Pharmaceutical composition for treating cancer

Abstract

[Problem] To develop a low-molecular-weight compound which exhibits high accumulation in cancer cells and can emit α rays, and to provide a pharmaceutical composition for treating cancer that contains the compound. [Solution] A pharmaceutical composition for cancer treatment comprising a compound represented by general formula (I) or a salt thereof.

Description

Pharmaceutical composition for cancer treatment

[0001] The present invention relates to a pharmaceutical composition for cancer treatment and a method for producing the same.

[0002] Radiopharmaceuticals can realize "radiotheranostics" (a portmanteau of therapeutics and diagnostics) that integrates diagnostic imaging and treatment by taking advantage of the differences in the physical properties (penetration power and linear energy transfer) of the radiation emitted from radionuclides and changing only the labeled nuclide of the drug (Non-Patent Documents 1 and 2).

[0003] In this approach, a radioactive diagnostic agent labeled with a highly biopermeable gamma-ray-emitting nuclide is first administered to a patient. Nuclear medicine imaging techniques such as PET and SPECT are then used to noninvasively visualize and quantify the expression of cancer-specific target molecules within the patient's body. For patients whose expression of the target molecule is confirmed, a radioactive therapeutic agent with a carrier moiety identical to the diagnostic agent's molecular structure but with a highly cytotoxic nuclide instead is then administered to the patient. This allows for more precise treatment of only those patients who are likely to respond to treatment (Non-Patent Document 2). This innovative approach allows for accurate understanding of each patient's pathology and the provision of optimal treatment. It is expected to serve as an important technological foundation for the realization of personalized medicine.

[0004] In the field of radiotheranostics, targeted alpha-ray therapy has attracted particular attention in recent years. Numerous cases have been reported in which alpha-ray therapy achieved significant therapeutic effects even in cases where resistance to conventional beta-ray therapy, a nuclear medicine treatment, was demonstrated (Non-Patent Documents 3 and 4). The reason alpha rays are effective is because they deliver a large amount of energy within a short range, resulting in a very high linear energy transfer (LET). This value is said to be approximately 500 times that of beta rays (β = 0.2 keV / μm vs. α = 100 keV / μm). This allows alpha rays to frequently induce DNA double-strand breaks and exhibit high cytotoxicity (Non-Patent Document 5). Furthermore, because their range is extremely short, only the length of a few cells, accumulating drugs only in targeted cancer cells offers the advantage of minimizing radiation exposure to surrounding normal tissue and reducing unnecessary damage (Non-Patent Document 6). These characteristics make radiotheranostics using alpha-ray-emitting nuclides a revolutionary next-generation cancer treatment, and further development is expected in the future.

[0005] As the usefulness of targeted alpha ray therapy is recognized, ２１１ Att (astatine) has been attracting attention as one of the most promising nuclides (Non-Patent Document 7). Astatine is an artificial element that was synthesized and discovered in 1940 using an accelerator called a cyclotron. Of the 32 isotopes discovered to date, ２１１ Only At has physical properties suitable for α-ray therapy, and in particular, its moderate half-life (7.2 hours) and simple radioactive decay pathway are superior to other α-ray nuclides (Non-Patent Document 8).

[0006] ２１１ The advantage of At half-life (7.2 hours) is that it is long enough for the synthesis, transport, and administration of the drug, but does not require long-term isolation of the patient after treatment (Non-Patent Document 9). ２０７ Since it does not produce harmful daughter isotopes before decaying to Pb, it avoids the toxicity to normal tissues caused by the release of daughter isotopes from the label carrier, which is a concern with other nuclides (Non-Patent Document 10). ２１１ ​Targeted α-ray therapy using At is superior in efficiency and safety to the therapy using other α-ray emitting nuclides, and expectations for its clinical application are growing.

[0007] Also, ２１１ Research and development of At-labeled therapeutic drugs has also been vigorously pursued, and various compounds, from low molecular weight to high molecular weight, have been used as carriers. ２１１ Many studies on At-labeled therapeutic drugs have been reported (Non-Patent Document 11). In the early studies, antibodies with high tumor selectivity were often used as carriers, but because antibodies accumulate slowly in tumor tissues, by the time a sufficient amount of antibody has accumulated in the target, ２１１ It has become clear that the radioactivity of At decays, and a high therapeutic effect cannot be expected (Non-Patent Documents 12 and 13). ２１１ While At-labeled antibodies circulate throughout the body, there is a risk of exposure of normal organs, and it has been suggested that exposure of bone marrow, which is particularly sensitive to radiation, may cause serious side effects such as bone marrow suppression (Non-Patent Document 9).

[0008] One approach to solving these problems is to use small molecule-based ２１１ The development of At-labeled drugs is attracting attention. Low molecular weight compounds are quickly excreted from the body, so they are tumor-selective. ２１１ If At can be accumulated, it will be possible to effectively irradiate tumors with radiation and reduce unnecessary exposure to normal tissues.

[0009] In fact, Na iodide symporter (NIS) is a therapeutic agent for differentiated thyroid cancer. ２１１ At (Non-Patent Documents 4, 14) and meta-AT, a therapeutic agent for pheochromocytoma that targets the norepinephrine transporter (NET), ２１１ At-astatobenzylguanidine (MABG) (Non-patent Documents 15, 16) has demonstrated a high therapeutic effect in tumor-bearing model mice, and a phase I clinical trial is currently underway in Japan. In addition, in basic research, we have been developing a novel inhibitor based on the structure of physiologically active substances and existing inhibitors. ２１１ At-labeled therapeutic drugs are also being developed (Non-patent Documents 17 and 18).

[0010] However, currently, there are few technologies for selectively accumulating small molecule drugs in cancer tissues, and their targets are mainly limited to transporters (NIS (Non-Patent Document 14), NET (Non-Patent Document 15), LAT1 (Non-Patent Documents 18, 19)) and receptors (PSMA (Non-Patent Document 20), mGluR (Non-Patent Document 21)). Targeting transporters poses challenges, such as the restriction of substrate recognition, which limits the drug structure, and targeting receptors poses challenges, such as the difficulty of concentrating sufficient radioactivity in cancer cells due to 1:1 binding. Furthermore, there are many patients for whom the above targets are not expressed, making treatment inapplicable.

[0011] In this context, we have developed a novel, small molecule-based, highly cancer-accumulating drug that targets a different biomarker from conventional drugs. ２１１ There is a need to develop drugs to treat Att.

[0012] Bodei, L., Herrmann, K., Schoer, H., Scott, A. M. & Lewis, J. S. Radiotheranostics in oncology: current challenges and emerging opportunities. Nat. Rev. Clin. Oncol. 19, 534-550 (2022).Lawal, I. O. et al. Advances in Radioligand Theranostics in Oncology. Mol. Diagn. The. 28, 265-289 (2024).Kratochwil, C. et al. 225Ac-PSMA-617 for PSMA-targeted a-radiation therapy of metastatic castration-resistant prostate cancer. J. Nucl. Med. 57, 1941-1944 (2016).Watabe, T. et al. Comparison of the Therapeutic Effects of [211At]NaAt and [131I]NaI in an NIS-Expressing Thyroid Cancer Mouse Model. Int. J. Mol. Sci. 23, 9434 (2022).Marcu, L., Bezak, E. & Allen, B. J. Global comparison of targeted alpha vs targeted beta therapy for cancer: In vitro, in vivo and clinical trials. Crit. Rev. Oncol. Hematol .123, 7-20 (2018).Salerno, K. E. et al. A Primer on Radiopharmaceutical Therapy. Int. J. of Radiat. Oncol.Biol.Phys. 115, 48-59 (2023).Corson, D. R. & Segre, E. Artificially Radioactive Element. Phys. Rev.1940, 58, 672-678.Eychenne, R., Cherel, M., Haddad, F., Guerard, F. & Gestin, J. F. Overview of the Most Promising Radionuclides for Targeted Alpha Therapy: The “Hopeful Eight”. Pharmaceutics 13, (2021).Dekempeneer, Y. et al. Targeted alpha therapy using short-lived alpha-particles and the promise of nanobodies as targeting vehicle. Expert Opin. Biol. Ther.16, 1035-1047 (2016).de Kruijff, R. M., Wolterbeek, H. T. & Denkova, A. G. A Critical Review of Alpha Radionuclide Therapy-How to Deal with Recoiling Daughters? Pharmaceuticals 8, 321-336 (2015).Albertsson, P. et al. Astatine-211 based radionuclide therapy: Current clinical trial landscape. Front Med (Lausanne) 9, 1076210 (2023).Palm, S. et al. Therapeutic Efficacy of Astatine-211-Labeled Trastuzumab on Radioresistant SKOV-3 Tumors in Nude Mice Int. J. Mol. Sci. 69, 572-579 (2007).Ayed, T. et al. At-labeled agents for alpha-immunotherapy: On the in vivo stability of astatine-agent bonds. Eur. J. Med. Chem.116, 156-164 (2016). Watanabe, N., Astatine treatment for RAI-refractory thyroid cancer. Journal of the Japanese Society of Endocrine Surgery, 40, 12-16. Ukon, N. et al. Human dosimetry of free 211At and meta-[211At]astatobenzylguanidine (211At-MABG) estimated using preclinical biodistribution from normal mice. EJNMMI Phys. 7, 1-14 (2020). Sudo, H. et al. Preclinical Evaluation of the Acute Radiotoxicity of the α-Emitting Molecular-Targeted Therapeutic Agent 211At-MABG for the Treatment of Malignant Pheochromocytoma in Normal Mice. Transl. Oncol. 12, 879-888 (2019). Aso, A. et al. Evaluation of Astatine-211-Labeled Fibroblast Activation Protein Inhibitor (FAPI): Comparison of Different Linkers with Polyethylene Glycol and Piperazine. Int. J. Mol. Sci. 24, 8701 (2023). Meyer, GJ et al. Synthesis and analysis of 2-[211At]-l-phenylalanine and 4-[211At]-l-phenylalanine and their uptake in human glioma cell cultures in-vitro. Applied Radiation and Isotopes 68, 1060-1065 (2010).Watabe, T. et al.Comparison of the Therapeutic Effects of [211At]NaAt and [131I]NaI in an NIS-Expressing Thyroid Cancer Mouse Model. Int J Mol Sci 23, (2022).Watabe, T. et al. Targeted Alpha Therapy Using Astatine (211At)-Labeled Phenylalanine: A Preclinical Study in Glioma Bearing Mice. Oncotarget vol. 11 www.oncotarget.com (2020).Watabe, T. et al. Targeted α-therapy using astatine (211 At)-labeled PSMA1, 5, and 6: a preclinical evaluation as a novel compound. Eur. J. Nucl. Med. Mol. Imaging 1, 3.

[0013] The present invention aims to develop a small molecule-based compound that exhibits high accumulation in cancer cells and is capable of emitting α-rays, and to provide a pharmaceutical composition for cancer treatment containing the compound. Another object of the present invention is to provide a method for producing such a compound.

[0014] The present inventors ２１１ As a completely new target biomarker for At-labeled therapeutic drugs, we focused on cancer-specific hydrolase activity, which was discovered in the course of fluorescent probe development research in our laboratory.

[0015] Our laboratory has designed and developed a group of activatable fluorescent probes that acquire strong fluorescence after reacting with target hydrolases by controlling the intramolecular spirocyclization equilibrium, and has applied them to cancer imaging. One of these, gGlu-HMRG, is a fluorescent probe that can detect the activity of γ-glutamyl transpeptidase (GGT), which is responsible for the γ-glutamyl group transfer reaction, and has been successfully used to image clinical specimens such as breast cancer. This is expected to be used as a tool for rapid intraoperative cancer diagnosis.

[0016] In addition, the fluorescent probe 4-CH developed by the laboratory of the present inventors ２ F-HMDiEtR-gGlu reacted with GGT to generate a reactive azaquinone methide, which became fluorescent upon attack by intracellular nucleophiles and self-immobilized, enabling washout-resistant tumor imaging.

[0017] Based on these findings, the present inventors have incorporated quinone methide chemistry into molecular design, ２１１ We believe that it is possible to develop a new therapeutic drug using At as a labeled nuclide. This will enable us to expand our target to completely different cancer biomarkers than before. ２１１ As a result of extensive research into the possibility of expanding the range of application of At drugs, the present invention was completed.

[0018] That is, the present invention has the following configurations: [1] A pharmaceutical composition for cancer treatment, comprising a compound represented by the following general formula (I) or a salt thereof: (wherein X is a fluorine atom, an ester group (—OC(═O)—R′), a carbonate group (—OCO ２ -R'), carbamate group (-OCONH-R'), phosphoric acid and its ester group (-OP(=O)(-OR')(-OR''), and sulfuric acid and its ester group (-OSO ２-OR'), wherein R' and R'' are each independently selected from a substituted or unsubstituted alkyl group or a substituted or unsubstituted aryl group; Y is -NH-CO-L, -NH-L', -OL'' or -OL''', wherein L, together with the C=O to which it is attached, constitutes an amino acid residue or a peptide, L' is a saccharide partial structure having a self-cleaving linker, or an amino acid residue or peptide having a self-cleaving linker; L'' is a saccharide partial structure; L''' is a saccharide partial structure having a self-cleaving linker, or an amino acid residue or peptide having a self-cleaving linker; wherein the saccharide partial structure is a structure obtained by removing one hydroxyl group from a saccharide, and R １ and R ２ are each independently selected from a hydrogen atom or a monovalent substituent; R ３ is a hydrogen atom or one to two identical or different monovalent substituents present on a benzene ring; R ４ is a hydrogen atom, or a substituent or molecule capable of changing pharmacokinetics, and the substituent or molecule may be bonded to the benzene ring via a linker; Z represents a single bond or a linking group.) [2] The pharmaceutical composition according to [1], wherein the substituent or molecule capable of changing pharmacokinetics is introduced into the benzene ring via a linker or directly. [3] The linker is an alkylene group (provided that one or more —CH ２ The pharmaceutical composition according to [1] or [2], wherein the linking group of Z is selected from the group consisting of an alkylene group (wherein - may be substituted by -O-, -S-, -NH-, or -CO-), an arylene (including heteroarylene), a cycloalkylene, an alkoxyl group, a polyethylene glycol chain, and a group formed by arbitrarily bonding two or more groups selected from these groups. [4] The pharmaceutical composition according to [1] or [2], wherein the linking group of Z is selected from the group consisting of an alkylene group (wherein one or more -CH ２The pharmaceutical composition according to any one of [1] to [3], wherein -Y in general formula (I) is selected from the group consisting of -C(R １ ) (R ２ The pharmaceutical composition according to any one of [1] to [4], wherein Y is bonded to X at the ortho- or para-position of the benzene ring. [6] The pharmaceutical composition according to any one of [1] to [5], wherein Y has a structure selected from the following: [7] The pharmaceutical composition according to any one of [1] to [6], wherein X is a fluorine atom or an ester group (—OCO—R′). [8] R １ and R ２ [9] The pharmaceutical composition according to any one of [1] to [7], wherein R is independently selected from a hydrogen atom or a fluorine atom. ３ The monovalent substituent is an alkyl group, an alkoxycarbonyl group (—CO—OR ａ ), nitro group, amino group, hydroxyl group, alkylamino group (-NHR ａ , -NR ａ ２ ), an alkoxy group (—OR ａ ), an ester group (—O—CO—R ａ ), a halogen atom, a boryl group, and a cyano group (R ａ is a substituted or unsubstituted alkyl group or a substituted or unsubstituted aryl group, R ａ If there are two or more R ａ may be the same or different), the pharmaceutical composition according to any one of [1] to [8].

[10] R ３

[11] The pharmaceutical composition according to any one of [1] to [9], wherein the monovalent substituent of R is an alkyl group or an alkoxycarbonyl group. ３

[12] The compound or salt thereof according to any one of [1] to [9], wherein the monovalent substituent of R is a halogen atom. ３At least one of the monovalent substituents of R is an alkyl group or an alkoxycarbonyl group, ３

[13] The compound or salt thereof according to any one of [1] to [9], wherein at least one of the monovalent substituents represented by R is a halogen atom. ３ and R ４ are all hydrogen atoms.

[14] The pharmaceutical composition according to any one of [1] to

[13] , which can accumulate in cancer cells by acting selectively on cells due to cancer cell-specific enzymatic activity.

[15] The pharmaceutical composition according to

[14] , wherein the enzyme is a peptidase or glycosidase.

[16] The pharmaceutical composition according to

[14] or

[15] , which is administered intravenously, intraperitoneally, or intratumorally to a subject.

[17] A compound of the following general formula (I): (wherein X is a fluorine atom, an ester group (—OC(═O)—R′), a carbonate group (—OCO ２ -R'), carbamate group (-OCONH-R'), phosphoric acid and its ester group (-OP(=O)(-OR')(-OR''), and sulfuric acid and its ester group (-OSO ２ -OR'), wherein R' and R'' are each independently selected from a substituted or unsubstituted alkyl group or a substituted or unsubstituted aryl group; Y is -NH-CO-L, -NH-L', -OL'' or -OL''', wherein L, together with the C=O to which it is attached, constitutes an amino acid residue or a peptide, L' is a saccharide partial structure having a self-cleaving linker, an amino acid residue or a peptide having a self-cleaving linker; L'' is a saccharide partial structure; L''' is a saccharide partial structure having a self-cleaving linker, or an amino acid residue or a peptide having a self-cleaving linker; wherein the saccharide partial structure is a structure obtained by removing one hydroxyl group from a saccharide, and R １ and R ２ are each independently selected from a hydrogen atom or a monovalent substituent; R ３ is a hydrogen atom or one to two identical or different monovalent substituents present on a benzene ring; R４ is a hydrogen atom, or a substituent or molecule capable of changing pharmacokinetics, and the substituent or molecule may be bonded to the benzene ring via a linker; and Z represents a single bond or a linking group. A method for preparing a compound represented by the following formula (Ia) or a salt thereof, comprising the steps of: (A) adding (In the formula, X, Y, Z, R １ ~R ４ is the same as defined in general formula (I), and E represents a halogen atom. ５ ) ３ Sn-Sn(R ５ ) ３ to obtain a precursor compound represented by the following formula (Ib); (In the formula, X, Y, Z, R １ ~R ４ is the same as defined in general formula (I), R ５ represents an alkyl group having 1 to 4 carbon atoms. (B) Adding to the precursor compound obtained in the step (A), ２１１ (C) a step of purifying the reaction product obtained in step (B) to obtain the compound represented by formula (I).

[18] The preparation method according to

[17] , wherein in step (C), the reaction product obtained in step (B) is purified by solid-phase extraction.

[19] The method according to

[17] or

[18] , wherein sodium ascorbate is added in step (B) and / or step (C).

[20] A method for diagnosing and treating a disease or a symptom that may lead to a disease, comprising the steps of: (a) administering a diagnostic agent comprising a compound represented by formula (II) or a salt thereof to a subject having or suspected of having a disease or symptom; (In the formula, X ａ represents a fluorine atom, an ester group (-OC(=O)-R'), a carbonate group (-OCO ２ -R'), carbamate group (-OCONH-R'), phosphoric acid and its ester group (-OP(=O)(-OR')(-OR''), and sulfuric acid and its ester group (-OSO ２—OR′), wherein R′ and R″ are each independently selected from a substituted or unsubstituted alkyl group or a substituted or unsubstituted aryl group; Y ａ is -NH-CO-L, -NH-L', -OL" or -OL"', wherein L, together with the C=O to which it is attached, constitutes an amino acid residue or a peptide, L' is a saccharide partial structure having a self-cleaving linker, an amino acid residue or a peptide having a self-cleaving linker; L" is a saccharide partial structure; L'" is a saccharide partial structure having a self-cleaving linker, or an amino acid residue or a peptide having a self-cleaving linker; wherein the saccharide partial structure is a structure obtained by removing one hydroxyl group from a saccharide, and R １ａ and R ２ａ are each independently selected from a hydrogen atom or a monovalent substituent; R ３ａ is a hydrogen atom or one to two identical or different monovalent substituents present on a benzene ring; R ４ａ is a hydrogen atom, or a substituent or molecule capable of changing pharmacokinetics, and the substituent or molecule may be bonded to the benzene ring via a linker; Z ａ represents a single bond or a linking group, and U represents １２５ I or １２３ (b) examining the presence of one or more selected from the group consisting of tumors, cancer cells, and cancer tissues in a target tissue or target organ of the subject by measuring radiation emitted from the radionuclide contained in the agent localized in the target tissue or target organ by a nuclear medicine examination; (c) administering a pharmaceutical composition for cancer treatment comprising a compound represented by the following general formula (I) or a salt thereof to a subject in whom the presence of one or more selected from the group consisting of tumors, cancer cells, and cancer tissues in the target tissue or target organ has been confirmed; (wherein X is a fluorine atom, an ester group (—OC(═O)—R′), a carbonate group (—OCO ２ -R'), carbamate group (-OCONH-R'), phosphoric acid and its ester group (-OP(=O)(-OR')(-OR''), and sulfuric acid and its ester group (-OSO２ -OR'), wherein R' and R'' are each independently selected from a substituted or unsubstituted alkyl group or a substituted or unsubstituted aryl group; Y is -NH-CO-L, -NH-L', -OL'' or -OL''', wherein L, together with the C=O to which it is attached, constitutes an amino acid residue or a peptide, L' is a saccharide partial structure having a self-cleaving linker, an amino acid residue or a peptide having a self-cleaving linker; L'' is a saccharide partial structure; L''' is a saccharide partial structure having a self-cleaving linker, or an amino acid residue or a peptide having a self-cleaving linker; wherein the saccharide partial structure is a structure obtained by removing one hydroxyl group from a saccharide, and R １ and R ２ are each independently selected from a hydrogen atom or a monovalent substituent; R ３ is a hydrogen atom or one to two identical or different monovalent substituents present on a benzene ring; R ４ is a hydrogen atom, or a substituent or molecule capable of changing pharmacokinetics, and the substituent or molecule may be bonded to the benzene ring via a linker; and Z represents a single bond or a linking group.

[21] The method according to

[20] , wherein the nuclear medicine examination is at least one selected from the group consisting of scintigraphy, SPECT (single photon emitter computed tomography), and PET (positron emitter tomography).

[22] The method according to

[20] or

[21] , further comprising the step of: (d) administering the diagnostic agent to the subject to which the pharmaceutical composition for cancer treatment was administered in step (c), and measuring by nuclear medicine examination the radiation emitted from the radionuclide contained in the diagnostic agent localized in the target tissue or target organ of the subject, thereby examining the presence of one or more selected from the group consisting of tumors, cancer cells, and cancer tissues in the target tissue or target organ, thereby assessing the therapeutic effect.

[23] A compound represented by the following general formula (I) or a salt thereof: (wherein X is a fluorine atom, an ester group (—OC(═O)—R′), a carbonate group (—OCO ２-R'), carbamate group (-OCONH-R'), phosphoric acid and its ester group (-OP(=O)(-OR')(-OR''), and sulfuric acid and its ester group (-OSO ２ -OR'), wherein R' and R'' are each independently selected from a substituted or unsubstituted alkyl group or a substituted or unsubstituted aryl group; Y is -NH-CO-L, -NH-L', -OL'' or -OL''', wherein L, together with the C=O to which it is attached, constitutes an amino acid residue or a peptide, L' is a saccharide partial structure having a self-cleaving linker, an amino acid residue or a peptide having a self-cleaving linker; L'' is a saccharide partial structure; L''' is a saccharide partial structure having a self-cleaving linker, or an amino acid residue or a peptide having a self-cleaving linker; wherein the saccharide partial structure is a structure obtained by removing one hydroxyl group from a saccharide, and R １ and R ２ are each independently selected from a hydrogen atom or a monovalent substituent; R ３ is a hydrogen atom or one to two identical or different monovalent substituents present on a benzene ring; R ４ represents a hydrogen atom, or a substituent or molecule capable of changing pharmacokinetics, and the substituent or molecule may be bonded to the benzene ring via a linker; and Z represents a single bond or a linking group.

[0019] According to the present invention, it is possible to provide a novel pharmaceutical composition for cancer treatment containing a low-molecular-weight compound that exhibits high accumulation in cancer cells and is capable of emitting α-rays. The pharmaceutical composition for cancer treatment according to the present invention is different from conventional low-molecular-weight compounds. ２１１ The At-labeled agent can target a different biomarker.

[0020] Glu-4-, an example of the compound of the present invention ２１１ A schematic diagram of the metabolic trapping mechanism of At-FMA is shown. A general conceptual diagram of radiotheranostics is shown. ２１１ The outline of the method for producing At is shown below. ２１１A schematic diagram of the enzymatic reaction of At-MA with GGT is shown. ２１１ The results of Radio-TLC analysis of At-FMA are shown below. ２１１ The stability of At-FMA is shown. ２１１ The results of HPLC analysis of the in vitro enzymatic reaction products of At-FMA are shown below. １２５ 1 shows the results of HPLC analysis of the in vitro enzymatic reaction products of I-MA. ２１１ The results of the retention evaluation (1) of At-FMA are shown below. ２１１ The results of the retention evaluation of At-FMA (2) are shown. SDS-PAGE and autoradiography of A549 cell lysate are shown. ２１１ The results of the colony assay (1) of At-FMA are shown below. ２１１ The results of the At-FMA colony assay (2) are shown below. ２１１ The results of evaluation of DNA damage markers using At-FMA are shown. The outline of treatment experiment 1 in Example 2 is shown. ２１１ 1 shows the evaluation results of the therapeutic effect of At-FMA. 2 shows the evaluation results of the weight change in therapeutic experiment 1. 3 shows the results of confirming the therapeutic effect by fluorescent imaging of the tumor in therapeutic experiment 1. 4 shows an outline of therapeutic experiment 2 in Example 2. ２１１ 1 shows the results of evaluating the therapeutic effect of At-FMA. 2 shows the results of evaluating the change in body weight in treatment experiment 2. 3 shows the results of confirming the therapeutic effect by fluorescent imaging of the tumor in treatment experiment 2.

[0021] In this specification, the term "halogen atom" refers to a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.

[0022] As used herein, "alkyl" may refer to any aliphatic hydrocarbon group that is linear, branched, or cyclic, or a combination thereof. The number of carbon atoms in an alkyl group is not particularly limited, and examples include 1 to 6 carbon atoms (C1-6), 1 to 10 carbon atoms (C1-10), 1 to 15 carbon atoms (C1-15), and 1 to 20 carbon atoms (C1-20). When the number of carbon atoms is specified, it means an "alkyl" having a carbon number within that range. For example, C1-8 alkyl includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neo-pentyl, n-hexyl, isohexyl, n-heptyl, n-octyl, and the like. As used herein, an alkyl group may have one or more optional substituents. Examples of such substituents include, but are not limited to, alkoxy groups, halogen atoms, amino groups, mono- or di-substituted amino groups, substituted silyl groups, and acyl. When an alkyl group has two or more substituents, they may be the same or different. The same applies to the alkyl moiety of other substituents containing an alkyl moiety (e.g., alkoxy groups, arylalkyl groups, etc.).

[0023] In this specification, when a functional group is defined as "optionally substituted," the type, substitution position, and number of substituents are not particularly limited, and when two or more substituents are present, they may be the same or different. Examples of substituents include, but are not limited to, alkyl groups, alkoxy groups, hydroxyl groups, carboxyl groups, halogen atoms, sulfo groups, amino groups, alkoxycarbonyl groups, and oxo groups. These substituents may further have a substituent. Examples of such substituents include, but are not limited to, halogenated alkyl groups and dialkylamino groups.

[0024] As used herein, "aryl" may refer to either a monocyclic or fused polycyclic aromatic hydrocarbon group, or an aromatic heterocycle containing one or more heteroatoms (e.g., oxygen, nitrogen, or sulfur atoms) as ring-constituting atoms. In this case, this may be referred to as a "heteroaryl" or a "heteroaromatic". Whether the aryl is a monocyclic or fused ring, it may be bonded at any available position. Non-limiting examples of monocyclic aryls include a phenyl group (Ph), a thienyl group (2- or 3-thienyl group), a pyridyl group, a furyl group, a thiazolyl group, an oxazolyl group, a pyrazolyl group, a 2-pyrazinyl group, a pyrimidinyl group, a pyrrolyl group, an imidazolyl group, a pyridazinyl group, a 3-isothiazolyl group, a 3-isoxazolyl group, a 1,2,4-oxadiazol-5-yl group, or a 1,2,4-oxadiazol-3-yl group. Non-limiting examples of fused polycyclic aryls include 1-naphthyl, 2-naphthyl, 1-indenyl, 2-indenyl, 2,3-dihydroinden-1-yl, 2,3-dihydroinden-2-yl, 2-anthryl, indazolyl, quinolyl, isoquinolyl, 1,2-dihydroisoquinolyl, 1,2,3,4-tetrahydroisoquinolyl, indolyl, isoindolyl, phthalazinyl, quinoxalinyl, benzofuranyl, 2,3-dihydrobenzofuran-1-yl, 2,3-dihydrobenzofuran-2-yl, 2,3-dihydrobenzothiophen-1-yl, 2,3-dihydrobenzothiophen-2-yl, benzothiazolyl, benzimidazolyl, fluorenyl, or thioxanthenyl. In this specification, an aryl group may have one or more optional substituents on its ring. Examples of such substituents include, but are not limited to, an alkoxy group, a halogen atom, an amino group, a mono- or di-substituted amino group, a substituted silyl group, or an acyl group. When an aryl group has two or more substituents, they may be the same or different. The same applies to the aryl moiety of other substituents containing an aryl moiety (e.g., an aryloxy group, an arylalkyl group, etc.).

[0025] In this specification, the term "alkoxy group" refers to a structure in which the alkyl group is bonded to an oxygen atom, and examples thereof include saturated alkoxy groups that are linear, branched, cyclic, or a combination thereof. Suitable examples include methoxy, ethoxy, n-propoxy, isopropoxy, cyclopropoxy, n-butoxy, isobutoxy, s-butoxy, t-butoxy, cyclobutoxy, cyclopropylmethoxy, n-pentyloxy, cyclopentyloxy, cyclopropylethyloxy, cyclobutylmethyloxy, n-hexyloxy, cyclohexyloxy, cyclopropylpropyloxy, cyclobutylethyloxy, and cyclopentylmethyloxy groups.

[0026] In the present specification, "alkylene" refers to a divalent group consisting of a linear or branched saturated hydrocarbon, and examples thereof include methylene, 1-methylmethylene, 1,1-dimethylmethylene, ethylene, 1-methylethylene, 1-ethylethylene, 1,1-dimethylethylene, 1,2-dimethylethylene, 1,1-diethylethylene, 1,2-diethylethylene, 1-ethyl-2-methylethylene, trimethylene, 1-methyltrimethylene, 2-methyltrimethylene, 1,1-dimethyltrimethylene, 1,2 2-dimethyltrimethylene, 2,2-dimethyltrimethylene, 1-ethyltrimethylene, 2-ethyltrimethylene, 1,1-diethyltrimethylene, 1,2-diethyltrimethylene, 2,2-diethyltrimethylene, 2-ethyl-2-methyltrimethylene, tetramethylene, 1-methyltetramethylene, 2-methyltetramethylene, 1,1-dimethyltetramethylene, 1,2-dimethyltetramethylene, 2,2-dimethyltetramethylene, 2,2-di-n-propyltrimethylene, and the like.

[0027] 1. Compounds Represented by General Formula (I) or Salts Thereof One embodiment of the present invention is a compound represented by the following general formula (I) or a salt thereof (hereinafter also referred to as "compounds of the present invention").

[0028] Because nuclides are constantly undergoing radioactive decay, there is concern that therapeutic drugs may increase the risk of side effects when circulating in the blood or when they accumulate non-specifically in tissues. Therefore, in the development of radioactive therapeutic drugs, it is desirable to use low molecular weight compounds that have the property of being excreted from the body relatively quickly as carriers for radionuclides. However, rapid excretion alone is not enough to expect a high therapeutic effect, so it is necessary to devise ways to make the radionuclide remain in tumor tissue. Therefore, in this study, we used an enzyme reaction as a trigger to ２１１ A small molecule designed to retain At in cells ２１１ We worked on developing At-labeled therapeutic drugs.

[0029] 18F-FDG is a representative example of a radiopharmaceutical that achieves a high tumor-to-background (T / B) ratio using a small-molecule nuclear medicine probe that is rapidly excreted from the body. In cancer cells with active glycolysis, the activity of glucose transporters (GLUTs) that transport sugars into the cells is increased, and 18F-FDG, a glucose analog, serves as a substrate for GLUTs and is taken up into cancer cells. However, unlike glucose, after the initial phosphorylation to FDG-6-phosphate by hexokinase, it cannot undergo further metabolism, resulting in the accumulation of radionuclides within cancer cells. This mechanism, which utilizes the metabolic activity unique to cancer tissue to promote the uptake of radionuclides into cancer tissue and further retain them within, is called "metabolic trapping." In the present invention, the principles of "quinone methide chemistry" were incorporated into the molecular design of the compounds of the present invention to achieve enzymatic activity-dependent metabolic trapping. (Aza)quinone methides are electrophilic intermediates with very short lifetimes that readily undergo Michael addition reactions with thiol groups to form covalent bonds.

[0030] Without intending to be bound by theory, one example of a compound of the present invention, Glu-4- ２１１ A schematic diagram of the specific metabolic trapping mechanism of At-FMA is shown in Figure 1. ２１１When the enzyme substrate site (γ-glutamyl group) contained in At-FMA is hydrolyzed by the target enzyme (GGT), fluorine is eliminated to generate an electrophilic azaquinone methide intermediate. This highly reactive azaquinone methide intermediate then rapidly forms a covalent bond with intracellular nucleophiles such as proteins, resulting in: ２１１ At accumulates in cells with high target enzyme activity. GGT is localized on the cell membrane, and its enzyme pocket faces outside the cell, so the azaquinone methide intermediate is generated outside the cell, but it is highly lipid-soluble and is presumed to be rapidly transferred into the target cell. On the other hand, it has poor membrane permeability before the enzyme reaction, so it does not accumulate in cells with low enzyme activity. In this way, Glu-4- ２１１ At-FMA can be selectively taken up by GGT activity and retained in cells.

[0031] In general formula (I), Y is an enzyme recognition site, a portion of which is cleaved by a cancer cell-specific enzymatic activity to induce the formation of a quinone methide. Y can be selected depending on the type of target enzyme. When the target enzyme, a cancer biomarker enzyme, is a peptidase, Y is selected from groups derived from amino acids and groups containing amino acids, and when the target enzyme is a glycosidase, Y is selected from groups derived from sugars.

[0032] In general formula (I), Y is -NH-CO-L, -NH-L', -OL'', or -OL''', where L is a partial structure of an amino acid. The partial amino acid structure of L means that L, together with the C=O to which it is bonded, constitutes an amino acid residue or a peptide. L' is a partial structure of a saccharide having a self-cleaving linker, or an amino acid residue or peptide having a self-cleaving linker. L'' is a partial structure of a saccharide. L''' is a partial structure of a saccharide having a self-cleaving linker, or an amino acid residue or peptide having a self-cleaving linker. The partial structure of a saccharide and self-cleaving linkers will be described in detail later.

[0033] As used herein, the term "amino acid" refers to any compound having both an amino group and a carboxyl group, including natural and unnatural amino acids. Neutral, basic, or acidic amino acids may be used. In addition to amino acids that function as neurotransmitters and other transmitters, amino acids that are components of polypeptide compounds such as physiologically active peptides (including dipeptides, tripeptides, tetrapeptides, and oligopeptides) and proteins may also be used, such as α-amino acids, β-amino acids, and γ-amino acids. Optically active amino acids are preferably used as amino acids. For example, while either D- or L-amino acids may be used for α-amino acids, it may be preferable to select optically active amino acids that function in living organisms.

[0034] As used herein, the term "amino acid residue" refers to a structure corresponding to the partial structure remaining after removing the hydroxyl group from the carboxyl group of an amino acid. Amino acid residues include α-amino acid residues, β-amino acid residues, and γ-amino acid residues. Preferred amino acid residues include the γ-glutamyl group of a GGT substrate, a dipeptide of a DPP4 substrate (a dipeptide consisting of an amino acid and proline), and dipeptides such as EK (Glu-Lys) and NA (Asn-Ala).

[0035] In this specification, the amino acid residue also includes the partial structure remaining after removing the hydroxyl group from the carboxyl group in the side chain of an amino acid, such as the above-mentioned γ-glutamyl group.

[0036] As used herein, "peptide" refers to a structure in which two or more amino acids are linked by peptide bonds. "Peptide residue" refers to a structure corresponding to the partial structure remaining after removing the hydroxyl group from the carboxyl group of the C-terminal amino acid constituting the peptide. Preferred peptides include the above-mentioned DPP4 substrate dipeptide (a dipeptide consisting of amino acid-proline; here, the amino acid is, for example, glycine, glutamic acid, or proline), KK, which is the target sequence of PSA, and the like.

[0037] The N-terminus of the monovalent substituent containing an amino acid residue or an oligopeptide residue may be protected. Examples of the protecting group include an acetyl group, a glutaryl group, a succinyl group, a tert-butoxycarbonyl group, and a benzyloxycarbonyl group, but other substituents may also be used.

[0038] L' is a partial structure of a saccharide having a self-cleaving linker, or an amino acid residue or peptide having a self-cleaving linker. L" is a partial structure of a saccharide. L'" is a partial structure of a saccharide having a self-cleaving linker, or an amino acid residue or peptide having a self-cleaving linker; here, the partial structure of a saccharide refers to a structure corresponding to the partial structure remaining after removing one hydroxyl group from a saccharide. The partial structure of a saccharide, together with the O to which L" is bound, constitutes a saccharide or a part of a saccharide. The partial structure of a saccharide, together with the O in the self-cleaving linker possessed by L', constitutes a saccharide or a part of a saccharide, and together with the O in the self-cleaving linker possessed by L'", constitutes a saccharide or a part of a saccharide. That is, when Y is -OL", this site corresponds to a substrate of a glycolytic enzyme. Furthermore, embodiments containing a self-cleaving linker that targets a glycolytic enzyme (target enzyme) are classified into a case where Y is -NH-L' and a case where Y is -OL''', and L', L''' are partial structures of a saccharide having a self-cleaving linker.

[0039] Examples of sugars include β-D-glucose, β-D-galactose, β-L-galactose, β-D-xylose, α-D-mannose, β-D-fucose, α-L-fucose, β-L-fucose, β-D-arabinose, β-L-arabinose, β-DN-acetylglucosamine, and β-DN-acetylgalactosamine, with β-D-galactose and β-DN-acetylgalactosamine being preferred.

[0040] The self-cleaving linker means a linker that is spontaneously cleaved and decomposed, and examples thereof include carbamate, urea, a para-aminobenzyloxy group, and an ester group (-CO-O-, -O-CO-).

[0041] Furthermore, the self-cleaving linker is preferably a benzyl group-type linker, a carbamate group-type linker, a carbonate group-type linker, or a combination of two or more of these.

[0042] In the case of a benzyl group-type linker, Y is -NH-L', and when glycosidase (sugar hydrolase) is targeted, it is combined with the nitrogen atom to form (partial structure of sugar) -O-Ph-CH ２ -NH- ＊ When aminopeptidase (protein hydrolase) is targeted, the compound can be combined with the nitrogen atom to form (amino acid residue or peptide)-CO-NH-Ph-CH ２ -NH- ＊ Here, Ph represents a substituted or unsubstituted benzene ring, and * represents the direction of bonding to the benzene ring in general formula (I). Similarly, in the case of a carbamate group-type linker, when Y is -NH-L' and glycosidase (sugar hydrolase) is targeted, it can be combined with the nitrogen atom to form (partial structure of sugar) -O-CO-HN- ＊ , (partial structure of sugar)-O-Ph-CH ２ —O—CO—HN— ＊ When targeting aminopeptidase (protein hydrolase), the hydroxyl group can be combined with the nitrogen atom to form (amino acid residue or peptide)-CO-NH-Ph-CH ２ —O—CO—NH— ＊ Similarly, as for the carbonate group type linker, when Y is -OL''' and L''' is a partial structure of a saccharide having a self-cleaving linker, it can be formed together with its oxygen atom to form (partial structure of saccharide) -O-CO-O- ＊ , (partial structure of sugar)-O-Ph-CH ２ -O-CO-O- ＊ When Y is -OL''' and aminopeptidase (protein hydrolase) is targeted, it can be formed together with the oxygen atom into (amino acid residue or peptide)-CO-NH-Ph-CH ２ -O-CO-O-＊ can be configured.

[0043] In one preferred aspect of the invention, Y has a structure selected from the following:

[0044] In general formula (I), X acts as a leaving group that is eliminated from the benzene ring when the enzyme recognition site of Y is partially cleaved by a cancer cell-specific enzyme activity, resulting in the formation of a quinone methide.

[0045] X is a fluorine atom, an ester group (-OC(=O)-R'), a carbonate group (-OCO ２ -R'), carbamate group (-OCONH-R'), phosphoric acid and its ester group (-OP(=O)(-OR')(-OR''), and sulfuric acid and its ester group (-OSO ２ —OR′), where R′ and R″ are each independently selected from a substituted or unsubstituted alkyl group or a substituted or unsubstituted aryl group.

[0046] X is preferably a fluorine atom or an ester group (-OCO-R'). Without intending to be bound by theory, when X is a fluorine atom or an ester group (-OC(=O)-R'), a quinone methide is rapidly formed upon cleavage of Y.

[0047] R １ and R ２ are each independently selected from a hydrogen atom or a monovalent substituent. Examples of the monovalent substituent include a halogen atom and an alkyl group having one or more carbon atoms (for example, an alkyl group having about 1 to 6 carbon atoms). １ and R ２ are preferably each independently selected from a hydrogen atom or a fluorine atom.

[0048] In general formula (I), -Y is -C(R １ ) (R ２ It is preferable that —Y and —C(R) are bonded to X at the ortho or para position of the benzene ring. １ ) (R ２) When X is in such a positional relationship on the benzene ring, a quinone methide structure can be formed when Y is cleaved.

[0049] R ３ R is a hydrogen atom or one to two identical or different monovalent substituents present on the benzene ring. ３ Examples of the monovalent substituent include an alkyl group having one or more carbon atoms (for example, an alkyl group having about 1 to 6 carbon atoms), an alkoxycarbonyl group (—C(═O)—OR ａ ), nitro group, amino group, hydroxyl group, alkylamino group (-NHR ａ , -NR ａ ２ ), an alkoxy group (—OR ａ ), an ester group (—O—CO—R ａ ), an amide group (—NHCOR ａ ), a halogen atom, a boryl group, and a cyano group. ａ is a substituted or unsubstituted alkyl group or a substituted or unsubstituted aryl group. ａ If there are two or more R ａ may be the same or different.

[0050] In one aspect of the compounds of the present invention, R ３ The monovalent substituent is an alkyl group (e.g., a methyl group) or an alkoxycarbonyl group (e.g., a methoxycarbonyl group). Introduction of an alkyl group, which is an electron-donating group, into the benzene ring is preferred because it results in excellent intracellular retention.

[0051] In one aspect of the compounds of the present invention, R ３ The monovalent substituent of R is a halogen atom (preferably an iodine atom). ３ When is a halogen atom (preferably an iodine atom), it is possible to enhance the trapping effect on cells.

[0052] In one aspect of the compounds of the present invention, R ３ at least one of the monovalent substituents of R is an alkyl group (e.g., a methyl group) or an alkoxycarbonyl group (e.g., a methoxycarbonyl group), ３At least one of the monovalent substituents is a halogen atom.

[0053] R ３ When R is a monovalent substituent as defined above, in particular an alkyl group, ３ The position of -C(R １ ) (R ２ ) The 5-position, which corresponds to the para-position of X, and / or the 4-position, which corresponds to the meta-position of X, are preferred.

[0054] In another aspect of the compounds of the invention, R ３ All of the atoms are hydrogen atoms.

[0055] R in general formula (I) ４ is a hydrogen atom, or a substituent or molecule that can change the pharmacokinetics.

[0056] The substituent or molecule capable of altering pharmacokinetics may be any substituent or molecule known to alter pharmacokinetics. Examples of such substituents or molecules include structures known to bind to serum albumin, such as substituted or unsubstituted biphenyl groups; monovalent or divalent substituents derived from bicyclic compounds (e.g., naphthalene, quinoline, etc.); dye molecules such as Evans Blue; and monovalent or divalent substituents derived from p-iodophenylbutyric acid. Here, a monovalent substituent derived from a bicyclic compound refers to a monovalent substituent (e.g., a naphthyl group) obtained by removing one hydrogen atom from a bicyclic compound, and a divalent substituent derived from a bicyclic compound refers to a divalent substituent obtained by removing two hydrogen atoms from a bicyclic compound. The monovalent or divalent substituent derived from a bicyclic compound may be unsubstituted or may have a substituent. Examples of these substituents include alkyl groups, alkoxy groups, hydroxyl groups, carboxyl groups, halogen atoms, sulfo groups, amino groups, alkoxycarbonyl groups, and oxo groups. Furthermore, substituents or molecules capable of altering pharmacokinetics also include groups formed by two or more of the same or different substituents or molecules listed above, optionally linked via a linking group. For example, substituents or molecules capable of altering pharmacokinetics include (but are not limited to) groups formed by two or more of the same or different substituted or unsubstituted biphenyl groups, optionally linked via a linking group; groups formed by two or more of the same or different substituted or unsubstituted naphthyl groups, optionally linked via a linking group; and groups formed by one or more substituted or unsubstituted biphenyl groups and one or more substituted or unsubstituted naphthyl groups (when there are two or more of either or both of these, they may be the same or different), optionally linked via a linking group.

[0057] The linking group may be any group that functions as a linker and is metabolically stable, but is preferably an alkylene group (provided that the alkylene group has one or more —CH ２- may be substituted with -O-, -S-, -NH-, or -CO-), arylene (including heteroarylene), cycloalkylene (for example, cyclohexylene), alkoxyl group, polyethylene glycol chain, and a group formed by arbitrarily bonding two or more groups selected from these groups.

[0058] By introducing a substituent or molecule capable of changing such pharmacokinetics into the benzene ring, it becomes easier to increase and / or adjust the half-life in blood, and the degree of freedom in the administration route can be increased when a pharmaceutical composition containing the compound of the present invention is administered to a subject.

[0059] The above-mentioned substituents or molecules capable of changing the pharmacokinetics can be introduced into the benzene ring via a linker or directly.

[0060] The linker may be an alkylene group (provided that the alkylene group has one or more —CH ２ - may be substituted with -O-, -S-, -NH-, or -CO-), arylene (including heteroarylene), cycloalkylene, alkoxyl group, polyethylene glycol chain, and a group formed by arbitrarily bonding two or more groups selected from these groups.

[0061] In one aspect of the compounds of the present invention, R ４ is a hydrogen atom.

[0062] In one aspect of the compounds of the present invention, R ３ and R ４ are all hydrogen atoms.

[0063] In general formula (I), Z represents a single bond or a linking group. When Z is a single bond, ２１１ This means that A is directly bonded to the benzene ring without a linking group.

[0064] The linking group may be any group that functions as a linker and is metabolically stable, but is preferably an alkylene group (provided that the alkylene group has one or more —CH ２The - may be substituted with -O-, -S-, -NH-, or -CO-. ), arylene (including heteroarylene), cycloalkylene (e.g., cyclohexylene), alkoxyl group, polyethylene glycol chain, and a group formed by arbitrarily bonding two or more groups selected from these groups. The number of carbon atoms in the alkylene group is not particularly limited, but is preferably 5 to 20, and more preferably 5 to 15. In addition, -CH in the alkylene group ２ Even when - is substituted with -O-, -S-, -NH-, or -CO-, these groups are considered to have one carbon atom and are included in the above-mentioned "number of carbon atoms in the alkylene group." In addition, arylene includes those that use a benzene ring as a linker, such as a phenylene group, and divalent linkers derived from aromatic and cyclic hydrocarbons containing heterocycles.

[0065] In one preferred embodiment of the compound of the present invention, the linking group is an alkylene group (provided that the alkylene group has one or more —CH ２ - may be substituted by -O-, -S-, -NH-, or -CO-.

[0066] ２１１ The position at which At-Z- is introduced is not particularly limited, but it is preferably bonded to the meta or para position of the benzene ring relative to Y, because this is metabolically stable and if At-Z- is too close to the enzyme recognition site, it may not serve as a substrate for the target enzyme.

[0067] Non-limiting examples of the compounds of the present invention are shown below, but the compounds of the present invention are not limited to these.

[0068] ​Unless otherwise specified, the compounds represented by general formula (I) also include stereoisomers such as their tautomers, geometric isomers (e.g., E-isomer, Z-isomer, etc.), and enantiomers. That is, when the compound represented by general formula (I) contains one or more asymmetric carbon atoms, the stereochemistry of the asymmetric carbon atoms can independently take either the (R) or (S) form, and the derivatives may exist as stereoisomers such as enantiomers or diastereoisomers. Therefore, the active ingredient of the nuclear medicine examination probe of the present invention can be any pure stereoisomer, any mixture of stereoisomers, or a racemate, and all of these are within the scope of the present invention.

[0069] 2. Method for Preparing the Compound of the Present Invention The method for preparing the compound represented by general formula (I) is not particularly limited, but it is preferable to prepare it by the following preparation method. That is, another embodiment of the present invention is a method for preparing a compound represented by general formula (I) or a salt thereof, which comprises the following steps: (A) adding a compound represented by the following formula (Ia): (In the formula, X, Y, Z, R １ ~R ４ is the same as defined in general formula (I), and E represents a halogen atom. ５ ) ３ Sn-Sn(R ５ ) ３ to obtain a precursor compound represented by the following formula (Ib); (In the formula, X, Y, Z, R １ ~R ４ is the same as defined in general formula (I), R ５ represents an alkyl group having 1 to 4 carbon atoms. (B) Adding to the precursor compound obtained in the step (A), ２１１ (C) a step of purifying the reaction product obtained in step (B) to obtain the compound represented by formula (I) (hereinafter also referred to as the "preparation method of the present invention").

[0070] ２１１ Considering that the physical half-life of At is short at 7.2 hours, ２１１ ​To achieve a high radiochemical yield (RCY) in the synthesis of At-labeled compounds, it is desirable to perform radiolabeling at the final stage of the synthesis process. In addition, simplifying the labeling procedure also reduces worker exposure. Therefore, in the present invention, it is important to use a compound of formula (Ib) as a labeled precursor so that the target compound of general formula (I) can be obtained in a single labeled synthesis step.

[0071] In step (A), Pd ２ (dba) ３ It is preferable to use

[0072] Used in step (B) ２１１ Attenuated ions can be obtained from bismuth (Bi) using an accelerator (cyclotron). Preferably, it can be obtained by the following two steps: Step 1: Irradiating a bismuth target with an α beam. Bismuth is irradiated with α particles accelerated to 28 MeV or higher by a cyclotron. ２０９ Bi+α→ ２１１ By the At + 2n reaction ２１１ If an alpha beam of 30 MeV or more hits the material, ２０９ Bi+α→ ２１０ At+3n occurs, ２１０ High bone marrow toxicity due to electronic decay of At ２１０ Since Po (half-life: 138 days) is generated, it is preferable that the beam energy be controlled with an accuracy of ±1%.

[0073] Step 2: Purification by dry separation method ２１１ The boiling point of At is 335°C, and the boiling point of Bi is 1564°C, so it is preferable to separate them by utilizing the difference in volatility. After α-beam irradiation, the Bi target is placed in a quartz tube and heated to about 850°C in an electric furnace. ２１１ At was passed through a fluororesin tube cooled to about -196°C together with a helium flow. ２１１ At is solidified and collected on the inner wall of the tube. The trapped astatine is then dissolved in a solvent such as chloroform and collected in a vial. The solvent in the collection vial is then blown off with nitrogen gas to obtain dry astatine.

[0074] In the preparation method of the present invention, it is preferable to add sodium ascorbate in step (B) and / or step (C). ２１１ At-labeled compounds are ２１１ The α- and γ-rays emitted by the decay of At decompose water molecules, and the resulting reactive oxygen species may damage the structure of the compound. ２１１ In At labeling, the use of a solvent containing sodium ascorbate as a quenching solvent or purification solvent can prevent the structure of the compound from being damaged by reactive oxygen species. In the preparation method of the present invention, it is more preferable to add sodium ascorbate in steps (B) and (C).

[0075] In the preparation method of the present invention, in step (C), the reaction product obtained in step (B) is preferably purified by solid-phase extraction, and more preferably by solid-phase extraction using Sep-Pak. This allows rapid purification, minimizing the time required for purification, and also achieving a high radiochemical yield (RCY), making it possible to obtain the target product in high yield.

[0076] For representative compounds included in general formula (I), synthesis methods using the preparation method of the present invention are specifically shown in the examples of this specification. Those skilled in the art can produce compounds included in general formula (I) by appropriately changing or modifying the starting materials, reaction reagents, reaction conditions, etc. as necessary, while referring to the examples of this specification and the following schemes.

[0077] 2. Pharmaceutical Composition for Cancer Treatment Another embodiment of the present invention is a pharmaceutical composition for cancer treatment comprising a compound represented by the following general formula (I) or a salt thereof (the compound of the present invention) (hereinafter also referred to as the "pharmaceutical composition of the present invention"). (wherein X is a fluorine atom, an ester group (—OC(═O)—R′), a carbonate group (—OCO ２ -R'), carbamate group (-OCONH-R'), phosphoric acid and its ester group (-OP(=O)(-OR')(-OR''), and sulfuric acid and its ester group (-OSO２ -OR'), wherein R' and R'' are each independently selected from a substituted or unsubstituted alkyl group or a substituted or unsubstituted aryl group; Y is -NH-CO-L, -NH-L', -OL'' or -OL''', wherein L, together with the C=O to which it is attached, constitutes an amino acid residue or a peptide, L' is a saccharide partial structure having a self-cleaving linker, an amino acid residue or a peptide having a self-cleaving linker; L'' is a saccharide partial structure; L''' is a saccharide partial structure having a self-cleaving linker, or an amino acid residue or a peptide having a self-cleaving linker; wherein the saccharide partial structure is a structure obtained by removing one hydroxyl group from a saccharide, and R １ and R ２ are each independently selected from a hydrogen atom or a monovalent substituent; R ３ is a hydrogen atom or one to two identical or different monovalent substituents present on a benzene ring; R ４ represents a hydrogen atom, or a substituent or molecule capable of changing pharmacokinetics, and the substituent or molecule may be bonded to the benzene ring via a linker; and Z represents a single bond or a linking group.

[0078] X, Y, Z, R １ ~R ４ The details of are the same as those described in detail for the compounds of the present invention.

[0079] A preferred embodiment of the pharmaceutical composition of the present invention is a pharmaceutical composition for cancer treatment used in α-ray therapy.

[0080] The pharmaceutical composition of the present invention can be accumulated in cancer cells by acting selectively on the cells, preferably through cancer cell-specific enzymatic activity.

[0081] In the pharmaceutical composition etc. of the present invention, the cancer cell-specific enzyme is preferably a peptidase or glycosidase. Examples of peptidases include γ-glutamyltranspeptidase (GGT), dipeptidyl peptidase IV (DPP-IV), PSA (puromycin-sensitive aminopeptidase), APN (aminopeptidase N), cathepsin B / L, calpain, etc. Examples of glycosidases include β-galactosidase, β-glucosidase, α-mannosidase, α-L-fucosidase, β-hexosaminidase, β-N-acetylgalactosaminidase, etc.

[0082] Without intending to be bound by theory, it is believed that the pharmaceutical composition of the present invention is taken up into cells in a cancer-specific enzyme activity-dependent manner, forms a covalent bond with an intracellular protein due to the generation of a quinone methide intermediate, and thereby acquires intracellular retention, and maintains high intracellular retention due to the formation of a covalent bond with an intracellular protein. ２１１ It is believed that At can be highly accumulated in cancer cells, thereby exerting an excellent therapeutic effect.

[0083] The pharmaceutical composition of the present invention can be administered into the body of a human or non-human animal (such as a mouse, rat, hamster, rabbit, cat, dog, cow, sheep, or monkey), and the α-rays emitted from the body act on one or more selected from the group consisting of tumors, cancer cells, and cancer tissues present in a target tissue or target organ, thereby achieving a therapeutic effect. Examples of diseases that can be treated with the pharmaceutical composition of the present invention include, but are not limited to, breast cancer, esophageal cancer, lung cancer, stomach cancer, colon cancer, small intestine cancer, pancreatic cancer, liver cancer, head and neck cancer, oral cancer, ovarian cancer, brain tumor, kidney cancer, prostate cancer, skin cancer, brain tumor, malignant melanoma, head and neck cancer, liver cancer, colon cancer, glioblastoma, sarcoma, bone cancer, brain cancer, head and neck cancer, skin cancer, thyroid cancer, bladder cancer, peritoneal dissemination, mesothelioma, meningioma, and sarcoma.

[0084] The pharmaceutical composition of the present invention can be suitably used for the treatment of primary cancer, metastatic cancer including peritoneal dissemination, blood cancer, sarcoma, and the like.

[0085] "Dissemination" refers to the process by which cancer cells break away from the site of initial development in the body and spread throughout the body via bodily fluids, or metastasize to other organs. "Peritoneal dissemination" refers to the state of cancer cells leaving the primary tumor and spreading to the peritoneum, a state also called "peritoneal metastasis." In the state of peritoneal dissemination, "malignant peritonitis" and "malignant ascites" may occur concomitantly. "Malignant ascites" refers to the seepage of blood components into the peritoneal cavity due to peritoneal dissemination of cancer, and is also called "malignant ascites" or "ascites."

[0086] Because complete surgical removal is difficult, chemotherapy with anticancer drugs is the primary treatment for peritoneal dissemination. The current standard chemotherapy regimen is paclitaxel plus carboplatin (TC). In recent years, accumulating case data and conducting clinical trials have established chemotherapy methods for peritoneal dissemination, and improvements have been proposed. However, current challenges have also become apparent. One issue is that sensitivity to chemotherapy varies depending on the histological type of cancer. Furthermore, chemotherapy is administered once a week over a long period of time, placing a significant burden on patients. While intraperitoneal administration in particular is expected to improve therapeutic efficacy, its invasive nature can reduce patients' quality of life. Alpha-ray therapy is considered effective in treating peritoneal dissemination, given these challenges. Because its antitumor effect is mediated by the physical action of alpha rays, there is little risk of reduced tumor growth suppression due to drug resistance. Therefore, it could be a new treatment option for cancers that are refractory to conventional chemotherapy. Furthermore, because it exhibits sustained therapeutic effects with a single dose, treatment can be completed in a short period of time with fewer doses, reducing the burden on patients.

[0087] Therefore, one preferred embodiment of the pharmaceutical composition of the present invention is a pharmaceutical composition for cancer treatment used in the treatment of peritoneal dissemination.

[0088] The pharmaceutical composition of the present invention may contain not only the compound represented by formula (I) but also a salt thereof, or a solvate or hydrate thereof. The salt is not particularly limited as long as it is a pharmaceutically acceptable salt, and examples thereof include base addition salts, acid addition salts, and amino acid salts. Examples of base addition salts include alkaline earth metal salts such as sodium salts, potassium salts, calcium salts, and magnesium salts, ammonium salts, and organic amine salts such as triethylamine salts, piperidine salts, and morpholine salts. Examples of acid addition salts include mineral acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; and organic acid salts such as methanesulfonic acid, benzenesulfonic acid, paratoluenesulfonic acid, acetic acid, propionate, tartaric acid, fumaric acid, maleic acid, malic acid, oxalic acid, succinic acid, citric acid, benzoic acid, mandelic acid, cinnamic acid, lactic acid, glycolic acid, glucuronic acid, ascorbic acid, nicotinic acid, and salicylic acid. Examples of amino acid salts include glycine salts, aspartates, glutamates, etc. Metal salts such as aluminum salts may also be used.

[0089] The type of solvent that forms the solvate is not particularly limited, but examples include solvents such as ethanol, acetone, and isopropanol.

[0090] Another embodiment of the present invention is a method for treating cancers such as primary cancers such as breast cancer, esophageal cancer, lung cancer, stomach cancer, colon cancer, small intestine cancer, pancreatic cancer, liver cancer, head and neck cancer, oral cancer, ovarian cancer, brain tumor, kidney cancer, prostate cancer, and skin cancer, metastatic cancers including peritoneal dissemination, blood cancer, and sarcoma in mammals, particularly humans, which comprises administering to a mammal in need of such treatment a pharmaceutical composition comprising an effective amount of a compound of the present invention represented by general formula (I) or a pharmaceutically acceptable salt thereof.

[0091] Although the prodrug-type anticancer agent or pharmaceutical composition of the present invention may be administered as the active ingredient, that is, the compound represented by general formula (I) or a pharmaceutically acceptable salt, hydrate, or solvate thereof, itself, it is generally desirable to administer it in the form of a pharmaceutical composition containing the above substance as the active ingredient and one or more formulation additives. The term "composition," as in pharmaceutical composition, encompasses not only a product containing an active ingredient and an inactive ingredient (a pharmaceutically acceptable excipient) that constitutes a carrier, but also any product that occurs directly or indirectly as a result of the association, complexation, or aggregation of any two or more components, or as a result of the dissociation of one or more components, or as a result of another type of reaction or interaction of one or more components.

[0092] As the active ingredient of the pharmaceutical composition of the present invention, two or more of the above compounds can be used in combination.

[0093] In addition, the pharmaceutical composition of the present invention contains ２１１ In addition to At-labeled compounds, ２１１ The composition may further contain a stabilizer for the purpose of stabilizing the bond between At and the carbon atom, suppressing decomposition of the compound, and increasing the stability of the compound in vivo. Such stabilizers are typically compounds having a reducing action, such as ascorbic acid, alkali metal ascorbate, alkali metal earth ascorbate, cysteine, glutathione, gentisic acid, glucose, etc. Preferred are ascorbic acid, alkali metal ascorbate, or alkali metal earth ascorbate, and more preferred are ascorbic acid or sodium ascorbate.

[0094] The type of pharmaceutical composition of the present invention is not particularly limited, and it can be formulated by mixing with a pharmaceutically acceptable carrier or diluent according to a known method. The dosage form is not particularly limited, and it can be a pharmaceutical composition for oral administration in the form of an injection, tablet, powder, granule, capsule, liquid, suppository, sustained-release agent, etc. These formulations are prepared according to conventional methods.

[0095] Liquid formulations may be dissolved or suspended in water or other suitable solvents before use. Tablets and granules may be coated by known methods. Injectable formulations are prepared by dissolving the compounds of the present invention in water, but may also be dissolved in physiological saline or glucose solution as needed, and buffers and preservatives may be added. They may be provided in any formulation for oral or parenteral administration. For example, they may be prepared as oral pharmaceutical compositions in the form of granules, fine granules, powders, hard capsules, soft capsules, syrups, emulsions, suspensions, or solutions; parenteral pharmaceutical compositions in the form of injections for intravenous, intramuscular, or subcutaneous administration, infusions, transdermal absorbents, transmucosal absorbents, nasal drops, inhalants, suppositories, and the like. Injectable formulations and infusions may be prepared in powdered forms, such as lyophilized forms, and then dissolved in an appropriate aqueous medium, such as physiological saline, before use. Furthermore, sustained-release formulations coated with polymers or the like may be administered directly into the brain.

[0096] The type of formulation additives used in the production of the pharmaceutical composition of the present invention, the ratio of the formulation additives to the active ingredient, or the production method of the pharmaceutical composition can be appropriately selected by those skilled in the art depending on the form of the composition. The formulation additives can be inorganic or organic substances, or solid or liquid substances, and can generally be blended in an amount of 1 to 90% by weight based on the weight of the active ingredient. Specific examples of such substances include lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium aluminometasilicate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch, calcium carboxymethylcellulose, ion exchange resins, methylcellulose, gelatin, gum arabic, hydroxypropyl cellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, light anhydrous silicic acid, magnesium stearate, talc, tragacanth, bentonite, Veegum, titanium oxide, sorbitan fatty acid esters, sodium lauryl sulfate, glycerin, fatty acid glycerin esters, purified lanolin, glycerogelatin, polysorbate, macrogol, vegetable oils, wax, liquid paraffin, white petrolatum, fluorocarbons, nonionic surfactants, propylene glycol, and water.

[0097] To prepare solid formulations for oral administration, the active ingredient is mixed with excipients such as lactose, starch, crystalline cellulose, calcium lactate, and anhydrous silicic acid to form a powder, or, if necessary, with binders such as sucrose, hydroxypropyl cellulose, and polyvinylpyrrolidone, and disintegrants such as carboxymethylcellulose and calcium carboxymethylcellulose, followed by wet or dry granulation to form granules. To prepare tablets, these powders and granules can be compressed directly or with the addition of lubricants such as magnesium stearate and talc. These granules or tablets can be coated with enteric-coated bases such as hydroxypropylmethylcellulose phthalate and methacrylic acid-methyl methacrylate polymer to form enteric-coated formulations, or coated with ethylcellulose, carnauba wax, and hydrogenated oil to form sustained-release formulations. To prepare capsules, the powder or granules can be filled into hard capsules, or the active ingredient can be dissolved directly or in glycerin, polyethylene glycol, sesame oil, olive oil, etc., and then coated with a gelatin membrane to form soft capsules.

[0098] To prepare an injection, the active ingredient may be dissolved in distilled water for injection, if necessary, together with a pH adjuster such as hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, sodium monohydrogen phosphate, sodium dihydrogen phosphate, etc., and an isotonic agent such as sodium chloride or glucose, and then sterile filtered and filled into an ampule, or mannitol, dextrin, cyclodextrin, gelatin, etc. may be added, followed by vacuum freeze-drying to produce an injection that is dissolved before use. Alternatively, the active ingredient may be emulsified in water with lecithin, polysorbate 80, polyoxyethylene hydrogenated castor oil, etc. to produce an emulsion for injection.

[0099] The pharmaceutical composition of the present invention may be administered locally or systemically. The route of administration can be determined appropriately depending on the condition of the subject, and may be prepared as a pharmaceutical composition for parenteral administration in the form of an injection, such as for intravenous administration, intraarterial administration, intradermal administration, intramuscular administration, intraperitoneal administration, or intratumoral administration. The pharmaceutical composition of the present invention is preferably administered intravenously, intraperitoneally, or intratumorally.

[0100] The dosage of the pharmaceutical composition of the present invention is preferably 1.0 MBq or less, more preferably 0.2 to 0.5 MBq, in terms of the radiation dose of the drug. When the dosage is within the above range, the therapeutic effect is sufficiently exhibited and side effects are minimal.

[0101] 3. Diagnostic and Treatment Methods of the Present Invention Another embodiment of the present invention is a method for diagnosing and treating a disease or a condition that may lead to a disease, comprising the steps of: (a) administering to a subject having or suspected of having the disease or condition a diagnostic agent comprising a compound represented by the following formula (II) or a salt thereof; (In the formula, X ａ represents a fluorine atom, an ester group (-OC(=O)-R'), a carbonate group (-OCO ２ -R'), carbamate group (-OCONH-R'), phosphoric acid and its ester group (-OP(=O)(-OR')(-OR''), and sulfuric acid and its ester group (-OSO ２ —OR′), wherein R′ and R″ are each independently selected from a substituted or unsubstituted alkyl group or a substituted or unsubstituted aryl group; Y ａ is -NH-CO-L, -NH-L', -OL" or -OL"', wherein L, together with the C=O to which it is attached, constitutes an amino acid residue or a peptide, L' is a saccharide partial structure having a self-cleaving linker, an amino acid residue or a peptide having a self-cleaving linker; L" is a saccharide partial structure; L'" is a saccharide partial structure having a self-cleaving linker, or an amino acid residue or a peptide having a self-cleaving linker; wherein the saccharide partial structure is a structure obtained by removing one hydroxyl group from a saccharide, R １ａ and R ２ａ are each independently selected from a hydrogen atom or a monovalent substituent; R ３ａ is a hydrogen atom or one to two identical or different monovalent substituents present on a benzene ring; R ４ａ is a hydrogen atom, or a substituent or molecule capable of changing pharmacokinetics, and the substituent or molecule may be bonded to the benzene ring via a linker; Z ａrepresents a single bond or a linking group, and U represents １２５ I or １２３ (b) examining the presence of one or more selected from the group consisting of tumors, cancer cells, and cancer tissues in a target tissue or target organ of the subject by measuring, by a nuclear medicine examination, the radiation emitted from the radionuclide contained in the agent localized in the target tissue or target organ of the subject; (c) administering a pharmaceutical composition for cancer treatment, comprising a compound represented by the following general formula (I) or a salt thereof, to a subject in whom the presence of one or more selected from the group consisting of tumors, cancer cells, and cancer tissues in the target tissue or target organ has been confirmed; (wherein X is a fluorine atom, an ester group (—OC(═O)—R′), a carbonate group (—OCO ２ -R'), carbamate group (-OCONH-R'), phosphoric acid and its ester group (-OP(=O)(-OR')(-OR''), and sulfuric acid and its ester group (-OSO ２ -OR'), wherein R' and R'' are each independently selected from a substituted or unsubstituted alkyl group or a substituted or unsubstituted aryl group; Y is -NH-CO-L, -NH-L', -OL'' or -OL''', wherein L, together with the C=O to which it is attached, constitutes an amino acid residue or a peptide, L' is a saccharide partial structure having a self-cleaving linker, an amino acid residue or a peptide having a self-cleaving linker; L'' is a saccharide partial structure; L''' is a saccharide partial structure having a self-cleaving linker, or an amino acid residue or a peptide having a self-cleaving linker; wherein the saccharide partial structure is a structure obtained by removing one hydroxyl group from a saccharide, and R １ and R ２ are each independently selected from a hydrogen atom or a monovalent substituent; R ３ is a hydrogen atom or one to two identical or different monovalent substituents present on a benzene ring; R ４is a hydrogen atom, or a substituent or molecule capable of changing pharmacokinetics, and the substituent or molecule may be bonded to the benzene ring via a linker; and Z represents a single bond or a linking group (hereinafter also referred to as the "diagnostic and therapeutic method of the present invention").

[0102] The diagnostic and therapeutic method of the present invention is one of so-called radiotheranostics, which integrates nuclear medicine diagnosis and therapy by utilizing the differences in the properties of radionuclides. Figure 2 shows a general conceptual diagram of radiotheranostics. The compound of formula (II) is a radionuclide １２５ I or １２３ Although the radionuclide is different from that of the compound of the present invention, the skeletal structure is the same. ａ By selecting the same or similar enzyme recognition site as the target, it is possible to establish the concept of radiotheranostics, which targets the same or similar cancer cell-specific enzyme activity, by combining a nuclear medicine diagnostic agent (nuclear medicine diagnostic probe) that targets the same or similar cancer cell-specific enzyme activity with a medical treatment drug (cancer drug).

[0103] In formula (II), X ａ , Y ａ , Z ａ , R １ａ ~R ４ａ For details of X, Y, Z, R of the compound or salt of general formula (I) described in detail in item 1, １ ~R ４ The details are the same as in

[0104] The diagnostic agent comprising the compound of formula (II) is preferably a nuclear medicine imaging agent.

[0105] As used herein, the term "nuclear medicine imaging diagnostic agent" refers to a drug containing the compound of formula (II) used in in vivo nuclear medicine testing, which is administered into the body and measures and images radiation (radioactive signals) emitted from the body from outside the body to evaluate or test the biological functions of organs or tissues, diagnose diseases, etc., or a drug containing the compound of formula (II) used in in vitro nuclear medicine testing, which is reacted in a test tube with a sample such as tissue or blood collected from the body to evaluate or test the biological functions of organs or tissues, diagnose diseases, etc. Examples of in vivo nuclear medicine testing include the above-mentioned methods using nuclear medicine imaging probes such as scintigraphy, SPECT (single photon emitter computed tomography), and PET (positron emitter computed tomography).

[0106] Furthermore, the diagnostic agent containing the compound of formula (II) is an imaging agent.

[0107] As used herein, "imaging" includes administering a compound of formula (II) containing a radionuclide (imaging probe) into the body and measuring and imaging radiation (radioactive signals) emitted from the body from outside the body. In another aspect, "imaging" includes measuring and imaging radiation (radioactive signals) emitted from a living body to which a compound of formula (II) containing a radionuclide (imaging probe) has been administered from outside the body. "Imaging" includes acquiring measurement data and / or image data for nuclear medicine imaging diagnosis.

[0108] A diagnostic agent containing a compound of formula (II) can be accumulated in cancer cells by acting selectively on the cells, preferably through a cancer cell-specific enzyme activity.

[0109] In diagnostic agents containing the compound of formula (II), the cancer cell-specific enzyme is preferably a peptidase or glycosidase. Examples of peptidases include γ-glutamyltranspeptidase (GGT), dipeptidylpeptidase IV (DPP-IV), PSA (puromycin-sensitive aminopeptidase), APN (aminopeptidase N), cathepsin B / L, and calpain. Examples of glycosidases include β-galactosidase, β-glucosidase, α-mannosidase, α-L-fucosidase, β-hexosaminidase, and β-N-acetylgalactosaminidase.

[0110] Diagnostic agents containing a compound of formula (II) may include not only the compound represented by formula (II) but also its salts, solvates, or hydrates thereof. The salts are not particularly limited as long as they are pharmaceutically acceptable, and examples thereof include base addition salts, acid addition salts, and amino acid salts. Examples of base addition salts include alkaline earth metal salts such as sodium salts, potassium salts, calcium salts, and magnesium salts, ammonium salts, and organic amine salts such as triethylamine salts, piperidine salts, and morpholine salts. Examples of acid addition salts include mineral acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; and organic acid salts such as methanesulfonic acid, benzenesulfonic acid, paratoluenesulfonic acid, acetic acid, propionate, tartaric acid, fumaric acid, maleic acid, malic acid, oxalic acid, succinic acid, citric acid, benzoic acid, mandelic acid, cinnamic acid, lactic acid, glycolic acid, glucuronic acid, ascorbic acid, nicotinic acid, and salicylic acid. Examples of amino acid salts include glycine salts, aspartates, glutamates, etc. Metal salts such as aluminum salts may also be used.

[0111] The type of solvent that forms the solvate is not particularly limited, but examples include solvents such as ethanol, acetone, and isopropanol.

[0112] In the diagnostic and therapeutic methods of the present invention, the nuclear medicine examination in step (b) is preferably at least one selected from the group consisting of scintigraphy, SPECT (single photon emitter computed tomography), and PET (positron emitter tomography).

[0113] Scintigraphy and SPECT involve, for example, measuring gamma rays emitted from a subject administered with a diagnostic agent containing a compound of formula (II) using a gamma camera. Measurement with a gamma camera involves, for example, measuring radiation (gamma rays) emitted from the radioactive nuclide of the administered compound over a fixed time period, preferably measuring the direction and quantity of radiation emitted over a fixed time period. The diagnostic and therapeutic methods of the present invention may further include representing the distribution of the measured radioactive compound obtained by measuring the radiation as a cross-sectional image, and reconstructing the obtained cross-sectional image.

[0114] PET may include, for example, counting by a PET detector gamma rays generated by annihilation of positrons and electrons from a subject administered with a diagnostic agent containing the compound of formula (II), and further including depicting the three-dimensional distribution of the positions of positron-emitting radionuclides based on the measurement results.

[0115] X-ray CT and / or MRI measurements may be performed in conjunction with measurements by scintigraphy, SPECT, or PET, thereby making it possible to obtain a fusion image by fusing, for example, an image (functional image) obtained by scintigraphy, SPECT, or PET with an image (morphological image) obtained by CT or MRI.

[0116] The diagnostic agent containing the compound of formula (II) may be administered locally or systemically. The route of administration can be appropriately determined depending on the condition of the subject, etc., and it can also be prepared as a pharmaceutical composition for parenteral administration in the form of an injection, for example, for intravenous administration, intraarterial administration, intradermal administration, intramuscular administration, intraperitoneal administration, or intratumoral administration.

[0117] The administration amount (dose) of the diagnostic agent containing the compound of formula (II) is not particularly limited, and it is sufficient to administer an amount sufficient to obtain the desired contrast for imaging, and can be, for example, 1 μg or less.

[0118] The diagnostic agent containing the compound of formula (II) may be prepared in the form of a solution or powder. These preparations are prepared according to conventional methods. Liquid preparations may be dissolved or suspended in water or other suitable solvents before use. Tablets and granules may be coated by known methods. Injections are prepared by dissolving the compound of the present invention in water, but may also be dissolved in physiological saline or glucose solution as needed, and buffers and preservatives may be added.

[0119] The diagnostic and therapeutic methods of the present invention may further comprise the step of (d) administering the diagnostic agent to the subject to which the pharmaceutical composition for cancer treatment was administered in step (c), and measuring by nuclear medicine examination the radiation emitted from the radionuclide contained in the diagnostic agent localized in the target tissue or target organ of the subject, thereby examining the presence of one or more selected from the group consisting of tumors, cancer cells, and cancer tissues in the target tissue or target organ, thereby assessing the therapeutic effect.

[0120] The present invention will be described in more detail below with reference to examples, but the scope of the present invention is not limited to these examples.

[0121] Materials and General Procedures. Reagents and solvents were of the highest grade and were supplied by Aldrich Chemical Co., Ltd., Tokyo Chemical Industry Co., Ltd., Fujifilm Wako Pure Chemical Industries, Ltd., Kanto Chemical Co., Ltd., Gibco, Invitrogen, Toyobo Co., Ltd., and Thermo Scientific Co., Ltd., and were used as is without purification. Reactions were monitored by TLC and ESI-MS or UPLC-MS. γ-Glutamyltranspeptidase from bovine kidney (product number 46556003) was purchased from Oriental Yeast Co., Ltd. gGlu-HMRG, compounds 1, 2, 3, and 4 were synthesized as described in the following papers 1 and 2. Paper 1: Ueo, H. et al. Rapid intraoperative visualization of breast lesions with γ-glutamyl hydroxymethyl rhodamine green. Sci Rep 5, (2015). Paper 2: Suzuki, H., Kanna53ka, K. & Uehara, T. Approaches to Reducing Normal Tissue Radiation from Radiolabeled Antibodies. Pharmaceuticals 17, 508 (2024).

[0122] Measurement equipment NMR spectrum was measured using a JEOL JNM-ECZ400S instrument. １ H NMR at 400 MHz; １３ C NMR was measured at 101 MHz. Mass spectra were measured using a JEOL JMS-T100LC AccuToF (ESI). Preparative HPLC of the compounds was performed using an HPLC system consisting of a pump (PU-2086 Plus, JASCO) and a detector (MD-201, JASCO) or a Biotage® Sfar C18 D (Biotage) with an eluent of C (H ２ O) and eluent D (CH ３CN) at a flow rate of 5 mL / min on an Inertsil™ ODS-3 5 mm (10.0 × 250 mm) column (GL Sciences Inc.). Preparative MPLC was performed on a silica gel column (silica gel 40 mm, Yamazen) using an MPLC system consisting of a pump and a detector (EPCLC AI-580S, Yamazen). Quantitation of absorbance in multiwell plates was performed using a multimode plate reader Envision® 2105 (PerkinElmet). LC / MS analysis was performed using a reversed-phase column (InertSustain C18 4.6 mm × 150 mm; GL Sciences) and a GABI* radioactivity-HPLC-flow monitor (Elysia-raytest) with eluent A (100 mM ammonium acetate) and eluent B (MeCN / H ２ Analysis was performed using a SHIMADZU Prominence LC-20AB HPLC system (Shimadzu Corporation) at a flow rate of 1 mL / min using a 1000 Hz chromatograph (O = 80 / 20). Autoradiography was performed using an Amersham Typhoon scanner (Cytiva) with an imaging plate (BAS-III; Fujifilm Corporation). In vivo luminescence images were obtained using an IVIS Lumina S5 imaging system (Summit Pharmaceuticals International Corporation). Ex vivo fluorescence images were obtained using a Maestro in-vivo imaging system (Cri Inc) with blue filter settings (excitation, 445-490 nm; emission, 515 nm long pass). Radioactivity was measured using a 2480 Wizard 2 gamma counter (Perkin Elmer) and a Curie Meter IGC-8 (ALOKA).

[0123] In this study, gGlu-4- ２１１A series of experiments was carried out, from the synthesis of At-FMA to the evaluation of its functionality in vivo. Each experimental method and result is explained in detail below. The production, synthesis, and purification of astatine were carried out at the Nuclear Chemistry Research and Development Laboratory of the Nishina Center for Accelerator-Based Science at RIKEN, while the subsequent evaluation of its drug properties was carried out at the Functional Diagnostics Development Division of the Advanced Medical Research Center at the National Cancer Center (Kashiwa).

[0124] 1. ２１１ Production of At 　２１１ At is an artificial element synthesized using a cyclotron. In this study, it was produced using the AVF cyclotron at the RIKEN Nishina Center for Accelerator-Based Science. ２１１ At was used. ２１１ The method for producing At is as follows.

[0125] Step 1: Alpha beam irradiation of bismuth target Alpha particles accelerated to 28.0 MeV in a cyclotron are used. ２０９ Irradiating a Bi metal target, ２０９ Bi+α→ ２１１ By the At + 2n reaction ２１１ If an alpha beam of 30 MeV or more hits the material, ２０９ Bi+α→ ２１０ At+3n occurs, ２１０ High bone marrow toxicity due to electronic decay of At ２１０ Po (half-life: 138 days) is generated, so the beam energy is controlled with an accuracy of ±1% (FIG. 3(A)).

[0126] Step 2: Purification by dry separation method ２１１ The boiling point of At is 335°C, and that of Bi is 1564°C, and these differences in volatility are utilized for separation. After α-beam irradiation, the Bi target is placed in a quartz tube and heated to 850°C in an electric furnace. ２１１ At was passed through a fluororesin tube cooled to -196°C together with a helium flow. ２１１ At is solidified and collected on the inner wall of the tube. The trapped astatine is then dissolved in chloroform and collected in a vial. The chloroform in the collection vial is then blown off with nitrogen gas, and the resulting dry astatine is supplied (Figure 3(B)).

[0127] FIG. 3(A) shows the 209Bi(α,2n)211At and 209Bi(α,3n)210At nuclear reactions resulting from irradiation of a natural bismuth target. ２１１ At and ２１０ The cross section of At production is shown (Feng et al., Hiromitsu Haba, Production of Radioisotopes for Nuclear Medicine Therapy at RIKEN. Drug Delivery System 35, 115-120 (2020)). Figure 3 (B) shows a schematic diagram of astatine production. After irradiation with a 4He ion beam, the target vessel is heated, ２１１ Only At (circles) evaporates and is chemically separated from the target, which is then transported by a helium stream and cooled and solidified within the tube.

[0128] [Synthesis Example 1] gGlu-4- ２１１ Synthesis of At-FMA According to the following synthesis scheme (Scheme 1), the compound of the present invention, gGlu-4- ２１１ At-FMA was synthesized.

[0129] <Scheme 1>

[0130] ２１１ Considering that the physical half-life of At is short at 7.2 hours, ２１１ To achieve a high radiochemical yield (RCY) in the synthesis of At-labeled compounds, it is desirable to perform radiolabeling at the final stage of the synthesis process. In addition, simplification of the labeling procedure also leads to a reduction in worker exposure. Therefore, it is possible to obtain the target compound gGlu-4- in a one-step labeled synthesis. ２１１ gGlu-4-SnMe was used as the labeling precursor to obtain At-FMA. ３ -FMA (compound 5) was prepared. Furthermore, rapid purification using solid-phase extraction with Sep-Pak enabled us to minimize the time required for purification. As a result, we achieved a radiochemical yield (RCY) of 65%, enabling us to obtain the target product in high yield. The results of the synthesis experiment for compound 5 are described below.

[0131] (1) Synthesis of Compound 5 ​

[0132] In a flask filled with argon gas, compound 4 (74.0 mg, 0.195 mmol) was dissolved in 3 mL of deoxygenated DMF. ３ Sn-SnMe ３ , 0.30 mL, 1.46 mmol) was added, and the solution was degassed using a vacuum pump and purged with argon gas three times. ２ (dba) ３ (89.3 mg, 0.0214 mmol) was added, followed by degassing and purging with argon three times. The reaction mixture was stirred at 60°C overnight, then filtered and evaporated. The residue was purified by column chromatography (silica gel, eluent: hexane / EtOAc = 50 / 50 for 10 min, then MeOH for 10 min). The collected fractions were concentrated in vacuo, and the residue was further purified by preparative HPLC (C / D = 80 / 20 to 0 / 100, 30 min) to give gGlu4-SnMe ３ -FMA (compound 5) (10.7 mg, 0.0342 mmol, 13%) was obtained as a white solid.

[0133] 1 H NMR (400 MHz, CD3OD): δ 7.53 (s, 1H), 7.47 (d, 1H, J = 7.8 Hz), 7.37 (d, 1H, J = 7.6 Hz), 5.37 (d, 2H, JH-F = 48.0 Hz), 3.64 (t, 1H, J = 6.0 Hz), 2.65 (t, 2H, J = 7.0 Hz), 2.18-2.23 (m, 2H), 0.27 (t, 9H, JSn-H = 27.9 Hz); 13 C NMR (100 MHz, CD3OD): δ 174.3, 173.8, 141.7, 137.8, 137.2, 136.7, 132,0, 126.5, 83.5(d, J CF= 163 Hz), 56.6, 33.3, 27.9, -9.9. HRMS: m / z Calcd for C 15 H 23 N2O3FNaSn [M+Na] + :441.06094, found 441.05955 (1.4 mDa).

[0134] (2) ２１１ At labeling Next, from compound 5, gGlu-4- ２１１ Obtain At-FMA (compound 6) ２１１ The protocol for At labeling is shown below.

[0135] ２１１ At labeling protocol A) Precursor (Glu-4-SnMe ３ -FMA) 100 μg dissolved in 10 μL MeOH B) ２１１ At activation: Add 100 μL of 10 mg / mL NCS + 1% AcOH + MeOH to 100 MBq of dry astatine. C) Add 60 MBq (60 μL) of astatine solution to the precursor-mixing container in A. D) 30-minute labeling. E) Add 450 μL of PBS + 0.6% sodium ascorbate (hereinafter referred to as SA) and quench for 3 minutes. F) Purification: (a) Sep-pak activation (MeCN or EtOH → PBS + 0.6% SA). (b) Load 5 mL of reaction solution: PBS + 0.6% SA. (c) Collect 5 tubes of 400 μL each using PBS + 0.6% SA / MeCN = 8 / 2 or PBS + 0.6% SA / EtOH = 6 / 4. G) Transfer from RIKEN to Cancer Center Hospital East.

[0136] In addition, ２１１ At-labeled compounds are ２１１ The alpha and gamma rays emitted by the decay of At break down water molecules, and the resulting reactive oxygen species can damage the structure of the compound. ２１１ In At labeling (reaction f), PBS + 0.6% SA was used as the quenching and purification solvents. As a result, even after 24 hours of labeling, the labeled compound showed high radiochemical purity (RCP) without significant decomposition.

[0137] [Reference Synthesis Example 1] gGlu-4- ２１１ Synthesis of At-MA Furthermore, gGlu-4- ２１１ gGlu-4- ２１１ At-MA labeling was performed.

[0138] <Scheme 2>

[0139] This compound does not have a leaving group at the benzyl position even when GGT reacts with the glutamic acid substrate site, so it is thought that it does not generate a quinone methide and does not exhibit intracellular retention (Figure 4). ２１１ At-FMA and gGlu-4- ２１１ The usefulness of "metabolic trapping using quinone methide chemistry" was evaluated by comparing At-MA with At-MA. The results of the synthesis experiments for Compounds 7 to 10 are described below.

[0140] (1) Synthesis of Compound 7

[0141] In an argon-filled flask fitted with a septum cap, 4-iodo-2-methylaniline (692 mg, 2.97 mmol) was dissolved in 20 mL of MeOH. To this solution was added 1-tert-butyl N-(tert-butoxycarbonyl)-L-glutamate (Boc-Glu-OtBu, 992 mg, 3.27 mmol) and DMTMM (816 mg, 3.27 mmol). The reaction mixture was stirred at room temperature for 4 hours. At the end of the reaction, the reaction mixture was quenched with H ２ The combined organic layers were treated with 200 ml of EtOAc / hexanes and extracted twice with EtOAc / hexanes. ２ SO ４ The mixture was dried at 77°C, filtered, and concentrated in vacuo. The resulting crude product was purified by column chromatography (silica gel, eluent: hexane / EtOAc = 90 / 10 to 70 / 30, 10 min) to give compound 7 (1.00 g, 4.50 mmol, 66%) as a white fluffy solid.

[0142] 1 H NMR (400 MHz, CDCl3): δ 8.31 (s, 1H), 7.67 (d, 1H, J = 8.3 Hz), 7.53 (s, 1), 7.50 (d, 1H, J = 8.3 Hz), 5.36 (d, 1H, J = 7.9 Hz), 5.33-5.38 (m, 1H), 4.21-4.26 (m, 1H), 2.27 (s, 3H), 2.24-2.34 (m, 1H), 1.90-2.01 (m, 1H), 1.46 (s, 9H), 1.45 (s, 9H);13 C NMR (100 MHz, CD3OD): δ171.3, 170.9, 139.2, 136.1, 136.6, 125.1, 89.0, 82.8, 80.5, 53.3, 40.0, 34.0, 31.0, 28.4, 28.0, 17.8 HRMS: m / z Calcd for C 21 H 31 NO2O5INa [M+Na] + :541.11753, found 541.12070 (3.0 mDa).

[0143] (2) Synthesis of Compound 8

[0144] Compound 7 (620.0 mg, 1.2 mmol) was dissolved in 2N HCl / AcOEt (18 mL). The solution was stirred at room temperature overnight. At the end of the reaction, the reaction mixture was concentrated in vacuo. The residue was purified by preparative HPLC (eluent A: 20% MeCN and 0.1% TFA in H NMR). ２ O, B:MeCN 0.1% TFA, A / B=80 / 20 to 0 / 100) to give gGlu-4I-MA (compound 8) (414.1 mg, 1.14 mmol, 95%) as a white solid.

[0145] 1 H NMR (400 MHz, CD3OD): δ7.63(d, 1H, J = 1.4 Hz), 7.53 (dd,1H, J = 8.3,1.4 Hz), 7.19 (d, 1H, J =8.3 Hz), 4.01 (t, 1H, J = 6.4 Hz), 2.73 (t, 2H, J = 6.9 Hz), 2.27-2.23 (m, 2H), 2.23 (s, 3H); 13 C NMR (100 MHz, DMSO-d6): δ 171.0, 170.3, 138.8, 136.5, 134.8, 134.3, 127.2, 90.0, 52.1, 31.4, 26.2, 17.6. HRMS : m / z Calcd for C 12 H 16 N2O3I [M+H] +: 363.02056, Found, 363.01997 (-0.6 mDa)

[0146] (3) Synthesis of Compound 9

[0147] In an argon-filled flask fitted with a septum cap, compound 8 (60 mg, 0.166 mmol) was dissolved in 3 mL of deoxygenated DMF. ３ Sn-SnMe ３ , 0.17 mL, 0.83 mmol) was added, and the solution was degassed using a vacuum pump and purged with argon gas three times. ２ (dba) ３ (151 mg, 0.166 mmol) was added, followed by degassing and purging with argon three times again. The reaction mixture was stirred at 60°C overnight, then filtered and evaporated. The crude product obtained was first purified by normal phase column chromatography (silica gel, eluent: hexane / EtOAc = 50 / 50, 10 min, then MeOH, 10 min). The collected fractions were concentrated in vacuo, and the residue was further purified by preparative HPLC (C / D = 80 / 20 to 0 / 100, 30 min) to give gGlu-4-SnMe ３ -MA Compound 9 (8.2 mg, 0.0342 mmol, 12%) was obtained as a white solid.

[0148] 1 H NMR (400 MHz, CD3OD): δ 7.32(s, 1H), 7.30 (d,1H, J=7.3 Hz), 7.28 (d, 1H, J=7.3 Hz), 3.64 (t, 1H, J = 6.0 Hz), 2.66 (td, 2H, J = 7.1, 2.3 Hz), 2.23 (s, 3H), 2.23-2.16 (m, 2H), 0.27 (t, 9H, JSn-H = 27.9 Hz); 13 C NMR (100 MHz, CD3OD): δ 173.9, 173.7, 141.2, 139.0, 136.9, 134.7, 133.6, 126.3, 55.6, 33.4, 28.0, 18.0, -9.9. HRMS: m / z Calcd for C 15H 24 N2O3NaSn [M+Na] + :423.07036, found 423.07110(-0.7 mDa).

[0149] [Example 1] (1) Confirmation of Radiochemical Purity (RCP) Next, gGlu-4- ２１１ RCP was measured for quality control of At-FMA. It is defined as RCP = (radioactivity found in a specific chemical form / total radioactivity of that nuclide) x 100 (%). The RCP measurement method avoids methods such as HPLC, which may cause impurities to adsorb to the column, and instead employs a method of development by TLC followed by detection by autoradiography (ARG) to ensure that all radioactivity is detected without omission. Specifically, the method is as follows:

[0150] (2) Quality control procedure gGlu-4- ２１１ The radiochemical purity (RCP) of At-FMA was assessed by C18 reverse-phase TLC (Sigma-Aldrich). 0.5 μL of approximately 30 kBq / 20 μL of gGlu-4-211At-FMA (hot) was spotted on the starting line, followed by H as the mobile phase. ２ The TLC plate was developed over a 3.5 cm section with 1:1 HCl / MeCN (4 mL total). The TLC plate was exposed to an imaging plate for 60 minutes and read using an Amersham Typhoon scanner (Cytiva). RCP was calculated by analyzing the resulting image files using Gel and Blot Analysis of ImageQuant TL software (Cytiva).

[0151] Figure 5 shows the results of the analysis of gGlu-4- in PBS + 0.6% SA / MeCN = 8 / 2 (purification solvent after labeling) after overnight incubation at 37°C. ２１１ This is an autoradiography (ARG) image of reversed-phase C-18 TLC when evaluating the RCP of At-FMA. ２１１The RCP was calculated by dividing the volume of the band of the At-labeled compound by the total volume of all bands, and it was found that the RCP remained at approximately 96% even one day after labeling.

[0152] From the above results, it is clear that gGlu-4- ２１１ Storing At-FMA is believed to be an effective method to prevent radiolysis and maintain the stability of the compound.

[0153] In the subsequent experiments, gGlu-4- was stored in PBS + 0.6% SA / MeCN = 8 / 2 until the in vitro experiments, and in PBS + 0.6% SA / EtOH = 6 / 4 for the cell experiments and mouse administration experiments. ２１１ At-FMA was used in part.

[0154] (3) Stability Evaluation Next, the stability of gGlu-4- ２１１ The stability of At-FMA was evaluated by the following procedure.

[0155] HPLC stability evaluation gGlu-4- ２１１ At-FMA (1.5 × 10 in 72 μL of PBS containing 0.6% SA / EOH (8:2, v / v) ３ 100 kBq) was incubated in 1.5 mL of DMEM supplemented with 10% FBS and 1% penicillin-streptomycin (PS) at 37°C for 1 and 18 hours. After incubation, 500 μL of the drug solution was transferred to an Amicon Ultra-0.5 filter (Merck Biopharma Co., Ltd.) and centrifuged at 14,000 × g for 10 minutes. Finally, 50 μL of the solution collected at the bottom of the filter device was injected for HPLC to evaluate the compound stability. The HPLC conditions were as follows: Eluent A, H2O containing 0.1 M ammonium acetate; ２ O; Eluent B, H ２ 80% acetonitrile in O; gradient elution, 70-0% A in 0-10 min, 0% A in 10-11 min, 0-70% A in 11-12.5 min, 70% A in 12.5-15 min; flow rate, 1 mL / min.

[0156] As a result, as shown in Figure 6, the peak of the target compound (retention time: 4.6 minutes) was clearly detected even 18 hours after the start of incubation in the cell culture medium. ２１１ It was confirmed that At-FMA remained stable for 18 hours even in a medium with a reduced concentration of sodium ascorbate, a radioprotectant. ２１１ The figure shows the radioactivity (unit: count) of gamma rays emitted from At. The value on the vertical axis after 18 hours is lower than that at 0 hours and 1 hour. ２１１ This is because the radioactivity decays according to the physical half-life of At.

[0157] (4) Enzyme Reaction Evaluation Based on the evaluations so far, gGlu-4- ２１１ It was confirmed that At-FMA was recovered stably with high purity. ２１１ To confirm whether At-FMA reacts with the target enzyme (GGT) and whether the reaction with GGT generates the active species azaquinone methide required for metabolic trapping, an in vitro enzymatic reaction with GGT was carried out and analyzed by HPLC. Then, gGlu-4-I-FMA (non-radioactive) was used as a standard sample, and the enzymatic reaction products were identified by comparing the peak retention times. Analysis of the enzymatic reaction products was performed as follows.

[0158] Product analysis of in vitro enzymatic reaction gGlu-4- ２１１ At-FMA (1.0 × 10) in 4.0 µL of PBS containing 0.6% SA / EtOH (8:2, v / v) ２ 100 kB) was dissolved in 95 μL of PBS containing 0.6% SA with or without 10 mM L-cysteine ​​containing 0.1% DMSO as a co-solvent. 0.1 units of GGT was then added and incubated at 37°C for 1 hour. The reaction was carried out under two conditions: with or without 10 mM L-cysteine ​​containing 0.1% DMSO as a co-solvent. Eluent A, 0.1 M ammonium acetate. ２ O; Eluent B, H ２80% acetonitrile in O; gradient elution, 95-0% A over 0-20 min; flow rate, 1 mL / min. Additionally, gGlu-4-I-FMA was analyzed under the same HPLC conditions for reference.

[0159] The results are shown in Figure 7. First, gGlu-4- ２１１ When At-FMA and GGT were reacted for 1 hour, H was produced as an azaquinone methide intermediate. ２ Furthermore, a peak was observed that was assumed to be 2-amino-5-astatobenzylalcohol to which O had been added. ２１１ When At-FMA and GGT were reacted for 1 hour, a new peak was detected in addition to 2-amino-5-astatobenzylalcohol. This peak is thought to be the peak of a compound to which L-cysteine ​​was added.

[0160] Next, we investigated the compound gGlu-4-, which does not generate quinone methide even when reacting with GGT. １２５ The enzyme reaction analysis for I-MA was also carried out using the same protocol. １２５ Not an I-labeled compound, ２１１ At-labeled gGlu-4- ２１１ Although the experiment should be conducted with At-MA, ２１１ At has a limited supply and a short physical half-life (7.2 hours), which limits the experiment time. Therefore, in this experiment, we used At, which is more easily available and has a longer half-life. １２５ I (half-life: 59.4 days) was used as a substitute. １２５ The I sign is ２１１ The same precursor as in the case of At labeling, gGlu-4-SnMe ３ As shown in Fig. 2-9, regardless of whether cysteine ​​was added or not, a new peak corresponding only to the enzyme reaction product, 4-iodo-2-methylaniline, was observed. １２５ It was revealed that I-MA does not react with nucleophilic species in the system after reacting with GGT. １２５ This suggests that the reaction of I-MA with GGT does not generate a quinone methide intermediate (Figure 8).

[0161] FIG. 8 shows the gGlu-4- １２５ 1 shows the results of HPLC analysis of the in vitro enzymatic reaction products of I-MA. １２５ I-MA (2.0×10 ２ 100 kBq) was incubated with 0.1 unit of GGT in PBS with or without 10 mM L-cysteine ​​containing 0.1% DMSO as a cosolvent at 37°C for 1 hour. (Eluent A: 0.1 M ammonium acetate in PBS) ２ O, eluent B:H ２ 80% acetonitrile in O, 70-0% A, 0-20 min, 1 mL / min)

[0162] (5) Evaluation of intracellular retention In vitro enzyme reaction analysis revealed that gGlu-4- ２１１ It was confirmed that At-FMA reacts with GGT to generate an azaquinone methide intermediate, which then reacts with nucleophilic species in the system. Based on these findings, experiments were conducted using cultured cells to verify the effectiveness of this molecular design at the cellular level. The intracellular retention was also examined under different conditions to evaluate the following points: (1) whether it is taken up into cells in a manner dependent on the activity of the cancer-specific enzyme (GGT); and (2) whether it forms a covalent bond with intracellular proteins through the generation of a quinone methide intermediate, thereby achieving intracellular retention.

[0163] Retention evaluation (1): Is it taken up into cells depending on GGT activity? ２１１ Intracellular concentrations of At-FMA after 3 hours of exposure ２１１ The radioactivity of At was quantified using a γ counter. The protocol for retention experiment (1) is shown in the upper part of Figure 9, and the details of the experimental conditions are described below.

[0164] Experiment for evaluating retention (1) A549-luc, SKOV3, and H226 cells were plated in 12-well plates at 0.67 × 10 ５ The cells were seeded at a density of 1.5 mL / well and incubated for 1 day. After incubation, the medium was removed and 1.5 mL of gGlu-4- ２１１At-FMA (40 kBq / mL) (±GGsTop) was added to each well. The cells were then incubated at 37°C for 3 hours. After incubation, the medium was collected as the "medium" fraction, and the cells were washed three times with 1 mL of medium and collected as the "wash." After washing, the cells were detached by adding 0.2 mL of 0.25% trypsin-EDTA solution and incubating at 37°C for 5 minutes. 0.8 mL of medium was added, and the entire 1 mL was collected as the "cell" fraction. The radioactivity of the three fractions was measured using a 2480 Wizard2 gamma counter (Perkin Elmer). The ratio of radioactivity in the cell fraction was calculated as a percentage of the sum of all fractions (cells, medium, and wash). Data are presented as the mean ± S.E. with n = 3 biological replicates per condition. Statistical analysis was performed using an unpaired, two-tailed t-test.

[0165] The measurement results are shown in the lower part of Figure 9. As a result, A549, which is a cell line with high GGT activity, showed a higher intracellular ２１１ It was revealed that the accumulation rate of At was more than six times higher. Furthermore, when GGsTop, a GGT inhibitor, was co-administered to A549, ２１１ The intracellular accumulation rate of gGlu-4-At was significantly reduced. ２１１ It was revealed that At-FMA is taken up into cells in a GGT activity-dependent manner. ２１１ p<0.01 compared to At-FMA exposed A549.

[0166] Retention evaluation (2): First, we examine whether the quinone methide intermediate forms a covalent bond with intracellular proteins and acquires intracellular retention. ２１１ To verify whether the generation of quinone methide intermediates is the reason for the intracellular retention of the At signal, we transfected gGlu-4- ２１１ At-FMA, gGlu-4- ２１１ At-MA, ２１１ Three types of At ２１１ The At compounds were exposed and their intracellular retention was compared. ２１１At was prepared by dissolving dry astatine in PBS + 0.6% SA. The protocol for the experiment to evaluate retention (2) is shown in the upper part of Figure 10, and the details of the experimental conditions are described below.

[0167] Experiment (2) for evaluation of retention Experiment (2) was carried out in the same manner as in Experiment (1). In this experiment, A549 cells were cultured in different ２１１ At-labeled drug compound (gGlu-4- ２１１ At-FMA, gGlu-4- ２１１ At-MA, ２１１ The mice were exposed to 100 kBq / mL of ATP (40 kBq / mL each). Data are presented as mean ± S.E. with n=3 biological replicates / condition. Statistical analysis was performed using unpaired two-tailed t-tests.

[0168] The measurement results are shown in the lower part of Figure 10. (** in the lower part of Figure 10: gGlu-4- ２１１ p<0.01 compared with At-FMA.) From FIG. 10, gGlu-4- ２１１ At-FMA exposure ２１１ The intracellular accumulation rate of At ２１１ Approximately 47 times that of At, gGlu-4- ２１１ The level was approximately six times higher than that of At-MA. A549 does not express NIS, a transporter that takes up halide ions. ２１１ Even if At is exposed ２１１ It is thought that the intracellular accumulation rate of At was low. ２１１ Regarding At-MA, in vitro enzyme reaction analysis showed that it reacts with GGT at gGlu-4- ２１１ It is similar to At-FMA, but does not generate an azaquinone methide intermediate after reaction with GGT. ２１１ It has been revealed that this is different from gGlu-4-At-FMA (Figs. 7 and 8). ２１１ At-FMA and gGlu-4- ２１１ At-MA ２１１ The difference in the intracellular accumulation rate of At is thought to be due to the presence or absence of the azaquinone methide intermediate after the GGT reaction. ２１１It was found that the specific and high accumulation of At-FMA in GGT-highly active cells requires both the ability to "react specifically with GGT activity" and the ability to "generate an azaquinone methide intermediate."

[0169] Furthermore, to examine whether the drug forms a covalent bond with proteins after generating the quinone methide intermediate, cell lysates were prepared and subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), followed by ARG. ２１１ Due to the limited number of experiments, this experiment was conducted using gGlu-4- １２５ The details of the experimental method are described below.

[0170] SDS-PAGE and autoradiography 1.5 × 10 A549-luc cells ５ The cells were seeded into a 12-well plate at a density of 1000 cells / mL (1 mL / well) and incubated for 1 day. After incubation, the medium was removed and 1.5 mL of gGlu-4- １２５ I-FMA (800 kBq / mL) was added to each well. The cells were then incubated at 37°C for 3 hours. After incubation, the medium was removed, and the cells were washed three times with 1 mL of PBS. Cells were lysed by adding CelLytic M (Sigma-Aldrich) for 15 minutes, followed by centrifugation at 15,000 × g for 15 minutes. The supernatant was collected, and the protein concentration was adjusted to a final protein concentration of 0.6 mg / mL using direct A280 quantification on a NanoDrop spectrophotometer (Thermo Fisher Scientific). For SDS-PAGE, 3 μL of the lysate was mixed with 9 μL of 4x Laemmli sample buffer (Bio-Rad) containing 10% β-mercaptoethanol and heated at 95°C for 5 minutes. Each sample and 5 μL of Precision Plus Protein Dual Color Standards (Bio-Rad) were loaded onto Mini-PROTEAN TGX Precast Gels (Bio-Rad). After electrophoresis, the gels were exposed to 200 V for 13 hours and scanned using an Amersham Typhoon scanner (Cytiva).

[0171] ARG is an imaging technique that visualizes the distribution of radioactive materials. In this experiment, as shown in Figure 11, the molecular weight range of proteins (10-250 kDa) was visualized. １２５ Radioactive signals from I were confirmed as multiple bands. １２５ It was suggested that I-FMA generates an azaquinone methide intermediate, then forms covalent bonds with multiple proteins in the cell, thereby acquiring intracellular retention.

[0172] Figure 11 shows SDS-PAGE and autoradiography of A549 cell lysate. The dark areas indicate the covalently bound azaquinone methide intermediate. １２５ I-labeled intracellular proteins are shown.

[0173] (6) Evaluation of cell proliferation ability Colony assay ２１１ It was revealed that At-FMA is selectively taken up into cells with high GGT activity. ２１１ A colony assay was performed to confirm whether At-FMA exhibits strong cytotoxicity against target cells. This assay is a common method for evaluating "proliferative death," in which cells lose their ability to divide after dividing several times following radiation exposure. The assay was performed under two conditions to verify the following two points: (1) whether At-FMA exhibits a cytostatic effect in a radiation dose-dependent and GGT activity-dependent manner; and (2) whether At-FMA retains its intracellular properties, thereby suppressing the growth of cells that do not retain their cellular properties. ２１１ Does it show superior cell proliferation inhibitory effects compared to At-labeled drugs?

[0174] Colony Assay (1): Confirmation of Radiation Dose-Dependent and GGT Activity-Dependent Cell Proliferation Inhibitory Effects The protocol was as follows (see the top panel of Figure 12). First, cultured cells were exposed to the drug for 4 hours, then detached and re-seeded for colony assay. After 10 days of culture, colonies were fixed and stained with crystal violet. Ethanol was added, and the cells were eluted with crystal violet. The absorbance at 544 nm was measured using a plate reader. The colony formation rate was calculated by comparing the absorbance of each group with the mean value of the 0 kBq / mL group, which was set to 100%. Details of the colony formation assay are described below.

[0175] Colony formation assay: A549 cells were plated in 12-well plates at 0.67 × 10 ６ The cells were seeded at a density of 1.5 mL / well and incubated for 1 day. After incubation, the medium was removed and 1.5 mL of gGlu-4- ２１１ At-FMA (0, 10, 40 kBq / mL) was added to each well. The cells were then incubated at 37°C for 3 hours. After incubation, the cells were washed three times with 1 mL of medium, and 0.2 mL of 0.25% trypsin-EDTA solution was added to detach the cells. The cells were then plated onto a 12-well plate at 1.0 x 10 ３ The cells were seeded at a density of 1.5 cells / mL. After 10 days of culture, the medium was carefully aspirated, and the cells were gently washed once with PBS(-). The colonies were then fixed with methanol for 30 minutes at room temperature. After fixation, the colonies were stained with 0.5% crystal violet solution for 60 minutes. Excess stain was removed by washing the plate with water. To quantify colony formation, the stained colonies were solubilized by adding 1 mL of ethanol, and the absorbance of the solubilized crystal violet was then measured at 544 nm using a plate reader. The colony formation rate was calculated by comparing the absorbance of each experimental group with the absorbance of the control group (0 kBq / mL) and set at 100%. Data are presented as mean ± S.E. with n = 3 biological replicates per condition. Statistical analysis was performed using an unpaired, two-tailed t-test.

[0176] 12, it was revealed that the colony formation rate decreased in a radiation dose-dependent manner. ２１１ When GGsTop, a GGT inhibitor, was added to At-FMA, the colony formation rate recovered, suggesting that toxicity is exerted intracellularly in a GGT activity-dependent manner.

[0177] Colony assay (2): Confirmation of differences in cytotoxicity depending on whether or not the cells are retained in the cells. Using the same protocol as in colony assay (1), gGlu-4- ２１１ At-FMA, gGlu-4- ２１１ At-MA, ２１１ Three types of At ２１１ The cells were exposed to At drugs and the cell proliferation inhibitory effects were compared.

[0178] Figure 13A) shows different ２１１ Figure 13B shows colonies of A549 cells cultured with At-drug. ２１１ At-FMA, gGlu-4- ２１１ At-MA, ２１１ The figure shows the viability of A549-luc cells cultured with At- for 4 hours. After staining, colonies were solubilized with ethanol, and the absorbance at 544 nm was measured. The absorbance of each group was compared with that of the 0 kBq / mL group (set as 100%) to calculate the colony formation rate. Data are the mean ± standard deviation (n = 3).

[0179] From Figure 13, it can be seen that other drugs ( ２１１ At, gGlu-4- ２１１ At-MA) rather than gGlu-4- ２１１ At-FMA showed stronger toxicity to A549 cells. Based on the results of retention evaluation (2) (Fig. 11), the drug with a high intracellular retention rate (gGlu-4- ２１１ At-FMA>gGlu-4- ２１１ It was revealed that the colony formation rate decreased as the concentration of α-MA increased. ２１１ It was suggested that a strong cell proliferation inhibitory effect could be obtained by accumulating At inside cells.

[0180] (7) Evaluation of DNA damage markers ２１１ It was revealed that the At-FMA single agent administration group had a higher cell proliferation inhibitory effect than the GGT inhibitor co-administration group. ２１１ It is thought that by taking At into the cells and irradiating the DNA with alpha rays, which have a short range, close to the DNA, many DNA double-strand breaks can be induced. Therefore, we performed fluorescent immunostaining using a DNA double-strand break detection kit, ２１１ We investigated whether the amount of DNA damage caused by the At-labeled drug varies depending on whether it is taken up into cells. The experimental conditions are described in detail below.

[0181] DNA double-strand break (DSB) detection: 0.5 × 10 A549-luc cells ５Cells were seeded at a density of 1000 cells / mL, and 100 μL was added to each well of a 96-well plate and incubated overnight. The medium was then removed, and the cells were fixed by gently adding 100 μL of 3.7% formaldehyde in PBS for 10 minutes at room temperature. The fixed cells were washed once with 200 μL of PBS(-). 100 μL of ice-cold 90% methanol was then added to each well and incubated at 4°C for 10 minutes. The cells were then washed once with 200 μL of PBS(-). To block nonspecific binding, 200 μL of blocking buffer (1% BSA in PBS) was added, and the cells were incubated for 30 minutes at room temperature on an orbital shaker. For immunostaining, 100 μL of 1X anti-phospho-histone H2A·X (Ser139) antibody solution was added to each well and incubated for 1 hour at room temperature on an orbital shaker. The wells were then gently washed five times with 200 μL of wash buffer (0.05% Tween-20 in PBS). Subsequently, 100 μL of 1X FITC-conjugated secondary antibody solution was added and incubated for 1 hour at room temperature on an orbital shaker. The wells were again washed five times with 200 μL of wash buffer. For nuclear counterstaining, Hoechst 33342 was added at a concentration of 5 μg / mL, 100 μL per well, and incubated for 10 minutes. The cells were then washed three times with PBS. Staining was visualized using a fluorescent microscope equipped with appropriate filters for GFP (for phospho-H2A.X) and Hoechst (for nuclear staining).

[0182] The results are shown in Figure 14. In the image of Figure 14, all cells are stained with Hoechst staining in blue, and cells that have undergone DNA double-strand breaks are specifically stained in green. Immunostaining was performed after 4 hours of exposure to the 211At-labeled drug, followed by overnight culture and removal of the drug. As expected, it was confirmed that DNA double-strand breaks had occurred in a greater number of cells in the GGsTop-untreated group compared to the GGsTop-added group. Normally, double-strand breaks caused by α-ray irradiation occur quickly, and are thought to be subsequently repaired by DNA repair mechanisms (non-homologous end joining and homologous recombination), but gGlu-4- ２１１In the At-FMA single-drug treatment group, the presence of double-strand breaks was confirmed even one day after drug exposure. ２１１ It has become clear that At-FMA exerts potent toxicity on target cells by utilizing metabolic trapping by GGT as the basis for its strategy to acquire intracellular retention.

[0183] [Example 2] Treatment experiment using peritoneal dissemination model mice From the evaluation results of Example 1, it was found that gGlu-4- ２１１ At-FMA is useful ２１１ Since it was suggested that the compound functions as an At-labeling agent, we next evaluated its antitumor effect using a tumor-bearing mouse model as an in vivo application using mice. Specifically, we evaluated the antitumor effect of the compound on a peritoneal dissemination mouse model. ２１１ The At-labeled drug was administered intraperitoneally to examine the therapeutic effect (FIG. 15).

[0184] (1) Treatment experiment 1: gGlu-4- ２１１ Comparison of At-FMA and vehicle (1-1) Treatment evaluation by measuring intraperitoneal luminescence value 0.5 / 1.0MBq gGlu-4- ２１１ At-FMA was administered to peritoneal dissemination model mice, and the therapeutic effect was tracked in comparison with the vehicle group. ２１１ A peritoneal dissemination model mouse was prepared one week before At drug administration. Specifically, A549-luc cells, which constitutively express luciferase, were administered into the peritoneal cavity of nude mice to promote tumor engraftment. The day before drug administration, luciferin was administered into the peritoneal cavity of each mouse, and the tumor luminescence intensity was measured using an in vivo imaging system (IVIS). Based on the measurement results, mice were assigned to each group so that the average luminescence intensity of each experimental group was equal. This procedure ensured that the tumor volume estimated from the luminescence value at the start of the experiment was equal between groups, ２１１ We have created conditions that allow us to accurately evaluate the effects of At drugs. ２１１At-FMA was administered at 0 / 0.5 / 1.0 MBq, and the therapeutic effect was tracked by measuring the intraperitoneal luminescence intensity for approximately 40 days after administration. The conditions for preparing the peritoneal dissemination model mice and details of treatment experiment 1 are described below.

[0185] Peritoneal metastasis model of A549-luc cells Female BALB / cAJcl-nu / nu mice (5 or 6 weeks old) were purchased from CLEA Japan. A549-luc-C8 cells (5 × 10 in PBS(-)) were used. ６ A total of 200 μL of 1000 cells / mL of the solution was injected intraperitoneally with a 27G × 1 / 2" needle (Terumo Corporation). Tumor growth was monitored by luminescence. 3 mg of D-luciferin potassium salt in 200 μL of PBS(-) was injected intraperitoneally per mouse, and luminescence from the abdomen was measured 15 minutes after injection with an IVIS Lumina S5 imaging system. All procedures were performed in accordance with the guidelines set for animal experiments by the Ethics Committee for Animal Experiments at the National Cancer Center of Japan.

[0186] ・gGlu-4- ２１１ Evaluation of the therapeutic effect of At-FMA First experiment: gGlu-4- ２１１ At-FMA was administered intraperitoneally to mice at a dose of 0 (control), 0.5 MBq, or 1.0 MBq in 200 μL of vehicle solution (ethanol / PBS + 0.6% sodium alginate (w / w) = 1 / 9). ２１１ At-FMA was administered intraperitoneally to mice at a dose of 0 (vehicle), 0.5, or 1.0 MBq ± 75 μM GGsTop. For bioluminescence imaging, 3 mg of D-luciferin potassium salt in 200 μL of PBS(-) was injected intraperitoneally twice a week. Abdominal luminescence was measured 15 minutes after injection using an IVIS Spectrum imaging system (Perkin Elmer). Body weight (g) was also monitored.

[0187] As a result, as shown in FIG. 16(A), the luminescence value of the vehicle group increased rapidly, while the luminescence value of gGlu-4- ２１１Both the At-FMA 0.5MBq and 1.0MBq groups maintained low luminescence values. Because luminescence values ​​did not decrease from before administration, the drug may not have caused tumor shrinkage under these conditions, but it was able to significantly suppress tumor growth. Furthermore, Figure 16 (B) shows that the 0.5MBq group had a higher average luminescence value than the 1.0MBq group, but this result is likely due to one individual in the 0.5MBq group exhibiting a high luminescence value, suggesting that there may be no substantial difference in therapeutic effect between the two doses.

[0188] (1-2) Body Weight Changes Regarding body weight changes (Fig. 17), the 1.0 MBq administration group showed a greater rate of weight loss than the 0.5 MBq group, suggesting that 1.0 MBq is more likely to cause side effects than 0.5 MBq. Therefore, considering both the therapeutic effect and side effects, 0.5 MBq of gGlu-4- ２１１ Administration of At-FMA is considered more preferable.

[0189] (1-3) Confirmation of Treatment Effect by Fluorescent Imaging of Tumors In addition to tracking the progression of luminescence values, mice were dissected 40 days after treatment and fluorescent imaging of disseminated cancer on the mesentery was performed. gGlu-HMRG was sprayed onto the mesentery, and fluorescent images were obtained 10 minutes later using the Maestro In Vivo Fluorescence Imaging System (see Figure 18). Figure 18 summarizes the mesentery imaging results of mice selected from each group that showed the median luciferin luminescence value. Details of the fluorescent imaging experiment are described below.

[0190] Ex Vivo Fluorescence Imaging of Peritoneal Dissemination Several days after administration of 40-42, mice were dissected and the mesentery was carefully spread. 10 μM gGlu-HMRG (500 μL in PBS) was applied to the exposed peritoneal surface. After 10 minutes of incubation at room temperature, fluorescence imaging was performed using a Maestro In-Vivo Imaging System (CRi Inc). The following imaging parameters were used: excitation filter 445-490 nm, emission filter 515 nm long pass, and exposure time 100 ms. Images were acquired using a blue filter set. The results are shown in Figure 18.

[0191] The left diagram of Figure 18 shows 0.5 and 1.0 MBqgGlu-4- ２１１ Bioluminescence images of tumor growth in a mouse model treated with At-FMA and vehicle are shown. Images were taken the day before treatment or 40 days after treatment. The color scale indicates luminescence intensity per pixel. The right image in Figure 18 shows images of peritoneal metastasis using gGlu-HMRG fluorescence imaging. The green color in the unmixed image indicates cancer cells. Images were acquired 10 minutes after application of gGlu-HMRG (10 μM, 500 μL) using a Maestro In-Vivo Imaging System (CRi Inc.). Imaging conditions: blue filter (excitation 445-490 nm, emission 515 nm long pass), exposure time: 100 ms.

[0192] In the vehicle group, numerous disseminated cancers (shown in green in the UNMIX image) were confirmed to be scattered on the mesentery, whereas gGlu-4- ２１１ The At-FMA administration group had significantly less disseminated cancer (Figure 18). Furthermore, in the vehicle group, cancer cell proliferation was confirmed not only on the mesentery but also in the diaphragm and liver, and some mice had serum ascites. On the other hand, no cancer proliferation or serum ascites was confirmed in other organs in the drug-administered group, demonstrating that the drug can suppress the proliferation of cancer cells not only in the mesentery but throughout the peritoneal cavity.

[0193] (2) Treatment experiment 2: Confirmation of GGT activity-dependent therapeutic effect (2-1) Treatment evaluation by measuring intraperitoneal luminescence value From treatment experiment 1, gGlu-4- ２１１It was revealed that At-FMA showed a higher therapeutic effect than the vehicle group. Regarding the radiation dose of the drug, it was found that the therapeutic effect was sufficiently exerted with 0.5 MBq administration, so it was confirmed whether the therapeutic effect was exhibited in a radiation dose-dependent manner at 0.5 MBq or less. Furthermore, to verify whether a high therapeutic effect could be obtained depending on GGT activity, the GGT inhibitors GGsTop and gGlu-4- ２１１ The therapeutic effect was compared with that of the group co-administered with At-FMA (FIG. 19). The results are shown in FIG.

[0194] Figure 20 shows the gGlu-4- in a xenograft mouse model of A549-luc peritoneal metastasis. ２１１ The therapeutic effect of At-FMA is shown. The relative tumor luminescence intensity of each group was quantified over time. A549-luc cells were intraperitoneally administered to female BALB / cAJcl-nu / nu mice to establish a peritoneal metastasis model. The treatment group included gGlu-4- ２１１ At-FMA was administered intraperitoneally as a single dose of 0 (vehicle), 0.23, or 0.45 MBq ± 75 μM GGsTop. Vehicle and treatment solutions were prepared in 200 μL of EtOH / PBS (1:9) containing 0.6% SA. Tumor burden was assessed by in vivo bioluminescence imaging for 42 days after treatment. Data are means ± standard deviation (n ≥ 3 per group).

[0195] As shown in Figure 20, gGlu-4- ２１１ At-FMA showed a higher therapeutic effect with 0.45 MBq than with 0.23 MBq, suggesting that the therapeutic effect is radiation dose-dependent. Furthermore, even with the same radioactivity, the GGT inhibitor co-administration group showed a higher gGlu-4- ２１１ The average luminescence value is increased compared to the group administered with At-FMA alone.

[0196] (2-2) Confirmation of therapeutic effect by fluorescent imaging of tumors Fluorescent imaging of disseminated cancer on the mesentery was performed using the same method as in Treatment Experiment 1. Figure 22 shows a summary of the mesentery imaging results for individuals selected from each group that showed the median luciferin luminescence value.

[0197] The left figure in Figure 22 shows 0.23 and 0.45 MBqgGlu-4-２１１ At-FMA, 0.45MBqgGlu-4- ２１１ Bioluminescence images of tumor growth in a mouse model treated with At-FMA+GGsTop and vehicle are shown. Images were taken the day before treatment or 42 days after treatment. The color scale indicates luminescence intensity per pixel. The right panel of Figure 22 shows peritoneal metastasis images taken using gGlu-HMRG fluorescence imaging. The green color in the unmixed image indicates cancer cells. Images were acquired 10 minutes after application of gGlu-HMRG (10 μM, 500 μL) using a Maestro In-Vivo Imaging System (CRi Inc.). Imaging conditions: blue filter (excitation 445-490 nm, emission 515 nm long pass), exposure time: 100 ms.

[0198] gGlu-4- ２１１ The At-FMA + GGsTop co-administration group showed a higher gGlu-4- ２１１ It can be seen that the number of cells disseminated onto the mesentery was greater than that in the At-FMA single-administration group. This tendency is consistent with the results of the luminescence value, and administration of GGsTop suppressed the growth of gGlu-4- ２１１ Therefore, in the mouse experiment, the therapeutic effect of At-FMA was also reduced. ２１１ It was revealed that At-FMA exhibits high toxicity in a GGT-dependent manner.

[0199] The drug used in this example targets gamma-glutamyl transpeptidase (GGT), whose activity has been confirmed to be elevated in cancer tissues, and selectively targets cells with high GGT activity. ２１１ To accumulate At, we incorporated quinone methide chemistry into our molecular design. Specifically, it is first specifically hydrolyzed by GGT to generate an electrophilic azaquinone methide intermediate, which then forms a covalent bond with intracellular nucleophiles such as proteins, resulting in the formation of a quinone methide intermediate. ２１１ It was assumed that At would be "metabolic trapped" inside the cells. In this way, the α-ray targeted therapeutic drug gGlu-4- ２１１We have newly designed At-FMA and succeeded in obtaining it with a high radiochemical yield (>60%). When this drug is applied to living cells, it has been found to accumulate in the cells in a GGT activity-dependent manner and inhibit cell proliferation. Furthermore, when this drug is administered intraperitoneally to a peritoneal dissemination model mouse, it exhibited a high antitumor effect that was dependent on GGT activity and drug concentration (dose) without any significant side effects such as weight loss. From these results, it is clear that gGlu-4- ２１１ At-FMA is a novel "metabolic trapping"-based therapeutic targeting tumor cell-selective enzyme activity. ２１１ It was revealed that the drug was an At-labeled drug.

Claims

1. A pharmaceutical composition for cancer treatment comprising a compound represented by the following general formula (I) or a salt thereof: (wherein X is a fluorine atom, an ester group (—OC(═O)—R′), a carbonate group (—OCO ２ -R'), carbamate group (-OCONH-R'), phosphoric acid and its ester group (-OP(=O)(-OR')(-OR''), and sulfuric acid and its ester group (-OSO ２ -OR'), wherein R' and R'' are each independently selected from a substituted or unsubstituted alkyl group or a substituted or unsubstituted aryl group; Y is -NH-CO-L, -NH-L', -OL'' or -OL''', wherein L, together with the C=O to which it is attached, constitutes an amino acid residue or a peptide, L' is a saccharide partial structure having a self-cleaving linker, or an amino acid residue or peptide having a self-cleaving linker; L'' is a saccharide partial structure; L''' is a saccharide partial structure having a self-cleaving linker, or an amino acid residue or peptide having a self-cleaving linker; wherein the saccharide partial structure is a structure obtained by removing one hydroxyl group from a saccharide, and R １ and R ２ are each independently selected from a hydrogen atom or a monovalent substituent; R ３ is a hydrogen atom or one to two identical or different monovalent substituents present on a benzene ring; R ４ represents a hydrogen atom, or a substituent or molecule capable of changing pharmacokinetics, and the substituent or molecule may be bonded to the benzene ring via a linker; and Z represents a single bond or a linking group.

2. The pharmaceutical composition according to claim 1, wherein the substituent or molecule capable of altering the pharmacokinetics is introduced into the benzene ring via a linker or directly.

3. The linker is an alkylene group (provided that the alkylene group has one or more —CH ２ The pharmaceutical composition according to claim 2, wherein - is optionally substituted with -O-, -S-, -NH-, or -CO-), arylene (including heteroarylene), cycloalkylene, alkoxyl group, polyethylene glycol chain, and a group formed by arbitrarily bonding two or more groups selected from these groups.

4. The linking group of Z is an alkylene group (provided that the alkylene group has one or more -CH ２ The pharmaceutical composition according to claim 1, wherein - is optionally substituted with -O-, -S-, -NH-, or -CO-), arylene (including heteroarylene), cycloalkylene, alkoxyl group, polyethylene glycol chain, and a group formed by arbitrarily bonding two or more groups selected from these groups.

5. -Y in general formula (I) is -C(R １ ) (R ２ 2. The pharmaceutical composition according to claim 1, wherein X is bonded to the benzene ring at the ortho or para position.

6. The pharmaceutical composition of claim 1, wherein Y has a structure selected from the following:

7. The pharmaceutical composition according to claim 1, wherein X is a fluorine atom or an ester group (-OCO-R').

8. R １ and R ２ The pharmaceutical composition according to claim 1 , wherein each of is independently selected from a hydrogen atom or a fluorine atom.

9. R ３ The monovalent substituent is an alkyl group, an alkoxycarbonyl group (—CO—OR ａ ), nitro group, amino group, hydroxyl group, alkylamino group (-NHR ａ , -NR ａ ２ ), an alkoxy group (—OR ａ ), an ester group (—O—CO—R ａ ), a halogen atom, a boryl group, and a cyano group (R ａ is a substituted or unsubstituted alkyl group or a substituted or unsubstituted aryl group, R ａ If there are two or more R ａ The pharmaceutical composition according to claim 1 , wherein 10. R ３ The pharmaceutical composition according to claim 9, wherein the monovalent substituent is an alkyl group or an alkoxycarbonyl group.

11. R ３ The compound or salt thereof according to claim 9, wherein the monovalent substituent represented by the formula: is a halogen atom.

12. R ３ At least one of the monovalent substituents of R is an alkyl group or an alkoxycarbonyl group, ３ 10. The compound or salt thereof according to claim 9, wherein at least one of the monovalent substituents represented by the formula: is a halogen atom.

13. R ３ and R ４ The compound or salt thereof according to claim 1, wherein all of are hydrogen atoms.

14. The pharmaceutical composition according to claim 1, which can accumulate in cancer cells by acting selectively on the cells through cancer cell-specific enzymatic activity.

15. The pharmaceutical composition of claim 14, wherein the enzyme is a peptidase or a glycosidase.

16. The pharmaceutical composition of claim 14, which is administered to a subject intravenously, intraperitoneally, or intratumorally.

17. A compound of the following general formula (I): (wherein X is a fluorine atom, an ester group (—OC(═O)—R′), a carbonate group (—OCO ２ -R'), carbamate group (-OCONH-R'), phosphoric acid and its ester group (-OP(=O)(-OR')(-OR''), and sulfuric acid and its ester group (-OSO ２ -OR'), wherein R' and R'' are each independently selected from a substituted or unsubstituted alkyl group or a substituted or unsubstituted aryl group; Y is -NH-CO-L, -NH-L', -OL'' or -OL''', wherein L, together with the C=O to which it is attached, constitutes an amino acid residue or a peptide, L' is a saccharide partial structure having a self-cleaving linker, an amino acid residue or a peptide having a self-cleaving linker; L'' is a saccharide partial structure; L''' is a saccharide partial structure having a self-cleaving linker, or an amino acid residue or a peptide having a self-cleaving linker; wherein the saccharide partial structure is a structure obtained by removing one hydroxyl group from a saccharide, and R １ and R ２ are each independently selected from a hydrogen atom or a monovalent substituent; R ３ is a hydrogen atom or one to two identical or different monovalent substituents present on a benzene ring; R ４ is a hydrogen atom, or a substituent or molecule capable of changing pharmacokinetics, and the substituent or molecule may be bonded to the benzene ring via a linker; and Z represents a single bond or a linking group. A method for preparing a compound represented by the following formula (Ia) or a salt thereof, comprising the steps of: (A) adding (In the formula, X, Y, Z, R １ ~R ４ is the same as defined in general formula (I), and E represents a halogen atom. ５ ) ３ Sn-Sn(R ５ ) ３ to obtain a precursor compound represented by the following formula (Ib); (In the formula, X, Y, Z, R １ ~R ４ is the same as defined in general formula (I), R ５ represents an alkyl group having 1 to 4 carbon atoms. (B) Adding to the precursor compound obtained in the step (A), ２１１ (C) a step of purifying the reaction product obtained in the step (B) to obtain the compound represented by formula (I).

18. The preparation method according to claim 17, wherein in step (C), the reaction product obtained in step (B) is purified by solid phase extraction.

19. The method according to claim 17, wherein sodium ascorbate is added in step (B) and / or step (C).

20. A method for diagnosing and treating a disease or a condition that may lead to a disease, comprising: (a) administering to a subject having or suspected of having the disease or condition a diagnostic agent comprising a compound represented by the following formula (II) or a salt thereof: (In the formula, X ａ represents a fluorine atom, an ester group (-OC(=O)-R'), a carbonate group (-OCO ２ -R'), carbamate group (-OCONH-R'), phosphoric acid and its ester group (-OP(=O)(-OR')(-OR''), and sulfuric acid and its ester group (-OSO ２ —OR′), wherein R′ and R″ are each independently selected from a substituted or unsubstituted alkyl group or a substituted or unsubstituted aryl group; Y ａ is -NH-CO-L, -NH-L', -OL" or -OL"', wherein L, together with the C=O to which it is attached, constitutes an amino acid residue or a peptide, L' is a saccharide partial structure having a self-cleaving linker, an amino acid residue or a peptide having a self-cleaving linker; L" is a saccharide partial structure; L'" is a saccharide partial structure having a self-cleaving linker, or an amino acid residue or a peptide having a self-cleaving linker; wherein the saccharide partial structure is a structure obtained by removing one hydroxyl group from a saccharide, and R １ａ and R ２ａ are each independently selected from a hydrogen atom or a monovalent substituent; R ３ａ is a hydrogen atom or one to two identical or different monovalent substituents present on a benzene ring; R ４ａ is a hydrogen atom, or a substituent or molecule capable of changing pharmacokinetics, and the substituent or molecule may be bonded to the benzene ring via a linker; Z ａ represents a single bond or a linking group, and U represents １２５ I or １２３ (b) examining the presence of one or more selected from the group consisting of tumors, cancer cells, and cancer tissues in a target tissue or target organ of the subject by measuring radiation emitted from the radionuclide contained in the agent localized in the target tissue or target organ by a nuclear medicine examination; (c) administering a pharmaceutical composition for cancer treatment comprising a compound represented by the following general formula (I) or a salt thereof to a subject in whom the presence of one or more selected from the group consisting of tumors, cancer cells, and cancer tissues in the target tissue or target organ has been confirmed; (wherein X is a fluorine atom, an ester group (—OC(═O)—R′), a carbonate group (—OCO ２ -R'), carbamate group (-OCONH-R'), phosphoric acid and its ester group (-OP(=O)(-OR')(-OR''), and sulfuric acid and its ester group (-OSO ２ -OR'), wherein R' and R'' are each independently selected from a substituted or unsubstituted alkyl group or a substituted or unsubstituted aryl group; Y is -NH-CO-L, -NH-L', -OL'' or -OL''', wherein L, together with the C=O to which it is attached, constitutes an amino acid residue or a peptide, L' is a saccharide partial structure having a self-cleaving linker, an amino acid residue or a peptide having a self-cleaving linker; L'' is a saccharide partial structure; L''' is a saccharide partial structure having a self-cleaving linker, or an amino acid residue or a peptide having a self-cleaving linker; wherein the saccharide partial structure is a structure obtained by removing one hydroxyl group from a saccharide, and R １ and R ２ are each independently selected from a hydrogen atom or a monovalent substituent; R ３ is a hydrogen atom or one to two identical or different monovalent substituents present on a benzene ring; R ４ is a hydrogen atom, or a substituent or molecule that can change pharmacokinetics, and the substituent or molecule may be bonded to the benzene ring via a linker; and Z represents a single bond or a linking group.

21. The method of claim 20, wherein the nuclear medicine examination is at least one selected from the group consisting of scintigraphy, SPECT (single photon emitter computed tomography), and PET (positron emitter tomography).

22. The method of claim 20, further comprising the step of (d) administering the diagnostic agent to the subject to which the pharmaceutical composition for cancer treatment was administered in step (c), and measuring by nuclear medicine examination the radiation emitted from the radionuclide contained in the diagnostic agent localized in the target tissue or target organ of the subject, thereby examining the presence of one or more selected from the group consisting of tumors, cancer cells, and cancer tissues in the target tissue or target organ, thereby determining the effectiveness of the treatment.

23. A compound represented by the following general formula (I) or a salt thereof: (wherein X is a fluorine atom, an ester group (—OC(═O)—R′), a carbonate group (—OCO ２ -R'), carbamate group (-OCONH-R'), phosphoric acid and its ester group (-OP(=O)(-OR')(-OR''), and sulfuric acid and its ester group (-OSO ２ -OR'), wherein R' and R'' are each independently selected from a substituted or unsubstituted alkyl group or a substituted or unsubstituted aryl group; Y is -NH-CO-L, -NH-L', -OL'' or -OL''', wherein L, together with the C=O to which it is attached, constitutes an amino acid residue or a peptide, L' is a saccharide partial structure having a self-cleaving linker, an amino acid residue or a peptide having a self-cleaving linker; L'' is a saccharide partial structure; L''' is a saccharide partial structure having a self-cleaving linker, or an amino acid residue or a peptide having a self-cleaving linker; wherein the saccharide partial structure is a structure obtained by removing one hydroxyl group from a saccharide, and R １ and R ２ are each independently selected from a hydrogen atom or a monovalent substituent; R ３ is a hydrogen atom or one to two identical or different monovalent substituents present on a benzene ring; R ４ represents a hydrogen atom, or a substituent or molecule capable of changing pharmacokinetics, and the substituent or molecule may be bonded to the benzene ring via a linker; and Z represents a single bond or a linking group.

Images