阅读说明：本技术 一种颗粒酶b结合化合物及其前体化合物和用途 (Granzyme B binding compound, precursor compound thereof and application ) 是由 宋少莉 许晓平 于 2021-04-30 设计创作，主要内容包括：本发明提供了通式(Ⅰ)～(Ⅳ)的化合物,其中通式(Ⅰ)-(Ⅱ)是通式(Ⅲ)-(Ⅳ)配合物的前体化合物,本发明通式(Ⅲ)的金属配合物可用于PET或SPECT成像方法测定免疫治疗疗效,为免疫治疗疗效评估提供新方法。本发明基于颗粒酶B抑制剂IEPD-CHO,设计合成了一种新的特异性靶向颗粒酶B的化合物及放射性金属配合物,能够定性和定量的反映免疫治疗直接生物标记物颗粒酶B的表达和活性,从而直接反映免疫疗效。(The invention provides compounds of general formulas (I) - (IV), wherein the general formulas (I) - (II) are precursor compounds of complexes of the general formulas (III) - (IV), and the metal complex of the general formula (III) can be used for determining the curative effect of immunotherapy by a PET or SPECT imaging method, thereby providing a new method for evaluating the curative effect of the immunotherapy. The invention designs and synthesizes a novel specific targeting granzyme B compound and a radioactive metal complex based on a granzyme B inhibitor IEPD-CHO, and can qualitatively and quantitatively reflect the expression and activity of an immunotherapy direct biomarker granzyme B so as to directly reflect the curative effect of immunity.)

1. A compound of formula (I) or a pharmaceutically acceptable salt, stereoisomer or tautomer thereof, wherein said formula (I) is:

wherein:

m is an integer of 0 to 10,

n is an integer of 0 to 10,

R₁and R₂Each independently selected from hydrogen, halogen, straight or branched carboxylic acid group, COOH, CHO, hydroxyl group, OC_1-6Alkyl radical, SC_1-6Alkyl radical, C_1-6Alkyl radical, C_1-6Halogenated alkyl, cycloalkyl, aryl, heterocyclic aryl or any one or more of various natural and unnatural amino acids;

R₃，R₅and R₇Each independently selected from hydrogen, C_1-6Alkyl radical, C_1-6Any one or more of halogenated alkyl;

R₄and R₆Each independently selected from hydrogen, halogen, amino acid side chain, COOH, CHO, hydroxyl, sulfydryl and OC_1-6Alkyl radical, SC_1-6Alkyl radical, C_1-6Alkyl radical, C_1-6Halogenated alkyl or any one or more of various natural and unnatural amino acids;

R₈and R₉Each independently selected from natural amino acids, unnatural amino acids, OC_1-6Alkyl radical, SC_1-6Alkyl radical, C_1-6Alkyl radical, C_1-6Any one or more of halogenated alkyl, cycloalkyl, aryl and heterocyclic aryl;

l is various metal chelating agents.

2. A compound of claim 1, wherein R is₁Is aldehyde group or monocyclic heteroaryl.

3. A compound according to claim 1 or 2, wherein R is₂Is acetoxy or hydrogen.

4. According to claim1 wherein R is as defined above₃、R₅And R₇Are all hydrogen.

5. A compound of claim 1, wherein R is₄Is a propanoyl or hydrogen.

6. A compound of claim 1, wherein R is₆Is butyl.

7. A compound of claim 1, wherein R is₈And R₉Independently selected from glycine or alanine, and m-0-10, n-0-10.

8. The compound of claim 1, wherein L is a variety of or substituted metal chelators.

9. A compound according to any one of claims 1 to 8, wherein R is₁Is formaldehyde group, R₂Is acetoxy, R₄Is propionic acid and R₆Is composed of

10. A compound according to any one of claims 1 to 8, wherein R is₁Is composed of

And R is₂Is hydrogen, R₄Acetoxy and R₆Is composed of

11. A compound of formula (ii) or a pharmaceutically acceptable salt, stereoisomer or tautomer thereof, wherein said formula (ii) is:

wherein:

m is an integer of 0 to 10,

n is an integer of 0 to 10,

R₁and R₂Each independently selected from hydrogen, halogen, straight or branched carboxylic acid group, COOH, CHO, hydroxyl group, OC_1-6Alkyl radical, SC_1-6Alkyl radical, C_1-6Alkyl radical, C_1-6Halogenated alkyl, cycloalkyl, aryl, heterocyclic aryl or any one or more of various natural and unnatural amino acids;

R₃，R₅and R₇Each independently selected from hydrogen, C_1-6Alkyl and C_1-6Any one or more of halogenated alkyl;

R₄and R₆Each independently selected from hydrogen, halogen, straight or branched carboxylic acid group, COOH, hydroxyl, mercapto, OC_1-6Alkyl radical, SC_1-6Alkyl radical, C_1-6Alkyl radical, C_1-6Halogenated alkyl or any one or more of various natural and unnatural amino acids;

R₈and R₉Each independently selected from natural amino acids, unnatural amino acids, OC_1-6Alkyl radical, SC_1-6Alkyl radical, C_1-6Alkyl radical, C_1-6Any one or more of halogenated alkyl, cycloalkyl, aryl and heterocyclic aryl;

l is various metal chelating agents.

12. A compound of claim 11, wherein R is₁Is aldehyde group or monocyclic heteroaryl.

13. A compound according to claim 11 or 12, wherein R is₂Is acetoxy or hydrogen.

14. A compound of claim 11, wherein R is₃，R₅And R₇Each is hydrogen.

15. A compound of claim 11, wherein R is₄Is a propanoyl or hydrogen.

16. A compound of claim 11, wherein R is₆Is butyl.

17. A compound of claim 11, wherein R is₈And R₉Independently selected from glycine or alanine, and m-0-10, n-0-10.

18. The compound of claim 11, wherein L is a variety of or substituted metal chelators.

19. A compound of claim 12, wherein R is₁Is formaldehyde group, R₂Is acetoxy, R₄Is hydrogen and R₆Is composed of

20. A compound of claim 12, wherein R is₁Is composed ofAnd R is₂Is hydrogen, R₄Is hydrogen and R₆Is composed of

21. A compound of formula (iii) or a pharmaceutically acceptable salt, stereoisomer or tautomer thereof, wherein said formula (iii) is:

wherein:

m is an integer of 0 to 10,

n is an integer of 0 to 10,

R₁and R₂Each independently selected from hydrogen, halogen, straight or branched carboxylic acid group, COOH, CHO, hydroxyl group, OC_1-6Alkyl radical, SC_1-6Alkyl radical, C_1-6Alkyl radical, C_1-6Halogenated alkyl, cycloalkyl, aryl, heterocyclic aryl or any one or more of various natural and unnatural amino acids;

R₃，R₅and R₇Each independently selected from hydrogen, C_1-6Alkyl and C_1-6Any one or more of halogenated alkyl;

R₄and R₆Each independently selected from hydrogen, halogen, straight or branched carboxylic acid group, COOH, hydroxyl, mercapto, OC_1-6Alkyl radical, SC_1-6Alkyl radical, C_1-6Alkyl radical, C_1-6Halogenated alkyl or various natural and non-natural amino acids,

R₈and R₉Each independently selected from natural amino acids, unnatural amino acids, OC_1-6Alkyl radical, SC_1-6Alkyl radical, C_1-6Alkyl radical, C_1-6Any one or more of halogenated alkyl, cycloalkyl, aryl and heterocyclic aryl;

l is various metal chelating agents;

m is a variety of radioisotopes, including⁶⁸Ga，⁶⁴Cu，⁸⁹Zr，⁹⁰Y，^99mTc，¹¹¹In，¹⁷⁷Lu，¹⁸⁶Re，¹⁸⁸Re，²⁰¹Tl，²⁰³Pb，²¹²Bi，²¹³Bi，²²⁵And any one or more of Ac.

22. A compound of claim 21, wherein R is₁Is aldehyde group or monocyclic heteroaryl.

23. A compound of claim 21, wherein R is₂Is acetoxy or hydrogen.

24. A compound of claim 21, wherein R is₃，R₅And R₇Each is hydrogen.

25. A compound of claim 21, wherein R is₄Is a propanoyl or hydrogen.

26. A compound of claim 21, wherein R is₆Is butyl.

27. A compound of claim 21, wherein R is₈And R₉Independently selected from glycine or alanine, and m-0-10, n-0-10.

28. The compound of claim 21, wherein L is a variety of or substituted metal chelators and M is a variety of metal radioisotopes or Al¹⁸F, conjugate.

29. A compound of claim 21, wherein R is₁Is formaldehyde group, R₂Is acetoxy, R₄Is propionic acid and R₆Is composed of

30. A compound of claim 21, wherein R is₁Is composed ofAnd R is₂Is hydrogen, R₄Acetoxy and R₆Is composed of

31. A compound of formula (iv), or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof, wherein said formula (iv) is:

wherein:

m is an integer of 0 to 10,

n is an integer of 0 to 10,

R₁and R₂Each independently selected from hydrogen, halogen, straight or branched carboxylic acid group, COOH, CHO, hydroxyl group, OC_1-6Alkyl radical, SC_1-6Alkyl radical, C_1-6Alkyl radical, C_1-6Halogenated alkyl, cycloalkyl, aryl, heterocyclic aryl or any one or more of various natural and unnatural amino acids;

R₃，R₅and R₇Each independently selected from hydrogen, C_1-6Alkyl and C_1-6Any one or more of halogenated alkyl;

R₄and R₆Each independently selected from hydrogen, halogen, straight or branched carboxylic acid group, COOH, hydroxyl, sulfydryl and OC_1-6Alkyl radical, SC_1-6Alkyl radical, C_1-6Alkyl radical, C_1-6Halogenated alkyl or any one or more of various natural and unnatural amino acids;

R₈and R₉Each independently selected from natural amino acids, unnatural amino acids, OC_1-6Alkyl radical, SC_1-6Alkyl radical, C_1-6Alkyl radical, C_1-6Any one or more of halogenated alkyl, cycloalkyl, aryl and heterocyclic aryl;

l is various metal chelating agents;

m is various radioactive isotopes or metal complexes;

further, the radioactive isotope comprises⁶⁸Ga，⁶⁴Cu，⁸⁹Zr，⁹⁰Y，^99mTc，¹¹¹In，¹⁷⁷Lu，¹⁸⁶Re，¹⁸⁸Re，²⁰¹Tl，²⁰³Pb，²¹²Bi，²¹³Bi，²²⁵Any one or more of Ac;

further, the metal complex includes Al¹⁸F。

32. The compound of claim 31, said R₁Is aldehyde group or monocyclic heteroaryl.

33. The compound of claim 31, said R₂Is acetoxy or hydrogen.

34. The compound of claim 31, said R₃，R₅And R₇Each is hydrogen.

35. The compound of claim 31, said R₄Is a propanoyl or hydrogen.

36. The compound of claim 31, said R₆Is butyl.

37. The compound of claim 31, said R₈And R₉Independently selected from glycine or alanine, m-0-10, n-0-10.

38. The compound of claim 31, wherein L is various or substituted metal chelators and M is a metal radioisotope.

39. The compound of claim 32, said R₁Is a formaldehyde group, and R₂Is acetoxy, R₄Is hydrogen, R₆Is composed of

40. The compound of claim 32, wherein R₁Is composed of And R is₂Is hydrogen, R_{4 is a}Hydrogen and R₆Is composed of

41. Use of a compound or complex of claims 1-40, or a pharmaceutically acceptable salt thereof, in the manufacture of an agent for imaging a patient.

42. Use of a compound or complex according to claims 1-40 or a pharmaceutically acceptable salt thereof for the manufacture of a reagent for the diagnosis of the therapeutic effect of immunotherapy of tumours.

43. Use of a compound or complex according to claims 1-40, or a pharmaceutically acceptable salt thereof, for the manufacture of an agent for the treatment of tumours.

Technical Field

The invention relates to the technical field of medicinal chemistry, in particular to a radioisotope-labeled granzyme B binding compound and a precursor compound thereof, and application of the compound in preparing a tracer and an imaging agent for evaluating the immunotherapy effect in nuclear medicine.

Background

Immunotherapy is a milestone innovation in the history of malignant tumor treatment, and compared with the traditional treatment means, immunotherapy has incomparable development advantages due to the unique curative effect and multiple cancer adaptability^[1]. Despite the successful application to a variety of tumors, immunotherapy remains challenging. Wherein followThe objective remission rates of patients vary greatly from tumor species to immunotherapy. Pembrolizumab (Pembrolizumab) is an example of a benefit in only 30%, 19%, 20% and 16% of patients with melanoma, non-small cell lung cancer, gastric cancer, and triple negative breast cancer, respectively. Therefore, the prediction of the population benefiting from immunotherapy is an urgent problem to be solved for tumor immunotherapy.

Predicting whether a patient benefits can be achieved by detection of pre-treatment relevant biomarkers or early efficacy assessment after treatment. At present, the genome and immune markers associated with tumor biopsy before treatment include tumor mutation load (TMB), microsatellite instability (MSI-H), mismatch repair gene and PD-L1 expression level and the like^[4]. However, the prediction threshold of these biomarkers is changed by different tumor species and immunotherapy, and there is no uniform standard, and in some tumors, the biomarkers have obvious overlap between responders and non-responders, and the problem of low immune response rate cannot be solved well. Analysis of relevant markers after immune system activation is more valuable than pre-treatment sample analysis after early immunotherapy compared to pre-treatment screening, since immune markers of various types after immune activation are more direct signals of tumor killing ability. And the immunotherapy scheme can be timely adjusted according to early immune response, so that individualized therapy is practically realized, and the response rate of immunotherapy is effectively improved. The molecular imaging method is one of the most effective methods for evaluating the curative effect in the early stage at present, the FDG PET/CT imaging which is most commonly used in clinic has proved the advantages of evaluating the curative effect in traditional therapies such as chemotherapy, radiotherapy and the like, however, a series of recent researches have exposed the limitation of evaluating the immune curative effect of FDG PET/CT in immunotherapy. Inflammatory cell infiltration by immunotherapy leads to a transient increase in tumor FDG, leading to false progression of the disease, misleading clinical diagnosis [7,8]. Therefore, a molecular imaging method reflecting the biomarkers directly related to the immune killing is urgently needed to effectively evaluate the immune curative effect and accurately screen the population benefiting from the immune therapy.

The killing and elimination of tumor cells by the immune system mainly depends on Cytotoxic T Lymphocytes (CTLs) and Natural Killer (NK) cellsThe most main tumor cell killing way of CTLs and NK cells is to enter tumor cells through granzyme released by the cells to activate Caspase-related enzymes, so as to cause Caspase cascade reaction, and finally to cause apoptosis^[9]. Among the various granzymes of the human body, granzyme B plays a dominant role. Therefore, monitoring the expression of granzyme B not only allows the determination of the activity of CTLs and NK cells, but also further reflects the effect of immunotherapy early. Granzyme B is the most directly relevant biomarker for immunotherapy.

Disclosure of Invention

The invention satisfies the specific compounds for granzyme B and their use as imaging agents, tracers and tumor therapeutic agents for granzyme B in nuclear medicine. The invention provides a novel granzyme B specific imaging agent, aiming at solving the problem of lack of accurate evaluation method for immunotherapy.

In order to solve the problems, the invention adopts the following technical scheme:

the present invention provides, in a first aspect, a compound represented by any one of the general formulae (I) to (IV),

wherein:

m is an integer of 0 to 10,

n is an integer of 0 to 10,

R₁and R₂Each independently selected from hydrogen, halogen, straight or branched carboxylic acid group, COOH, CHO, hydroxyl group, OC_1-6Alkyl radical, SC_1-6Alkyl radical, C_1-6Alkyl radical, C_1-6Halogenated alkyl, cycloalkyl, aryl, heterocyclic aryl or any one or more of various natural and unnatural amino acids;

further, said R₁Is an aldehyde group; further, said R₁Is a formaldehyde group;

or further, said R₁Is monocyclic heteroaryl; further, R₁Is composed of

Further, said R₂Is hydrogen or acetoxy;

R₃，R₅and R₇Each independently selected from hydrogen, C_1-6Alkyl radical, C_1-6Any one or more of halogenated alkyl; further, said R₃，R₅And R₇Each is hydrogen;

R₄and R₆Each independently selected from hydrogen, halogen, straight or branched carboxylic acid group, COOH, hydroxyl, mercapto, OC_1-6Alkyl radical, SC_1-6Alkyl radical, SC_1-6Alkyl radical, C_1-6Alkyl radical, C_1-6Halogenated alkyl or any one or more of various natural and unnatural amino acids;

further, said R₄Is propanoyl or hydrogen;

further, said R₆Is butyl or

R₈And R₉Each independently selected from natural amino acids, unnatural amino acids, OC_1-6Alkyl radical, SC_1-6Alkyl radical, C_1-6Alkyl radical, C_1-6Any one or more of halogenated alkyl, cycloalkyl, aryl and heterocyclic aryl; further, said R₈And R₉Independently selected from any one of glycine or alanine;

l is various metal chelating agents or substituted metal chelating agents;

m is a variety of radioisotopes, including⁶⁸Ga，⁶⁴Cu，⁸⁹Zr，⁹⁰Y，^99mTc，¹¹¹In，¹⁷⁷Lu，¹⁸⁶Re，¹⁸⁸Re，²⁰¹Tl，²⁰³Pb，²¹²Bi，²¹³Bi，²²⁵Ac and metal complex Al¹⁸F；

A second aspect of the invention provides the use of any one of the compounds or complexes described above, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for imaging a patient;

the third aspect of the present invention provides the use of any one of the compounds or complexes or pharmaceutically acceptable salts thereof in the preparation of a reagent for the diagnosis of the therapeutic effect of immunotherapy of tumors;

in a fourth aspect, the present invention provides the use of any one of the compounds or complexes described above, or a pharmaceutically acceptable salt thereof, in the manufacture of an agent for the treatment of a tumour.

If not otherwise stated, in the present invention, the term "alkyl" by itself or as part of another molecule refers to a straight or branched chain or cyclic hydrocarbon group, or combinations thereof, which may be fully saturated, mono or polyunsaturated, and may include divalent and polyvalent groups. An "alkyl" residue is preferably C1 to C10 and may be unsubstituted or substituted (e.g., with halogen). Preferred alkyl residues are methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl or n-octyl and the like. The same applies to the corresponding cycloalkyl compounds preferably having 3 to 10 carbon atoms, for example cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like. Unsaturated alkyl is alkyl having one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, ethenyl, 2-propenyl, 2-butenyl, 2-isopentenyl, 2- (butadienyl), 2, 4-pentadienyl, 3- (1, 4-pentadienyl), ethynyl, 1-and 3-propynyl, 3-butynyl, and higher homologs and isomers. Unless otherwise indicated, the term "alkyl" is also intended to include those derivatives of alkyl groups, such as "heteroalkyl," haloalkyl, "and" higher alkyl.

As used herein, the term "aryl" refers to a closed ring structure having at least one ring with a conjugated pi-electron system and includes carbocyclic aryl and heterocyclic aryl (or "heteroaryl" or "heteroaromatic") groups. Carbocyclic or heterocyclic aromatic groups may contain 5 to 20 ring atoms. The above terms include covalently linked monocyclic or fused-ring polycyclic (i.e., rings that share adjacent pairs of carbon atoms) groups. The aromatic group may be unsubstituted or substituted. Non-limiting examples of "aromatic" or "aryl" groups include phenyl, 1-naphthyl, 2-biphenyl, 3-biphenyl, 4-biphenyl, anthracenyl, and phenanthrenyl. The substituents for each of the above aryl and heteroaryl ring systems are selected from the group of acceptable substituents described herein (e.g., alkyl, carbonyl, carboxyl, or halogen). When used in conjunction with other terms (including, but not limited to, aryloxy, arylthioxy, aralkyl), the term "aryl" includes aryl and heteroaryl rings. Thus, the term "aralkyl" or "alkaryl" is intended to include those groups in which an aryl group is attached to an alkyl group (including, but not limited to, benzyl, phenethyl, pyridylmethyl and the like), including those alkyl groups in which a carbon atom (including, but not limited to, methylene) has been replaced with a heteroatom, by way of example only, an oxygen atom. Examples of such aryl groups include, but are not limited to, phenoxymethyl, 2-pyridyloxymethyl, 3- (1-naphthoxy) propyl, and the like.

"heteroaryl" refers to an aryl group containing at least one heteroatom selected from N, O and S; wherein the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen atoms may be optionally quaternized. Heteroaryl groups may be substituted or unsubstituted. The heteroaryl group may be attached to the remainder of the molecule through a heteroatom. Non-limiting examples of suitable groups include 1-pyrrolyl, 2-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-benzothiazolyl, 5-benzothiazolyl, 6-benzothiazolyl, 2-benzothiazolyl, 3-isoxazolyl, 3-oxazolyl, 3-isoxazolyl, 4-oxazolyl, 4-thiazolyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyrimidyl, 4-benzothiazolyl, and the like, 7-benzothiazolyl, purinyl, 2-benzimidazolyl, 4-indolyl, 5-indolyl, 6-indolyl, 7-indolyl, 1-isoquinolinyl, 5-isoquinolinyl, 2-quinoxalinyl, 5-quinoxalinyl, 2-quinolyl, 3-quinolyl, 4-quinolyl, 5-quinolyl, 6-quinolyl, 7-quinolyl, or 8-quinolyl.

The term "amino acid" refers to naturally occurring and unnatural amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Natural amino acids are the 20 common amino acids in their D-or L-form (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine) as well as pyrrolysine and selenocysteine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, by way of example only, an off-carbon (ex-carbon) that is bound to a hydrogen, a carboxyl group, an amino group, and an R group. Such analogs may have modified R groups (by way of example, norleucine) or may have modified peptide backbones, while still retaining the same basic chemical structure as a naturally occurring amino acid. Non-limiting examples of amino acid analogs include homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Amino acids may be referred to herein by their name, their commonly known three letter symbols, or by the single letter symbol (recommended by the IUPAC-IUB biochemical nomenclature commission). By "unnatural amino acid" is meant an amino acid that is not one of the 20 common amino acids or pyrrolysine or selenocysteine. Other terms that may be used synonymously with the term "unnatural amino acid" are "non-naturally encoded amino acid", "unnatural amino acid", "non-naturally occurring amino acid" or "artificial amino acid". The term "unnatural amino acid" includes, but is not limited to, amino acids that have been modified by naturally encoded amino acids in their backbones or side chains. In some embodiments, the unnatural amino acid comprises a carbonyl group, an acetyl group, an aminooxy group, a hydrazine group, a hydrazide group, a semicarbazide group, an azide group, or an alkyne group.

Within the meaning of the present invention, all residues are considered combinable unless otherwise indicated in the definition of the residue. All conceivable subgroups thereof are considered to be disclosed.

According to the invention, all chiral C atoms should have D-and/or L-configuration; furthermore, combinations within a compound should be possible, i.e. some chiral C atoms may be in the D-configuration and other chiral C atoms may be in the L-configuration.

Suitable chelating agents include, but are not limited to, 1,4, 7-triazacyclononane triacetic acid (NOTA), 1,4, 7-triazacyclononane-4, 7-diacetic acid (NODA), 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid (DOTA), 1,4, 7-triazacyclononane-1-glutaric acid-4, 7-diacetic acid (NODAGA), Ethylene Diammonium Tetraacetic Acid (EDTA), Diethylene Triamine Pentaacetic Acid (DTPA), cyclohexyl-1, 2-diamine tetraacetic acid (CDTA), ethylene glycol-O, O '-bis (2-aminoethyl) -N, N, N', N '-tetraacetic acid (EGTA), N, N-bis (hydroxybenzyl) -ethylene diamine-N, N' -diacetic acid (HBED), Triethylenetetramine Hexaacetic Acid (TTHA), hydroxyethylenediamine triacetic acid (HEDTA) and 1,4,8, 11-tetraazacyclotetradecane-N, N ', N ", N'" -tetraacetic acid (TETA), 1,4,7, 10-tetraaza-1, 4,7, 10-tetrakis- (2-carbamoylmethyl) -cyclododecane (TCMC) and deferoxamine B (DFO). In some embodiments, the chelating agent is p-isothiocyanatobenzyl-1, 4, 7-triazacyclononane triacetic acid (p-SCN-Bn-NOTA).

Advantageous effects

The compounds of the general formulae (I) to (II) according to the invention are precursor compounds of the complexes of the general formulae (III) to (IV).

The metal complex of the general formulas (III) to (IV) can be used for determining the curative effect of immunotherapy by a PET or SPECT imaging method, and provides a new method for evaluating the curative effect of immunotherapy. The invention designs and synthesizes a novel specific targeting granzyme B compound and a radioactive metal complex based on a granzyme B inhibitor IEPD-CHO, and can qualitatively and quantitatively reflect the expression and activity of an immunotherapy direct biomarker granzyme B so as to directly reflect the curative effect of immunity.

Drawings

FIG. 1 is HPLC ultraviolet spectrum and mass spectrum of GSI-1

FIG. 2 is⁶⁸Radioactive HPLC chromatogram of Ga-GSI-1

FIG. 3 is Al¹⁸Radioactive HPLC chromatogram of F-GSI-1

FIG. 4 is a drawing showing⁶⁸Ga-GSI-1 and Al¹⁸In vitro stability of F-GSI-1

FIG. 5 shows the model B16F10 before and after treatment⁶⁸Ga-GSI-1PET/CT continuous imaging

FIG. 6 shows the normal combination blocking group after model PD-L1 treatment in B16F10⁶⁸Ga-GSI-1PET/CT imaging verification of granzyme B specificity

FIG. 7 is a graph of immunohistochemical and conventional tumor volume methods for assessing therapeutic efficacy of B16F10 model

Detailed Description

Examples

The following examples illustrate the invention in more detail, but are not to be construed in any way as limiting the invention to only the illustrated embodiments.

Example 1: synthesis of NOTA-Gly-Gly-Gly-Ile-Glu-Pro-Asp-CHO (GSI-1)

By adopting a solid-phase synthesis method, 2-CTC resin is taken as a solid phase, Fmoc-Asp (OBn) -OH, Fmoc-Pro-OH, Fmoc-Glu (OBn) -OH, Fmoc-Ile-OH and Fmoc-Gly-OH are sequentially added, HOBt and DIC are taken as catalysts in each amidation reaction, 20% pyridine is adopted for Fmoc protection removal in each step, p-SCN-Bn-NOTA is added after the last Gly is connected, and after the reaction is finished, a mixture consisting of trifluoroacetic acid, triisopropylsilane and water (95/2.5/2.5) is used for removing protection and the 2-CTC resin to obtain (OBn) NOTA-Gly-Gly-Ile-Glu (OBn) -Pro-Asp (OBn) -OH. And reducing the carboxyl of Asp into aldehyde group, removing the protection of benzyl to obtain NOTA-Gly-Gly-Gly-Ile-Glu-Pro-Asp-CHO, and purifying the product by using a semi-preparative liquid phase. The final product was characterized by HPLC (FIG. 1a) and mass spectrometry (FIG. 1b), the relevant spectra are shown in FIG. 1.

Example 2:⁶⁸Ga-NOTA-Gly-Gly-Gly-Ile-Glu-Pro-Asp-CHO(⁶⁸synthesis of Ga-GSI-1)

⁶⁸Ga-NOTA-Gly-Gly-Gly-Ile-Glu-Pro-Asp-CHO(⁶⁸The specific reaction route of Ga-GSI-1) is as follows:

adding 600 μ l of 0.5M sodium acetate into 20 μ g GSI-1, and adding⁶⁸GaCl₃Eluting with about 10mCi, reacting the mixed solution at 40 deg.C for 10min, performing solid phase extraction with C-18 column, and eluting with 1ml ethanol to obtain the final product⁶⁸Ga-GSI-1. The radiochemical purity of the product is more than 99 percent through the detection of radio-HPLC, and a related analysis spectrogram is shown in figure 2.

Example 3: al (Al)¹⁸F-NOTA-Gly-Gly-Gly-Ile-Glu-Pro-Asp-CHO(Al¹⁸Synthesis of F-GSI-1)

Al¹⁸F-NOTA-Gly-Gly-Gly-Ile-Glu-Pro-Asp-CHO(Al¹⁸The specific synthetic reaction route of the F-GSI-1) is as follows:

produced by accelerator bombardment¹⁸Transferring F ions to QMA column for enrichment, washing QMA column with 2mL ultrapure water after transferring, and then 0.4M KHCO₃0.2mL of solution × 2 elution¹⁸And F ions. Taking 100 μ L¹⁸The F ion solution (about 10mCi) was mixed with 10. mu.L of glacial acetic acid and then 100. mu.L of 2mM AlCl was added₃Solution, incubated at room temperature for 5 minutes; then 160. mu.L GSI-1 (1. mu.g/. mu.L) and 530. mu.L of 0.1M NaOAc solution are added to make up the total volume of 1 mL; heating and reacting for 15 minutes at 105 ℃; after the reaction is finished, adding 1mL of 0.1M NaOAc solution for dilution, then carrying out solid phase extraction through a C-18 small column, washing with 2 times of 5mL of injection water, and finally eluting with 1mL of ethanol to obtain the product Al¹⁸F-GSI-1. The radiochemical purity of the product is more than 95 percent by radio-HPLC analysis, and the analysis spectrogram is shown in figure 3.

Example 4: experiment of in vitro stability of Complex

Taking a certain amount⁶⁸Ga-GSI-1 and Al¹⁸F-GSI-1 is added into PBS and serum of fresh mice respectively, the radiochemical purity is measured at different time points, and the stability of the two complexes is detected. The results are shown in figure 4 of the drawings,⁶⁸Ga-GSI-1 andAl¹⁸the stability of F-GSI-1 is shown in FIG. 4a and FIG. 4b, respectively.⁶⁸Ga-GSI-1 can maintain over 90 percent of radiochemical purity after 3 hours, while Al¹⁸F-GSI-1 can still maintain over 90 percent of radiochemical purity after 6 hours.

Example 5:⁶⁸evaluation of tumor immunotherapy efficacy by Ga-GSI-1 imaging

A murine melanoma B16F10 model is established on a normal Balb/c mouse, and the tumor highly expresses PD-L1. 12B 16F10 model mice were randomly divided into 2 groups of 6 mice each. The experimental group received PD-L1 monoclonal antibody treatment (10mg/kg) once every 3 days; the control group was given an equivalent volume of saline at the same time point. All mice were dosed prior to dosing⁶⁸Ga-GSI-1PET/CT imaging as baseline, then 1 day after each treatment⁶⁸The imaging results of Ga-GSI-1PET/CT are shown in FIG. 5.

⁶⁸Ga-GSI-1PET/CT imaging shows that no obvious radioactive uptake exists in the tumor of the control group and the experimental group before treatment. The radioactive uptake of the experimental group tumors increased dramatically with the depth of treatment; while there was only a slight increase in the radioactive uptake by the control tumors. Such a clear difference between the experimental and control groups could confirm that the uptake should be caused by treatment with PD-L1 monoclonal antibody. T/M data of tumor/contralateral muscle was obtained by quantitative calculation using PET/CT self-contained software. The results show that the T/M ratios of the experimental group and the control group are 1.06 +/-0.05 and 1.01 +/-0.15 respectively when the experimental group and the control group are subjected to baseline imaging, and the tumor uptake and the background are consistent. After 3 days of treatment, the T/M ratio in the experimental group increased to 6.68. + -. 0.35, whereas that in the control group was 1.71. + -. 0.18, which was only slightly higher. The T/M ratio of the experimental group 12 days after the treatment is as high as 13.24 +/-0.96, and the result is far higher than that reported in the literature⁶⁸Ga-GZP (Larimer BM, et al. cancer Res,2017,77, 2318). Tumor models in the literature are treated 12 days after immunotherapy,⁶⁸the quantitative results of Ga-GZP PET/CT imaging show that the T/M ratio is only 1.90 +/-0.55. The quantitative results show that,⁶⁸Ga-GSI-1 is preferred⁶⁸Ga-GZP is more sensitive and can predict the curative effect of immunity earlier.

To prove⁶⁸Ga-GSI-1 is indeed specific targeting granzyme B, we refer to IEPD-CHOInhibitors blocking experiments were performed. Blocking experimental mice were injected with granzyme B inhibitor IEPD-CHO (1000-fold molar equivalent) half an hour in advance, normal imaging and blocking imaging of the same mice are shown in FIG. 6, and results from tumor sites indicated by arrows in the figure show that excessive granzyme B inhibitor completely shields the tumor from radioactive uptake, confirming that⁶⁸Ga-GSI-1 does specifically target granzyme B.

To further confirm that the T cells were reactivated after PD-L1 treatment, releasing more lethal granzyme B, after the last imaging, 1 mouse was randomly selected from the experimental group and the control group, respectively, and dissected, and then the tumors were taken out for immunohistochemical analysis. Immunofluorescence results showed that there was a high amount of granzyme B expression in the experimental group of tumors, whereas the control group of tumors had only sporadic granzyme B expression (fig. 7 a). The tumor size of the mouse is continuously tracked and observed in the whole experimental period, and the result shows that the tumor growth of the PD-L1 monoclonal antibody treatment group is slow, the tumor size reaches the peak value at the 9 th day, then the tumor begins to shrink and regress, and part of the tumors in the mouse completely disappear, thereby showing the excellent immunotherapy effect of the PD-L1 monoclonal antibody. Whereas the saline treated group showed rapid tumor growth until the mice died (fig. 7 b).

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.