Functionalized peptides for directed PD-L1 expression in vivo

Abstract

The invention provides a coupling compound, a radiotracer and a radiopharmaceutical containing the coupling compound, and application of the coupling compound and the radiotracer and the radiopharmaceutical in imaging of PD-L1 expression. This allows determining whether the target patient is suitable for treatment using an immune checkpoint inhibitor therapy. Pharmaceutical compositions, kits, methods of treatment, and methods of synthesis are also provided.

Description

Technical Field

[0001] This invention relates to the field of molecular imaging, and more particularly to the field of imaging the expression of immune checkpoint inhibitor receptors using nuclear imaging methods (e.g., positron emission tomography). Background Technology

[0002] Cancer cells can regulate their interaction with the immune system and the immune system's recognition of them by overexpressing certain cell surface proteins. One such protein, PD-L1, is recognized by the receptor PD-1 expressed by immune cells, which prevents immune cells from attacking cancer cells. Through this mechanism, cancer cells can evade the attack of the immune system. By blocking either PD-1 or PD-L1 proteins with immune checkpoint inhibitors (ICIs), thereby inhibiting this cell signaling pathway, the anti-cancer activity of the immune system can be restored. Therefore, these ICIs (usually antibodies) are increasingly being successfully applied to cancer immunotherapy. [1] Despite the significant success of immune checkpoint inhibitors across a wide range of tumor types, only a small percentage of cancer patients respond to treatment in most tumor families. Therefore, there is an urgent need for predictive biomarkers to screen patients receiving these extremely expensive therapies before treatment begins. This will maximize response rates and prevent unnecessary side effects for patients who may not respond.

[0003] In this context, the most obvious biomarker is to assess the expression of core regulators of the signaling axis addressed by these inhibitors. Therefore, all pharmaceutical companies have evaluated PD-1 and PD-L1 expression as potential predictive biomarkers in ICI treatment. While the presence / absence of PD-1 expression has no predictive value, PD-L1 expression in tumors (and within inflammatory tumors) has been found to be a reliable predictor of response in many clinical scenarios and different tumor entities. [2] Currently, the determination of PD-L1 status for treatment decisions is performed histologically using tissue material (usually biopsies, and occasionally resected specimens). However, biopsies (typically no larger than 1 mm) are often clearly not representative of metastatic tumors with multiple lesions several centimeters in diameter. Furthermore, PD-L1 expression in tumors can be highly heterogeneous, so the actual location of the biopsy specimen can have a strong and unpredictable impact on the overall analysis results. These factors can lead to a significant proportion of erroneous treatment decisions when treatment plans are based on tumor PD-L1 expression measured in tissue specimens. Finally, obtaining a biopsy sample (or resected specimen) is an invasive procedure. However, since no other technical methods are currently available, assessing PD-L1 status in biopsy specimens before treatment is currently the standard of care for many important tumor entities, such as lung cancer, head and neck cancer, and kidney / bladder cancer.

[0004] Clearly, a non-invasive, three-dimensional whole-body mapping method for PD-L1 expression, which could address the aforementioned shortcomings of current best practices, would be highly desirable.

[0005] Existing technology Non-invasive nuclear imaging targeting PD-L1 expression is of great importance for clinical decision-making and patient management, and has been the subject of extensive research. Most of these methods are based on radiolabeled antibodies or proteins. However, they typically exhibit slow pharmacokinetics and significant background, meaning they can accumulate undesirably in non-target tissues. Generally, small molecules or peptides are preferred as radiotracers for imaging because they are typically characterized by good tissue penetration, enabling rapid target uptake and excretion, which is advantageous for diagnostic imaging protocols in clinical settings.

[0006] A recent report describes a PD-L1-binding peptide called WL12. This peptide is functionalized at its N-terminus with the chelator DOTAGA (DOTAGA-WL12) and labeled with the radionuclides copper-64 and gallium-68 for PET imaging. [3,4] However, in preclinical imaging experiments using xenograft mice transplanted with human tumor cell lines expressing PD-L1, the corresponding radiotracers exhibited unsatisfactory properties. Specifically, they showed unnecessarily high uptake in some organs (e.g., liver, lung, and / or intestine) and slow and / or incomplete clearance from the blood pool, making them unsuitable for clinical translation. Another study investigated the PD-L1 binding ability of conjugates containing WL12 derivatives and modified with hydrophilicity. [5] Summary of the Invention

[0007] In view of the above-mentioned state of the technology, the present invention aims to provide a coupling compound and a radiotracer and / or radiopharmaceutical containing the coupling compound, wherein the coupling compound and the tracer and / or radiopharmaceutical exhibit better clinical translational applicability, especially with higher enrichment in tumor tissues, which in particular means avoiding high uptake in some organs (e.g., liver, lungs and / or intestines) and / or clearing from the blood pool more quickly or more effectively.

[0008] The aforementioned objectives are achieved by the conjugate compound of appended claim 1, the radiotracer of appended claim 3, and the radiopharmaceutical of appended claim 6. Preferred embodiments of the invention are specified in appended claims 2, 4, 5, 7, and 8. The invention also provides a pharmaceutical composition as specified in appended claim 9, comprising the radiotracer or radiopharmaceutical of the invention and one or more excipients. On the other hand, a kit according to appended claim 10 is provided, the kit comprising the conjugate compound of the invention. In other aspects, the invention provides the radiotracer or pharmaceutical composition of the invention for imaging cells carrying PD-L1 (as specified in appended claim 11), for determining the susceptibility of cancer patients to immune checkpoint inhibitor therapy (as specified in appended claim 12), or for use in methods of treating cancer patients (as specified in appended claim 13). The invention also provides the radiopharmaceutical as specified in appended claim 14, for use in methods of treating cancer patients. The present invention also provides a method for treating cancer patients with immune checkpoint inhibitors, the method comprising the steps of: determining susceptibility to immune checkpoint inhibitor treatment using a radiotracer of the present invention, and then administering immune checkpoint inhibitor therapy or a radiopharmaceutical of the present invention to the cancer patient. The present invention provides an intermediate suitable for synthesizing the coupling compound described herein, and a corresponding method for its preparation. These aspects of the invention are defined in appended claims 15, 16, and 17. A multimeric compound or a precursor thereof as defined in appended claim 18 is also provided.

[0009] In specific embodiments, the present invention also provides coupling compounds, radiotracers, radiopharmaceuticals, pharmaceutical compositions, kits, uses, treatments, intermediates, and preparation methods closely related to specific embodiments of the present invention, as described below. Detailed Implementation Attached Figure Description

[0011] Figure 1(A) and 1(B)The in vitro biodistribution of Ga-68-TRAP-WL12 in MDA-MB231 tumor-bearing SCID mice is shown at 60 min (n = 6) and 120 min (n = 4) post-injection (data are presented as mean ± standard deviation).

[0012] Figure 2 The tumor-to-organ ratio of Ga-68-TRAP-WL12 in MDA-MB231 tumor-bearing SCID mice is shown at 60 min and 120 min post-injection (data are expressed as mean ± standard deviation), which was calculated from in vitro biodistribution data.

[0013] Figure 3 The kinetics of tissue activity distribution of Ga-68-TRAP-WL12 in MDA-MB231 tumor-bearing SCID mice are shown, derived from the target region integral of dynamic PET scans (n ​​= 4, data are expressed as mean ± standard deviation).

[0014] Figure 4(A) and 4(B) Representative examples of positron emission tomography (PET) scans (maximum intensity projection) of MDA-MB231 tumor-bearing SCID mice at 60 min and 120 min after injection of Ga-68-TRAP-WL12 are shown.

[0015] Figure 5(A) and 5(B) This study presents in vitro biodistribution data for Ga-68-TRAP-WL12 and Ga-68-DOTAGA-WL12 in MDA-MB231 tumor-bearing SCID mice at 60 min post-injection, along with a comparison of calculated tumor-to-organ ratios. Data for Ga-68-DOTAGA-WL12 were obtained from the literature (De Silva et al., Molecular Pharmaceutics 2018, 15, 3946). Collected from 3952).

[0016] the term In this document, the term "radioactive tracer" refers to a chemical compound comprising a radionuclide that emits gamma radiation or positrons and can be used as an imaging agent in single-photon emission computed tomography (SPECT) or positron emission tomography (PET). While the radionuclide may also emit other types of radiation, in the context of this document, the corresponding radioisotope-labeled compound is still referred to as a radioactive tracer because the emission of gamma photons or positrons is relevant to the application.

[0017] This article uses the term "radiopharmaceutical" to better distinguish it from "radiotracer," referring to chemical compounds containing a radionuclide that emits alpha or beta radiation (typically producing ≥10 alpha or beta particles per 100 decays). These compounds are commonly used in radionuclide therapy. While the radionuclide may also emit other types of radiation, in the context of this article, the labeled compound of the corresponding radioisotope is still referred to as a radiopharmaceutical because the emission of alpha or beta radiation is relevant to the application. Although a radionuclide may emit alpha or beta radiation, as well as additional gamma radiation or positrons, and thus theoretically could serve as both a radiotracer and a radiopharmaceutical, this dual use is not very meaningful in practice: emitting alpha or beta radiation at a therapeutically effective dose typically precludes any imaging applications for such radionuclides. Therefore, these radionuclides are primarily used as radiopharmaceuticals. On the other hand, if the alpha or beta radiation dose is low enough that imaging is practically feasible, this would preclude therapeutic applications. Therefore, if a radionuclide emits alpha or beta radiation, and additional gamma radiation or positrons, then it should be considered a radiopharmaceutical if the emitted dose of alpha or beta radiation is high enough for therapeutic purposes. Otherwise, it should be considered a radioactive tracer.

[0018] As used herein, the term "radionium" refers to a radioactive isotope of an element that emits at least one of positrons, gamma radiation, alpha radiation, or beta radiation. The half-life of the radionuclide used in this invention is typically in the range of 30 min to 12 days, more preferably 45 min to 10 days.

[0019] As used herein, the term "patient" refers to a human or animal subject. In a preferred embodiment of the invention, the patient is a human. A human can be an adult or a child. A patient is characterized by a condition in which PD-L1 expression is upregulated or at least suspected to be upregulated, typically cancer.

[0020] The terms "treatment" and "administration of treatment," etc., are intended to define treatment of a patient, typically a patient requiring treatment (e.g., a cancer patient), which includes administering to the patient the radiopharmaceutical described in this invention, or a pharmaceutical composition or kit containing the radiopharmaceutical described in this invention. There are no particular limitations on the method of administration, dosage form, dose, and administration interval; any suitable method may be determined by a technician based on literature and / or conventional experimental methods, selectively taking into account the condition, weight, age, sex, other medications, etc., of one or more specific patients.

[0021] In the context of this specification, all disclosures of coupling compounds, radiotracers, radiopharmaceuticals, and intermediate compounds described herein should also be understood as disclosures of the corresponding compounds in the form of pharmaceutically acceptable salts, solvates, and / or polymorphs. The invention is not limited in this respect, provided that the compound is suitable for the pharmaceutical field, i.e., pharmaceutically acceptable (the requirement of pharmaceutical acceptability applies only indirectly to intermediate compounds, as they must be suitable for the preparation of pharmaceutically acceptable coupling compounds, radiotracers, and / or radiopharmaceuticals).

[0022] Unless otherwise specified, all terms shall be interpreted in accordance with their usual meaning as reflected in standard textbooks, encyclopedias, etc. For example, the term "peptide" is used to characterize compounds in which at least two amino acids are linked by peptide bonds (amide bonds).

[0023] In this specification, the singular forms “a,” “an,” and “the” mean that the corresponding plural forms are also included, unless the context otherwise specifies. Thus, for example, submitting “compound” or “the said compound” includes two or more compounds. The word “comprise” should be interpreted as inclusive rather than exclusive. Similarly, the terms “contain,” “containing,” “include,” and “or” should be interpreted as inclusive, allowing for additional unmentioned items, unless the context otherwise specifies. However, the use of these terms should also be understood to indicate that there may be no other items disclosed. In other words, a disclosure using the term “comprising” should, in a sense, be understood as a disclosure “substantially consisting of” or “consisting of” the listed items. Likewise, the methods disclosed herein may include one or more additional steps not specifically disclosed herein, but in one respect, these additional steps are not present according to the terms “substantially consisting of” or “consisting of”. The term “and / or” used in the context of “X and / or Y” should be interpreted as “X,” “Y,” or “X and Y.” The terms “example” and “for example” used in this document, especially when listed thereafter, are exemplary and illustrative only and should not be considered exclusive or comprehensive.

[0024] Compounds of the present invention This invention relates to conjugates of PD-L1 binding peptides (such as WL12) and their derivatives with radionuclide chelators and other functional moieties to form compounds such as TRAP (TRAP-WL12) or DOTPI (DOTPI-WL12) for labeling with positron emitter gallium-68 or other potentially useful radionuclides, or for achieving other useful diagnostic or therapeutic effects.

[0025] In the broadest sense, this invention relates to coupling compounds characterized by the following formulas Y0 and X0:

[0026] Formula Y0

[0027] Formula X0 Where, in equation Y0 and equation X0: FM The representative is a functional part selected from the imaging active part and the therapeutic active part.

[0028] h It can take the value 0 or 1; i It can take the value 0 or 1; k It can take the value 1, 2, or 3; g It can take the value 1, 2, or 3; A Representatives selected –(CH2) n - , –(CH2CH2O) m -(CH2) p - or –(OCH2CH2) m - The groups, in which This indicates binding to the cyclic peptide moiety. Indicates combination to B , where n is selected from the range of 1 to 6; m is selected from the range of 1 to 6, preferably 1 to 4 or preferably 3 to 5; p is 1 or 2; B Represents the selected group, such that part of ABC for or ; C Representatives selected α –(CH2) n’ - β , α –(CH2CH2O) m’ - β or α -(CH2) p’ –(OCH2CH2) m’ - β The group, where α represents the group bound to... B β represents binding to FM binding, where n' is selected from the range of 1 to 6; m' is selected from the range of 1 to 6, preferably 1 to 4 or preferably 3 to 5; p' is 1 or 2; D The group represents a group selected from –H, –CH3, –CH2–COOH, –CH2–SO3H, –CH2–P(H)(O)(OH) or –CH2–P(O)(OH)2; E The representatives are selected from –CH(CH3)2, –COOH, –CH2–COOH, –CH2–CH2–COOH, –SO3H, –CH2–SO3H, –CH2–P(H)(O)(OH), or –CH2–P(O)(OH)2; X 1 X 2 X 3 X 4 and X 5 Either both are –C(H)=, or X 1 X 2 X 3 X 4 and X 5 One of them is an aromatic nitrogen atom (–N=), and the rest are all –C(H)=; X 6 X 7 X 8 X 9 and X 10 Either both are –C(H)=, or X 6 X 7 X 8 X 9 and X 10 One of them is an aromatic nitrogen atom (–N=), and the rest are all –C(H)=; X 11 X 12 X 13 and X 14They were chosen together to adopt one of the configurations listed in the table below, where –C(H) = represents an aromatic ring carbon atom with hydrogen, –C = represents an aromatic ring carbon atom without hydrogen, and –N = represents an aromatic ring nitrogen atom:

[0029] X 15 It is –H or –OH, where X 15 When it is –OH, it reacts with X 15 The stereochemistry of adjacent C atoms is S or R; X 16 It is –H or –OH, where X 16 When it is –OH, it reacts with X 16 The stereochemistry of adjacent C atoms is S or R; X 17 (If present) can be –H, –OH, –(CH2)-OH, –SH, –(CH2)-SH, or –(CH2)-S-CH3; X 18 It can be –(CH2)–, –O–, –S–, –N(H)–, –N(CH3)–, –S(O)–, –S(O)2–, or X 18 This represents a direct covalent bond between adjacent carbon atoms.

[0030] For clarity, B and C (Bold and italic text) do not represent boron atoms and carbon atoms respectively within the scope of this article, but only have the meanings defined above.

[0031] The remaining variable groups are defined relative to Equation 1 as follows.

[0032] In the first implementation scheme, the functional part FM Derived from the chelating group TRAP, a possible coupling compound of the present invention is characterized by the following formula Y1:

[0033] Formula Y1, in A , B , C , D , E , g , h , i , k X 1 X 2 X 3 X 4 X 5 X 6 X 7X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 and X 18 The meaning is the same as the definition of Y0 in the above formula.

[0034] In the above formula Y1, n and n' can be chosen independently of each other. However, it is preferable to choose n and n' such that the range of n+n' is 3 to 7, more preferably 4 to 6.

[0035] Similarly, m and m' can be chosen independently of each other. However, it is preferable to choose m and m' such that the range of m+m' is 2 to 12, more preferably 4 to 8.

[0036] It can also be in B One side has an alkylene group, in B The other side has polyoxyethylene groups. In this case, m or m' and n or n' are selected such that m+n' or m'+n is preferably in the range of 3 to 9, more preferably in the range of 4 to 7.

[0037] In a preferred aspect of the first embodiment, the coupling compound (TRAP-WL12) of the present invention is characterized by the following formula Y1a: .

[0038] Formula Y1a.

[0039] In another preferred aspect of the first embodiment, the coupling compound is characterized by the following formula Y1b: .

[0040] Formula Y1b.

[0041] In another preferred aspect of the first embodiment, the coupling compound is characterized by the following formula Y1c: .

[0042] Formula Y1c.

[0043] In another preferred aspect of the first embodiment, the coupling compound is characterized by the following formula Y1d: .

[0044] Formula Y1d.

[0045] Nuclide that emits positrons 68 Ga、64 Cu、 66 Ga, or 18 F (preferably with high-charge cations such as Al) 3+ It exists in the form of metal complexes, as described by WJ McBride et al. (EJNMMI Res.2013; 3: 36; doi:10.1186 / 2191-219X-3-36), hereinafter referred to as Al. 18 F); and gamma emitters 67 Ga or 99 mTc, or β emitter 67 Cu can be chelated to the TRAP moiety of the coupling compound of the present invention to obtain the radiotracer or radiopharmaceutical described in the first embodiment of the present invention. For this purpose, the radionuclides are preferably in their most stable ionic form (e.g., Ga). 3+ and Cu 2+ The conjugate compound is used in aqueous solution with a suitable counterion (preferably chloride ion). Of course, it is preferable to use the preferred conjugate compound of the first embodiment as described above to form the radiotracer or radiopharmaceutical of the first embodiment. If a conjugate compound having the structure of formula Y1a is combined with... 68 When used with Ga, the radioactive tracer in this case is sometimes referred to as Ga-68-TRAP-WL12.

[0046] Suitable nonmetallic radionuclides that emit positrons or gamma radiation include, for example: 123 I, 124 I or 125 I. Suitable nonmetallic radionuclides that emit α or β radiation, for example: 131 I or 211 At.

[0047] All conjugates of the present invention can also be directly labeled with suitable radionuclides by mixing the radionuclide solution and the conjugate in separate vials, preferably containing other necessary excipients, such as buffers, radiolysis protection compounds, or stabilizers. Such vials (before the addition of the radionuclide) are sometimes referred to as kits or single-vial kits. The compounds of the present invention, along with the necessary buffers, radiolysis protection compounds, and stabilizers, can also be provided in separate vials, and the contents of these vials are then transferred to a single vial for the labeling reaction. Such ready-to-use vial combinations are sometimes referred to as multi-vial kits. The present invention also relates to such kits containing the conjugate compounds of the present invention, including single-vial kits and multi-vial kits.

[0048] For all kits, the radionuclide complex formation reaction is preferably carried out between 20 and 120°C. 68 The preferred time for Ga complexation is 1 to 15 minutes, or 1 to 60 minutes for other radionuclides. These preferred temperatures and times apply even if the complexation is performed independently of a kit.

[0049] Such kits can be used manually or automatically for processing before, during, and / or after the formation of complexes with radionuclides. Furthermore, all components for preparing radiolabeled compounds using the coupling compounds of the present invention can be contained in a combination of vials, tubing, manifolds, and separation cassettes, sometimes referred to as kits, preferably for use with a robotic system for radiolabeling (commonly referred to as a synthesis module). Such kits are preferably provided as single-use, sterile, ready-to-use packaging.

[0050] The resulting chelate can be used without further purification, or it can be further purified by conventional purification methods (such as RP-HPLC or solid phase extraction) before further use. 68 Ga can be obtained from 68 Ge / 68 Ga is provided by elution from a Ga generator, preferably with a nominal activity of 1-4 GBq, for example, by Eckert & Ziegler (Berlin, Germany), ITM (Garching, Germany), Monrol (Turkey), Rosatom / Isotope (Russia), PARS Isotopes (Iran), iTHEMBA (South Africa), or IRE Elit (Belgium). Prior to complexation, a suitable buffer can be used to... 68 The pH of the Ga aqueous solution is adjusted to a range of 1 to 7, preferably 2 to 3. 68 Ga can also be synthesized using a cyclotron, with either a solid or liquid target. The resulting chelates can have very high activity, for example, up to 300 GBq when using a solid target.

[0051] In the second embodiment, the coupling compound of the present invention contains a DOTPI chelating moiety instead of a TRAP moiety. DOTPI is a larger homologue of TRAP and has an additional binding unit. Therefore, it is more likely to bind larger-sized alternative radionuclides. The coupling compound of the second embodiment is characterized by the following formula Y2:

[0052] Formula Y2 in A , B , C , D , E ,g , h , i , k X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 and X 18 The meaning is the same as the definition of Y0 in the above formula.

[0053] In the above formula Y2, n and n' can be chosen independently of each other. However, it is preferable to choose n and n' such that n+n' is in the range of 3 to 7, more preferably 4 to 6.

[0054] Similarly, m and m' can be chosen independently of each other. However, it is preferable to choose m and m' such that m+m' is in the range of 2 to 12, more preferably 4 to 8.

[0055] It is also possible to have an alkylene group on one side of B, in B The other side has polyoxyethylene groups. In this case, m or m' and n or n' are selected such that m+n' or m'+n is preferably in the range of 3 to 9, more preferably in the range of 4 to 7.

[0056] In a preferred aspect of the second embodiment, the coupling compound is characterized by the following formulas: Y2a, Y2b, Y2c, or Y2d:

[0057] Equation Y2a,

[0058] Formula Y2b,

[0059] Formula Y2c,

[0060] Formula Y2d.

[0061] The coupling compound of the second embodiment can be used with Al... 18 F, 43 Sc、 44Sc、 99m Tc, 111 In、 155 A radionuclide of Tb, or other suitable radionuclide emitting positrons or gamma radiation, forms a chelate to form the radioactive tracer described in the second embodiment of the invention. Suitable nonmetallic radionuclides emitting positrons or gamma radiation include, for example,... 123 I, 124 I or 125 I. Furthermore, the coupling compound of the second embodiment can be used with a compound selected from... 47 Sc、 90 Y、 149 Tb, 161 Tb, 177 Lu、 212 Pb, 212 Bi、 213 Bi、 223 Ra、 225 Ac、 227 The radioactive nuclide of Th, a metal cation, or other suitable metal ions or non-metallic radionuclides emitting α or β radiation, forms a chelate to form the radiopharmaceutical of the second embodiment of the present invention. Suitable non-metallic radionuclides emitting α or β radiation are, for example,... 131 I or 211 At. The radiotracers and radiopharmaceuticals of the second embodiment of the present invention can be prepared and further used as described in the first embodiment above, except that the coupling compounds of formula Y1, Y1a, or Y1b are replaced by coupling compounds of formula Y2, Y2a, or Y2b. Unless otherwise specified or required by context, all information provided herein with respect to the first embodiment (e.g., information regarding kits or purification) applies in a similar manner to the second embodiment, and vice versa.

[0062] In a third embodiment, the coupling compound of the present invention is characterized by the following formulas Y3 and Y4, i.e., it is a coupling compound according to formula Y0, wherein... i = 0、 h = 1, FM It is TRAP or DOTPI, and the meanings of all other variables are the same as those outlined in the definition of equation Y0:

[0063] Formula Y3,

[0064] Formula Y4.

[0065] In a preferred aspect of the third embodiment, the coupling compound is characterized by one of the following formulas: Y3-1 and Y4-1.

[0066] Formula Y3-1,

[0067] Formula Y4-1.

[0068] in D and E It has the meaning defined above for formula Y0; X 18 Selected from –O–, –S–, or covalent bonds; g It can take the value 1 or 2; k It can take the values ​​1, 2, or 3; and C Representative – (CH2) n’ - where n' is selected from the range of 1 to 6, preferably 1 to 3, more preferably 1 to 2, and most preferably 1.

[0069] More specifically, regarding equations Y3-1 and Y4-1, D Represents a group selected from –CH3 and –CH2–COOH; E Represents a group selected from –CH(CH3)2 and –CH2–COOH; X 18 Selected from –O–, –S–, or covalent bonds; g It can take the value 1 or 2; k It can take the values ​​1, 2, or 3; and C Representative – (CH2) n’ - where n' is selected from the range of 1 to 3, preferably 1 to 2, and more preferably 1.

[0070] More specifically, regarding equations Y3-1 and Y4-1, D E represents the –CH3 group, and E represents the –CH2–COOH group, or D Represents the –CH2–COOH group, and E Represents the –CH(CH3)2 group; X 18 Selected from –O–, –S–, or covalent bonds; g It can take the value 1 or 2; k It can take the values ​​1, 2, or 3; and C Representative – (CH2) n’ - where n' is selected from the range of 1 to 3, preferably 1 to 2, and more preferably 1.

[0071] In a preferred aspect of the third embodiment, the coupling compound is characterized by the following formulas: Y3a, Y3b, Y3c, Y3d, Y3e, Y3f, Y3g, Y3h, Y4a, Y4b, Y4c, Y4d, Y4e, Y4f, Y4g, or Y4h.

[0072] Formula Y3a,

[0073] Formula Y3b,

[0074] Formula Y3c,

[0075] Formula Y3d,

[0076] Formula Y3e,

[0077] Formula Y3f,

[0078] Formula Y3g,

[0079] Formula Y3h,

[0080] Formula Y4a,

[0081] Formula Y4b,

[0082] Formula Y4c,

[0083] Formula Y4d,

[0084] Formula Y4e,

[0085] Formula Y4f,

[0086] Formula Y4g,

[0087] Formula Y4h.

[0088] In the fourth embodiment, the coupling compound is represented by the above formulas Y0 and X0, and the functional part... FMThis refers to a functional portion selected from the imaging-active and therapeutic-active portions, but excluding the portions specified in the first to third embodiments described above. Examples include portions containing one or more of the following: chromophores, especially fluorescent or luminescent groups; radiolabeled precursor groups; chelating agents; magnetic resonance imaging agents; enzyme inhibitors; chemotherapeutic agents, especially cell inhibitors, topoisomerase inhibitors, alkylating agents, antimetabolites, antimicrotubule agents, cytotoxic antibiotics, taxanes, intercalating agents, platinum compounds, mitotic inhibitors, tyrosine kinase inhibitors; peptides, especially peptide receptor ligands; functional building blocks for surface grafting, especially in medical devices, nanoparticles, micelles, magnetic particles, or quantum dots; functional proteins, such as antibodies, antibody fragments, nanobodies, or affinity molecules; pharmacokinetic modifiers, especially albumin binders, sugars, oligosaccharides, and polysaccharides; and metal-containing or metal-free compounds suitable for photodynamic therapy, especially compounds that generate reactive oxygen species upon irradiation with light (visible or near-infrared wavelengths).

[0089] In the fifth embodiment of the above formula Y0 and / or X0, the cyclic peptide portion, the linker, and the functional portion FM As mentioned above, but on the premise of functional parts FM Having included as described above FM The structure of a portion (e.g., TRAP or DOTPI or other imaging-active or therapeutic-active portion); but additionally having one or more portions capable of chemically reacting to form one or more covalent bonds with other chemical portions. In this case, compounds according to formulas Y0 and X0 can serve as “building blocks,” for example, for preparing polymers as described below.

[0090] In this implementation scheme, it is preferred that... FM Representing the portions that can undergo coupling reactions as described above, coupling reactions are widely referred to as "click chemistry" (e.g., as described in Bauer D et al., Nat Protocols 2023;18:1659; and Bauer D et al., Bioconjugate Chem. 2023;34:1925), particularly terminal alkynes; terminal azides; strained alkynes such as dibenzo-azacyclooctyne (DBCO or DIBAC, sometimes also called aza-dibenzo-azacyclooctyne (ADIBO)), 4-dibenzo-azacyclooctyne alcohol (DIBO), bicyclic [6.1.0]nonyne (BCN), difluorobenzo-azacyclooctyne (DIFO), and 4,8-diazacyclononyne (DACN); tetrazine; trans-cyclooctene; and pinacolyl borane.

[0091] The chemically reactive portion can be attached to any location of the remaining FM portion. Preferably, the remaining FM portion comprises TRAP or DOTPI, and one or two (or three in the case of DOTPI) chemically reactive portions are bonded to the remaining FM portion by the corresponding number of free carboxyl groups of TRAP or DOTPI.

[0092] In the sixth embodiment of the above formula Y0 and / or X0, the cyclic peptide portion, the linker, and the functional portion FM As mentioned above, but on the premise of functional parts FM Having included as described above FM The structure of a portion (e.g., TRAP or DOTPI or other imaging-active or therapeutically active portion), but additionally having one or more portions, each of which contains A , B and / or C Type A, B, and / or C structural elements and cyclic peptides, all of which conform to the above definition. The one or more additional portions (each containing a cyclic peptide and type A, B, and / or C structural elements) may be the same as or different from each other, as long as they all conform to the above definition. In a specific embodiment, the functional portion... FM It can carry one or more of the formula -NH-(- C - B ) h -(- A -C(=O)-NH) i -(CH2) k -X 18 -(CH2) g - The other parts of the cyclic peptide, as specified above for formula Y0. If FM If the compound contains a TRAP moiety, one or two other such moieties can be attached to the central chelate moiety by forming amide bonds with a corresponding number of free carboxyl groups; if FM If a structure contains a DOTPI moiety, one, two, or three other moieties can be attached by forming amide bonds with a corresponding number of free carboxyl groups. Therefore, such structures (referred to as polymers) contain more than one of the aforementioned cyclic peptides. The preferred number of cyclic peptide moieties in such polymers is 2, 3, and 4.

[0093] The seventh embodiment relates to the synthesis of the conjugates of the present invention and the synthesis of intermediates that can be used for the synthesis. For the synthesis of the conjugates of the present invention, the peptide is provided in a modified form suitable for chelating the TRAP or DOTPI moiety via click chemical attachment. This modified form is an intermediate suitable for the manufacturing method of the present invention. These intermediates of the present invention are specified in the following formulas Y5, Y6, Y7, Y8, X5, X6, X7, and X8:

[0094] Formula Y5,

[0095] Formula Y6,

[0096] Formula Y7,

[0097] Formula Y8,

[0098] Formula X5,

[0099] Formula X6,

[0100] Formula X7,

[0101] Formula X8, If any of the following exist in equations Y5, Y6, Y7, Y8, X5, X6, X7, and X8... A , D , E , g , k X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 and X 18The meaning is the same as that specified in the definitions of Y0 and X0 above.

[0102] Preferred intermediates are characterized by the following formulas: Y5a, Y5b, Y5c, Y5d, Y6a, Y6b, Y6c, Y6d, Y7a, Y7b, Y7c, Y7d, Y8a, Y8b, Y8c, and Y8d.

[0103] Formula Y5a,

[0104] Formula Y5b,

[0105] Formula Y5c,

[0106] Formula Y5d,

[0107] Formula Y6a,

[0108] Formula Y6b,

[0109] Formula Y6c,

[0110] Formula Y6d,

[0111] Formula Y7a,

[0112] Formula Y7b,

[0113] Formula Y7c,

[0114] Formula Y7d,

[0115] Formula Y8a,

[0116] Formula Y8b,

[0117] Formula Y8c,

[0118] Formula Y8d.

[0119] According to the present invention, intermediates of formulas Y5, Y5a, Y5b, Y5c, Y5d, Y6, Y6a, Y6b, Y6c, or Y6d can be obtained by reacting peptide WL12 or suitable derivatives thereof with suitable reagents containing azido groups and reagents containing alkynyl groups, respectively. For example, the above intermediates Y5a and Y6a can be prepared using 5-azidopentanoic acid or pent-4-alkynic acid, respectively. Suitable reaction conditions are shown in Example 1 below. WL12 is shown as formula 9 below. It is commercially available. Suitable derivatives of WL12 having E (not isopropyl) and D (not methyl) groups can be obtained by using a standard peptide synthesis method (Fmoc strategy) with appropriately modified amino acid monomers instead of leucine and N-methyltryptophan.

[0120]

[0121] Formula 9.

[0122] The intermediates of formulas Y5, X5, Y5a, Y5b, Y5c, Y5d, Y7, X7, Y7a, Y7b, Y7c, and Y7d can react with TRAP or DOTPI intermediates containing an alkyne group (hereinafter referred to as TRAP-alkynes and DOTPI-alkynes). TRAP-alkynes are derived from the structural formula TRAP-NH- C -C Characterized by CH, DOTPI-alkynes are derived from the structural formula DOTPI-NH- C -C CH characterization. TRAP and DOTPI are molecules described in Figure 1, for example, by A. Wurzer et al. (Front.Chem.2018 Apr 10;6:107. doi: 10.3389 / fchem.2018.00107). C - Alkynes and C - The bonding of the azide moieties occurs via amide bonds, which are bonded to... C The terminal nitrogen atom forms with one of the carboxyl groups of TRAP and DOTPI. C As defined above with respect to formula Y0. Preferred variants of TRAP-alkyne and DOTPI-alkyne intermediates are listed below: and .

[0123] Intermediates according to formulas Y7, X7, Y7a, Y7b, Y7c, and Y7d are of particular interest because they can be synthesized from commercially available amino acid building blocks using standard peptide synthesis methods (Fmoc strategy). Specifically, intermediates Y7a, Y7b, Y7c, and Y7d can be obtained using the commercially available non-natural amino acid azidolysine (CAS No. 159610-92-1) or suitable derivatives suitable for peptide synthesis reactions (such as Fmoc-azidolysine (CAS 159610-89-6)). Intermediates according to formulas Y7, Y7a, Y7b, Y7c, and Y7d can be directly functionalized by click chemistry (CuAAC) without attaching separate functional linkers, for example, as described above, synthesizing intermediates according to formulas Y5a and Y5b from WL12 or suitable derivatives thereof.

[0124] The intermediates of formulas Y6, X6, Y6a, Y6b, Y6c, Y6d, Y8, X8, Y8a, Y8b, Y8c, and Y8d react with a TRAP or DOTPI intermediate containing an azide group (hereinafter referred to as TRAP-azides and DOTPI-azides). TRAP-azides are derived from the structural formula TRAP-NH- C Characterized by -N3, DOTPI-azides are derived from the structural formula DOTPI-NH- C -N3 characterization, where C - The bonding of the azide moiety occurs via an amide bond, which is bonded to... C The terminal nitrogen atom forms with one of the carboxyl groups of TRAP and DOTPI, respectively. C As defined above with respect to formula Y0. Preferred variants of the TRAP-azide and DOTPI-azide intermediates are listed below: ,and .

[0125] Intermediates according to formulas Y8, X8, Y8a, Y8b, Y8c, and Y8d are of particular interest because they can be synthesized from commercially available amino acid building blocks using standard peptide synthesis methods (Fmoc strategy). Specifically, intermediates Y8a, Y8b, Y8c, and Y8d can be obtained using the commercially available amino acid building block O-propynylserine (CAS No. 1379150-93-2) or suitable derivatives suitable for peptide synthesis reactions (such as Fmoc-O-propynylserine (CAS 1354752-75-2)). Intermediates according to formulas Y8, Y8a, Y8b, Y8c, and Y8d can be directly functionalized by click chemistry (CuAAC) without attaching separate functional linkers, for example, as described above, synthesizing intermediates according to formulas Y5a and Y5b from WL12 or suitable derivatives thereof.

[0126] The CuAAC reaction of intermediates Y5, X5, Y5a, Y5b, Y5c, Y5d, Y6, X6, Y6a, Y6b, Y6c, Y6d, Y7, X7, Y7a, Y7b, Y7c, Y7d, Y8, X8, Y8a, Y8b, Y8c, and Y8d with building blocks containing alkynes or azide groups (where applicable; the azide group on one molecule must always match the alkyne group on another molecule, and vice versa) is obviously not limited to TRAP-alkynes, DOTPI-alkynes, TRAP-azides, and DOTPI-azides, but can react with any molecule or functional building block that contains or is equipped with alkynes or azide groups. The following are categories of compounds of particular interest: chromophores, especially fluorescent or luminescent molecules; radiolabeled precursor groups; chelating agents; magnetic resonance imaging agents; enzyme inhibitors; chemotherapeutic agents, especially cell inhibitors, topoisomerase inhibitors, alkylating agents, antimetabolites, antimicrotubule agents, cytotoxic antibiotics, taxanes, intercalating agents, platinum compounds, mitotic inhibitors, tyrosine kinase inhibitors; peptides, especially peptide receptor ligands; functional building blocks for surface grafting, especially in medical devices, nanoparticles, micelles, magnetic particles, or quantum dots; functional proteins, such as antibodies, antibody fragments, nanobodies, or affinities; pharmacokinetic modifiers, especially albumin binders, sugars, oligosaccharides, and polysaccharides; metal-containing or metal-free compounds suitable for photodynamic therapy, especially compounds that generate reactive oxygen species when irradiated with light (in the visible or near-infrared wavelength range), or any combination thereof. The alkyne- or azide-containing building blocks are used for CuAAC coupling to intermediates Y5, X5, Y5a, Y5b, Y5c, Y5d, Y6, X6, Y6a, Y6b, Y6c, Y6d, Y7, X7, Y7a, Y7b, Y7c, Y7d, Y8, X8, Y8a, Y8b, Y8c, and Y8d, where the alkyne moiety has tissue-specific parameters related to the molecule's biological activity or other functional parts, such as the presence of enzymes, pH, and oxygen partial pressure.

[0127] The pharmaceutical composition of the present invention The present invention also provides a pharmaceutical composition comprising the radiotracer or radiopharmaceutical described herein. The pharmaceutical composition further comprises one or more excipients. The excipients may be appropriately selected by a person skilled in the art according to the intended use, method of administration, etc. Excipients commonly used in the pharmaceutical compositions of preferred embodiments of the present invention are buffering substances, radiodegradation protective compounds, and / or stabilizers.

[0128] Applications of this invention In preclinical trials, the radiotracer Ga-68-TRAP-WL12 of the preferred embodiment of the present invention exhibited higher uptake in tumor tissue, significantly lower uptake in many organs, and significantly accelerated blood clearance compared to the prior art structures Ga-68-DOTAGA-WL12 or Cu-64-DOTAGA-WL12. The conjugate of the first embodiment allows for faster radiolabeling, and the resulting radiotracer has higher molar activity compared to the corresponding antibody-based radiotracers and their precursors.

[0129] Therefore, the radiotracer (e.g., Ga-68-TRAP-WL12) and its precursor conjugate of the first embodiment of the present invention represent a peptide PD-L1 imaging agent with excellent properties. Based on these excellent properties, the present invention also provides a radiotracer of the first embodiment for imaging PD-L1-carrying cells. The imaging can be performed using positron emission tomography (PET), single-photon emission computed tomography (SPECT), or planar scintillation. If necessary, it can be performed in vivo in a patient. This generally refers to cancer patients, and more specifically, cancer patients considering immune checkpoint inhibitor (ICI) therapy, particularly ICI therapy targeting the PD-1 / PD-L1 axis. The imaging is preferably performed by quantifying the uptake of the radiotracer in tumor tissue and other tissues to assess the expression status of PD-L1 in the patient.

[0130] In the context of this invention, ICI therapy particularly includes the use of agents selected from pembrolizumab, nivolumab, cemiplimab, spartalizumab, atezolizumab, durvalumab, avelumab, or any other substance (including antibodies or small molecules) that interferes with the interaction with PD-1 / PD-L1, such as those described in WO 2014 / 151634 A1, WO 2017 / 176608 A1, or WO 2018 / 237153 A1.

[0131] The types of cancer that can be treated, imaged, assessed for treatment sensitivity, or otherwise subjected to the uses and methods of this invention are naturally any type of cancer for which ICI therapy has been authorized and / or tested in clinical trials and / or otherwise published in scientific or patent literature and / or used in clinical practice. It particularly includes any cancer on the surface of cancer cells where PD-L1 upregulation is present or at least suspected. On the other hand, it particularly includes melanoma, especially metastatic melanoma, non-small cell lung cancer, renal cell carcinoma, Hodgkin's lymphoma, head and neck cancer, urothelial carcinoma, colorectal cancer, hepatocellular carcinoma, small cell lung cancer, esophageal cancer, malignant pleural mesothelioma, gastric cancer, cervical cancer, hepatocellular carcinoma, Merkel cell carcinoma, endometrial cancer, squamous cell carcinoma, bladder cancer, breast cancer, and basal cell carcinoma.

[0132] This invention also provides the radiotracer described herein for determining the susceptibility of cancer patients to ICI therapy (particularly ICI therapy targeting the PD-1 / PD-L1 axis). Sensitivity can be determined by the following steps: (i) Administering a radioactive tracer to the patient. This can be done by intravenous infusion or injection.

[0133] (ii) Perform PET scan, SPECT scan or scintillation scan on the patient.

[0134] (iii) The quantitative uptake value obtained in the scan is compared with a reference uptake value. Specifically, according to (ii), the signal intensity in the patient scan can be quantified in one or more target regions. The obtained signal intensity value can then be compared with a reference value, preferably with a reference value for the same target region, which is predetermined such that similar or higher signal intensities indicate susceptibility to ICI treatment. For a specific target radiotracer, the reference value is determined empirically based on measurements from cancer patients known to be sensitive (or insensitive) to ICI treatment.

[0135] (iv) If the signal intensity of the patient scan in at least one target region is equal to or higher than the signal intensity of the reference scan in the same region, then sensitivity to ICI treatment is confirmed. Alternatively, if the quantized signal intensity value of the patient scan in at least one target region is equal to or higher than the signal intensity of the reference scan in the same region, then susceptibility to ICI treatment is confirmed. Otherwise, if the signal intensity or quantized signal intensity value in all target regions is lower than the corresponding signal intensity or signal intensity value in the reference scan, then the patient is not sensitive to ICI treatment.

[0136] In one aspect of the above method, the radioactive tracer applied in step (i) is a radioactive tracer based on a coupling compound according to formula Y1 or Y3 of the first embodiment, preferably Y1a, Y1b, Y1c, Y1d, Y3a, Y3b, Y3c, Y3d, Y3e, Y3f, Y3g, or Y3h, and the radionuclide is selected from... 68 Ga、 66 Ga、 64 Cu, Al 18 F; or a radioactive tracer based on a coupling compound of formula Y2 or Y4 according to the second embodiment, preferably Y2a, Y2b, Y2c, Y2d, Y4a, Y4b, Y4c, Y4d, Y4e, Y4f, Y4g, or Y4h, wherein the radionuclide is selected from Al. 18 F, 43 Sc or 44 Step Sc; Step (ii) is accomplished by performing a PET scan.

[0137] In another aspect of the above method, the radioactive tracer applied in step (i) is a radioactive tracer based on a coupling compound of formula Y1 or Y3 according to the first embodiment, preferably of formula Y1a, Y1b, Y1c, Y1d, Y3a, Y3b, Y3c, Y3d, Y3e, Y3f, Y3g or Y3h, and selected from... 67 Ga or 99m Radionuclides of Tc; or radiotracers based on coupling compounds of formula Y2 or Y4 according to the second embodiment, preferably of formula Y2a, Y2b, Y2c, Y2d, Y4a, Y4b, Y4c, Y4d, Y4e, Y4f, Y4g, or Y4h, and selected from... 111 In、 155 Tb or 99m The radionuclide of Tc; step (ii) is performed by SPECT scanning or scintillation imaging.

[0138] There are no particular limitations on the dosage of the radioactive tracer. Those skilled in the art can determine the appropriate dosage based on the known characteristics of the target radionuclide and the sensitivity of the scanner. For example, for 68 The commonly used dosage for Ga is currently 80–300 MBq, but next-generation PET scanners are expected to have higher sensitivity, so dosages as low as 5 MBq may be sufficient. Therefore, possible dosage ranges are 0.1 to 6000 MBq, such as 1–1000 MBq, 2–500 MBq, 4–400 MBq, or 5–300 MBq. However, the present invention is not limited to any of the above ranges.

[0139] Step (ii) is typically started 15-90 minutes after step (i), preferably 30-60 minutes after step (i).

[0140] On the other hand, the present invention provides a radiotracer of the present invention for use in a method of treating cancer patients with ICI therapy (particularly ICI therapy targeting the PD-1 / PD-L1 axis). The method includes the following steps: The first step in determining whether a cancer patient is sensitive to the aforementioned ICI therapy; The second step involves administering treatment with immune checkpoint inhibitors only if the cancer patient is confirmed to be sensitive to ICI therapy in the first step.

[0141] Other applications of the radiotracer of the present invention include monitoring the efficacy of anticancer therapies. This use involves performing steps (i) and (ii) above once or multiple times after the initiation of anticancer treatment. The obtained scan images (or quantitative data obtained from the scans) can then be compared to determine: (a) Despite anticancer treatment, if an increase in the volume and / or signal intensity of tissue with PD-L1 upregulation is detected during treatment, the disease has progressed; (b) Disease remission is indicated by a decrease in the volume and / or signal intensity of PD-L1-upregulated tissues detected during treatment; or (c) If the imaging or the resulting quantitative data does not show a clear trend, the condition is stable.

[0142] In a specific aspect of the first embodiment of the invention, a coupling compound of formula Y1 or Y3, preferably formula Y1a, Y1b, Y1c, Y1d, Y3a, Y3b, Y3c, Y3d, Y3e, Y3f, Y3g, or Y3h, is coupled with... 68 Ga forms chelates. 68 Ga, being a radioactive tracer, is a positron emitter and can therefore be used as a peptide PD-L1 imaging agent, as described above, via PET imaging. Using this imaging technique, this aspect of the first embodiment of the invention provides the following uses: imaging PD-L1 expression, determining the susceptibility of cancer patients to ICI therapy, monitoring the efficacy of anticancer therapies, and in methods for treating cancer patients using ICI therapy as described above.

[0143] In one specific aspect of the first embodiment of the present invention, a coupling compound of formula Y1 or Y3, preferably formula Y1a, Y1b, Y1c, Y1d, Y3a, Y3b, Y3c, Y3d, Y3e, Y3f, Y3g, or Y3h, is coupled with... 64 Cu, Al 18 F or66 Ga forms chelates. These contain 64 Cu, Al 18 F or 66 Ga, as a radioactive tracer, is also a positron emitter and can therefore be used as a peptide PD-L1 imaging agent, as described in the first embodiment of the present invention above. Thus, this aspect of the first embodiment of the present invention provides the same uses as described above, namely, imaging PD-L1 expression, determining the susceptibility of cancer patients to ICI therapy, monitoring the efficacy of anticancer therapies, and methods for treating cancer patients with ICI therapy, the only difference being the use of [a specific ingredient] in the first embodiment. 64 Cu, Al 18 F or 66 Ga is a radioactive tracer.

[0144] In one aspect of the second embodiment of the invention, a coupling compound of formula Y2 or Y4, preferably formula Y2a, Y2b, Y2c, Y2d, Y4a, Y4b, Y4c, Y4d, Y4e, Y4f, Y4g, or Y4h, is coupled with Al. 18 F, 43 Sc or 44 Sc forms chelates. These contain Al. 18 F, 43 Sc or 44 Sc, being a radioactive tracer, is also a positron emitter and can therefore be used as a peptide PD-L1 imaging agent as described in the first embodiment of the present invention. Therefore, this aspect of the second embodiment of the present invention provides the same uses as the first embodiment, namely, imaging PD-L1 expression based on PET, determining the susceptibility of cancer patients to ICI therapy, monitoring the efficacy of anticancer therapies, and methods for treating cancer patients with ICI therapy, the only difference being that the coupling compound in the second embodiment uses an Al-containing compound. 18 F, 43 Sc or 44 Sc is a radioactive tracer.

[0145] In another aspect of the first embodiment of the invention, a coupling compound of formula Y1 or Y3, preferably formula Y1a, Y1b, Y1c, Y1d, Y3a, Y3b, Y3c, Y3d, Y3e, Y3f, Y3g, or Y3h, is coupled with... 67 Ga or 99m Tc forms chelates. These contain 67 Ga or 99mThe radiotracer of Tc is a gamma emitter, and therefore can be used as a peptide PD-L1 imaging agent in the same manner as the radiotracer of the first embodiment of the present invention described above, the difference being that imaging is performed by SPECT or planar scintillation imaging. Therefore, this aspect of the first embodiment of the present invention provides the same uses as described above, namely, imaging PD-L1 expression, determining the susceptibility of cancer patients to ICI therapy, monitoring the efficacy of anticancer therapies, and methods for treating cancer patients with ICI therapy, the only difference being the use of a PD-L1-containing... 67 Ga or 99m Radioactive tracers of Tc and the use of SPECT or planar scintillation imaging.

[0146] In another aspect of the second embodiment of the invention, a coupling compound of formula Y2 or Y4, preferably formula Y2a, Y2b, Y2c, Y2d, Y4a, Y4b, Y4c, Y4d, Y4e, Y4f, Y4g, or Y4h, is coupled with... 111 In、 155 Tb or 99m Tc forms chelates. These contain 111 In、 155 Tb or 99m The radiotracer of Tc is a gamma emitter, and therefore can be used as a peptide PD-L1 imaging agent in the same manner as the radiotracer of the first embodiment of the present invention described above. The difference is that the conjugate compound of the second embodiment is used, and the imaging is performed by SPECT or planar scintillation imaging. Therefore, this aspect of the second embodiment of the present invention provides the same uses as the first embodiment described above, namely, imaging PD-L1 expression, determining the susceptibility of cancer patients to ICI therapy, monitoring the efficacy of anticancer therapy, and methods for treating cancer patients with ICI therapy. The only difference is that the radiotracer of the second embodiment contains a conjugate compound. 111 In、 155 Tb or 99m Tc tracer.

[0147] In another aspect of the first embodiment of the invention, a coupling compound of formula Y1 or Y3, preferably formula Y1a, Y1b, Y1c, Y1d, Y3a, Y3b, Y3c, Y3d, Y3e, Y3f, Y3g, or Y3h, is coupled with... 67 Cu or other suitable metal ion radionuclides emitting alpha or beta radiation form chelates, thereby providing a radiopharmaceutical for therapeutic use. That is, the radiopharmaceutical of this other aspect of the first embodiment is suitable for treating cancer patients, particularly those with cancers associated with high levels of PD-L1 expression. The treatment method includes administering the radiopharmaceutical to the cancer patient.

[0148] In another aspect of the second embodiment of the present invention, a coupling compound of formula Y2 or Y4, preferably formula Y2a, Y2b, Y2c, Y2d, Y4a, Y4b, Y4c, Y4d, Y4e, Y4f, Y4g, or Y4h, is coupled with... 47 Sc、 90 Y、 149 Tb, 161 Tb, 177 Lu、 212 Pb, 212 Bi、 213 Bi、 225 Ac、 227 Th or other suitable metal ion radionuclides emitting alpha or beta radiation form chelates, thereby providing a radiopharmaceutical for therapeutic use. That is, this other aspect of the second embodiment of the radiopharmaceutical is suitable for treating cancer patients, particularly those with cancers associated with high PD-L1 expression. The treatment method includes administering the radiopharmaceutical to the cancer patient.

[0149] The treatable cancer types are as described above, preferably selected from the list above. Radioactive tracers of the first or second embodiment (i.e., containing Al as described above) can be used. 18 F, 43 Sc、 44 Sc、 64 Cu、 66 Ga、 67 Ga、 68 Ga、 111 In、 155 Tb or 99m (using a radioactive tracer of Tc) to determine an individual patient's susceptibility to radiopharmaceutical treatment of this other aspect of the first or second embodiment. Therefore, according to a preferred embodiment, the therapeutic use of this other aspect of the radiopharmaceutical in the first and second embodiments mentioned above comprises the following two steps: a first step, in which the patient's susceptibility is determined as described above; and a second step, in which the radiopharmaceutical of this other aspect of the first or second embodiment is administered to the patient.

[0150] To avoid ambiguity, the above description of the application of the radiotracers and / or radiopharmaceuticals of the present invention in various methods (particularly imaging methods, methods for determining treatment sensitivity, methods for monitoring treatment efficacy, treatment methods, etc.) should also be understood as a description of the respective methods themselves. All these methods using the radiotracers and / or radiopharmaceuticals of the present invention are also embodiments of the present invention.

[0151] Specific implementation scheme of the present invention Compounds of specific embodiments of the present invention In specific embodiments, the present invention relates to conjugates of PD-L1 binding peptides (e.g., WL12) and their derivatives with radionuclide chelators and other functional portions to form compounds (e.g., TRAP (TRAP-WL12) or DOTPI (DOTPI-WL12)) for labeling with positron emitter gallium-68 or other potentially useful radionuclides, or for achieving other useful diagnostic or therapeutic effects.

[0152] In the broadest sense, specific embodiments of the present invention relate to coupling compounds characterized by the following formula 0:

[0153] Formula 0 In equation 0, FM The i represents the functional portion selected from the imaging and therapeutic active components, and can be 0 or 1. The remaining variable groups are as defined with respect to Equation 1.

[0154] In the first embodiment, the functional part FM Derived from the chelating group TRAP, the coupling compounds of the specific embodiments of the present invention are characterized by the following Formula 1:

[0155] Formula 1 Wherein, in equation 0 and / or 1: A Representatives selected –(CH2) n - , –(CH2CH2O) m -(CH2) p - or –(OCH2CH2) m - The groups, in which This indicates binding to the cyclic peptide moiety. This indicates a combination to B, where n is selected from the range of 1 to 6; m is selected from the range of 1 to 6, preferably 1 to 4 or preferably 3 to 5; and p is 1 or 2. B Represents the selected group, such that part of ABC is or , C Representatives selected α –(CH2) n’ -β , α –(CH2CH2O) m - β or α -(CH2) p’ –(OCH2CH2) m’ - β The group, wherein α represents binding to B, β represents binding to the chelate, and n' is selected from the range of 1 to 6; m' is selected from the range of 1 to 6, preferably 1 to 4 or preferably 3 to 5; p' is 1 or 2.

[0156] D It represents a group selected from –CH3, –CH2–COOH, –CH2–SO3H, –CH2–P(H)(O)(OH) or –CH2–P(O)(OH)2.

[0157] E The group represents a group selected from –CH(CH3)2, –COOH, –CH2–COOH, –CH2–CH2–COOH, –SO3H, –CH2–SO3H, –CH2–P(H)(O)(OH) or –CH2–P(O)(OH)2. k It can take the value 1, 2 or 3.

[0158] In Equation 1 above, n and n' can be chosen independently of each other. However, it is preferable to choose n and n' such that n+n' is in the range of 3 to 7, more preferably 4 to 6.

[0159] Similarly, m and m' can be chosen independently of each other. However, it is preferable to choose m and m' such that m+m' is in the range of 2 to 12, more preferably 4 to 8.

[0160] It can also be in B One side has an alkylene group, in B The other side has polyoxyethylene groups. In this case, m or m' and n or n' are selected such that m+n' or m'+n is preferably in the range of 3 to 9, more preferably in the range of 4 to 7.

[0161] In a preferred aspect of the first embodiment, the coupling compound (TRAP-WL12) of the specific embodiment of the present invention is characterized by the following formula 1a: .

[0162] Equation 1a.

[0163] In another preferred aspect of the first embodiment, the coupling compound is characterized by the following formula 1b: .

[0164] Equation 1b.

[0165] In another preferred aspect of the first embodiment, the coupling compound is characterized by the following formula 1c: .

[0166] Equation 1c.

[0167] In another preferred aspect of the first embodiment, the coupling compound is characterized by the following formula 1d: .

[0168] Formula 1d.

[0169] Nuclide that emits positrons 68 Ga、 64 Cu、 66 Ga, or 18 F (preferably with high-charge cations such as Al) 3+ It exists in the form of metal complexes, as described by WJ McBride et al. (EJNMMI Res.2013; 3: 36; doi:10.1186 / 2191-219X-3-36), hereinafter referred to as Al. 18 F); and gamma emitters 67 Ga or 99 mTc, or β emitter 67 Cu can be chelated to the TRAP moiety of the coupling compound of the present invention to obtain the radiotracer or radiopharmaceutical described in the first embodiment of the present invention. For this purpose, the radionuclides are preferably in their most stable ionic form (e.g., Ga). 3+ and Cu 2+ The conjugate compound is used in aqueous solution with a suitable counterion (preferably chloride ion). Of course, it is preferable to use the preferred conjugate compound of the first embodiment as described above to form the radiotracer or radiopharmaceutical of the first embodiment. If a conjugate compound having the structure of formula Y1a is combined with... 68 When used with Ga, the radioactive tracer in this case is sometimes referred to as Ga-68-TRAP-WL12.

[0170] All conjugates of the present invention can also be directly labeled with suitable radionuclides by mixing the radionuclide solution and the conjugate in separate vials, preferably containing other necessary excipients, such as buffers, radioprotective compounds, or stabilizers. Such vials (before the addition of the radionuclide) are sometimes referred to as kits or single-vial kits. The compounds of the present invention, along with the necessary buffers, radioprotective compounds, and stabilizers, can also be provided in separate vials, and the contents of the vials are then transferred to a single vial for the labeling reaction. Such ready-to-use vial combinations are sometimes referred to as multi-vial kits. The present invention also relates to such kits containing the conjugate compounds of the present invention, including single-vial kits and multi-vial kits.

[0171] For all kits, the radionuclide complex formation reaction is preferably carried out between 20 and 120°C. 68 The preferred time for Ga complexation is 1 to 15 minutes, or 1 to 60 minutes for other radionuclides. These preferred temperatures and times apply even if the complexation is performed independently of a kit.

[0172] Such kits can be used manually or automatically for processing before, during, and / or after the formation of complexes with radionuclides. Furthermore, all components for preparing radiolabeled compounds using the coupling compounds of the present invention can be contained in a combination of vials, tubing, manifolds, and separation cassettes, sometimes referred to as kits, preferably for use with a robotic system for radiolabeling (commonly referred to as a synthesis module). Such kits are preferably provided as single-use, sterile, ready-to-use packaging.

[0173] The resulting chelate can be used without further purification, or it can be further purified by conventional purification methods (such as RP-HPLC or solid phase extraction) before further use. 68 Ga can be obtained from 68 Ge / 68 Ga is provided by elution from a Ga generator, preferably with a nominal activity of 1-4 GBq, for example, by Eckert & Ziegler (Berlin, Germany), ITM (Garching, Germany), Monrol (Turkey), Rosatom / Isotope (Russia), PARS Isotopes (Iran), iTHEMBA (South Africa), or IRE Elit (Belgium). Prior to complexation, a suitable buffer can be used to... 68 The pH of the Ga aqueous solution is adjusted to a range of 1 to 7, preferably 2 to 3. 68 Ga can also be synthesized using a cyclotron, with either a solid or liquid target. The resulting chelates can have very high activity, for example, up to 300 GBq when using a solid target.

[0174] In the second embodiment, the coupling compound of the present invention contains a DOTPI chelating moiety instead of a TRAP moiety. DOTPI is a larger homologue of TRAP and has an additional binding unit. Therefore, it is more likely to bind larger-sized alternative radionuclides. The coupling compound of the second embodiment is characterized by the following formula 2:

[0175] Formula 2 in A , B , C , D , E ,and k The meaning is the same as that specified in the first implementation plan above.

[0176] In a preferred aspect of the second embodiment, the coupling compound is characterized by formula 2a, 2b, 2c or 2d:

[0177] Equation 2a,

[0178] Equation 2b

[0179] Equation 2c,

[0180] Equation 2d.

[0181] The coupling compound of the second embodiment can be used with Al... 18 F, 43 Sc、 44 Sc、 99m Tc, 111 In、 155 Tb radionuclides, or other suitable radionuclides emitting positrons or gamma radiation, form chelates to form the radiotracer described in the second embodiment of the present invention. Furthermore, the coupling compound of the second embodiment can be used with a variety of radionuclides selected from... 47 Sc、 90 Y、 149 Tb, 161 Tb, 177 Lu、 212 Pb, 212 Bi、 213 Bi、 225 Ac、 227The metal cation of a radionuclide of Th, or other metal ion radionuclide emitting α or β radiation, forms a chelate to form the radiopharmaceutical of the second embodiment of the present invention. The radiotracer and radiopharmaceutical of the second embodiment of the present invention can be prepared and further used as described in the first embodiment above, except that the coupling compound of formula 1, 1a, or 1b is replaced by a coupling compound of formula 2, 2a, or 2b. Unless otherwise specified or required by the context, all information provided herein with respect to the first embodiment (e.g., information regarding kits or purification) applies in a similar manner to the second embodiment, and vice versa.

[0182] In the third embodiment, the coupling compound of the specific embodiment of the present invention is characterized by the following formulas 3 and 4, that is, it is a coupling compound of formula 0, wherein i = 0:

[0183] Formula 3,

[0184] Formula 4.

[0185] In equations 3 and 4 above, C , D , E and k The meaning is the same as that specified in the first implementation plan above.

[0186] In a preferred aspect of the third embodiment, the coupling compound is characterized by the following formulas 3a, 3b, 4a, or 4b:

[0187] Equation 3a,

[0188] Equation 3b

[0189] Equation 4a,

[0190] Equation 4b.

[0191] In the fourth embodiment, the coupling compound is represented by the above formula 0, and the functional part... FMThis refers to a functional portion selected from the imaging-active and therapeutic-active portions, but excluding the portions specified in the first to third embodiments described above. Examples include portions containing one or more of the following: chromophores, especially fluorescent or luminescent groups; radiolabeled precursor groups; chelating agents; magnetic resonance imaging agents; enzyme inhibitors; chemotherapeutic agents, especially cell inhibitors, topoisomerase inhibitors, alkylating agents, antimetabolites, antimicrotubule agents, cytotoxic antibiotics, taxanes, intercalating agents, platinum compounds, mitotic inhibitors, tyrosine kinase inhibitors; peptides, especially peptide receptor ligands; functional building blocks for surface grafting, especially in medical devices, nanoparticles, micelles, magnetic particles, or quantum dots; functional proteins, such as antibodies, antibody fragments, nanobodies, or affinity molecules; pharmacokinetic modifiers, especially albumin binders, sugars, oligosaccharides, and polysaccharides; metal-containing or metal-free compounds suitable for photodynamic therapy, especially compounds that generate reactive oxygen species upon irradiation with light (visible or near-infrared wavelengths).

[0192] To synthesize the coupling compounds of the embodiments of the present invention, the peptides are provided in a modified form suitable for click chemical attachment to the chelating moiety TRAP or DOTPI. The modified form is an intermediate suitable for the manufacturing method of the embodiments of the present invention. The intermediates of the embodiments of the present invention are specified in formulas 5, 6, and 7:

[0193] Equation 5,

[0194] Formula 6,

[0195] Equation 7, In equations 5, 6, and 7 A , D , E and k The meaning is the same as that specified in the first implementation plan above.

[0196] Preferred intermediates are characterized by the following formulas 5a, 5b, 6a, 6b, 7a, and 7b:

[0197] Equation 5a,

[0198] Equation 5b

[0199] Equation 6a,

[0200] Equation 6b

[0201] Equation 7a,

[0202] Formula 7b.

[0203] According to specific embodiments of the present invention, the intermediates of formulas 5, 5a, 5b, 6, 6a, or 6b can be obtained by reacting peptide WL12 or suitable derivatives thereof with suitable reagents containing azido groups and reagents containing alkynyl groups, respectively. For example, the intermediates of formulas 5a and 6a can be prepared using 5-azidopentanoic acid or pent-4-alkynic acid, respectively. Suitable reaction conditions are shown in Example 1 below. WL12 is shown as formula 8 below. It is commercially available. Suitable derivatives of WL12 having E (not isopropyl) and D (not methyl) groups can be obtained by using a standard peptide synthesis method (Fmoc strategy) and replacing leucine and N-methyltryptophan with appropriately modified amino acid monomers.

[0204]

[0205] Formula 8.

[0206] The intermediates of formulas 5, 5a, 5b, 7, 7a, and 7b react with TRAP- or DOTPI- intermediates containing an alkyne group (hereinafter referred to as TRAP-alkynes and DOTPI-alkynes). TRAP-alkynes are derived from the structural formula TRAP-NH- C -C Characterized by CH, DOTPI-alkynes are derived from the structural formula DOTPI-NH- C -C CH characterization. TRAP and DOTPI are molecules described in Figure 1, for example, by A. Wurzer et al. (Front. Chem. 2018 Apr 10;6:107. doi: 10.3389 / fchem.2018.00107). C - Alkynes and C - The bonding of the azide moieties occurs via amide bonds, which are bonded to... C The terminal nitrogen atom forms with one of the carboxyl groups of TRAP and DOTPI. C As defined above with respect to formula Y0. Preferred variants of TRAP-alkyne and DOTPI-alkyne intermediates are listed below: and .

[0207] Intermediates synthesized according to Formulas 7, 7a, and 7b are of particular interest because they can be synthesized from commercially available amino acid building blocks using standard peptide synthesis methods (Fmoc strategy). Specifically, in peptide synthesis, 7a and 7b can be obtained using the commercially available non-natural amino acid azidolysine (CAS No. 159610-92-1) or suitable derivatives suitable for peptide synthesis reactions (such as Fmoc-azidolysine (CAS 159610-89-6)). Intermediates according to Formulas 7, 7a, and 7b can be directly functionalized by click chemistry (CuAAC) without attaching separate functional linkers, for example, as described above, synthesizing intermediates according to Formulas 5a and 5b from WL12 or suitable derivatives thereof.

[0208] The intermediates of formulas 6, 6a, and 6b react with TRAP- or DOTPI- intermediates (hereinafter referred to as TRAP-azides and DOTPI-azides), each containing an azide group. TRAP-azides are derived from the structural formula TRAP-NH- C Characterized by -N3, DOTPI-azides are derived from the structural formula DOTPI-NH- C -N3 characterization, where C - The bonding of the azide moiety occurs via an amide bond, which is bonded to... C The terminal nitrogen atom forms with one of the carboxyl groups of TRAP and DOTPI, respectively. C As defined above with respect to formula Y0. Preferred variants of the TRAP-azide and DOTPI-azide intermediates are listed below: ,and .

[0209] The CuAAC reaction of intermediates 5, 5a, 5b, 6, 6a, 6b, 7, 7a, and 7b with building blocks containing alkynes or azide groups (where applicable; the azide group on one molecule must always match the alkyne group on another molecule, and vice versa) is obviously not limited to TRAP-alkynes, DOTPI-alkynes, TRAP-azides, and DOTPI-azides, but can react with any molecule or functional building block that contains or is equipped with alkynes or azide groups. The following are categories of compounds of particular interest: chromophores, especially fluorescent or luminescent molecules; radiolabeled precursor groups; chelating agents; magnetic resonance imaging agents; enzyme inhibitors; chemotherapeutic agents, especially cell inhibitors, topoisomerase inhibitors, alkylating agents, antimetabolites, antimicrotubule agents, cytotoxic antibiotics, taxanes, intercalating agents, platinum compounds, mitotic inhibitors, tyrosine kinase inhibitors; peptides, especially peptide receptor ligands; functional building blocks for surface grafting, especially in medical devices, nanoparticles, micelles, magnetic particles, or quantum dots; functional proteins, such as antibodies, antibody fragments, nanobodies, or affinities; pharmacokinetic modifiers, especially albumin binders, sugars, oligosaccharides, and polysaccharides; metal-containing or metal-free compounds suitable for photodynamic therapy, especially compounds that generate reactive oxygen species when irradiated with light (in the visible or near-infrared wavelength range), or any combination thereof. The alkyne- or azide-containing building blocks used for CuAAC coupling to intermediates 5, 5a, 5b, 6, 6a, 6b, 7, 7a, and 7b may also be equipped with a branch linker, or a functional linker, between its alkyne portion and the biologically active or other functional portion of the molecule, which selectively cleaves according to tissue-specific parameters such as the presence of an enzyme, pH, oxygen partial pressure, etc.

[0210] Pharmaceutical compositions according to specific embodiments of the present invention A further embodiment of the present invention provides a pharmaceutical composition comprising a radiotracer or radiopharmaceutical according to the present invention, as described herein. The pharmaceutical composition further includes one or more excipients. These excipients may be appropriately selected by a person skilled in the art based on the intended use, method of administration, etc. Excipients commonly used in the pharmaceutical compositions of preferred embodiments of the present invention are buffering substances, radiodegradation protective compounds, and / or stabilizers.

[0211] Uses of specific embodiments of the present invention In preclinical trials, the radiotracer Ga-68-TRAP-WL12 of a preferred aspect of the first embodiment of the present invention exhibited higher uptake in tumor tissue, significantly lower uptake in many organs, and significantly accelerated blood clearance compared to the prior art structures Ga-68-DOTAGA-WL12 or Cu-64-DOTAGA-WL12. The conjugate of the first embodiment allows for faster radiolabeling, and the resulting radiotracer has higher molar activity compared to the corresponding antibody-based radiotracers and their precursors.

[0212] Therefore, the radiotracer (e.g., Ga-68-TRAP-WL12) and precursor conjugate of the first embodiment of the present invention represent a peptide PD-L1 imaging agent with excellent properties. Based on these excellent properties, the present invention also provides the radiotracer of the first embodiment for imaging PD-L1-carrying cells. The imaging can be performed using positron emission tomography (PET), single-photon emission computed tomography (SPECT), or planar scintillation scanning. In vivo trials in patients may be conducted if necessary. This generally refers to cancer patients, and more specifically, cancer patients considering immune checkpoint inhibitor (ICI) therapy, particularly ICI therapy targeting the PD-1 / PD-L1 axis. The imaging is preferably performed by quantifying the uptake of the radiotracer in tumor tissue and other tissues to assess the patient's PD-L1 expression status.

[0213] ICI therapy in the context of specific embodiments of the present invention particularly includes therapy using agents selected from: pembrolizumab, nivolumab, cimiprimab, spartalizumab, atezolizumab, durvalumab, avelumab, or any other substance (including antibodies or small molecules) that interferes with PD-1 / PD-L1 interaction, such as those described in WO2014 / 151634 A1, WO 2017 / 176608 A1, or WO 2018 / 237153 A1.

[0214] The types of cancer that can be treated, imaged, assessed for treatment sensitivity, or otherwise subjected to the uses and methods of this invention are naturally any type of cancer for which ICI therapy has been authorized and / or tested in clinical trials and / or otherwise published in scientific or patent literature and / or used in clinical practice. It particularly includes any cancer on the surface of cancer cells where PD-L1 upregulation is present or at least suspected. On the other hand, it particularly includes melanoma, especially metastatic melanoma, non-small cell lung cancer, renal cell carcinoma, Hodgkin's lymphoma, head and neck cancer, urothelial carcinoma, colorectal cancer, hepatocellular carcinoma, small cell lung cancer, esophageal cancer, malignant pleural mesothelioma, gastric cancer, cervical cancer, hepatocellular carcinoma, Merkel cell carcinoma, endometrial cancer, squamous cell carcinoma, bladder cancer, breast cancer, and basal cell carcinoma.

[0215] Specific embodiments of the present invention further provide a radiotracer of the present invention for determining the sensitivity of cancer patients to ICI therapy (particularly ICI therapy targeting the PD-1 / PD-L1 axis). Sensitivity can be determined through the following steps: (v) Administer a radioactive tracer to the patient. This can be done by intravenous infusion or injection.

[0216] (vi) Perform PET scans, SPECT scans, or scintillation scans on the patient.

[0217] (vii) The quantitative uptake value obtained in the scan is compared with a reference uptake value. Specifically, according to (ii), the signal intensity in the patient scan can be quantified in one or more target regions. The obtained signal intensity value can then be compared with a reference value, preferably with a reference value for the same target region, which is predetermined such that similar or higher signal intensities indicate susceptibility to ICI treatment. For a specific target radiotracer, the reference value is determined empirically based on measurements in cancer patients known to be sensitive (or insensitive) to ICI treatment.

[0218] (viii) If the signal intensity of the patient scan in at least one target region is equal to or higher than the signal intensity of the reference scan in the same region, then sensitivity to ICI treatment is confirmed. Alternatively, if the quantized signal intensity value of the patient scan in at least one target region is equal to or higher than the signal intensity of the reference scan in the same region, then susceptibility to ICI treatment is confirmed. Otherwise, if the signal intensity or quantized signal intensity value in all target regions is lower than the corresponding signal intensity or signal intensity value in the reference scan, then the patient is not sensitive to ICI treatment.

[0219] In one aspect of the above method, the radioactive tracer applied in step (i) is a radioactive tracer based on a coupling compound according to formula 1 or 3 of the first embodiment, preferably formula 1a, 1b, 1c, 1d, 3a or 3b, and selected from... 68 Ga、 66 Ga、 64 Cu, Al 18 Radionuclide of F; or radiotracer based on a coupling compound of formula 2 or 4 according to the second embodiment, preferably formula 2a, 2b, 2c, 2d, 4a or 4b, and selected from Al. 18 F, 43 Sc or 44 The radionuclide of Sc; step (ii) is accomplished by performing a PET scan.

[0220] In another aspect of the above method, the radioactive tracer applied in step (i) is a radioactive tracer based on a coupling compound according to formula 1 or formula 3 of the first embodiment, preferably formula 1a, 1b, 1c, 1d, 3a or 3b, and selected from... 67 Ga or 99m Radionuclides of Tc; or radiotracers of formula 2 or 4 based on coupling compounds of the second embodiment, preferably formula 2a, 2b, 2c, 2d, 4a or 4b, and selected from... 111 In、 155 Tb or 99m The radionuclide of Tc; step (ii) is performed by SPECT scanning or scintillation imaging.

[0221] There are no particular limitations on the dosage of the radioactive tracer. Those skilled in the art can determine the appropriate dosage based on the known characteristics of the target radionuclide and the sensitivity of the scanner. For example, for 68 The commonly used dosage for Ga is currently 80–300 MBq, but next-generation PET scanners are expected to have higher sensitivity, so dosages as low as 5 MBq may be sufficient. Therefore, possible dosage ranges are 0.1 to 6000 MBq, such as 1–1000 MBq, 2–500 MBq, 4–400 MBq, or 5–300 MBq. However, the present invention is not limited to any of the above ranges.

[0222] Step (ii) is typically started 15-90 minutes after step (i), preferably 30-60 minutes after step (i).

[0223] On the other hand, a specific embodiment of the present invention provides a radiotracer for use in a method of treating cancer patients using ICI therapy (particularly ICI therapy targeting the PD-1 / PD-L1 axis). This method includes the following steps: The first step in determining whether a cancer patient is sensitive to the aforementioned ICI therapy; The second step involves administering treatment with immune checkpoint inhibitors only if the cancer patient is confirmed to be sensitive to ICI therapy in the first step.

[0224] A further application of the radiotracer in a specific embodiment of the present invention is for monitoring the efficacy of anticancer therapy. This use involves performing steps (i) and (ii) above once or multiple times after the initiation of anticancer treatment. The obtained scan images (or quantitative data obtained from the scans) can then be compared to determine: (d) Despite anticancer treatment, if an increase in the volume and / or signal intensity of tissue with PD-L1 upregulation is detected during treatment, the disease has progressed; (e) Disease remission is indicated by a decrease in the volume and / or signal intensity of PD-L1-upregulated tissues detected during treatment; or (f) If the imaging or the resulting quantitative data does not show a clear trend, the condition is stable.

[0225] In a specific aspect of the first embodiment of the present invention, a coupling compound of formula 1 or 3, preferably formula 1a, 1b, 1c, 1d, 3a or 3b, with 68 Ga forms chelates. 68 Ga, being a radioactive tracer, is a positron emitter and therefore can be used as a peptide PD-L1 imaging agent, as described above, through PET imaging. Using this imaging technique, this aspect of a first embodiment of the present invention provides the following uses: imaging PD-L1 expression, determining the susceptibility of cancer patients to ICI therapy, monitoring the efficacy of anticancer therapies, and utilizing the methods described above for treating cancer patients with ICI therapy.

[0226] In a specific aspect of a first embodiment of the present invention, a coupling compound of formula 1 or 3, preferably formula 1a, 1b, 1c, 1d, 3a or 3b, is used with... 64 Cu, Al 18 F or 66 Ga forms chelates. These contain 64 Cu, Al 18 F or 66Ga radioactive tracers are also positron emitters, and therefore can be used as peptide PD-L1 imaging agents as described in the first embodiment of the present invention above. Therefore, this aspect of the first embodiment of the present invention provides the same uses as described above, namely, imaging PD-L1 expression, determining the susceptibility of cancer patients to ICI therapy, monitoring the efficacy of anticancer therapies, and methods for treating cancer patients with ICI therapy, the only difference being that the first embodiment uses a substance containing... 64 Cu, Al 18 F or 66 Ga is a radioactive tracer.

[0227] In one aspect of a second embodiment of the present invention, a coupling compound of formula 2 or 4, preferably formula 2a, 2b, 2c, 2d, 4a or 4b, is coupled with Al 18 F, 43 Sc or 44 Sc forms chelates. These contain Al. 18 F, 43 Sc or 44 Sc, as a radioactive tracer, is also a positron emitter and can therefore be used as a peptide PD-L1 imaging agent, as described in the first embodiment of the present invention. Therefore, this aspect of the second embodiment of the present invention provides the same uses as the first embodiment, namely, imaging PD-L1 expression based on PET, determining the susceptibility of cancer patients to ICI therapy, monitoring the efficacy of anticancer therapies, and methods for treating cancer patients with ICI therapy. The only difference is that the coupling compound in the second embodiment uses an Al-containing compound. 18 F, 43 Sc or 44 Sc is a radioactive tracer.

[0228] In another aspect of the first embodiment of the present invention, a coupling compound of formula 1 or 3, preferably formula 1a, 1b, 1c, 1d, 3a or 3b, with 67 Ga or 99m Tc forms chelates. These contain 67 Ga or 99mThe radiotracer of Tc is a gamma emitter, and therefore can be used as a peptide PD-L1 imaging agent in the same manner as the radiotracer in the first embodiment of the present invention described above, the difference being that imaging is performed via SPECT or planar scintillation imaging. Therefore, this aspect of the first embodiment of the present invention provides the same uses as described above, namely, imaging PD-L1 expression, determining the susceptibility of cancer patients to ICI therapy, monitoring the efficacy of anticancer therapies, and methods for treating cancer patients with ICI therapy, the only difference being the use of a PD-L1-containing... 67 Ga or 99m Radioactive tracers of Tc and the use of SPECT or planar scintillation imaging.

[0229] In another aspect of the second embodiment of the present invention, a coupling compound of formula 2 or 4, preferably formula 2a, 2b, 2c, 2d, 4a or 4b, with 111 In、 155 Tb or 99m Tc forms chelates. These contain 111 In、 155 Tb or 99m The radiotracer of Tc is a gamma emitter, and therefore can be used as a peptide PD-L1 imaging agent in the same manner as the radiotracer of the first embodiment of the present invention described above. The difference is that the conjugate compound of the second embodiment is used, and imaging is performed by SPECT or planar scintillation imaging. Therefore, this aspect of the second embodiment of the present invention provides the same uses as the first embodiment described above, namely, imaging PD-L1 expression, determining the susceptibility of cancer patients to ICI therapy, monitoring the efficacy of anticancer therapy, and methods for treating cancer patients with ICI therapy. The only difference is that the radiotracer of the second embodiment contains a conjugate compound. 111 In、 155 Tb or 99m Tc tracer.

[0230] In another aspect of the first embodiment of the present invention, a coupling compound of formula 1 or 3, preferably formula 1a, 1b, 1c, 1d, 3a or 3b, with 67 Cu or other suitable metal ion radionuclides that emit alpha or beta radiation form chelates, thereby providing a radiopharmaceutical for therapeutic purposes. That is, the radiopharmaceutical of this aspect of the first embodiment is suitable for treating cancer patients, particularly those with cancers associated with high levels of PD-L1 expression. The treatment method includes administering the radiopharmaceutical to the cancer patient.

[0231] In other aspects of the second embodiment of the specific embodiments of the present invention, coupling compounds of formula 2 or formula 4, preferably formula 2a, formula 2b, formula 2c, formula 2d, formula 4a or formula 4b, with 47 Sc、 90 Y、 149 Tb, 161 Tb, 177 Lu、 212 Pb, 212 Bi、 213 Bi、 225 Ac、 227 Th or other suitable metal-ion radionuclides emitting alpha or beta radiation form chelates, thereby providing a radiopharmaceutical for therapeutic use. That is, the radiopharmaceutical of this aspect of the second embodiment is suitable for treating cancer patients, particularly those with cancers associated with high PD-L1 expression. The treatment method includes administering the radiopharmaceutical to the cancer patient.

[0232] The treatable cancer types are as described above, preferably selected from the above list. A radioactive tracer of the first or second embodiment (i.e., containing Al as described above) can be used. 18 F, 43 Sc、 44 Sc、 64 Cu、 66 Ga、 67 Ga、 68 Ga、 111 In、 155 Tb or 99m (using a radioactive tracer of Tc) to determine the sensitivity of an individual patient to the radiopharmaceutical of the first or second embodiment. Therefore, according to a preferred embodiment, the therapeutic use of the radiopharmaceutical in the further embodiments of the first and second embodiments mentioned above comprises the following two steps: a first step, in which the patient's sensitivity is determined as described above; and a second step, in which the radiopharmaceutical of the other aspect of the first or second embodiment is administered to the patient.

[0233] To avoid ambiguity, the above descriptions of the use of the radiotracers and / or radiopharmaceuticals of the specific embodiments of the present invention in various methods (especially imaging methods, methods for determining treatment sensitivity, methods for monitoring treatment efficacy, treatment methods, etc.) should also be understood as descriptions of the respective methods themselves. All methods using the radiotracers and / or radiopharmaceuticals of the specific embodiments of the present invention are also embodiments described in the specific embodiments of the present invention.

[0234] The specific implementation schemes of this invention are numbered. The specific embodiments of the present invention also relate to the following numbered implementation methods: 1. A coupling compound having the structure shown in Formula 0, Formula 1, Formula 2, Formula 3 or Formula 4, or a pharmaceutically acceptable salt thereof:

[0235] Equation 0;

[0236] Formula 1;

[0237] Formula 2;

[0238] Equation 3; and

[0239] Equation 4; If it exists, then in equations 0, 1, 2, 3, and 4, FM The representative is a functional portion selected from the imaging-active portion and the therapeutic-active portion; i You can choose 1 or 2; A Representatives selected –(CH2) n - , –(CH2CH2O) m -(CH2) p - or –(OCH2CH2) m - The groups, in which This indicates the position to which the expression is joined to the left-hand side of equation 0, 1, or 2 above. Indicates combination to B Location; B Represents the selected group, such that part of ABC is or , C Representatives selected α –(CH2) n’ - β , α –(CH2CH2O) m’ - β or α -(CH2) p’–(OCH2CH2) m’ - β The group, where α represents the group bound to... B The position of β indicates the position to which it is attached to the right-hand side of the above equation 0, 1, 2, 3, or 4; D Represents a group selected from –CH3, –CH2–COOH, –CH2–SO3H, –CH2–P(H)(O)(OH) or –CH2–P(O)(OH)2; E The group represents a group selected from –CH(CH3)2, –COOH, –CH2–COOH, –CH2–CH2–COOH, –SO3H, –CH2–SO3H, –CH2–P(H)(O)(OH), or –CH2–P(O)(OH)2; k It can take the value 1, 2, or 3; Where n and n' are independently selected from the range of 1 to 6, m and m' are independently selected from the range of 1 to 6, preferably 1 to 4 or preferably 3 to 5, and p and p' are 1 or 2.

[0240] 2. The coupling compound according to embodiment 1 or a pharmaceutically acceptable salt thereof, wherein the coupling compound is characterized by a structure selected from the following formulas: 1a, 1b, 1c, 1d, 2a, 2b, 2c, 2d, 3a, 3b, 4a, and 4b. , Equation 1a; , Equation 1b; , Formula 1c; , Formula 1d;

[0241] Equation 2a;

[0242] Equation 2b;

[0243] Equation 2c;

[0244] Equation 2d;

[0245] Equation 3a;

[0246] Equation 3b;

[0247] Equation 4a; and

[0248] Equation 4b.

[0249] 3. A radioactive tracer comprising a coupling compound or a pharmaceutically acceptable salt thereof as described in embodiment 1 or 2, and a metal-ion radionuclide that emits positrons and / or gamma radiation.

[0250] 4. The radiotracer according to embodiment 3, comprising a coupling compound or pharmaceutically acceptable salt according to embodiment 1 or 2 and a radionuclide, wherein the coupling compound has formula 1, 1a, 1b, 1c or 1d, and the radionuclide is selected from... 68 Ga、 66 Ga、 67 Ga、 64 Cu, Al 18 F and 99m Tc.

[0251] 5. The radiotracer according to embodiment 3, comprising the coupling compound according to embodiment 1 or 2 or a pharmaceutically acceptable salt thereof and a radionuclide, wherein the coupling compound has formula 2, 2a, 2b, 2c or 2d, and the radionuclide is selected from Al. 18 F, 43 Sc、 44 Sc、 99m Tc, 111 In and 155 Tb.

[0252] 6. A radiopharmaceutical comprising a coupling compound or a pharmaceutically acceptable salt thereof as described in embodiment 1 or 2, and a metal-ion radionuclide emitting alpha or beta radiation.

[0253] 7. The radiopharmaceutical according to embodiment 6, comprising the coupling compound according to embodiment 1 or 2 or a pharmaceutically acceptable salt thereof, and 67 Cu, the coupling compound having formula 1, 1a, 1b, 1c or 1d.

[0254] 8. A radiopharmaceutical according to embodiment 6, comprising a coupling compound or a pharmaceutically acceptable salt thereof according to embodiment 1 or 2, and a radionuclide, wherein the coupling compound has formula 2, 2a, 2b, 2c or 2d, and the radionuclide is selected from... 47 Sc、90 Y、 149 Tb, 161 Tb, 177 Lu、 212 Pb, 212 Bi、 213 Bi、 225 Ac or 227 Th.

[0255] 9. A pharmaceutical composition comprising a radiotracer according to any one of embodiments 3, 4 or 5, or alternatively a radiopharmaceutical according to any one of embodiments 6, 7 or 8, and one or more excipients, preferably comprising one or more excipients selected from buffer substances, radiodegradation protective compounds and stabilizers.

[0256] 10. A kit comprising at least one container containing a coupling compound or a pharmaceutically acceptable salt thereof according to embodiment 1 or 2, and one or more excipients, preferably comprising one or more excipients selected from buffering substances, radiation-protecting compounds and stabilizers, wherein the excipients may be present in the same or different containers.

[0257] 11. The radiotracer according to embodiment 3, 4 or 5, or the pharmaceutical composition containing the radiotracer according to embodiment 9, for imaging cells carrying PD-L1.

[0258] 12. A radioactive tracer according to embodiment 3, 4 or 5, or a pharmaceutical composition containing a radioactive tracer according to embodiment 9, for determining the susceptibility of cancer patients to immune checkpoint inhibitor therapy.

[0259] 13. A method of using a radioactive tracer according to embodiment 3, 4, or 5, or a pharmaceutical composition containing a radioactive tracer according to embodiment 9, for treating a cancer patient, wherein the method comprises: The first step in determining the susceptibility of cancer patients to immune checkpoint inhibitor therapy is to use a radiotracer as described in embodiment 3, 4, or 5, or a pharmaceutical composition containing a radiotracer as described in embodiment 9; and The second step involves administering treatment containing an immune checkpoint inhibitor only if the cancer patient's susceptibility to ICI therapy has been confirmed in the first step.

[0260] 14. A method of treating a cancer patient with a radiopharmaceutical according to embodiment 6, 7 or 8 or a pharmaceutical composition containing a radiopharmaceutical according to embodiment 9.

[0261] 15. A radiopharmaceutical according to embodiment 6, 7 or 8, or a radiopharmaceutical-containing pharmaceutical composition according to embodiment 9, used in the method according to embodiment 14, the method comprising: The first step in determining the susceptibility of cancer patients to immune checkpoint inhibitor therapy is to use a radioactive tracer as described in embodiment 3, 4 or 5 or a pharmaceutical composition containing a radioactive tracer as described in embodiment 9. The second step of treatment, which involves administering a radiopharmaceutical according to embodiment 6, 7 or 8, or a pharmaceutical composition containing a radiopharmaceutical according to embodiment 9, is performed only if the susceptibility of the cancer patient to ICI therapy has been confirmed in the first step.

[0262] 16. An intermediate compound for synthesizing the coupling compound of embodiment 1, characterized by formula 5, formula 6 or formula 7:

[0263] Equation 5,

[0264] Formula 6,

[0265] Equation 7, Among them, in equations 5, 6 and 7 A , D , E and k The meaning is the same as that defined in Implementation Scheme 1, for example, especially selected from intermediates of the following formulas 5a, 5b, 6a, 6b, 7a and 7b:

[0266] Equation 5a,

[0267] Equation 5b

[0268] Equation 6a,

[0269] Equation 6b

[0270] Equation 7a, and

[0271] Formula 7b.

[0272] 17. A method for synthesizing the coupling compound according to embodiment 1 or 2, the method comprising: reacting an intermediate compound (formula 5 or 7) according to embodiment 16 with a chelate precursor selected from TRAP-alkynes and DOTPI-alkynes; or reacting an intermediate compound (formula 6) according to embodiment 16 with a chelate precursor selected from TRAP-azides and DOTPI-azides, wherein the TRAP-alkyne is formed by the structural formula TRAP-NH- C -C Characterized by CH, DOTPI-alkynes are derived from the structural formula DOTPI-NH- C -C CH characterization; TRAP-azides are characterized by the structural formula TRAP-NH-C-N3; DOTPI-azides are characterized by the structural formula DOTPI-NH-C-N3. C- Alkynes and C - The azide moiety is bonded to TRAP and DOTPI via amide bonds, respectively, the amide bonds being attached to... C One of the terminal nitrogen atoms forms with one of the carboxyl groups of TRAP and DOTPI; wherein, C As defined in Scheme 1 above, TRAP-alkyne, DOTPI-alkyne, TRAP-azide, and DOTPI-azide are preferably selected from the following compounds: , , ,and .

[0273] Example Example 1: Synthesis Steps Overview Unless otherwise stated, all commercially available reagents and solvents are of analytical grade and can be used without further purification. Cu(OAc)2 H2O, 4-pentyneic acid, diisopropylamine (DIPEA), and sodium ascorbate were purchased from Sigma Aldrich (Darmstadt, Germany). 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) was purchased from Macrocyclics (Plaino, Texas, USA). HATU was purchased from Bachem Holding AG (Bubdendorf, Switzerland). WL12 was purchased from CPC Scientific Inc. (Sunnyvale, California, USA). The structures of the TRAP-alkynes used are shown below. They were synthesized according to the previously described method. [6] Analytical and preparative HPLC were performed on Shimadzu gradient systems, each equipped with an SPD-20A dual-wavelength UV / Vis detector (λ1 = 220 nm, λ2 = 254 nm). For analysis, Nucleosil was used. ® A 100-5 C18 column (125 × 4.6 mm, 5 μm particle size) was used, with a flow rate of 1.0 mL / min, and linear gradient elution was performed over 15 or 20 minutes. Acetonitrile (JTBaker) was used. ® Supergradient HPLC grade was used, with a mixture of acetonitrile (5% H2O added) and purified water (from a Millipore system) as the eluent, containing 0.1% trifluoroacetic acid; gradient A: acetonitrile concentration increased from 15% to 65% within 15 minutes, and gradient B: acetonitrile concentration increased from 0% to 40% within 20 minutes. Preparative HPLC was performed using a Multospher 100 RP 18-5μ column (250 × 10 mm, 5 μm particle size) at a flow rate of 5.0 mL / min and a run time of 15 minutes. Mass spectra were acquired using an expressionL CMS mass spectrometer (Advion, Ithaca, USA) equipped with an electrospray ionization source and a quadrupole analyzer.

[0274]

[0275] The structure of the TRAP-alkyne used synthesis

[0276] Equation 2. Structural formula and calculation data of WL12-azides WL12-Azide. HBTU (1.96 mg, 5.17 µmol, 1.0 equivalent) was added to a solution of HOBt (699 µg, 5.17 µmol, 1.0 equivalent), 5-azidopentanoic acid (2.75 mg, 7.24 µmol, 1.4 equivalent), and DIPEA (10.35 µl, 1.34 mg, 10.35 µmol, 2 equivalent) dissolved in DMF (800 µl). The solution was stirred at room temperature for 10 min, and then added dropwise to a stirred solution of WL12 peptide (9.74 mg, 5.17 µmol, 1.0 equivalent) and DIPEA (10.35 µl, 1.34 mg, 10.35 µmol, 2 equivalent) in DMF (200 µl). The reaction was carried out with stirring at room temperature for 60 min. The crude product was precipitated in cold diethyl ether and washed with cold diethyl ether to remove any coupling reagents. ESI-MS (positive ion mode): m / z: = 1339.4 [2M+3H] 3+ , 1004.7 [M+2H] 2+ , 670.3 [M+3H] 3+ RP-HPLC (gradient: within 15 min, in H2O containing 10–90% MeCN solution, all containing 0.1% TFA), t R = 9.8 min. The resulting product does not require purification for subsequent CuAAC reactions.

[0277]

[0278] Equation 1: Structural formula and calculation data of TRAP-WL12 TRAP-WL12 was prepared by dissolving WL12-azide (10.38 mg, 5.17 µmol, 1.0 equivalent), sodium ascorbate (51.2 mg, 258 µmol, 50 equivalent), and TRAP-alkyne (4.78 mg, 7.75 µmol, 1.5 equivalent) in a tert-butanol:water (1:3) solution (800 µL). Copper(II) acetate (1.24 mg, 6.20 µmol, 1.2 equivalent) was added to the solution, immediately forming a brown precipitate. This precipitate dissolved after vortexing for 1 min, yielding a clear green solution. The solution was heated in a 60°C water bath for 1 hour, then diluted with water to 2 mL. Nota (31.4 mg, 104 µmol, 20.0 equivalent) was added. The pH of the solution was adjusted to 2.2–2.4 with 1M HCl aqueous solution, and the solution was heated in a 60°C water bath for 1 hour. The reaction mixture was directly purified by preparative RP-HPLC (gradient: 20–70% MeCN in H2O, both containing 0.1% TFA, over 15 min, followed by washing the phase with 100% MeCN containing 0.1% TFA for 5 min): t R = 15.4 min. ESI-MS (positive ion mode): m / z: = 1312.4 [M+2H] 2+ , 876.3 [M+3H] 3+ .

[0279] Example 2: Experimental Procedure for Compound Evaluation Metal complexes and radiochemistry The radiochemical purity of the radiometal-doped and labeled compounds was determined by radiometric thin-layer chromatography on silica-impregnated ITLC paper (Agilent, Santa Clara, USA; eluent: 0.1 M trisodium citrate, or a 1:1 (v / v) mixture of 1 M ammonium acetate and methanol). LabLogic systems The analysis was performed using the scan-RAM radiometric-TLC detector from the company (Brandon, USA). 68 Ga marking uses a fully automated field system (GallElut from Lindach Scintomics, Germany). + ) as described above. [7] In short, the SnO2 matrix is ​​used to... 68 Ge / 68 Ga generator (IThemba LABS, South Africa; 1.25 mL, eluent: 1M HCl aqueous solution containing approximately 500 MBq) 68Ga) was adjusted to pH 2 by adding HEPES aqueous buffer (450 µL, 2.7 M) and used for labeling 5 nmol of TRAP-WL12 at 95°C for 2 minutes. The radiolabeled peptide was captured in SepPak. ® The product was loaded onto a C8 light solid phase extraction (SPE) column, which was washed with 10 mL of water. The product was eluted with 2 mL of 50% EtOH aqueous solution. After ethanol evaporation, the labeling efficiency was determined by radiometric TLC, consistently showing ≥98%.

[0280] Determining the value of logD To determine the partition coefficient of n-octanol-PBS (log D) 7.4 Combine 500 µL of n-octanol and 500 µL of phosphate buffer in a 1.5 mL Eppendorf tube. Add approximately 1 MBq of... 68 Ga-TRAP-WL12 was subjected to vigorous vortexing for 3 minutes. The sample was centrifuged (13000 rpm, 5 min), and the radioactivity in 200 µL of organic phase and 20 µL of aqueous phase was quantitatively analyzed using a gamma counter. Through eight independent experiments, log D = –2.66 ± 0.07 (mean ± standard deviation) was calculated.

[0281] Cell lines and animal models All animal experiments were conducted in accordance with German General Animal Welfare Regulations and institutional animal care and use guidelines. MDA-MB-231 human breast adenocarcinoma cells (HTB-26; American Type Culture Collection) were cultured in RPMI 1640 containing 10% FBS. To establish a tumor xenograft model, 5 × 10⁶ cells were cultured in Matrigel (Cultrex BME, PathClear Type 3; Trevigen, GENTAUR GmbH). 6 MDA-MB-231 cells were inoculated into 6- to 10-week-old female CB17 severely combined immunodeficient mice (Charles River). When the tumors grew to a diameter of 10-14 mm (10-14 weeks post-inoculation), the mice were used for biodistribution or PET studies.

[0282] PET imaging Mice were anesthetized with isoflurane and then intravenously administered Ga-68-TRAP-WL12. The administered activity per mouse ranged from 12–16 MBq (200–400 pmol, depending on production and administration time). PET imaging was performed on a Siemens Inveon small animal PET system, with dynamic acquisitions for 90 min under isoflurane anesthesia, or single-frame acquisitions for 15 min at 60 or 120 min post-injection. Data were reconstructed using Siemens Inveon Research Workspace software with the three-dimensional ordered subset expectation maximum (OSEM3D) algorithm, without scattering and attenuation corrections. For kinetic analysis, regions of interest (ROIs) were manually defined.

[0283] Biological distribution For biodistribution studies, 3–6 MBq (between 70–180 pmol) were used. 68 Ga-TRAP-WL12 was injected into the tail vein. Mice were sacrificed 60 or 120 minutes after injection, blood samples were collected, and the target organs were dissected. A 2480 WIZARD was used. 2 An automated gamma counter (PerkinElmer, USA) was used to quantify the activity in recombinant tissue samples. The injection dose per gram of tissue (%ID / g) was calculated based on organ weight and counted activity.

[0284] Example 3: Test Results In SCID mice subcutaneously xenografted with the MDA-MB231 human papillary carcinoma cell line expressing PD-L1 (see figure), the biodistribution of Ga-68-TRAP-WL12 showed the highest uptake in tumors (aside from the activity present in the kidneys, which is associated with excretion). Tumor uptake could be blocked, indicating its target specificity. Other uptake in all organs and tissues could not be blocked and were therefore considered independent of PD-L1 expression. Decreased activity in the blood pool and organs was observed at 120 min post-injection compared to 60 min post-injection, indicating clearance from the blood pool and elution from non-target tissues over time.

[0285] The retention of activity within the tumor resulted in a significantly increased tumor-to-organ ratio at 120 minutes post-injection compared to 60 minutes post-injection. Figure 2 Dynamic PET imaging results ( Figure 3This observation was confirmed by the results showing that Ga-68-TRAP-WL12 was retained in the tumor, while its clearance from the blood pool and liver was observed to be either fast or slow.

[0286] PET imaging of MDA-MB231 tumor xenograft mice was performed using Ga-68-TRAP-WL12 as a radiotracer. Figure 4(A) and 4(B) The tumor outline was clearly visible. Local uptake was observed in the kidneys and bladder due to renal excretion; local uptake was also observed in the liver due to nonspecific accumulation. Tumor outline and image contrast were improved at 120 min post-injection compared to 60 min post-injection due to clearance from non-target tissues.

[0287] The in vivo properties of Ga-68-TRAP-WL12 are superior to those of the previously published gallium-68-labeled WL12 derivative Ga-68-DOTAGA-WL12. [4] The purpose of using TRAP chelators instead of DOTAGA for gallium-68 complexation was to improve the hydrophilicity of the resulting radiopharmaceutical. Based on past experience, lower excretion via the hepatobiliary route and higher excretion via the renal route were expected. In addition, faster blood clearance was expected to result in lower uptake in all tissues, including tumors. As shown in Figure 5(A) (left), all non-target-related uptake was indeed reduced (except for the kidneys). Unexpectedly, the study found increased tumor uptake, resulting in a significantly higher tumor-to-organ ratio for Ga-68-TRAP-WL12 compared to Ga-68-DOTAGA-WL12 (Figure 5(B) (right)). Due to this unexpected discovery, Ga-68-TRAP-WL12 appears to be more suitable for imaging PD-L1 expression in vivo, thus representing a PD-L1 imaging agent with significant market potential for improvement.

[0288] References [1]A. Ribas, JD Wolchok. Cancer immunotherapy using checkpointblockade. Science 2018, 359 , 1350–1355. [2]X. Shen, B. Zhao, Efficacy of PD-1 or PD-L1 inhibitors and PD-L1 expression status in cancer: meta-analysis. BMJ 2018, 362 , k3529. [3] S. Chatterjee, W. G. Lesniak, M. S. Miller, A. Lisok, E.Sikorska, B. Wharram, D. Kumar, M. Gabrielson, M. G. Pomper, S. B. Gabelli,S. Nimmagadda. Rapid PD-L1 detection in tumors with PET using a highlyspecific peptide. Biochem. Biophys. Res. Commun. 2017, 483 , 258–263. [4] R. A. De Silva, D. Kumar, A. Lisok, S. Chatterjee, B. Wharram, K.V. Rao, R. Mease, R. F. Dannals, M. G. Pomper, S. Nimmagadda. Peptide-Based 68 Ga-PET Radiotracer for Imaging PD-L1 Expression in Cancer. Mol. Pharmaceutics 2018, 15 , 3946 3952. [5]A. Mishra, D. Kumar, K. Gupta, G. Lo and, A. K. Sharma, D. S.Banka, R. F. Hobbs, R. F. Dannals, S. P. Rowe, E. Gabrielson, and S.Nimmagadda. Gallium-68–labeled Peptide PET Quanti es Tumor Exposure of PD-L1Therapeutics. Clin Cancer Res2023, doi: 10.1158 / 1078-0432.CCR-22-1931 [6] D. Reich, A. Wurzer, M. Wirtz, V. Stiegler, P. Spatz, J. Pollmann, H.-J. Wester, J. Notni. Dendritic poly-chelator frameworks for multimeric bioconjugation. Chem. Commun. 2017, 53 , 2586 2589. [7] J. Notni, J. Simecek, P. Hermann, HJ Wester. TRAP, a powerful and versatile framework for gallium-68 radiopharmaceuticals. Chem. Eur. J. 2011, 17 , 14718–14722.

Claims

1. A coupling compound having the following structure or a pharmaceutically acceptable salt thereof, said structure being as shown in formula Y0, X0, Y1, Y2, Y3 or Y4: Formula Y0; Formula X0; Formula Y1; Formula Y2; Formula Y3; and Formula Y4; in, In each of equations Y0, X0, Y1, Y2, Y3, and Y4, if there exists, FM The representative is a functional portion selected from the imaging-active portion and the therapeutic-active portion; h It can take the value 0 or 1; i It can take the value 0 or 1; k It can take the value 1, 2, or 3; g It can take the value 1, 2, or 3; A Representatives selected –(CH2) n - , –(CH2CH2O) m -(CH2) p - or –(OCH2CH2) m - The groups, in which This indicates the position of the part that is incorporated into the left side of equation 0, equation 1, or equation 2 above. Indicates combination to B Location; B The representative makes part of ABC as or Selected groups, C Representatives selected α –(CH2) n’ - β , α –(CH2CH2O) m’ - β or α -(CH2) p’ –(OCH2CH2) m’ - β The group, where α represents the group bound to... B The position of β, and β represents the position of the part that is attached to the right side of the above formulas Y0, X0, Y1, Y2, Y3 or Y4; D Represents a group selected from –H, –CH3, –CH2–COOH, –CH2–SO3H, –CH2–P(H)(O)(OH), or –CH2–P(O)(OH)2; E The group represents a group selected from –CH(CH3)2, –COOH, –CH2–COOH, –CH2–CH2–COOH, –SO3H, –CH2–SO3H, –CH2–P(H)(O)(OH), or –CH2–P(O)(OH)2; X 1 , X 2 , X 3 , X 4 and X 5 are each -C(H)=, or one of X 1 , X 2 , X 3 , X 4 and X 5 is an aromatic nitrogen atom (-N=) and the others are -C(H)=; X 6 X 7 X 8 X 9 and X 10 Both are –C(H)=, or X 6 X 7 X 8 X 9 and X 10 One of them is an aromatic nitrogen atom (–N=) and the rest are –C(H)=; X 11 X 12 X 13 and X 14 The common selection is to make X contain 11 X 12 X 13 and X 14 The residues are selected from Phe, Tyr, m-Tyr, 3-Pal, 4-Pal and DOPA as specified in the following table: X 15 It is –H or –OH, where X 15 When it is –OH, it reacts with X 15 The stereochemistry of adjacent C atoms is S or R; X 16 It is –H or –OH, where X 16 When it is –OH, it reacts with X 16 The stereochemistry of adjacent C atoms is S or R; If it exists, then X 17 Selected from –H, –OH, –(CH2)-OH, –SH, –(CH2)-SH and –(CH2)-S-CH3; X 18 Selected from –(CH2)–, –O–, –S–, –N(H)–, –N(CH3)–, –S(O)–, –S(O)2–, or X 18 Represents a direct covalent bond between adjacent carbon atoms; Wherein n and n' are independently selected from 1 to 6; and wherein m and m' are independently selected from 1 to 6, preferably 1 to 4 or preferably 3 to 5; and p and p' are independently 1 or 2.

2. The coupling compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the coupling compound is characterized by a structure selected from the following formulas: Y1a, Y1b, Y1c, Y1d, Y2a, Y2b, Y2c, Y2d, Y3a, Y3b, Y4a, and Y4b. ， Formula Y1a; ， Formula Y1b; ， Formula Y1c; ， Formula Y1d; ， Formula Y2a; ， Formula Y2b; Formula Y2c; Formula Y2d; Formula Y3a; Formula Y3b; Formula Y3c, Formula Y3d, Formula Y3e, Formula Y3f, Formula Y3g, Formula Y3h, Formula Y4a; Formula Y4b, Formula Y4c, Formula Y4d, Formula Y4e, Formula Y4f, Formula Y4g, and Formula Y4h.

3. A radioactive tracer comprising the coupling compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof, and a radionuclide emitting positrons and / or gamma radiation.

4. The radiotracer of claim 3, comprising the coupling compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof and a radionuclide, wherein the coupling compound has the formula Y1a, Y1b, Y1c, Y1d, Y3a, Y3b, Y3c, Y3d, Y3e, Y3f, Y3g or Y3h, and the radionuclide is selected from... 68 Ga、 66 Ga、 67 Ga、 64 Cu, Al 18 F and 99m Tc.

5. The radiotracer of claim 3, comprising the coupling compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof and a radionuclide, wherein the coupling compound has the formula Y2a, Y2b, Y2c, Y2d, Y4a, Y4b, Y4c, Y4d, Y4e, Y4f, Y4g or Y4h, and the radionuclide is selected from Al. 18 F, 43 Sc、 44 Sc、 99m Tc, 111 In and 155 Tb.

6. A radiopharmaceutical comprising a coupling compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, and a radionuclide emitting alpha or beta radiation.

7. The radiopharmaceutical of claim 6, comprising the coupling compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof, and 67 Cu, the coupling compound having the formula Y1a, Y1b, Y1c, Y1d, Y3a, Y3b, Y3c, Y3d, Y3e, Y3f, Y3g or Y3h.

8. The radiopharmaceutical according to claim 6, comprising the conjugate according to claim 1 or 2 or a pharmaceutically acceptable salt thereof and a radionuclide, said conjugate having the formula Y2a, Y2b, Y2c, Y2d, Y4a, Y4b, Y4c, Y4d, Y4e, Y4f, Y4g or Y4h, said radionuclide being selected from... 47 Sc、 90 Y、 149 Tb, 161 Tb, 177 Lu、 212 Pb, 212 Bi、 213 Bi、 223 Ra、 225 Ac, or 227 Th.

9. A pharmaceutical composition comprising a radiotracer according to any one of claims 3, 4 or 5, or alternatively a radiopharmaceutical according to any one of claims 6, 7 or 8, and one or more excipients, preferably comprising one or more excipients selected from buffer substances, radiodegradation protective compounds and stabilizers.

10. A kit comprising at least one container, said at least one container containing a coupling compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, and one or more excipients, preferably comprising one or more excipients selected from buffering substances, radiation-protecting compounds, and stabilizers, wherein, The excipients may be present in the same or different containers.

11. The radiotracer according to claim 3, 4 or 5, or the pharmaceutical composition containing the radiotracer according to claim 9, for imaging of cells carrying PD-L1.

12. The radiotracer according to claim 3, 4 or 5, or the pharmaceutical composition containing the radiotracer according to claim 9, used to determine the susceptibility of cancer patients to treatment with immune checkpoint inhibitors.

13. A method of applying the radioactive tracer according to claim 3, 4, or 5, or a pharmaceutical composition containing the radioactive tracer according to claim 9, to a patient with cancer, the method comprising: The first step in determining the susceptibility of cancer patients to treatment with immune checkpoint inhibitors is to use the radiotracer as described in claim 3, 4 or 5 or the pharmaceutical composition containing the radiotracer as described in claim 9. as well as The second step involves administering a treatment containing an immune checkpoint inhibitor only if the cancer patient's susceptibility to ICI therapy has been confirmed in the first step.

14. The radiopharmaceutical according to claim 6, 7 or 8, or the pharmaceutical composition containing a radiopharmaceutical according to claim 9, in a method of treating a cancer patient.

15. A radiopharmaceutical according to claim 6, 7, or 8, or a pharmaceutical composition containing a radiopharmaceutical according to claim 9, for use in the application according to claim 14, the method comprising: The first step in determining the susceptibility of cancer patients to treatment with immune checkpoint inhibitors using the radiotracer according to claim 3, 4, or 5, or the pharmaceutical composition containing the radiotracer according to claim 9; and The second step involves administering treatment containing a radiopharmaceutical as claimed in claim 6, 7, or 8, or a pharmaceutical composition containing a radiopharmaceutical as claimed in claim 9, only if the susceptibility of the cancer patient to ICI therapy has been confirmed in the first step.

16. An intermediate compound characterized by formula Y5, Y6, Y7, Y8, X5, X6, X7, or X8: Formula Y5, Formula Y6, Formula Y7, Formula Y8, Formula X5, Formula X6, Formula X7, Formula X8, in, If any of the following exist in equations Y5, Y6, Y7, Y8, X5, X6, X7, and X8, then A , D , E , g , k X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 and X 18 The meaning is the same as that defined in claim 1, and the intermediate is, in particular, selected from the following intermediates: Y5a, Y5b, Y5c, Y5d, Y6a, Y6b, Y6c, Y6d, Y7a, Y7b, Y7c, Y7d, Y8a, Y8b, Y8c, and Y8d. Formula Y5a, Formula Y5b, Formula Y5c, Formula Y5d, Formula Y6a, Formula Y6b, Formula Y6c, Formula Y6d, Formula Y7a, Formula Y7b Formula Y7c, Formula Y7d, Formula Y8a, Formula Y8b, Formula Y8c, Formula Y8d.

17. A method for synthesizing the coupling compound according to claim 1 or 2, the method comprising: The intermediate compound of formula Y5 or Y7 according to claim 16 is reacted with a chelate precursor selected from TRAP-alkynes and DOTPI-alkynes; or the intermediate compound of formula Y6 or Y8 according to claim 16 is reacted with a chelate precursor selected from TRAP-azides and DOTPI-azides, wherein the TRAP-alkyne is derived from the formula TRAP-NH- C -C CH characterization, DOTPI- alkyne is represented by the formula DOTPI-NH- C -C CH characterization, TRAP-azides are derived from the formula TRAP-NH- C -N3 characterization, and DOTPI-azides are characterized by the structural formula DOTPI-NH- C -N3 characterization, where C- Alkynes and C - The azide moiety is bonded to TRAP and DOTPI via amide bonds, respectively, the amide bonds being attached to... C One of the terminal nitrogen atoms forms with one of the carboxyl groups of TRAP and DOTPI; and in which C As defined in claim 1 above, TRAP-alkyne, DOTPI-alkyne, TRAP-azide, and DOTPI-azide are preferably selected from the following compounds: ， ， ,as well as .

18. The coupling compound according to claim 1, wherein, The compound is characterized by formula Y0 or formula X0, but the functional part thereof FM It additionally has one or more portions capable of undergoing click chemistry, said click chemistry resulting in the formation of one or more covalent bonds bonded to other chemical portions, or wherein, FM It additionally has one or more portions, each of which comprises the contents of claim 1. A , B and / or C Type structural elements and cyclic peptides.

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