Targeting compounds and methods of production thereof

Abstract

Targeted compounds and methods of producing the same are disclosed. The present invention relates to a compound of formula (I) based on a sarcophagine. The compounds can complex radioisotopes, such as 64-copper, and thus are useful in radioimaging and radiotherapy of cancer, such as colorectal cancer. The compounds function via a pre-targeted imaging method in which the tetrazine is chemically reacted with a functional group, such as trans-cyclooctene, pre-bound to a ligand, such as a tumor-specific antibody, via a click chemistry. # imgabs0 #

Description

[0001] This application is a divisional application of the application with the application date of April 11, 2019, application number 201980024850.6, and invention title "Targeting Compounds and Methods for Their Production".

[0002] Field

[0003] The present invention relates to targeting compounds and methods for their production. Background

[0005] Ligands containing radioisotopes are commonly used in radiotherapy and diagnostic imaging. The ligand acts by directly targeting a specific site, such as a specific receptor on a cell. In the case where the ligand is intended for the treatment or imaging of cancer or a similar proliferative disease, the site to be targeted can be a receptor expressed on the surface of a tumor or cancer cell. Ideally, a radiolabeled ligand is administered, where the ligand rapidly and directly selectively binds to the targeted receptor and excludes other sites. This is a rare situation. Typically, the radiolabeled ligand is administered and remains in the subject's circulatory system, where only a small fraction of the ligand binds to the receptor target. The rate of ligand binding can be poor, where sufficient binding is achieved several hours after administration. In the case where the ligand remains in the circulatory system, the radioisotope can be delivered to unwanted sites and cause unwanted damage (due to the emitted radioactivity) or side effects.

[0006] Ideally, the ligand itself should exhibit good stability and not undergo decomposition in the body. This typically poses a problem because the functional groups present in the ligand may be prone to oxidation and subsequent oxidative decomposition. In addition, the ligand complexed with the radioisotope should also exhibit good stability. In the case where the ligand complex decomposes in the body, the radioisotope is then released and carried throughout the subject, where the radioactivity emitted by the radioisotope causes damage in unwanted regions.

[0007] In the case where a radiolabeled ligand is administered for the purpose of diagnostic imaging, the ligand should ideally selectively bind to the desired site, and any unbound ligand should be cleared from the patient. This creates sufficient contrast between the tumor site (e.g., where the site to be imaged is a tumor) and the background (where the presence of radioactivity indicates a tumor). When sufficient contrast is obtained, particularly by minimizing background radiation or radiation due to circulating unbound radioisotopes, then an image of appropriate quality can be obtained. In the case where the radioisotope is released from the ligand by ligand degradation or other means, the radioisotope travels via the circulation rather than binding to the tumor (or other) site and contributes to background radiation in any image taken. This reduces the quality and contrast of the image, which in turn reduces the quality of the information available for diagnostic purposes. While this can be overcome by administering a larger amount of ligand, using such a large amount of radioisotope may be harmful to the subject in order to encourage greater binding at the desired site.

[0008] An alternative to the direct binding described above is through the use of an intermediate binding compound. Instead, this intermediate binding compound binds to the target site and contains functional groups that selectively react with complementary functional groups on a ligand that is subsequently administered. There is a need for ligands that can be radiolabeled and have the ability to bind to an intermediate target site in the body, where the ligands can be used for the purposes of radiotherapy and diagnostic imaging.

[0009] Overview

[0010] The present invention is based on the discovery that there are compounds that can be used as ligands, yet do not necessarily target the intended site itself. Instead, the ligand targets an intermediate compound that initially binds to the intended site and also has functional groups that can selectively react with the ligand itself. The advantage of this method is that the reaction between the ligand and the intermediate compound attached to the intended site can be a reaction in which a covalent bond is formed, which allows for a more secure attachment of the ligand. The complementary functional groups on the ligand and the intermediate compound can react with each other quickly and selectively. Since the ligand typically contains a radioisotope, the ability of the ligand to react covalently with a specific functional group, rather than covalently with a specific receptor (which may be expressed in other non-intended locations), the subsequent localization of the ligand and the radioisotope can be improved due to reduced diffusion of the ligand away from the target site.

[0011] In one aspect of the invention, there is provided a compound of formula (I) or a salt thereof:

[0012]

[0013] Wherein:

[0014] R 1is a linking group; and

[0015] R 2 is H, optionally substituted alkyl, optionally substituted aryl, or optionally substituted heteroaryl.

[0016] In an embodiment, the compound of formula (I) is complexed with a radioisotope.

[0017] In another aspect of the present invention, there is provided a composition comprising a compound of formula (I) or a salt thereof according to an earlier aspect.

[0018] In another aspect of the present invention, there is provided a kit comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0019]

[0020] The kit comprises:

[0021] a container of a compound of formula (I) or a pharmaceutically acceptable salt thereof;

[0022] a container of a solution comprising a Cu radioisotope; and

[0023] instructions for use for preparing an aqueous formulation of a compound of formula (I) or a pharmaceutically acceptable salt thereof complexed with a Cu radioisotope.

[0024] In an embodiment, the present invention provides a kit according to an earlier aspect, wherein the kit further comprises a container of an antibody comprising a functional group that reacts with the tetrazine moiety of the compound of formula (I).

[0025] In yet another aspect of the present invention, there is provided a method for radioimaging a tumor or cancer in a subject, the method comprising the steps of:

[0026] i) administering to the subject a compound that can bind to the tumor or cancer site and comprises a functional group that reacts with the tetrazine moiety

[0027] portion;

[0028] ii) allowing the unbound compound administered in step i) to clear from

[0029] the subject; and

[0030] iii) administering to the subject a compound of formula (I), wherein the compound is complexed with a radioisotope selected from the group consisting of 60 Cu, 61 Cu, 64 Cu, and 67 Cu.

[0031] In another aspect of the present invention, there is provided a method for treating a tumor or cancer in a subject, the method comprising the steps of:

[0032] i) administering to the subject a compound that can bind to the tumor or cancer site and that comprises a functional group reactive with a tetrazine moiety

[0033] portion;

[0034] ii) allowing the compound administered in step i) that has not bound to the tumor or cancer site to clear from

[0035] the subject; and

[0036] iii) administering to the subject a compound of formula (I), wherein the compound is complexed with a radioisotope selected from the group consisting of 60 Cu, 61 Cu, 64 Cu and 67 Cu.

[0037] In another aspect of the present invention, there is provided the use of a compound as defined in an earlier aspect or a composition as defined in an earlier aspect in the manufacture of a medicament for treating cancer or for radioimaging a subject.

[0038] In a further aspect of the present invention, there is provided a combination comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0039]

[0040] and a compound that can bind to the tumor or cancer site and that comprises a functional group reactive with the tetrazine moiety of the compound of formula (I).

[0041] DETAILED DESCRIPTION

[0042] The inventors have found that the compounds of formula (I) can be used as ligands for radiolabeled compounds.

[0043]

[0044] The compounds of formula (I) can be radiolabeled and then used as part of a pretargeting method for the treatment and / or imaging of cancer. The pretargeting method requires the prior addition to the subject of an intermediate compound, wherein the intermediate compound binds to the target site and also comprises a moiety to which the compound of formula (I) binds selectively and tightly. This eliminates the need to bind the radiolabeled compound directly to the target site and also eliminates any harmful side effects resulting from the introduction of the radioisotope via a ligand that shows limited binding to the target site.

[0045] The intermediate compound first introduced into the subject must selectively bind to the target site. Examples of such intermediate compounds include antibodies that target antigens expressed on the surface of the target site. The compound is administered to the subject and allowed to circulate in the body for a certain period of time to allow the compound to bind to the target site. Since no radioisotope is administered at this time, even if the binding of the intermediate compound to the target site is slow, no unwanted side effects caused by the radioisotope are observed. The intermediate compound is allowed to be eliminated from the subject, which labels the target site in the subject with a moiety to which the compound of formula (I) can bind.

[0046] The compound of formula (I) contains a tetrazine moiety that can participate in a "click-type" reaction, i.e., a cycloaddition reaction, with a coupling partner having a suitable functional group. In this case, the coupling partner with the functional group is on the surface of the target site and is installed by administering the intermediate compound described above. The cycloaddition reaction produces a "click" adduct that is stable and results in excellent binding of the compound of formula (I) to the target site through the intermediate compound.

[0047] The tetrazine on the compound of formula (I) can participate in a click-type reaction with a trans-cyclooctene moiety. The cycloaddition reaction that occurs between the tetrazine and the trans-cyclooctene is an inverse electron demand Diels-Alder (IEDDA) reaction, where the tetrazine acts as the diene and the double bond of the trans-cyclooctene acts as the dienophile. The cycloaddition between the tetrazine and the trans-cyclooctene is known to be fast, with a rate constant k > 30,000 M -1 s -1 . After allowing the intermediate compound containing the trans-cyclooctene moiety to clear, the compound of formula (I) labeled with a radioisotope is administered to the subject. Considering the rapidity of the cycloaddition between the tetrazine and the trans-cyclooctene, the administered compound of formula (I) binds to the trans-cyclooctene installed on the surface of the target site. This results in the delivery of the radioisotope to the target site.

[0048] The compound of formula (I) contains a methyl-capped macrocyclic ligand. The macrocyclic ligand is more commonly known as sarcophagine or 3,6,10,13,16,19-hexaazabicyclo[6.6.6]icosane and complexes a variety of transition metal ions. The inventors have found that the compound of formula (I) containing sarcophagine and tetrazine can be successfully used in the pre-targeting strategy described above for radiotherapy and diagnostic imaging of tumors. In addition, the inventors have found that the nature of the group (denoted as R 1 ) that links the tetrazine moiety to the remainder of the molecule and the substituent group (denoted as R 2 ) at the terminal position of the tetrazine can be altered.

[0049]

[0050] Linking group R 1 Ensure that the distance between the target and the radioisotope complexed in sarcophagine is optimal for the target site. Ideally, the linking group R 1 has a length such that the radioisotope contained in the ligand of formula (I) is at an appropriate distance from the target site. The appropriate distance can vary depending on the nature of the target site, the nature of the intermediate compound that can react with the compound of formula (I), and the nature of the radioisotope. In addition, the nature of the linking group should be such that it does not participate in any side reactions, either with other functional groups in the compound or in vivo. In some embodiments, R 1 is an optionally substituted alkylene linking group. In some embodiments, R 1 is an optionally substituted arylene linking group. In an embodiment, R 1 is a benzylidene (-CH2C6H4-) group.

[0051] Thus, the compound of formula (I) can have the structure of formula (I'), where R 1 is a benzylidene group:

[0052]

[0053] where R 2 is as defined above.

[0054] Substituent R 2 is at the terminal position of the tetrazine. The steric and / or electronic properties of the substituent R 2 can affect the rate of the click-type reaction between the tetrazine and the coupling partner (e.g., the trans-cyclooctene moiety). In an embodiment, R 2 is H. In another embodiment, R 2 is an optionally substituted alkyl group. In another embodiment, R 2 is an optionally substituted aryl group. In an embodiment, R 2 is an optionally substituted heteroaryl group.

[0055] As used throughout the specification, the term "optionally substituted" means that the group may or may not be further substituted or fused (to form a fused polycyclic system) by one or more non-hydrogen substituent groups. In certain embodiments, the substituent groups are one or more groups independently selected from the group consisting of: halogen, ═O, ═S, -CN, -NO2, -CF3, -OCF3, alkyl, alkenyl, alkynyl, haloalkyl, haloalkenyl, haloalkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, heteroarylalkyl, arylalkyl, cycloalkylalkenyl, heterocycloalkylalkenyl, arylalkenyl, heteroarylalkenyl, cycloalkylheteroalkyl, heterocycloalkylheteroalkyl, arylheteroalkyl, heteroarylheteroalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkoxycycloalkyl, alkoxyheterocycloalkyl, alkoxyaryl, alkoxyheteroaryl, alkoxycarbonyl, alkylaminocarbonyl, alkenoxy, alkynoxy, cycloalkoxy, cycloalkenoxy, heterocycloalkoxy, heterocycloalkenoxy, aryloxy, phenoxy, benzyloxy, heteroaryloxy, arylalkoxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, alkylsulfinyl, arylsulfinyl, aminosulfinylaminoalkyl, -C(═O)OH, -C(═O)Ra, -C(═O)ORa, -C(═O)NR a R b 、-C(═NOH)R a 、-C(═NR a )NR b R c 、-NR a R b 、-NR a C(═O)R b 、-NR a C(═O)OR b 、-NR a C(═O)NR b R c 、-NR a C(═NR b )NR c R d 、-NR a SO2R b 、-SR a 、-SO2NR a R b 、-OR a 、-OC(═O)NR a R b 、-OC(═O)R a and acyl, where R a, R b , R c and R d each independently is selected from the group consisting of: H, C1-C 12 alkyl, C1-C 12 haloalkyl, C2-C 12 alkenyl, C2-C 12 alkynyl, C2-C 10 heteroalkyl, C3-C 12 cycloalkyl, C3-C 12 cycloalkenyl, C2-C 12 heterocycloalkyl, C2-C 12 heterocycloalkenyl, C6-C 18 aryl, C1-C 18 heteroaryl and acyl, or any two or more of R a , R b , R c and R d form a heterocyclic ring system having 3 to 12 ring atoms when combined together with the atoms to which they are attached.

[0056] In some embodiments, each optional substituent is independently selected from the group consisting of: halogen, ═O, ═S, -CN, -NO2, -CF3, -OCF3, alkyl, alkenyl, alkynyl, haloalkyl, haloalkenyl, haloalkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkoxyaryl, alkoxyheteroaryl, alkenoxy, alkynoxy, cycloalkoxy, cycloalkenoxy, heterocycloalkoxy, heterocycloalkenoxy, aryloxy, heteroaryloxy, arylalkyl, heteroarylalkyl, arylalkoxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, aminoalkyl, -COOH, -SH and acyl.

[0057] Examples of particularly suitable optional substituents include F, Cl, Br, I, CH3, CH2CH3, OH, OCH3, CF3, OCF3, NO2, NH2 and CN.

[0058] "Alkyl" as a group or part of a group refers to a straight-chain or branched aliphatic hydrocarbon group, preferably C1-C 12 alkyl, more preferably C1-C 10 alkyl, most preferably C1-C6, unless otherwise specified. Examples of suitable straight-chain and branched C1-C6 alkyl substituents include methyl, ethyl, n-propyl, 2-propyl, n-butyl, sec-butyl, tert-butyl, hexyl and the like. The group can be a terminal group or a bridging group.

[0059] "Aryl", as a group or part of a group, means (i) an optionally substituted monocyclic aromatic carbocyclic or fused polycyclic aromatic carbocyclic (ring structure with ring atoms all being carbon), preferably each ring having from 5 to 12 atoms. Examples of aryl groups include phenyl, naphthyl and similar groups; (ii) an optionally substituted partially saturated bicyclic aromatic carbocyclic moiety, where a phenyl group and a C5-C7 cycloalkyl group or a C5-C7 cycloalkenyl group are fused together to form a ring structure, such as tetrahydronaphthyl, indenyl or indanyl. The group can be a terminal group or a bridging group. Generally, an aryl group is a C6-C 18 aryl group.

[0060] "Heteroaryl", alone or as part of a group, refers to a group containing an aromatic ring (preferably a 5- or 6-membered aromatic ring), which has one or more heteroatoms as ring atoms in the aromatic ring, and the remaining ring atoms are carbon atoms. Suitable heteroatoms include nitrogen, oxygen and sulfur. Examples of heteroaryl include thiophene, benzothiophene, benzofuran, benzimidazole, benzoxazole, benzothiazole, benzisothiazole, naphtho[2,3-b]thiophene, furan, isoindolizine, xantholene, phenoxatine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, tetrazole, indole, isoindole, 1H-indazole, purine, quinoline, isoquinoline, phthalazine, naphthyridine, quinoxaline, cinnoline, carbazole, phenanthridine, acridine, phenazine, thiazole, isothiazole, phenothiazine, oxazole, isoxazole, furazan, phenoxazine, 2-pyridyl, 3-pyridyl or 4-pyridyl, 2-quinolyl, 3-quinolyl, 4-quinolyl, 5-quinolyl or 8-quinolyl, 1-isoquinolyl, 3-isoquinolyl, 4-isoquinolyl or 5-isoquinolyl, 1-indolyl, 2-indolyl or 3-indolyl, and 2-thienyl or 3-thienyl. Heteroaryl groups are generally C1-C6 heteroaryl groups. The group can be a terminal group or a bridging group.

[0061] Examples of the compounds of formula (I) include:

[0062]

[0063]

[0064] The compounds of the present invention can be complexed with radioisotopes. In some embodiments, the radioisotope is an isotope of copper. In some embodiments, the radioisotope is selected from the group consisting of 60 Cu, 61 Cu, 64 Cu and 67 Cu.

[0065] Without wishing to be bound by theory, it has been found that the compounds described herein offer one or more advantages over similar compounds known in the art. The compounds of the invention comprising a sarcophagine cage with a terminal methyl substituent and a single propyl linker adjacent to the sarcophagine have shown good retention of radioisotopes complexed with the ligand, such that the ligand-radioisotope complexes generally show greater stability. It is believed that the combination of the methyl group and the absence of an aminobenzylidene linker in the compound provides the advantages now observed. One advantage of the increased retention of the radioisotope includes a reduction in side effects and unwanted damage to other parts of the subject due to any free radioisotope released from the ligand. Another advantage is that the radioisotope is used and delivered more effectively to the intended site of action, where the radioisotope is not released unnecessarily. Yet another advantage is that once administered, the ligand-radioisotope is available for a longer time than similar compounds.

[0066] The compounds and complexes of the invention can be administered in a manner that makes the compounds available for the desired applications, such as for radiotherapy or diagnostic imaging. Those skilled in the art of preparing such types of compositions or formulations will be aware of the appropriate forms and modes of administration, which depend on the compound, the condition to be treated, and other circumstances.

[0067] The compounds and complexes of the present invention can be administered alone or in the form of pharmaceutical compositions in combination with pharmaceutically acceptable carriers, diluents or excipients. Although the compounds and complexes of the present invention are effective alone, they can be formulated and administered in the form of pharmaceutically acceptable salts because the salt forms of the compounds can exhibit better stability, solubility or crystallinity. The compounds and complexes of the present invention are generally used in the form of compositions containing the compound or complex, and their formulations depend on the mode of administration and the purpose or condition to be treated. Another advantage of the compounds of the present invention is that they are very stable in formulations or compositions when compared to similar compounds. In this regard, when compared to similar compounds, formulations of the compounds of the present invention complexed with radioisotopes undergo less radiolysis. Due to the energy released during the spontaneous radioactive decay of the radioisotope, radiolysis causes the breaking of bonds in the ligand. This then results in the release of free radioisotope, which can then circulate and damage nearby tissues. In addition to the unwanted tissue damage, radiolysis results in a decrease in the overall efficacy of the radioisotope-ligand complex. The inventors have found that radiolabeled complexes of the compounds of the present invention can have the required stability in pharmaceutical formulations without the need to add one or more stabilizers to the formulation. Without wishing to be bound by theory, the inventors believe that the overall structure of the compounds of the present invention provides the stability observed in the corresponding radiolabeled complexes upon formulation.

[0068] The pharmaceutical compositions of the present invention for parenteral injection include pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions immediately before use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. For example, suitable fluidity can be maintained by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants.

[0069] Although the compounds and complexes of the present invention can exhibit a certain degree of stability in pharmaceutical formulations or compositions in the absence of any stabilizers, the formulations can still contain one or more excipients. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of microbial action can be ensured by including a variety of antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of injectable pharmaceutical forms can be effected by including agents that delay absorption such as aluminum monostearate and gelatin.

[0070] For example, an injectable preparation can be sterilized by filtration through a bacteria-retaining filter or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium immediately before use.

[0071] According to another aspect of the present invention, there is provided a kit comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0072]

[0073] The kit comprises:

[0074] a container of the compound of formula (I) or a pharmaceutically acceptable salt thereof;

[0075] a container of a solution containing a Cu radioisotope; and

[0076] instructions for use for preparing an aqueous preparation of the compound of formula (I) or a pharmaceutically acceptable salt thereof complexed with a Cu radioisotope.

[0077] The kit of the present invention may further comprise a container consisting of an excipient and / or a stabilizer and instructions for use for adding said excipient and / or stabilizer.

[0078] Use of the compounds of the present invention

[0079] The compounds of the present invention can be particularly useful for diagnostic and therapeutic purposes in medicine. Complexes with ligands and isotopes bearing appropriate targeting moieties can be used to localize and treat specific tissue types. For such complexes that are considered suitable for use in in vivo diagnosis and therapy, in addition to the solubility and stability properties required for the complex in solution, the complex must also exhibit appropriate kinetic, stability, and clearance properties under physiological conditions. As used herein, the term "complex" can refer to a ligand-metal ion complex, where the metal ion is a radioisotope, or alternatively, the metal ion is a non-radioactive isotope. As previously described, the use of the compounds of the present invention for diagnostic and therapeutic purposes relies on the introduction of an intermediate compound that binds to the target site and also binds to the compound of formula (I).

[0080] Accordingly, the present invention provides a method for radioimaging, a method for diagnosing a disease in a subject, or a method for treating a disease in a subject, comprising administering to the subject an effective amount of a compound as defined herein. The inventors have found that the compounds of the present invention can be used in a method for radioimaging, a method for diagnosis, or a method for treating cancer.

[0081] As used herein, the term "cancer" broadly encompasses a class of neoplastic diseases characterized by abnormal cell growth with the potential to invade or spread to other parts of the body. These are contrasted with benign tumors, which do not spread to other parts of the body, and thus the definition as used herein includes all malignant (cancerous) disease states. Accordingly, the term encompasses the treatment of tumors.

[0082] Thus, the term "tumor" is generally used to define any malignant cancerous or precancerous cell growth and can include leukemia, but particularly refers to solid tumors or carcinomas such as melanoma, colon cancer, lung cancer, ovarian cancer, skin cancer, breast cancer, pancreatic cancer, pharyngeal cancer, brain cancer, prostate cancer, CNS cancer, and kidney cancer (and other cancers).

[0083] The radioisotope-ligand complexes of the present invention can comprise radioisotopes such as 64 Cu. 64 The Cu isotope has a half-life of approximately 12.7 hours and decays by both positron emission and β decay, which makes 64 the use of Cu-labeled complexes suitable for use in multiple modalities of radioimaging. In particular, 64 the decay characteristics and half-life of Cu make this radioisotope a favorable choice for use in positron emission tomography (PET) and single photon emission computed tomography (SPECT). The radioisotope-ligand complexes of the present invention can comprise radioisotopes such as 61 Cu. 61 The Cu isotope has a half-life of approximately 3 hours and decays by positron emission, which makes 61 the use of Cu-labeled complexes suitable for use in multiple modalities of radioimaging. The radioisotope-ligand complexes of the present invention can also comprise radioisotopes such as 67 Cu. 67 The Cu isotope has a half-life of approximately 61.8 hours and decays by β emission, which makes 67 the use of Cu-labeled complexes suitable for use in SPECT imaging. 67 Cu-labeled complexes can also be suitable for use in radiotherapy treatment.

[0084] In an embodiment, the present invention provides a method for radioimaging a tumor or cancer in a subject, the method comprising the steps of:

[0085] i) administering to the subject a compound that can bind to the tumor or cancer site and comprises a functional group that reacts with a tetrazine moiety

[0086] portion;

[0087] ii) allowing the compound administered in step i) to clear from the subject; and

[0088] iii) administering to the subject a compound of formula (I), wherein the compound is complexed with a radioisotope selected from the group consisting of 60 Cu, 61 Cu, 64 Cu and 67 Cu.

[0089] Administering an effective amount of a compound of formula (I) and a Cu radioisotope, such as 60 Cu, 61 Cu, 64 Cu or 67 Cu, can result in the binding of the ligand and the radioisotope to an intermediate compound, wherein the intermediate compound binds to the target site. The target site can be the surface of a tumor. In an embodiment, the present invention provides a method for radioimaging, comprising administering to a subject a compound of formula (I) and Cu ions. In an embodiment, a formulation comprising a compound of formula (I) and 64 Cu or 67 Cu ions can be used in a method for radioimaging. For example, after administering the intermediate compound, a subject administered a compound of formula (I) and a Cu radioisotope is monitored by PET or SPECT, allowing visualization and subsequent detection of the tumor site. The visualization information obtained by radioimaging can provide information related to the location of any such tumor site. This provides information related to the location of the tumor, if present. Repeated imaging at a later time point allows monitoring of the clearance of the radioisotope-ligand complex, which enables the calculation of dosimetry estimates. Those skilled in the art will understand that the amount administered for the purpose of facilitating radioimaging can vary and will subsequently depend on the nature of the subject and the intended imaging site.

[0090] The compounds of the present invention can be administered to a subject for the purposes of radioimaging, diagnosis or treatment. The administration is by a parenteral route, wherein administration by intravenous injection is preferred. Alternatively, the formulations of the present invention can be provided by intra-arterial or other routes for delivery to the systemic circulation. The subject administered the compound is then placed in a PET scanner, and an image is obtained showing the localization of the radioisotope-ligand complex and subsequently the location of any tumors. This then allows for the diagnosis and detection of tumors.

[0091] In an embodiment, the present invention provides the use of a compound of formula (I) in a method for radioimaging of a tumor or cancer. Those skilled in the art will understand that the information obtained from the radioimaging of a subject can be used in the diagnosis of the tumor or cancer of the subject. In an embodiment, the present invention provides a method for diagnosing a tumor or cancer. In an embodiment, the present invention provides a method for diagnosing colorectal cancer.

[0092] In an embodiment, the present invention provides a method for treating a tumor or cancer in a subject, the method comprising the steps of:

[0093] i) administering to the subject a compound that can bind to the tumor or cancer site and comprises a functional group that reacts with the tetrazine moiety

[0094] portion;

[0095] ii) allowing the compound administered in step i) to clear from the subject; and

[0096] iii) administering to the subject a compound of formula (I), wherein the compound is complexed with a radioisotope selected from the group consisting of 60 Cu,

[0097] 61 Cu, 64 Cu and 67 Cu.

[0098] As described above, the compound of formula (I) binds to an intermediate compound that has bound to a target site. Examples of intermediate compounds are compounds containing a trans-cyclooctene group, as such a functional group can react with the tetrazine of the compound of formula (I) in a cycloaddition reaction that firmly attaches the compound of formula (I) to the intermediate compound. The intermediate compound also contains a functional group that binds to a specific tumor or cancer site by recognizing a specific receptor or analogue that is characteristic of the tumor or cancer. In this regard, the intermediate compound can be an antibody that binds to an antibody expressed on the surface of a tumor or cancer site. In the case where the intermediate compound is an antibody, it can be modified to contain a functional group that reacts with a tetrazine, such as a trans-cyclooctene functional group. For diagnostic or therapeutic purposes, the intermediate compound is first administered to a subject and allowed to bind to the targeted tumor or cancer site. In the case where a fraction of the administered intermediate compound remains unbound to the cancer or tumor site, the remaining portion of the unbound intermediate compound is then allowed to clear from the subject's circulation. The compound of formula (I) is then administered to the subject and selectively targets the intermediate compound bearing a functional group that reacts with the tetrazine moiety in the compound of formula (I). In allowing the unbound portion of the intermediate compound to clear from the subject's circulation, the subsequent administration of the compound of formula (I) is ultimately only attached and localized at the position where the intermediate compound is located, rather than in the systemic circulation. This improves the specificity of the radioisotope delivered with the compound of formula (I). In the case where the intermediate compound contains a trans-cyclooctene moiety, a click-type reaction occurs between the trans-cyclooctene and the tetrazine in the compound of formula (I), which firmly attaches the compound of formula (I) to the intermediate compound. In the case where the compound of formula (I) contains a radioisotope, it is then localized at the targeted tumor or cancer site. Products resulting from the spontaneous decay of the radioisotope are detected and can generate an image showing the location of the radioisotope. Alternatively, or concurrently, the radioactive decay products can be used to treat the tumor or cancer that has caused the initial expression of the antigen.

[0099] In the case where the intermediate compound is an antibody, it can be modified to contain a functional group that reacts with the tetrazine of the compound of formula (I). An example of an antibody that can be modified to contain such a functional group (e.g., a trans-cyclooctene group) is huA33, a monoclonal antibody that recognizes the A33 antigen. The A33 antigen is typically expressed in cancers such as colorectal cancer. In the case where the intermediate compound is a modified trans-cyclooctene-huA33 antibody, the compound of formula (I) can be used to detect and diagnose colorectal cancer by radioimaging. In addition, the compound of formula (I) can also be used to treat colorectal cancer.

[0100] In an embodiment, the compound containing a functional group that reacts with the tetrazine moiety is an antibody.

[0101] Thus, in an embodiment, the present invention provides a method for radioimaging a tumor or cancer in a subject, the method comprising the steps of:

[0102] i) administering to the subject an antibody comprising a trans-cyclooctene group;

[0103] ii) allowing the antibody administered in step i) to clear from the subject; and

[0104] iii) administering to the subject a compound of formula (I), wherein the compound is complexed with a radioisotope selected from the group consisting of 60 Cu, 61 Cu, 64 Cu and 67 Cu.

[0105] In another embodiment, the present invention provides a method for treating a tumor or cancer in a subject, the method comprising the steps of:

[0106] i) administering to the subject an antibody comprising a trans-cyclooctene group;

[0107] ii) allowing the antibody administered in step i) to clear from the subject; and

[0108] iii) administering to the subject a compound of formula (I), wherein the compound is complexed with a radioisotope selected from the group consisting of 60 Cu, 61 Cu, 64 Cu and 67 Cu;

[0109] wherein the cancer is colorectal cancer.

[0110] In the case where the compound of formula (I) is complexed with a Cu radioisotope, administering a complex of the compound of formula (I) and Cu ions can treat the tumor or cancer. As discussed above, the compound of formula (I) binds to an intermediate compound administered prior to the compound of formula (I), wherein an intermediate compound comprising, for example, a trans-cyclooctene group first binds to the tumor or cancer. The binding of the compound of formula (I) and the Cu radioisotope brings the radioisotope very close to the target site. Since the Cu radioisotope undergoes radioactive decay, the decay products can be used to treat the tumor or cancer.

[0111] In an embodiment, the radioimaging is positron emission tomography (PET). In an embodiment, the radioisotope is 64 Cu. In another embodiment, the radioisotope is 67Cu. When the compound of formula (I) is complexed with a radioisotope and used in a method for radioimaging a subject, for example, an intermediate compound containing a trans-cyclooctene group first binds to the site to be imaged. The binding of the compound of formula (I) and the Cu radioisotope brings the radioisotope very close to the imaging site. When the Cu radioisotope undergoes radioactive decay, the decay products are detected and allow the image to be developed.

[0112] According to another embodiment of the present invention, there is provided a combination of a compound of formula (I) or a pharmaceutically acceptable salt thereof and a compound comprising a functional group that reacts with the tetrazine moiety of the compound of formula (I):

[0113]

[0114] A combination of a compound of formula (I) and a compound comprising a functional group that reacts with the tetrazine moiety of the compound of formula (I) can be provided such that these compounds are administered sequentially to allow the compound comprising the functional group that reacts with tetrazine to bind to a selected site.

[0115] Any reference in this specification to any prior publication (or information derived therefrom) or to any thing known is not and should not be taken as an admission or acknowledgment or any form of implication that the prior publication (or information derived therefrom) or the thing known forms part of the common general knowledge in the field to which this specification pertains.

[0116] Throughout this specification and the appended claims, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to mean including the stated integer or step or group of integers or steps but not excluding any other integer or step or group of integers or steps. Examples

[0117] 1. Synthesis of the compound of formula (I)

[0118]

[0119] The above compound of formula (I) can be synthesized by coupling an amino-substituted sarcophagine with a phthalimide ester derivative of a propyl linker / tetrazine functional group. The coupling reaction may require a coupling agent, such as a peptide coupling agent. The coupling reaction may also require one or more bases to proceed.

[0120]

[0121] Optionally, the compounds of formula (I) above can be synthesized between a carboxylic acid derivative of a linker-bound sarcophagine and the corresponding amine under standard peptide conditions.

[0122] 2. Synthesis of the complex of formula (I)

[0123] The copper(II) ion complex of formula (I) can be prepared by adding a solution of a copper(II) salt to a solution of a compound of formula (I).

[0124] The present invention also relates to the following items:

[0125] 1. A compound of formula (I) or a salt thereof,

[0126]

[0127] wherein:

[0128] R 1 is a linker group; and

[0129] R 2 is H, optionally substituted alkyl, optionally substituted aryl or optionally substituted heteroaryl.

[0130] 2. The compound according to item 1, wherein R 1 comprises an arylene group.

[0131] 3. The compound according to item 2, wherein R 1 is a benzylidene group.

[0132] 4. The compound according to any one of items 1 to 3, wherein R 2 is H.

[0133] 5. The compound according to any one of items 1 to 4, wherein the compound is:

[0134]

[0135] 6. The compound according to any one of items 1 to 5, wherein the compound coordinates with Cu ions.

[0136] 7. The compound according to item 6, wherein the Cu ions are selected from the group consisting of 60 Cu, 61 Cu, 64 Cu and 67 Cu.

[0137] 8. A composition comprising the compound according to any one of items 1 to 7.

[0138] 9. Kit, comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0139]

[0140]

[0141] The kit comprises:

[0142] A container for the compound of formula (I) or a pharmaceutically acceptable salt thereof;

[0143] A container for a solution containing a Cu radioisotope; and

[0144] Instructions for use for preparing an aqueous formulation of the compound of formula (I) or a pharmaceutically acceptable salt thereof complexed with a Cu radioisotope.

[0145] 10. A method for treating a tumor or cancer in a subject, the method comprising the steps of:

[0146] i) Administering to the subject a compound capable of binding to the tumor or cancer site and comprising a functional group reactive with a tetrazine moiety;

[0147] ii) Allowing the compound administered in step i) to clear from the subject; and

[0148] iii) Administering to the subject a compound of formula (I), wherein the compound is complexed with a radioisotope selected from the group consisting of 60 Cu, 61 Cu, 64 Cu and 67 Cu.

[0149] 11. A method for radioimaging a tumor or cancer in a subject, the method comprising the steps of:

[0150] i) Administering to the subject a compound capable of binding to the tumor or cancer site and comprising a functional group reactive with a tetrazine moiety;

[0151] ii) Allowing the compound administered in step i) to clear from the subject; and

[0152] iii) Administering to the subject a compound of formula (I), wherein the compound is complexed with a radioisotope selected from the group consisting of 60 Cu, 61 Cu, 64 Cu and 67 Cu.

[0153] 12. The method according to item 10 or 11, wherein the compound capable of binding to a tumor or cancer site comprises a trans-cyclooctene group.

[0154] 13. The method according to item 12, wherein the compound comprising a trans-cyclooctene group is an antibody.

[0155] 14. The method according to any one of items 10 to 13, wherein the cancer is colorectal cancer.

[0156] 15. Use of the compound according to any one of items 1 to 7 in the preparation of a medicament for treating cancer or for radiographically imaging a subject.

[0157] 16. A combination comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0158]

[0159] and a compound capable of binding to a tumor or cancer site and comprising a functional group reactive with the tetrazine moiety of the compound of formula (I).

[0160] 17. The combination according to item 16, wherein the compound capable of binding to a tumor or cancer site further comprises a trans-cyclooctene group.

Claims

1. A compound of formula (I) or a salt thereof, wherein: R 1 is a linking group; and R 2 is H, optionally substituted alkyl, optionally substituted aryl or optionally substituted heteroaryl.

2. The compound according to claim 1, wherein R 1 comprises an arylene group.

3. The compound according to claim 2, wherein R 1 is a benzylidene group.

4. The compound according to any one of claims 1 to 4, wherein R 2 is H.

5. The compound according to any one of claims 1 to 5, wherein the compound is:

6. The compound according to any one of claims 1 to 6, wherein the compound coordinates with Cu ions.

7. The compound according to claim 7, wherein the Cu ion is selected from the group consisting of 60 Cu, 61 Cu, 64 Cu, and 67 Cu.

8. A composition comprising the compound according to any one of claims 1 to 8.

9. A kit comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof: The kit comprises: A container of the compound of formula (I) or a pharmaceutically acceptable salt thereof; A container of a solution containing a Cu radioisotope; and Instructions for use for preparing an aqueous formulation of the compound of formula (I) or a pharmaceutically acceptable salt thereof complexed with a Cu radioisotope.

10. A method for treating a tumor or cancer in a subject, the method comprising the steps of: i) administering to the subject a compound capable of binding to the tumor or cancer site and comprising a functional group reactive with a tetrazine moiety; ii) allowing the compound administered in step i) to clear from the subject; and (iii) administering to the subject a compound of formula (I), wherein the compound is complexed with a radioisotope selected from the group consisting of 60 Cu, 61 Cu, 64 Cu and 67 Cu.

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