Bone-targeted alendronate-propranolol as well as preparation method and application thereof

Abstract

The invention discloses bone targeted alendronate-propranolol as well as a preparation method and application thereof, the bone targeted alendronate-propranolol has good in-vitro bone targeting property, dissociates and releases a propranolol active compound in an in-vivo bone microenvironment, can inhibit osteoclasts, and has certain protectiveness on bone tissues. Meanwhile, tumor growth after breast cancer bone metastasis can be inhibited, and a novel treatment method is expected to be provided for breast cancer bone metastasis. The synthesis route and the post-treatment method are safe, simple and convenient, the used synthesis raw materials and solvents are cheap and easy to obtain, and the yield of the intermediate compound and the target product is relatively high. The invention also provides application of the bone-targeted alendronate-propranolol in preparation of drugs for treating breast cancer bone metastasis.

Description

Technical Field

[0001] The invention relates to bone-targeted propranolol and a preparation method and application thereof, and in particular to bone-targeted alendronic acid-propranolol and a preparation method and application thereof. Background Art

[0002] Metastasis is one of the important causes of death in cancer patients. Bone metastasis of cancer refers to the transfer of cancer cells to bone tissue through the blood circulation and the formation of new tumors. Epidemiology shows that breast cancer, lung cancer, liver cancer, etc. are all malignant tumors that are prone to bone metastasis. Among them, breast cancer mediates osteolytic bone metastasis, and its late-stage bone metastasis rate is as high as 65% to 75%, and the 5-year overall survival rate is as low as 22.8%. Once bone metastasis occurs, it is almost impossible to cure, and it will cause varying degrees of bone pain, pathological fractures, spinal cord compression, and hypercalcemia. These are a series of bone complications caused by bone metastasis of malignant tumors, namely bone-related events (SREs), which bring great pain to patients.

[0003] Previous studies by our research group have shown that the sympathetic nerves in the bone marrow microenvironment of mice under stress are excited, and the expression of osteopontin (OPN, an intracellular protein) in bone marrow cells is increased, which promotes the bone metastasis of breast cancer.

[0004] Propranolol is a non-selective beta-blocker, commonly used in the clinic to treat arrhythmias, hypertension, etc. Studies have shown that propranolol can inhibit bone metastasis of breast cancer, but many types of cells and tissues express beta-receptors, which makes the use of propranolol have systemic effects, leading to greater toxic and side effects of propranolol's systemic effects.

[0005] Therefore, it is of great significance to develop targeted inhibitors of breast cancer bone metastasis. Summary of the invention

[0006] The purpose of the present invention is to provide a bone-targeted alendronic acid-propranolol, which is expected to provide a new treatment method with less toxic and side effects for breast cancer bone metastasis.

[0007] The purpose of the present invention is achieved by the following methods: The bone-targeted alendronic acid-propranolol provided by the present invention has a chemical structure as shown in formula (AP): (AP).

[0008] The beneficial effects of the present invention are reflected in that the bone-targeted alendronic acid-propranolol provided by the present invention has good in vitro bone targeting, and dissociates and releases the propranolol original drug in the bone microenvironment in vivo, and the bone-targeted alendronic acid-propranolol can inhibit osteoclasts and has a certain protective effect on bone tissue. At the same time, it can also inhibit the growth of tumors after breast cancer bone metastasis, and is expected to provide a new treatment method for breast cancer bone metastasis.

[0009] The present invention also provides a method for preparing bone-targeted alendronic acid-propranolol, and the synthetic route is as follows: ; Wherein, step a is: propranolol reacts with di-tert-butyl dicarbonate to generate compound 1; Step b is: compound 1 reacts with 4-nitrophenyl chloroformate to generate compound 2; Step c is: compound 2 reacts with alendronic acid to generate compound 3.

[0010] Step d is: compound 3 is deprotected under the condition of hydrogen chloride methanol to generate compound AP.

[0011] The step a comprises: dissolving propranolol hydrochloride and sodium bicarbonate in water, dissolving di-tert-butyl dicarbonate in dioxane, slowly dropping the aqueous solution into the dioxane solution and stirring at room temperature for 12 h until completion is shown by TLC; filtering under reduced pressure, washing with water, and drying to obtain compound 1.

[0012] The step b is as follows: compound 1 is dissolved in dichloromethane, the solution is cooled to 0°C, and anhydrous triethylamine solution is added; 4-nitrophenyl chloroformate is dissolved in dichloromethane, and slowly added to the above solution at 0°C within 15 minutes; after the dropwise addition is completed, the reaction solution is slowly heated to room temperature, stirred at room temperature for 24 hours, and TLC shows that the reaction is complete; the reaction solution is extracted with a saturated sodium carbonate aqueous solution, the water layer is discarded, the dichloromethane layer is dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure, and purified by a silica gel column to obtain compound 2.

[0013] The step c comprises: dissolving compound 2 and Cs2CO3 in dioxane, dissolving alendronic acid and Cs2CO3 in water, slowly dropping the aqueous solution into the dioxane solution at room temperature within 15 minutes, and after the dropping is completed, placing the reaction solution in a 60°C oil bath and heating and stirring for 24 hours, and TLC shows that the reaction is complete; after the reaction solution is cooled, adding a mixed solution of water and ethyl acetate, standing and stratifying, extracting the water layer with ethyl acetate, discarding the ethyl acetate layer, adding 2 M HCl aqueous solution to the water layer until pH = 5, extracting with ethyl acetate again, adding 2 M HCl aqueous solution to the water layer to completely precipitate, and then extracting with dichloromethane, combining the dichloromethane layers and drying with anhydrous magnesium sulfate, and concentrating under reduced pressure to obtain compound 3.

[0014] The step d is as follows: dissolving compound 3 in a 4M methanolic hydrogen chloride solution, stirring at room temperature for 2 hours, and after the mass spectrum shows that compound 3 disappears, adding a 4M aqueous solution of NaOH to completely precipitate it, filtering under reduced pressure and washing the precipitate with methanol, and drying in an oven at 50° C. to obtain a compound represented by formula (AP).

[0015] The synthetic route and post-processing method of the present invention are safe and simple, the synthetic raw materials and solvents used are cheap and easily available, and the yields of intermediate compounds and target products are high.

[0016] In view of the fact that the bone-targeted alendronic acid-propranolol provided by the present invention can inhibit osteoclasts, has a certain protective effect on bone tissue, and can also inhibit the growth of tumors after breast cancer bone metastasis, the present invention also proposes its use in the preparation of drugs for treating tumor bone metastasis, especially in drugs for treating breast cancer bone metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 These are the ESI-MS spectra of compounds 1, 2, 3, and AP, wherein: A shows the ESI-MS spectrum of compound 1, B shows the ESI-MS spectrum of compound 2, C shows the ESI-MS spectrum of compound 3, and D shows the ESI-MS spectrum of compound AP.

[0018] Figure 2 It is the high performance liquid chromatogram of compound 1, 2, 3 and AP, wherein: A shows the spectrum of compound 1, B shows the spectrum of compound 2, C shows the spectrum of compound 3, and D shows the spectrum of compound AP.

[0019] Figure 3 This is a diagram of the binding kinetics of propranolol hydrochloride, AP and hydroxyapatite (HAP). (A) shows the results of measurement at 210 nm, and (B) shows the results of measurement at 290 nm.

[0020] Figure 4 AP is cleaved and releases propranolol in the mouse bone microenvironment. (A) shows the content of propranolol in the bone marrow interstitial fluid of the AP group, (B) shows the content of propranolol in the bone marrow cell lysate of the AP group, (C) shows the content of propranolol in the bone marrow interstitial fluid of the propranolol hydrochloride group, and (D) shows the content of propranolol in the bone marrow cell lysate of the propranolol hydrochloride group.

[0021] Figure 5 AP is a factor in the regulation of mouse bone marrow OPN + The role of cells, as shown in the figure (A) is the detection of OPN in mouse bone marrow cells by flow cytometry + A typical flow chart of the ratio, (B) shows OPN in mouse bone marrow cells+ Typical images of cells, (C) shows OPN in mouse bone marrow cells + Statistical results of cells.

[0022] Figure 6 AP inhibits osteoclasts and increases bone mass. (A) shows TRAP staining of mouse femur sections after 48 days (20×), (B) shows the ratio of osteoclast number to bone surface, (C) shows the ratio of osteoclast surface to bone surface, (D) shows H&E staining of mouse femur paraffin sections after 48 days (5×), and (E) shows the ratio of trabecular volume to bone volume.

[0023] Figure 7 It is the survival period of mice after E0771-Luc cells were injected into the tibia bone marrow cavity.

[0024] Figure 8 These are the IVIS results of mouse tibia bone marrow cavity injection of E0771-Luc cells and treatment with different drugs. (A) shows the BLI monitoring of tumor burden 7, 12, 24, and 35 days after injection of breast cancer cells, (B) shows the tumor bioluminescence values ​​of each group 7 days after injection of E0771-Luc cells, (C) shows the tumor bioluminescence values ​​of each group 35 days after injection of E0771-Luc cells, and (D) shows the changes in tumor growth rate of each group.

[0025] Fig. 9 These are the results of biochemical indicators of peripheral blood serum of mice after AP administration. (A) shows the changes in ALT content, (B) shows the changes in AST content, (C) shows the changes in BUN content, and (D) shows the changes in CREA content. DETAILED DESCRIPTION

[0026] The present invention is further illustrated by specific examples. Various processes and methods not described in detail are conventional methods known in the art, and reagents used without indicating the source and specification are all commercially available analytically pure or chemically pure, and the instruments and experimental mice used can be purchased commercially.

[0027] Example 1: Preparation of bone-targeted alendronic acid-propranolol This embodiment includes the following steps: (1) Synthesis of intermediate compound 1 Propranolol hydrochloride (5 g, 16.9 mmol, 1 eq) and sodium bicarbonate (2.6 g, 31 mmol, 2.2 eq) were dissolved in water (30 ml) and added to a 100 ml single-necked round-bottom flask. Di-tert-butyl dicarbonate (3.72 g, 16.9 mmol, 1 eq) was dissolved in dioxane (15 ml) and added to a 50 ml constant pressure dropping funnel. The aqueous solution was slowly added dropwise to the dioxane solution within 15 min, stirred at room temperature for 12 h, and TLC showed completion. After vacuum filtration, the mixture was washed with water and dried in an oven at 50°C for 4 h to obtain white solid compound 1 (5.2 g, 85.67%). 1 H NMR (600 MHz, Chloroform-d) δ 8.23 ​​(d, J= 7.7 Hz, 1H), 7.81 (d, J = 7.6 Hz, 1H), 7.52 – 7.42(m, 3H), 7.38 (t, J = 7.9Hz, 1H), 6.85 (d, J = 7.6 Hz, 1H), 5.12 (s, 1H), 4.36 – 3.96 (m, 4H), 3.53(s, 2H), 1.51 (s, 9H), 1.24 (d, J = 6.9 Hz, 3H), 1.15 (d,J = 6.3 Hz, 3H). 13 CNMR (151 MHz, Chloroform-d) δ 154.30, 134.65, 127.74, 126.54, 126.04, 125.60,125.38, 121.80, 120.74, 104.93, 80.90, 72.32,70.00, 48.86, 47.28, 28.61,20.64. (2) Synthesis of intermediate compound 2 Compound 1 (3 g, 8.35 mmol, 1 eq) was dissolved in dichloromethane (20 ml) dried over molecular sieves and added to a 100 ml single-necked round-bottom flask. The solution was cooled to 0 °C and anhydrous triethylamine solution (2.32 ml, 16.7 mmol, 2 eq) was added. 4-Nitrophenyl chloroformate (2.02 g, 10.02 mmol, 1.2 eq) was dissolved in dichloromethane (20 ml) dried over molecular sieves and added to a 50 ml constant pressure drop funnel. At 0 °C, the mixture was slowly added to the above solution over 15 min. After the addition was completed, the reaction solution was slowly heated to room temperature and stirred at room temperature for 24 h. TLC showed that the reaction was complete. The reaction solution was extracted with saturated aqueous sodium carbonate solution (15 ml×3), the aqueous layer was discarded, the dichloromethane layer was dried over anhydrous magnesium sulfate and filtered, concentrated under reduced pressure, and purified by silica gel column (dichloromethane: petroleum ether = 6:1) to obtain a light yellow solid compound 2 (2.26 g, 51.60%). 1 H NMR (600MHz, Chloroform-d) δ 8.25 (dd, J = 11.8, 8.7 Hz, 3H), 7.82 (d, J = 8.1 Hz, 1H), 7.52 (ddd, J = 8.1, 6.7, 1.3 Hz, 1H), 7.49 – 7.44 (m, 2H), 7.38 (t, J =7.9 Hz, 1H), 7.34 (d, J = 9.1 Hz, 2H), 6.82 (d, J = 8.0 Hz, 1H), 5.61 (s,1H), 4.34 (dd, J =10.6, 6.3 Hz, 2H), 4.29 – 4.04 (m, 1H), 3.80 – 3.52 (m,2H), 1.51 (d, J = 3.4 Hz, 9H), 1.26 (d, J = 6.8 Hz, 3H), 1.21 (d, J = 6.7 Hz,3H).13C NMR (151 MHz,Chloroform-d) δ 155.65, 154.09, 152.48, 145.60, 134.70,127.74, 126.78, 125.86, 125.62, 125.44, 122.00, 121.92, 121.25, 104.87,80.57, 67.78, 29.84, 28.60. (3) Synthesis of intermediate compound 3 Compound 2 (0.58 g, 1.11 mmol, 1 eq) and C S2CO3 (1.08 g, 3.33 mmol, 3 eq) was dissolved in dioxane (10 ml) and added to a 50 ml single-necked round-bottom flask. Alendronic acid (0.276 g, 1.11 mmol, 1 eq) and C S2 CO3 (0.72 g, 2.22 mmol, 2 eq) was dissolved in water (10 ml) and added to a 50 ml constant pressure drip funnel. At room temperature, the aqueous solution was slowly added dropwise to the dioxane solution within 15 min. After the addition was completed, the reaction solution was placed in a 60°C oil bath and heated and stirred for 24 h. TLC showed that the reaction was complete. After the reaction solution was cooled, a mixed solution of water and ethyl acetate (40 ml, H2O:EA=1:1) was added. After standing and stratification, the aqueous layer was extracted with ethyl acetate (10 ml×3). The ethyl acetate layer was discarded, and 2 M HCl aqueous solution was added to the aqueous layer until pH = 5. After extraction with ethyl acetate (10 ml×3) again, 2 M HCl aqueous solution was added to the aqueous layer to complete the precipitation. After extraction with dichloromethane (10 ml×3), the dichloromethane layers were combined and dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain white solid compound 3 (0.37 g, 52.86%). No further purification was required, and the next step reaction was carried out directly.

[0028] (4) Synthesis of target compounds Compound 3 (0.36 g, 0.57 mmol, 1 eq) was dissolved in 4M methanolic hydrogen chloride solution (5 ml, 20 mmol, 35 eq), added to a 20 ml single-necked round-bottom flask, and stirred at room temperature for 2 h. After the mass spectrum showed that compound 3 disappeared, 4M NaOH aqueous solution was added to completely precipitate it. After vacuum filtration, the precipitate was washed with methanol and dried in an oven at 50 °C to obtain a white solid compound - bone-targeted alendronic acid-propranolol (0.13 g, 41.67%). 1H NMR (600 MHz, Methanol-d4) δ7.57 – 7.53 (m, 2H), 7.36 (t, J = 7.9 Hz, 1H), 6.33 (s, 1H), 6.29 (t, J = 4.4Hz, 1H), 5.07 – 5.04 (m, 1H), 4.26 – 4.19 (m, 2H), 3.08 – 3.05 (m, 1H), 3.05– 2.98 (m, 2H), 2.69 (q, J = 6.8 Hz, 1H), 2.24 (t, J = 6.7 Hz,2H), 1.80 –1.75 (m, 2H), 1.14 (d, J = 5.3 Hz, 3H), 1.09 (d, J = 5.1 Hz, 3H).13C NMR (151MHz, Common NMR Solvents) δ 156.43, 154.47, 134.62, 126.85,126.42, 125.68,125.63, 125.58, 123.69, 122.39, 107.36, 78.63, 78.00, 77.37, 70.32, 68.25,48.90, 47.51, 40.52, 40.48, 40.44, 31.38, 31.34, 31.29, 23.35,23.28, 23.21,20.93. The structural identification and purity analysis of compounds 1, 2 and AP are as follows. Since compound 3 was directly subjected to the next step reaction without further purification, NMR analysis was not performed on compound 3.

[0029] MS and 1 H-NMR detection: Weigh appropriate amounts of compounds 1, 2, 3 and AP, dissolve them in 50% methanol water, and inject them into the mass spectrometer for analysis. Weigh appropriate amounts of compounds 1 and 2, dissolve them in CDCl3, weigh AP, dissolve them in CD3OD 1 H-NMR and 13 C-NMR analysis. Results are shown in Figure 1 , Table 1.

[0030] HPLC detection of compounds: Weigh appropriate amounts of compounds 1, 2, 3 and AP, dissolve them in 50% methanol water, inject them into liquid chromatography for analysis, and determine the retention time and purity of the compounds. Figure 2 And as shown in Table 1.

[0031] Table 1 Spectral data of intermediate compounds and target compounds in, Figure 2 HPLC elution conditions were: 0.1% methanol (A)-0.1% formic acid aqueous solution (B), gradient elution: 0~2 min, 5% A; 2 min~25 min, 5% A~100% A; 25 min~30 min, 100% A; 30 min~35 min, 100% A~5% A.

[0032] The above results prove that compound AP was successfully synthesized.

[0033] Example 2: Bone targeting of alendronic acid-propranolol and release of propranolol in bone 2.1 Methods for determining AP bone targeting in vitro The propranolol hydrochloride solution was scanned by UV-visible light using NanoDrop to determine the maximum absorption wavelength λ max Weigh propranolol hydrochloride and AP, dilute with 1 ml PBS, place one part in a 1.5 ml centrifuge tube, add HAP (3 mg, 30 eq), shake at 37 °C, 220 rpm to obtain a suspension. The other part was not added with HAP as a control. After 0, 0.25, 0.5, 1, 4, 8, 14, 24 h, centrifuge at 6000 rpm for 3 min, and detect the supernatant at λ using NanoDrop. max The absorbance at .

[0034] The percentage of binding to HAP was calculated as follows: HPA binding rate = [(OD without HAP - OD with HAP) / (OD without HAP)] × 100%, where OD represents optical density.

[0035] 2.2 Determination of propranolol release from AP in the bone microenvironment in vivo Detection conditions: Mass spectrometry conditions: Multiple reaction monitoring (MRM) mode was used: ion pairs were m / z 261.0→116.2, 216.0→158.1, 216.0→184.0. Chromatographic conditions: Analytical column: phenomenex Kinetex XB-C18, (2.6 μm, 50×2.0 cm); mobile phase: methanol (A): 0.1% formic acid aqueous solution (B), gradient elution: 0~0.3 min, 70% A; 0.3 min~1 min, 70% A~5% A; 1 min~5 min, 5% A; flow rate: 0.4 ml / min; column temperature 40 ℃; injection volume 5 μl.

[0036] Collection and pretreatment of biological samples: 20% β-cyclodextrin (β-CD) was used to prepare 0.90 mg / ml APi.p. solution and 0.5 mg / ml propranolol hydrochloride ip solution. Female C57BL / 6J mice were randomly divided into propranolol hydrochloride group and AP group, with 3 mice in each group. They were fasted for 12 h before administration. The mice were weighed and ip-administered at 10 μl / g. After 24 h, they were killed and the right leg bones were taken to separate the bone marrow interstitial fluid and bone marrow cells. 300 μl of pre-cooled HPLC-grade methanol solution was added to the bone marrow cells, vortexed and mixed, placed on ice, ultrasonically disrupted for 3 min, centrifuged at 14000 r for 10 min, and the supernatant was separated to obtain bone marrow cell lysate. 100 μl of bone marrow interstitial fluid and bone marrow cell lysate were taken respectively, added with 300 μl of HPLC grade methanol solution, vortexed and mixed, centrifuged at 14000 r for 10 min, and 100 μl of supernatant was placed in an injection vial for HPLC-MS / MS analysis.

[0037] Results UV full wavelength scanning showed that the maximum absorption wavelengths of propranolol hydrochloride were approximately 210 nm and 290 nm. After adding HAP, the OD value of propranolol hydrochloride did not change significantly at these two wavelengths, while the OD value of AP decreased significantly. At 15 minutes, the binding rate of AP and HAP reached 70%; after 8 hours, AP was basically completely bound to HAP. During the entire 24 hours, the binding rate of propranolol hydrochloride and HAP was basically 0 (e.g. Figure 3 The results show that HAP adsorbs AP quickly and completely, suggesting that AP has good bone targeting.

[0038] The mice were weighed and ip administered with propranolol hydrochloride (10 mg / kg) or AP (18.10 mg / kg). 24 h later, no propranolol peak was found in the bone marrow interstitial fluid and bone marrow cell lysate of the propranolol hydrochloride group, while it was detected in the AP group (e.g. Figure 4 As shown in Figure 2 ). This further indicates that propranolol cannot accumulate in the bone microenvironment because it does not have bone targeting. After AP is targeted to the bone in vivo, it may break down to form the original drug due to the action of the microenvironment pH and enzymes, thereby achieving the purpose of local bone tissue accumulation of propranolol.

[0039] Example 3: Inhibitory effect of bone-targeted alendronic acid-propranolol on breast cancer bone metastasis 3.1 AP for bone marrow OPN + Cell Inhibition Research Methods 3.1.1 Dosage: AP ip solution with concentrations of 1.81, 0.905, 0.362, 0.181, and 0.091 mg / ml was prepared with 20% β-cyclodextrin (β-CD). Female mice were randomly divided into 6 groups, 3 mice in each group, and injected intraperitoneally with 10 μl / g of the above AP solutions of different concentrations. The control group (0 mg / ml AP) was injected with 10 μl / g of 20% β-CD solution twice a week for a total of 6 weeks.

[0040] 3.1.2 Preparation of flow cytometry samples of bone marrow cells of AP-administered mice: After the mice were administered, they were killed by dislocation of the neck, and bone marrow cells of the right hind limb of the mice were taken for flow cytometry detection. After cell technology, the cells were resuspended with cell fixation and permeabilization buffer, perforated at 4℃ for 20min, and then washed with 1ml FACS buffer, and then added with OPN primary antibody solution for staining at 4℃ for 30min. After that, they were washed with 1ml FACS buffer, and then added with OPN secondary antibody solution, and incubated at 37℃ for 1h. After that, they were washed with 1ml FACS buffer and placed in a flow detection tube, and stored at 4℃ in the dark until the flow detection machine.

[0041] 3.2 Research methods on the protective effect of AP on bone tissue under stress 3.2.1 Dosage method: First, a mouse chronic restraint stress (CIS) model was established: 40 female mice were randomly divided into 4 groups, 10 mice in each group. The mice were placed in a 50 ml syringe with a hole, and the mice were restrained with a rubber stopper to prevent them from moving freely for a total of 2 hours, 6 days a week, for a total of 6 weeks. 1mg / ml, 0.84 mg / ml, 1.81mg / ml propranolol hydrochloride, alendronic acid, and AP ip injection were prepared with 20% β-CD solution. The mice were weighed and injected at 10 μl / g. The control group was injected with 20% β-CD solution. The mice were restrained twice a week for a total of 6 weeks.

[0042] 3.2.2 Construction of bone metastasis model: At the end of the third week of restraint, mice were anesthetized and injected with 2×10 5 E0771-Luc breast cancer cells.

[0043] 3.2.3 Hematoxylin-eosin (H&E) staining of mouse leg bones: After the modeling was completed, the mice were killed and the right leg bones were decalcified with 10% EDTA and embedded in paraffin. The paraffin blocks of the leg bones of mice in different groups were sliced ​​and placed in a 37 °C oven for 30 min after slicing. Then they were dewaxed: xylene for 4 min × 3 times, alcohol (100%, 95%, 85%, 75%) for 2 min at each concentration, rinsed with running water for 2 min, then stained with hematoxylin for 50 s, rinsed with running water for 3-5 min, then washed in 95% alcohol, stained with eosin for 50 s, then 95% alcohol for 3 min, 100% alcohol for 1 min × 2 times, xylene for 4 min × 3 times, and finally sealed with neutral resin.

[0044] 3.2.4 Tartrate resistant acidphosphatase (TRAP) staining of mouse leg bones: Add 200 ml of TRAP buffer to the antigen retrieval box and keep it warm at 37°C. Tissue dewaxing and rehydration: xylene × 3 × 6 min, alcohol (100%, 95%, 85%, 75%) for 2 min at each concentration, and place in tap water. Place the slide in a humidified box, drop the prepared naphthol bisphosphate substrate buffer solution on the slide, and incubate at 37°C for 45 min. Mix 4 ml of sodium nitrite solution and parafuchsin stain for 30 s, let it stand for 2 min, then add it to 200 ml buffer kept at 37°C, and place the slide in the box. Incubate at room temperature for 5-10 min, rinse with distilled water, stain the nucleus with hematoxylin for 30 s, and rinse with tap water for 4-5 min. Dehydrate in alcohol gradient for 2 min each, after 100% alcohol, place in xylene and remove the sealant from xylene.

[0045] 3.3 Study on the inhibitory effect of AP on breast cancer bone metastasis under stress 3.3.1 Dosage: The method for constructing the mouse CIS model is the same as that in Section 3.2.1 of this section. 0.5 mg / ml, 0.42 mg / ml, and 0.91 mg / ml of propranolol hydrochloride, alendronic acid, and AP ip injection were prepared with 20% β-CD solution. The mice were weighed and injected at 10 μl / g. The control group was injected with 20% β-CD solution. The injection was given twice a week from the beginning of restraint for a total of 6 weeks.

[0046] 3.3.2 Construction of bone metastasis model: After the third week of restraint, mice were anesthetized and injected with 1×10 5 E0771-Luc breast cancer cells.

[0047] 3.3.3 In vivo imaging of mice to monitor tumor changes: Take an appropriate amount of Luc substrate (D-Luciferinpotassium salt) solid and dissolve it in sterile PBS to prepare a 10 mg / ml substrate solution. Each mouse is injected with 200 μl ip. After 10 minutes, it is anesthetized with isoflurane and placed in IVIS for photography. After the tumor injection, IVIS is used to monitor tumor changes every week.

[0048] 3.4 Results 3.4.1 AP for bone marrow OPN + Inhibitory effect on cells After 6 weeks of AP administration, the results of mouse flow cytometry showed that with the increase of AP concentration, the expression of OPN in mouse bone marrow increased. + The number of cells gradually decreased, and the AP effect reached its maximum at 9.05 mg / kg. There was a statistically significant difference between 9.05 mg / kg and 18.10 mg / kg compared with the control group (e.g. Figure 5 The results show that AP has the effect of inhibiting breast cancer bone metastasis at these two concentrations.

[0049] 3.4.2 Protective effect of AP on mouse bone tissue The mice were restrained for 6 weeks before being killed. The right femur was embedded in paraffin and sliced ​​for TRAP and H&E staining. TRAP staining showed that compared with the control group and propranolol hydrochloride group, the number of osteoclasts / bone surface area (Oc.n / BS) and the surface area of ​​osteoclasts / bone surface area (Oc.S / BS%) in the alendronic acid group and the AP group were reduced, indicating that osteoclasts were inhibited. The results of H&E staining showed that the bone volume fraction (BV / TV) in the alendronic acid group and the AP group increased, indicating that the bone mass in these two groups increased (e.g. Figure 6 In general, AP has the effect of inhibiting osteoclasts and has a certain protective effect on bone tissue, which also reflects that AP has bone targeting in vivo to a certain extent.

[0050] 3.4.3 The inhibitory effect of AP on breast cancer tumors in bones and its effect on the survival of mice E0771-Luc breast cancer cells were injected into the bilateral tibia bone marrow cavity of CIS-treated mice. The results showed that mice in each group died during the entire experiment, and even all mice in the alendronic acid group died. The survival time of the AP group was improved compared with that of the β-CD group and the propranolol hydrochloride group (e.g. Figure 7 as shown).

[0051] After E0771-Luc was injected into the tibia bone marrow cavity of mice, IVIS detection was performed on days 7, 12, 24, and 35. Since all mice in the alendronic acid group died, the IVIS results compared the bioluminescence intensity of the β-CD group, propranolol hydrochloride group, and AP group. It was found that on day 7, although the bioluminescence of the propranolol hydrochloride group and the AP group was stronger than that of the β-CD group, there was no statistical significance among the three groups. On day 35, the bioluminescence intensity of the β-CD group and the propranolol hydrochloride group was significantly stronger than that of the AP group, indicating that the tumor in the AP group was the smallest and tumor growth was inhibited.

[0052] At the same time, the effect of AP on tumor growth rate was observed. The bioluminescence intensity of tumor cells in vivo imaged by mice on the 7th day after E0771-Luc tumor cell injection was used as the baseline value to compare the tumor growth rate. The results showed that there was no difference in the luminescence intensity of tumor cells in each group on the 7th day, and then all groups grew slowly until the 24th day. However, there was a significant difference on the 35th day: the tumor growth of mice in the control group and propranolol hydrochloride group was significantly accelerated, while the tumor of mice in the AP group not only did not grow further, but was reduced compared with the 24th day (such as Figure 8 These results all indicate that AP administration can inhibit the growth of tumors after bone metastasis.

[0053] Example 4: Evaluation of the hepato-renal toxicity of bone-targeted alendronate-propranolol 4.1 Evaluation method of AP hepatotoxicity and renal toxicity: APi.p. solutions with concentrations of 1.81, 0.905, 0.362, 0.181, and 0.091 mg / ml were prepared with 20% β-cyclodextrin (β-CD). Female mice were randomly divided into 6 groups, 3 mice in each group, and 10 μl / g of AP solutions of different concentrations were intraperitoneally injected. The control group (0 mg / ml AP) was injected with 10 μl / g 20% ​​β-CD solution twice a week for a total of 6 weeks. After the last week, the mice were bled, and the obviously hemolyzed samples were discarded, and the serum was analyzed. The biochemical indices were tested as follows: alanine aminotransferase (AST), aspartate aminotransferase (ALT), urea nitrogen (BUN), and creatinine (CRE).

[0054] Results After 6 weeks of AP administration, the biochemical test of mouse serum showed that AST had a dose-dependent increase trend with AP, and the AST value at the maximum AP concentration (18.10 mg / kg) was statistically different from the AST value of the control group. However, when the AP concentration was in the range of 0-18.10 mg / kg, the ALT, BUN and CRE levels of mouse serum did not show dose-dependent changes, and there was no significant difference compared with the control group (such as Fig. 9 These results suggest that AP has weak hepatotoxicity and renal toxicity and has a certain degree of safety.

Claims

1. Bone-targeted alendronic acid-propranolol, characterized in that: Its chemical structure is shown in formula (AP): (AP).

2. The method for preparing the bone-targeted alendronic acid-propranolol according to claim 1, characterized in that: The synthetic route is as follows: ； Wherein, step a is: propranolol reacts with di-tert-butyl dicarbonate to generate compound 1; Step b is: compound 1 reacts with 4-nitrophenyl chloroformate to generate compound 2; Step c is: compound 2 reacts with alendronic acid to generate compound 3; Step d is: compound 3 is deprotected under the condition of hydrogen chloride methanol to generate compound AP.

3. Use of the bone-targeted alendronic acid-propranolol according to claim 1 in inhibiting tumor bone metastasis.

4. The use according to claim 3, characterized in that: The tumor is breast cancer.

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