Bone-targeted alendronic acid-propranolol and its preparation method and application

Abstract

The present invention discloses bone-targeted alendronate-propranolol and its preparation method and application. The bone-targeted alendronate-propranolol has good in vitro bone targeting and dissociates and releases the propranolol original drug in the bone microenvironment in vivo, which 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. The synthesis route and post-processing method of the present invention are safe and simple, the synthetic raw materials and solvents used are cheap and easy to obtain, and the intermediate compounds and target products have high yields. The present invention also proposes the application of bone-targeted alendronate-propranolol in the preparation of drugs for treating breast cancer bone metastasis.

Description

Technical Field

[0001] The present 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 a major cause of death in cancer patients. Bone metastasis of cancer refers to the transfer of cancer cells through the bloodstream to bone tissue and the formation of new tumors. Epidemiological studies have shown that breast cancer, lung cancer, and liver cancer are all malignant tumors prone to bone metastasis. Breast cancer mediates osteolytic bone metastasis, with a high late-stage bone metastasis rate of 65% to 75% and a low 5-year overall survival rate of 22.8%. Once bone metastasis occurs, it is virtually incurable and can cause varying degrees of bone pain, pathological fractures, spinal cord compression, and hypercalcemia. These are all skeletal complications of malignant tumor bone metastasis, known as skeletal-related events (SREs), which bring great suffering 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 clinically to treat arrhythmias, hypertension, and other conditions. Studies have shown that propranolol can inhibit bone metastasis of breast cancer. However, many cell and tissue types express beta-receptors, which can lead to systemic effects of propranolol and potentially significant toxic side 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:

[0008] The bone-targeted alendronate-propranolol provided by the present invention has a chemical structure as shown in formula (AP):

[0009] (AP).

[0010] The beneficial effects of the present invention are reflected in the fact that the bone-targeted alendronate-propranolol provided by the present invention has good bone targeting in vitro and dissociates and releases the original propranolol drug in the bone microenvironment in vivo. Bone-targeted alendronate-propranolol can inhibit osteoclasts and have a certain protective effect on bone tissue. It can also inhibit tumor growth after breast cancer bone metastasis, and is expected to provide a new treatment method for breast cancer bone metastasis.

[0011] The present invention also provides a preparation method of bone-targeted alendronic acid-propranolol, and the synthesis route is as follows:

[0012] ;

[0013] Wherein, step a is: propranolol reacts with di-tert-butyl dicarbonate to generate compound 1;

[0014] Step b is: compound 1 reacts with 4-nitrophenyl chloroformate to generate compound 2;

[0015] Step c is: compound 2 reacts with alendronic acid to generate compound 3.

[0016] Step d is: compound 3 is deprotected under hydrogen chloride methanol conditions to generate compound AP.

[0017] The step a comprises: dissolving propranolol hydrochloride and sodium bicarbonate in water, dissolving di-tert-butyl dicarbonate in dioxane, slowly adding the aqueous solution dropwise to the dioxane solution and stirring at room temperature for 12 hours until TLC shows completion; filtering under reduced pressure, washing with water, and drying to obtain compound 1.

[0018] The step b comprises: dissolving compound 1 in dichloromethane, cooling the solution to 0°C, and adding anhydrous triethylamine solution; dissolving 4-nitrophenyl chloroformate in dichloromethane and slowly adding the solution to the solution at 0°C over 15 minutes; after the dropwise addition is completed, slowly warming the reaction solution to room temperature and stirring at room temperature for 24 hours, and TLC shows that the reaction is complete; extracting the reaction solution with a saturated sodium carbonate aqueous solution, discarding the aqueous layer, and drying the dichloromethane layer with anhydrous magnesium sulfate, filtering, concentrating under reduced pressure, and purifying on a silica gel column to obtain compound 2.

[0019] 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 over 15 minutes, and after the dropwise addition is complete, placing the reaction solution in a 60°C oil bath and heating with stirring for 24 hours, until TLC indicates the reaction is complete; after the reaction solution is cooled, adding a mixed solution of water and ethyl acetate, allowing the solution to stand for stratification, extracting the aqueous layer with ethyl acetate, discarding the ethyl acetate layer, adding a 2 M HCl aqueous solution to the aqueous layer until the pH is 5, extracting again with ethyl acetate, adding a 2 M HCl aqueous solution to the aqueous layer to complete precipitation, and then extracting with dichloromethane, combining the dichloromethane layers, drying over anhydrous magnesium sulfate, and concentrating under reduced pressure to obtain compound 3.

[0020] 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. After vacuum filtration, the precipitate is washed with methanol and dried in an oven at 50° C. to obtain a compound represented by formula (AP).

[0021] 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.

[0022] Given that the bone-targeted alendronate-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

[0023] 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.

[0024] Figure 2 These are the high performance liquid chromatograms of compounds 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.

[0025] 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.

[0026] Figure 4AP decomposes and releases propranolol in the bone microenvironment of mice. In the figure, (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.

[0027] 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 flow cytometry detection of OPN in mouse bone marrow cells + 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 + Cell statistics results.

[0028] Figure 6 AP inhibits osteoclasts and increases bone mass. (A) shows TRAP staining of mouse femur sections 48 days after treatment (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 paraffin sections of mouse femurs 48 days after treatment (5×), and (E) shows the ratio of trabecular volume to bone volume.

[0029] Figure 7 It is the survival period of mice after E0771-Luc cells were injected into the tibial bone marrow cavity.

[0030] Figure 8 Figure 3 shows the IVIS results of mouse tibial bone marrow cavity injection of E0771-Luc cells and treatment with different drugs. (A) shows BLI monitoring of tumor burden 7, 12, 24, and 35 days after breast cancer cell injection. (B) shows the tumor bioluminescence values ​​of each group 7 days after E0771-Luc cell injection. (C) shows the tumor bioluminescence values ​​of each group 35 days after E0771-Luc cell injection. (D) shows the changes in tumor growth rate in each group.

[0031] Figure 9 These are the results of peripheral blood serum biochemical indexes of mice after AP administration. In the figure, (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

[0032] The present invention is further illustrated below by means of specific examples. Various processes and methods not described in detail are conventional methods known in the art. All reagents used without indicating their source or specifications were commercially available as analytical or chemically pure. All instruments and experimental mice used were commercially available.

[0033] Example 1: Preparation of bone-targeted alendronate-propranolol

[0034] This embodiment includes the following steps:

[0035] (1) Synthesis of intermediate compound 1

[0036] 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 over 15 minutes. The mixture was stirred at room temperature for 12 hours until completion by TLC. The product was filtered under reduced pressure, washed with water, and dried in an oven at 50°C for 4 hours to yield Compound 1 (5.2 g, 85.67%) as a white solid. 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.

[0037] (2) Synthesis of intermediate compound 2

[0038] Compound 1 (3 g, 8.35 mmol, 1 eq) was dissolved in molecular sieve-dried dichloromethane (20 ml) 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. Nitrophenyl 4-chloroformate (2.02 g, 10.02 mmol, 1.2 eq) was dissolved in molecular sieve-dried dichloromethane (20 ml) and added to a 50 ml constant pressure dripping funnel. The mixture was slowly added to the above solution over 15 minutes at 0°C. After the addition was complete, the reaction solution was slowly warmed to room temperature and stirred at room temperature for 24 hours. TLC indicated the reaction was complete. The reaction solution was extracted with saturated aqueous sodium carbonate solution (15 ml × 3), the aqueous layer was discarded, and the dichloromethane layer was dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure, and purified on a 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.

[0039] (3) Synthesis of intermediate compound 3

[0040] Compound 2 (0.58 g, 1.11 mmol, 1 eq) and CS2 CO3 (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 dripping funnel. The aqueous solution was slowly added dropwise to the dioxane solution at room temperature over 15 minutes. After the addition was complete, the reaction solution was heated and stirred in an oil bath at 60°C for 24 hours. TLC indicated the reaction was complete. After cooling, a mixture of water and ethyl acetate (40 ml, H₂O:EA = 1:1) was added. After standing for separation, the aqueous layer was extracted with ethyl acetate (10 ml x 3). The ethyl acetate layer was discarded, and the aqueous layer was added with 2 M aqueous HCl to pH = 5. After extraction with ethyl acetate (10 ml x 3), the aqueous layer was completely precipitated with 2 M aqueous HCl. The reaction was then extracted with dichloromethane (10 ml x 3). The combined dichloromethane layers were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to yield compound 3 (0.37 g, 52.86%) as a white solid. This was carried on to the next step without further purification.

[0041] (4) Synthesis of target compound

[0042] Compound 3 (0.36 g, 0.57 mmol, 1 eq) was dissolved in 4 M methanolic hydrogen chloride (5 ml, 20 mmol, 35 eq) in a 20 ml single-necked round-bottom flask and stirred at room temperature for 2 h. After the mass spectrum showed the disappearance of compound 3, 4 M aqueous sodium hydroxide was added to completely precipitate it. The precipitate was filtered under reduced pressure, washed with methanol, and dried in a 50°C oven to obtain a white solid compound—bone-targeted alendronate-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.

[0043] The structural identification and purity analysis of compounds 1, 2, and AP are as follows. Since compound 3 was directly used for the next reaction without further purification, NMR analysis was not performed on compound 3.

[0044] 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.

[0045] HPLC detection of compounds: Weigh appropriate amounts of compounds 1, 2, 3 and AP and 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.

[0046] Table 1 Spectral data of intermediate compounds and target compounds

[0047]

[0048] in, Figure 2 The 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.

[0049] The above results demonstrate that compound AP was successfully synthesized.

[0050] Example 2: Bone targeting of alendronate-propranolol and release of propranolol in bone

[0051] 2.1 Determination of AP bone targeting in vitro

[0052] The propranolol hydrochloride solution was scanned by UV-visible light at all wavelengths using NanoDrop to determine the maximum absorption wavelength λ max Propranolol hydrochloride and AP were weighed and diluted with 1 ml PBS respectively. A portion was placed in a 1.5 ml centrifuge tube, and HAP (3 mg, 30 eq) was added. The suspension was shaken at 37 °C and 220 rpm to obtain a suspension. The other portion was not added with HAP as a control. After 0, 0.25, 0.5, 1, 4, 8, 14, and 24 h, the supernatant was centrifuged at 6000 rpm for 3 min, and the supernatant was detected by NanoDrop at λ max The absorbance at .

[0053] 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.

[0054] 2.2 Determination of propranolol release from AP in the bone microenvironment in vivo

[0055] Detection conditions: Mass spectrometry conditions: Multiple reaction monitoring (MRM) mode was used with ion transitions m / z 261.0→116.2, 216.0→158.1, and 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 in water (B); Gradient elution: 70% A (0–0.3 min); 70% A (0.3 min–1 min); 5% A (1 min–5 min); Flow rate: 0.4 ml / min; Column temperature: 40°C; Injection volume: 5 μl.

[0056] Biological sample collection and pretreatment: A 0.90 mg / ml solution of AP (i.p.) and a 0.5 mg / ml solution of propranolol hydrochloride (i.p.) were prepared in 20% β-cyclodextrin (β-CD). Female C57BL / 6J mice were randomly divided into a propranolol hydrochloride group and an AP group, with three mice in each group. The mice were fasted for 12 hours before administration. Mice were weighed and administered i.p. at a rate of 10 μl / g. Twenty-four hours later, the mice were sacrificed and the right femur was removed for separation of bone marrow interstitial fluid and bone marrow cells. 300 μl of pre-chilled HPLC-grade methanol was added to the bone marrow cells, vortexed, placed on ice, sonicated for 3 minutes, and centrifuged at 14,000 r / min for 10 minutes. 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 to 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.

[0057] Results

[0058] UV full-wavelength scanning showed that the maximum absorption wavelengths of propranolol hydrochloride were approximately 210 nm and 290 nm. After the addition of HAP, the OD values ​​of propranolol hydrochloride at these two wavelengths did not change significantly, while the OD value of AP decreased significantly. At 15 minutes, the binding rate of AP to HAP reached 70%; after 8 hours, AP was almost completely bound to HAP. For the entire 24 hours, the binding rate of propranolol hydrochloride to HAP was essentially zero (e.g., Figure 3 This demonstrates that HAP adsorbs AP quickly and completely, suggesting that AP has good bone targeting.

[0059] 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 both the AP group (e.g. Figure 4 This further suggests that propranolol cannot accumulate in the bone microenvironment due to its inherent lack of bone targeting. However, after AP is targeted to the bone in vivo, it may be broken down into the original drug due to the action of the microenvironmental pH and enzymes, thereby achieving the goal of localized bone tissue accumulation of propranolol.

[0060] Example 3: Inhibitory effect of bone-targeted alendronate-propranolol on breast cancer bone metastasis

[0061] 3.1 AP vs. Bone Marrow OPN + Cell Inhibitory Effects Research Methods

[0062] 3.1.1 Administration: AP solutions were prepared with 20% β-cyclodextrin (β-CD) at concentrations of 1.81, 0.905, 0.362, 0.181, and 0.091 mg / ml. Female mice were randomly divided into six groups of three mice each and received intraperitoneal injections of 10 μl / g of the AP solutions at these concentrations. A control group (0 mg / ml AP) received 10 μl / g of 20% β-CD solution twice weekly for six weeks.

[0063] 3.1.2 Preparation of Flow Cytometry Samples for Bone Marrow Cells from AP-Administered Mice: After administration, mice were sacrificed by cervical dislocation, and bone marrow cells from the right hind limb of the mice were collected for flow cytometry. After cell culture, the cells were resuspended in cell fixation and permeabilization buffer, perforated at 4°C for 20 minutes, and then washed with 1 ml of FACS buffer. OPN primary antibody solution was then added and stained at 4°C for 30 minutes. After staining, the cells were washed with 1 ml of FACS buffer, and then OPN secondary antibody solution was added. The cells were incubated at 37°C for 1 hour. After washing, the cells were placed in a flow cytometer tube and stored in the dark at 4°C until analysis on the flow cytometer.

[0064] 3.2 Research methods on the protective effect of AP on bone tissue under stress

[0065] 3.2.1 Administration: First, a chronic restraint stress (CIS) model was established in mice. Forty female mice were randomly divided into four groups of 10 mice each. Mice were placed in a 50 ml syringe with a perforated syringe and restrained with a rubber stopper to prevent free movement for 2 hours, 6 days a week for 6 weeks. Propranolol hydrochloride, alendronic acid, and AP solutions at 1 mg / ml, 0.84 mg / ml, and 1.81 mg / ml were prepared in a 20% β-CD solution. Mice were weighed and injected at a rate of 10 μl / g. A control group received a 20% β-CD solution. This was administered twice weekly from the start of restraint for a total of 6 weeks.

[0066] 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.

[0067] 3.2.3 Hematoxylin-eosin (H&E) staining of mouse femurs: After modeling, mice were sacrificed, and right femurs were removed and decalcified with 10% EDTA before being embedded in paraffin. Paraffin blocks of femurs from mice in different groups were sectioned and baked in a 37°C oven for 30 min. Dewaxing followed by three cycles of xylene for 4 min, followed by 2 min of each concentration of alcohol (100%, 95%, 85%, and 75%). After rinsing with running water for 2 min, the sections were stained with hematoxylin for 50 s, rinsed with running water for 3-5 min, and then rinsed once in 95% alcohol and eosin for 50 s. The sections were then incubated with 95% alcohol for 3 min, 100% alcohol for 1 min twice, and xylene for 4 min three times. Finally, the sections were mounted with neutral resin.

[0068] 3.2.4 Tartrate-resistant acidphosphatase (TRAP) staining of mouse leg bones: Add 200 ml of TRAP buffer to an antigen retrieval box and incubate at 37°C. Tissue dewaxing and rehydration: Xylene (3 x 6 min), alcohol (100%, 95%, 85%, 75%) for 2 min each, then place in tap water. Place the slide in a humidified chamber and drop the prepared naphthol bisphosphate substrate buffer solution onto the slide. Incubate at 37°C for 45 min. Mix 4 ml each of sodium nitrite solution and parafuchsin stain for 30 s. Let stand for 2 min, then add to 200 ml of buffer incubated at 37°C. Place the slide in the chamber. Incubate at room temperature for 5-10 min. Rinse with distilled water, stain the nuclei with hematoxylin for 30 s, and rinse in tap water for 4-5 min. Dehydrate in alcohol gradient for 2 min each, then place in xylene after 100% alcohol, and remove the sealant from xylene.

[0069] 3.3 Study on the inhibitory effect of AP on breast cancer bone metastasis under stress

[0070] 3.3.1 Administration: The mouse CIS model was established using the same method as described in Section 3.2.1 of this section. Propranolol hydrochloride, alendronic acid, and AP were prepared with 20% β-CD solution at 0.5 mg / ml, 0.42 mg / ml, and 0.91 mg / ml, respectively, for intravenous injection. After weighing mice, the injection was administered at a rate of 10 μl / g. A control group received an injection of 20% β-CD solution. Injections were administered twice weekly from the start of restraint for a total of 6 weeks.

[0071] 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.

[0072] 3.3.3 In vivo imaging of mouse tumor changes: Dissolve an appropriate amount of Luc substrate (D-Luciferin potassium salt) in sterile PBS to prepare a 10 mg / ml substrate solution. Administer 200 μl per mouse intraperitoneally. After 10 minutes, anesthetize with isoflurane and place the mouse in an IVIS for imaging. Tumor changes were monitored weekly using IVIS following tumor injection.

[0073] 3.4 Results

[0074] 3.4.1 AP vs. Bone Marrow OPN + Cell inhibition

[0075] After 6 weeks of AP administration, the flow cytometry results of mice showed that with the increase of AP concentration, the OPN expression 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.

[0076] 3.4.2 Protective effect of AP on mouse bone tissue

[0077] Mice were restrained for 6 weeks before being sacrificed. The right femurs were paraffin-embedded and sectioned for TRAP and H&E staining. TRAP staining showed that compared with the control and propranolol hydrochloride groups, the alendronate and AP groups had decreased osteoclast number / bone surface area (Oc.n / BS) and osteoclast surface area / bone surface area (Oc.S / BS%), indicating that osteoclasts were inhibited. H&E staining showed that the bone volume fraction (BV / TV) increased in the alendronate and AP groups, indicating that bone mass increased in both groups (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 to some extent that AP has bone targeting in vivo.

[0078] 3.4.3 Inhibitory Effect of AP on Bone Breast Cancer Tumors and Its Effect on Mouse Survival

[0079] E0771-Luc breast cancer cells were injected into the bilateral tibial bone marrow cavity of CIS-treated mice. The results showed that mice in all groups died during the entire experiment, and even all mice in the alendronate group died. However, the survival time of the AP group was improved compared with the β-CD group and the propranolol hydrochloride group (e.g. Figure 7 shown).

[0080] After E0771-Luc was injected into the tibial bone marrow cavity of mice, IVIS was performed on days 7, 12, 24, and 35. Since all mice in the alendronate group died, IVIS results compared the bioluminescence intensity of the β-CD, propranolol hydrochloride, and AP groups. While the bioluminescence intensity of the propranolol hydrochloride and AP groups was higher than that of the β-CD group on day 7, there was no statistically significant difference between the three groups. On day 35, the bioluminescence intensity of the β-CD and propranolol hydrochloride groups was significantly higher than that of the AP group, indicating that the AP group had the smallest tumors and inhibited tumor growth.

[0081] At the same time, the effect of AP on tumor growth rate was observed. The bioluminescence intensity of tumor cells in mice on day 7 after injection of E0771-Luc tumor cells 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 at day 7. After that, all groups grew slowly until day 24, but there was no difference. However, a significant difference appeared at day 35: 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 actually decreased compared to day 24 (such as Figure 8 These results indicate that AP administration can inhibit the growth of tumors after bone metastasis.

[0082] Example 4: Evaluation of Hepatotoxicity and Renal Toxicity of Bone-Targeted Alendronate-Propranolol

[0083] 4.1 AP hepatotoxicity and renal toxicity evaluation method:

[0084] Api.p. solutions of 1.81, 0.905, 0.362, 0.181, and 0.091 mg / ml were prepared using 20% ​​β-cyclodextrin (β-CD). Female mice were randomly divided into six groups of three mice each and received intraperitoneal injections of 10 μl / g of the various AP concentrations. A control group (0 mg / ml AP) received 10 μl / g of 20% β-CD solution twice weekly for six weeks. After the final week, mice were bled, and samples with significant hemolysis were discarded. Serum was collected for analysis. Biochemical parameters were measured: alanine aminotransferase (AST), aspartate aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine (CRE).

[0085] Results

[0086] After 6 weeks of AP administration, serum biochemical tests of mice showed that AST increased in a dose-dependent manner. The AST value at the maximum AP concentration (18.10 mg / kg) was statistically different from that of the control group. However, when the AP concentration was in the range of 0-18.10 mg / kg, the serum ALT, BUN, and CRE levels of mice did not show dose-dependent changes and showed no significant differences compared with the control group (e.g. Figure 9 These results suggest that AP has weak hepatotoxicity and renal toxicity and is relatively safe.

Claims

1. Bone-targeted alendronate-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: reacting compound 2 with alendronic acid to generate compound 3; Step d is: compound 3 is deprotected under hydrogen chloride methanol conditions to generate compound AP.

3. Use of the bone-targeted alendronate-propranolol according to claim 1 in the preparation of a drug for inhibiting tumor bone metastasis.

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

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