Radioactive marker, precursor compound thereof, preparation method and application

Abstract

The precursor compound of the radioactive marker shown in the formula (I), the radioactive marker or the pharmaceutically acceptable salt of the radioactive marker have the advantages that the water solubility of the radioactive marker is high, the in-vitro stability at room temperature is good, the plasma protein binding rate is high, and the high and long-time skeleton uptake is shown in the in-vivo distribution in mice and the imaging of New Zealand rabbits; animal experiments show that the imaging effect is better than that of 99mTc-MDP. # imgabs0 #

Description

[0001] This application is a divisional application based on the parent application with application number 202280072109.9. The application date of the parent application is January 6, 2022. The parent application claims the priority of the domestic priority application (application number 202111419244.X) filed on November 26, 2021. Technical Field

[0002] The invention belongs to the technical field of nuclear medicine, and in particular relates to a radioactive marker, a precursor compound thereof, and a preparation method and application thereof. Background Art

[0003] Bone is a common site for distant metastasis of malignant tumors, and its incidence is second only to lung and liver. Early diagnosis and treatment of metastatic bone tumors can effectively improve the prognosis and quality of life of patients.

[0004] At present, the treatment methods for metastatic bone tumors mainly include radiotherapy, chemotherapy, radionuclide therapy, bisphosphonate therapy, analgesia and palliative surgery. Targeted radionuclide therapy for metastatic bone tumors can significantly relieve bone pain and kill tumor cells with little toxicity and side effects. It is a safe and effective treatment method. The radioactive drugs commonly used in the treatment of metastatic bone tumors in China include 89 SrCl2, 153 Sm-EDTMP, which can significantly relieve bone pain and reduce the incidence of bone-related events, but both need to be produced by reactors, are relatively expensive, and 89 SrCl2 cannot be used for imaging, so its use is limited. There is an urgent need for more effective, cheap and easily available therapeutic radiopharmaceuticals in clinical practice.

[0005] 68 Ga, 177 Lu, 225 Ac, 64 Cu, 211 At is a medical isotope with excellent nuclear properties. 68 Ga is through 68 Ge / 68 Ga generator is available, with convenient source and low cost; it can emit β + X-rays are used for PET imaging. 177 Lu, 225 Ac, 64 Cu, 211 At are all purchased from abroad, and the supply channels are smooth and stable. 177The half-life (T1 / 2) of Lu is 6.7 days. It emits three energy β-particles of 497keV (78.6%), 384keV (9.1%), and 176keV (12.2%), which can be used for treatment. It also emits γ rays of 113keV (6.4%) and 208keV (11%), which are suitable for in vivo positioning imaging. 225 Ac emits α rays, which have higher energy transmission line density, higher ray energy, shorter range, higher relative biological effect, and the strongest killing effect on tumor cells. Its half-life (T1 / 2) is 9.9 days. 213 The half-life (T1 / 2) of Bi is 46min. 64 Cu nuclide has become a research hotspot in the field of PET molecular probes and integrated diagnostic and therapeutic drugs due to its suitable half-life (12.7h), unique decay properties (β+ decay, β- decay, electron capture), and the ability to form complexes with a variety of ligands. 211 The average energy of α rays released by At decay is 6.8MeV, the range in tissue is 55-88um, and its half-life is 7.2h, which has strong clinical application value. 99m Tc-MDP is the most commonly used bone imaging agent in clinical practice. However, as a single-photon imaging agent, the localization and display of lesions are far inferior to positron imaging agents. Phosphonates (HEDP) are drugs commonly used in clinical studies for the imaging and targeted treatment of metastatic bone tumors. However, as a first-generation nitrogen-free bisphosphonate, the effect strength of HEDP is significantly lower than that of second- and third-generation nitrogen-containing bisphosphonates, such as alendronic acid and ibandronate. Therefore, providing drugs with better imaging effects, better therapeutic effects, and both imaging and treatment of metastatic bone tumors has become an urgent problem to be solved by those skilled in the art. Summary of the invention

[0006] One of the purposes of the present invention is to provide a precursor compound shown in Formula I, which can obtain a drug for both imaging and treating metastatic bone tumors after radionuclide labeling.

[0007] The second object of the present invention is to provide a radioactive label represented by Formula II, which is obtained by labeling the compound of Formula I with a nuclide.

[0008] The third object of the present invention is to provide a method for preparing the precursor compound shown in Formula I.

[0009] A fourth object of the present invention is to provide a method for preparing the radioactive marker represented by formula II.

[0010] A fifth object of the present invention is to provide an application of the precursor compound shown in Formula I.

[0011] A sixth object of the present invention is to provide an application of the radioactive marker represented by formula II.

[0012] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0013] The present invention provides a radioactive marker precursor compound having a structure as shown in Formula I or a pharmaceutically acceptable salt thereof,

[0014]

[0015] The radioactive label of the compound of formula I or a pharmaceutically acceptable salt thereof provided by the present invention has a structure as shown in formula II:

[0016]

[0017] Where A is a nuclide, preferably 68 Ga, 177 Lu, 225 Ac, 64 Cu, 211 At.

[0018] The present invention creatively combines the chelating agent DOTA with ibandronic acid to form a new compound DOTA-ibandronic acid. 68 Ga, 177 Lu, 225 Ac, 64 Cu, 211 When labeled with At, the amount of precursor used is much lower than that of ibandronate alone. We provide a variety of radionuclide-labeled DOTA-ibandronate, which is a new drug that has both bone imaging and the ability to treat metastatic bone tumors. This drug can better exert the effects of radionuclides. 68 Ga, 177 Lu, 225 Ac, 64 Cu, 211 The imaging and therapeutic effects of At and DOTA-ibandronic acid on metastatic bone tumors create an effect of 1 plus 1 greater than 2.

[0019] In some embodiments of the present invention, the radiochemical purity is greater than or equal to 95%.

[0020] The method for preparing the precursor compound of the radioactive marker represented by Formula I provided by the present invention comprises the following steps:

[0021] S1. Compound 1 reacts with tert-butyl acrylate to generate compound 2;

[0022] S2. HCl / EtOAc is added dropwise to the EtOAc solution of compound 2 to react to generate compound 3;

[0023] S3. Compound 3 was added to pHCl, POCl3 and H3PO3 were added under nitrogen protection, heated to react, and after cooling, the chlorobenzene solution was poured out, H2O was added, heated and stirred to generate compound 4;

[0024] S4. DOTA-NHS-ester solution was added to the H2O solution of compound 4, followed by the addition of triethylamine to react to produce a compound of formula I;

[0025] The reaction formula is as follows:

[0026]

[0027] The method for preparing the radioactive marker of formula II provided by the present invention comprises: reacting the compound of formula I with a radioactive nuclide salt solution to obtain the radioactive marker of formula II.

[0028] In some embodiments of the present invention, the radionuclide is 68 Ga, 177 Lu or 64 Cu, the solution of the compound of formula I, the sodium acetate solution, and the radioactive nuclide salt solution are mixed evenly, the pH value of the mixed solution is adjusted, reacted, the pH value is adjusted again, sterilized, and filtered to obtain;

[0029] When the radionuclide is 225 Ac, the solution of the compound of formula I, the sodium citrate solution, the sodium ascorbate solution and the radioactive nuclide salt solution are mixed uniformly, the pH value of the mixed solution is adjusted, reacted, the pH value is adjusted again, sterilized, filtered, and the mixture is obtained;

[0030] When the radionuclide is 211 At, the solution of the compound of formula I, the sodium borate solution, and the radioactive nuclide salt solution are mixed uniformly, the pH value of the mixed solution is adjusted, reacted, the pH value is adjusted again, sterilized, and filtered to obtain.

[0031] In some embodiments of the present invention, when the radionuclide is 68 Ga, 20-30 μg of the compound of formula I was added to 0.8-1.4 ml of 0.25 M sodium acetate solution; then 20 mCi of 68 Ga salt solution; mix evenly, adjust the pH value of the mixed solution to 4-7, preferably 5; react at 80-100°C, preferably 95°C; the reaction time is 10-30min, preferably 15min; after the reaction, adjust the pH value to 5; the concentration of the solution of the compound of formula I is 1mg / ml; 68 The concentration of Ga salt solution is 5mCi / ml~10mCi / ml;

[0032] When the radionuclide is 177Lu, 20-30 μg of the compound of formula I was added to 0.8-1.4 ml of a 0.25 M sodium acetate solution, followed by the addition of 2 mCi of 177 Lu salt solution; mix evenly, adjust the pH value of the mixed solution to 4-7, preferably 5; react at 80-100°C, preferably 95°C; the reaction time is 10-30min, preferably 15min; after the reaction, adjust the pH value to 4.5; the concentration of the solution of the compound of formula I is 1mg / ml; 177 The concentration of Lu salt solution is 10mCi / ml~20mCi / ml;

[0033] When the radionuclide is 225 Ac, 20-30 μg of the compound of formula I was added to 0.8-1.4 ml of 0.1 M sodium ascorbate solution and 0.8-1.4 ml of 0.1 M sodium citrate solution, followed by the addition of 0.01 mCi of 225 Ac salt solution; mix evenly, adjust the pH value of the mixed solution to 4-7, preferably 5; react at 80-100°C, preferably 95°C; the reaction time is 10-30min, preferably 15min; after the reaction, adjust the pH value to 4.5; the concentration of the solution of the compound of formula I is 1mg / ml; 225 The concentration of Ac salt solution is 0.01mCi / ml to 0.02mCi / ml;

[0034] When the radionuclide is 64 Cu, 20-30 μg of the compound of formula I was added to 0.8-1.4 ml of 0.25 M sodium acetate solution; then 5 mCi of 64 Cu salt solution; mix evenly, adjust the pH value of the mixed solution to 4-7, preferably 5; react at 80-100°C, preferably 95°C; the reaction time is 10-30min, preferably 15min; after the reaction, adjust the pH value to 4; the concentration of the solution of the compound of formula I is 1mg / ml; 64 The concentration of Cu salt solution is 5mCi / ml~10mCi / ml;

[0035] When the radionuclide is 211 At 20-30 μg of the compound of formula I was added to 0.8-1.4 ml of a 0.25 M sodium borate solution; then 1 mCi of 211 At salt solution; mix evenly, adjust the pH value of the mixed solution to 4-7, preferably 5; react at 80-100°C, preferably 95°C; the reaction time is 10-30min, preferably 15min; after the reaction, adjust the pH value to 7; the concentration of the solution of the compound of formula I is 1mg / ml; 211The concentration of the At salt solution is 1.0 mCi / ml to 2.0 mCi / ml.

[0036] The invention provides an application of the compound of formula I in the preparation of a drug for both imaging and treating metastatic bone tumors.

[0037] The invention provides an application of the compound of formula II in the preparation of a drug for both imaging and treating metastatic bone tumors.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] The invention has a scientific design and a simple method. The invention creatively combines ibandronic acid with a chelating agent to obtain DOTA-ibandronic acid, and then uses a radioactive nuclide 68 Ga, 177 Lu, 225 Ac, 64 Cu, 211 At labeled DOTA-ibandronic acid to obtain 68 Ga, 177 Lu, 225 Ac, 64 Cu, 211 At-DOTA-ibandronic acid. 68 Ga, 177 Lu, 225 Ac, 64 Cu, 211 At-DOTA-ibandronic acid has high water solubility, good in vitro stability at room temperature, high plasma protein binding rate, and shows high and long-term bone uptake in the in vivo distribution in mice and imaging in New Zealand rabbits. It is a bone imaging agent with excellent performance and a radiopharmaceutical for the treatment of metastatic bone tumors. Pharmacodynamic experiments show that 68 Ga, 177 Lu, 225 Ac, 64 Cu, 211 At-DOTA-ibandronic acid has a higher effect than the corresponding group after diagnosis or treatment. 68 GaCl3, 177 LuCl3, 225 AcCl3, 64 CuCl2、Na 211 At or DOTA-ibandronic acid, indicating 68 Ga, 177 Lu, 225 Ac, 64 Cu, 211 Feasibility and effectiveness of At-DOTA-ibandronic acid in the diagnosis and treatment of bone metastases from malignant tumors.

[0040] Preparation of the present invention 68 Ga, 177 Lu, 225 Ac, 64 Cu, 211 The labeling method of At-DOTA-ibandronic acid is simple, the reaction time is short, the labeling yield is high, the amount of precursor used is very small (microgram level), and the ratio of lesion target to non-target (T / N value) is very high, which can reach more than 10 times. According to literature reports, a T / N ratio greater than 4-5 has a high potential therapeutic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Attached Figure 1 For Test Example 5 68 PET images of Ga-DOTA-IBA New Zealand rabbits at 1h and 3h;

[0042] Attached Figure 2 For Test Example 6 177 SPECT whole-body static images of Lu-DOTA-IBA New Zealand rabbits at 1d, 3d, 5d, and 7d;

[0043] Attached Figure 3 For Test Example 7 225 -SPECT whole-body static images of DOTA-IBA New Zealand rabbits at 1d, 3d, and 5d.

[0044] Attached Figure 4 HPLC chart of the precursor compound shown in Formula I;

[0045] Attached Figure 5 It is the LC-MS chart of the precursor compound shown in Formula I. DETAILED DESCRIPTION

[0046] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0047] Example 1

[0048] This embodiment discloses a method for preparing a precursor compound represented by formula I of the present invention, and its synthetic route is:

[0049]

[0050] S1. Preparation of Compound 2

[0051]

[0052] Add tert-butyl acrylate (6.34 g, 49.4 mmol) to compound 1 (5.00 g, 24.7 mmol). Stir the reaction at 25°C for 3 hours. TLC monitoring shows that the reaction is complete and new spots are generated. The reaction solution is concentrated to obtain a colorless oil compound 2 (7.90 g, yield: 96.7%). The crude product is used directly in the next step without purification.

[0053] S2. Preparation of Compound 3

[0054]

[0055] Under ice bath conditions, HCl / EtOAc (4M, 20.0mL) was added dropwise to a solution of compound 2 (1.90g, 5.75mmol) in EtOAc (2.00mL). The reaction solution was stirred at 25°C for 2 hours. LC-MS detected that the reaction of the raw material was complete. The reaction solution was concentrated in vacuo to obtain a colorless oily crude compound 3 (800mg, yield: 79.9%), which was used directly in the next step without further purification.

[0056] S3. Preparation of Compound 4

[0057]

[0058] Compound 3 (800 mg, 3.24 mmol, HCl salt) was added to pHCl (100 mL), and POCl3 (9.93 g, 64.7 mmol) and H3PO3 (5.31 g, 64.7 mmol) were added under nitrogen protection. The reaction was heated to 80°C and stirred for 16 hours. After cooling, the chlorobenzene solution was poured out, H2O (20.0 mL) was added, and stirred at 80°C for 5 hours. LC-MS monitored the mass of the product, the reaction solution was filtered and concentrated under vacuum, and the residue was purified by prep-HPLC (YMC-Actus Triart Diol-HILIC, 150*30 mm, 5 um, Mobile phase: 0.2% CH3COOH, CH3CN) was purified to obtain white solid compound 4 (90.0 mg, 8.68% yield)

[0059] S4. Preparation of Compound Carbs of Formula I

[0060]

[0061] To a solution of compound 4 (60.0 mg, 187 umol) in H2O (200 uL), a solution of DOTA-NHS-ester (141 mg, 281 umol) in DMF (200 uL) was added, followed by the addition of triethylamine (114 mg, 1.12 mmol). The reaction solution was stirred at 25 °C for 16 hours. LC-MS monitored the mass of the product. The reaction solution was filtered and concentrated, and the residue was purified by prep-HPLC (Hilic, 0.2% CH3COOH). White solid compound I Carbs (4.00 mg, 3.02% yield, 86.8% purity) was obtained.

[0062] The HPLC diagram of the compound of formula I is shown in the attached Figure 4 As shown, LC-MS pictures are shown in the attached Figure 5 shown.

[0063] Example 2

[0064] This example discloses the preparation 68 When Ga-DOTA-ibandronic acid is used, the dosage of DOTA-ibandronic acid is investigated, specifically:

[0065] Take 8 EP tubes, add 5μg, 10μg, 15μg, 20μg, 25μg, 30μg, 35μg, 40μg of DOTA-ibandronic acid (1mg / ml) to each tube in turn, add 1ml of sodium acetate solution (0.25M) to each tube, and then add freshly rinsed 68 4 ml of GaCl3 eluent (5 mci / ml) was adjusted to pH 5 with 0.25 molar sodium acetate solution and 0.01 molar hydrochloric acid, and then fully shaken and mixed. The mixture was reacted in a 95°C metal bath for 15 min. After the reaction was completed and cooled to room temperature, the pH value of each tube was adjusted to 4-6, and sterilized and filtered using a 0.22 μm filter membrane. Thin layer paper chromatography (TLC) was then used to determine 68 The radiochemical purity of Ga-DOTA-ibandronic acid is shown in Table 1:

[0066] Table 1 Radiochemical purity results at different DOTA-ibandronic acid dosages

[0067]

[0068] It can be seen from Table 1 that when other conditions are fixed, when the dosage of DOTA-ibandronic acid is 20-30 μg, 68 The radiochemical purity of Ga-DOTA-ibandronic acid is the best. When the dosage is lower or higher than this range, the radiochemical purity will gradually decrease.

[0069] Example 3

[0070] This example investigates the pH value of the present invention, specifically:

[0071] Take 7 EP tubes and add 25 μg of DOTA-ibandronic acid (1 mg / ml), 1 ml of sodium acetate solution (0.25 M), and freshly washed 68 4 ml of GaCl3 eluent (5 mci / ml) was adjusted to pH 2, 3, 4, 5, 6, 7, and 8 with 0.25 molar sodium acetate solution and 0.1 molar hydrochloric acid, and then fully shaken and mixed. The mixture was reacted in a 95°C metal bath for 15 min. After the reaction was completed and cooled to room temperature, it was sterilized and filtered using a 0.22 μm filter membrane. Thin layer paper chromatography (TLC) was then used to determine 68 The radiochemical purity of Ga-DOTA-ibandronic acid is shown in Table 2:

[0072] Table 2 Radiochemical purity results at different pH

[0073] Serial number pH Radiochemically pure 1 2 47％ 2 3 89％ 3 4 92％ 4 5 98％ 5 6 95％ 6 7 27％ 7 8 18％

[0074] As can be seen from Table 2, when the pH value is 5, the radiochemical purity of DOTA-ibandronic acid is the best, when the pH value is higher or lower than 5, the radiochemical purity gradually decreases, and when the pH value is higher than 6, the radiochemical purity decreases rapidly.

[0075] Example 4

[0076] This example investigates the amount of sodium acetate used in the present invention, specifically:

[0077] Take 8 EP tubes and add 0.2ml, 0.4ml, 0.6ml, 0.8ml, 1.0ml, 1.2ml, 1.4ml, and 1.6ml of sodium acetate solution (0.25M) to each tube in sequence, and add 25μg of DOTA-ibandronic acid (1mg / ml) and freshly rinsed 68 4 ml of GaCl3 eluent (5 mci / ml) was adjusted to pH 5 with 0.25 molar sodium acetate solution and 0.1 molar hydrochloric acid, respectively, and fully shaken and mixed. The mixture was reacted in a 95°C metal bath for 15 min. After the reaction was completed and cooled to room temperature, the pH value of each tube was adjusted to 5, and sterilized and filtered using a 0.22 μm filter membrane. Thin layer paper chromatography (TLC) was then used to determine 68 The radiochemical purity of Ga-DOTA-ibandronic acid is shown in Table 3 below:

[0078] Table 3 Radiochemical purity results under different dosages of sodium acetate solution (0.25M)

[0079]

[0080]

[0081] It can be seen from Table 3 that, under the condition that other conditions remain unchanged, when the amount of sodium acetate solution is 0.8-1.4ml, 68 The radiochemical purity of Ga-DOTA-ibandronic acid is the best. When the dosage is lower or higher than this range, the radiochemical purity will gradually decrease.

[0082] Example 5

[0083] This example investigates the reaction temperature and time in the present invention, specifically:

[0084] Take 18 EP tubes and set them into 3 groups: A (room temperature: 25±2℃), B (60℃), and C (95℃). Take 6 tubes from each group and mark them as A / B / C. ①-⑥ Each tube was pre-added with 25 μg of DOTA-ibandronic acid (1 mg / ml), 1 ml of sodium acetate solution (0.25 M) and freshly washed 68 4 ml of GaCl3 eluent (5 mci / ml) was adjusted to pH 5 with 0.25 molar sodium acetate solution and 0.1 molar hydrochloric acid, respectively, and fully shaken and mixed. Then, each group of 6 tubes was reacted at the temperature of each group for 5 min, 10 min, 15 min, 20 min, 25 min, and 30 min in sequence. After the reaction was completed and cooled to room temperature, the pH value of each tube was adjusted to 5, and sterilized and filtered with a 0.22 μm filter membrane. Then, thin layer paper chromatography (TLC) was used for determination. 68 The radiochemical purity of Ga-DOTA-ibandronic acid is shown in Table 4 below:

[0085] Table 4 Radiochemical purity results at different temperatures and times

[0086] Temperature / Time 5min 10min 15min 20min 25min 30min 25±2℃ 40％ 52％ 56％ 65％ 70％ 72％ 60℃ 56％ 65％ 79％ 81％ 84％ 85％ 95℃ 78％ 87％ 96％ 94％ 94％ 93％

[0087] It can be seen from Table 4 that when other conditions remain unchanged, when the reaction is carried out at 95°C for 15 min, 68 The radiochemical purity of Ga-DOTA-ibandronic acid is the best. If the reaction time is too long, too short or the temperature is too low, the radiochemical purity will decrease.

[0088] Example 6

[0089] This embodiment discloses the present invention 177 The preparation method of Lu-DOTA-ibandronic acid is specifically as follows:

[0090] Take DOTA-ibandronic acid (25ug) and add 1ml sodium acetate solution (0.25M) and then add 0.2ml freshly washed 177LuCl3 eluate (about 2.0 mci) was adjusted to pH 5 with 0.25 M sodium acetate solution and 0.01 M hydrochloric acid, reacted in a 95°C metal bath for 15 minutes, taken out and adjusted to pH 4.5 with 0.04 M hydrochloric acid, and the reaction solution was passed through a CM column (to remove free 177 Lu), and then the product is filtered through a sterile filter membrane to obtain 177 Lu-DOTA-ibandronic acid.

[0091] Example 7

[0092] This embodiment discloses the present invention 225 Ac-DOTA-ibandronic acid is prepared in a new method, specifically:

[0093] Take DOTA-ibandronic acid (25ug) and add 1ml ascorbic acid solution (0.1M) and 1ml sodium citrate solution (0.1M), then add 100ul freshly washed 225 The AcCl3 eluate (about 0.01 mci) was adjusted to pH 4.5 with 0.04 M hydrochloric acid, reacted in a 95°C metal bath for 15 minutes, taken out and adjusted to pH 4.5 with 0.04 M hydrochloric acid, and then the product was filtered through a sterile filter membrane to obtain 225 Ac-DOTA-ibandronic acid.

[0094] Example 8

[0095] This embodiment discloses the present invention 64 The new preparation method of Cu-DOTA-ibandronic acid is as follows:

[0096] Take DOTA-ibandronic acid (25ug) and add 1ml sodium acetate solution (0.25M) and then add 1ml freshly washed 64 The CuCl2 eluate (about 5 mci) was adjusted to pH 4 with 0.04 M hydrochloric acid, reacted in a 95°C metal bath for 15 minutes, taken out and adjusted to pH 4 with 0.04 M hydrochloric acid, and then the product was filtered through a sterile filter membrane to obtain 64 Cu-DOTA-ibandronic acid.

[0097] Example 9

[0098] This embodiment discloses the present invention 211 At-DOTA-ibandronic acid is prepared in a novel method, specifically:

[0099] Take DOTA-ibandronic acid (25ug), add 1ml sodium borate solution (0.25M) and then add 1ml freshly washed Na 211At eluate (about 1.0 mci), adjust the pH to about 7, react in a metal bath at 95°C for 15 minutes, adjust the pH to about 7, and then filter the product through a sterile filter membrane to obtain 211 At-DOTA-ibandronic acid.

[0100] Test Examples 1-5 used 68 Ga-DOTA-ibandronic acid was prepared according to the method No. 5 in Table 1 of Example 2.

[0101] Test Example 1

[0102] This test example discloses the 68 The in vitro stability test of Ga-DOTA-ibandronic acid is as follows:

[0103] Take 4 EP tubes and mark them as ①-④. Add 0.1ml of normal saline to ① and ② respectively, add 0.1ml of fresh human serum diluted 10 times to ③ and ④ respectively, and then add freshly prepared 68 Ga-DOTA-ibandronic acid 1.0mci, No. ① and No. ③ were placed at room temperature (25±2℃), No. ② and No. ④ were placed in a 37℃ metal bath for incubation, and the radiochemical purity of No. ①-④ was determined by thin layer paper chromatography (TLC) at 10min, 30min, 1h, 2h, 3h, and 4h, respectively. The results are shown in Table 5 below.

[0104] Table 5 Different treatment methods and treatment time 68 Radiochemical purity of Ga-DOTA-ibandronic acid

[0105] 10min 30min 1h 2h 3h 4h Room temperature (normal saline) 98％ 97％ 95％ 95％ 95％ 93％ Room temperature (serum) 99％ 99％ 99％ 96％ 96％ 93％ 37℃ (normal saline) 99％ 96％ 96％ 92％ 90％ 87％ 37℃(Serum) 99％ 97％ 96％ 92％ 88％ 82％

[0106] From Table 5, we can see that 68 Ga-DOTA-ibandronic acid has good in vitro stability at room temperature. Its radiochemical purity is still greater than 90% after being placed in physiological saline and serum for 4 hours at room temperature.

[0107] Test Example 2

[0108] This test example discloses the 68 The lipid-water distribution coefficient of Ga-DOTA-ibandronic acid was investigated as follows:

[0109] Take three 5 ml centrifuge tubes and label them A-①, A-②, and A-③ respectively. Add 0.5 ml of n-octanol, 0.485 ml of deionized water, and 0.25 ml of about 0.5 mci of freshly prepared 68Ga-DOTA-ibandronic acid was shaken with a vortex mixer for 20 minutes, then centrifuged at 2000r / min for 5 minutes, and 0.1ml of the upper liquid (organic phase) was collected into 3 corresponding test tubes (numbered B-①, B-②, and B-③, respectively), and 0.1ml of the lower liquid (aqueous phase) was collected into 3 corresponding test tubes (numbered C-①, C-②, and C-③, respectively). The total radioactivity counts of the organic phase and the aqueous phase were determined using a γ counter, and the lipid-water partition coefficient lgP was calculated using the formula.

[0110] The results show that the present invention 68 The lipid-water partition coefficient lgP of Ga-DOTA-ibandronic acid is -2.33, indicating that the marker has high water solubility and low lipid solubility.

[0111] Test Example 3

[0112] This test example discloses the 68 The plasma protein binding rate of Ga-DOTA-ibandronic acid was investigated as follows:

[0113] Take 1 ml of fresh human plasma anticoagulated with heparin for later use. Take 3 EP tubes numbered A-①, A-②, and A-③, and add 0.1 ml of fresh human plasma and 0.25 ml of about 0.5 mci of freshly prepared 68 Ga-DOTA-ibandronic acid, and then incubate in a 37°C metal bath for 30 minutes. Take three 5ml centrifuge tubes and number them B-①, B-②, and B-③, respectively, and transfer the incubated solutions to the corresponding centrifuge tubes, then add 1ml of 25% trichloroacetic acid solution to each centrifuge tube, mix thoroughly and centrifuge at 2000r / min for 5 minutes, collect the supernatant into three corresponding test tubes (numbered C-①, C-②, and C-③ respectively), repeat centrifugation and collection of supernatant 3 times. Use a γ counter to measure the total radioactivity counts in the precipitate in the centrifuge tube and the supernatant in the test tube, and calculate the plasma protein binding rate (PPB).

[0114] The results show that the present invention 68 The PPB of Ga-DOTA-ibandronic acid in plasma after incubation for 30 minutes was 81%. The higher the plasma protein binding rate, the longer the marker stays in the body, the less likely it is to be cleared, and the better the imaging effect can be achieved.

[0115] Test Example 4

[0116] This test example discloses the 68 The in vivo distribution of Ga-DOTA-ibandronic acid in mice was investigated, specifically:

[0117] Twenty healthy Kunming mice (body weight 18-22 g) were randomly divided into five groups, with four mice in each group, half male and half female. The mice were injected via the tail vein with 0.5 mci / 0.1 ml of freshly prepared 68 Ga-DOTA-ibandronic acid, the mice were decapitated after 15min, 30min, 1h, 2h, and 4h, respectively, and the carotid blood, brain, heart, liver, spleen, lung, kidney, stomach, small intestine, muscle, and femur tissues were weighed and measured with a gamma counter. After time decay correction, the percentage injected dose rate per gram of tissue (%ID / g) was calculated. 68 The in vivo distribution results of Ga-DOTA-ibandronic acid in mice are shown in Table 6 below:

[0118] Table 6 68 The results of the in vivo distribution of Ga-DOTA-ibandronic acid in mice

[0119]

[0120] It can be seen from Table 6 that the bones 68 The uptake rate of Ga-DOTA-ibandronic acid was high, reaching a maximum value (8.365% ID / g) at 2 hours. Thereafter, the uptake in bones decreased with time, but at 4 hours, the percentage of injected dose per gram of bone tissue was still 4.268% ID / g, which was significantly higher than that of other organs and tissues. 68 Ga-DOTA-ibandronic acid has a stronger effect on bones and a longer residence time, which can achieve a more ideal imaging effect.

[0121] Test Example 5

[0122] This test example discloses the 68 Ga-DOTA-ibandronic acid imaging was investigated at different time points in New Zealand rabbits, specifically:

[0123] A New Zealand rabbit weighing about 2 kg was injected with freshly prepared 68 Ga-DOTA-ibandronic acid was about 1.0 mci / 0.2 ml, and PET whole-body imaging was performed 1 hour and 3 hours after injection ( Figure 1 ).

[0124] Depend on Figure 1 It can be seen that 1 hour after the injection, the kidneys and bladder were clearly visible, the chest cavity and soft tissues were more obvious, the whole body bone images were clearer, and the limb joints were clearly visible; Figure 2 It shows that as time goes by, the chest and soft tissue shadows gradually fade, the kidneys gradually become lighter and gradually excrete into the bladder, the bladder shadow gradually becomes thicker, and the bladder shadow gradually disappears with the excretion of urine. 68Ga-DOTA-ibandronic acid is an imaging agent with rapid soft tissue clearance, high bone uptake, and excretion through the urinary system. It also stays in the bone for a relatively long time. If it is used in patients with metastatic bone tumors, it will achieve better imaging effects.

[0125] Test Example 6

[0126] The method prepared according to Example 6 177 Lu-DOTA-ibandronic acid imaging was investigated at different time points in New Zealand rabbits, specifically:

[0127] A New Zealand rabbit weighing about 2 kg was injected with freshly prepared 177 Lu-DOTA-ibandronic acid was about 2.0 mci / 0.2 ml. PET whole-body imaging was performed at 1, 3, 5, and 7 days after injection. Figure 2 ).

[0128] Test Example 7

[0129] The method prepared according to Example 7 225 Ac-DOTA-ibandronic acid imaging was investigated at different time points in New Zealand rabbits, specifically:

[0130] A New Zealand rabbit weighing about 2 kg was injected into the ear vein with the freshly prepared 225 Ac-DOTA-ibandronic acid (about 0.01 mci / 2 ml) was injected and PET whole-body imaging was performed at 1, 3, and 5 days after injection. Figure 3 ).

[0131] Test Example 8

[0132] This test example discloses the 177 The therapeutic effect of Lu-DOTA-ibandronic acid in tumor-bearing mice (PC-3, H1975, MDA-MB-231 bone metastasis models) was investigated. 177 Lu-DOTA-ibandronic acid was prepared according to the method of Example 6.

[0133] (1) Establishment of tumor-bearing mouse model: 8 SPF male nude mice and 4 SPF female nude mice were prepared. After isoflurane anesthesia, the skin around the left knee joint was disinfected. The left hind limb was bent and an insulin needle (29G needle) was used to vertically insert the tibia from the tibial fossa concave point and slowly rotated into the bone marrow cavity. 25 μl (about 2×10 6 )PC-3 (4 males), H1975 (4 males), and MDA-MB-231 (4 females) cell culture medium; then disinfected, the holes were sealed with sterile bone wax, and the animals were placed on a 37°C hot plate to rewarm, and after they recovered, they were returned to the cage for continued feeding.

[0134] (2) Tumor-bearing mouse model testing: Four weeks after inoculation, 12 nude mice were scanned using Micro-CT. It was observed that each nude mouse had varying degrees of bone destruction and soft tissue swelling, indicating that the bone metastasis model was successfully established.

[0135] (3) Different treatment methods and grouping: Twelve tumor-bearing mice with successful modeling were randomly divided into 4 groups. 177 Lu-DOTA-ibandronic acid was injected into two groups with low and high radioactivity (100μCi, 500μCi), with 3 mice in each group; the blank control group was injected with normal saline and was divided into small and large volume groups (10μl, 50μl), with 3 mice in each group.

[0136] (4) Treatment effect test and results: Free walking pain behavior scores were performed on days 0, 7, 14, 21, 28, and 35, and used as the main indicator of bone pain relief in tumor-bearing mice. At the corresponding time points, the tumor-bearing mice were placed in a transparent, smooth, flat-bottomed plastic box. The tumor-bearing mice were allowed to move freely in the box and were observed after 30 minutes of adaptation. Pain behavior scores were performed according to the following standards: 0 points, normal movement, the movements of the hind limbs on the model side and the control side were the same; 1 point, mild lameness of the hind limbs on the model side; 2 points, the degree of lameness of the hind limbs on the model side was between 1 and 3 points; 3 points, severe lameness of the hind limbs on the model side; 4 points, the hind limbs on the model side were completely unable to move and could not touch the ground. The results are shown in Table 7 below:

[0137] Table 7 Free walking pain behavior scores of tumor-bearing mice in each group under different treatment methods and treatment time

[0138]

[0139] Note: () represents the 1st, 2nd, and 3rd tumor-bearing mice in each group, and / represents the tumor-bearing mice have died.

[0140] As can be seen from Table 7, taking day 0 as the baseline, a high-activity dose was given 177 At week 5 after Lu-DOTA-ibandronic acid (500 μCi) treatment, the average decrease in pain behavior scores of free walking in surviving tumor-bearing mice was the largest among all groups, and the number of survivors was relatively large; using different active doses 177 The average reduction in pain behavior scores of free walking in tumor-bearing mice that survived at week 5 of Lu-DOTA-ibandronic acid treatment was higher than that of the corresponding control group.

[0141] The above results show that high-activity doses of 177 Lu-DOTA-ibandronic acid (500 μCi) showed the greatest relief of bone pain and the highest survival rate within 5 weeks of administration. 177The efficacy of Lu-DOTA-ibandronate treatment was higher than that of the corresponding control group, indicating that 177 Feasibility and effectiveness of Lu-DOTA-ibandronic acid in the treatment of bone metastases from malignant tumors.

[0142] Based on the above embodiments 1-9 and test examples 1-8, 68 Ga / 177 Lu / 225 Ac / 64 Cu / 211 The labeling method of At-DOTA-ibandronic acid is simple, the reaction time is short, the labeling yield is high, it has high water solubility, good in vitro stability at room temperature, high plasma protein binding rate, and shows high and long-term bone uptake in the in vivo distribution in mice and imaging in New Zealand rabbits, indicating that the preparation invented by the institute is a bone imaging agent with excellent performance and a therapeutic radiopharmaceutical for metastatic bone tumors.

[0143] The embodiments described above are part of the embodiments of the present invention, rather than all of the embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims

1. A precursor compound of a radioactive marker having a structure as shown in Formula I or a pharmaceutically acceptable salt thereof, 2. A radiolabeled compound of formula I or a pharmaceutically acceptable salt thereof, characterized in that: The structure is shown in Formula II, Wherein A is a radionuclide, preferably 68 Ga, 177 Lu, 225 Ac, 64 Cu, 211 At.

3. The radioactive marker or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: A 177 Lu.

4. The radioactive marker or a pharmaceutically acceptable salt thereof according to claim 2 or 3, characterized in that: The radiochemical purity is greater than or equal to 95%.

5. The radioactive marker or a pharmaceutically acceptable salt thereof according to claim 4, characterized in that: Its radiochemical purity is 95-99%.

6. The method for preparing a precursor compound of a radioactive marker according to claim 1, characterized in that: The following steps are involved: S1. Compound 1 reacts with tert-butyl acrylate and stirs to generate compound 2; S2. HCl / EtOAc is added dropwise to the EtOAc solution of compound 2 to react to generate compound 3; S3. Compound 3 was added to PhCl, POCl3 and H3PO3 were added under nitrogen protection, heated to react, and after cooling, the chlorobenzene solution was poured out, H2O was added, heated and stirred to generate compound 4; S4. DOTA-NHS-ester solution was added to the H2O solution of compound 4, followed by the addition of triethylamine to react to produce a compound of formula I; The reaction formula is as follows:

7. The method for preparing a radioactive marker according to any one of claims 2 to 5, characterized in that: include: The compound of formula I is reacted with a radioactive nuclide salt solution to obtain a radioactive label represented by formula II.

8. The preparation method according to claim 7, characterized in that: The radionuclides are 68 Ga, 177 Lu or 64 Cu, the solution of the compound of formula I, the sodium acetate solution, and the radioactive nuclide salt solution are mixed evenly, the pH value of the mixed solution is adjusted, reacted, the pH value is adjusted again, sterilized, and filtered to obtain; When the radionuclide is 225 Ac, the solution of the compound of formula I, the sodium citrate solution, the sodium ascorbate solution and the radioactive nuclide salt solution are mixed uniformly, the pH value of the mixed solution is adjusted, reacted, the pH value is adjusted again, sterilized, filtered, and the mixture is obtained; When the radionuclide is 211 At, the solution of the compound of formula I, the sodium borate solution, and the radioactive nuclide salt solution are mixed uniformly, the pH value of the mixed solution is adjusted, reacted, the pH value is adjusted again, sterilized, and filtered to obtain.

9. The preparation method according to claim 8, characterized in that: When the radionuclide is 68 Ga, 20-30 μg of the compound of formula I was added to 0.8-1.4 ml of 0.25 M sodium acetate solution; then 20 mCi of 68 Ga salt solution; mix evenly, adjust the pH value of the mixed solution to 4-7; react at 80-100°C; the reaction time is 10-30min; after the reaction, adjust the pH value to 5; the concentration of the solution of the compound of formula I is 1mg / ml; 68 The concentration of Ga salt solution is 5mCi / ml~10mCi / ml; When the radionuclide is 177 Lu, 20-30 μg of the compound of formula I was added to 0.8-1.4 ml of a 0.25 M sodium acetate solution, followed by the addition of 2 mCi of 177 Lu salt solution; mix well, adjust the pH value of the mixed solution to 4-7; react at 80-100°C; the reaction time is 10-30min; after the reaction, adjust the pH value to 4.5; the concentration of the solution of the compound of formula I is 1mg / ml; 177 The concentration of Lu salt solution is 10mCi / ml~20mCi / ml; When the radionuclide is 225 Ac, 20-30 μg of the compound of formula I was added to 0.8-1.4 ml of 0.1 M sodium ascorbate solution and 0.8-1.4 ml of 0.1 M sodium citrate solution, followed by the addition of 0.01 mCi of 225 Ac salt solution; mix well, adjust the pH value of the mixed solution to 4-7; react at 80-100°C; the reaction time is 10-30min; after the reaction, adjust the pH value to 4.5; the concentration of the solution of the compound of formula I is 1mg / ml; 225 The concentration of Ac salt solution is 0.01mCi / ml~0.02mCi / ml; When the radionuclide is 64 Cu, 20-30 μg of the compound of formula I was added to 0.8-1.4 ml of 0.25 M sodium acetate solution; then 5 mCi of 64 Cu salt solution; mix well, adjust the pH value of the mixed solution to 4-7; react at 80-100°C; the reaction time is 10-30min; after the reaction, adjust the pH value to 4; the concentration of the solution of the compound of formula I is 1mg / ml; 64 The concentration of Cu salt solution is 5mCi / ml~10mCi / ml; When the radionuclide is 211 At 20-30 μg of the compound of formula I was added to 0.8-1.4 ml of a 0.25 M sodium borate solution; then 1 mCi of 211 At salt solution; mix well, adjust the pH value of the mixed solution to 4-7; react at 80-100°C; the reaction time is 10-30min; after the reaction, adjust the pH value to 7; the concentration of the solution of the compound of formula I is 1mg / ml; 211 The concentration of the At salt solution is 1.0 mCi / ml to 2.0 mCi / ml.

10. Use of the compound of formula I as claimed in claim 1 in the preparation of a drug for both imaging and treating metastatic bone tumors.

11. Use of the compound of formula II as claimed in any one of claims 2 to 5 in the preparation of a drug for both imaging and treating metastatic bone tumors.

12. A compound or a pharmaceutically acceptable salt thereof, which is a compound of formula I labeled with a nuclide 13. Use of the compound according to claim 12 or a pharmaceutically acceptable salt thereof in the preparation of a drug for both imaging and treating metastatic bone tumors.

14. The following compounds:

15. A method for preparing compound 2, comprising the step of reacting compound 1 to produce compound 2, preferably comprising the step of reacting compound 1 with tert-butyl acrylate to produce compound 2, 16. A method for preparing compound 3, comprising the step of reacting compound 2 to produce compound 3, Preferably, the step of adding HCl / EtOAc dropwise to the EtOAc solution of compound 2 is included.

17. A method for preparing compound 4, comprising the step of reacting compound 3 to generate compound 4, preferably comprising the following steps: adding compound 3 to PhCl, adding POCl3 and H3PO3 under nitrogen protection, heating for reaction, pouring out the chlorobenzene solution after cooling, adding H2O, heating and stirring to generate compound 4; 18. A method for preparing a compound of formula I, comprising the step of reacting compound 4 to produce a compound of formula I, preferably comprising the steps of: adding a DOTA-NHS-ester solution to a H2O solution of compound 4, followed by adding triethylamine to react to produce a compound of formula I,

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