0049] Step 2: 1 M aqueous NaOH solution was added dropwise to a suspension of (4-amino-1- hydroxy-1-phosphonobutyl)phosphonic acid (3.88 g, 15.59 mmol) until a clear solution was formed. N-(2,2-dimethyl-4-oxothiazolidin-3-yl)acetamide (2.7 g, 15.59 mmol) was then dissolved in acetonitrile (40 mL) and added in four portions of 15 minutes each. The reaction mixture was stirred at 25 °C overnight. The crude product was purified by reverse phase (FA) to obtain [10-hydroxy-2,5-dioxo-10-phosphonooxy-4-(2-mercaptopropan-2-yl)-3,6-diaza-10-yl] phosphonic acid (6000 mg, 14.21 mmol, 91.15%) as a white solid.

[0050] Step 3: [10-hydroxy-2,5-dioxo-10-phosphonooxy-4-(2-mercaptopropan-2-yl)-3,6-diaza-10- yl] phosphonic acid (1.00 g, 2.37 mmol) was dissolved in water (5 mL) and 4 M aqueous sodium nitrite solution (0.62 mL, 2.49 mmol) was added dropwise at 0 °C, followed by the addition of 1 M hydrochloric acid (2.37 mL, 2.37 mmol) dropwise at 0 °C. The mixture was stirred at 0 °C for 30 minutes. The product was purified by filtration, recrystallization and drying to obtain ALD-SNAP (ALD / NO) (800 mg, 1.77 mmol, 74.86%) as a green solid.

[0051] Figure 1The results of LCMS and HNMR show that ALD-SNAP (ALD / NO) is successfully prepared.

[0052] As Figure 2 shown, ALP is covalently linked with S-nitroso-N-acetyl-penicillamine (SNAP) and contains phosphate group and nitroso group.

[0053] Example 2 ALP Qualification

[0054] ALP Qualification Experiment: Before fixing the sample, wash the sample 1-2 times with PBS buffer along the wall of the culture dish; use 400 microliters of paraformaldehyde to fix the sample in the refrigerator for 1-2 hours; after fixation, wash 2-3 times with PBS; prepare the staining reagent (Biyun Tian kit): prepare the alkaline phosphatase staining reagent (10 ml system), including buffer 10 ml, 33 microliters of BCIP, 66 microliters of NBT, and 400 microliters of liquid addition, note that it is carried out at room temperature and in the dark; stain the sample, after staining is completed, wash the sample 1-2 times with PBS or distilled water; check the staining result every 20 minutes, and take a microscope photo according to the staining trend after about 2 hours.

[0055] Through alkaline phosphatase (ALP) staining as Figure 3 shown, we can observe the results under different treatment conditions, and the samples treated with ALD and NAP show lighter ALP staining at concentrations of 0 μΜ, 5 μΜ and 20 μΜ. This indicates that under these conditions, the ALP activity is relatively low, which may be due to the lack of exogenous NO supply, resulting in insufficient activation of ALP in cells. SNAP and SNAP-ALD (provide exogenous NO): In contrast, in samples treated with SNAP and SNAP-ALD, especially at concentrations of 5 μΜ and 20 μΜ, the staining appears more obvious. This suggests that the presence of exogenous NO may promote the activation of ALP, resulting in increased ALP activity. Therefore, NO may play a role in promoting ALP expression under these conditions, thereby affecting cell differentiation or mineralization. Comparative analysis: 5 μΜ and 20 μΜ: With the supply of NO (SNAP and SNAP-ALD), the depth of ALP staining significantly increases, especially at 20 μΜ, indicating that a higher concentration of NO further enhances ALP activity.

[0056] The presence of exogenous NO (such as SNAP and SNAP-ALD) significantly enhances ALP activity, suggesting that NO may promote ALP expression or activation through some mechanism, thereby affecting cell differentiation or mineralization. ALD and NAP exhibit lower ALP activity in the absence of exogenous NO, further supporting the important role of NO in regulating ALP activity.

[0057] Example 3 ALP Quantification

[0058] The experimental steps for ALP quantification are as follows:

[0059] 1. PBS washing: After taking out the sample, wash it with PBS solution, being careful not to wash away the cells.

[0060] 2. Lysis: Lyse with 350 μL 1% Triton for 4 hours and place in a 4°C refrigerator (adjust the volume according to different samples).

[0061] 3. BCA standard curve: Prepare seven 1.5 mL EP tubes and add 100 μL of 1% Triton in sequence. Add 100 μL of BSA standard solution (2 mg / mL) to the first tube and dilute tube by tube until the sixth tube. Do not add liquid to the last tube.

[0062] 4. Preparation of blank group and enzyme-labeled standard group in group F: Add 108 μL 1% Triton X-ray dilution and 2 μL enzyme-labeled standard solution, mix well, and add 30 μL to a 96-well plate. Add 30 μL 1% Triton X-ray dilution to the blank group.

[0063] 5. Preparation of experimental group: Mix solution 1 and solution 2 at a ratio of 1:1, pipette the lysate into a 96-well plate, and then add 100 μL of the mixed solution.

[0064] 6. Preparation of BCA experimental group: Mix solution A and solution B at a ratio of 50:1, add 25 μL of sample to a 96-well plate, and add 200 μL of the A+B mixture (also to the 7 wells of the standard curve).

[0065] 7. Incubation: Place in a 37°C incubator and incubate for 30 minutes.

[0066] 8. Add detection solution: Add 150 μL detection solution to each well.

[0067] 9. Determine the OD value: The OD value of ALP is 520nm, and the OD value of BCA is 562nm.

[0068] ALP=AKP / BCA*20

[0069] Through the above steps, the ALP activity in the sample can be quantitatively detected. Figure 4As shown in the figure, at a concentration of 5 μM, the production of ALP increased, but there was no significant difference between the groups. At a concentration of 20 μM, SNAP and SNAP-ALD significantly increased the production of alkaline phosphatase, especially SNAP-ALD, which produced the highest amount of alkaline phosphatase. Compared with ALD and NAP, SNAP and SNAP-ALD showed significant differences. After 7 days of treatment, exogenous NO provided by SNAP and SNAP-ALD significantly promoted the production of alkaline phosphatase, especially at higher concentrations. This further demonstrates that the production of exogenous NO by SNAP and the twin drug SNAP-ALD plays a key role in the production of alkaline phosphatase.

[0070] Example 4 Mineralization Experiment

[0071] To detect the degree of mineralization of the samples, a mineralization experiment was conducted with the following steps: 1. PBS washing: wash the samples three times with PBS; 2. Fixation: add 350 μL of paraformaldehyde and fix at 4°C for 30 minutes; 3. Ultrapure water washing: wash three times with ultrapure water; 4. Alizarin red staining: add 200 μL of alizarin red dye solution and stain at room temperature for 20 minutes, then wash with ultrapure water (observe under a stereomicroscope); 5. 16-alkylpyrazine chloride treatment: add 200 μL of 10% alkylpyrazine chloride solution and shake for 15 minutes; 6. OD value determination: after sampling, add to a 96-well plate and measure the OD value at a wavelength of 540 nm.

[0072] like Figure 5 The qualitative results of Alizarin Red shown in the figure show that at a concentration of 0 μM, all groups had similar staining results, with less calcium deposition and a lighter disc color. At a concentration of 5 μM, the staining results of the SNAP and SNAP-ALD groups were significantly darker than those of the ALD and NAP groups, indicating more calcium deposition. The SNAP-ALD group had the darkest color, indicating the greatest amount of calcium deposition. At a concentration of 20 μM, the SNAP and SNAP-ALD groups still showed darker staining, especially the SNAP-ALD group, indicating the greatest amount of calcium deposition. Figure 6 As shown, at a concentration of 0 μM, the absorbance of the four drugs was nearly identical, indicating no significant difference in calcium deposition between the groups in the absence of exogenous NO. At a concentration of 5 μM, the absorbance of the SNAP and SNAP-ALD groups was significantly higher than that of the ALD and NAP groups, indicating that calcium deposition increased in the presence of exogenous NO. The SNAP-ALD group had the most significant effect, with statistical analysis demonstrating significant differences from the other groups. At a concentration of 20 μM, the SNAP-ALD group exhibited the highest calcium deposition, still significantly higher than the ALD and NAP groups, and was significantly different from the other groups.

[0073] Both quantitative and qualitative results clearly demonstrated that the presence of exogenous NO (SNAP and SNAP-ALD) significantly increased calcium deposition in cells, particularly at higher concentrations (20 μM). The SNAP-ALD group showed the most pronounced effect, demonstrating its potent effect in promoting calcium deposition. Compounds that do not provide exogenous NO (ALD and NAP) did not exhibit a significant calcium deposition-enhancing effect under the same conditions. This suggests that exogenous NO plays a crucial role in promoting the formation of calcium deposits.

[0074] Example 5 TRAP staining

[0075] To detect osteoclast activity, TRAP staining (Tartrate-Resistant Acid Phosphatase Staining) is performed using the following steps: 1. Sample Fixation: Fix the cell or tissue sample with 4% paraformaldehyde (PFA) at room temperature for 10-20 minutes to ensure that the cell structure remains intact. 2. PBS Washing: Wash the sample three times with PBS (phosphate-buffered saline) for approximately 5 minutes each time to remove the fixative. 3. Preparation of TRAP Staining Solution: Prepare the TRAP staining solution. 4. Staining Reaction: Add the staining solution to the sample and incubate in the dark at room temperature or 37°C for 30 minutes to 2 hours until a red or purple-red precipitate appears, indicating TRAP activity. 5. Stop Reaction: After staining, wash the sample one or two times with distilled water or PBS to stop the reaction. Observation: Observe the results using a light microscope. TRAP-positive cells (osteoclasts) typically stain red or purple-red, reflecting high TRAP activity.

[0076] Figure 7 Qualitative results showed that: Control (C) group: no Rankl was added, showing a negative control, with basically no purple staining area, indicating that no osteoclasts were formed. + Rankl group: showed significant purple staining, indicating that Rankl induced the formation of osteoclasts. ALD group: 5μM and 20μM: the staining area was further reduced, showing a concentration-dependent inhibitory effect on osteoclast formation, which is consistent with the result that ALD has mineralization ability in Example 4; NAP group: at concentrations of 0μM, 5μM and 20μM, TRAP staining showed a large number of osteoclasts, indicating that NAP did not inhibit osteoclast formation; SNAP group: all concentrations showed more purple staining, indicating that the exogenous NO provided by SNAP did not inhibit osteoclast formation; SNAP-ALD group: 5μM and 20μM: the number of osteoclasts decreased, especially at 20μM, showing a significant decrease, indicating that at higher concentrations, the SNAP-ALD combination exhibited an anti-osteoclast effect similar to ALD. Figure 8Quantitative results showed that: +Rankl vs. Control: +Rankl significantly increased the number of TRAP-positive osteoclasts, validating the osteoclast-inducing effect of Rankl. ALD group: Compared with the +Rankl group, ALD significantly reduced the number of TRAP-positive osteoclasts at concentrations of 5 μM and 20 μM, and especially at 20 μM, it almost completely inhibited osteoclast formation, confirming the anti-osteoclast activity of ALD. NAP group: The coverage of TRAP-positive cells at all concentrations was similar to that of the +Rankl group, indicating that NAP was unable to provide exogenous NO and did not exhibit significant anti-osteoclast activity. SNAP group: The coverage of TRAP-positive cells was high at all concentrations, indicating that exogenous NO provided by SNAP did not significantly inhibit osteoclast formation. SNAP-ALD group: At concentrations of 5 μM and 20 μM, especially at 20 μM, SNAP-ALD significantly reduced the number of TRAP-positive cells, demonstrating a strong anti-osteoclast effect, which may be due to the action of ALD, which was not completely offset by the NO provided by SNAP.

[0077] ALD exhibited concentration-dependent anti-osteoclastogenic effects. NAP showed no anti-osteoclastogenic activity. Exogenous NO provided by SNAP failed to inhibit osteoclast formation. SNAP-ALD exhibited significant anti-osteoclastogenic effects at higher concentrations (particularly 20 μM), likely due to the dominant effect of ALD. These results demonstrate that different drugs and combinations have different effects on osteoclast formation, with ALD exhibiting significant anti-osteoclastogenic activity, while SNAP and NAP had no significant effects when used alone.

[0078] Example 6 Bone Targeting Experiment

[0079] To test the ability of twin drugs to target hydroxyapatite in bone, the following experiment was conducted. 10 ml of 100 μM ALD, NAP, SNAP, and SNAP-ALD were prepared respectively. 1 μM EDTA and 100 mg of hydroxyapatite were added to each group. The mixture was stirred for 1 hour using a magnetic stirrer in the dark, and centrifuged at 8000 rpm for 10 minutes using a high-speed centrifuge. The supernatant was removed and mixed with Griess reagent in a 1:1 ratio. After 15 minutes in the dark, the mixture was washed with water. Figure 9As shown, only SNAP and SNAP-ALD groups showed color, indicating that ALD and NAP could not provide exogenous NO. Then, each group of hydroxyapatite was washed twice with normal saline, centrifuged at 8000r / min for 5min, the supernatant was discarded, 10ml of normal saline and 100ul of a mixture of 10mM GSH and SeCA were added to catalyze the release of NO. The supernatant of each group was taken, griess was added, and the mixture was protected from light for 15min. It can be seen that only SNAP-ALD showed color. Because ALD has bone targeting, SNAP and SNAP-ALD can release NO to change the color of the griess reagent. Therefore, it can be judged that SNAP-ALD can target hydroxyapatite to bone and release NO. Figure 10 As shown, the supernatant of each group was used to detect NO release using Griess reagent. The theoretical release amount of 100 μM SNAP-ALD in 10 ml was 10 μM, and the actual release amount was about 3.5 μM. Therefore, the bone targeting rate of SNAP-ALD was about 35%.

[0080] The preferred embodiments of the present invention have been described in detail above. It should be understood that numerous modifications and variations based on the concepts of the present invention are possible without inventive effort by those skilled in the art. Therefore, any technical solution that can be derived by one skilled in the art through logical analysis, reasoning, or limited experimentation based on the concepts of the present invention and the prior art should be within the scope of protection defined by the claims.

Claims

1. A compound having a structure of Formula I or a pharmaceutically acceptable salt thereof, the general formula of which is as follows:

2. A method for preparing the compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The following steps are involved: Step 1: 2-amino-3-methyl-3-mercaptobutyric acid is dissolved in pyridine, and then acetic anhydride is added dropwise at 0°C. The reaction mixture is stirred at 25°C overnight. After the reaction is completed, the mixture is adjusted to pH <7 with 1M hydrochloric acid and extracted three times with dichloromethane. The organic phases are combined, washed with saturated brine, dried over hydrated sodium sulfate, filtered, and concentrated to dryness under reduced pressure to obtain a crude product. The crude product is mixed with petroleum ether and methanol, and the suspension is separated by filtration. The filter cake is washed with petroleum ether and dried under reduced pressure to obtain N-(2,2-dimethyl-4-oxothiane-3-yl)acetamide as a white solid; Step 2: A 1M NaOH aqueous solution was added dropwise to the (4-amino-1-hydroxy-1-phosphonobutyl)phosphonic acid suspension until a clear solution was formed, and then N-(2,2-dimethyl-4-oxothiane-3-yl)acetamide was dissolved in acetonitrile and added four times over 15 minutes each time. The reaction mixture was stirred at 25°C overnight, and the crude product was purified by reverse phase to obtain a white solid [10-hydroxy-2,5-dioxo-10-phosphono-4-(2-mercaptopropan-2-yl)-3,6-diaza-10-yl]phosphonic acid; Step 3: Dissolve [10-hydroxy-2,5-dioxo-10-phosphono-4-(2-mercaptoprop-2-yl)-3,6-diaza-10-yl]phosphonic acid in water, then add 4 M sodium nitrite aqueous solution dropwise at 0°C, then add 1 M hydrochloric acid dropwise at 0°C, and the mixture is stirred at 0°C for 30 minutes. The product is purified by filtration, recrystallization and drying to obtain a green solid ALD-SNAP.

3. A pharmaceutical composition comprising the compound according to claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

4. The pharmaceutical composition according to claim 3, characterized in that The pharmaceutical composition can be formulated as an injectable fluid, aerosol, cream, gel, pill, capsule, syrup, transdermal patch, or excipient.

5. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof and / or the pharmaceutical composition according to any one of claims 3 to 4 in the preparation of a medicament for treating orthopedic diseases or atherosclerosis.

6. The use according to claim 5, characterized in that The orthopedic diseases include osteosclerosis, osteoporosis, fractures, bone defects, and femoral head necrosis.

7. The use according to claim 6, characterized in that The orthopedic disease is osteoporosis.

8. The use according to claim 5, characterized in that The treatment of orthopedic diseases is to promote the proliferation and differentiation of bone cells and the regeneration of bone tissue; the treatment of atherosclerosis is to promote the proliferation and migration of endothelial cells and promote angiogenesis.

9. The use according to claim 5 or 6, characterized in that: The orthopedic disease is induced by diabetes.

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