Bone-targeted ALD / NO twin drug

Abstract

The invention relates to the field of orthopedics, in particular to a bone-targeted ALD / NO twin drug, and provides a compound with a structure shown in a formula I or pharmaceutically acceptable salt of the compound. The compound or the pharmaceutically acceptable salt thereof can improve osteoblast activity and promote calcium deposition without generating an inhibiting effect on the function of osteoclast, the compound has great significance on regeneration and repair of damaged bone tissue, accurate delivery of NO gas molecules in local tissue at the implant-bone tissue surface interface is achieved, and the compound has a good application prospect. Wide application prospects are realized.

Description

Technical Field

[0001] The present invention relates to the field of orthopedics, and in particular to a bone-targeted ALD / NO twin drug. Background Art

[0002] NO is an important gaseous molecular signaling molecule. It is not only a key factor in maintaining the health of the vascular system, but also an important signaling molecule that affects the physiological process of bone metabolism. 1 A large number of studies have shown that NO can promote the proliferation and migration of endothelial cells, thereby promoting the sprouting of new blood vessels from existing blood vessels. 2 It can also regulate vascular smooth muscle cells to dilate blood vessels through the cGMP signaling pathway, provide sufficient blood supply to 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 glycolytic pathway of osteoblasts and promote osteoblast proliferation and differentiation. 4 Current research evidence shows that NO produced by bone tissue H-type vascular endothelial cells can activate the NOTCH signaling pathway in a variety of ways. 5,6 , regulating angiogenesis and osteogenesis in bone tissue. However, with aging, the degeneration of H-type blood vessels in bone tissue decreases, resulting in a decrease in NO production in the endothelial cells of bone tissue, which not only affects angiogenesis during bone damage repair, but also affects bone metabolism, which is an important cause of bone loss. 7 Exogenous NO can effectively promote bone tissue regeneration, improve or even reverse osteoporosis. 8 .

[0003] However, NO is chemically active and can be easily oxidized to nitrite (NO2 - ) and loses its activity. Its effective diffusion distance is limited to only about ≈100μm, and its half-life is 1-3 seconds. It is enriched in bone tissue, and it is challenging to effectively deliver exogenous NO. Therefore, the core issue of promoting NO gas therapy to 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 local tissues at the implant-bone tissue interface.

[0004] Although many clinical studies on the topical application of bisphosphonates in periodontal and implant settings have shown that it is an important therapeutic strategy to improve implant bone integration, clinical applications are still challenging because bisphosphonates inhibit both osteoclast activity and osteoblast function. Therefore, technicians in this field are committed to developing a new 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 forsurface synergistic catalytic modication:Therapeutic gas generation andbiomolecule 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 angiogenicsprouting and retinal neovascularization through the regulation ofendothelial 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-elutinghydrogel coating suppresses neointimal hyperplasia on vascular stent.Naturecommunications 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 modulatesbone anabolism through regulation ofosteoblast glycolysis anddifferentiation.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 oxideactivates notch signaling and promotes stem-like character in PDGF-inducedglioma 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 preventsaortic valve calcification by S-nitrosylation ofUSP9X to activate NOTCHsignaling.Science Advances2021,7(6),eabe3706.https: / / pubmed.ncbi.nlm.nih.gov / 33547080 / .

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

[0013] 8. Lin, YJ; Chen, CC; Chi, NW; et al., In Situ Self-Assembling MicellarDepots that Can Actively Trap and Passively Release NO with Long-LastingActivity to Reverse Osteoporosis. Adv Mater 2018,30(22),e1705605.https: / / pubmed.ncbi.nlm.nih.gov / 29665153 / . Summary of the invention

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

[0015] To achieve the above object, the present invention provides a compound having a structure of Formula I or a pharmaceutically acceptable salt thereof, the general formula of which is as follows:

[0016]

[0017] In a preferred embodiment of the present invention, the present invention provides a method for preparing a compound of formula I, comprising the following steps:

[0018] Step 1: dissolving 2-amino-3-methyl-3-mercaptobutyric acid in pyridine, then adding acetic anhydride dropwise at 0°C, stirring the reaction mixture at 25°C overnight, adjusting the mixture to pH <7 with 1M hydrochloric acid after the reaction, and extracting three times with dichloromethane, combining the organic phases, washing with saturated brine, drying with hydrated sodium sulfate, filtering and concentrating to dryness under reduced pressure to obtain a crude product, mixing the crude product with petroleum ether and methanol, separating the suspension by filtration, washing the filter cake with petroleum ether and drying under reduced pressure to obtain a white solid N-(2,2-dimethyl-4-oxothiane-3-yl)acetamide;

[0019] 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 for 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;

[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 sodium nitrite aqueous solution dropwise at 0°C, then add 1M hydrochloric acid dropwise at 0°C, the mixture is stirred at 0°C for 30 minutes, and the product is purified by filtration, recrystallization and drying to obtain a green solid ALD-SNAP.

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

[0022] Preferably, the pharmaceutical composition can be formulated into 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 invention, the present invention provides use of the compound or pharmaceutically acceptable salt thereof and pharmaceutical composition thereof in the preparation of a method for treating orthopedic diseases.

[0024] Preferably, the orthopedic diseases include osteosclerosis, osteoporosis, fractures, bone defects, and femoral head necrosis.

[0025] Preferably, the orthopedic disease is osteoporosis.

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

[0027] In a preferred embodiment of the present invention, the present invention provides the compound of the present invention or its pharmaceutically acceptable salt and pharmaceutical composition thereof, characterized in that they are used for treating orthopedic diseases or atherosclerosis.

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

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

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

[0031] Preferably, the drug or preparation is administered orally or by injection as a once-daily dose.

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

[0033] The outstanding features and beneficial effects of the present invention are:

[0034] The compounds described in the present invention can enhance the activity of osteoblasts and promote calcium deposition without inhibiting the function of osteoclasts. The compounds described in the present invention are of great significance for the regeneration and repair of damaged bone tissue, and can achieve precise delivery of NO gas molecules in local tissues at the implant-bone tissue surface interface, and have broad application prospects.

[0035] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Schematic diagram of the ALD / NO twin drug synthesis.

[0037] Figure 2 It is the structural formula of ALD / NO twin drugs.

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

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

[0040] Figure 5 This is the qualitative result of Alizarin red staining.

[0041] Figure 6 This is the quantitative result of Alizarin red staining.

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

[0043] Figure 8 This is the quantitative result of TRAP staining.

[0044] Fig. 9 This is the color reaction result of ALD, NAP, SNAP and SNAP-ALD after adding Griess reagent.

[0045] Fig.10 This is a graph showing the results of NO catalytic release rate after ALD, NAP, SNAP and SNAP-ALD and hydroxyapatite targeting. DETAILED DESCRIPTION

[0046] The following describes several preferred embodiments of the present invention with reference to the drawings in the specification, so that the technical content is clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.

[0047] Example 1 Preparation and Characterization of ALD / NO Twin Drugs

[0048] Step 1: 2-amino-3-methyl-3-mercaptobutyric acid (20 g, 134.04 mmol) was dissolved in pyridine (80 ml), and then 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 1M hydrochloric acid and extracted with dichloromethane (extracted 3 times). The organic phases were 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 was mixed and stirred with 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 a white solid N-(2,2-dimethyl-4-oxothiane-3-yl)acetamide (6000 mg, 34.64 mmol, 25.84%).

[0049] Step 2: 1M NaOH aqueous 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-oxothiane-3-yl)acetamide (2.7 g, 15.59 mmol) was then dissolved in acetonitrile (40 ml) and added four times over 15 minutes each. The reaction mixture was stirred at 25°C overnight. The crude product was purified by reverse phase (FA) to give [10-hydroxy-2,5-dioxo-10-phosphono-4-(2-mercaptopropane-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-phosphono-4-(2-mercaptopropane-2-yl)-3,6-diaza-10-yl]phosphonic acid (1.00 g, 2.37 mmol) was dissolved in water (5 ml), and then 4M sodium nitrite aqueous solution (0.62 ml, 2.49 mmol) was added dropwise at 0°C, and then 1M hydrochloric acid (2.37 ml, 2.37 mmol) was added dropwise at 0°C, and the mixture was stirred at 0°C for 30 minutes. The product was purified by filtration, recrystallization and drying to obtain green solid ALD-SNAP (ALD / NO) (800 mg, 1.77 mmol, 74.86%).

[0051] Figure 1A schematic diagram of the synthesis of ALD / NO twin drugs is shown, and the results of LCMS and HNMR indicate that ALD-SNAP (ALD / NO) was successfully prepared.

[0052] like Figure 2 As shown, alendronate (ALD) and S-nitroso-N-acetyl-penicillamine (SNAP) are linked by covalent bonds and contain a phosphate group and a nitro group.

[0053] Example 2 ALP qualitative

[0054] ALP qualitative 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 and place the sample in the refrigerator for 1-2 hours; after fixation, wash it 2-3 times with PBS; prepare staining reagent (Biyuntian test kit): prepare alkaline phosphatase staining reagent (10ml system), including 10ml buffer, 33 microliters BCIP, 66 microliters NBT, and 400 microliters of added liquid, and make sure to perform it at room temperature and away from light; stain the sample, and after staining, wash the sample 1-2 times with PBS or double distilled water; check the staining results every 20 minutes, and take pictures under a microscope according to the staining trend after about 2 hours.

[0055] Through Figure 3 As shown in the alkaline phosphatase (ALP) staining, we can observe the results under different treatment conditions. At concentrations of 0μM, 5μM, and 20μM, samples treated with ALD and NAP all showed lighter ALP staining. This indicates that under these conditions, ALP activity is relatively low, probably because there is no exogenous NO supply, resulting in insufficient activation of ALP in the cells. SNAP and SNAP-ALD (providing exogenous NO): The comparison shows that in samples treated with SNAP and SNAP-ALD, especially at concentrations of 5μM and 20μM, 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: 5μM and 20μM: With the supply of NO (SNAP and SNAP-ALD), the depth of ALP staining increased significantly, especially at 20μM, indicating that higher concentrations of NO further enhanced ALP activity.

[0056] The presence of exogenous NO (such as SNAP and SNAP-ALD) significantly enhanced the activity of ALP, indicating that NO may promote the expression or activation of ALP through some mechanism, thereby affecting the differentiation or mineralization process of cells. ALD and NAP showed 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: Use 350 μL 1% Trion for 4 hours and place in a 4°C refrigerator (adjust the amount according to different samples).

[0061] 3. BCA standard curve: prepare 7 1.5mL EP tubes, add 100μL 1% Trion in sequence, add 100μL BSA standard solution (2mg / mL) to the first tube, dilute tube by tube until the 6th tube, and 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% Trion and 2 μL enzyme-labeled standard solution and mix well, take 30 μL and add to 96-well plate. Add 30 μL 1% Trion to the blank group.

[0063] 5. Preparation of experimental group: Mix solution 1 and solution 2 in 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 an incubator at 37°C 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, 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 had the highest production of alkaline phosphatase. SNAP and SNAP-ALD showed significant differences compared with ALD and NAP. 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 illustrates 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] In order to detect the mineralization degree of the sample, a mineralization experiment was carried out, and the steps are as follows: 1. PBS washing: wash the sample 3 times with PBS; 2. Fixation: add 350 μL of paraformaldehyde and fix at 4°C for 30 minutes; 3. Ultrapure water washing: wash 3 times with ultrapure water; 4. Alizarin red staining: add 200 μL of alizarin red dye solution, stain at room temperature for 20 minutes, and then wash with ultrapure water (observe under a stereomicroscope); 5. 16-alkyl pyrazine chloride treatment: add 200 μL of 10% alkyl pyrazine 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 0μM concentration: the staining results of all groups were similar, with less calcium deposition and a lighter color on the disk surface. At 5μM concentration: the staining results of the SNAP and SNAP-ALD groups were significantly darker than those of the ALD and NAP groups, showing more calcium deposition. The SNAP-ALD group had the darkest color, indicating the largest amount of calcium deposition. At 20μM concentration: the SNAP and SNAP-ALD groups still showed darker staining, especially the SNAP-ALD group, indicating the largest amount of calcium deposition. Figure 6 As shown, at 0μM concentration: the absorbance of the four drugs is almost the same, indicating that there is no significant difference in the amount of calcium deposition in each group without the action of exogenous NO. At 5μM concentration: the absorbance of the SNAP and SNAP-ALD groups is significantly higher than that of the ALD and NAP groups, indicating that calcium deposition increases under the condition of exogenous NO. Among them, the effect of the SNAP-ALD group is the most significant, and statistical analysis shows that it is significantly different from other groups. At 20μM concentration: the amount of calcium deposition in the SNAP-ALD group is still significantly higher than that in the ALD and NAP groups, showing the highest amount of calcium deposition, and there is a significant difference between it and other groups.

[0073] Both quantitative and qualitative results clearly show that in the presence of exogenous NO (SNAP and SNAP-ALD), the amount of calcium deposition in cells increased significantly, especially at higher concentrations (20 μM). Among them, the effect of the SNAP-ALD group was the most significant, indicating that the drug has a strong effect on promoting calcium deposition. However, compounds that cannot provide exogenous NO (ALD and NAP) did not show a significant calcium deposition enhancement effect under the same conditions. This shows that exogenous NO plays an important role in promoting the formation of calcium deposition.

[0074] Example 5 TRAP staining

[0075] TRAP staining (Tartrate-Resistant Acid Phosphatase Staining) is performed to detect the activity of osteoclasts. The steps are as follows: 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 with PBS (phosphate buffered saline) 3 times, about 5 minutes each time, and remove the fixative. 3. Preparation of TRAP staining working solution: Prepare TRAP staining solution, 4. Staining reaction: Add the staining working solution to the sample and incubate at room temperature or 37°C in the dark for 30 minutes to 2 hours until a red or purple-red precipitate appears, indicating TRAP activity, 5. Stop the reaction: After the staining is completed, wash the sample 1-2 times with distilled water or PBS to stop the reaction. Observation: Use an optical microscope to observe the results. TRAP-positive cells (osteoclasts) usually show red or purple-red staining, reflecting high TRAP activity.

[0076] Figure 7 Qualitative results showed that: Control (C) group: Rankl was not added, showing a negative control, with basically no purple staining area, indicating that osteoclasts were not 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 reacted in Example 4 has mineralization ability; NAP group: At concentrations of 0μM, 5μM and 20μM, TRAP staining showed a large number of osteoclasts, indicating that NAP did not inhibit the formation of osteoclasts; SNAP group: All concentrations showed more purple staining, indicating that the exogenous NO provided by SNAP did not inhibit the formation of osteoclasts; 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 showed an anti-osteoclast effect similar to ALD. Figure 8Quantitative results showed that: +Rankl v. Control: +Rankl significantly increased the number of TRAP-positive osteoclasts, verifying 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 5μM and 20μM, especially at a concentration of 20μM, which almost completely inhibited the formation of osteoclasts, confirming the anti-osteoclast ability of ALD; NAP group: The coverage rate of TRAP-positive cells at each concentration was similar to that of the +Rankl group, indicating that NAP could not provide exogenous NO and did not show significant anti-osteoclast ability; SNAP group: The coverage rate of TRAP-positive cells at each concentration was high, indicating that the exogenous NO provided by SNAP did not significantly inhibit the formation of osteoclasts; SNAP-ALD group: At concentrations of 5μM and 20μM, especially at a concentration of 20μM, SNAP-ALD significantly reduced the number of TRAP-positive cells, showing a strong anti-osteoclast effect, which may be due to the effect of ALD, and the NO provided by SNAP did not completely offset this effect.

[0077] ALD exhibited concentration-dependent anti-osteoclastogenic effects. NAP did not exhibit anti-osteoclastogenic ability. Exogenous NO provided by SNAP failed to inhibit osteoclast formation. SNAP-ALD exhibited significant anti-osteoclastogenic effects at higher concentrations (especially 20 μM), probably due to the dominant effect of ALD. These results show the different effects of different drugs and combinations on osteoclastogenesis, with ALD exhibiting significant anti-osteoclastogenic ability, while SNAP and NAP were not significantly effective when used alone.

[0078] Example 6 Bone Targeting Experiment

[0079] To detect 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 hydroxyapatite were added to each group and stirred for 1 h using a magnetic stirrer under dark conditions. The mixture was centrifuged at 8000 r / min for 10 min using a high-speed centrifuge. The supernatant was removed and mixed with Griess reagent in a 1:1 ratio. After 15 min of dark protection, the mixture was stirred for 1 h using a magnetic stirrer. Fig. 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 saline, centrifuged at 8000r / min for 5min, the supernatant was discarded, 10ml of saline and 100ul of a mixture of 10mM GSH and SeCA were added to catalyze NO release, and 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. Fig.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 specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined 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: dissolving 2-amino-3-methyl-3-mercaptobutyric acid in pyridine, then adding acetic anhydride dropwise at 0°C, stirring the reaction mixture at 25°C overnight, adjusting the mixture to pH <7 with 1M hydrochloric acid after the reaction, and extracting three times with dichloromethane, combining the organic phases, washing with saturated brine, drying with hydrated sodium sulfate, filtering and concentrating to dryness under reduced pressure to obtain a crude product, mixing the crude product with petroleum ether and methanol, separating the suspension by filtration, washing the filter cake with petroleum ether and drying under reduced pressure to obtain a white solid N-(2,2-dimethyl-4-oxothiane-3-yl)acetamide; 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 for 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-mercaptopropan-2-yl)-3,6-diaza-10-yl]phosphonic acid in water, then add 4M sodium nitrite aqueous solution dropwise at 0°C, then add 1M hydrochloric acid dropwise at 0°C, the mixture is stirred at 0°C for 30 minutes, and 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 claims 3-4 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. Preferably, the orthopedic disease is osteoporosis; optionally, the treatment of orthopedic diseases is to promote osteocyte proliferation and differentiation, and bone tissue regeneration; the treatment of atherosclerosis is to promote endothelial cell proliferation and migration, and promote angiogenesis.

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

8. The compound according to claim 1 or a pharmaceutically acceptable salt thereof and / or the pharmaceutical composition according to claims 3-4, characterized in that: Used to treat orthopedic diseases or atherosclerosis.

9. The compound or pharmaceutically acceptable salt thereof and / or pharmaceutical composition according to claim 8, characterized in that: The orthopedic diseases include osteosclerosis, osteoporosis, fractures, bone defects, and femoral head necrosis. Preferably, the orthopedic disease is osteoporosis; optionally, the treatment of orthopedic diseases is to promote osteocyte proliferation and differentiation, and bone tissue regeneration; the treatment of atherosclerosis is to promote endothelial cell proliferation and migration, and promote angiogenesis.

10. The compound or pharmaceutically acceptable salt thereof and / or pharmaceutical composition according to claim 8 or 9, characterized in that: The orthopedic disease is induced by diabetes.

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