Compounds

Abstract

The present invention relates to radiolabeled compounds, precursor compounds and reference compounds, and pharmaceutical compositions comprising said radiolabeled compounds. The present invention also relates to said radiolabeled compounds for use in positron emission tomography (PET). The present invention also relates to methods for radiolabeling a precursor compound to form said radiolabeled compound, and to methods for preparing a precursor compound or a reference compound.

Description

Technical Field

[0001] This invention relates to radiolabeled compounds, precursor compounds, and reference compounds, as well as pharmaceutical compositions comprising said radiolabeled compounds. A further aspect of the invention relates to radiolabeled compounds for use in diagnostic methods performed on humans or animals using positron emission tomography (PET). Other aspects of the invention relate to methods for radiolabeling precursor compounds to form radiolabeled compounds, and methods for preparing precursor compounds or reference compounds. Background Technology

[0002] Positron emission tomography (PET) is a non-invasive nuclear imaging technique that uses radiolabeled molecules to detect the expression of targets or monitor metabolic processes in the body.

[0003] Single-photon emission computed tomography (SPECT) is a nuclear imaging modality that produces three-dimensional images of the distribution of a radioactive tracer emitting gamma rays within the body to assess organ function and physiology.

[0004] Functional proteins, such as antibodies, single-chain variable fragments (SCFVs), antigen-binding fragments (Fabs), nanobodies, and cytokines, are emerging as lead molecules for the development of PET / SPECT tracers and radioimmunotherapy agents due to their excellent binding affinity and high specificity to their receptors. However, a key challenge in converting such molecules into PET / SPECT tracers and radioimmunotherapy agents is to "site-specifically" conjugate PET / SPECT and therapeutic radionuclides at desired sites within the molecules, ensuring that their affinity and specificity remain unaffected.

[0005] Conventional site-specific conjugation of functional proteins involves using a maleimide-based radiolabeled prosthetic group via a cysteine ​​residue bound to the C-terminus of the target protein. In this technique, the sortase A substrate LPXTG motif must be genetically engineered into the target protein to introduce a cysteine ​​residue (Morgan, HE; ​​Turnbull, WB; Webb, ME; Challenges in the use of sortase and other peptide ligases for site-specific protein modification; Chem. Soc. Rev. 2022, 51, 4121-4145), adding another layer of complexity to obtaining the target protein.

[0006] There remains a need for methods to use site-specific radiolabeled compounds for conjugation with natural proteins. Natural proteins are proteins purified from their natural sources without any chemical or bioengineering alterations. Site-specific radiolabeling methods for conjugating natural proteins would avoid expensive and time-consuming genetic engineering methods. Summary of the Invention

[0007] One aspect of the present invention provides a radiolabeled compound of formula (I):

[0008] (I)

[0009] in: R 1 R² is independently selected from halogens and –SR³, wherein R³ is independently selected from C. 1-12 Alkyl, C 1-12 alkenyl, C 1-12 alkynyl group and C 6-10 Aryl; L represents a direct bond or connector, where the connector is C. 1-20 Alkylene, the C 1-20 The alkylene group is unsubstituted or selected from hydroxyl, -COOH, C 1-12 Alkoxy, C 1-12 Alkyl and hydroxy-C 1-12 -One or more substituents of the alkyl group are used, wherein zero or one to ten carbon atoms in the alkylene chain are selected from C 6-10 aryl, -O-, -S-, -NR 4 -、-C(O)NR 4 -、-NR 4The intervals between C(O)-, -C(O)-, -OC(O)- and -C(O)O- are partially replaced, where R 4 Is it hydrogen or C? 1-12 Alkyl, and wherein the C 6-10 The arylene moiety is unsubstituted or selected from hydroxyl, C 1-12 Alkyl and C 1-12 Alkoxy groups may be substituted with one, two, three, or four substituents; X is absent or is PEG. 2-20 Group; and Q Indicates 18 The part marked with F; Or its pharmaceutically acceptable salt.

[0010] Another aspect of the present invention provides a reference compound of formula (II):

[0011] (II)

[0012] in: R 1 R², L, and X are as defined above for radiolabeled compounds of formula (I); and Q indicates that chelation is involved. 19 F-Al complex 19 The part marked with F; Or its pharmaceutically acceptable salt.

[0013] Another aspect of the present invention provides a precursor compound of formula (III):

[0014] (III)

[0015] in: R 1 R², L, and X are as defined above for radiolabeled compounds of formula (I); and Y indicates the ability to chelate. 18 Part of the F-Al complex; Or its pharmaceutically acceptable salt.

[0016] Another aspect of the present invention provides a pharmaceutical composition comprising a radiolabeled compound of formula (I) as defined above and a pharmaceutically acceptable carrier.

[0017] Another aspect of the present invention provides a method for preparing a precursor compound of formula (III), the method comprising the following steps according to the reaction scheme: a) Reacting a compound of formula (V) with a compound of formula (VI) via a coupling reaction to form a compound of formula (IV); and b) Deprotecting the compound of formula (IV) to form the compound of formula (III);

[0018] Where R 1 R², L and X are as defined above for radiolabeled compounds of formula (I); Y is defined above for the precursor compound of formula (III); L 1 L² and L² are parts that can be coupled together to form group L; and Y p It is a portion containing at least one protecting group, which can be deprotected to provide group Y.

[0019] Another aspect of the present invention provides a method for preparing a radiolabeled compound of formula (I), the method comprising radiolabeling a precursor compound of formula (III).

[0020] Another aspect of the present invention provides a method for preparing a reference compound of formula (II), the method comprising reacting a precursor compound of formula (III) with a fluoride-19 source and Al according to the following reaction scheme. 3+ The source reaction is used to form the reference compound of formula (II):

[0021] Where R 1 R², L and X are as defined above for radiolabeled compounds of formula (I); Y is as defined above for the precursor compound of formula (III); and Q is defined above for the reference compound of formula (II).

[0022] Another aspect of the present invention provides a radiolabeled compound of formula (I) for use in a diagnostic method performed on a human or animal body using positron emission tomography (PET).

[0023] Throughout this specification and claims, the terms “comprise” and “contain,” and variations thereof (e.g., “comprising” and “comprises”), mean “including, but not limited to,” and do not exclude other components, integers, or steps. Furthermore, unless the context otherwise requires, the singular encompasses the plural: in particular, where the indefinite article is used, unless the context otherwise requires, the specification should be understood to cover both the plural and the singular.

[0024] Preferred features of each aspect of the invention may be described as in conjunction with any other aspect. Within the scope of this application, it is expressly intended that the various aspects, embodiments, examples, and alternatives set forth in the foregoing paragraphs, claims, and / or the following description and drawings, particularly their respective features, may be employed independently or in any combination. That is, all embodiments and / or features of any embodiment may be combined in any manner and / or combination unless such features are incompatible. Attached Figure Description

[0025] One or more embodiments of the invention will now be described by way of example only with reference to the accompanying drawings, in which: Figure 1 Display in bold [ 18 HPLC purification of the F]AlF-NOTA-dibromomaleimide reaction mixture.

[0026] Figure 2 show[ 18 HPLC co-elution of F]AlF-NOTA-dibromomaleimide with its non-radioactive reference compound.

[0027] Figure 3 Showing purified [ 18 HPLC quality control of F]AlF-NOTA-dibromomaleimide.

[0028] Figure 4 show[ 18 Tumor cell uptake of F]AlF-NOTA-NM-02. Samples blocked by pretreatment with 100X natural NM-02. Data shown are mean ± SD of three independent experiments performed in triplicate.

[0029] Figure 5 show[ 18Cellular uptake of F]AlF-NOTA-IL-4DE. Blocked samples were pretreated with 100X of native IL-4DE. Data shown are mean ± SD of independent experiments, where n=5 for T4-CAR T cells expressing 4αβ; n=5 for inactive, non-transduced T cells; n=5 for PBMCs; and n=3 for SKOV3 and 3HN cancer cells.

[0030] Figure 6 The injections shown at 5, 20, 40, 80, and 120 minutes for reconstruction had [ 18 PET images of mice with F]AlF-NOTA-HSA. Most [ 18 F]AlF-NOTA-HSA remained in the bloodstream 120 minutes after intravenous injection, which was due to Figure 6 The very high level of radioactive accumulation in the left ventricular cavity indicated by the PET image shown. Detailed Implementation

[0031] The present invention provides or utilizes radiolabeled compounds.

[0032] Suitablely, the radiolabeled compound is a compound of formula (I):

[0033] (I)

[0034] in: R 1 R² is independently selected from halogens and –SR³, wherein R³ is independently selected from C. 1-12 Alkyl, C 1-12 alkenyl, C 1-12 alkynyl group and C 6-10 Aryl; L represents a direct bond or connector, where the connector is C. 1-20 Alkylene, the C 1-20 The alkylene group is unsubstituted or selected from hydroxyl, -COOH, C 1-12 Alkoxy, C 1-12 Alkyl and hydroxy-C 1-12 -One or more substituents of the alkyl group are used, wherein zero or one to ten carbon atoms in the alkylene chain are selected from C 6-10 aryl, -O-, -S-, -NR 4 -、-C(O)NR 4 -、-NR 4 The intervals between C(O)-, -C(O)-, -OC(O)- and -C(O)O- are partially replaced, where R 4 Is it hydrogen or C? 1-12 Alkyl, and wherein the C 6-10The arylene moiety is unsubstituted or selected from hydroxyl, C 1-12 Alkyl and C 1-12 Alkoxy groups may be substituted with one, two, three, or four substituents; X is absent or is PEG. 2-20 Group; and Q Indicates 18 The part marked with F; Or its pharmaceutically acceptable salt.

[0035] The inventor developed 18 F-labeled disubstituted maleimide compounds comprising (i) a disubstituted maleimide moiety that allows for site-specific bioconjugation of proteins via intramolecular disulfide bridges, and (ii) fluorine-18 labeling that allows for PET imaging. Compounds of formula (I) can be referred to as F-labeled disubstituted maleimide compounds. 18 F is a biological conjugation reagent.

[0036] Fluorine-18 has a half-life of about 110 minutes, which is compatible with the in vivo biological half-life of small proteins such as nanobodies and cytokines.

[0037] In one implementation, Q Indicates chelation 18 F-Al complex 18 The F-marked portion contains chelates. 18 F-Al complex 18 The F-marked portion is known in the art and may include, for example, chemical groups, such as [ 18 F]AlF-NOTA, [ 18 F]AlF-NODA, [ 18 F]AlF-NODAGA and [ 18 F]AlF-RESCA.

[0038] In the preferred embodiment, Q It indicates that it is selected from the following scriptures 18 The part marked with F: , and .

[0039] In one implementation, Q It indicates that it has the formula (Q) -A) 18 The part marked with F.

[0040] In another implementation, Q It indicates that it has the formula (Q) -B1) or (Q -B2) 18 The part marked with F. Q For example, it can have the formula (Q) -B1). Or, Q For example, it can have the formula (Q) -B2).

[0041] The compound of formula (I) contains a disubstituted maleimide moiety. The substituent is R... 1 And R² indicates. R 1 R² is independently selected from halogens and –SR³, wherein R³ is independently selected from C. 1-12 Alkyl, C 1-12 alkenyl, C 1-12 alkynyl group and C 6-10 Aryl.

[0042] It should be understood that R 1 R² can be the same as or different from R².

[0043] Preferably, R³ is C 6-10 Aryl. More preferably, R³ is phenyl (–Ph).

[0044] In the preferred embodiment, R 1 R² is independently selected from –Br, –I, and –SPh. More preferably, R 1 R² is –Br.

[0045] In one implementation, L represents a connector, wherein the connector is C 1-20 Alkylene, the C 1-20 The alkylene group is unsubstituted or selected from hydroxyl, -COOH, C 1-12 Alkoxy, C 1-12 Alkyl and hydroxy-C 1-12 -One or more substituents of the alkyl group are used, wherein zero or one to ten carbon atoms in the alkylene chain are selected from C 6-10 aryl, -O-, -S-, -NR 4 -、-C(O)NR 4 -、-NR 4 The intervals between C(O)-, -C(O)-, -OC(O)- and -C(O)O- are partially replaced, where R 4 Is it hydrogen or C? 1-12 Alkyl, and wherein the C 6-10 The arylene moiety is unsubstituted or selected from hydroxyl, C 1-12 Alkyl and C 1-12 The alkoxy group may be substituted with one, two, three, or four substituents.

[0046] Preferably, L represents a connector, wherein the connector is C 1-20 Alkylene, wherein 1 to 10 carbon atoms in the alkylene chain are selected from C 6-10 aryl, -C(O)NR 4 -、-NR 4 The intervals between C(O)-, -C(O)-, -OC(O)- and -C(O)O- are partially replaced, where R 4 Is it hydrogen or C? 1-12 Alkyl, and wherein the C 6-10 The aryl portion is unsubstituted.

[0047] More preferably, L represents a connector, wherein the connector is C 1-20 Alkylene, wherein 1 to 10 carbon atoms in the alkylene chain are selected from C 6-10 The spaces between aryl groups, -C(O)NH- and -NHC(O)- are partially replaced, and wherein the C 6-10 The aryl portion is unsubstituted.

[0048] In one implementation, L has the following formula: –(CH2) n –NH–C(O)–(CH2) m – or –(CH2) n –C(O)–NH–(CH2) m –, Where n is an integer from 0 to 18, m is an integer from 0 to 18, and n+m≤18.

[0049] In one embodiment, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18. Preferably, n is an integer from 1 to 10, 1 to 6, 1 to 4, 2 to 10, 2 to 6, 2 to 4, or 2 to 3. More preferably, n is 2.

[0050] In one embodiment, m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18. Preferably, m is an integer from 1 to 10, 1 to 6, 1 to 4, 1 to 3, or 1 to 2. More preferably, m is 1.

[0051] In another embodiment, L has the following formula:

[0052] Where p is an integer from 0 to 18, and q is an integer from 0 to 18, and p+q≤18.

[0053] In one implementation, p is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18. Preferably, p is an integer from 1 to 10, 1 to 6, 1 to 4, 2 to 10, 2 to 6, 2 to 4, or 2 to 3.

[0054] In one implementation, q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18. Preferably, q is an integer from 1 to 10, 1 to 6, 1 to 4, 1 to 3, or 1 to 2. More preferably, q is 1.

[0055] In yet another implementation, L has the following formula:

[0056] Where r is an integer from 0 to 18, and s is an integer from 0 to 18, and r+s≤18.

[0057] In one implementation, r is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18. Preferably, r is an integer from 1 to 10, 1 to 6, 1 to 4, 2 to 10, 2 to 6, 2 to 4, or 2 to 3.

[0058] In one embodiment, s is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18. Preferably, s is an integer from 1 to 10, 1 to 6, 1 to 4, 1 to 3, or 1 to 2. More preferably, s is 2.

[0059] In one implementation, X does not exist.

[0060] In the alternative implementation, X exists. When X exists, X is PEG. 2-20 Group. PEG 2-20 Groups can have the following formula: –(O–CH2–CH2) x – or –(CH2–CH2–O) x –, Where x is an integer from 2 to 20.

[0061] In one implementation, X exists when L is a direct bond.

[0062] In a preferred embodiment, the compound of formula (I) is a compound of the following formula:

[0063] This compound can be called [ 18F]AlF-NOTA-dibromomaleimide.

[0064] Another aspect of the present invention provides a reference compound of formula (II):

[0065] (II)

[0066] Where R 1 R², L, and X are as defined above for radiolabeled compounds of formula (I); and Q represents compounds containing chelates. 19 F-Al complex 19 The F-labeled portion; or its pharmaceutically acceptable salt.

[0067] The reference compound of formula (II) can be used as a reference compound for the radiolabeled compound of formula (I), wherein the reference compound has a substitute for the one in formula (I). 18 F atoms (radioactive label) 19 F atoms (more commonly referred to as F atoms). Reference compounds can, for example, help identify the radiolabeled compound.

[0068] Appropriately, Q may represent a selection from the following: , and .

[0069] In one implementation, Q represents a part having formula (QA).

[0070] In another embodiment, Q represents a portion having formula (Q-B1) or (Q-B2). Q may, for example, have formula (Q-B1). Alternatively, Q may, for example, have formula (Q-B2).

[0071] Preferably, the reference compound of formula (II) is a compound of the following formula:

[0072] Another aspect of the present invention provides a precursor compound of formula (III):

[0073] (III)

[0074] Where R 1 R², L, and X are as defined above for radiolabeled compounds of formula (I); and Y indicates the ability to chelate. 18 F-Al complexes (and / or those capable of chelating) 19 A portion of an F-Al complex; or a pharmaceutically acceptable salt thereof.

[0075] The precursor compound of formula (III) can be used as a precursor compound of the radiolabeled compound of formula (I).

[0076] The precursor compound of formula (III) can be used as a precursor compound of the reference compound of formula (II).

[0077] Y indicates the ability to chelate. 18 The F-Al complex portion. This type of portion can also chelate. 19 F-Al complex.

[0078] Appropriately, Y may represent a selection from the following: , and .

[0079] In one implementation, Y represents a part having the formula (YA).

[0080] In another embodiment, Y represents a portion having formula (Y-B1) or (Y-B2). Y may, for example, have formula (Y-B1). Alternatively, Y may, for example, have formula (Y-B2).

[0081] Preferably, the precursor compound of formula (III) is a compound of the following formula:

[0082] Another aspect of the present invention provides a pharmaceutical composition comprising a radiolabeled compound of formula (I) as defined above and a pharmaceutically acceptable carrier.

[0083] In one embodiment, the pharmaceutical composition comprises a radiolabeled compound of formula (I), wherein Q Having the formula (Q) -A).

[0084] In another embodiment, the pharmaceutical composition comprises a radiolabeled compound of formula (I), wherein Q Having the formula (Q) -B1); radiolabeled compounds of formula (I), wherein Q Having the formula (Q) -B2); or combinations thereof. Radiolabeled compounds of formula (I) (where Q Having the formula (Q) -B1)) and radiolabeled compounds of formula (I) (where Q Having the formula (Q) -B2)) can exist, for example, as a racemic mixture.

[0085] Another aspect of the present invention provides a method for preparing a precursor compound of formula (III), the method comprising the following steps according to the reaction scheme: c) Reacting a compound of formula (V) with a compound of formula (VI) via a coupling reaction to form a compound of formula (IV); and d) Deprotecting the compound of formula (IV) to form the compound of formula (III);

[0086] Where R 1 R², L and X are as defined above for radiolabeled compounds of formula (I); Y is defined above for the precursor compound of formula (III); L 1 L² and L² are parts that can be coupled together to form group L; and Y p It is a portion containing at least one protecting group, which can be deprotected to provide group Y.

[0087] L 1 L² and L² are the parts that can be coupled together to form the group L.

[0088] The coupling reaction in step (a) can be, for example, amide coupling. In such embodiments, L 1 One of L² has a –NH2 end group, and L 1 And another one in L² has a –COOH end group, which can couple together to form an amide bond.

[0089] In one implementation, L 1 It has the formula –(CH2) n –NH2, and L² has the formula HOOC–(CH2). m – where n and m are as defined above.

[0090] In another implementation, L 1 It has the formula –(CH2) n –COOH, and L² has the formula H₂N–(CH₂). m – where n and m are as defined above.

[0091] Y p It is a portion containing at least one protecting group, and Y p It can be deprotected to provide group Y. For example, group Y p It may contain at least one protecting group that protects heteroatoms present in group Y, such as, for example, oxygen atoms.

[0092] Appropriately, Y p It can be selected from the following parts: , and .

[0093] Where R 10 R 11 R 12 R 13 and R 14 It is a protecting group. Any suitable protecting group can be used. Each protecting group can be the same or different. Protecting groups can be selected from, for example, – t Bu, –Bn, –CMe2Ph and 4-methoxybenzyl.

[0094] In one implementation, Y p It indicates that it has the formula (Y) p -A) part.

[0095] In another implementation, Y p It indicates that it has the formula (Y) p -B1) or (Y p -B2) part. Y p For example, it can have the formula (Y) p -B1). Or, Y p For example, it can have the formula (Y) p -B2).

[0096] Another aspect of the present invention provides a method for preparing a radiolabeled compound of formula (I), the method comprising radiolabeling a precursor compound. Preferably, the precursor compound may be a precursor compound of formula (III).

[0097] The radiolabeled compound of formula (I) contains 18 F is a radioactive isotope. 18 F-fluoride is a commonly used radioactive isotope for PET.

[0098] Suitably, the method includes the step of reacting a precursor compound of formula (III) with a radiolabeling reagent containing a fluoride-18 source according to the following reaction scheme to form a radiolabeled compound of formula (I):

[0099] Where R 1 R², L, X and Q As defined above for the radiolabeled compound of formula (I); and Y as defined above for the precursor compound of formula (III).

[0100] In one embodiment, the radiolabeling reagent comprises a fluoride-18 source and Al 3+ source.

[0101] The fluoride-18 source can be, for example, K. 18 F, Na 18 F, Li 18 F, Cs 18 F、tBu4N 18 F、Et4N 18 F and / or combinations thereof. Preferably, the fluoride-18 source is K. 18 F.

[0102] Al 3+ The source can be, for example, AlCl3, AlBr3, AlI3, Al(CH3CO2)3 and / or combinations thereof. Preferably, Al 3+ The source is AlCl3.

[0103] Another aspect of the present invention provides a method for preparing a reference compound of formula (II), the method comprising reacting a precursor compound of formula (III) with a fluoride-19 source and Al according to the following reaction scheme. 3+ The source reaction is used to form the reference compound of formula (II):

[0104] Where R 1 R², L and X are as defined above for radiolabeled compounds of formula (I); Y is as defined above for the precursor compound of formula (III); and Q is defined above for the reference compound of formula (II).

[0105] Another aspect of the invention provides a radiolabeled compound of formula (I) as defined above for use in a diagnostic method performed on a human or animal body using positron emission tomography (PET).

[0106] Appropriately, diagnostic methods may include labeling proteins with radiolabeled compounds of formula (I).

[0107] Preferably, the protein is a natural protein.

[0108] Suitablely, the protein may be selected from antibodies, single-chain fragment variable (SCFV), antigen-binding fragments (Fab), nanobodies, and / or cytokines.

[0109] In a preferred embodiment, the diagnostic method includes labeling a HER2-targeting nanobody with a radiolabeled compound of formula (I). Suitably, the HER2-targeting nanobody may be NM-02.

[0110] In one implementation, the diagnostic method is a method for diagnosing cancer.

[0111] Preferably, the diagnostic method can be a method for identifying the location of the tumor.

[0112] definition

[0113] It should be understood that the wavy lines (as shown below) in any chemical structure or part represented herein indicate the attachment points of that structure or part.

[0114]

[0115] As used in this article, the term "hydrogen" or "hydrogen atom" refers to the –H portion.

[0116] As used in this article, the terms “halogenated,” “halogen,” or “halogen atom” refer to the –F, –Cl, –Br, or –I moiety.

[0117] As used in this article, the term "hydroxyl" or "hydroxyl" refers to the –OH moiety.

[0118] prefix "C" x “C” x-y "" indicates the number or range of carbon atoms present in the group. Therefore, the term "C" 1-12 "Alkyl" refers to an alkyl group having 1 to 12 carbon atoms.

[0119] The term "alkyl" refers to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a saturated hydrocarbon compound having, for example, 1 to 20 carbon atoms, which can be straight-chain, branched, or cyclic. Therefore, the term "alkyl" includes the following cycloalkyl subclasses. Examples of alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), propyl (C3), butyl (C4), pentyl (C5), hexyl (C6), heptyl (C7), octyl (C8), nonyl (C9), and decyl (C1). 10 Examples of straight-chain alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), n-propyl (C3), n-butyl (C4), n-pentyl (C5), n-hexyl (C6), and n-heptyl (C7). Examples of branched-chain alkyl groups include, but are not limited to, isopropyl (C3), isobutyl (C4), sec-butyl (C4), tert-butyl (C4), isopentyl (C5), and neopentyl (C5).

[0120] The term "cycloalkyl" refers to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a cyclic saturated hydrocarbon compound having, for example, 3 to 20 carbon atoms. "Cycloalkyl" includes monocyclic and polycyclic compounds, including bicyclic compounds. Examples of monocyclic cycloalkyls include, but are not limited to, cyclopropyl (C3), cyclobutyl (C4), cyclopentyl (C5), cyclohexyl (C6), cycloheptyl (C7), and methylcyclopropyl (C4). Cycloalkyls include bicyclic molecules in which two rings share one, two, or three or more atoms. The term "fused cycloalkyl" refers to a bicyclic cycloalkyl compound in which each ring shares two adjacent atoms with the other ring. The second ring of a bicyclic cycloalkyl compound can be selected from saturated, unsaturated, and aromatic rings. Examples of saturated polycyclic hydrocarbon compounds include, but are not limited to, thujane (C4). 10 ), carane (C 10 ), pinane (C 10 ), Bornane (C 10 Norcarane (C7), Norpinane (C7), Norbornane (C7), Adamantane (C7) 10 ) and decalin (C 10 ).

[0121] The term "alkenyl" refers to a monovalent hydrocarbon moiety obtained by removing a hydrogen atom from a carbon atom of a (partially) unsaturated hydrocarbon compound having one or more carbon-carbon double bonds and, for example, having 2 to 20 carbon atoms, which can be straight-chain, branched, or cyclic. Therefore, the term "alkenyl" includes the following cycloalkenyl subclasses. Examples of alkenyl groups include, but are not limited to, vinyl (vinyl, -CH=CH2), 1-propenyl (-CH=CH-CH3), and 2-propenyl (allyl, -CH-CH=CH2).

[0122] The term "cycloalkenyl" refers to a monovalent cyclic hydrocarbon moiety obtained by removing hydrogen atoms from carbon atoms of a cyclic (partially) unsaturated hydrocarbon compound containing one or more carbon-carbon double bonds and, for example, having 3 to 20 carbon atoms. "Cycloalkenyl" includes monocyclic and polycyclic compounds, including bicyclic compounds. Examples of unsaturated monocyclic hydrocarbon compounds include, but are not limited to, cyclopropene (C3), cyclobutene (C4), cyclopentene (C5), cyclohexene (C6), methylcyclopropene (C4), and dimethylcyclopropene (C5). Examples of unsaturated polycyclic hydrocarbon compounds include, but are not limited to, camphene (C6). 10 ), limonene (C 10 ) and pinene (C 10 ).

[0123] The term "alkynyl" refers to a monovalent hydrocarbon moiety obtained by removing a hydrogen atom from a carbon atom of a (partially) unsaturated hydrocarbon compound having one or more carbon-carbon triple bonds and, for example, having 2 to 20 carbon atoms. This moiety can be straight-chain, branched, or cyclic. Therefore, the term "alkynyl" includes the following cycloalkynyl subclasses. Examples of alkynyl groups include, but are not limited to, ethynyl (ethinyl, -C≡CH) and 2-propynyl (propynyl, -CH2-C≡CH).

[0124] The term "cycloalkynyl" refers to a monovalent cyclic hydrocarbon moiety obtained by removing a hydrogen atom from a carbon atom of a cyclic (partially) unsaturated hydrocarbon compound containing one or more carbon-carbon triple bonds and, for example, having 2 to 20 carbon atoms. "Cycloalkynyl" includes monocyclic and polycyclic compounds, including bicyclic compounds.

[0125] The term "aryl" refers to the monovalent moiety obtained by removing a hydrogen atom from a ring atom of an aromatic compound; this moiety can be, for example, a monocyclic or bicyclic group. Aromatic compounds from which aryl groups are derived can contain a full-carbon ring structure or can be heteroaromatic compounds containing one or more heteroatoms in their ring structure. Therefore, the term "aryl" includes the following heteroaryl subclasses. Aryl groups having a full-carbon ring structure can, for example, have 3 to 20 carbon atoms. Examples of aryl groups include, but are not limited to, phenyl (derived from benzene) and naphthyl (derived from naphthalene).

[0126] The term "heteroaryl" refers to the monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heteroaryl compound, which can be, for example, a monocyclic or bicyclic group. The heteroaryl moiety may, for example, contain one or more N, O, S, or P atoms, and may, for example, contain 1 to 20 carbon atoms. Examples of heteroaryl moieties include, but are not limited to, pyridinyl (or pyridyl, derived from pyridine), pyrimidinyl (derived from pyrimidine), pyrazinyl (derived from pyrazine), pyrroleyl (derived from pyrrole), imidazolyl (derived from imidazolium), pyrazolyl (derived from pyrazole), furanyl (derived from furan), thiophene (derived from thiophene), oxazolyl (derived from oxazole), isoxazolyl (derived from isoxazole), and thiazolyl (derived from thiazolyl).

[0127] The term "heterocyclic group" refers to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound, which can be, for example, a monocyclic or bicyclic group. Heterocyclic groups can, for example, contain one or more N, O, S, or P atoms, and can, for example, contain 1 to 20 carbon atoms.

[0128] The term "alkoxy" or "alkoxyl" refers to an alkyl-oxy group, where the alkyl group is as defined above. Examples of alkoxy groups include, but are not limited to, -OMe (methoxy), -OEt (ethoxy), and -O (...).n Pr) (n-propoxy), -O( i Pr) (isopropoxy), -O( n Bu) (n-butoxy group), -O( s Bu) (sec-butoxy), -O( i Bu) (isobutyroxy) and -O( t Bu) (tert-butoxy).

[0129] The term "acyl" refers to a group represented by the general formula –C(O)-alkyl, such as –C(O)-alkyl.

[0130] The term "alkylene" refers to a divalent hydrocarbon moiety obtained by removing two hydrogen atoms from the same carbon atom of a saturated hydrocarbon compound having, for example, 1 to 20 carbon atoms, or by removing one hydrogen atom from each of two different carbon atoms of the saturated hydrocarbon compound, which can be straight-chain, branched, or cyclic. Therefore, the term "alkylene" includes the following cycloalkylene subclasses. Examples of straight-chain alkylenes include, but are not limited to, -CH2- (methylene), -CH2CH2- (ethylene), -CH2CH2CH2- (propylene), and -CH2CH2CH2CH2- (butylene). Examples of branched alkylenes include, but are not limited to, -CH(CH3)-, -CH(CH3)CH2-, and -CH(CH3)CH2CH2-.

[0131] The term "cycloalkylene" refers to a divalent moiety obtained by removing two hydrogen atoms from the same carbon atom of a cyclic saturated hydrocarbon compound having, for example, 3 to 20 carbon atoms, or by removing one hydrogen atom from each of two different carbon atoms of the cyclic saturated hydrocarbon compound. "Cycloalkylene" includes monocyclic and polycyclic compounds, including bicyclic compounds. Examples of cyclic alkylenes include, but are not limited to, cyclopentylenes (e.g., cyclopentyl-1,3-ene) and cyclohexylenes (e.g., cyclohexyl-1,4-ene).

[0132] The term "alkenyl" refers to a divalent hydrocarbon moiety obtained by removing two hydrogen atoms from the same carbon atom of a (partially) unsaturated hydrocarbon compound having one or more carbon-carbon double bonds and, for example, having 2 to 20 carbon atoms, or by removing one hydrogen atom from each of two different carbon atoms of the (partially) unsaturated hydrocarbon compound, which can be straight-chain, branched, or cyclic. Therefore, the term "alkenyl" includes the following cyclic alkenyl subclasses. Examples of straight-chain alkenyl groups include, but are not limited to, -CH=CH- (vinylene), -CH=CHCH2-, -CH2-CH=CH2-, and -CH=CHCH2CH2-. Examples of branched alkenyl groups include, but are not limited to, -C(CH3)=CH-, -C(CH3)=CHCH2-, and -CH=CHCH(CH3)-.

[0133] The term "cycloene-olefin" refers to a divalent moiety obtained by removing two hydrogen atoms from the same carbon atom of a cyclic (partially) unsaturated hydrocarbon compound having one or more carbon-carbon double bonds and, for example, having 3 to 20 carbon atoms, or by removing one hydrogen atom from each of two different carbon atoms of the cyclic (partially) unsaturated hydrocarbon compound. "Cycloene-olefin" includes monocyclic and polycyclic compounds, including bicyclic compounds. Examples of cycloene-olefins include, but are not limited to, cyclopentenyl (e.g., 4-cyclopenten-1,3-olefin) and cyclohexenyl (e.g., 2-cyclohexen-1,4-olefin; 3-cyclohexen-1,2-olefin; 2,5-cyclohexadiene-1,4-olefin).

[0134] The term "ynynyl" refers to a divalent moiety obtained by removing two hydrogen atoms from the same carbon atom of a (partially) unsaturated hydrocarbon compound having one or more carbon-carbon triple bonds and, for example, having 2 to 20 carbon atoms, or by removing one hydrogen atom from each of two different carbon atoms of the (partially) unsaturated hydrocarbon compound, which can be straight-chain, branched, or cyclic. Therefore, the term "ynynyl" includes the following subclass of cyclic ynynyl groups.

[0135] The term "cycloynylene" refers to a divalent moiety obtained by removing two hydrogen atoms from the same carbon atom of a cyclic (partially) unsaturated hydrocarbon compound containing one or more carbon-carbon triple bonds and having, for example, 3 to 20 carbon atoms, or by removing one hydrogen atom from each of two different carbon atoms of the cyclic (partially) unsaturated hydrocarbon compound. "Cycloynylene" includes monocyclic and polycyclic compounds, including bicyclic compounds.

[0136] The term "arylene" refers to a divalent moiety obtained by removing two hydrogen atoms from the same carbon atom of an aromatic compound or by removing one hydrogen atom from each of two different carbon atoms of the aromatic compound; this moiety can be, for example, a monocyclic or bicyclic group. Aromatic compounds from which arylene groups are derived can contain a full-carbon ring structure or can be heteroaromatic compounds containing heteroatoms in their ring structure. Therefore, the term "arylene" includes the following subclasses of heteroarylene. Arolene groups having a full-carbon ring structure can, for example, have 3 to 20 carbon atoms.

[0137] The term "hybrid aryl" refers to a divalent moiety obtained by removing two hydrogen atoms from the same ring atom of a heteroaromatic compound or by removing one hydrogen atom from each of two different ring atom of the heteroaromatic compound. This moiety can be, for example, a monocyclic or bicyclic group. The hybrid aryl moiety can, for example, contain one or more N, O, S, or P atoms, and can, for example, contain 1 to 20 carbon atoms.

[0138] The term "PEG" refers to the divalent moiety obtained by removing two hydrogen atoms from two atoms of a PEG (polyethylene glycol) chain, which, for example, has 2 to 20 –(O–CH2–CH2)– repeating units. The prefix "PEG" x PEG x-y "" indicates the number of repeating units present in the group, or a range of repeating units. Therefore, the term "PEG" 2-20 "Group" refers to a PEG chain with 2 to 20 repeating units, corresponding to the formula –(O–CH2–CH2). x – or –(CH2–CH2–O) x – where x is an integer from 2 to 20.

[0139] Examples of PEG groups include, but are not limited to, PEG2 (n is 2), PEG3 (n is 3), PEG4 (n is 4), PEG5 (n is 5), PEG6 (n is 6), PEG7 (n is 7), PEG8 (n is 8), PEG9 (n is 9), and PEG 10 (n is 10), PEG 11 (n is 11), PEG 12 (n is 12), PEG 13 (n is 13), PEG 14 (n is 14), PEG 15 (n is 15), PEG 16 (n is 16), PEG 17 (n is 17), PEG 18 (n is 18), PEG 19 (n is 19) and PEG 20 (n is 20).

[0140] The term "substituent" refers to a chemical moiety that is covalently attached to a parent group or, where appropriate, fused to a parent group.

[0141] The phrase "optionally substituted" refers to a parent group that can be unsubstituted or can be substituted by one or more (e.g., one or two) substituents. Substituents on an "optionally substituted" group can be selected, for example, from alkyl, alkenyl, alkynyl, aryl, heteroaryl and heterocyclic groups; carboxylic acids and carboxylate ions; carboxylic esters; carbamates; alkoxy groups; ketones and aldehydes; aminos and amides; –OH; –CN; –NO2; and halogens.

[0142] As used herein, the term "protecting group" refers to a group that can protect a functional group (e.g., a heteroatom, such as an oxygen atom), which can be removed after the reaction in which protection is applied without affecting the rest of the molecule. Protecting groups are well known and listed in standard textbooks such as Kocienski PJ, Protecting Groups, 3rd ed., Georg Thieme Verlag, New York, 2005; and Greene TW, Wuts PGM, Protective Groups In Organic Synthesis, 3rd ed., John Wiley & Sons, New York, 1998.

[0143] As used in this article, the term "small protein" can refer to proteins such as nanobodies and cytokines. A small protein can be, for example, a protein of about 15 kDa.

[0144] Certain compounds may exist in one or more specific geometric, enantiomer, diastereomer, tautomer, or conformational forms. Unless otherwise stated, references to a particular compound include all such isomer forms, including (all or part) racemic mixtures and other mixtures. Methods for preparing and isolating such isomer forms are known in the art.

[0145] Pharmaceutically acceptable salt forms include pharmaceutically acceptable acidic / anionic or basic / cationic salts.

[0146] Examples of pharmaceutically acceptable acidic / anionic salts include acetates, benzenesulfonates, benzoates, bicarbonates, tartrates, bromides, calcium edetate, camphorsulfonates, carbonates, chlorides, citrates, dihydrochlorides, edetate, ethanedisulfonate, estolate, ethanesulfonate, fumarate, gluconate, gluconate, glutamate, glycolyllarsanilate, and hexylresorcinol. (esorcinate), hydrobromide, hydrochloride, hydroxynaphthyl carboxylate, iodide salts, hydroxyethyl sulfonate, lactate, lactobionate, malate, maleate, malonate, mandelate, methanesulfonate, methyl sulfate, mucilage, naphthalene sulfonate, nitrate, dihydroxynaphthyl carboxylate, pantothenate, phosphate / bisphosphate, polygalacturonic acid salt, salicylate, stearate, basic acetate, succinate, sulfate, hydrogen sulfate, tannate, tartrate, teoclate, toluenesulfonate, and triethyl iodide salts.

[0147] Examples of pharmaceutically acceptable basic / cationic salts include sodium, potassium, calcium, magnesium, diethanolamine, N-methyl-D-glucosamine, L-lysine, L-arginine, ammonium, ethanolamine, piperazine, and triethanolamine.

[0148] If the compound is anionic, or has a functional group that can be anionic, it can form a salt with a suitable cation. Examples of suitable inorganic cations include alkali metal ions, such as Na+. + and K + Alkaline earth metal cations, such as Ca 2 + and Mg 2+ and other cations, such as Al 3+ Examples of suitable organic cations include ammonium ions (i.e., NH4+). + ) and substituted ammonium ions (e.g., NH3R) + NH2R 2+ NHR 3+ NR 4+ (where R is an alkyl group).

[0149] If a compound is cationic, or has a functional group that can be cationic, it can form a salt with a suitable anion. Examples of suitable inorganic anions include those derived from the following inorganic acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, nitrous acid, phosphoric acid, and phosphorous acid. Examples of suitable organic anions include those derived from the following organic acids: 2-acetoxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, edemanic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheponic acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxynaphthoic acid, hydroxyethanesulfonic acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, methanesulfonic acid, viscous acid, oleic acid, oxalic acid, palmitic acid, dihydroxynaphthoic acid, pantothenic acid, phenylacetic acid, benzenesulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, p-aminobenzenesulfonic acid, tartaric acid, toluenesulfonic acid, and valeric acid.

[0150] If a compound has both a cationic functional group or a functional group that can become a cationic group, and an anionic functional group or a functional group that can become an anionic group, then the compound can exist as a zwitterion.

[0151] The following non-restrictive examples are provided for illustrative purposes only.

[0152] Example

[0153] abbreviation

[0154] NOTA stands for 1,4,7-triazacyclononane-1,4,7-triacetic acid.

[0155] 1. Synthetic Chemistry

[0156] Precursor compound 5 (N-[2-(NOTA-amido)ethyl]-3,4-dibromo-maleimide, abbreviated as NOTA-dibromo-maleimide) and its non-radioactive reference compound 6 ([ 19 F]AlF-NOTA-3,4-dibromomaleimide, abbreviated as [ 19 F]AlF-NOTA-dibromomaleimide).

[0157]

[0158] Option 1

[0159] N-(methoxycarbonyl)-3,4-dibromomaleimide (compound 1) 1

[0161] 3,4-Dibromomaleimide (1.00 g, 3.90 mmol) and N 1-Methylmorpholine (0.43 mL, 3.90 mmol) was dissolved in THF (35 mL). Next, methyl chloroformate (0.3 mL, 3.90 mmol) was added to the reaction mixture, and the mixture was stirred at room temperature for 20 minutes. The crude mixture was then diluted with DCM (40 mL) and partitioned in water. The combined organic layers were dried over MgSO4 and concentrated under vacuum to give the title compound (1.09 g, 90%) as a pink crystalline solid. 1 HNMR (400 MHz, CDCl3) δ 4.00 (s, 3H); 13 C NMR (101 MHz, CDCl3) δ 158.25,145.94, 130.43, 53.80

[0162] N-(2-Boc-aminoethyl)-3,4-dibromomaleimide (compound 2) 2

[0164] N-(methoxycarbonyl)-3,4-dibromomaleimide (1) (1.00 g, 3.50 mmol) was dissolved in DCM (10 mL). A solution of N-Boc-ethylenediamine (0.50 g, 3.50 mmol) in DCM (10 mL) was added dropwise, and the resulting reaction mixture was stirred at room temperature for 2 hours. Next, the crude reaction mixture was quenched with water (20 mL) and extracted with DCM (3 x 50 mL). The combined organic layers were dried over MgSO4, concentrated under vacuum, and purified by rapid column chromatography, eluting with DCM / MeOH (0 to 10% methanol over 20 column volumes) to give the title compound (348 mg, 25%) as a white powder. 1 H NMR (400 MHz, CDCl3) 3.67 (m, 2H), 3.32–3.25 (m, 2H), 1.33 (s, 9H). 13 C NMR (101 MHz, CDCl3) δ 163, 155, 128, 79, 39, 38, 27.

[0165] N-(2-Aminoethyl)-3,4-dibromo-maleimide (Compound 3) 3

[0167] N-(2-Boc-aminoethyl)-3,4-dibromomaleimide (2) (300 mg, 0.7 mmol) was dissolved in a 1:1 DCM / TFA solution (2 mL) and stirred at room temperature for 2 hours. The reaction mixture was then concentrated under vacuum to give the title compound (273 mg, 95%) as a white powder. 1 H NMR (400 MHz, D2O) δ 3.90 (m, 2H), 3.21(m, 2H) 13 C NMR (101 MHz, DO) δ 166, 129, 38, 37.

[0168] N-{2-[(tert-butyl)2-NOTA-amido]ethyl]-3,4-dibromo-maleimide (compound 4) 4

[0170] Nota(tBu)2 (50 mg, 0.12 mmol) and EEDQ (35 mg, 0.14 mmol) were dissolved in DMF (300 mL) and stirred at room temperature for 30 minutes. Next, a solution of N-(2-aminoethyl)-3,4-dibromo-maleimide (3) (40 mg, 0.10 mmol) in DMF (200 mL) was added to the mixture and stirred at room temperature overnight. The reaction mixture was concentrated under vacuum. The crude mixture was purified by HPLC using a semi-preparative Zorbax SB-300 C18 using solvent B (solvent A: H2O / 0.1% FA, solvent B: MeCN / 0.1% FA) for 30 minutes to give the title compound (31 mg, 45%) as a yellow powder. 1 H NMR (400 MHz, CDCl3) δ 9.05 (t, J = 5.8 Hz,1H), 4.10 (s, 2H), 3.81 – 3.76 (m, 2H), 3.58 (d, J = 3.6 Hz, 4H), 3.47 (d, J= 3.9 Hz, 2H), 3.32 (s, 2H), 3.21 (s, 4H), 3.14 (s, 2H), 2.95 (d, J = 14.0Hz, 4H), 1.46 (s, 18H); 13 C NMR (101 MHz, CDCl3) δ 170, 167, 164, 130, 82, 58,56, 52, 51, 48, 39, 38, 28; HR-MS (EI, m / z) [M+H] + :C 26 H 42 79 Br2N5O7 + The calculated value is 694.1451; the measured value is 694.1465.

[0171] N-[2-(NOTA-amido)ethyl]-3,4-dibromo-maleimide (compound 5) 5

[0173] N-{2-[(tert-butyl)2-NOTA-amido]ethyl]-3,4-dibromo-maleimide (4) (30.0 mg, 0.04 mmol) was dissolved in a 1:1 DCM / TFA solution (2 mL) and stirred at room temperature for 2 hours. The reaction mixture was then concentrated under vacuum. The crude mixture was purified by HPLC using a semi-preparative Zorbax SB-300 C18 instrument within 30 minutes to 5% to 95% of solvent B (solvent A: H2O / 0.1% FA, solvent B: MeCN / 0.1% FA), yielding the title compound (17.4 mg, 75%) as a yellow powder. 1 ¹H NMR (400 MHz, methanol-d⁴) δ 3.83 (s, 4H), 3.76 (s, 2H), 3.45 (s, 2H), 3.40 (s, 2H), 3.14 (s, 8H), 2.86 (s, 4H); 13 C10 NMR (101 MHz, methanol-d4) δ 172, 171, 164, 129, 59, 55, 50, 49, 49, 39, 38; HR-MS (EI, m / z) [M+H] + :C 18 H 26 79 Br2N5O7 + The calculated value is 582.01299; the measured value is 582.0210.

[0174] [ 19 F]AlF-NOTA-3,4-dibromomaleimide (compound 6) 6

[0176] N-[2-(NOTA-amido)ethyl]-3,4-dibromo-maleimide (5) (1.0 mg, 1.71 mmol) was dissolved in NaOAc buffer (20 mL, 0.10 M, pH 4.5) and reacted with AlCl3. The mixture was prepared by mixing 6H₂O (5.10 mmol, 51 mL, 0.10 M in 0.10 M pH 4.5 NaOAc buffer). After adding 51 mL of 157 M NaF dissolved in 0.1 M pH 4.5 NaOAc buffer, the resulting mixture was stirred at room temperature for 5 minutes. The reaction mixture was stirred at 100 °C for 30 minutes and then cooled to room temperature. The crude mixture was purified by HPLC using a semi-preparative Zorbax SB-300 C18 instrument using the following method: 5% to 95% solvent B (solvent A: H₂O / 0.1% FA, solvent B: MeCN / 0.1% FA) within 30 minutes to give the title compound (0.3 mg, 30%) as a white powder. HR-MS (EI, m / z) [M+H] + :C 18 H 24 Al 79 Br2 19 FN5O7 + The calculated value is 625.9842; the measured value is 625.9842.

[0177] 2. Radiochemistry

[0178] According to the following scheme 2, use Al 18 F / NOTA chelation chemistry, radiosynthesis of radiolabeled compound 7 with a moderate radiochemical yield of 51 ± 7% (n=3) 18 The total production time is approximately 60 minutes (F]AlF-NOTA-dibromomaleimide).

[0179]

[0180] Option 2

[0181] [ 18 F]AlF-NOTA-3,4-dibromomaleimide (compound 7) 7

[0183] Water 18 F-fluoride (approximately 500 MBq) was captured in a QMA filter cartridge (Waters Sep-Pak Lightweight, pretreated with 10 mL of water) and released into a 5 mL Wheaton vial with 0.9% saline (300 mL). Glacial acetic acid (10 mL) and AlCl3 were then added. 6H2O (0.24 mmol, 48 mL, 5.0 mM in 0.10 M pH 4.5 NaOAc buffer) was added to the reaction vial. N-[2-(NOTA-amido)ethyl]-3,4-dibromo-maleimide (5) (0.50 mg, 0.86 mmol) in EtOH (200 mL) was added to the resulting solution. The reaction mixture was heated at 70 °C for 25 min and then cooled to room temperature. The crude mixture was purified by HPLC using a semi-preparative HPLC column with 5% to 95% solvent B (solvent A: 0.01 M NH4OAc, solvent B: MeCN) over 30 min. The HPLC retention time of the title compound was 10.45 min. It was collected from the HPLC and diluted to 10% acetonitrile aqueous solution. 18 F]AlF-NOTA-3,4-dibromomaleimide was retained on a Sep-PakC-18 lightweight filter cartridge (pre-activated with 5 mL of methanol followed by 5 mL of water). The filter cartridge was washed with 2 mL of water and then eluted with 1.0 mL of ethanol. 18 F]AlF-NOTA-3,4-dibromomaleimide. Ethanol was removed with N2, and the radioactive residue was dissolved in DMSO for bioconjugation.

[0184] Radiolabeled compound 7 ([ 18 The identity of [F]AlF-NOTA-dibromomaleimide was confirmed by HPLC co-elution with its non-radioactive reference compound 2, such as Figures 1-3 As shown.

[0185] 3. Bioconjugation with NM-02

[0186] Subsequently, the HER2-targeting nanobody NM-02 was reduced with an equivalent of TCEP, followed by sequential reaction with radiolabeled compound 7 ([ 18 F]AlF-NOTA-dibromomaleimide) and an equivalent of its non-radioactive reference compound 6 are tandemly conjugated. Scheme 3 shows the radiolabeled compound 7 ([ 18 Bioconjugation of F]AlF-NOTA-dibromomaleimide with NM-02.

[0187] 7

[0189] Option 3

[0190] NM-02 (120 μg, 30 μL, 8.0 nmol) and TCEP (8.0 nmol, 1.0 equivalent) were incubated in DMSO (3.4 μL) at 37 °C for 1 hour. The pH of the reaction mixture was increased to pH 8.0 by adding borate buffer (0.5 μL, 1.0 M, pH 8.5). 18 F]AlF-NOTA-3,4-dibromomaleimide (7) (approximately 10 MBq) was added to the reactants and incubated at 37 °C for 15 min. Then, cold reference compound (6) (1.0 equivalent relative to NM-02) was added to the reactants and incubated for a final 15 min. The reactants were then diluted to 500 μL with PBS and purified using a PD MiniTrap G-25 column. 18 F]AlF-NOTA-NM-02 was eluted in PBS in 100 μL fractions.

[0191] After size exclusion purification [ 18 The bioconjugation efficiency of F]AlF-NOTA-NM-02 was 40±8% (n=3).

[0192] 4. [ 18 Tumor cell uptake of F]AlF-NOTA-NM-02

[0193] use[ 18 Both HER2-positive SKBR3 and MDA-MB-231 breast cancer cells were treated with F]AlF-NOTA-NM-02. Blocking studies were also conducted by pretreating SKBR3 cells with 100 times the amount of natural NM-02.

[0194] SKBR3 and MDA-MB-231 breast cancer cells (1x10) 6 Both were washed with PBS and resuspended in 200 µL PBS + 3% BSA. [Add] 18 F]AlF-NOTA-NM-02 (5.0 nM) was added and incubated with cells at 37°C for 1 h. For the blocking study, [F]AlF-NOTA-NM-02 (5.0 nM) was added to the cells. 18 Before incubating [F]AlF-NOTA-NM-02 (5.0 nM), NM-02 (500.0 nM) was incubated with tumor cells at 37°C for 1 h. [F]AlF-NOTA-NM-02 (5.0 nM) was then incubated with tumor cells. 18 After incubation with F]AlF-NOTA-NM-02, cells were washed twice with PBS and transferred to fresh Eppendorf tubes. Cell viability was counted in 1282 CompuGamma CS (LKB-Wallac) samples.

[0195] Figure 4 show[ 18 Tumor cell uptake of F]AlF-NOTA-NM-02. Samples blocked by pretreatment with 100X natural NM-02. Data shown are mean ± SD of three independent experiments performed in triplicate.

[0196] SKBR3 cells against [ 18 The uptake of F]AlF-NOTA-NM-02 is approximately 14%. Most of this uptake can be blocked by natural NM-02. HER2-negative MDA-MB-231 breast cancer cells, however, are affected by […]. 18 The intake of F]AlF-NOTA-NM-02 is very low.

[0197] New 18 F-Bioconjugation reagent [ 18 F]AlF-NOTA-dibromomaleimide (compound 7) specifically labeled the HER2-targeting nanobody NM-02 via its reduced intramolecular disulfide bridge site. 18 F]AlF-NOTA-dibromomaleimide-labeled NM-02 has been shown to be specifically taken up by HER2-positive SKBR3 human breast cancer cells.

[0198] 5. Bioconjugation with IL-4DE

[0199] IL-4DE was reduced with one equivalent of TCEP, followed by sequential reaction with radiolabeled compound 7 ([ 18 F]AlF-NOTA-dibromomaleimide) and an equivalent of its non-radioactive reference compound 6 are tandemly conjugated. Scheme 4 shows the radiolabeled compound 7 ([ 18 Bioconjugation of F]AlF-NOTA-dibromomaleimide with IL-4DE.

[0200] 7

[0202] Option 4

[0203] After size exclusion purification [ 18 The bioconjugation efficiency of [F]AlF-NOTA-IL-4DE was 53±5% (n=3). Purified [ 18 F]AlF-NOTA-IL-4DE was stable in PBS at 37°C for at least 4 hours. 18 No oligomer formation was observed during the generation of F]AlF-NOTA-IL-4DE.

[0204] 6. Cellular uptake of [18F]AlF-NOTA-IL-4DE

[0205] The study investigated the effects of 4αβ-expressing T4-CAR T cells, inactivated, non-transduced T cells, peripheral blood mononuclear cells (PBMCs), ErbB-positive IL-4 receptor-expressing SKOV3 ovarian cancer and HN3 head and neck cancer cells on […]. 18 In vitro uptake of F]AlF-NOTA-IL-4DE.

[0206] T4-CAR T cells expressing 4αβ, inactivated, non-transduced T cells, PBMCs, SKOV3, or HN3 cancer cells were washed in PBS and then rinsed at 5 x 10⁻⁶ ppm. 6 Resuspend the cells in 200 μL of PBS at a concentration of [number of cells / mL]. 18 F]AlF-NOTA-IL-4DE (50 KBq, 1 nM) was added to the cells and incubated in the dark at 37°C for 30 min. Cells were washed twice with PBS, the supernatant was retained in separate Ependorf tubes and resuspended in fresh culture medium, and then gamma counting was performed to determine labeling efficiency. Cell labeling efficiency was calculated as [radioactivity in cell pellet / (radioactivity in cell pellet + radioactivity in supernatant)] x 100%.

[0207] Figure 5 show[ 18 Cellular uptake of F]AlF-NOTA-IL-4DE. Blocked samples were pretreated with 100X of native IL-4DE. Data shown are mean ± SD of independent experiments, where n=5 for T4-CAR T cells expressing 4αβ; n=5 for inactive, non-transduced T cells; n=5 for PBMCs; and n=3 for SKOV3 and 3HN cancer cells.

[0208] For T4-CAR T cells expressing 4αβ, [ 18 The uptake of F]AlF-NOTA-IL-4DE was approximately 37%. In contrast, inactivated, non-transduced T cells, PBMCs, SKOV3, and HN3 cancer cells showed only negligible uptake. 18 F]AlF-NOTA-IL-4DE uptake. Therefore, [ 18 F]AlF-NOTA-IL-4DE shows selective uptake by T4-CAR T cells expressing 4αβ.

[0209] 7. Biological conjugation with HSA

[0210] Human serum albumin (HSA) was reduced with one equivalent of TCEP, and then successively reacted with [ 18 F]AlF-NOTA-dibromomaleimide (compound 7) and an equivalent of its non-radioactive reference compound (compound 6) are conjugated in tandem. After size exclusion purification, [18 The bioconjugation efficiency of F]AlF-NOTA-HSA was 66±3% (n=5). 18 F]AlF-NOTA-HSA was co-eluted with natural HSA, with a retention time of 6.49 minutes.

[0211] 8. [ 18 PET imaging of F]AlF-NOTA-HSA

[0212] In healthy BALB / c mice (n=3), the drug was administered via intravenous (IV) injection. 18 F]AlF-NOTA-HSA (approximately 10 MBq). A 120-minute dynamic positron emission tomography (PET) scan was performed following intravenous injection.

[0213] Healthy BALB / c mice (n=3) were anesthetized with 2% isoflurane in oxygen. The animals (n=3) were injected via the lateral tail vein. 18 [F]AlF-NOTA-IL-HSA (approximately 10 MBq). Immediately afterward, a 120-minute dynamic PET scan was performed using a NanoScan PET (Mediso, Budapest, Hungary) scanner. All PET data were reconstructed using the Monte Carlo-based full 3D iterative algorithm Tera-Tomo (Mediso Medical Imaging Systems, Budapest, Hungary). The raw PET data were reconstructed using reconstruction settings (4 iterations, 6 subsets, 0.4 × 0.4 × 0.4 mm³ voxel size) and intergranular scattering correction. All reconstructed data were analyzed using VivoQuant software (v3.0, inviCRO, LLC, Boston, USA). At the end of the PET scan, all animals were euthanized via cervical dislocation.

[0214] Figure 6 PET images of mice reconstructed at 5, 20, 40, 80, and 120 minutes are shown. Most [ 18 F]AlF-NOTA-HSA remained in the bloodstream 120 minutes after intravenous injection, which was due to Figure 6 The very high level of radioactive accumulation in the left ventricular cavity indicated by the PET image shown.

Claims

1. A radiolabeled compound of formula (I): (I) in: R 1 R² is independently selected from halogens and –SR³, wherein R³ is independently selected from C. 1-12 Alkyl, C 1-12 alkenyl, C 1-12 alkynyl group and C 6-10 Aryl; L represents a direct bond or connector, where the connector is C. 1-20 Alkylene, the C 1-20 The alkylene group is unsubstituted or selected from hydroxyl, -COOH, C 1-12 Alkoxy, C 1-12 Alkyl and hydroxy-C 1-12 -One or more substituents of the alkyl group are used, wherein zero or one to ten carbon atoms in the alkylene chain are selected from C 6-10 aryl, -O-, -S-, -NR 4 -、-C(O)NR 4 -、-NR 4 The intervals between C(O)-, -C(O)-, -OC(O)- and -C(O)O- are partially replaced, where R 4 Is it hydrogen or C? 1-12 Alkyl, and wherein the C 6-10 The arylene moiety is unsubstituted or selected from hydroxyl, C 1-12 Alkyl and C 1-12 Alkoxy groups may be substituted with one, two, three, or four substituents; X is absent or is PEG. 2-20 Group; and Q Indicates 18 The part marked with F; Or its pharmaceutically acceptable salt.

2. The radiolabeled compound according to claim 1, wherein Q Indicates chelation 18 F-Al complex 18 The part marked with F.

3. The radiolabeled compound according to claim 1 or 2, wherein Q It indicates that it is selected from the following scriptures 18 The part marked with F: , and .

4. The radiolabeled compound according to claim 3, wherein Q It indicates that it has the formula (Q) -A) 18 The part marked with F.

5. The radiolabeled compound according to any one of the preceding claims, wherein R 1 R² is independently selected from –Br, –I, and –SPh.

6. The radiolabeled compound according to claim 5, wherein R 1 It is –Br and R² is –Br.

7. The radiolabeled compound according to any one of the preceding claims, wherein L represents a linker, and wherein the linker is C 1-20 Alkylene, wherein 1 to 10 carbon atoms in the alkylene chain are selected from C 6-10 aryl, -C(O)NR 4 -、-NR 4 The intervals between C(O)-, -C(O)-, -OC(O)- and -C(O)O- are partially replaced, where R 4 Is it hydrogen or C? 1-12 Alkyl, and wherein the C 6-10 The aryl portion is unsubstituted.

8. The radiolabeled compound according to claim 7, wherein the linker is C 1-20 Alkylene, wherein 1 to 10 carbon atoms in the alkylene chain are selected from C 6-10 The spaces between aryl groups, -C(O)NH- and -NHC(O)- are partially replaced, and wherein the C 6-10 The aryl portion is unsubstituted.

9. The radiolabeled compound according to any one of the preceding claims, wherein L has the following formula: –(CH2) n –NH–C(O)–(CH2) m – or –(CH2) n –C(O)–NH–(CH2) m –, Where n is an integer from 0 to 18, m is an integer from 0 to 18, and n+m≤18.

10. The radiolabeled compound according to claim 9, wherein n is 2 and m is 1.

11. The radiolabeled compound according to any one of claims 1-8, wherein L has the following formula: Where p is an integer from 0 to 18, and q is an integer from 0 to 18, and p+q≤18.

12. The radiolabeled compound according to claim 11, wherein q is 1.

13. The radiolabeled compound according to any one of claims 1-8, wherein L has the following formula: Where r is an integer from 0 to 18, and s is an integer from 0 to 18, and r+s≤18.

14. The radiolabeled compound according to claim 13, wherein s is 2.

15. The radiolabeled compound according to any one of claims 1-10, wherein the compound of formula (I) is a compound of the following formula: 。 16. A reference compound of formula (II): (II) in: R 1 R², L, and X are as defined in any one of claims 1-15; and Q indicates that chelation is involved. 19 F-Al complex 19 The part marked with F; Or its pharmaceutically acceptable salt.

17. The reference compound according to claim 16, wherein Q represents a portion selected from: , and .

18. The reference compound according to claim 17, wherein Q represents a portion having the formula (QA).

19. The reference compound according to any one of claims 16-18, wherein the compound of formula (II) is a compound of the following formula: 。 20. A precursor compound of formula (III): (III) in: R 1 R², L, and X are as defined in any one of claims 1-15; and Y indicates the ability to chelate. 18 Part of the F-Al complex; Or its pharmaceutically acceptable salt.

21. The precursor compound according to claim 20, wherein Y represents a portion selected from: , and .

22. The precursor compound according to claim 21, wherein Y represents a portion having the formula (YA).

23. The precursor compound according to any one of claims 20-22, wherein the compound of formula (III) is a compound of the following formula: 。 24. A pharmaceutical composition comprising a radiolabeled compound according to any one of claims 1-15 and a pharmaceutically acceptable carrier.

25. A method for preparing a radiolabeled compound according to any one of claims 1-15, the method comprising radiolabeling a precursor compound according to any one of claims 20-23.

26. The method according to claim 25, wherein the method comprises the following steps: The precursor compound of formula (III) is reacted with a radiolabeling reagent containing a fluoride-18 source according to the following reaction scheme to form the radiolabeled compound of formula (I): Where R 1 R², L, X and Q As defined in any one of claims 1-15; and Y is as defined in any one of claims 20-23.

27. The method of claim 26, wherein the radiolabeling reagent comprises a fluoride-18 source and Al. 3+ source.

28. The radiolabeled compound according to any one of claims 1-15, used in a diagnostic method performed on a human or animal body using positron emission tomography (PET).

29. The radiolabeled compound for use according to claim 28, wherein the diagnostic method comprises labeling a protein with the radiolabeled compound according to any one of claims 1-15.

30. The radiolabeled compound for use according to claim 29, wherein the protein is a natural protein.

31. The radiolabeled compound for use according to claim 29 or 30, wherein the protein is selected from antibodies, single-chain variable fragments (SCFV), antigen-binding fragments (Fab), nanobodies, and / or cytokines.

32. The radiolabeled compound for use according to any one of claims 28 to 31, wherein the diagnostic method comprises labeling a nanobody targeting HER2 with the radiolabeled compound according to any one of claims 1 to 15.

33. The radiolabeled compound for use according to claim 32, wherein the HER2-targeting nanobody is NM-02.

34. The radiolabeled compound for use according to any one of claims 28 to 33, wherein the diagnostic method is a method for diagnosing cancer.

35. The radiolabeled compound for use according to any one of claims 28 to 34, wherein the diagnostic method is a method for identifying the location of a tumor.

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