Melanoma targeted radiopharmaceutical as well as preparation and application thereof

Abstract

The invention provides a compound shown as a formula I, or a pharmaceutically acceptable salt thereof, or a precursor compound and an isotope compound thereof, or a stereoisomer and a tautomer thereof. The compound has high affinity with melanoma, strong targeting specificity, low non-specific uptake in liver and kidney, and high tumor / non-tumor ratio. Besides, the probe precursor can be selectively chelated with different radioactive diagnosis or treatment nuclides, and is used for early diagnosis of malignant tumors, Parkinson's diseases and other major diseases through single photon emission computed tomography, and guidance of treatment scheme selection, curative effect monitoring and radioactive treatment. The compound has the advantages of simple synthesis, easily available raw materials and good stability, and can be popularized and applied as a novel melanoma targeted radiopharmaceutical. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of single-photon emission computed tomography (SPECT), and in particular relates to a melanoma-targeted radioactive drug and its preparation and application. Background Art

[0002] According to data from the National Cancer Institute in the United States in 2023, melanoma has become the fifth most common cancer worldwide. In my country, the annual growth rate of melanoma is between 3% and 5%. Although melanoma only accounts for 3% of skin cancer diagnoses, it accounts for 75% of skin cancer-related deaths. Malignant melanoma is highly invasive and fatal, prone to regional lymph node metastasis and distant metastasis. Its prognosis is closely related to early diagnosis, staging, and classification. The five-year survival rate for early-stage melanoma can reach 95%, while in advanced stages, it is less than 30%. Therefore, early diagnosis and detection of melanoma and implementation of effective interventions are of paramount importance.

[0003] Melanoma is common on the skin. Currently, clinical diagnosis of melanoma mainly relies on visual examination, tissue pathology biopsy and imaging examination. Compared with CT and MRI, nuclear medicine imaging technology represented by SPECT / CT and PET / CT has more obvious advantages in early screening of tumors. It can perform whole-body imaging and judgment in one scan with high sensitivity and strong specificity. 18 F-FDG is effective in advanced melanoma, but its sensitivity and specificity for stage I and II melanoma and small metastatic lesions still need to be improved, and it also has the risk of false positives for inflammatory lesions.

[0004] In recent years, researchers have found that melanin is overexpressed in most tumor patients and has become a specific target for diagnosis and treatment. CN107556236 B discloses an isotope-substituted positron emission tomography agent targeting melanoma, its preparation method and use. The compound uses benzamide and its derivatives as targeting ligands, and is labeled with radioactive 18 F is used to prepare high-affinity diagnostic probes for melanoma imaging. 18 F has a half-life of 110 minutes and requires rapid radiochemical synthesis. At the same time, the popularity of PET / CT in China is still not high enough, which limits its large-scale clinical promotion. 99m Tc-labeled radiopharmaceuticals account for 80% of clinical radiopharmaceuticals and have a half-life of 6.02 hours. 99 Mo / 99m After the Tc generator is eluted, it can be prepared by kit 99m Tc labeled probe is easy to promote and use. 186 W / 188 The sodium perrhenate solution obtained by eluting the Re generator has a half-life of 16.9 hours and a 2.12 MeV β - The rays are extremely suitable for treatment, and 15% of the 0.155MeV gamma rays can also be used for imaging at the same time and for tumor treatment monitoring research.

[0005] Therefore, there is an urgent need to develop a new type of tumor-targeting radioligand and its derivatives that can be 99m Tc / 188 After Re labeling, a high-affinity and high-specificity tumor-targeting radioactive probe is obtained. Summary of the Invention

[0006] The purpose of the present invention is to provide a new type of tumor-targeting radioligand and its derivatives, which can be 99m Tc / 188 After Re labeling, a high-affinity and high-specificity tumor-targeting radioactive probe is obtained, specifically relating to a melanoma-targeting radioactive drug and its preparation and application.

[0007] In the first aspect of the present invention, there is provided a compound of formula I, or a pharmaceutically acceptable salt thereof, or a precursor compound, an isotope compound, or a stereoisomer or tautomer thereof,

[0008]

[0009] in,

[0010] M is a nuclide or its oxide;

[0011] Rc is a substituted or unsubstituted C1-C12 alkyl group or

[0012] L is selected from the group consisting of: -(CH2) n -L1-(CH2) m -、-(CH2) n -L1-(phenylene)-L2-(CH2) m -、-(CH2) n -L1-L2-(CH2) m -, C1-C12 alkylene, C1-C12 alkoxy, -(CH2CH2O)-, divalent amino acid; wherein L1 and L2 are each independently selected from the following group: -CO-, -COO-, -CONH-, -S-, 5-10 membered heterocyclic group, 5-6 membered heteroaryl, phenyl; and said L may be optionally substituted;

[0013] R1 and R2 are the same or different and are independently selected from the following groups: H, C 2-4 Aldehyde, substituted or unsubstituted C1-8 Alkyl, substituted or unsubstituted C 1-8 Alkoxy, R a (COO)-、R a (OCO)-、R a (CONH)-、R a (NHCO)-, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted 4-10 membered heterocyclyl, or substituted or unsubstituted C6-C10 aryl;

[0014] Alternatively, R1 and R2 together with the nitrogen atom to which they are attached are unsubstituted or replaced by one or more R h substituted 3-8 membered heterocyclic group; wherein each of the R h Each is independently selected from the following group: hydroxyl, halogen, C2-C4 aldehyde (C1-C3 alkyl-C(O)-), C1-C12 alkyl, C1-C12 alkoxy, C3-C6 cycloalkyl, 4-8 membered heterocyclic group; the heterocyclic group refers to a cyclic group having 1-3 heteroatoms selected from N and O on the ring;

[0015] R a and R b are the same or different and are each independently selected from the group consisting of: H, substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted C 6-10 Aryl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 4-8 membered heterocyclic group;

[0016] R d and R f are the same or different and are each independently selected from the following group: H, ═O, C1-C12 alkyl, C1-C12 alkoxy, C1-C6 alkyl ester, C6-12 aryl ester, C1-C6 alkylamide, C6-C12 arylamide, C1-C12 alkylcarboxyl;

[0017] R g With R c are the same or different, and the R g Selected from: none, H, =O, substituted or unsubstituted C1-C12 alkyl, C1-C12 alkoxy, C1-C6 alkyl ester, C6-12 aryl ester, C1-C6 alkyl amide, C6-C12 aryl amide, C1-C12 alkyl carboxyl, or

[0018] n, m, p are 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0019] The nuclide is a radioactive nuclide or a non-radioactive nuclide;

[0020] The radionuclide is selected from the group consisting of99m Tc, 188 Re、 186 Re、 90 Y. 131 I. 153 Sm, 177 Lu, 211 At 212 Pb, 223 Ra, 225 Ac、Al[ 18 F]、 51 Mn, 52m Mn, 52g Mn, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 86 Y. 89 Zr, 111 In, 123 I. 124 I. 125 I. 166 Ho, 213 Bi, 227 Th;

[0021] The non-radioactive nuclide is selected from the group consisting of: Re, Mn, Si, Pt, Pb, Nd, Gd, Yb, Er, Cu, Y, Fe, and Tm;

[0022] The substitution refers to the substitution of one or more hydrogen atoms on the group by a substituent selected from the group consisting of hydroxyl, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-6 Cycloalkyl, 4-6 membered heterocyclic group; wherein the ring skeleton of the heteroaryl or heterocyclic group has 1-3 heteroatoms selected from N and O; the C 3-6 The cycloalkyl group and the 4- to 6-membered heterocyclic group may optionally further have 1-3 halogen or alkyl substituents.

[0023] In another preferred embodiment, the L is selected from the following group: -(CH2) n -L1-(CH2) m -、-(CH2) n -L1-(phenylene)-L2-(CH2) m -、-(CH2) n -L1-L2-(CH2) m-, C1-C10 alkylene, C1-C10 alkoxy, -(CH2CH2O)-, divalent amino acid; wherein, L1 and L2 are each independently selected from the following group: -CO-, -CONH-, 5-10 membered heterocyclyl, 5-6 membered heteroaryl, phenyl; and the L can be optionally substituted.

[0024] In another preferred embodiment, in the compound, R1 and R2 are the same or different and are independently selected from the following groups: H, substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted C 1-6 Alkoxy, R a (COO)-、R a (OCO)-、R a (CONH)-、R a (NHCO)-, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 4-6 membered heterocyclyl, or substituted or unsubstituted C6-C10 aryl;

[0025] Alternatively, R1 and R2 together with the nitrogen atom to which they are attached are unsubstituted or replaced by one or more R h substituted 3-8 membered heterocyclic group; wherein each of the R h Each is independently selected from the following groups: hydroxyl, halogen, C2-C4 aldehyde (C1-C3 alkyl-C(O)-), C1-C10 alkyl, C1-C10 alkoxy, C3-C6 cycloalkyl, 4-8 membered heterocyclic group; the heterocyclic group refers to a ring group having 1-3 heteroatoms selected from N and O on the ring.

[0026] In another preferred embodiment, the radionuclide is selected from the following group: 99m Tc, 188 Re、 186 Re、 90 Y. 131 I. 153 Sm, 177 Lu, 211 At 212 Pb, 223 Ra, 225 Ac、Al[ 18 F]、 51 Mn, 52m Mn, 52g Mn, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 86 Y. 89 Zr, 111 In, 123 I. 124 I. 125 I.166 Ho, 213 Bi, 227 Th;

[0027] The non-radioactive nuclide is selected from the group consisting of: Re, Mn, Si, Pt, Pb, Nd, Gd, Yb, Er, Cu, Y, Fe, and Tm.

[0028] In another preferred embodiment, the compound has a structure of Formula II:

[0029]

[0030] in,

[0031] M is a nuclide or its oxide;

[0032] L is selected from the group consisting of: -(CH2) n -L1-(CH2) m -、-(CH2) n -L1-(phenylene)-L2-(CH2) m -、-(CH2) n -L1-L2-(CH2) m -, C1-C12 alkylene, C1-C12 alkoxy, -(CH2CH2O)-, divalent amino acid; wherein L1 and L2 are each independently selected from the following group: -CO-, -COO-, -CONH-, -S-, 5-10 membered heterocyclic group, 5-6 membered heteroaryl, phenyl; and said L may be optionally substituted;

[0033] R1 and R2 are the same or different and are independently selected from the following groups: H, C 2-4 Aldehyde, substituted or unsubstituted C 1-8 Alkyl, substituted or unsubstituted C 1-8 Alkoxy, R a (COO)-、R a (OCO)-、R a (CONH)-、R a (NHCO)-, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted 4-10 membered heterocyclyl, or substituted or unsubstituted C6-C10 aryl;

[0034] Alternatively, R1 and R2 together with the nitrogen atom to which they are attached are unsubstituted or replaced by one or more R h substituted 3-8 membered heterocyclic group; wherein each of the R hEach is independently selected from the following group: hydroxyl, halogen, C2-C4 aldehyde (C1-C3 alkyl-C(O)-), C1-C12 alkyl, C1-C12 alkoxy, C3-C6 cycloalkyl, 4-8 membered heterocyclic group; the heterocyclic group refers to a cyclic group having 1-3 heteroatoms selected from N and O on the ring;

[0035] R a Selected from the group consisting of H, substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted C 6-10 Aryl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 4-8 membered heterocyclic group;

[0036] n, m are 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0037] The nuclide is a radioactive nuclide or a non-radioactive nuclide; the radioactive nuclide is selected from the group consisting of 99m Tc, 188 Re、 186 Re、 90 Y. 131 I. 153 Sm, 177 Lu, 211 At 212 Pb, 223 Ra, 225 Ac、Al[ 18 F]、 51 Mn, 52m Mn, 52g Mn, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 86 Y. 89 Zr, 111 In, 123 I. 124 I. 125 I. 166 Ho, 213 Bi, 227 Th;

[0038] The non-radioactive nuclide is selected from the group consisting of: Re, Mn, Si, Pt, Pb, Nd, Gd, Yb, Er, Cu, Y, Fe, and Tm;

[0039] The substitution refers to the substitution of one or more hydrogen atoms on the group by a substituent selected from the group consisting of hydroxyl, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-6Cycloalkyl, 4-6 membered heterocyclic group; wherein the ring skeleton of the heteroaryl or heterocyclic group has 1-3 heteroatoms selected from N and O; the C 3-6 The cycloalkyl group and the 4- to 6-membered heterocyclic group may optionally further have 1-3 halogen or alkyl substituents.

[0040] In another preferred embodiment, the compound of formula I is selected from the following compounds:

[0041]

[0042]

[0043]

[0044] In a second aspect of the present invention, there is provided a method for preparing the compound of formula (I) as described in the first aspect, the method comprising the steps of:

[0045] 1) Synthesis of ligand skeleton AADT-COOH:

[0046] 2-(tritylthio)ethylamine and bromoacetyl bromide are mixed and reacted in chloroform to obtain N-[2-(trityl)thioethyl][2-(trityl)thioethylamino]acetamide after purification;

[0047] N-[2-(trityl)thioethyl][2-(trityl)thioethylamino]acetamide is reacted with ethyl bromobutyrate to obtain ethyl 4-((2-oxo-2-((2-(triphenylthio)ethyl)amino)ethyl)(2-(triphenylthio)ethyl)amino)butanoate; the ethyl ester is then removed using a methanol-water system of NaOH, and the mixture is separated and purified by preparative liquid phase separation to obtain 4-((2-oxo-2-((2-(triphenylthio)ethyl)amino)ethyl)(2-(triphenylthio)ethyl)amino)butyric acid (AADT-COOH);

[0048] 2) Synthesis of ligand molecules:

[0049] The AADT-COOH obtained in the previous step and the commercially purchased N-containing small molecule L-NR1R2 were dissolved in anhydrous acetonitrile, and appropriate amounts of N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl) urea hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIPEA) were added. The reaction was stirred at room temperature overnight. After the reaction was completed, the target ligand molecule was purified and separated by preparative liquid chromatography;

[0050]

[0051] Alternatively, the compound 2 obtained in step 1) and the bromine-containing small molecule Br-L-NR1R2 are dissolved in anhydrous acetonitrile, and appropriate amounts of cesium carbonate and potassium iodide are added. The mixture is heated to react overnight, and the target ligand molecule is obtained by separation and purification through preparative liquid phase separation;

[0052]

[0053] Subsequently, the trityl-protected ligand was deprotected and titrated with triethylsilane until it became colorless; the isolated product was redissolved for the preparation of 99m Tc-labeled compounds;

[0054] 3) 99m Preparation of Tc-CX complex:

[0055] Prepared by sodium glucoheptonic acid (CH2OH(CHOH)5COOH) exchange reaction 99m Tc-C1 complex, the specific steps are as follows:

[0056] First, Na 99m The TcO4 eluent was added to the freeze-dried drug box containing sodium glucoheptonic acid, stannous chloride and urea, and the solution was allowed to stand for 1 h. 99m TcO-GH intermediate solution;

[0057] Then add the CX ligand to the above 99m The TcO-GH intermediate solution is reacted to obtain 99m Tc-C1 complex.

[0058] In the third aspect of the present invention, there is provided a use of a compound of formula I as described in the first aspect, or a pharmaceutically acceptable salt thereof, or a precursor compound, an isotope compound, or a stereoisomer or tautomer thereof for preparing an imaging agent.

[0059] In another preferred embodiment, the imaging agent is used to monitor the imaging ability of the probe at the cellular, tissue, or living body levels.

[0060] In another preferred embodiment, the imaging agent is incubated with tumor cells or tissues or administered in vivo to monitor the imaging ability of the probe at the cellular, tissue, or in vivo levels.

[0061] In another preferred embodiment, the imaging agent is used for clinical diagnosis of tumor sites with high melanin expression.

[0062] In another preferred embodiment, the imaging is performed using molecular imaging techniques and radionuclide therapy.

[0063] In another preferred embodiment, the molecular imaging is selected from the group consisting of PET imaging, SPECT imaging, optical imaging, MRI imaging, and multimodal imaging in combination therewith. More preferably, the molecular imaging is performed by single-photon emission computed tomography (SPECT).

[0064] In another preferred embodiment, the imaging agent is selected from the following group: cancer contrast agents, tumor contrast agents, and Parkinson's disease contrast agents.

[0065] In another preferred embodiment, the tumor is a tumor associated with high expression of melanin.

[0066] In another preferred embodiment, the cancer is selected from the group consisting of primary tumors and metastatic lesions of one or more of melanoma, basal cell carcinoma, cutaneous squamous cell carcinoma, non-melanoma skin cancer, Kaposi's sarcoma, or Merkel cell carcinoma.

[0067] In another preferred embodiment, the metastatic lesion is selected from the following group: lymph node metastasis lesion, liver metastasis lesion, lung metastasis lesion, brain organ metastasis lesion, or bone metastasis lesion.

[0068] In the fourth aspect of the present invention, there is provided a use of a compound of formula I as described in the first aspect, or a pharmaceutically acceptable salt thereof, or a precursor compound, an isotope compound, or a stereoisomer or tautomer thereof, for preparing a radiopharmaceutical that specifically targets melanoma.

[0069] In another preferred embodiment, the radiopharmaceutical is used for in vivo clinical treatment of primary and metastatic melanoma lesions.

[0070] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 Probes are shown 99m Radioactive HPLC spectrum of Tc-C-13.

[0072] Figure 2 Probes are shown 99m Radioactive HPLC spectrum of Tc-C-13 incubated with normal saline for 6 h.

[0073] Figure 3 Probes are shown 99mCellular uptake of Tc-C-13 in different cell lines and at different times (p<0.001, ***).

[0074] Figure 4 Probes are shown 99m Tumor / non-tumor ratio of Tc-C-13 in B16F10 tumor-bearing mice.

[0075] Figure 5 Probes are shown 99m SPECT / CT imaging of Tc-C-13 in B16F10 and A375 tumor-bearing mice.

[0076] Figure 6 Probes are shown 99m SPECT / CT imaging of Tc-C-13 in B16F10 lung metastasis model mice.

[0077] Figure 7 Probes are shown 188 Radioactive HPLC spectrum of Re-C-13.

[0078] Figure 8 Probes are shown 188 Radioactive HPLC spectrum of Re-C-13 incubated with normal saline for 24 h. DETAILED DESCRIPTION

[0079] After extensive and in-depth research and a large number of experimental screenings, the inventors unexpectedly discovered for the first time that the compound shown in Formula I, or its pharmaceutically acceptable salt, or its precursor compound, isotopic compound, or its stereoisomer, or tautomer, discovered by the present invention, has high affinity for melanoma, strong targeting specificity, low nonspecific uptake in the liver and kidney, and a high tumor / non-tumor ratio. In addition, the probe precursor of the present invention can selectively chelate different radioactive diagnostic or therapeutic nuclides and can be used for single-photon emission computed tomography for early diagnosis of major diseases such as malignant tumors and Parkinson's disease, guiding treatment regimen selection, efficacy monitoring, and radiotherapy. The compound of the present invention is simple to synthesize, the raw materials are readily available, and the stability is good. It can be promoted and applied as a new type of melanoma-targeted radiopharmaceutical. On this basis, the inventors completed the present invention.

[0080] Terminology

[0081] In the present invention, "the compound of the present invention", "the compound of formula I of the present invention", "the compound of formula 99m Tc-CX series of small molecule probes", "the present invention 188 "Re-CX series small molecule probes" can be used interchangeably to refer to small molecule probes that can be used as imaging agents through the preparation method of the present invention. 99m Tc-CX and integrated diagnosis and treatment 188Re-CX series of small molecule probes.

[0082] In the present invention, nat Re refers to the non-radioactive metal Re, which is the following 188 RC-13 and 188 The cold labeled reference substance of Re-C-15 can be directly expressed as Re or nat Re.

[0083] Unless otherwise specified, in all compounds of the present invention, each chiral carbon atom may be optionally in the R configuration or the S configuration, or a mixture of the R configuration and the S configuration.

[0084] The term "C1-C12 alkyl" refers to a straight or branched chain alkyl group having 1 to 12 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or the like.

[0085] The term "C1-C6 alkyl" refers to a straight or branched chain alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or the like.

[0086] The term "C1-C12 alkoxy" refers to a straight or branched alkoxy group having 1 to 12 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, or the like.

[0087] The term "C2-C6 ester group" refers to a ROC(=O)- group having 2 to 6 carbon atoms, such as -COOCH3, -COOC2H5, -COOC3H7, -COOC4H9, or the like.

[0088] The term "C6-C12 arylester group" refers to a ROC(=O)- group having an aromatic group of 6 to 12 carbon atoms, such as -Ar-OC(=O), or a similar group.

[0089] The term "C1-C6 alkylamide" refers to a RC(=O)-NH- group having 1 to 6 carbon atoms, such as -CC(=O)-NH-, or similar groups.

[0090] The term "C6-C12 arylamide" refers to a RC(=O)-NH- group having an aromatic group of 6 to 12 carbon atoms, such as -Ar-C(=O)-NH-, or a similar group.

[0091] The term "C3-C12 cycloalkyl" refers to a cycloalkyl group having 3 to 12 carbon atoms, such as cyclopropyl, or the like.

[0092] The term "5-10 membered heterocyclic group" refers to a group formed by losing a hydrogen atom from a 5- to 10-membered saturated ring having 1-3 heteroatoms selected from the group consisting of N, S, and O; for example, a pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, or a 3- to 10-membered heterocyclic group having a bridged ring structure, or a 3- to 10-membered heterocyclic group having a spirocyclic structure, or similar groups.

[0093] The term "C6-C10 aryl" refers to a group formed by a 6- to 10-membered aryl group losing a hydrogen atom; for example, phenyl, naphthyl, or the like.

[0094] The term "halogen" refers to F, Cl, Br and I.

[0095] Unless otherwise specified, the structural formulas described in the present invention are intended to include all isomeric forms (such as enantiomers, diastereomers and geometric isomers (or conformers): for example, R, S configurations containing asymmetric centers, (Z), (E) isomers of double bonds and (Z), (E) conformers. Therefore, single stereochemical isomers of the compounds of the present invention or mixtures of their enantiomers, diastereomers or geometric isomers (or conformers) are all within the scope of the present invention.

[0096] The term "tautomer" refers to structural isomers of different energies that can interconvert across a low energy barrier. For example, proton tautomers (i.e., prototropy) include interconversions via proton migration, such as 1H-indazole and 2H-indazole, and 1H-benzo[d]imidazole and 3H-benzo[d]imidazole. Valence tautomers include interconversions via reorganization of some of the bonding electrons.

[0097] Herein, the form "C1-C6" indicates that the group may have 1 to 6 carbon atoms, such as 1, 2, 3, 4 or 5 carbon atoms.

[0098] Compounds of the present invention

[0099] In the present invention, the raw materials of chemical synthesis reagents are all commercially available commodities with a wide range of sources.

[0100] Formula I compound of the present invention also includes its all possible stereoisomers separately, and it is the form of any mixture of any ratio of single stereoisomer or described stereoisomer (for example R-isomer or S-isomer, or E-isomer or Z-isomer).In addition, described formula I compound can also exist in the form of tautomer. Formula I compound of the present invention includes its all possible tautomers separately, and it is the form of any mixture of any ratio of single stereoisomer or described tautomer.All these isomers and their mixture are included in the present invention.

[0101] In the present invention, the desired compounds can be prepared by conventional methods in the art. Exemplary preparation methods are as follows:

[0102] 1) Synthesis of ligand skeleton AADT-COOH:

[0103] 2-(tritylthio)ethylamine and bromoacetyl bromide are mixed and reacted in chloroform to obtain N-[2-(trityl)thioethyl][2-(trityl)thioethylamino]acetamide after purification;

[0104] N-[2-(trityl)thioethyl][2-(trityl)thioethylamino]acetamide is reacted with ethyl bromobutyrate to obtain ethyl 4-((2-oxo-2-((2-(triphenylthio)ethyl)amino)ethyl)(2-(triphenylthio)ethyl)amino)butanoate; the ethyl ester is then removed using a methanol-water system of NaOH, and the mixture is separated and purified by preparative liquid phase separation to obtain 4-((2-oxo-2-((2-(triphenylthio)ethyl)amino)ethyl)(2-(triphenylthio)ethyl)amino)butyric acid (AADT-COOH);

[0105] 2) Synthesis of ligand molecules:

[0106] The AADT-COOH obtained in the previous step and the commercially purchased N-containing small molecule L-NR1R2 were dissolved in anhydrous acetonitrile, and appropriate amounts of N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl) urea hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIPEA) were added. The reaction was stirred at room temperature overnight. After the reaction was completed, the target ligand molecule was purified and separated by preparative liquid chromatography;

[0107]

[0108] Alternatively, the N-[2-(trityl)thioethyl][2-(trityl)thioethylamino]acetamide obtained in step 1) and the bromine / chlorine-containing small molecule L-NR1R2 are dissolved in anhydrous acetonitrile, and appropriate amounts of cesium carbonate and potassium iodide are added. The reaction is heated overnight, and the target ligand molecule is obtained by separation and purification via preparative liquid phase separation;

[0109]

[0110] Subsequently, 1-5 mg of the trityl-protected ligand was dissolved in 1-5 mL of trifluoroacetic acid (TFA) for deprotection, and the resulting yellow solution was titrated with triethylsilane until it became colorless; the trifluoroacetic acid was dried by rotary evaporation and then further dried by vacuum drying; the dried residue was then redissolved in argon-saturated methanol for subsequent preparation 99m Tc-labeled compounds;

[0111] 3)99m Preparation of Tc-C1 complex:

[0112] Prepared by sodium glucoheptonic acid (CH2OH(CHOH)5COOH) exchange reaction 99m Tc-C1 complex, the specific steps are as follows:

[0113] First, 37-370 MBq of Na 99m Add TcO4 eluent to a freeze-dried drug box containing 5-10 mg of sodium glucoheptonic acid, 1-20 μg of stannous chloride, and 1-10 mg of urea, shake well, and let stand for five minutes after the solid is completely dissolved. 99m TcO-GH intermediate solution;

[0114] Then, add C1 ligand at a concentration of 1-10 g / L to the above 99m The TcO-GH intermediate solution is heated at 60-100°C for 15-30 minutes to obtain 99m Tc-C1 complex.

[0115] Radiopharmaceuticals of the present invention

[0116] Medical imaging has entered the era of molecular imaging. The research on molecular imaging includes two aspects: imaging equipment and molecular probes. The development of new radioactive probes is the core of the development of molecular imaging. Single photon emission computed tomography (SPECT) is an imaging technique at the molecular level of living organisms. Compared with traditional imaging techniques, it can provide more and more detailed physiological and biochemical information. In recent years, integrated diagnosis and treatment radiopharmaceuticals that combine diagnosis and treatment have become one of the most promising directions for clinical transformation. This type of drug uses radionuclides as paired imaging and therapeutic agents, targeting molecules that are highly expressed specifically in tumors, and delivers the nuclides to the tumor site through high-affinity ligands to kill the tumor. Compared with single diagnostic or therapeutic drugs, it has the advantages of reducing systemic toxicity and improving therapeutic effects. At present, the most commonly used diagnostic drugs for clinical SPECT are mainly 99m Tc and its labeled compounds, so the development of new radionuclide drugs is of great significance.

[0117] In the present invention, the compound represented by Formula I, or a pharmaceutically acceptable salt thereof, or a precursor compound, an isotope compound, or a stereoisomer or a tautomer thereof is used to prepare an imaging agent.

[0118]

[0119] Preferably, the compound of the present invention is selected from the following group: 99m Tc-CX series of small molecule probes, 188 Re-CX series of small molecule probes,186 Re-CX series of small molecule probes.

[0120] Preferably, the imaging agent of the present invention is used to monitor the imaging ability of the probe at the cellular, tissue, or in vivo levels.

[0121] Preferably, the imaging agent is incubated with tumor cells or tissues or administered in vivo to monitor the imaging ability of the probe at the cellular, tissue or in vivo levels.

[0122] Preferably, the imaging agent is used for clinical diagnosis of tumor sites with high melanin expression.

[0123] In this invention, the probe precursor can selectively chelate different radioactive diagnostic or therapeutic nuclides and can be used for single-photon emission computed tomography (SPECT) imaging to diagnose malignant tumors, Parkinson's disease, and other major diseases early, guide treatment selection, monitor efficacy, and conduct radiotherapy. The compound described in this invention is simple to synthesize, uses readily available raw materials, and exhibits excellent stability, making it promising for application as a novel melanoma-targeting radiopharmaceutical.

[0124] Compared with the prior art, the present invention has the following beneficial effects:

[0125] 1. The present invention uses a freeze-dried medicine kit containing sodium glucoheptanoate to carry out 99m Tc labeling has high radiochemical yield and radiochemical purity, simple synthesis, and readily available raw materials. It can be used as a new melanoma targeted imaging agent for clinical promotion and application.

[0126] 2. The present invention has found 99m The Tc-CX series of small molecule probes are radioactive probes targeting melanoma. 18 F and 68 The Ga-labeled melanoma probe has a half-life of 6.02 h and good in vitro stability, making it more suitable for clinical diagnosis of melanoma and metastatic lesions.

[0127] 3. The present invention has found 99m The SPECT / CT imaging results of the Tc-CX series of small molecule probes show that they can specifically target tumor sites with high melanin expression, with high tumor / blood and tumor / muscle contrast, high tumor uptake and tumor / organ uptake ratios, and long retention time, which helps clinicians diagnose the occurrence of melanoma and monitor metastatic lesions.

[0128] 4. The present invention has found 99m The Tc-CX series of small molecule probes can also be further used with radioactive 188 Re was labeled to obtain therapeutic probes for the treatment of melanoma.

[0129] 5. The present invention introduces different linking groups, which can further improve the pharmacokinetic properties of the probe, especially the clearance kinetics from non-tumor tissues.

[0130] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0131] Unless otherwise specified, the raw materials, reagents, methods, etc. used in the examples are conventional raw materials, reagents, and methods in the art.

[0132] Raw materials: All compounds used were purchased commercially unless otherwise specified.

[0133] Example 1: Compound 99m Synthesis of Tc-C-1

[0134]

[0135] Compound 1 (1 g, 3.13 mmol) was dissolved in 20 mL of chloroform, and triethylamine (1.6 mL, 11.6 mmol) was added. Bromoacetyl bromide (96 μL, 1.16 mmol) was slowly added dropwise in an ice bath. After a half-hour of reaction at low temperature, the ice bath was removed and the mixture was stirred at room temperature for 48 hours. After completion of the reaction as monitored by TLC, the solvent was removed by rotary evaporation. The residue was extracted three times with 100 mL of ethyl acetate. The organic phases were combined and dried to afford crude product 2. Purification by silica gel column chromatography (petroleum ether:ethyl acetate = 1:1) afforded 1.9 g of pure compound 2 in a 90% yield. 1 H NMR (600 MHz, Chloroform-d) δ 7.42 (t, J = 7.6 Hz, 12H), 7.30–7.27 (m, 13H), 7.22 (d, J = 19.4 Hz, 6H), 3.09 (q, J = 6.4 Hz, 2H), 3.05 (s, 2H), 2.47 (t, J = 6.3 Hz, 2H), 2.37 (dt, J = 11.9, 6.3 Hz, 4H), 1.28 (s, 1H). ESI-MS: Calculated: 678.95; Found: 679.49 [M+H], 243.13 [CPh3+].

[0136] Compound 2 (500 mg, 0.73 mmol), cesium carbonate (700 mg, 2.14 mmol), and potassium iodide (130 mg, 0.78 mmol) were dissolved in 10 mL of anhydrous acetonitrile. Ethyl bromobutyrate (520 μL, 3.70 mmol) was added dropwise. Under nitrogen, the mixture was heated at reflux at 80°C overnight. The reaction was monitored for completion by TLC. The solvent was removed by rotary evaporation, and the product was purified by silica gel column chromatography (dichloromethane:methanol = 10:1) to obtain 490 mg of pure compound 3 in an 84% yield. 1 H NMR(600MHz,Chloroform-d)δ7.68(s,1H),7.41(dt,J=9.1,3.2Hz,12H),7.30–7.26(m,14H),7.25–7.19(m,6H),4.10(q,J=7.1Hz,2H),3.10–3.01(m,4H ),2.52(s,2H),2.47(d,J=7.2Hz,2H),2.42(d,J=7.0Hz,2H),2.38(t,J=6.6 Hz, 2H), 2.23 (t, J = 7.0 Hz, 2H), 1.68 (t, J = 7.6 Hz, 2H), 1.24 (t, J = 7.1 Hz, 3H). ESI-MS: calcd: 792.34; found: 793.52 [M+H], 243.18 [CPh3+].

[0137] Compound 3 (500 mg, 0.63 mmol) was dissolved in 10 mL of methanol-water (1:1) and NaOH (50 mg, 1.25 mmol) was added. The mixture was stirred and heated at 50°C under nitrogen overnight. The reaction was complete as monitored by TLC. Purification by silica gel column chromatography (dichloromethane:methanol = 95:5) afforded 350 mg of pure compound 4 in a 72% yield. 1 H NMR (500 MHz, Chloroform-d) δ 7.40–7.35 (m, 12H), 7.24 (dd, J = 8.4, 6.7 Hz, 12H), 7.21–7.15 (m, 6H), 3.01 (q, J = 6.3 Hz, 2H), 2.86 (s, 2H), 2.37 (td, J = 6.8, 4.2 Hz, 4H), 2.30 (t, J = 7.3 Hz, 2H), 2.25 (td, J = 6.8, 3.6 Hz, 4H), 1.63 (p, J = 7.3 Hz, 2H). ESI-MS: calcd: 765.04; found: 765.66 [M+H], 243.22 [CPh3+].

[0138] Compound 4 (100 mg, 0.13 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (HATU, 150 mg, 0.39 mmol) were dissolved in 10 mL of anhydrous DMF. N,N-diethyl-1,2-ethylenediamine hydrochloride (45 μL, 0.2 mmol) and N,N-diisopropylethylamine (DIPEA, 70 μL, 0.39 mmol) were added. The mixture was stirred at room temperature under nitrogen overnight. The reaction was complete as monitored by TLC. Purification by preparative liquid chromatography afforded 51 mg of pure compound 5 in a 46% yield. 1 HNMR(500MHz,Chloroform-d)δ7.41–7.35(m,13H),7.31–7.25(m,14H),7.21(dt,J=14.4,7.3Hz,6H),3.56(d,J=21.8Hz,4H),3.19–3.06(m,6 H),3.03(q,J=6.5Hz,2H),2.82(t,J=6.4Hz,2H),2.74–2.55(m,4H),2.33(q,J=6.3,5.7Hz,4H),1.70(t,J=6.5Hz,2H),1.27(t,J=7.3Hz,6H). ESI-MS: calculated: 863.24; found: 863.59 [M+H], 243.22 [CPh3+].

[0139] Compound 5 (20 mg, 0.02 mmol) was dissolved in 5 mL of trifluoroacetic acid and shaken for 2 minutes. Deprotection treatment was then performed, and the resulting yellow solution was titrated with triethylsilane until it became colorless. The trifluoroacetic acid was dried by rotary evaporation and then further dried in a vacuum dryer to obtain the deprotected precursor compound 6 for subsequent labeling. ESI-MS: Calculated: 378.59; Found: 377.67 [M].

[0140] Take a freeze-dried drug box of sodium stannous glucoheptonic acid for injection and inject 370MBq of Na into the vial. 99m The TcO4 eluent was thoroughly shaken and dissolved on an oscillator and allowed to stand for five minutes to obtain 99m TcO-GH intermediate solution. Then, add compound 6 with a concentration of 10g / L to the above 99m The TcO-GH intermediate solution was heated at 75°C for 20 minutes to obtain 99m Tc-C-1 complex.

[0141] After labeling, samples were taken for TLC spotting. Development was performed in acetone and ammonium acetate-methanol, respectively, followed by drying and scanning on a radioactive TLC detector to calculate the labeling yield. Simultaneously, 5 μL of the reaction solution was sampled for radiochemical purity monitoring by radioactive HPLC.

[0142] Example 2: Compound 99m Synthesis of Tc-C-2

[0143]

[0144] Compound 4 (100 mg, 0.13 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (HATU, 150 mg, 0.39 mmol) were dissolved in 10 mL of anhydrous DMF. N,N-dipropylethane-1,2-diamine (35 μL, 0.2 mmol) and N,N-diisopropylethylamine (DIPEA, 70 μL, 0.39 mmol) were added. The mixture was stirred at room temperature under nitrogen overnight. The reaction was complete as monitored by TLC. Purification by preparative liquid chromatography afforded 82 mg of pure compound 7 in a 70% yield. ESI-MS: Calcd: 891.29; Found: 892.26 [M+H], 243.18 [CPh3+].

[0145] Compound 7 (20 mg, 0.02 mmol) was dissolved in 5 mL of trifluoroacetic acid and shaken for 2 minutes for deprotection. The resulting yellow solution was titrated with triethylsilane until it became colorless. The trifluoroacetic acid was dried on a rotary evaporator and then further dried in a vacuum dryer to obtain the deprotected precursor compound 8 for subsequent labeling.

[0146] Take a freeze-dried drug box of sodium stannous glucoheptonic acid for injection and inject 370MBq of Na into the vial. 99m The TcO4 eluent was thoroughly shaken and dissolved on an oscillator and allowed to stand for five minutes to obtain 99m TcO-GH intermediate solution. Then, add compound 8 with a concentration of 10g / L to the above 99m The TcO-GH intermediate solution was heated at 75°C for 20 minutes to obtain 99m Tc-C-2 complex.

[0147] Example 3: Compound 99m Synthesis of Tc-C-3

[0148]

[0149] Compound 4 (100 mg, 0.13 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (HATU, 150 mg, 0.39 mmol) were dissolved in 10 mL of anhydrous DMF. N,N-dimethylethylenediamine hydrochloride (21 μL, 0.2 mmol) and N,N-diisopropylethylamine (DIPEA, 70 μL, 0.39 mmol) were added. The mixture was stirred at room temperature under nitrogen overnight. The reaction was complete as monitored by TLC. Purification by preparative liquid chromatography afforded 82 mg of pure compound 9 in a 77% yield. ESI-MS: Calcd: 835.18; Found: 836.82 [M+H], 243.27 [CPh3+].

[0150] Compound 9 (20 mg, 0.02 mmol) was dissolved in 5 mL of trifluoroacetic acid and shaken for 2 minutes. Deprotection was then performed, and the resulting yellow solution was titrated with triethylsilane until it became colorless. The trifluoroacetic acid was dried on a rotary evaporator and then further dried in a vacuum dryer to obtain the deprotected precursor compound 10 for subsequent labeling.

[0151] Take a freeze-dried drug box of sodium stannous glucoheptonic acid for injection and inject 370MBq of Na into the vial. 99m The TcO4 eluent was thoroughly shaken and dissolved on an oscillator and allowed to stand for five minutes to obtain 99m TcO-GH intermediate solution. Then, compound 10 with a concentration of 10 g / L was added to the above 99m The TcO-GH intermediate solution was heated at 75°C for 20 minutes to obtain 99m Tc-C-3 complex.

[0152] Example 4: Compound 99m Synthesis of Tc-C-4

[0153]

[0154] Compound 4 (100 mg, 0.13 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (HATU, 150 mg, 0.39 mmol) were dissolved in 10 mL of anhydrous DMF. N,N-dimethyl-2-(piperazin-1-yl)ethanamine (35 μL, 0.19 mmol) and N,N-diisopropylethylamine (DIPEA, 70 μL, 0.39 mmol) were added. The mixture was stirred at room temperature under nitrogen overnight. The reaction was complete as monitored by TLC. Purification by preparative liquid chromatography afforded 81 mg of pure compound 11 in a 69% yield. ESI-MS: Calcd: 904.29; Found: 905.04 [M+H], 243.36 [CPh3+].

[0155] Compound 11 (20 mg, 0.02 mmol) was dissolved in 5 mL of trifluoroacetic acid and shaken for 2 minutes. Deprotection was then performed, and the resulting yellow solution was titrated with triethylsilane until it became colorless. The trifluoroacetic acid was dried on a rotary evaporator and then further dried in a vacuum dryer to obtain the deprotected precursor compound 12 for subsequent labeling.

[0156] Take a freeze-dried drug box of sodium stannous glucoheptonic acid for injection and inject 370MBq of Na into the vial. 99m The TcO4 eluent was thoroughly shaken and dissolved on an oscillator and allowed to stand for five minutes to obtain 99m TcO-GH intermediate solution. Then, compound 12 with a concentration of 10 g / L was added to the above 99m The TcO-GH intermediate solution was heated at 75°C for 20 minutes to obtain 99m Tc-C-4 complex.

[0157] Example 5: Compound 99m Synthesis of Tc-C-5

[0158]

[0159] Compound 4 (100 mg, 0.13 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (HATU, 150 mg, 0.39 mmol) were dissolved in 10 mL of anhydrous DMF. N,N-dimethyl-p-phenylenediamine dihydrochloride (29 μL, 0.19 mmol) and N,N-diisopropylethylamine (DIPEA, 73 μL, 0.39 mmol) were added. The mixture was stirred at room temperature under nitrogen overnight. The reaction was complete as monitored by TLC. Purification by preparative liquid chromatography afforded 73 mg of pure compound 13 in a 63% yield. ESI-MS: Calcd: 897.25; Found: 897.80 [M+H], 243.31 [CPh3+].

[0160] Compound 13 (20 mg, 0.02 mmol) was dissolved in 5 mL of trifluoroacetic acid and shaken for 2 minutes. Deprotection was then performed, and the resulting yellow solution was titrated with triethylsilane until it became colorless. The trifluoroacetic acid was dried on a rotary evaporator and then further dried in a vacuum dryer to obtain the deprotected precursor compound 14 for subsequent labeling.

[0161] Take a freeze-dried drug box of sodium stannous glucoheptonic acid for injection and inject 370MBq of Na into the vial. 99m The TcO4 eluent was thoroughly shaken and dissolved on an oscillator and allowed to stand for five minutes to obtain 99m TcO-GH intermediate solution. Then, compound 14 with a concentration of 10 g / L was added to the above 99m The TcO-GH intermediate solution was heated at 75°C for 20 minutes to obtain 99m Tc-C-5 complex.

[0162] Example 6: Compound 99m Synthesis of Tc-C-6

[0163]

[0164] 2-Methoxy-5-nitrobenzoic acid (1.5 g, 7.6 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (HATU, 4.35 g, 11.4 mmol) were dissolved in 30 mL of dichloromethane. N,N-dimethylethylenediamine (1 mL, 9.1 mmol) and N,N-diisopropylethylamine (DIPEA, 3.97 mL, 22.8 mmol) were added. Under nitrogen, the mixture was stirred and heated at 60°C overnight. The reaction was complete as monitored by TLC. Purification by silica gel column chromatography (dichloromethane:methanol = 95:5) afforded 1.63 g of pure compound M2 in 80% yield. ESI-MS: Calculated: 267.29; Found: 268.45 [M+H].

[0165] Compound M2 (1 g, 9.8 mmol) was dissolved in methanol-tetrahydrofuran and an appropriate amount of Pd / C was added. The reaction was stirred overnight at room temperature under hydrogen reduction. The reaction was complete as monitored by TLC. The filtrate was filtered and dried to give crude 4-amino-N-(2-(dimethylamino)ethyl)-2-methoxybenzamide M3. Purification by silica gel column chromatography (dichloromethane:methanol = 90:10) afforded 0.84 g of pure compound M3 in a 95% yield. ESI-MS: Calculated: 237.30; Found: 238.12 [M+H].

[0166] Compound 4 (300 mg, 0.39 mmol), compound M3 (138 mg, 0.58 mmol), and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (HATU, 450 mg, 1.17 mmol) were dissolved in 15 mL of dichloromethane. N,N-diisopropylethylamine (DIPEA, 204 μL, 1.17 mmol) was added and the mixture was stirred at room temperature under nitrogen overnight. The reaction was complete as monitored by TLC. Purification by preparative liquid chromatography afforded 332 mg of pure compound 15 in 86% yield. ESI-MS: Calcd: 984.33; Found: 984.93.19 [M+H], 243.31 [CPh3+].

[0167] Compound 15 (20 mg, 0.02 mmol) was dissolved in 5 mL of trifluoroacetic acid and shaken for 2 minutes. Deprotection was then performed, and the resulting yellow solution was titrated with triethylsilane until it became colorless. The trifluoroacetic acid was dried on a rotary evaporator and then further dried in a vacuum dryer to obtain the deprotected precursor compound 16 for subsequent labeling.

[0168] Take a freeze-dried drug box of sodium stannous glucoheptonic acid for injection and inject 370MBq of Na into the vial. 99mThe TcO4 eluent was thoroughly shaken and dissolved on an oscillator and allowed to stand for five minutes to obtain 99m TcO-GH intermediate solution. Then, compound 16 with a concentration of 10 g / L was added to the above 99m The TcO-GH intermediate solution was heated at 75°C for 20 minutes to obtain 99m Tc-C-6 complex.

[0169] Example 7: Compound 99m Synthesis of Tc-C-7

[0170]

[0171] Compound 4 (300 mg, 0.39 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (HATU, 450 mg, 1.17 mmol) were dissolved in 15 mL of dichloromethane. N,N-diisopropylethylamine (DIPEA, 340 μL, 1.17 mmol) and 2-(aminomethyl)-1-ethylpyrrolidine (80 μL, 0.58 mmol) were added. The mixture was stirred at room temperature under nitrogen overnight. The reaction was complete as monitored by TLC. Purification by preparative liquid chromatography afforded 223 mg of pure compound 17 in a 65% yield. ESI-MS: Calcd: 875.25; Found: 875.83 [M+H], 243.31 [CPh3+].

[0172] Compound 17 (20 mg, 0.02 mmol) was dissolved in 5 mL of trifluoroacetic acid and shaken for 2 minutes. Deprotection was then performed, and the resulting yellow solution was titrated with triethylsilane until it became colorless. The trifluoroacetic acid was dried on a rotary evaporator and then further dried in a vacuum dryer to obtain the deprotected precursor compound 18 for subsequent labeling.

[0173] Take a freeze-dried drug box of sodium stannous glucoheptonic acid for injection and inject 370MBq of Na into the vial. 99m The TcO4 eluent was thoroughly shaken and dissolved on an oscillator and allowed to stand for five minutes to obtain 99m TcO-GH intermediate solution. Then, compound 18 with a concentration of 10 g / L was added to the above 99m The TcO-GH intermediate solution was heated at 75°C for 20 minutes to obtain 99m Tc-C-7 complex.

[0174] Example 8: Compound 99m Synthesis of Tc-C-8

[0175]

[0176] Compound 4 (300 mg, 0.39 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (HATU, 450 mg, 1.17 mmol) were dissolved in 15 mL of dichloromethane. N,N-diisopropylethylamine (DIPEA, 340 μL, 1.17 mmol) and N-(2-aminoethyl)morpholine (80 μL, 0.58 mmol) were added. The mixture was stirred at room temperature under nitrogen overnight. The reaction was complete as monitored by TLC. Purification by preparative liquid chromatography afforded 206 mg of pure compound 19 in a 60% yield. ESI-MS: Calcd: 877.22; Found: 877.81 [M+H], 243.27 [CPh3+].

[0177] Compound 19 (20 mg, 0.02 mmol) was dissolved in 5 mL of trifluoroacetic acid and shaken for 2 minutes. Deprotection was then performed, and the resulting yellow solution was titrated with triethylsilane until it became colorless. The trifluoroacetic acid was dried on a rotary evaporator and then further dried in a vacuum dryer to obtain the deprotected precursor compound 20 for subsequent labeling.

[0178] Take a freeze-dried drug box of sodium stannous glucoheptonic acid for injection and inject 370MBq of Na into the vial. 99m The TcO4 eluent was thoroughly shaken and dissolved on an oscillator and allowed to stand for five minutes to obtain 99m TcO-GH intermediate solution. Then, compound 20 with a concentration of 10 g / L was added to the above 99m The TcO-GH intermediate solution was heated at 75°C for 20 minutes to obtain 99m Tc-C-8 complex.

[0179] Example 9: Compound 99m Synthesis of Tc-C-9

[0180]

[0181] Compound 4 (100 mg, 0.13 mmol), 4-amino-1-methylpiperidine dihydrochloride (37 mg, 0.20 mmol), and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (HATU, 150 mg, 0.39 mmol) were dissolved in 10 mL of anhydrous DMF. N,N-diisopropylethylamine (DIPEA, 102 μL, 0.58 mmol) was added and the mixture was stirred at room temperature under nitrogen overnight. The reaction was complete as monitored by TLC. Purification by preparative liquid chromatography afforded 75 mg of pure compound 21 in a 67% yield. ESI-MS: Calcd: 861.25; Found: 861.79 [M+H], 243.27 [CPh3+].

[0182] Compound 21 (20 mg, 0.02 mmol) was dissolved in 5 mL of trifluoroacetic acid and shaken for 2 minutes. Deprotection was then performed, and the resulting yellow solution was titrated with triethylsilane until it became colorless. The trifluoroacetic acid was dried on a rotary evaporator and then further dried in a vacuum dryer to obtain the deprotected precursor compound 22 for subsequent labeling.

[0183] Take a freeze-dried drug box of sodium stannous glucoheptonic acid for injection and inject 370MBq of Na into the vial. 99m The TcO4 eluent was thoroughly shaken and dissolved on an oscillator and allowed to stand for five minutes to obtain 99m TcO-GH intermediate solution. Then, compound 22 with a concentration of 10 g / L was added to the above 99m The TcO-GH intermediate solution was heated at 75°C for 20 minutes to obtain 99m Tc-C-9 complex.

[0184] Example 10: Compound 99m Synthesis of Tc-C-10

[0185]

[0186] Compound 4 (100 mg, 0.13 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (HATU, 150 mg, 0.39 mmol) were dissolved in 10 mL of anhydrous DMF. 1-Amino-4-methylpiperazine (23 μL, 0.19 mmol) and N,N-diisopropylethylamine (DIPEA, 102 μL, 0.58 mmol) were added. The mixture was stirred at room temperature under nitrogen overnight. The reaction was complete as monitored by TLC. Purification by preparative liquid chromatography afforded 69 mg of pure compound 23 in a 62% yield. ESI-MS: Calcd: 862.21; Found: 863.91 [M+H], 243.27 [CPh3+].

[0187] Compound 23 (20 mg, 0.02 mmol) was dissolved in 5 mL of trifluoroacetic acid and shaken for 2 minutes. Deprotection was then performed, and the resulting yellow solution was titrated with triethylsilane until it became colorless. The trifluoroacetic acid was dried on a rotary evaporator and then further dried in a vacuum dryer to obtain the deprotected precursor compound 24 for subsequent labeling.

[0188] Take a freeze-dried drug box of sodium stannous glucoheptonic acid for injection and inject 370MBq of Na into the vial. 99m The TcO4 eluent was thoroughly shaken and dissolved on an oscillator and allowed to stand for five minutes to obtain 99m TcO-GH intermediate solution. Then, compound 24 with a concentration of 10 g / L was added to the above 99m The TcO-GH intermediate solution was heated at 75°C for 20 minutes to obtain 99m Tc-C-10 complex.

[0189] Example 11: Compound 99m Synthesis of Tc-C-11

[0190]

[0191] Compound 2 (40 mg, 0.06 mmol), N-BOC-4-bromoethylpiperazine (52 mg, 0.17 mmol), cesium carbonate (58 mg, 0.18 mmol), and potassium iodide (10 mg, 0.06 mmol) were dissolved in anhydrous acetonitrile and stirred at 80°C under nitrogen overnight. The reaction was complete as monitored by TLC. Purification by preparative liquid chromatography afforded 23 mg of pure compound 25 in a 44% yield. ESI-MS: Calculated: 891.25; Found: 891.95 [M+H], 243.36 [CPh3+].

[0192] Compound 25 (10 mg, 0.01 mmol) was dissolved in 5 mL of trifluoroacetic acid and shaken for 2 minutes. Deprotection was then performed, and the resulting yellow solution was titrated with triethylsilane until it became colorless. The trifluoroacetic acid was dried on a rotary evaporator and then further dried in a vacuum dryer to obtain the deprotected precursor compound 26 for subsequent labeling.

[0193] Take a freeze-dried drug box of sodium stannous glucoheptonic acid for injection and inject 370MBq of Na into the vial. 99m The TcO4 eluent was thoroughly shaken and dissolved on an oscillator and allowed to stand for five minutes to obtain 99m TcO-GH intermediate solution. Then, compound 26 with a concentration of 10 g / L was added to the above 99m The TcO-GH intermediate solution was heated at 75°C for 20 minutes to obtain 99m Tc-C-11 complex.

[0194] Example 12: Compound 99m Synthesis of Tc-C-12

[0195]

[0196] Compound 2 (40 mg, 0.06 mmol), tert-butyl 4-(3-bromopropyl)piperazine-1-carboxylate (55 mg, 0.17 mmol), cesium carbonate (58 mg, 0.18 mmol), and potassium iodide (10 mg, 0.06 mmol) were dissolved in anhydrous acetonitrile and stirred at 80°C under nitrogen overnight. The reaction was complete as monitored by TLC. Purification by preparative liquid chromatography afforded 21 mg of pure compound 27 in a 40% yield. ESI-MS: Calculated: 905.27; Found: 906.12 [M+H], 243.12 [CPh3+].

[0197] Compound 27 (10 mg, 0.01 mmol) was dissolved in 5 mL of trifluoroacetic acid and shaken for 2 minutes. Deprotection was then performed, and the resulting yellow solution was titrated with triethylsilane until it became colorless. The trifluoroacetic acid was dried on a rotary evaporator and then further dried in a vacuum dryer to obtain the deprotected precursor compound 28 for subsequent labeling.

[0198] Take a freeze-dried drug box of sodium stannous glucoheptonic acid for injection and inject 370MBq of Na into the vial. 99m The TcO4 eluent was thoroughly shaken and dissolved on an oscillator and allowed to stand for five minutes to obtain 99m TcO-GH intermediate solution. Then, compound 28 with a concentration of 10 g / L was added to the above 99mThe TcO-GH intermediate solution was heated at 75°C for 20 minutes to obtain 99m Tc-C-12 complex.

[0199] Example 13: Compound 99m Synthesis of Tc-C-13

[0200]

[0201] Compound 2 (200 mg, 0.29 mmol), 2-bromo-N,N-dimethylethylamine hydrobromide (687 mg, 2.9 mmol), cesium carbonate (480 mg, 1.47 mmol), and potassium iodide (49 mg, 0.29 mmol) were dissolved in anhydrous acetonitrile and stirred at 80°C under nitrogen overnight. The reaction was complete as monitored by TLC. Purification by preparative liquid chromatography afforded 132 mg of pure compound 29 in a 60% yield. ESI-MS: Calcd: 750.08; Found: 750.80 [M+H], 243.36 [CPh3+].

[0202] Compound 29 (10 mg, 0.01 mmol) was dissolved in 5 mL of trifluoroacetic acid and shaken for 2 minutes. Deprotection was then performed, and the resulting yellow solution was titrated with triethylsilane until it became colorless. The trifluoroacetic acid was dried on a rotary evaporator and then further dried in a vacuum dryer to obtain the deprotected precursor compound 30 for subsequent labeling.

[0203] Take a freeze-dried drug box of sodium stannous glucoheptonic acid for injection and inject 370MBq of Na into the vial. 99m The TcO4 eluent was thoroughly shaken and dissolved on an oscillator and allowed to stand for five minutes to obtain 99m TcO-GH intermediate solution. Then, compound 30 with a concentration of 10 g / L was added to the above 99m The TcO-GH intermediate solution was heated at 75°C for 20 minutes to obtain 99m Tc-C-13 complex.

[0204] Example 14: Compound 99m Synthesis of Tc-C-14

[0205]

[0206] Compound 2 (200 mg, 0.29 mmol), 2-bromo-N-ethyl-N-methylethanamine hydrobromide (728 mg, 2.9 mmol), cesium carbonate (480 mg, 1.47 mmol), and potassium iodide (166 mg, 1 mmol) were dissolved in anhydrous acetonitrile and stirred at 80°C under nitrogen overnight. The reaction was complete as monitored by TLC. Purification by preparative liquid chromatography afforded 130 mg of pure compound 31 in a 58% yield. ESI-MS: Calculated: 764.10; Found: 764.70 [M+H], 243.12 [CPh3+].

[0207] Compound 31 (10 mg, 0.01 mmol) was dissolved in 5 mL of trifluoroacetic acid and shaken for 2 minutes. Deprotection was then performed, and the resulting yellow solution was titrated with triethylsilane until it became colorless. The trifluoroacetic acid was dried on a rotary evaporator and then further dried in a vacuum dryer to obtain the deprotected precursor compound 32 for subsequent labeling.

[0208] Take a freeze-dried drug box of sodium stannous glucoheptonic acid for injection and inject 370MBq of Na into the vial. 99m The TcO4 eluent was thoroughly shaken and dissolved on an oscillator and allowed to stand for five minutes to obtain 99m TcO-GH intermediate solution. Then, compound 32 with a concentration of 10 g / L was added to the above 99m The TcO-GH intermediate solution was heated at 75°C for 20 minutes to obtain 99m Tc-C-14 complex.

[0209] Example 15: Compound 99m Synthesis of Tc-C-15

[0210]

[0211] Compound 2 (200 mg, 0.29 mmol), 3-bromo-N,N-dimethyl-1-propylamine hydrobromide (728 mg, 2.9 mmol), cesium carbonate (480 mg, 1.47 mmol), and potassium iodide (166 mg, 1 mmol) were dissolved in anhydrous acetonitrile and stirred at 80°C under nitrogen overnight. The reaction was complete as monitored by TLC. Purification by preparative liquid chromatography afforded 130 mg of pure compound 33 in a 58% yield. ESI-MS: Calculated: 764.10; Found: 764.70 [M+], 243.12 [CPh3+].

[0212] Compound 33 (10 mg, 0.01 mmol) was dissolved in 5 mL of trifluoroacetic acid and shaken for 2 minutes. Deprotection was then performed, and the resulting yellow solution was titrated with triethylsilane until it became colorless. The trifluoroacetic acid was dried on a rotary evaporator and then further dried in a vacuum dryer to obtain the deprotected precursor compound 34 for subsequent labeling.

[0213] Take a freeze-dried drug box of sodium stannous glucoheptonic acid for injection and inject 370MBq of Na into the vial. 99m The TcO4 eluent was thoroughly shaken and dissolved on an oscillator and allowed to stand for five minutes to obtain 99m TcO-GH intermediate solution. Then, compound 34 with a concentration of 10 g / L was added to the above 99m The TcO-GH intermediate solution was heated at 75°C for 20 minutes to obtain 99m Tc-C-15 complex.

[0214] Example 16: Compound 99m Synthesis of Tc-C-16

[0215]

[0216] Compound 2 (100 mg, 0.14 mmol), N-(3-bromopropyl)-N-butylbutan-1-amine hydrobromide (488 mg, 1.47 mmol), cesium carbonate (480 mg, 0.66 mmol), and potassium iodide (73 mg, 0.43 mmol) were dissolved in anhydrous acetonitrile and stirred at 80°C under nitrogen overnight. The reaction was complete as monitored by TLC. Purification by preparative liquid chromatography afforded 130 mg of pure compound 35 in a 58% yield. ESI-MS: Calcd: 848.27; Found: 848.96 [M+H].

[0217] Compound 35 (10 mg, 0.01 mmol) was dissolved in 5 mL of trifluoroacetic acid and shaken for 2 minutes. Deprotection was then performed, and the resulting yellow solution was titrated with triethylsilane until it became colorless. The trifluoroacetic acid was dried on a rotary evaporator and then further dried in a vacuum dryer to obtain the deprotected precursor compound 36 for subsequent labeling.

[0218] Take a freeze-dried drug box of sodium stannous glucoheptonic acid for injection and inject 370MBq of Na into the vial. 99m The TcO4 eluent was thoroughly shaken and dissolved on an oscillator and allowed to stand for five minutes to obtain 99m TcO-GH intermediate solution. Then, compound 36 with a concentration of 10 g / L was added to the above 99mThe TcO-GH intermediate solution was heated at 75°C for 20 minutes to obtain 99m Tc-C-16 complex.

[0219] Example 17: Compound 99m Synthesis of Tc-C-17

[0220]

[0221] Compound 2 (300 mg, 0.44 mmol), 4-(2-bromoethyl)morpholine hydrobromide (121 mg, 4.4 mmol), cesium carbonate (72 mg, 2.2 mmol), and potassium iodide (22 mg, 2.2 mmol) were dissolved in anhydrous acetonitrile and stirred at 80°C under nitrogen overnight. The reaction was complete as monitored by TLC. Purification by preparative liquid chromatography afforded 172 mg of pure compound 37 in a 49% yield. ESI-MS: Calculated: 792.11; Found: 792.75 [M+H], 243.31 [CPh3+].

[0222] Compound 37 (10 mg, 0.01 mmol) was dissolved in 5 mL of trifluoroacetic acid and shaken for 2 minutes. Deprotection was then performed, and the resulting yellow solution was titrated with triethylsilane until it became colorless. The trifluoroacetic acid was dried on a rotary evaporator and then further dried in a vacuum dryer to obtain the deprotected precursor compound 38 for subsequent labeling.

[0223] Take a freeze-dried drug box of sodium stannous glucoheptonic acid for injection and inject 370MBq of Na into the vial. 99m The TcO4 eluent was thoroughly shaken and dissolved on an oscillator and allowed to stand for five minutes to obtain 99m TcO-GH intermediate solution. Then, compound 38 with a concentration of 10 g / L was added to the above 99m The TcO-GH intermediate solution was heated at 75°C for 20 minutes to obtain 99m Tc-C-17 complex.

[0224] Example 18: Compound 99m Synthesis of Tc-C-18

[0225]

[0226] Compound 2 (300 mg, 0.44 mmol), 4-3'-bromopropylmorpholine hydrobromide (128 mg, 4.4 mmol), cesium carbonate (72 mg, 2.2 mmol), and potassium iodide (22 mg, 2.2 mmol) were dissolved in anhydrous acetonitrile and stirred at 80°C under nitrogen overnight. The reaction was complete as monitored by TLC. Purification by preparative liquid chromatography afforded 185 mg of pure compound 39 in a 52% yield. ESI-MS: Calcd: 806.14; Found: 806.79 [M+H], 243.36 [CPh3+].

[0227] Compound 39 (10 mg, 0.01 mmol) was dissolved in 5 mL of trifluoroacetic acid and shaken for 2 minutes. Deprotection was then performed, and the resulting yellow solution was titrated with triethylsilane until it became colorless. The trifluoroacetic acid was dried on a rotary evaporator and then further dried in a vacuum dryer to obtain the deprotected precursor compound 40 for subsequent labeling.

[0228] Take a freeze-dried drug box of sodium stannous glucoheptonic acid for injection and inject 370MBq of Na into the vial. 99m The TcO4 eluent was thoroughly shaken and dissolved on an oscillator and allowed to stand for five minutes to obtain 99m TcO-GH intermediate solution. Then, compound 40 with a concentration of 10 g / L was added to the above 99m The TcO-GH intermediate solution was heated at 75°C for 20 minutes to obtain 99m Tc-C-18 complex.

[0229] Example 19: Compound 99m Synthesis of Tc-C-19

[0230]

[0231] Compound 2 (300 mg, 0.44 mmol), 5-iodopent-1-yne (128 mg, 4.4 mmol), and cesium carbonate (72 mg, 2.2 mmol) were dissolved in anhydrous acetonitrile and stirred at 80°C under nitrogen overnight. The reaction was complete as monitored by TLC. Purification by preparative liquid chromatography afforded 185 mg of pure compound 41 in a 52% yield. ESI-MS: Calculated: 745.06; Found: 746.23 [M+H], 243.21 [CPh3+].

[0232] Compound 41 (50 mg, 0.062 mmol), sodium ascorbate (6.14 mg, 0.031 mmol), copper sulfate (2.46 mg, 0.015 mmol), and azidoacetic acid (25 mg, 0.248 mmol) were dissolved in 2 mL of N,N-dimethylformamide and stirred at 70°C overnight under nitrogen. The reaction was complete as monitored by TLC. Purification by preparative liquid chromatography afforded 30 mg of pure compound 42 in a 58% yield. ESI-MS: Calculated: 832.09; Found: 832.81 [M+H], 243.13 [CPh3+].

[0233] Compound 42 (20 mg, 0.024 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (HATU, 150 mg, 0.036 mmol) were dissolved in 10 mL of anhydrous DMF. N,N-dimethyl-1,2-ethylenediamine hydrochloride (12.5 μL, 0.072 mmol) and N,N-diisopropylethylamine (DIPEA, 14 μL, 0.072 mmol) were added. The mixture was stirred at room temperature under nitrogen overnight. The reaction was monitored for completion by TLC spot chromatography. Purification by preparative liquid chromatography afforded 13 mg of pure compound 43 in a 62% yield. ESI-MS: Calcd: 902.23; Found: 903.37 [M+H], 243.14 [CPh3+].

[0234] Compound 43 (10 mg, 0.01 mmol) was dissolved in 5 mL of trifluoroacetic acid and shaken for 2 minutes. Deprotection was then performed, and the resulting yellow solution was titrated with triethylsilane until it became colorless. The trifluoroacetic acid was dried on a rotary evaporator and then further dried in a vacuum dryer to obtain the deprotected precursor compound 44 for subsequent labeling.

[0235] Take a freeze-dried drug box of sodium stannous glucoheptonic acid for injection and inject 370MBq of Na into the vial. 99m The TcO4 eluent was thoroughly shaken and dissolved on an oscillator and allowed to stand for five minutes to obtain 99m TcO-GH intermediate solution. Then, compound 44 with a concentration of 10 g / L was added to the above 99m The TcO-GH intermediate solution was heated at 75°C for 20 minutes to obtain 99m Tc-C-19 complex.

[0236] Example 20: Compound nat Synthesis of Re-C-13

[0237]

[0238] 0.5 mg of the precursor compound 30 obtained after deprotection was dissolved in methanol-water, and 28 mg (0.1 mmol) of sodium gluconate heptanoate and 100 μL of a 50 mg / mL stannous chloride (0.1 mmol) dilute hydrochloric acid solution were added. 15 mg of NaReO4 was added, and NaOH was added to adjust the pH to 7. The reaction solution was heated at 75°C for 1 hour, then cooled to room temperature, and NaOH was added to adjust the pH to 8. Purification by preparative liquid phase gave a purple-red solid powder. nat Re-C-13, yield 30%. 1 H NMR(500MHz,Chloroform-d)δ4.93(d,J=16.7Hz,1H),4.61–4.55(m,1H),4.26(d,J=16.7Hz,1H),4.11–4.05(m,1H),3.97(dt,J=14.6,5.6Hz,1H),3. 68(dt,J=14.6,5.9Hz,1H),3.47–3.36(m,2H),3.29–3.12(m,2H),2.92–2. 85(m,1H),2.72(q,J=6.4,6.0Hz,2H),2.31(s,6H),1.61(d,J=4.7Hz,1H). ESI-MS: calcd: 464.62; found: 464.4 and 466.3 [M+H].

[0239] Example 21: Compound nat Synthesis of Re-C-15

[0240]

[0241] 0.5 mg of the precursor compound 34 obtained after deprotection was dissolved in methanol-water, and 28 mg (0.1 mmol) of sodium gluconate heptanoate and 100 μL of a 50 mg / mL stannous chloride (0.1 mmol) dilute hydrochloric acid solution were added. 15 mg of NaReO4 was added, and NaOH was added to adjust the pH to 7. The reaction solution was heated at 75°C for 1 hour, then cooled to room temperature, and NaOH was added to adjust the pH to 8. Purification by preparative liquid phase gave a purple-red solid powder. nat Re-C-15, yield 25%. 1H NMR(500MHz,D2O)δ4.92(d,J=16.9Hz,1H),4.39(dd,J=6.6,2.9Hz,1H),4.24(d,J=16.9Hz,1H),4 .09–4.01(m,1H),3.94(ddd,J=13.6,11.6,5.5Hz,1H),3.63–3.51(m,1H),3.42(td,J=13.3,3.7H z, 1H), 3.34 (dd, J = 12.8, 3.6 Hz, 1H), 3.17–3.05 (m, 4H), 2.93 (dd, J = 13.7, 4.4 Hz, 1H), 2.84 (s, 6H), 2.35–2.15 (m, 2H), 1.70 (td, J = 13.0, 4.5 Hz, 1H). ESI-MS: calcd: 478.64; found: 478.21 and 480.23 [M+H].

[0242] Example 22: Compound 188 Synthesis of Re-C-13

[0243] Take a 1.5 mL EP tube, add 100 μL of sodium glucosyl heptanoate (100 mg / mL), 100 μL of dilute hydrochloric acid solution of stannous chloride (6 mg / mL), 50 μL of ascorbic acid (10 mg / mL) and 10-100 μg of the deprotected precursor 30, and add 37 MBq of Na 188 The ReO4 eluent was placed in a metal bath at 100°C and heated for 30 minutes. Then it was taken out and cooled to room temperature to obtain 188 Re-C-13.

[0244] Example 23: Compound 188 Synthesis of Re-C-14

[0245] Take a 1.5 mL EP tube, add 100 μL of sodium glucoheptanoate (100 mg / mL), 100 μL of dilute hydrochloric acid solution of stannous chloride (6 mg / mL), 50 μL of ascorbic acid (10 mg / mL) and 10-100 μg of the deprotected precursor 32, and add 37 MBq of Na 188 The ReO4 eluent was placed in a metal bath at 100°C and heated for 30 minutes. Then it was taken out and cooled to room temperature to obtain 188 Re-C-14.

[0246] Example 24: Compound 188 Synthesis of Re-C-15

[0247] Take a 1.5 mL EP tube, add 100 μL of sodium glucoheptanoate (100 mg / mL), 100 μL of stannous chloride (6 mg / mL) in dilute hydrochloric acid, 50 μL of ascorbic acid (10 mg / mL) and 10-100 μg of the deprotected precursor 34, and add 37 MBq of Na 188 The ReO4 eluent was placed in a metal bath at 100°C and heated for 30 minutes. Then it was taken out and cooled to room temperature to obtain 188 Re-C-15.

[0248] Example 25: Compound 188 Synthesis of Re-C-16

[0249] Take a 1.5 mL EP tube, add 100 μL of sodium glucosyl heptanoate (100 mg / mL), 100 μL of stannous chloride (6 mg / mL) in dilute hydrochloric acid, 50 μL of ascorbic acid (10 mg / mL) and 10-100 μg of the deprotected precursor 36, and add 37 MBq of Na 188 The ReO4 eluent was placed in a metal bath at 100°C and heated for 30 minutes. Then it was taken out and cooled to room temperature to obtain 188 Re-C-16.

[0250] Example 26: Probe 99m Performance determination of Tc-C-13

[0251] 1. 99m Radiochemical purity identification of Tc-C-13 complex:

[0252] Radioactive high performance liquid chromatography (HPLC) was used to identify the radiochemical purity of the labeled substance: pure water containing 0.1% trifluoroacetic acid (phase A) and acetonitrile (phase B) were used as mobile phases, and the elution gradient was shown in Table 1 below.

[0253] Table 1 HPLC elution gradient

[0254]

[0255] like Figure 1 As shown, HPLC results showed that 99m The retention time of the Tc-C-13 complex was 9.47 min, and the radiochemical purity was greater than 95%.

[0256] 2. 99m Determination of lipid-water partition coefficient of Tc-C-13 complex

[0257] Take 100 μL of radioactive probe (740 KBq / mL), add 400 μL of water or normal saline, mix with 500 μL of n-octanol, shake on a vortex for 5 minutes, and then centrifuge at 1400 rpm for 6 minutes. After taking out, take 100 μL of organic phase and aqueous phase respectively, count with a γ-counter, and calculate the lipid-water partition coefficient Log P = Log (radioactivity of organic phase / radioactivity of aqueous phase). Three groups of independent repeated experiments were set up in parallel, and the results were expressed as mean ± SD. 99m Log P of Tc-C-13 = -1.63 ± 0.12, Log D pH7.4 =-1.8±0.08, indicating 99m Tc-C-13 is a hydrophilic substance.

[0258] 3. Probe 99m Stability determination of Tc-C-13

[0259] Mark the 99m Tc-C-13 was incubated with saline or FBS at 37°C for 6 h. Figure 2 As shown, the radiochemical purity of the product was greater than 95% as determined by HPLC, indicating that the product had good in vitro stability.

[0260] Example 27: Probe 99m Tc-C-13 cellular uptake experiment

[0261] 2×10 6 B16F10 melanoma cells were cultured in six-well plates using DMEM medium. After the cells adhered to the plate and grew, 2 mmol / L L-tyrosine was added or not 24 hours before the assay, serving as the experimental and control groups (n=8). After the cells entered the logarithmic phase of growth, the old medium was discarded and 2 μCi L-tyrosine was added to each well of the six-well plate. 99m After incubation in a serum-free medium containing Tc-C-13 probe in a 37°C incubator for 1, 2, and 4 hours, the supernatant was aspirated into a radioimmunoassay tube, washed three times with 500 μL / well PBS, and the supernatant was combined. 500 μL / well NaOH (1 M) was added to a six-well plate to lyse the cells for five minutes, and the adherent cells were collected by pipetting. 500 μL / well PBS was added to wash three times, and the cell suspension was combined. The radioactivity of the supernatant and the lower cell suspension was measured using a γ-counter, and the cellular uptake percentage was calculated as 100 [cpm (precipitate)] / [cpm (precipitate) + cpm (supernatant)].

[0262] To verify whether the probe binds specifically to cells, 2 μCi 99mHalf an hour before the addition of serum-free medium to the Tc-C-13 probe, 200 times the non-radioactive technetium-labeled precursor was added to the cells and incubated in a 37°C incubator for half an hour as the blocking group (n=8). Subsequent measurement experiments were the same as above, and the percentage of cell uptake was calculated according to the above method, as shown in Figure 2. Figure 3 As shown, it was found that the uptake rate of B16F10 cells at 1 h was significantly reduced after blocking, indicating that 99m Tc-C-13 specifically binds to B16F10 cells.

[0263] Example 28: Probe 99m Biodistribution of Tc-C-13 in B16F10 and A375 tumor-bearing mice

[0264] First, B16F10 and A375 tumor-bearing mouse models with high and low expression of melanoma were established, and 100 μCi (100 μL) of probe was administered via the tail vein. 99m Tc-C-13 probe was used. Mice were killed at different time points (1h, 3h, and 6h). The tumor, blood, heart, lung, liver, spleen, pancreas, stomach, brain, intestine, kidney, skin, muscle, bone, eye and other tissues were removed. The radioactivity of each tissue was measured by γ-counter counting. The percentage injected dose per gram (%ID / g) and tumor / non-tumor ratio were calculated. The tumor / non-tumor ratio of the probe in B16F10 tumor-bearing mice was 44.8%, 14.3%, 16.7%, 18.7% and 20.9%, respectively. Figure 4 Each time point was repeated for 5 mice in each group, and the results are shown in Table 2. The biodistribution results showed that the probe 99m Tc-C-13 has a higher uptake and good retention in melanoma sites with high melanin expression, with high tumor / blood and tumor / muscle ratios, which are significantly different from tumors with low melanin expression, indicating that it can specifically target and identify tumors with high and low melanin expression.

[0265] Table 2 Probes 99m Biodistribution of Tc-C-13 in B16F10 and A375 tumor-bearing mice

[0266]

[0267]

[0268] Example 29: Probe 99m SPECT / CT imaging of Tc-C-13 in B16F10 and A375 tumor-bearing mice

[0269] First, the B16F10 and A375 tumor-bearing mouse models with high and low expression of melanoma were established, and 250 μCi (100 μL) of probe were administered via the tail vein. 99mTc-C-13 probe was used and SPECT / CT imaging was performed 1 hour, 3 hours and 6 hours after administration under isoflurane anesthesia. Figure 5 The results showed that the probe accumulated at high concentrations in tumor sites with high melanin expression, but had almost no uptake in the A375 model with low melanin expression. Furthermore, the probe could still clearly show the tumor outline after 6 hours, indicating that its target retention time was long. 99m The Tc-C-13 probe is mainly metabolized in the intestine, with little accumulation in the liver and kidneys, and has the potential for further development.

[0270] Example 30: Probe 99m SPECT / CT imaging of Tc-C-13 in B16F10 lung metastasis model mice

[0271] First, a lung metastasis model of melanoma B16F10 was established. 10×10 4 B16F10 cells were injected into C57 / BL6 mice via the tail vein, and SPECT / CT imaging was performed on the model mice on days 14 and 21. 250 μCi (100 μL) of probe was administered to each mouse via the tail vein. 99m Tc-C-13, SPECT / CT imaging was performed 1 hour and 3 hours after administration of isoflurane anesthesia. Figure 6 As shown, the three views of the SPECT imaging results of the 21-day lung metastasis model mouse showed 99m The Tc-C-13 probe has a strong uptake in the lungs of model mice. The imaging results of isolated organs show that the lung lesions have a high radioactive signal. Further HE section results also prove the presence of melanoma metastasis in the lungs. This shows that 99m The Tc-C-13 probe has the ability to identify melanoma lung metastases and is expected to be used for the early detection of melanoma metastatic lesions.

[0272] Example 31: Probe 188 Determination of properties of Re-C-13

[0273] 1. 188 Radiochemical purity identification of Re-C-13 complex:

[0274] Radioactive high performance liquid chromatography (HPLC) was used to identify the radiochemical purity of the labeled substance: pure water containing 0.1% trifluoroacetic acid (phase A) and acetonitrile (phase B) were used as mobile phases, and the elution gradient was shown in Table 3 below.

[0275] Table 3 HPLC elution gradient

[0276]

[0277] like Figure 7 As shown, HPLC results showed that 188 The retention time of the Re-C-13 complex was 9.52 min, and the radiochemical purity was greater than 95%.

[0278] 2. 188 Determination of lipid-water partition coefficient of Re-C-13 complex

[0279] Take 100 μL of radioactive probe (740 KBq / mL), add 400 μL of water or normal saline, mix with 500 μL of n-octanol, shake on a vortex for 5 minutes, then centrifuge at 1400 rpm for 6 minutes. After taking out, take 100 μL of organic phase and aqueous phase respectively, count with a γ-counter, and calculate the lipid-water partition coefficient Log P = Log (radioactivity of organic phase / radioactivity of aqueous phase). Three groups of independent repeated experiments were set up in parallel, and the results were expressed as mean ± SD. 188 Log P of Re-C-13 = -1.38 ± 0.04, Log D pH7.4 =-1.44±0.16, indicating 188 Re-C-13 is a hydrophilic substance.

[0280] 3. Probe 188 Stability determination of Re-C-13

[0281] Mark the 188 Re-C-13 was incubated with saline or FBS at 37°C for 24 h. Figure 8 As shown, the radiochemical purity of the product was greater than 95% as determined by HPLC, indicating that the product had good in vitro stability.

[0282] Example 32 Probe 188 Biodistribution of Re-C-13 in B16F10 tumor-bearing mice

[0283] First, a B16F10 tumor-bearing mouse model with high melanoma expression was established, and 10 μCi (100 μL) of the probe was administered via the tail vein. 188 Re-C-13 probe, mice were killed at 1 hour and 4 hours, and tumor, blood, heart, lung, liver, spleen, pancreas, stomach, brain, intestine, kidney, skin, muscle, bone, eye and other tissues were removed. The radioactivity of each tissue was measured by γ-counter counting, and the percentage of injected dose per gram (%ID / g) and tumor / non-tumor ratio were calculated. Each time point was repeated for 5 mice in each group. The results are shown in Table 4. The biodistribution results showed that the probe 188Re-C-13 has a high uptake in melanoma sites with high melanin expression, with high tumor / blood and tumor / muscle ratios. Over time, the probe is well retained in the tumor site and rapidly metabolized in non-tumor targeted organs, indicating that the probe has the potential to be further used in melanoma treatment.

[0284] Table 4 Probes 188 Biodistribution of Re-C-13 in B16F10 tumor-bearing mice

[0285]

[0286]

[0287] In summary, the compound series of small molecule probes of the present invention, especially 99m The Tc-CX series of small molecule probes can specifically target tumor sites with high melanin expression, with high tumor / blood and tumor / muscle contrast, high tumor uptake and tumor / organ uptake ratios, and long retention time, which helps clinicians diagnose the occurrence of melanoma and monitor metastatic lesions. 99m Compared with the reported small molecule probes of Tc-CX series 18 F and 68 The Ga-labeled melanoma probe has a half-life of 6.02 h and good in vitro stability, making it more suitable for clinical diagnosis of melanoma and metastatic lesions. 188 The Re-CX series of small molecule probes are similar to those reported 131 Compared with the I-labeled melanoma probe, the half-life is 16.9h and it is easy to prepare. It is expected to further explore its potential for melanoma treatment and has good prospects for clinical translation.

[0288] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A compound of formula I, or a pharmaceutically acceptable salt thereof, or a precursor compound, isotope compound, or a stereoisomer or tautomer thereof, in, M is a nuclide or its oxide; Rc is a substituted or unsubstituted C1-C12 alkyl group or L is selected from the group consisting of: -(CH2) n -L1-(CH2) m -、-(CH2) n -L1-(phenylene)-L2-(CH2) m -、-(CH2) n -L1-L2-(CH2) m -, C1-C12 alkylene, C1-C12 alkoxy, -(CH2CH2O)-, divalent amino acid; wherein L1 and L2 are each independently selected from the following group: -CO-, -COO-, -CONH-, -S-, 5-10 membered heterocyclic group, 5-6 membered heteroaryl, phenyl; and said L may be optionally substituted; R1 and R2 are the same or different and are independently selected from the following groups: H, C 2-4 Aldehyde, substituted or unsubstituted C 1-8 Alkyl, substituted or unsubstituted C 1-8 Alkoxy, R a (COO)-、R a (OCO)-、R a (CONH)-、R a (NHCO)-, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted 4-10 membered heterocyclyl, or substituted or unsubstituted C6-C10 aryl; Alternatively, R1 and R2 together with the nitrogen atom to which they are attached are unsubstituted or replaced by one or more R h substituted 3-8 membered heterocyclic group; wherein each of the R h Each is independently selected from the following group: hydroxyl, halogen, C2-C4 aldehyde (C1-C3 alkyl-C(O)-), C1-C12 alkyl, C1-C12 alkoxy, C3-C6 cycloalkyl, 4-8 membered heterocyclic group; the heterocyclic group refers to a cyclic group having 1-3 heteroatoms selected from N and O on the ring; R a and R b are the same or different and are each independently selected from the group consisting of: H, substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted C 6-10 Aryl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 4-8 membered heterocyclic group; R d and R f are the same or different and are each independently selected from the following group: H, ═O, C1-C12 alkyl, C1-C12 alkoxy, C1-C6 alkyl ester, C6-12 aryl ester, C1-C6 alkylamide, C6-C12 arylamide, C1-C12 alkylcarboxyl; R g With R c are the same or different, and the R g Selected from: none, H, =O, substituted or unsubstituted C1-C12 alkyl, C1-C12 alkoxy, C1-C6 alkyl ester, C6-12 aryl ester, C1-C6 alkyl amide, C6-C12 aryl amide, C1-C12 alkyl carboxyl, or n, m, p are 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; The nuclide is a radioactive nuclide or a non-radioactive nuclide; The radionuclide is selected from the group consisting of 99m Tc, 188 Re、 186 Re、 90 Y. 131 I. 153 Sm, 177 Lu, 211 At 212 Pb, 223 Ra, 225 Ac、Al[ 18 F]、 51 Mn, 52m Mn, 52g Mn, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 86 Y. 89 Zr, 111 In, 123 I. 124 I. 125 I. 166 Ho, 213 Bi, 227 Th; The non-radioactive nuclide is selected from the group consisting of: Re, Mn, Si, Pt, Pb, Nd, Gd, Yb, Er, Cu, Y, Fe, and Tm; The substitution refers to the substitution of one or more hydrogen atoms on the group by a substituent selected from the group consisting of hydroxyl, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-6 Cycloalkyl, 4-6 membered heterocyclic group; wherein the ring skeleton of the heteroaryl or heterocyclic group has 1-3 heteroatoms selected from N and O; the C 3-6 The cycloalkyl group and the 4- to 6-membered heterocyclic group may optionally further have 1-3 halogen or alkyl substituents.

2. The compound of formula I according to claim 1, or a pharmaceutically acceptable salt thereof, or a precursor compound, an isotope compound, or a stereoisomer or tautomer thereof, wherein: Said L is selected from the following group: -(CH2) n -L1-(CH2) m -、-(CH2) n -L1-(phenylene)-L2-(CH2) m -、-(CH2) n -L1-L2-(CH2) m -, C1-C10 alkylene, C1-C10 alkoxy, -(CH2CH2O)-, divalent amino acid; wherein, L1 and L2 are each independently selected from the following group: -CO-, -CONH-, 5-10 membered heterocyclyl, 5-6 membered heteroaryl, phenyl; and the L can be optionally substituted.

3. The compound of formula I according to claim 1, or a pharmaceutically acceptable salt thereof, or a precursor compound, an isotope compound, or a stereoisomer or tautomer thereof, characterized in that: In the compound, R1 and R2 are the same or different and are independently selected from the following groups: H, substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted C 1-6 Alkoxy, R a (COO)-、R a (OCO)-、R a (CONH)-、R a (NHCO)-, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 4-6 membered heterocyclyl, or substituted or unsubstituted C6-C10 aryl; Alternatively, R1 and R2 together with the nitrogen atom to which they are attached are unsubstituted or replaced by one or more R h substituted 3-8 membered heterocyclic group; wherein each of the R h Each is independently selected from the following groups: hydroxyl, halogen, C2-C4 aldehyde (C1-C3 alkyl-C(O)-), C1-C10 alkyl, C1-C10 alkoxy, C3-C6 cycloalkyl, 4-8 membered heterocyclic group; the heterocyclic group refers to a ring group having 1-3 heteroatoms selected from N and O on the ring.

4. The compound of formula I according to claim 1, or a pharmaceutically acceptable salt thereof, or a precursor compound, an isotope compound, or a stereoisomer or tautomer thereof, wherein: The compound has the structure of Formula II: in, M is a nuclide or its oxide; L is selected from the group consisting of: -(CH2) n -L1-(CH2) m -、-(CH2) n -L1-(phenylene)-L2-(CH2) m -、-(CH2) n -L1-L2-(CH2) m -, C1-C12 alkylene, C1-C12 alkoxy, -(CH2CH2O)-, divalent amino acid; wherein L1 and L2 are each independently selected from the following group: -CO-, -COO-, -CONH-, -S-, 5-10 membered heterocyclic group, 5-6 membered heteroaryl, phenyl; and said L may be optionally substituted; R1 and R2 are the same or different and are independently selected from the following groups: H, C 2-4 Aldehyde, substituted or unsubstituted C 1-8 Alkyl, substituted or unsubstituted C 1-8 Alkoxy, R a (COO)-、R a (OCO)-、R a (CONH)-、R a (NHCO)-, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted 4-10 membered heterocyclyl, or substituted or unsubstituted C6-C10 aryl; Alternatively, R1 and R2 together with the nitrogen atom to which they are attached are unsubstituted or replaced by one or more R h substituted 3-8 membered heterocyclic group; wherein each of the R h Each is independently selected from the following group: hydroxyl, halogen, C2-C4 aldehyde (C1-C3 alkyl-C(O)-), C1-C12 alkyl, C1-C12 alkoxy, C3-C6 cycloalkyl, 4-8 membered heterocyclic group; the heterocyclic group refers to a cyclic group having 1-3 heteroatoms selected from N and O on the ring; R a Selected from the group consisting of H, substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted C 6-10 Aryl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 4-8 membered heterocyclic group; n, m are 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; The nuclide is a radioactive nuclide or a non-radioactive nuclide; the radioactive nuclide is selected from the group consisting of 99m Tc, 188 Re、 186 Re、 90 Y. 131 I. 153 Sm, 177 Lu, 211 At 212 Pb, 223 Ra, 225 Ac、Al[ 18 F]、 51 Mn, 52m Mn, 52g Mn, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 86 Y. 89 Zr, 111 In, 123 I. 124 I. 125 I. 166 Ho, 213 Bi, 227 Th; The non-radioactive nuclide is selected from the group consisting of: Re, Mn, Si, Pt, Pb, Nd, Gd, Yb, Er, Cu, Y, Fe, and Tm; The substitution refers to the substitution of one or more hydrogen atoms on the group by a substituent selected from the group consisting of hydroxyl, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-6 Cycloalkyl, 4-6 membered heterocyclic group; wherein the ring skeleton of the heteroaryl or heterocyclic group has 1-3 heteroatoms selected from N and O; the C 3-6 The cycloalkyl group and the 4- to 6-membered heterocyclic group may optionally further have 1-3 halogen or alkyl substituents.

5. The compound of formula I according to claim 1, or a pharmaceutically acceptable salt thereof, or a precursor compound, an isotope compound, or a stereoisomer or tautomer thereof, characterized in that: The compound of formula I is selected from the following compounds:

6. A method for preparing a compound of formula (I) as claimed in claim 1, characterized in that: The method comprises the steps of: 1) Synthesis of ligand skeleton AADT-COOH: 2-(tritylthio)ethylamine and bromoacetyl bromide are mixed and reacted in chloroform to obtain N-[2-(trityl)thioethyl][2-(trityl)thioethylamino]acetamide after purification; N-[2-(trityl)thioethyl][2-(trityl)thioethylamino]acetamide is reacted with ethyl bromobutyrate to obtain ethyl 4-((2-oxo-2-((2-(triphenylthio)ethyl)amino)ethyl)(2-(triphenylthio)ethyl)amino)butanoate; the ethyl ester is then removed using a methanol-water system of NaOH, and the mixture is separated and purified by preparative liquid phase separation to obtain 4-((2-oxo-2-((2-(triphenylthio)ethyl)amino)ethyl)(2-(triphenylthio)ethyl)amino)butyric acid (AADT-COOH); 2) Synthesis of ligand molecules: The AADT-COOH obtained in the previous step and the commercially purchased N-containing small molecule L-NR1R2 were dissolved in anhydrous acetonitrile, and appropriate amounts of N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl) urea hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIPEA) were added. The reaction was stirred at room temperature overnight. After the reaction was completed, the target ligand molecule was purified and separated by preparative liquid chromatography; Alternatively, the compound 2 obtained in step 1) and the bromine-containing small molecule Br-L-NR1R2 are dissolved in anhydrous acetonitrile, and appropriate amounts of cesium carbonate and potassium iodide are added. The mixture is heated to react overnight, and the target ligand molecule is obtained by separation and purification through preparative liquid phase separation; Subsequently, the trityl-protected ligand was deprotected and titrated with triethylsilane until it became colorless; the isolated product was redissolved for the preparation of 99m Tc-labeled compounds; 3) 99m Preparation of Tc-CX complex: Prepared by sodium glucoheptonic acid (CH2OH(CHOH)5COOH) exchange reaction 99m Tc-C1 complex, the specific steps are as follows: First, Na 99m The TcO4 eluent was added to the freeze-dried drug box containing sodium glucoheptonic acid, stannous chloride and urea, and the solution was allowed to stand for 1 h. 99m TcO-GH intermediate solution; Then add the CX ligand to the above 99m The TcO-GH intermediate solution is reacted to obtain 99m Tc-C1 complex.

7. The use of the compound of formula I according to claim 1, or a pharmaceutically acceptable salt thereof, or a precursor compound, an isotope compound, or a stereoisomer or tautomer thereof, characterized in that: Used to prepare imaging agents.

8. The use according to claim 7, characterized in that The imaging agent is used for clinical diagnosis of tumor sites with high melanin expression.

9. The use according to claim 7, characterized in that The imaging agent is selected from the group consisting of cancer contrast agents, tumor contrast agents, and Parkinson's disease contrast agents.

10. The use of the compound of formula I according to claim 1, or a pharmaceutically acceptable salt thereof, or a precursor compound, an isotope compound, or a stereoisomer or tautomer thereof, wherein: Used to prepare radiopharmaceuticals that specifically target melanoma.

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