Photoreactive gold nanocluster and intermediate

Abstract

To provide a gold nanocluster and its intermediate that release bioactive molecules, such as anticancer agents, in response to light exposure.SOLUTION: The present invention provides a gold nanocluster combined with a group represented by formula (II) (where L1, L2, and L3 independently represent a linker group; X represents a divalent group containing a 1,2,3-triazole ring; Y represents a targeting ligand; Q represents O, NH, or a single bond; D1 represents a bioactive portion).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to gold nanoclusters and their intermediates that release bioactive molecules such as anticancer drugs in response to irradiation with red light, which has high biological permeability. The present invention provides a new nanomedicine that binds to specific pathological sites such as cancer, releases bioactive molecules such as anticancer drugs when irradiated with light, and exhibits bioactivity such as anticancer action. [Background technology]

[0002] Chemical reactions induced by light irradiation are being actively studied in the fields of life science, material science, and organic synthetic chemistry because the time and space of light irradiation can be easily controlled. In particular, photooxidation reactions, in which highly reactive singlet oxygen is generated by irradiating a photosensitizer with light and the substrate is oxidized, are also used in the total synthesis of natural organic compounds (Non-Patent Document 1). In the field of medicine, photodynamic therapy, in which singlet oxygen is generated by irradiating a photosensitizer that selectively accumulates in tumors, causing necrosis of tumor tissue, is used in cancer treatment (Non-Patent Document 2).

[0003] The present inventors have developed a photooxidation reaction that releases biologically active carboxylic acids and alcohol compounds from 3-acylindolizine, a type of aromatic heterocyclic compound (Scheme 1) (Patent Document 1, Non-Patent Documents 3-5). In this reaction, singlet oxygen generated by short-term irradiation of a photosensitizer with red light is used to decompose the indolizine ring and release biologically active molecules. Such photoreactions are expected to be applied to pharmaceutical uncaging and drug delivery.

[0004] [ka] [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2021 / 177343

Non-licensed literature

[0006] [Non-licensed document 1] Ghogare, AA; Greer, A. Chem. Rev. 2016, 116, 19994.

Non-licensed Document 2

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Fashion 8

Number 9

Wood 10

Fashion 11

Wood 12

Wood 13

[0007] However, although the lifetime of singlet oxygen is long in organic solvents such as acetonitrile (t 1 / 2 = 80 μs), and shorter in water (t 1 / 2= 3 μs) (Non-Patent Document 6). For this reason, the efficiency of photoreaction mediated by singlet oxygen in water tends to be low. In addition, when photoreaction is performed in a biological environment such as inside a cell, the photoreaction efficiency decreases due to dilution of the two components, indolizine and photosensitizer. Furthermore, when considering application to drug delivery, it is necessary to link indolizine with a targeting ligand that binds to the target pathological tissue. Therefore, in order to realize a photoreaction system that functions in a biological environment, a new methodology is needed to appropriately assemble indolizine, photosensitizer, and targeting ligand. [Means for solving the problem]

[0008] In light of this background, the present inventors have considered using metal nanoparticles as carriers for the photoreaction of indolizine. To date, drug delivery systems have been developed that release biologically active compounds from metal nanoparticles using external stimuli such as hydrolysis reactions, enzyme reactions, pH changes, ionic strength changes, light, heat, ultrasound, and magnetic fields (Non-Patent Documents 7 and 8). In the current field of nanomedicine, there is a demand for the development of methodologies for efficiently producing uniform nanomaterials and improving their medicinal efficacy. The present inventors have focused on gold nanoclusters (approximately 2 nm) which have a small particle size among metal nanoparticles. These gold nanoclusters have a high tendency to accumulate in pathological tissues such as tumors, but are excreted appropriately from the body due to their small size, and have excellent biocompatibility (Non-Patent Document 9).

[0009] The present inventors have developed new photoreactive nanoparticles by utilizing the photoreaction of indolizine derivatives. Specifically, indolizine derivatives and photosensitizers were regularly arranged on gold nanoclusters with uniform molecular size. To realize such a gold nanocluster system with uniform structure and arrangement, the gold nanocluster surface was modified with a single peptide having azide and amino groups that can be applied to click reactions and bioconjugation reactions (see the formula below). Next, the obtained gold nanoclusters were used as carriers to modify indolizine derivatives, photosensitizers, and targeting ligands via click reactions and bioconjugation reactions (see the formula below).

[0010] [ka]

[0011] The synthesized gold nanoclusters were irradiated with red light, and the amount of bioactive compounds released from the gold nanoclusters, such as anticancer drugs, was quantified using a liquid chromatography mass spectrometer (LC / MS / MS). To demonstrate that the gold nanoclusters actually function in a cell system, the light irradiation-dependent cytocidal effect on human lung cancer cells was examined.

[0012] The present invention has been completed based on the above findings, and provides the following. [1] Formula (II)

[0013] [ka]

[0014] (In the formula, L 1 , L 2 , and L 3 are each independently a linker group; X is a divalent group containing a 1,2,3-triazole ring; Y is a targeting ligand; Q is O, NH, or a single bond; D 1 is the biologically active moiety) A gold nanocluster having a group represented by the formula: [2] The gold nanocluster according to [1], wherein Y is a group containing a cyclic RGD peptide. [3] D 1 The gold nanocluster according to [1] or [2], wherein the biologically active moiety is derived from an anticancer drug. [4] Q is O and D 1 is represented by the following formula (XV)

[0015] [ka]

[0016] The gold nanocluster according to any one of [1] to [3], wherein the group is represented by the following formula: [4a] D 1 The gold nanocluster according to any one of [1] to [3], wherein the is a biologically active moiety derived from an anticancer drug and capable of releasing the anticancer drug upon exposure to light. [4b] X is the formula

[0017] [ka]

[0018] (Wherein, * represents L 1 (represents the bond position with The gold nanocluster according to any one of [1] to [4] and [4a], wherein the gold nanocluster is a divalent group represented by the following formula: [5] Further, the formula (I)

[0019] [ka]

[0020] (In the formula, Z is a photosensitizer moiety; L 1 and L 2 are each independently a linker group; X is a divalent group containing a 1,2,3-triazole ring; Y is a targeting ligand. The gold nanocluster according to any one of [1] to [4], [4a], and [4b], wherein the gold nanocluster is bonded to a group represented by the following formula: [6] A gold nanocluster according to [5], wherein Z is a group derived from pyropheopherbide a. [7] Z is formula (XVI)

[0021] [ka]

[0022] The gold nanocluster according to [5], wherein the group is represented by the formula: [8] A gold nanocluster according to any one of [5] to [7], wherein Y is a group containing a cyclic RGD peptide. [8a] X is the formula

[0023] [ka]

[0024] (Wherein, * represents L 1 (represents the bond position with The gold nanocluster according to any one of [5] to [8], wherein the gold nanocluster is a divalent group represented by the following formula: [9] Formula (III)

[0025] [ka]

[0026] (In the formula, L 1 and L 2 are each independently a linker group; X is a divalent group containing a 1,2,3-triazole ring; Y is a targeting ligand. A gold nanocluster having a group represented by the formula:

[10] The gold nanocluster according to [9], wherein Y is a group containing a cyclic RGD peptide. [10a] X is of formula

[0027] [ka]

[0028] (Wherein, * represents L 1 (represents the bond position with The gold nanocluster according to [9] or

[10] , wherein the gold nanocluster is a divalent group represented by the following formula:

[11] Formula (IV)

[0029] [ka]

[0030] (In the formula, L 1 is a linker group) A gold nanocluster having a group represented by the formula:

[12] Formula (I)

[0031] [ka]

[0032] (In the formula, L 1 and L 2 are each independently a linker group; X is a divalent group containing a 1,2,3-triazole ring; Y is a targeting ligand; Z is a photosensitizer moiety. A gold nanocluster having a group represented by the formula:

[13] A pharmaceutical composition comprising a gold nanocluster described in any one of [1] to [8], [4a], [4b], [8a], and

[12] and a pharma- ceutical acceptable carrier.

[14] An anti-cancer composition comprising a gold nanocluster described in any one of [3], [4], and [4a] and a pharma- ceutical acceptable carrier.

[15] Formula (IV)

[0033] [ka]

[0034] (In the formula, L 1 is a linker group) The method for producing gold nanoclusters having attached thereto groups represented by formula (V)

[0035] [ka] (In the formula, L 1 is as defined above) or a salt thereof with tetrachloroauric acid or a salt thereof, or a hydrate thereof, in the presence of a metal phenoxide. Effect of the Invention

[0036] The inventors have succeeded in developing a photoreaction system that rapidly releases bioactive molecules such as anticancer drugs by incorporating photoreactive indolizine, photosensitizers, and targeting ligands into small gold nanoclusters. The gold nanocluster surface is modified with a bifunctional peptide that has an azide group and an amino group that can be used for molecular binding, so that different functional molecules can be uniformly assembled. This indicates that by introducing each function into the gold nanocluster in an appropriate arrangement, the photoreaction mediated by singlet oxygen proceeds smoothly within the cell. In addition, since the active species of this photoreaction is singlet oxygen, a synergistic effect with photodynamic therapy can be expected.

[0037] Furthermore, gold nanoclusters can generate singlet oxygen under external stimuli such as X-rays and ultrasound irradiation, and can be used to release bioactive compounds using external stimuli such as X-rays and ultrasound irradiation other than light. [Brief description of the drawings]

[0038] [Figure 1] Figure 1 shows TEM images of gold nanoclusters 1 (A), 6 (B), 9 (C) and 10 (D). [Diagram 2] Figure 2 shows the IR spectra of gold nanoclusters 1' (A), 1 (B), 4 (C) and 6 (D). [Diagram 3] FIG. 3 is a graph showing the amount of combretastatin A4 (CA4) released versus time of irradiation of gold nanocluster 9 with red light (660 nm). [Figure 4]FIG. 4 is a graph showing the cytotoxicity of gold nanoclusters 9-11 to A549 cells in the dark (A) and under light irradiation (B). [Diagram 5] FIG. 5 shows a production scheme for gold nanocluster 1 (Scheme 2). [Figure 6] FIG. 6 shows a synthesis scheme for gold nanoclusters 4 and 6 (Scheme 3). [Figure 7] FIG. 7 shows the synthesis scheme (Scheme 4) of gold nanoclusters 9-11. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] The present inventors designed gold nanocluster 1, which is modified with a single peptide 2 containing a thiol group with high affinity for gold atoms, an azide group and a primary amino group with a triethylene glycol spacer for molecular linkage, and a highly hydrophilic carboxyl group, with the aim of uniformly assembling different functional molecules on the surface of the gold nanocluster (Scheme 2, Figure 5). However, peptides with thiol and azide groups are generally difficult to synthesize (Non-Patent Document 10). This is because when deprotecting a thiol group protected with a trityl (Trt) group, the azide group is easily reduced to an amino group by a thiol additive such as ethanedithiol, which is used to trap the trityl group. The present inventors used dithiothreitol (DTT) as a trapping agent for the Trt group, referring to the synthesis of peptide nucleic acids with thiol groups by Diederichsen et al. (Non-Patent Document 10), and obtained peptide 2 in a 52% yield after purification by high performance liquid chromatography (Scheme 2). In a typical synthesis of gold nanoclusters coordinated with thiols, chloroauric acid (HAuCl4) is reduced with sodium borohydride (NaBH4) in the presence of thiol ligands to obtain gold nanoclusters with small particle size (Non-Patent Document 11). However, under such harsh conditions, the azide group is reduced to an amino group (Non-Patent Document 12). In fact, when the inventors attempted to synthesize gold nanocluster 1 using NaBH4, IR spectrum measurement revealed that the azide group had almost disappeared (Figure 2A, 1'). In addition, clustering was attempted under heating conditions with reference to the synthesis method of gold nanoclusters protected with glutathione (Non-Patent Document 13), but the target product was not obtained. To realize the synthesis of gold nanocluster 1, the inventors referred to the synthesis method of gold nanoclusters protected with proteins or peptides containing tyrosine (Non-Patent Document 14). In the synthesis of these gold nanoclusters, the phenoxide group of the tyrosine residue acts as a reducing agent for chloroauric acid under basic conditions, resulting in the formation of gold clusters. The inventors have demonstrated that even peptides that do not have a phenoxide group, such as peptide 2, can produce gold nanocluster 1 by adding phenol under basic conditions (Scheme 2).

[0040] It is known that the efficiency of cellular uptake of metal nanoparticles can be improved by introducing a targeting ligand that binds to a specific receptor on the cell surface into the metal nanoparticle (Non-Patent Document 15). For the same purpose, the present inventors have targeted integrin α, which is highly expressed in cancer cells, to v Cyclic RGD peptide 5, which specifically binds to β3, was introduced onto gold nanoclusters by copper-catalyzed azide-alkyne cycloaddition (CuAAC) (Scheme 3, compound 6, Figure 6).

[0041] Furthermore, indolizine 7 and pyropheophorbide a derivative 8 bearing active ester groups were introduced by condensation with the amino groups of gold nanoclusters 6 (Scheme 4, Figure 7).

[0042] The present invention will be described in detail below. In this specification, gold nanoclusters refer to metal clusters containing gold atoms. The average particle size of the gold nanoclusters of the present invention is preferably 5 nm or less, more preferably 4 nm or less, and even more preferably 3 nm or less. The average particle size of the gold nanoclusters of the present invention is preferably 0.5 nm or more, more preferably 1 nm or more, and even more preferably 1.5 nm or more. In a preferred embodiment, the average particle size of the gold nanoclusters of the present invention is preferably 0.5 nm to 5 nm, more preferably 1 nm to 4 nm, and even more preferably 1.5 nm to 3 nm. In this specification, the average particle size refers to the average value of particle sizes measured by TEM (transmission electron microscope) imaging.

[0043] In this specification, the peptide portion consisting of L-aspartic acid, L-cysteine, and L-lysine in the formula may be referred to as a “tripeptide portion.” The tripeptide portion is bound to the gold nanocluster via the thiol group (—SH) of the L-cysteine ​​residue.

[0044] D 1 is a biologically active moiety. As used herein, a biologically active moiety refers to a portion of a biologically active molecule that contains a functional group selected from the group consisting of -OH, -NH2, and -COOH, excluding the functional group. When Q is O, the biologically active molecule is D1 When Q is NH, the bioactive molecule is D 1 When Q is a single bond, the biologically active molecule is D 1 The functional group is -COOH. The biologically active molecule is not particularly limited as long as it contains a functional group selected from the group consisting of -OH, -NH2, and -COOH and has biological activity. Examples of the biologically active molecule include low molecular weight drugs, peptides, proteins, DNA, RNA, mRNA, synthetic peptides, synthetic oligonucleotides, etc. The biologically active molecule is preferably a low molecular weight drug.

[0045] The low molecular weight drug is not particularly limited as long as it shows biological activity when administered to mammals including humans. The low molecular weight drug preferably has a molecular weight of 5,000 or less, more preferably a molecular weight of 1,500 or less, and even more preferably a molecular weight of 1,000 or less.

[0046] Examples of biologically active molecules include, but are not limited to, anticancer agents, anti-HBV agents (anti-hepatitis B virus agents), anti-HIV agents, etc. Suitable examples of anticancer agents include combretastatin A4 (CA4), monomethylauristatin F, monomethylauristatin E, etc.

[0047] D 1 The biologically active portion derived from an anticancer drug is preferably a biologically active portion derived from an anticancer drug. The biologically active portion derived from an anticancer drug refers to a portion of the anticancer drug molecule excluding a functional group selected from the group consisting of -OH, -NH2, and -COOH. When Q is O, the anticancer drug molecule is D 1 -OH. When Q is NH, the anticancer drug molecule is D 1 -NH2. When Q is a single bond, the anticancer drug molecule is D 1 -COOH.

[0048] Q is O, NH, or a single bond. Preferably, Q is O or a single bond, and more preferably, Q is O.

[0049] X is a divalent group containing a 1,2,3-triazole ring. In this specification, the divalent group containing a 1,2,3-triazole ring means a divalent group formed by a click reaction between an azide and an alkyne. Examples of the click reaction include copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC) and strain-promoted azide-alkyne cycloaddition reaction (SPAAC).

[0050] An example of X is the formula

[0051] [ka]

[0052] (Wherein, * represents L 1 (represents the bond position with X is preferably a divalent group represented by the formula

[0053] [ka]

[0054] (Wherein, * represents L 1 (represents the bond position with It is a divalent group represented by the formula:

[0055] Y is a targeting ligand. The targeting ligand has the property of binding to a specific protein present on the surface of a target cell. By introducing a targeting ligand into a gold nanocluster, it is possible to selectively deliver the gold nanocluster bound to a biologically active moiety to the target cell. As the targeting ligand, a targeting ligand known in the field of drug delivery systems (DDS) can be used. As the targeting ligand, a peptide, a low molecular weight compound, an antibody, a protein, an aptamer, a sugar, etc. are mentioned. As the targeting ligand, a low molecular weight ligand is preferable, and a peptide or a low molecular weight compound having a molecular weight of 1,000 or less is preferable.

[0056] An example of a targeting ligand is integrin α, which is highly expressed in cancer cells. v Examples of the targeting ligand include, but are not limited to, cyclic RGD peptides that specifically bind to β3, folic acid, anisamide, hyaluronic acid, glucose, lactose, PSMA ligands, and the like. As the targeting ligand, a group containing a cyclic RGD peptide is preferable. Examples of the cyclic RGD peptide include Cyclo(RGDfK) (f is D-Phe), and the like. Cyclo(RGDfK) is a peptide that is linked to the linker L via the amino group of the side chain of the lysine residue. 2 For example, when the targeting ligand is a group comprising Cyclo(RGDfK), Y can be linked to the formula

[0057] [ka]

[0058] It is a group represented by the following formula:

[0059] Z is a photosensitizer moiety. In this specification, the photosensitizer moiety refers to a portion of a photosensitizer that contains a functional group selected from the group consisting of -OH, -NH2, and -COOH, but does not include the functional group. The photosensitizer moiety Z may include a suitable linker. A photosensitizer is a compound that can generate singlet oxygen by irradiation with light.

[0060] Examples of photosensitizers include porphyrins such as pyropheophorbide a, pheophorbide a, talaporfin, verteporfin, porfimer, temoporfin, chlorin e6, HPPH (Photochlor), hematoporphyrin, protoporphyrin IX, IR700, and indocyanine green, phthalocyanines, cyanine dyes, etc. Among these, pyropheophorbide a or pheophorbide a is preferred, and pyropheophorbide a is more preferred.

[0061] When the photosensitizer has a carboxy group, the amino group of the side chain of L-lysine in the tripeptide moiety and the carboxy group of the photosensitizer can be bonded by an amide bond. The photosensitizer may be bonded to the amino group via a suitable linker.

[0062] When the photosensitizer is pyropheophorbide a, the photosensitizer moiety Z is a group derived from pyropheophorbide a and has the formula

[0063] [ka]

[0064] When the photosensitizer is pheophorbide a, the photosensitizer moiety Z is a group derived from pheophorbide a and has the formula

[0065] [ka]

[0066] It is a group represented by the following formula:

[0067] L 1 , L 2 , and L 3 are each independently a linker group. A linker group refers to a divalent group that connects two moieties. Linker groups include -O-, -NH-, -C(=O)-, -C(=O)NH-, -C(=O)O-, -(O-CH2CH2) n and the like.

[0068] L 1 is not particularly limited as long as it is a linker group that can link the N-terminus of L-aspartic acid in the tripeptide portion to the azide group (-N3). 1 An example of this is the formula *-C(=O)-CH2-(O-CH2CH2) m -** (In the formula, m is 2, 3, or 4, preferably m is 3 or 4, and more preferably m is 3, * represents the binding position of the tripeptide moiety to the N-terminus of L-aspartic acid, and ** represents the binding position of the azide group (-N3) or X.) Examples of the divalent group include a divalent group represented by the following formula:

[0069] L 2 is not particularly limited as long as it is a linker group that can link the alkyne moiety used in the click reaction to the targeting ligand Y. 2 Examples include: *-(CH2) p -C(=O)-** (In the formula, p is 1, 2 or 3, and preferably, p is 2, * represents the bonding position to X, and ** represents the bonding position to Y.) Examples of the divalent group include a divalent group represented by the following formula:

[0070] L 3 is not particularly limited as long as it is a linker group that can bond to the amino group in the side chain of L-lysine in the tripeptide portion via a carbonyl group (C(=O)) and link the indolizine ring to the amino group in the side chain of L-lysine in the tripeptide portion. 3 Examples include: *-O-(CH2) q -** (In the formula, q is 2 or 3, and preferably q is 3, * represents the bonding position to the indolizine ring, and ** represents the bonding position to C(=O).) Examples of the divalent group include a divalent group represented by the following formula:

[0071] In a gold nanocluster in which a group represented by formula (I) and a group represented by formula (II) are bonded (hereinafter sometimes referred to as gold nanocluster (Ia)), the ratio (molar ratio) of the group represented by formula (II) to the group represented by formula (I) (II):(I) is preferably 1:0.2-5, more preferably 1:0.5-3, and even more preferably 1:1-2.

[0072] The total amount of the group represented by formula (I) and the group represented by formula (II) per 1 mg of the gold nanocluster (Ia) is preferably 10 to 250 mol / mg, more preferably 175 to 225 nmol / mg.

[0073] The amount of the group represented by formula (III) relative to 1 mg of gold nanocluster to which the group represented by formula (III) is bonded is preferably 10 to 250 nmol / mg, and more preferably 175 to 226 nmol / mg.

[0074] The amount of the group represented by formula (IV) per 1 mg of gold nanocluster to which the group represented by formula (IV) is bonded is preferably 10 to 250 nmol / mg, and more preferably 175 to 225 nmol / mg.

[0075] Manufacturing method The method for producing the gold nanocluster and intermediate of the present invention will be described. In this specification, room temperature is about 10° C. to about 35° C. In the following formula,

[0076] [ka]

[0077] indicates the gold nanocluster portion (the core of the nanocluster, which is composed of gold atoms). The structure of the gold nanocluster is shown diagrammatically, and there are multiple groups on the gold nanocluster that bond via -S-.

[0078] [ka]

[0079] (In the formula, L 1 is a linker group, SP is a solid phase, Boc is a tert-butoxycarbonyl group, Trt is a trityl group, and t-Bu is a tert-butyl group. The azide compound represented by formula (V) or a salt thereof can be prepared by simultaneously deprotecting the compound of formula (VI) and cleaving it from the solid phase. The solid phase is not limited as long as it is used in peptide synthesis, and for example, Wang resin can be used. Deprotection and cleavage from the solid phase can be carried out in the presence of trifluoroacetic acid, triisopropylsilane (TIS), and dithiothreitol (DTT). The amount of trifluoroacetic acid used is, for example, 200 to 400 equivalents, and preferably 250 to 350 equivalents, relative to the compound of formula (VI). The amount of TIS and DTT used is, for example, 1 to 5 equivalents, preferably 1 to 2 equivalents, relative to the compound of formula (VI). The reaction can be carried out in an aqueous solution. The reaction can be carried out, for example, at room temperature to 60° C., and is preferably carried out at room temperature. The reaction time is, for example, 5 minutes to 24 hours, and preferably 30 minutes to 4 hours. Examples of salts of the azide compound of formula (V) include salts with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, etc., and salts with organic acids such as trifluoroacetic acid, acetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc. Trifluoroacetic acid salts are preferred. Compounds of formula (VI) can be prepared by standard peptide synthesis techniques using commercially available reagents and solid phase.

[0080] [ka]

[0081] (In the formula, L 1 is a linker group) A gold nanocluster represented by formula (IVa) (a gold nanocluster having a group represented by formula (IV) bonded thereto) can be produced by reacting an azide compound represented by formula (V) or a salt thereof with tetrachloroauric acid (III) or a salt thereof, or a hydrate thereof, in the presence of a base. Examples of tetrachloroauric(III) acid and its salts and hydrates include tetrachloroauric(III) acid and its hydrates, sodium tetrachloroaurate(III) and its hydrates, and potassium tetrachloroaurate(III) and its hydrates, and preferably tetrachloroauric(III) acid tetrahydrate. Examples of the base include inorganic salts such as sodium hydroxide, potassium hydroxide, sodium carbonate, and potassium carbonate; metal alkoxides such as sodium methoxide, sodium ethoxide, potassium t-butoxide, and lithium t-butoxide; and metal phenoxides such as sodium phenoxide and potassium phenoxide. Of these, metal phenoxides are preferred, and sodium phenoxide is more preferred. The amount of tetrachloroauric(III) acid or a hydrate thereof used is, for example, 0.1 to 1 equivalent, and preferably 0.4 to 0.6 equivalent, relative to the azide compound of the formula (V) or a salt thereof. The amount of the base used is, for example, 10 to 100 equivalents, and preferably 20 to 40 equivalents, relative to the azide compound of the formula (V) or a salt thereof. The reaction can be carried out in an aqueous solution. The reaction can be carried out, for example, at room temperature to 60°C, and is preferably carried out at 40°C to 60°C. The reaction time is, for example, 30 minutes to 4 days, and preferably 1 to 72 hours. The reaction is preferably carried out in an atmosphere of an inert gas such as argon or nitrogen, and more preferably in an argon atmosphere.

[0082] [ka]

[0083] (In the formula, R 4is a group that reacts with an azide group to form a divalent group containing a 1,2,3-triazole ring, and the other symbols are as defined above. A gold nanocluster represented by formula (IIIa) (a gold nanocluster having a group represented by formula (III) bonded thereto) can be produced by subjecting a gold nanocluster represented by formula (IVa) (a gold nanocluster having a group represented by formula (IV) bonded thereto) and an alkyne compound represented by formula (VII) to a click reaction. R 4 An example of this is the formula

[0084] [ka]

[0085] Examples of the group include a group represented by the following formula: The click reaction can be performed under normal conditions, for example, copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC) or strain-promoted azide-alkyne cycloaddition reaction (SPAAC). When the click reaction is CuAAC, the reaction can be carried out in the presence of a copper catalyst. As the copper catalyst, copper(II) salt and ascorbate can be used. As the copper(II) salt, copper(II) sulfate or its hydrate can be used. As the ascorbate, sodium ascorbate can be used. To promote the reaction, the reaction can be carried out in the presence of a water-soluble ligand. Examples of the water-soluble ligand include tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), tris(2-benzimidazolylmethyl)amine, etc. The amount of the alkyne compound represented by formula (VII) used is, for example, 0.3 to 3 micromoles, and preferably 2 to 3 micromoles, per 1 mg of the gold nanocluster represented by formula (IVa). The amount of copper (II) salt used is, for example, 0.1 to 1 micromol, preferably 0.5 to 0.7 micromol, per 1 mg of the gold nanocluster represented by formula (IVa). The amount of ascorbate used is, for example, 0.5 to 2 micromoles, and preferably 1 to 1.5 micromoles, per 1 mg of the gold nanocluster represented by formula (IVa). The amount of the water-soluble ligand used is, for example, 0.5 to 5 micromoles, and preferably 2.5 to 3.5 micromoles, per 1 mg of the gold nanocluster represented by formula (IVa). The reaction can be carried out in an aqueous solution or a phosphate buffer solution. The reaction can be carried out, for example, at room temperature to 60°C, and is preferably carried out at room temperature. The reaction time is, for example, 5 minutes to 48 hours, and preferably 30 minutes to 24 hours. The reaction is preferably carried out under an atmosphere of an inert gas such as argon or nitrogen, and more preferably under an argon atmosphere.

[0086] [ka]

[0087] [ka]

[0088] (In the formula, X 1 is a halogen atom such as a chlorine atom or N-succinimidyloxy, and in the formula (Ia), some of the R 5 is a group represented by the following formula (VIIIa), and the remaining R 5 is a group represented by the following formula (IXa):

[0089] [ka]

[0090] The other symbols are as defined above.

[0091] A gold nanocluster represented by formula (IA) (a gold nanocluster having a group represented by formula (II) bonded thereto, hereinafter sometimes referred to as gold nanocluster (IA)) can be produced by reacting a gold nanocluster represented by formula (IIIa) (a gold nanocluster having a group represented by formula (III) bonded thereto) with an indolizine activated ester represented by formula (VIII).

[0092] The amount of the indolizine activated ester represented by formula (VIII) used is, for example, 0.5 to 2 micromoles, and preferably 1 to 1.5 micromoles, per 1 mg of the gold nanocluster represented by formula (IIIa).

[0093] A gold nanocluster represented by formula (IB) (a gold nanocluster having a group represented by formula (I) bonded thereto, hereinafter sometimes referred to as gold nanocluster (IB)) can be produced by reacting a gold nanocluster represented by formula (IIIa) (a gold nanocluster having a group represented by formula (III) bonded thereto) with an activated ester represented by formula (IX).

[0094] The amount of the activated ester represented by formula (IX) used is, for example, 0.5 to 2 micromoles, and preferably 1 to 1.5 micromoles, per 1 mg of the gold nanocluster represented by formula (IIIa).

[0095] A gold nanocluster represented by formula (Ia) (a gold nanocluster having a group represented by formula (I) and a group represented by formula (II) bonded thereto) can be produced by reacting a gold nanocluster represented by formula (IIIa) (a gold nanocluster having a group represented by formula (III) bonded thereto) with an indolizine activated ester represented by formula (VIII), and then reacting the resulting compound with an activated ester represented by formula (IX).

[0096] The amount of the indolizine activated ester represented by formula (VIII) used is, for example, 0.5 to 2 micromoles, and preferably 1 to 1.5 micromoles, per 1 mg of the gold nanocluster represented by formula (IIIa). The amount of the activated ester represented by formula (IX) used is, for example, 0.5 to 2 micromoles, and preferably 1 to 1.5 micromoles, per 1 mg of the gold nanocluster represented by formula (IIIa). The ratio (molar ratio) of the indolizine activated ester represented by formula (VIII) to the activated ester represented by formula (IX), (VIII):(IX), is preferably 1:0.2-5, more preferably 1:0.5-3, and even more preferably 1:1-2.5.

[0097] The reaction for obtaining gold nanocluster (IA), (Ia), or (IB) from the gold nanocluster represented by formula (IIIa) can be carried out in the presence of a base. Examples of the base include sodium hydrogen carbonate. The reaction can be carried out in an organic solvent such as dimethyl sulfoxide, water, or a mixture thereof. The reaction can be carried out, for example, at room temperature to 60°C, and is preferably carried out at room temperature. The reaction time is, for example, 5 minutes to 48 hours, and preferably 30 minutes to 36 hours. The reaction is preferably carried out in an atmosphere of an inert gas such as argon or nitrogen, and more preferably in an argon atmosphere.

[0098] The indolizine activated ester represented by formula (VIII) can be prepared according to the scheme shown below.

[0099] [ka]

[0100] (In the formula, X 2 is a halogen atom such as a chlorine atom, and the other symbols are as defined above. The indolizine compound represented by formula (XIV) can be produced by reacting the indolizine compound represented by formula (X) with a compound represented by formula (XI), (XII), or (XIII). The reaction can be carried out according to the method described in WO 2021 / 177343. The indolizine activated ester represented by formula (VIII) can be produced by deprotecting the tert-butyl ester of the indolizine compound represented by formula (XIV) to convert it to a carboxylic acid compound, and converting the resulting carboxylic acid compound to an activated ester compound. For example, the indolizine activated ester can be produced by deprotecting the tert-butyl ester of the indolizine compound represented by formula (XIV) with trifluoroacetic acid to convert it to a carboxylic acid compound, and reacting the resulting carboxylic acid compound with N-hydroxysuccinimide in the presence of a base and a condensing agent.

[0101] The pharmaceutical composition of the present invention contains gold nanocluster (IA), (Ia), or (IB) and a pharma- ceutical acceptable carrier. The anti-cancer composition of the present invention is 1 comprises a gold nanocluster (IA) or (Ia), which is a biologically active moiety derived from an anticancer drug, and a pharma- ceutically acceptable carrier.

[0102] When the gold nanocluster (IA), (Ia), or (IB) is administered to humans or non-human animals, it can be safely administered orally or parenterally as a pharmaceutical composition, such as an oral preparation (e.g., powder, granules, tablets, capsules), a parenteral preparation (e.g., injection), or a suppository (e.g., rectal suppository, vaginal suppository), by itself or in combination with a pharma- ceutical acceptable carrier, excipient, diluent, etc. These preparations can be prepared by conventional methods.

[0103] Injections include subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, drip infusions, etc. Injections can be made into aqueous injections by standard methods by dissolving, suspending, or emulsifying the gold nanocluster (IA), (Ia), or (IB) together with a solubilizer (e.g., β-cyclodextrins), a dispersant (e.g., carboxymethylcellulose, sodium alginate), a preservative (e.g., methylparaben, propylparaben, benzyl alcohol, chlorobutanol), an isotonic agent (e.g., sodium chloride, glycerin, sorbitol, glucose), etc. In addition, they can be made into oily injections by dissolving, suspending, or emulsifying in vegetable oils (e.g., olive oil, sesame oil, peanut oil, cottonseed oil, corn oil), propylene glycol, etc.

[0104] Orally administered agents can be produced by appropriately adding excipients (e.g., lactose, sucrose, starch), disintegrants (e.g., starch, calcium carbonate), binders (e.g., starch, gum arabic, carboxymethylcellulose, polyvinylpyrrolidone, hydroxypropylcellulose), or lubricants (e.g., talc, magnesium stearate, polyethylene glycol) to the gold nanocluster (IA), (Ia), or (IB), compressing the mixture, and then coating the mixture with hydroxypropylmethylcellulose, etc., as necessary. Suppositories can be produced by mixing the gold nanocluster (IA), (Ia), or (IB) with non-irritating excipients (e.g., polyethylene glycol, glycerides of higher fatty acids).

[0105] The dosage of gold nanocluster (IA), (Ia), or (IB) varies depending on age, body weight, symptoms, dosage form, administration method, administration period, etc., but for example, it is usually 0.01 to 100 mg / kg, preferably 0.05 to 50 mg / kg, and more preferably 0.1 to 10 mg / kg per day per patient (adult, body weight approximately 60 kg), administered orally or parenterally in one or several divided doses.

[0106] Gold nanoclusters (IA), (Ia), and (IB) can be used for photodynamic therapy (PDT). For example, when gold nanoclusters (IA) and (Ia) are administered in vivo, they are delivered to the target site by the targeting ligand Y and accumulate there. Here, in the presence of a photosensitizer such as pyropheophorbide a, rose bengal, or methylene blue, singlet oxygen is generated by irradiating light to decompose the indolizine ring, which then releases a biologically active molecule (D 1 -OH, D 1 -NH2 or D 1 -COOH) is released, exerting the desired biological activity at the target site. If the target site is cancer tissue, using an anticancer drug as the biologically active molecule can be expected to have a selective therapeutic effect on the target cancer tissue. If the gold nanocluster does not have a photosensitizer portion, the gold nanocluster can be administered together with a photosensitizer. In addition, when gold nanoclusters (IB) are irradiated with light, singlet oxygen is generated by the photosensitizer portion that they themselves possess, allowing them to selectively exert cytotoxicity at the target site.

[0107] Furthermore, the gold nanocluster (Ia) can be used for photodynamic therapy (PDT). A pharmaceutical composition containing the gold nanocluster (Ia) is injected into a living body. The gold nanocluster (Ia) is selectively delivered to the target tissue by the targeting ligand Y. By irradiating the target tissue with light, singlet oxygen is generated from the photosensitizer, which decomposes the indolizine ring, and the bioactive molecule (D) is released from the gold nanocluster (Ia). 1 -OH, D 1 -NH2 or D 1 -COOH) is released. Singlet oxygen generated from the photosensitizer is cytotoxic to cancer cells, so D 1 When the biologically active moiety is derived from an anticancer drug, a synergistic therapeutic effect can be expected by combining the biologically active molecule (anticancer drug) with a photosensitizer.

[0108] The light irradiation can be performed using a light source capable of generating singlet oxygen from the photosensitizer. Red light having high biological permeability is preferable, and for example, light in the wavelength region of 600 to 800 nm, preferably 600 to 700 nm, can be used. When the photosensitizer is phthalocyanine, rose bengal, or fluorescein, light in the wavelength region of 400 to 500 nm can be used. EXAMPLES

[0109] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples. The following abbreviations are used in the following examples and test examples. Fmoc: 9-fluorenylmethyloxycarbonyl Boc: tert-butoxycarbonyl Trt: Trityl DMF: N,N-dimethylformamide HATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate HOAt: 1-hydroxy-7-azabenzotriazole t-Bu: tert-butyl 1 H NMR: Proton nuclear magnetic resonance THF: tetrahydrofuran HRMS: High resolution mass spectrometry ESI: electrospray ionization TLC: Thin Layer Chromatography EtOAc: ethyl acetate GPC: Gel Permeation Chromatography DMSO: Dimethyl sulfoxide THPTA: Tris(3-hydroxypropyltriazolylmethyl)amine EDTA: Ethylenediaminetetraacetic acid TEM: Transmission electron microscope

[0110] Example 1 The production schemes for gold nanoclusters are shown in Figures 5 to 7 (Schemes 2 to 4). 1. Reagents Pyropheophorbide a was purchased from Frontier Scientific and used as is without purification. Combretastatin A4 (CA4) was purchased from BLD Pharm and used as is without purification. Cyanine dye 3 (Scheme 3) was purchased from Broad Pharm and used as is without purification. Reagents used in other synthesis experiments were commercially available from reagent companies and used as is without purification unless otherwise specified. Cyclic RGD peptide 5 (Scheme 3) was synthesized with reference to the literature (Non-Patent Document 17).

[0111] 2. Synthesis of small molecules (Schemes 2 and 4) (13S,16R,19S)-1-Azido-19-carboxy-13-(carboxymethyl)-16-(mercaptomethyl)-11,14,17-trioxo-3,6,9-trioxa-12,15,18-triazatricosane-23-aminium trifluoroacetate (2) A 10 cm column was attached to a multi-solid phase synthesizer (Kokusan Chemical, KMS-3). Fmoc-L-Lys(Boc)-Wang Resin (100-200 mesh, 0.56 mmol / g for Lys, 893 mg, 0.50 mmol for Lys) and DMF (5.0 mL) were added to the column and stirred at room temperature for 1 hour. The DMF was discarded, and a mixture of DMF / piperidine (6.0 mL / 1.5 mL) was added, and the suspension was stirred at room temperature for 10 minutes. The resin was washed 10 times with DMF (3.0 mL). To the resin was added a solution of Fmoc-L-Cys(trt)-OH (879 mg, 1.50 mmol, 3 equiv), HATU (570 mg, 1.50 mmol, 3 equiv), HOAt (204 mg, 1.50 mol, 3 equiv) in DMF (5.0 mL) and a solution of N,N-diisopropylethylamine (523 μL, 3.00 mmol, 6 equiv) in DMF (2.0 mL). The suspension was stirred at room temperature for 1.5 h. The reaction solution was discarded and the resin was washed 10 times with DMF (3.0 mL). To this resin was added a solution of Fmoc-L-Asp(t-Bu)-OH (626 mg, 1.50 mmol, 3 equiv), HATU (570 mg, 1.50 mmol, 3 equiv), HOAt (204 mg, 1.50 mol, 3 equiv) in DMF (5.0 mL) and a solution of N,N-diisopropylethylamine (523 μL, 3.00 mmol, 6 equiv) in DMF (2.0 mL). The suspension was stirred at room temperature for 1.5 h. The reaction solution was discarded and the resin was washed 10 times with DMF (3.0 mL). To this resin was added a solution of 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)acetic acid (350 mg, 1.50 mmol, 3 equiv), HATU (570 mg, 1.50 mmol, 3 equiv), HOAt (204 mg, 1.50 mol, 3 equiv) in DMF (5.0 mL) and a solution of N,N-diisopropylethylamine (523 μL, 3.00 mmol, 6 equiv) in DMF (2.0 mL). The suspension was stirred at room temperature for 1.5 h.The reaction solution was discarded, and the resin was washed 10 times with DMF (3.0 mL) and 5 times with dichloromethane (3.0 mL). A mixed solution of trifluoroacetic acid (9.4 mL), water (0.30 mL), triisopropylsilane (0.10 mL), and DL-dithiothreitol (300 mg) was added to the resin. The suspension was gently stirred at room temperature for 1.5 hours. After the reaction was completed, the suspension was filtered, and the filtrate was collected in a 50 mL tube while washing with trifluoroacetic acid (1.0 mL) three times. Argon gas was blown into the filtrate, and it was concentrated to about 1 mL. Diethyl ether (40 mL) was added to the residue, and the mixture was left to stand at -20°C for 30 minutes. The obtained white solid was collected by centrifugation. The white solid was decanted twice with diethyl ether (20 mL). The white solid was purified by high performance liquid chromatography (column: COSMOSIL 5C18-AR-II, 20×250 mm; developing solvent: HO / MeCN + 0.1v / v% trifluoroacetic acid = 95 / 5→55 / 45, flow rate: 10 mL / min). The fraction containing the target product was lyophilized.

[0112] Yield 181 mg (0.261 mmol, 52%); colorless solid; melting point 158°C (decomposition); 1 H NMR (400 MHz, CDCl3): δ 4.43 (dd, J = 6.8, 6.0 Hz, 1H), 4.29 (dd, J = 9.6, 5.2 Hz, 1H), 4.09-4.00 (m, 2H), 3.67-3.61 (m, 11H), 3.40 (t, J = 4.8 Hz, 2H), 2.93-2.76 (m, 6H), 1.87-1.80 (m, 1H), 1.73-1.67 (m, 1H), 1.63-1.55 (m, 2H), 1.41-1.29 (m, 2H); 13 C NMR (100 MHz, CDCl3): δ 175.1, 174.1, 172.8, 172.1, 171.6, 163.0 (q, 1 J C-F = 35.5 Hz), 116.3 (q, 2 J C-F= 293.2 Hz), 70.4, 69,7, 69.5, 69.4, 69.2, 55.6, 52.5, 50.2, 49.7, 39.2, 35.3, 29.8, 26.1, 25.2, 22.0; 19 F NMR (CDCl3): δ -75.5; IR (ZnSe) 2932, 2016, 1714, 1643, 1518, 1180, 1134, 721 cm -1 ; HRMS (ESI, m / z): [M - CF3CO2 - ] + calcd for C 21 H 38 N7O 10 S + , 580.2395; found 580.2396.

[0113] (Z)-2-Methoxy-5-(3,4,5-trimethoxystyryl)phenyl 6-(4-(tert-butoxy)-4-oxobutoxy)-2-methoxyindolizine-3-carboxylate (S1) A solution of triphosgene (496 mg, 0.167 mmol, 0.667 equiv) in THF (0.50 mL) was placed in a 4 mL vial and cooled to 0°C. A solution of combretastatin A4 (158 mg, 0.499 mmol, 2 equiv) in THF (1.0 mL) was added dropwise to this solution. Then, a solution of N,N-diisopropylethylamine (87.1 μL, 0.500 mmol, 2 equiv) in THF (0.50 mL) was added dropwise to this solution. The solution was returned to room temperature and stirred for 20 hours under an argon atmosphere. After the reaction was completed, the reaction solution was diluted with ethyl acetate (15 mL) and washed three times with water (15 mL). The organic layer was dried over sodium sulfate, filtered, and the filtrate was concentrated. The residue was dissolved in THF (1.0 mL) and added to a 4 mL vial. The solution was cooled to 0°C, and a solution of tert-butyl 4-((2-methoxyindolizin-6-yl)oxy)butanoate (72.3 mg, 0.250 mmol, 1 equiv) in THF (1.0 mL) was added dropwise. The solution was returned to room temperature and stirred under an argon atmosphere for 20 hours. After the reaction was completed, the reaction solution was diluted with ethyl acetate (15 mL) and washed three times with water (15 mL). The organic layer was dried over sodium sulfate, filtered, and the filtrate was concentrated. The residue was purified by silica gel flash column chromatography (developing solvent, hexane / ethyl acetate = 1 / 0 → 1 / 1). The obtained compound was further purified by GPC column chromatography.

[0114] Yield 57.7 mg (89.1 μmol, 35.7%); colorless solid; melting point 53.0-56.4℃; TLC R f = 0.51 (hexane / EtOAc = 2 / 1); 1H NMR (400 MHz, CDCl3): δ 9.13 (d, J = 2.0 Hz, 1H), 7.24 (m, 2H), 7.17 (dd, J = 8.4, 2.0 Hz, 1H), 6.91-6.87 (m, 2H), 6.59 (s, 2H), 6.49-6.40 (m, 2H), 6.01 (s, 1H), 3.97-3.94 (m, 5H), 3.82 (s, 3H), 3.81 (s, 3H), 3.75 (s, 6H), 2.40 (t, J = 8.0 Hz, 2H), 2.09-2.02 (m, 2H), 1.44 (s, 9H); 13 C NMR (100 MHz, CDCl3): δ 172.6, 159.8, 157.9, 153.1, 151.3, 148.4, 139.6, 137.3, 133.6, 132.8, 130.1, 129.1, 129.0, 127.6, IR (ZnSe) 2936, 1727, 1680, 1580, 1504, 1454, 1418, 1267, 1240, 1126, 1090, 1009, 808 cm -1 HRMS (ESI, m / z): [M + H + ] + calcd for C 36 H 42 NO 10 + , 648.2803; found 648.2808.

[0115] (Z)-2-Methoxy-5-(3,4,5-trimethoxystyryl)phenyl 6-(4-((2,5-dioxopyrrolidin-1-yl)oxy)-4-oxobutoxy)-2-methoxyindolizine-3-carboxylate (7) S1 (32.4 mg, 49.9 μmol, 1 equiv) and dichloromethane (1.0 mL) were added to a 20 mL vial and cooled to 0°C. Trifluoroacetic acid (1.0 mL) was added dropwise to this solution. The solution was returned to room temperature and stirred under an argon atmosphere for 3 hours. After the reaction was completed, the reaction solution was distilled off and the residue was dried in vacuum.

[0116] The residue was dissolved in a mixture of dichloromethane (0.40 mL) and THF (0.2 mL), and N-hydroxysuccinimide (11.5 mg, 99.9 μmol, 2 equiv), N,N-diisopropylethylamine (17.4 μL, 99.9 μmol, 2 equiv), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (19.2 mg, 100 μmol, 2 equiv) were added to the solution. The reaction solution was stirred at room temperature for 20 hours under an argon atmosphere. After the reaction was completed, the reaction solution was diluted with ethyl acetate (15 mL) and washed three times with water (15 mL). The organic layer was dried over sodium sulfate, filtered, and the filtrate was concentrated. The residue was purified by silica gel flash column chromatography (developing solvent, hexane / ethyl acetate = 1 / 0→0 / 1). The obtained compound was further purified by GPC column chromatography.

[0117] Yield 13.6 mg (19.8 μmol, 39.6%); colorless solid; melting point 71.5-74.7℃; TLC R f = 0.45 (hexane / EtOAc = 1 / 1); 1H NMR (400 MHz, CDCl3): δ 9.14 (d, J = 2.0 Hz, 1H), 7.28-7.23 (m, 2H), 7.16 (d, J = 8.8, 2.0 Hz, 1H), 6.93 (dd, J = 9.6, 2.4 Hz, 1H), 6.88 (d, J = 8.8 Hz, 1H), 6.59 (s, 2H), 6.49-6.40 (m, 2H), 6.02 (s, 1H), 4.04 (t, J = 6.0 Hz, 2H), 3.95 (s, 3H), 3.82 (s, 3H), 3.81 (s, 3H), 3.74 (s, 6H), 2.86-2.82 (m, 6H), 2.25-2.18 (m, 2H); 13 C NMR (100 MHz, CDCl3): δ 169.3, 168.4, 159.7, 157.9, 153.1, 151.3, 148.2, 139.6, 137.3, 133.7, 132.8, 130.1, 129.2, 129.0, IR (ZnSe) 2938, 1738, 1674, 1504, 1454, 1418, 1350, 1267, 1206, 1088, 1007, 806 cm -1 HRMS (ESI, m / z): [M + H + ] + calcd for C 36 H 37 N2O 12 + , 689.2341; found 689.2346.

[0118] Pyropheophorbide a N-hydroxysuccinimidyl ester (8) Pyropheophorbide a (10.7 mg, 10.0 μmol, 1 equiv), DMSO (0.50 mL), and N,N-diisopropylethylamine (3.5 μL, 20 μmol, 1 equiv) were added to a 4 mL vial. N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (12.0 mg, 39.9 μmol, 2 equiv) was added to the solution. The solution was stirred at room temperature under an argon atmosphere for 20 hours. After the reaction was completed, the reaction solution was neutralized by adding formic acid (0.8 μL, 21 μmol, 1 equiv). Next, this solution was purified by high performance liquid chromatography (column, COSMOSIL 5C18-AR-II, 10 × 250 mm; developing solvent, H2O / MeCN+0.1v / v% formic acid = 1 / 1→0 / 1, flow rate 5 mL / min). Yield 9.6 mg (15 μmol, 76%); purple solid; melting point 212°C (decomposition); 1 H NMR (400 MHz, CDCl3): δ 9.37 (s, 1H), 9.26 (s, 1H), 8.49 (s, 1H), 7.88 (dd, J = 17.6, 11.6 Hz, 1H), 5.16-5.02 (m, 2H), 4.45-4.39 (m, 1H), 4.34-4.32 (m, 1H), 3.59-3.53 (m, 5H), 3.31 (s, 3H), 3.11 (s, 3H), 2.85-2.68 (m, 5H), 2.57-2.51 (m, 1H), 2.24-2.15 (m, 1H), 1.73 (d, J = 7.6 Hz, 3H), 1.58 (t, 3H), 0.31 (s, 1H), -1.85 (s, 1H); 13C NMR (100 MHz, CDCl3): δ 196.4, 171.3, 169.2, 168.5, 160.0, 154.9, 149.2, 145.1, 141.8, 138.0, 136.4, 136.1, 131.9, 130.8, IR (ZnSe) 2866, 1738, 1683, 1616, 1499, 1347, 1204, 981cm -1 HRMS (ESI, m / z): [M + H + ] + calcd for C 37 H 38 N5O5 + , 632.2867; found 632.2870. The spectral data were consistent with literature values ​​(Non-Patent Document 18).

[0119] 3. Synthesis of Gold Nanoclusters Gold nanocluster 1 (Scheme 2) In a 50 mL eggplant flask, a solution of tetrachloroauric acid tetrahydrate (51.5 mg, 125 μmol, 0.5 equiv) in water (7.5 mL) was added to a solution of peptide 2 (173 mg, 249 μmol, 1 equiv) in water (7.5 mL). The solution was stirred at room temperature for 30 minutes under an argon atmosphere. A solution of phenol (706 mg, 750 μmol, 30 equiv) and NaOH (300 mg, 750 μmol, 30 equiv) in water (7.5 mL) was added to the solution. The solution was stirred at 50°C for 48 hours under an argon atmosphere. After the reaction was completed, the reaction solution was neutralized by adding formic acid (28.8 μL, 751 μmol, 30 equiv). The solution was then added to an ultrafiltration filter unit (Amicon Ultra, NMWL 10000) and ultrafiltration was performed five times. The concentrated aqueous solution was lyophilized to give a black solid. Yield: 45.2 mg

[0120] Study on synthesis of gold nanocluster 1 using NaBH4 (1') In a 4 mL vial, tetrabutylammonium bromide (2.4 mg, 4.4 μmol, 1.1 equiv) was added to a solution of tetrachloroaurate(III) tetrahydrate (1.7 mg, 4.1 μmol, 1.0 equiv) in methanol (0.15 mL). The solution was stirred for 20 minutes at room temperature under an argon atmosphere. Next, a solution of peptide 2 (13.9 mg, 20.0 μmol, 5 equiv) in methanol (0.30 mL) was added to the solution. The solution was stirred for 30 minutes at 0° C. under an argon atmosphere. Next, a solution of sodium borohydride (3.0 mg, 80 μmol, 20 equiv) in water (0.60 mL) was added to the solution. The solution was stirred for 18 hours at room temperature under an argon atmosphere. After the reaction was completed, the reaction solution was diluted with water (12 mL). The solution was passed through a syringe filter (22 μm), and the filtrate was added to an ultrafiltration filter unit (Amicon Ultra, NMWL 3000). Ultrafiltration was performed five times. The concentrated aqueous solution was freeze-dried to obtain a black solid. Yield: 2.5 mg

[0121] Study on synthesis of gold nanocluster 1 by heating In a 20 mL vial, a solution of tetrachloroauric acid tetrahydrate (8.8 mg, 21 μmol, 1.0 equiv) in water (0.70 mL) was added to a solution of peptide 2 (22.2 mg, 32.0 μmol, 1.5 equiv) in water (10 mL). The solution was stirred at 70°C for 24 hours under an argon atmosphere. After the reaction was completed, the reaction solution was added to an ultrafiltration filter unit (Amicon Ultra, NMWL 3000) and ultrafiltration was performed five times. The concentrated aqueous solution was freeze-dried. However, almost no compound was obtained, indicating that gold nanoclusters had not been synthesized.

[0122] Gold nanocluster 4 (Scheme 3) In a 4 mL vial, a solution of cyanine dye 3 (3.9 mg, 3.9 μmol) in water (300 μL) was added to a solution of gold nanocluster 1 (1.6 mg) in water (300 μL). The solution was stirred at room temperature for 20 hours under an argon atmosphere. After the reaction was completed, water (14 mL) was added to the reaction solution. The solution was added to an ultrafiltration filter unit (Amicon Ultra, NMWL 10000) and ultrafiltration was performed five times. The concentrated aqueous solution was freeze-dried to obtain a blue solid. Yield: 3.2 mg

[0123] The obtained gold nanocluster 1 was found to have a highly uniform particle size of about 2.68 nm by transmission electron microscopy (TEM) (Figure 1A). In addition, IR spectroscopy of gold nanocluster 1 showed a peak at 2106 cm derived from the azide group. -1 The presence of azide groups was confirmed by the absorption of 1 (Fig. 2B). The absorption due to the azide groups disappeared completely when 1 was reacted with cyanine dye 3 bearing a dibenzocyclooctyne (DBCO) group (Fig. 2C, 4). It can be seen that the azide groups with triethylene glycol spacers in gold nanocluster 1 exhibit high reactivity in the strain-promoted azide-alkyne cycloaddition reaction (Scheme 3). Cyanine dyes absorb at 649 nm, a wavelength at which gold nanoclusters do not absorb. Therefore, the amount of cyanine dye 3 introduced into gold nanocluster 4 was quantified to be 198 nmol / mg. Since the click reaction proceeded quantitatively, it is considered that almost the same amount of azide groups was present in nanocluster 1.

[0124] Gold nanocluster 6 (Scheme 3) A solution of gold nanocluster 1 (9.6 mg), cyclic RGD peptide 5 (18.3 mg, 22.9 μmol) and aminoguanidine hydrochloride (2.5 mg, 23 μmol) in water (3.3 mL) was added to a 4 mL vial. In a 4 mL vial, a solution of THPTA (12.4 mg, 28.5 μmol) in water (750 μL) was added to a solution of copper(II) sulfate pentahydrate (1.4 mg, 5.6 μmol) in water (150 μL). Then, a solution of sodium L-ascorbate (2.3 mg, 12 μmol) in water (300 μL) was added to this solution. The resulting aqueous solution of copper(I) catalyst was added to a solution of gold nanocluster 1 (9.6 mg), cyclic RGD peptide 5 (18.3 mg, 22.9 μmol) and aminoguanidine hydrochloride (2.5 mg, 23 μmol) in water (3.3 mL) in another 4 mL vial. The solution was stirred at room temperature under an argon atmosphere for 20 hours. After the reaction was completed, water (9.0 mL) and an aqueous solution of EDTA (1.0 mL, 0.5 M, pH 8.0) were added to the reaction solution. This solution was added to an ultrafiltration filter unit (Amicon Ultra, NMWL 10000) and ultrafiltration was performed five times. The concentrated aqueous solution was freeze-dried to obtain a black solid. Yield: 10.6 mg

[0125] Integrin α, which is highly expressed in cancer cells v Cyclic RGD peptide 5, which specifically binds to β3, was introduced onto gold nanoclusters via copper-catalyzed azide-alkyne cycloaddition (CuAAC) (Scheme 3, compound 6). IR measurements of product 6 showed that the azide group was completely consumed, suggesting that peptide 5 was quantitatively introduced (Figure 2D, 6).

[0126] Gold nanocluster 9 (Scheme 4) In a 4 mL vial, an aqueous solution of sodium bicarbonate (700 μL, 100 mM, 700 μmol) was added to the gold nanocluster 6 (1.5 mg). A DMSO solution of indolizine 7 (350 μL, 1.43 mM, 501 nmol) was added to this solution. The solution was stirred at room temperature under an argon atmosphere for 6 hours. Next, a DMSO solution of porphyrin 8 (350 μL, 2.86 mM, 1.00 μmol) was added to this solution. The solution was stirred at room temperature under an argon atmosphere for 20 hours. After the reaction was completed, DMSO (3.5 mL) and water (10.5 mL) were added to the reaction solution. The solution was added to an ultrafiltration filter unit (Amicon Ultra, NMWL 10000) and ultrafiltration was performed five times. The concentrated aqueous solution was lyophilized to obtain a purple solid. Yield: 1.9 mg

[0127] Indolizine 7 and pyropheophorbide a derivative 8 bearing an active ester group were introduced by condensation with the amino group of gold nanocluster 6 (Scheme 4). The absorbance at 678 nm of the product 9 indicated that it contained 143 nmol / mg of pyropheophorbide a derivative.

[0128] Gold nanocluster 10 (Scheme 4) In a 4 mL vial, an aqueous solution of sodium bicarbonate (700 μL, 100 mM, 700 μmol) was added to gold nanocluster 6 (1.5 mg). A DMSO solution of porphyrin 8 (350 μL, 2.86 mM, 1.00 μmol) was added to this solution. The solution was stirred at room temperature under an argon atmosphere for 20 hours. After the reaction was completed, DMSO (3.5 mL) and water (10.5 mL) were added. The solution was added to an ultrafiltration filter unit (Amicon Ultra, NMWL 10000) and ultrafiltration was performed five times. The concentrated aqueous solution was lyophilized to obtain a purple solid. Yield: 2.1 mg

[0129] Gold nanocluster 11 (Scheme 4) In a 4 mL vial, an aqueous solution of sodium bicarbonate (700 μL, 100 mM, 700 μmol) was added to gold nanocluster 6 (1.5 mg). A DMSO solution of indolizine 7 (700 μL, 2.86 mM, 2.00 μmol) was added to this solution. The solution was stirred at room temperature for 20 hours under an argon atmosphere. After the reaction was completed, DMSO (3.5 mL) and water (10.5 mL) were added to the reaction solution. The solution was added to an ultrafiltration filter unit (Amicon Ultra, NMWL 10000) and ultrafiltration was performed five times. The concentrated aqueous solution was lyophilized to obtain a black solid. Yield: 1.4 mg

[0130] 4. Quantification of CA4 released from gold nanoclusters 9 The gold nanoclusters were irradiated with 660 nm red light in phosphate-buffered saline (PBS) and the amount of the anticancer drug combretastatin A4 (CA4) released from 9 was quantified.

[0131] A phosphate buffered saline solution (0.05 mg / mL, 150 μL) containing gold nanocluster 9 was added to a 4 mL vial. The 4 mL vial was placed in an EvoluChem PhotoRedOx Box photoreactor equipped with a Kessil H160 Tuna Flora LED lamp (red channel). The reaction solution in the 4 mL vial was irradiated with light for 0-300 s at room temperature under air while being stirred with a rotor and magnetic stirrer. A calibration curve for CA4 was created using a Thermo Fisher Scientific DIONEX Ultimate 3000 ultra-performance liquid chromatography (UPLC)-equipped Q Exactive Orbitrap mass spectrometry (LC / MS / MS) system, and the amount of CA4 produced in the reaction solution after the reaction was completed was quantified. The results are shown in Figure 3.

[0132] The release of CA4 was almost saturated 5 minutes after irradiation, and the amount released was 81.1 nmol / mg (Figure 3). This indicated that the gold nanocluster 9 contained about 81.1 nmol / mg of indolizine derivative with CA4 caged. Thus, the two components introduced via the amino groups were found to be present in gold nanocluster 9 in a total amount of about 224 nmol / mg. This is almost consistent with the amount of azide groups (198 nmol / mg) present in the gold nanocluster. Since the gold nanocluster was modified with a single peptide 2, it can be seen that the azide groups and amino groups are present in a ratio of about 1:1. In gold nanocluster 10, which does not contain an indolizine derivative, the amount of pyropheophorbide A introduced was 209 nmol / mg (Scheme 4). This indicates that the maximum amount of molecules that can be introduced via the amino groups of the nanocluster is approximately 200 nmol / mg. In addition, in gold nanocluster 11, which does not contain pyropheophorbide A, CA4 is not released. This indicates that pyropheophorbide a in the gold nanoclusters is essential for the photoreaction of indolizine.

[0133] 5. TEM measurement TEM imaging of the gold nanoclusters was performed using a JEOL field emission transmission electron microscope (JEM-2100F). The measurement sample was obtained by placing a 95% ethanol solution of gold nanoclusters (0.05 mg / mL) on a copper grid with a carbon support film (STEM HRC-C10) and drying it in vacuum. The results are shown in Figure 1. The average particle sizes of gold nanoclusters 1 (A), 6 (B), 9 (C) and 10 (D) measured by TEM were 2.68±0.58 nm, 2.15±0.44 nm, 1.68±0.46 nm and 2.00±0.58 nm, respectively. TEM measurements confirmed that the particle sizes of gold nanoclusters 1, 6, 9 and 10 were almost unchanged by the series of click reactions and condensation reactions (Figure 1).

[0134] Test Example Cell experiments Integrin α vThe light-induced cytotoxicity of gold nanoclusters was examined by methylthiazolyl tetrazolium (MTT) assay using A549 cells derived from human lung cancer, which express a large amount of β3 and are highly sensitive to CA4 (Non-Patent Document 16).

[0135] A549 cells (human alveolar basal epithelial adenocarcinoma cells) were purchased from the RIKEN Cell Materials Research Center and were passaged at least three times before use. A549 cells (approximately 5 × 10 4 A549 cells were seeded in each well of a 96-well plate and cultured at 37°C for 24 hours in a humidified CO2 (5%) atmosphere. The adhesion of A549 cells to the bottom of the well was confirmed by optical microscopy. The medium in the well was discarded, and D-10 medium (100 μL) containing gold nanocluster 9-11 (0-20 μg / mL) was added and cultured at 37°C for 1 hour in a humidified CO2 (5%) atmosphere. The medium containing the sample was discarded and replaced with fresh D-10 medium (100 μL) without the sample. Next, the 96-well plate containing the cells was irradiated with a red LED (660 nm) for 5 minutes. For light irradiation, a Kessil H160 Tuna Flora LED lamp (red channel) was used, and the optical path length from the light source to the 96-well plate was set to 5 cm. After light irradiation, the cells were cultured at 37°C for 20 hours in a humidified CO2 (5%) atmosphere. The cell viability was evaluated using the MTT Cell Proliferation Assay Kit (Cayman Chemical Co.), and the results are shown in FIG.

[0136] In the cell experiments, A549 cells were cultured for 1 hour in DMEM medium supplemented with fetal bovine serum albumin (FBS, 10%) containing gold nanoclusters, and the medium was replaced with fresh medium before light irradiation to eliminate the effect of gold nanoclusters not taken up by the cells. In a control experiment in the dark without light irradiation, it was found that gold nanoclusters 9-11 showed almost no cytotoxicity (Figure 4A). On the other hand, under the condition of irradiating with 660 nm red light for 5 minutes, gold nanoclusters 9 and 10 showed concentration-dependent photocytotoxicity, while non-photoreactive nanocluster 11 showed almost no toxicity (Figure 4B). The phototoxicity of gold nanocluster 10 is thought to be due to the photodynamic effect of pyropheophorbide a. Gold nanocluster 9 showed more significant cytotoxicity, suggesting a synergistic effect between the release of CA4 and the photodynamic effect (Figure 4B). [Industrial Applicability]

[0137] The gold nanoclusters of the present invention are useful as medicines because they are selectively delivered to the target pathological site, release bioactive molecules such as anticancer drugs upon irradiation with light, and exhibit bioactivity such as anticancer effects. In particular, gold nanoclusters that can be used in photodynamic therapy are provided. Also provided are intermediates and methods for producing the intermediates that are useful for producing gold nanoclusters.

Claims

1. Formula (II) 【Chemistry 1】 (In the formula, L １ , L ２ , and L ３ are each independently a linker group; X is a divalent group containing a 1,2,3-triazole ring; Y is a targeting ligand; Q is O, NH, or a single bond; D １ is the biologically active moiety) A gold nanocluster having a group represented by the formula:

2. The gold nanocluster of claim 1, wherein Y is a group comprising a cyclic RGD peptide.

3. D １ The gold nanocluster of claim 1, wherein is a biologically active moiety derived from an anticancer drug.

4. Q is O and D １ is represented by the following formula (XV) 【Chemistry 2】 The gold nanocluster according to claim 1, which is a group represented by the formula:

5. Further, the formula (I) 【Chemistry 3】 (In the formula, Z is a photosensitizer moiety; L １ and L ２ are each independently a linker group; X is a divalent group containing a 1,2,3-triazole ring; Y is a targeting ligand. The gold nanocluster according to claim 1 , having a group represented by the following formula:

6. The gold nanocluster of claim 5, wherein Z is a group derived from pyropheopherbide a.

7. Z is of formula (XVI) 【Chemistry 4】 The gold nanocluster according to claim 5, which is a group represented by the formula:

8. The gold nanocluster of claim 5, wherein Y is a group comprising a cyclic RGD peptide.

9. Formula (III) 【Chemistry 5】 (In the formula, L １ and L ２ are each independently a linker group; X is a divalent group containing a 1,2,3-triazole ring; Y is a targeting ligand. A gold nanocluster having a group represented by the formula:

10. The gold nanocluster of claim 9, wherein Y is a group comprising a cyclic RGD peptide.

11. Formula (IV) 【Chemistry 6】 (In the formula, L １ is a linker group) A gold nanocluster having a group represented by the formula:

12. Formula (I) 【Chemistry 7】 (In the formula, L １ and L ２ are each independently a linker group; X is a divalent group containing a 1,2,3-triazole ring; Y is a targeting ligand; Z is a photosensitizer moiety. A gold nanocluster having a group represented by the formula:

13. A pharmaceutical composition comprising the gold nanocluster of any one of claims 1 to 8 and 12 and a pharma- ceutically acceptable carrier.

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