Compound, its manufacturing method, composite and short-wave infrared fluorescent agent

Abstract

Provided is a new compound that enables bio-imaging in the short wavelength infrared range. A compound according to one embodiment of the present invention is represented by formula (1). In formula (1), n represents an integer of 3-5, X represents a salt of a sulfonate group, or a reactive crosslinking group for a molecular identification agent.

Description

[Technical Field]

[0001] The present invention relates to a compound, a method for producing the same, a composite, and a short-wave infrared fluorescent agent. [Background technology]

[0002] A widely used bioimaging technique for non-invasively visualizing living organisms is the visualization of blood vessels, tumors, etc. using fluorescent agents that emit near-infrared light with wavelengths of 700 to 900 nm. Indocyanine green (ICG), which emits near-infrared fluorescence, is known as an active ingredient in fluorescent agents that can be used in bioimaging.

[0003] On the other hand, near-infrared light is easily scattered and absorbed by tissues. Therefore, in biological imaging using fluorescent agents that emit near-infrared light, it is difficult to clearly visualize microstructures located, for example, more than 1 cm below the surface of the target organism or biological tissue. Therefore, fluorescent agents that emit short-wave infrared light with a wavelength of 900 to 1400 nm, which is less likely to scatter, are needed. In this regard, it is also known that the fluorescence of ICG extends into the wavelength range of short-wave infrared light (see, for example, Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Setsuko Tsuboi and Takashi Jin, RSC Advances, pp. 28171-28179, 2020 Summary of the Invention [Problem to be solved by the invention]

[0005] However, the fluorescence intensity of ICG in the short-wave infrared region is low. Therefore, it can be difficult to clearly visualize minute structures located more than 1 cm below the surface of the living body when using short-wave infrared bioimaging with fluorescent agents containing ICG. Therefore, there is a need for fluorescent agents that can be used in short-wave infrared bioimaging technology.

[0006] An object of one aspect of the present invention is to provide a novel technique that enables biological imaging in the short-wave infrared region. [Means for solving the problem]

[0007] In order to solve the above problems, a compound according to one embodiment of the present invention is represented by the following formula (1): In the following formula (1), n ​​represents an integer of 3 to 5, and X represents a salt of a sulfonic acid group or a crosslinking group reactive to a molecular recognition agent.

[0008] [ka]

[0009] In order to solve the above problems, a complex according to one aspect of the present invention is formed by binding a molecular recognition agent to the compound of the present invention via a residue of a reactive crosslinking group in the compound.

[0010] Furthermore, in order to solve the above-mentioned problems, a short-wave infrared fluorescent agent according to one embodiment of the present invention contains one or both of the compound of the present invention and the complex of the present invention.

[0011] Furthermore, in order to solve the above-mentioned problems, a method for producing a compound represented by the following formula (1) according to one embodiment of the present invention includes a first step of synthesizing a first compound represented by the following formula (1a), a second step of replacing the anilino group in formula (1a) with a structure represented by the following formula (1b), and a third step of replacing the phenylimino group in formula (1a) with a structure represented by the following formula (1c): In the following formula, n represents an integer of 3 to 5, and X represents a salt of a sulfonic acid group or a crosslinking group reactive to a molecular recognition agent.

[0012] [ka] [Effects of the Invention]

[0013] According to one aspect of the present invention, a novel technique that enables biological imaging in short-wave infrared can be provided. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 shows the relationship between the concentration of Compound 4 and cell viability in an example of the present invention. [Figure 2] FIG. 1 shows the relationship between the concentration of Compound 10 and cell viability in an example of the present invention. [Figure 3] FIG. 1 shows photographs showing bright-field images and short-wave infrared fluorescent images of the lower limbs and abdomen of a hairless mouse in which short-wave infrared fluorescent agent 1 in an example of the present invention was used as an optical contrast agent. [Figure 4] FIG. 1 shows photographs showing bright-field images and short-wave infrared fluorescent images of the lower limbs and abdomen of a hairless mouse in which short-wave infrared fluorescent agent 2 in an example of the present invention was used as an optical contrast agent. [Figure 5] FIG. 1 shows photographs showing bright-field images and short-wave infrared fluorescent images of the top of the head of a hairless mouse using short-wave infrared fluorescent agent 1 and short-wave infrared fluorescent agent 2 in examples of the present invention and short-wave infrared fluorescent agent C1 as a comparative example as optical contrast agents. [Figure 6] FIG. 1 shows photographs showing bright-field images and short-wave infrared fluorescent images of the lower limbs and abdomen of a hairless mouse in which short-wave infrared fluorescent agent 3 in an example of the present invention was used as an optical contrast agent. [Figure 7] FIG. 10 is a photograph showing a visual field image and a short-wave infrared fluorescent image of the lower limbs and abdomen of a hairless mouse in which short-wave infrared fluorescent agent 4 in an example of the present invention was used as an optical contrast agent. [Figure 8]FIG. 1 shows photographs showing bright-field images and short-wave infrared fluorescent images of the top of the head of a hairless mouse using short-wave infrared fluorescent agents 3 and 4 of the examples of the present invention and short-wave infrared fluorescent agent C2 of the comparative example as optical contrast agents. [Figure 9] FIG. 1 shows the fluorescence spectra of conjugates 1 and 2 in an example of the present invention. [Figure 10] FIG. 1 shows photographs showing a bright-field image and a short-wave infrared fluorescent image of a tumor area in a cancer-bearing mouse in which short-wave infrared fluorescent agent 5 in an example of the present invention was used as an optical contrast agent. [Figure 11] 1A and 1B are photographs showing bright-field images and short-wave infrared fluorescence images of the cancer tumor, heart, kidney, spleen, and liver of a cancer-bearing mouse in which short-wave infrared fluorescent agent 5 in an example of the present invention was used as an optical contrast agent. [Figure 12] FIG. 1 shows photographs showing a bright-field image and a short-wave infrared fluorescent image of a tumor area in a cancer-bearing mouse in which short-wave infrared fluorescent agent 6 in an example of the present invention was used as an optical contrast agent. [Figure 13] 1A and 1B are photographs showing bright-field images and short-wave infrared fluorescence images of the cancer tumor, heart, kidney, spleen, and liver of a cancer-bearing mouse in which short-wave infrared fluorescent agent 6 in an example of the present invention was used as an optical contrast agent. [Figure 14] FIG. 1 shows photographs showing a bright-field image and a short-wave infrared fluorescent image of a tumor area in a cancer-bearing mouse in which short-wave infrared fluorescent agent 7 in an example of the present invention was used as an optical contrast agent. [Figure 15] FIG. 1 shows photographs showing bright-field images and short-wave infrared fluorescence images of the cancer tumor, heart, kidney, spleen, and liver of a cancer-bearing mouse in which short-wave infrared fluorescent agent 7 in an example of the present invention was used as an optical contrast agent. [Figure 16] 1A and 1B are photographs showing bright-field images and short-wave infrared fluorescent images of tumor areas in tumor-bearing mice in which short-wave infrared fluorescent agent 8 and short-wave infrared fluorescent agent 9 were used as optical contrast agents in an example of the present invention, respectively. [Figure 17] 1A and 1B are photographs showing bright-field images and short-wave infrared fluorescent images of the tumor area of ​​a cancer-bearing mouse in which short-wave infrared fluorescent agent 10 and short-wave infrared fluorescent agent 11 in an example of the present invention were used as optical contrast agents, respectively. [Figure 18] FIG. 1 shows photographs showing bright-field images and short-wave infrared fluorescence images of tumor areas in tumor-bearing mice injected with and not injected with Kadcyla, taken using short-wave infrared fluorescent agent 12 and short-wave infrared fluorescent agent 13 as optical contrast agents in an example of the present invention. [Figure 19] FIG. 1 shows photographs showing bright-field images and short-wave infrared fluorescent images of the tumor area of ​​a cancer-bearing mouse injected with Kadcyla after a specific period of time using short-wave infrared fluorescent agent 13 as an optical contrast agent in an embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0015] Hereinafter, embodiments of the present invention will be described in detail.

[0016] [Compound] The compound according to an embodiment of the present invention is represented by the following formula (1).

[0017] [ka]

[0018] In formula (1), n ​​represents an integer of 3 to 5. n can be appropriately determined from the viewpoint of enabling the compound of formula (1) to emit short-wave infrared fluorescence and exhibit sufficient water solubility. From the above viewpoint, n is preferably 3 or 4.

[0019] In the embodiments of the present invention, "short-wave infrared" refers to electromagnetic waves having a wavelength of 900 nm or more and 1400 nm or less. The compound of formula (1) has a peak fluorescence intensity in the short-wave infrared wavelength region depending on its structure (e.g., n). Therefore, the wavelength for detecting the fluorescence of the compound of formula (1) may be appropriately determined within a range in which the compound of formula (1) emits sufficiently high-intensity fluorescence in the short-wave infrared region. Furthermore, the wavelength of the excitation light for the fluorescence of the compound of formula (1) may be any wavelength that excites the compound of formula (1). The excitation wavelength can be appropriately determined, for example, from the range of 900 to 1100 nm depending on the structure of the compound of formula (1) (e.g., n) or the depth of the detection target site, etc.

[0020] In formula (1), X represents a salt of a sulfonic acid group or a reactive crosslinking group for a molecular recognition agent. The salt of the sulfonic acid group is not limited and may be, for example, an alkali metal salt, more specifically, a sodium salt.

[0021] The reactive crosslinking group is a functional group that serves to bond with a molecular recognition agent. The bond with the molecular recognition agent may be any suitable bond depending on the application of the compound of formula (1), and may be, for example, a covalent bond or a hydrogen bond. Furthermore, the bond between the reactive crosslinking group and the molecular recognition agent may be in a form in which the molecular recognition agent is bonded to the reactive crosslinking group, or in a form in which a part of the reactive crosslinking group is detached and a part of the reactive crosslinking group is bonded to the molecular recognition agent.

[0022] From the viewpoint of stability and ease of handling, the reactive crosslinking group is preferably a functional group that undergoes crosslinking with the molecular recognition agent under mild conditions, such as mixing at room temperature, and from this viewpoint, the reactive crosslinking group is preferably one or more organic groups selected from the group consisting of an N-hydroxysuccinimide ester group, a maleimide group, an alkynyl group, and an azide group.

[0023] The alkynyl group is not limited, but from the viewpoint of water solubility of the compound of formula (1), it is preferable that the number of carbon atoms is 5 or less. Examples of the alkynyl group include an ethynyl group and a propynyl group.

[0024] The ester structure of the N-hydroxysuccinimide ester group may be any structure that can be bonded to a linear hydrocarbon group or can be derived from an organic group containing a linear hydrocarbon group, such as an N-hydroxysuccinimide carbonyl group. In addition to the organic group described above, the reactive crosslinking group may further have other molecular structures that can constitute the compound of formula (1), as long as the effects of this embodiment can be achieved. Reactive crosslinking groups other than the N-hydroxysuccinimide ester group may also further contain structures (e.g., alkyl groups, amide groups, etc.) that can be bonded to a linear hydrocarbon group or can be derived from an organic group containing a linear hydrocarbon group.

[0025] The reactive crosslinking group can easily bind to a molecular recognition agent having an amino group, a sulfhydryl group, an azide group, or an alkynyl group, and is therefore preferred from the viewpoint of easily and accurately introducing a biological tissue-recognizing moiety into the compound of formula (1).

[0026] Alternatively, a reactive crosslinking group may be introduced into the compound of formula (1) by attaching a linker substance used to attach a fluorescent substance to an amino acid. In this case, a reactive crosslinking group capable of binding to an amino group of a molecular recognition agent may be introduced into the compound of formula (1). Examples of linker substances include 6-aminocaproic acid, 2-aminoadipic acid, 3-aminoadipic acid, 4-aminobutyric acid, 5-aminovaleric acid, 7-aminoheptanoic acid, 8-aminooctanoic acid, 11-aminoundecanoic acid, 12-aminododecanoic acid, 2-aminobenzoic acid, 3-aminobenzoic acid, and 4-aminobenzoic acid.

[0027] A molecular recognition agent is a component having a molecular structure capable of recognizing a specific molecule. The molecular recognition agent may be any component having a molecular structure capable of binding to a specific molecule, and may be one or more types. The molecular recognition agent also has a site available for binding to a reactive crosslinking group. Examples of such sites include amino groups, sulfhydryl groups, azide groups, and alkynyl groups. Examples of molecular recognition agents include peptides, proteins, nucleic acid derivatives, antibodies, fragments of such antibodies capable of binding to antigens, and cells.

[0028] The compound of formula (1) as described above can be more specifically represented by any of formulas (2) to (9) below.

[0029] [ka]

[0030] The compound of formula (1) is an organic dye that emits short-wave infrared fluorescence. As mentioned above, such organic dyes are expected to be applied to biological imaging, and for this reason, compounds with various molecular skeletons have been investigated as short-wave infrared fluorescent agents (e.g., "Jin T, ECS J. Solid State Sci. Technol, 8, R9-R13 (2019)"). Thus, compounds with short-wave infrared fluorescence due to the fluorescent properties specific to the molecular skeleton have been investigated.

[0031] In fluorescent compounds, a portion of the energy absorbed from excitation light is generally converted into molecular vibrational energy and consumed. It is believed that the larger the molecular structure, the greater the rate at which the absorbed energy is converted and consumed. Based on this general trend, it is expected that the longer the chain structure of ICG, the lower the fluorescence emission efficiency.

[0032] However, the compound of formula (1) has a similar structure to ICG, but a longer chain structure than ICG, while exhibiting a high luminescence efficiency in short-wave infrared fluorescence that is equal to or higher than the near-infrared fluorescence of ICG. Therefore, despite having a larger molecular structure than ICG, the compound of formula (1) is a unique compound that does not fit the general trend described above.

[0033] [Method for producing compounds] The compound of formula (1) can be produced by a production method including the following three steps, steps 1 to 3. In the following description of the production method, the meanings of the letters in the general formula are the same as those of the compound described above.

[0034] The first step is a step of synthesizing a first compound represented by the following formula (1a): As described above, n represents an integer of 3 to 5.

[0035] [ka]

[0036] The compound of formula (1a) can be synthesized by reacting a linear diene aldehyde in the presence of a phosphorus compound to appropriately extend the linear diene structure, followed by reaction with aniline. The synthesis can be carried out efficiently by appropriately adjusting the temperature. In extending the linear diene structure, the terminal aldehyde of the linear diene structure in the intermediate product may be temporarily replaced with another structure such as a carboxylic acid ester or alcohol, taking into account the reactivity, solubility, or stability of the intermediate product.

[0037] The second step is a step of replacing the anilino group in formula (1a) with a structure represented by the following formula (1b).

[0038] [ka]

[0039] The anilino group in formula (1a) can be replaced with the structure of formula (1b) by reacting a compound represented by the following formula (1b1) with the compound of formula (1a) or a compound in which the phenylimino group in formula (1a) has been replaced with the structure represented by the following formula (1c) in the presence of acetic anhydride and sodium acetate. The compound of formula (1b1) can be obtained by reacting 1,1,2-trimethyl-1H-benzo[e]indole with 1,4-butanesultone.

[0040] [ka]

[0041] The third step is a step of replacing the phenylimino group in formula (1a) with a structure represented by the following formula (1c).

[0042] [ka]

[0043] When X is a salt of a sulfonic acid group, the phenylimino group in formula (1a) can be replaced with the structure of formula (1c) in the same manner as in the replacement of the anilino group in formula (1a) described above, using as a raw material a compound of formula (1a) or a compound in which the phenylimino group in formula (1a) is replaced with a structure represented by formula (1b).

[0044] When X is the reactive crosslinking group described above, the phenylimino group in formula (1a) can be replaced with the structure of formula (1c) by first replacing the phenylimino group with the structure represented by formula (1c1) below, and then bonding the reactive crosslinking group to the terminal carboxyl group, or by replacing the carboxyl group with a reactive crosslinking group.

[0045] [ka]

[0046] The replacement of the phenylimino group in formula (1a) with the structure represented by formula (1c1) can be carried out in the same manner as the replacement of the anilino group in formula (1a) described above, for example, by using a bromine salt of a compound in which the sulfonic acid group of formula (1b1) has been replaced with a carboxyl group. The reaction of introducing a reactive crosslinking group into the carboxyl group after replacement with the structure represented by formula (1c1) can be carried out by applying a known method depending on the desired reactive crosslinking group.

[0047] In the above production method, the order of the second step and the third step can be appropriately determined from the viewpoint of the solubility of the raw materials or the product, the reactivity of the raw materials, etc., within the range in which the target compound of formula (1) can be produced.

[0048] For example, when the target compound is a compound of formula (2), i.e., when n in the above formula is 3 and X is a salt of a sulfonic acid group, the second step and the third step may be carried out simultaneously. The second step and the third step described above can be carried out simultaneously, for example, by reacting the compound of formula (1a) with two or more equivalents of the compound of formula (1b1) under the conditions of the second step. By carrying out the second step and the third step simultaneously, the compound of formula (1) can be produced more easily and is advantageous from the viewpoint of suppressing a decrease in yield due to an increase in the number of steps.

[0049] When the target compound is a compound of formula (3), i.e., when n in the above formula is 4 and X is a salt of a sulfonic acid group, it is preferable to carry out the third step after the second step. Also, when the target compound is a compound of formula (4), i.e., when n in the above formula is 3 and X is a reactive crosslinking group, it is preferable to carry out the third step after the second step. In these cases, the third step can be carried out using the product of the second step as a raw material. Carrying out the third step after the second step in the production of a compound of formula (3) or a compound of formula (4) is preferable from the viewpoint of increasing the yield of substitution of the phenylimino group in formula (1a) in the third step.

[0050] When the target compound is a compound of formula (5), i.e., when n in the above formula is 4 and X is a reactive crosslinking group, it is preferable to carry out the third step before the second step. In this case, the second step can be carried out using the product of the third step as a raw material. Carrying out the third step before the second step in the production of the compound of formula (5) is preferable from the viewpoint of increasing the yield in the replacement of each of the anilino group and the phenylimino group in formula (1a).

[0051] The above-described production method may further include steps other than the first to third steps described above, as long as the effects of this embodiment can be obtained. For example, the above-described production method may further include a step of purifying the product of each step. Such a purification step can be appropriately performed by a known method such as washing with a solvent or column chromatography.

[0052] In the above production methods, the compounds of the respective formulae can be synthesized using known techniques, but it is preferable to select synthesis conditions appropriately from the viewpoints of reactivity and productivity.

[0053] For example, the synthesis of the compound of formula (1a), the second and third steps, are accelerated by high temperatures. On the other hand, raising the reaction temperature may result in the production of the target compound having a chain structure of the desired length and by-products having a chain structure shorter than that in approximately equal proportions.

[0054] The target compound and the by-products have similar physical properties, such as polarity, and it is easily expected that it would be difficult to separate the target compound from a mixture thereof. In fact, the physical properties are similar, and it is difficult to isolate the target compound from the mixture.

[0055] In the synthesis reaction of the compound of formula (1a), the second and third steps, lowering the reaction temperature slows the reaction rate, but it is possible to synthesize the target compound at a higher rate. For example, as shown in the examples described below, if the reaction to lengthen the chain structure is carried out at room temperature, it is possible to efficiently obtain a highly pure target compound, although the reaction rate is not high. Thus, in the above-mentioned production method, while utilizing known techniques, it is possible to obtain the target compound with high efficiency by appropriately adjusting synthesis conditions such as the reaction temperature as necessary from the standpoint of reactivity and productivity.

[0056] Here, the above-described synthetic reaction in an embodiment of the present invention will be described in more detail with specific examples. In the synthesis of ICG, which has traditionally been used in bioimaging, the anilino group of a compound of formula (1a) where n is 2 is typically replaced with a sulfonic acid compound represented by formula (1b), and then the structure of formula (1c) is introduced into the sulfonic acid compound. Corresponding to the manufacturing method of the compound of this embodiment, the second and third steps described above are performed in this order. In the synthesis of ICG, the solubility of the product significantly decreases as the synthesis proceeds, especially when the substitution with the sulfonic acid compound is performed. For this reason, in the usual synthesis method of ICG, the synthetic reaction to introduce the compound of formula (1b) and the structure of formula (1c) is performed at a higher temperature.

[0057] On the other hand, in the embodiment of the present invention, the carbon chain (linear polyene chain) in the compound of formula (1a) used is longer than in the synthesis of ICG. In particular, the compound of formula (1a) used in the synthesis of the compound of formula (5) (ICG-C11-NHS) has an n of 4, resulting in an even longer linear polyene chain. For this reason, in the production method of this embodiment, the stability of the compound of formula (1a) is significantly lower than that of the compound of formula (1a) used in the synthesis of ICG. Therefore, if the synthesis of the compound of formula (1c) after the introduction of the compound of formula (1b) is performed under high temperature conditions, a by-product with two fewer carbon atoms in the polyene chain than the target compound is produced at a high ratio (approximately a target compound:by-product ratio of 1:1). Because the target compound and the by-product have very similar polarities, obtaining the target compound by separating them is undesirable from the viewpoint of yield.

[0058] Therefore, as described above, it is preferable to carry out the third step prior to the second step, particularly in the synthesis of the compound of formula (5). By carrying out such synthesis, a product with higher solubility in a solvent is produced by the third step carried out first, and as a result, it becomes possible to carry out the subsequent second step at room temperature or lower. This suppresses the production of by-products in the second step, making it possible to obtain essentially only the target compound. Although the reaction rate of the target compound is slightly low, this synthesis method using such an order of steps is preferable from the viewpoint of obtaining the target compound with high purity.

[0059] [Complex] In the conjugate of an embodiment of the present invention, the molecular recognition agent is bound to the compound via a residue of the reactive crosslinking group in the compound of the present embodiment described above. The meanings of the reactive crosslinking group and the molecular recognition agent are as described above in the description of the compound of the embodiment of the present invention. From the perspective of application to molecular imaging, the molecular recognition agent is preferably a component capable of binding to a specific site in a living body. From this perspective, among the above-mentioned examples, the molecular recognition agent is preferably an antibody or a fragment of the antibody that has antigen-binding ability. A conjugate bound to such an antibody or fragment is useful as a fluorescent marker for specific biological tissues such as tumors. Furthermore, the conjugate can be used to detect tumors and confirm pharmacological or therapeutic effects on tumors by short-wave infrared molecular imaging.

[0060] [Short-wave infrared fluorescent agent] A shortwave infrared fluorescent agent according to an embodiment of the present invention is either the compound of formula (1) or the complex described above, or a composition containing the compound. The shortwave infrared fluorescent agent according to an embodiment of the present invention may contain one or more compounds of formula (1), and may contain one or more complexes. Since the compound of formula (1) or the complex containing the compound has the property of emitting shortwave infrared light, the shortwave infrared fluorescent agent according to an embodiment of the present invention can be used for shortwave infrared fluorescence imaging.

[0061] The short-wave infrared fluorescent agent of this embodiment may further contain components other than the compound and complex of formula (1) as long as the effects of this embodiment are obtained. For example, since the short-wave infrared fluorescent agent is usually used in living organisms, it can be used as a solution in phosphate buffered saline.

[0062] It is also known that the stability of ICG and its conjugates in aqueous solution is improved in the presence of bovine serum albumin (BSA), and that the fluorescence emission intensity of ICG and its conjugates is increased in the presence of BSA (see, for example, "Takashi Jin et al., Med. Chem. Commun. 2016, 7, 623-631"). The same is true for the short-wave infrared fluorescent agent in an embodiment of the present invention. That is, it is preferable for the short-wave infrared fluorescent agent in an embodiment of the present invention to contain BSA from the viewpoints of increasing the stability of the compound and conjugate in aqueous solution and increasing the fluorescence emission intensity of short-wave infrared.

[0063] The concentration of the compound or complex of this embodiment in the shortwave infrared fluorescent agent may be any concentration that achieves sufficient fluorescence emission intensity depending on the application of the shortwave infrared fluorescent agent. For example, in the case of biological imaging or molecular imaging, the concentration of the compound or complex of this embodiment in the shortwave infrared fluorescent agent is preferably 0.1 μM or more, more preferably 1 μM or more, and even more preferably 10 μM or more, from the viewpoint of forming a sufficiently clear image in shortwave infrared biological imaging or molecular imaging. Furthermore, in the case of biological imaging or molecular imaging, the concentration of the compound or complex of this embodiment in the shortwave infrared fluorescent agent may be 100 μM or less, 10 μM or less, or 1 μM or less, from the above viewpoint.

[0064] The content of BSA in the short-wave infrared fluorescent agent may be determined appropriately within a range that provides the effect of increasing the fluorescence emission intensity, and from this viewpoint, it may be 1 mg / mL or more and 100 mg / mL or less.

[0065] In addition to BSA, any component that forms micelles with the short-wave infrared fluorescent agent can be expected to have the same effect as when used in combination with BSA. From this perspective, examples of similar components other than BSA include human serum albumin (HSA), phospholipids such as lecithin, and long-chain fatty acids.

[0066] [Application] The shortwave infrared fluorescent agent according to an embodiment of the present invention functions as an excellent optical contrast agent in fluorescent imaging of living organisms using shortwave infrared light in the wavelength range of 900 nm or greater. In fluorescent imaging of living organisms, the wavelength of excitation light and the wavelength of fluorescent detection can be appropriately determined from the ranges described above in the description of the compound. Furthermore, by setting multiple different combinations of excitation wavelengths and fluorescent detection wavelengths, imaging of multiple biological tissues or biomolecules at different depths can be performed with a single administration of the shortwave infrared fluorescent agent to a living organism.

[0067] Furthermore, the short-wave infrared fluorescent agent containing the complex according to the embodiment of the present invention can be easily prepared and can be used as an optical contrast agent for molecular imaging.

[0068] By using the shortwave infrared fluorescent agent according to an embodiment of the present invention as an optical contrast agent, it is possible to non-invasively image blood vessels, including those in the brain, image lymph nodes, and detect cancer tumors with high sensitivity using shortwave infrared light.

[0069] Furthermore, the short-wave infrared fluorescent agent in an embodiment of the present invention can also be used for drug evaluation using antibody-drug conjugates (ADCs), for example, by conjugating a drug component to the molecular recognition agent in the above-mentioned complex.

[0070] When used for biological imaging, the short-wave infrared fluorescent agent can be administered to the subject to be imaged by a known method appropriate for the subject, for example, by direct administration to the area to be imaged or by administration into the blood vessels of the living body, such as by intravenous injection.

[0071] [Action and effect] The compound of formula (1) in an embodiment of the present invention is an ICG analogue whose molecular structure is similar to that of clinically used ICG. ICG is currently used as a pharmaceutical for human retinal angiography, liver function tests (ICG tests), and the like. The compound of formula (1) in this embodiment has a molecular structure similar to that of ICG, and, like ICG, has substantially no cytotoxicity as shown in the examples below, and can be used in the form of an aqueous solution. Furthermore, the compound of formula (1) can be conjugated to an antibody in the same manner as ICG, and can be used in a form suitable for short-wave infrared imaging, similar to conventional ICG.

[0072] Furthermore, quantum dots with a final hydrodynamic diameter of less than 5 nm are known to be rapidly and efficiently excreted in urine and removed from the body (see, for example, "Hak Soo Choil et al., NATURE BIOTECHNOLOGY VOLUME 25 NUMBER 10 OCTOBER 2007"). Therefore, the compound of formula (1) of this embodiment also has a sufficiently high renal clearance that it is substantially excreted outside the body. Thus, the compound of formula (1), which is an analog of ICG, is highly safe for the body.

[0073] In addition, the compound of formula (1) has a sulfone group. Therefore, it is highly water-soluble and dissolves in physiological buffer solutions. Furthermore, because it is a highly water-soluble organic molecule, it is easily excreted from the body when introduced into the body.

[0074] The compound of formula (1) can be excited with light of long wavelengths (900 nm or longer) in the near-infrared region. Furthermore, in aqueous solution, it has sufficient short-wave infrared fluorescence intensity for fluorescent imaging of living organisms. The short-wave infrared wavelength region is known to be weakly absorbed and scattered by biological tissues, and to have weak autofluorescence from biological tissues. Therefore, the compound of formula (1) can realize fluorescent imaging at deeper locations than can be observed with fluorescent imaging of ICG in living organisms.

[0075] Furthermore, because the excitation wavelength of the compound of formula (1) is longer than that of ICG, the absorption of the excitation light by biological tissue is weaker than in the fluorescent imaging of ICG in vivo. Therefore, by applying the compound of formula (1) to fluorescent imaging in vivo, the influence of fluorescent imaging on biological tissue can be further reduced.

[0076] Furthermore, when the compound of formula (1) has a reactive crosslinking group such as an N-hydroxysuccinimide ester group, it can be easily bound to a biomolecule such as an antibody, and can easily form a conjugate as a short-wave infrared fluorescent label. This facilitates the development of an optical contrast agent for molecular imaging, and also enables non-invasive molecular imaging in the short-wave infrared region.

[0077] Furthermore, the compound of formula (1) can be exposed to excitation light for a relatively short time, and therefore, by applying the compound of formula (1) to fluorescent imaging of a living body, the fluorescence of the compound of formula (1) and the detection operation of the living body tissue using the fluorescence can be efficiently performed.

[0078] 〔summary〕 As is clear from the above description, the compound according to the embodiment of the present invention is represented by the aforementioned formula (1). Therefore, a novel compound that enables short-wave infrared biological imaging is provided.

[0079] The reactive crosslinking group in formula (1) may contain one or more organic groups selected from the group consisting of an N-hydroxysuccinimide ester group, a maleimide group, an alkynyl group, and an azide group. This configuration is even more effective from the viewpoint of stably obtaining a conjugate in good yield when the reactive crosslinking group binds to a biological molecular recognition agent such as an antibody to form a conjugate.

[0080] In formula (1), n ​​may be 3 or 4. This configuration is even more effective in terms of achieving sufficient water solubility and sufficient short-wave infrared fluorescence intensity.

[0081] The compound of formula (1) may be a compound represented by any one of the above formulas (2) to (9). This configuration is suitable from the viewpoint of application to short-wave infrared biological imaging and molecular imaging.

[0082] In the conjugate according to an embodiment of the present invention, a molecular recognition agent is bound to a compound according to an embodiment of the present invention via a residue of a reactive crosslinking group in the compound, and the conjugate can be used for short-wave infrared biological imaging and molecular imaging.

[0083] In this complex, the molecular recognition agent may be an antibody or a fragment of the antibody having antigen-binding ability. In one example, the antibody or fragment thereof specifically binds to an antigen contained in the biological tissue that is the target of molecular imaging. This configuration is even more effective from the perspective of achieving molecular imaging of biological tissue using short-wave infrared light.

[0084] A short-wave infrared fluorescent agent according to an embodiment of the present invention contains one or both of a compound according to an embodiment of the present invention and a conjugate according to an embodiment of the present invention, and thus can be used for short-wave infrared biological imaging and molecular imaging.

[0085] A method for producing a compound according to an embodiment of the present invention includes a first step of synthesizing a first compound represented by formula (1a), a second step of replacing the anilino group in formula (1a) with a structure represented by formula (1b), and a third step of replacing the phenylimino group in formula (1a) with a structure represented by formula (1c). Thus, this production method can produce a novel compound (compound of formula (1)) that enables shortwave infrared bioimaging.

[0086] In the method for producing a compound according to an embodiment of the present invention, when n in the formula is 3 and X is a salt of a sulfonic acid group, the second step and the third step may be carried out at the same time. This configuration is more effective from the viewpoint of more easily producing the compound of formula (2) and from the viewpoint of suppressing a decrease in yield due to an increase in the number of steps.

[0087] In the method for producing a compound according to an embodiment of the present invention, when n in the aforementioned formula is 4 and X is a salt of a sulfonic acid group, the third step may be carried out after the second step. This configuration is more effective in increasing the yield of the substitution of the phenylimino group in formula (1a) in the third step in the production of the compound of formula (3).

[0088] In the method for producing a compound according to an embodiment of the present invention, when n in the aforementioned formula is 3 and X is a reactive crosslinking group, the third step may be carried out after the second step. This configuration is also more effective in increasing the yield of the substitution of the phenylimino group in formula (1a) in the third step in the production of the compound of formula (4).

[0089] In the method for producing a compound according to an embodiment of the present invention, when n in the aforementioned formula is 4 and X is a reactive crosslinking group, the third step may be carried out before the second step. This configuration is more effective in increasing the yield in the replacement of the anilino group and the phenylimino group in formula (1a) in the production of the compound of formula (5).

[0090] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention. [Example]

[0091] An embodiment of the present invention will be described below.

[0092] [Reagent purchase source and compound identification method] Commercially available solvents and chemicals used in the following examples were purchased from Sigma-Aldrich, Tokyo Chemical Industry Co., Ltd., and Fujifilm Wako Pure Chemical Industries, Ltd.

[0093] The synthesis reactions in the following examples were monitored by thin-layer chromatography (TLC) on silica gel plates (0.2 mm, Merck 60 F-254). In the following examples, column chromatography was carried out using silica gel (Wakogel (registered trademark of Fujifilm Wako Pure Chemical Industries, Ltd.) N60, spherical, 38-100 μm).

[0094] In the following examples: 1 H NMR (500 MHz) spectra were measured at 25°C using a Varian-Inova-500 NMR instrument (Varian). CDCl3, CD3OD, or DMSO-d6 were used as the solvent. Chemical shifts (δ) are measured in ppm, and bond constants (J) are expressed in hertz (Hz) relative to CDCl3 (δ 7.27), CD3OD (δ 3.31, 4.85), DMSO-d6 (δ 2.50), and tetramethylsilane. The following abbreviations are used to denote signal multiplicities: s = singlet; d = doublet; t = triplet; q = quartet; m = multiplet; dd = doublet-doublet;

[0095] Also, in the following examples, high-resolution mass spectra were obtained using a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific, Massachusetts, USA).

[0096] Example 1: Synthesis of ICG-C9 ICG-C9, a compound of the present invention, was synthesized according to the synthesis scheme shown below.

[0097] [ka]

[0098] [Compound 1] As Compound 1, 1,1,2-trimethyl-1H-benzo[e]indole (Fujifilm Wako Pure Chemical Industries, Ltd.) was prepared.

[0099] [Compound 2] A mixture of 1,1,2-trimethyl-1H-benzo[e]indole (1) (2 g, 9.6 mmol) and 1,4-butanesultone (2.6 g, 19.1 mmol) was stirred at 130 °C for 4 hours. The resulting solid was collected by vacuum filtration, thoroughly washed with acetone, and dried under vacuum. Compound 2 was thus obtained as a pale blue solid (2.85 g, 86% yield). The mass spectrometry (MS) and nuclear magnetic resonance (NMR) analysis results for compound 2 are shown below. High-resolution mass spectrometry (m / z): Molecular formula C 19 H 23 NO3S [MH] + against Calculated value: 344.1344 Actual value: 344.131 1 H NMR (DMSO-d6,500MHz): δ=8.37(1H,d,J=8.6Hz),8.28(1H,d,J=9.2Hz),8.19-8.21(2H,m),7.77(1H,t,J=7.3Hz),7.72(1H,t, J=7.6Hz),4.60(2H,t,J=7.8Hz),2.94(3H,s),2.51-2.53(2H,m),2.03(2H,quintet,J=7.6Hz),1.76-1.80(2H,m),1.75(6H,s)

[0100] [Compound 3] The synthesis scheme of compound 3 is shown below.

[0101] [ka]

[0102] Phosphoryl chloride (8 g, 52 mmol) was added dropwise to a mixture of dimethylformamide (10 mL, 130 mmol) and methanol (0.75 mL) at 0 °C under an inert atmosphere with stirring. The reaction temperature was raised to 50 °C, and 2,4-hexadienal 1 (2.5 g, 26 mmol) was added dropwise. The reaction mixture was stirred at 50 °C for approximately 4 h, cooled to room temperature, added to aqueous sodium perchlorate (5% aqueous solution), and extracted with dichloromethane (80 mL). The organic phase was filtered through sodium sulfate. Aniline (4.8 g, 52 mmol) was added dropwise to the dichloromethane phase with stirring at room temperature, and the mixture was stirred overnight. The resulting precipitate was collected by filtration and washed several times with dichloromethane. Pure compound 3 was thus obtained as a blue solid (6.8 g, 78% yield). Mass spectrometry (MS) and nuclear magnetic resonance (NMR) analysis of compound 3 are shown below. High-resolution mass spectrometry (m / z): Molecular formula C 19 H 18 N2 [M+H] + against Calculated value: 275.1542 Actual value: 275.1534 1 H NMR(CD3OD,500MHz):δ=8.30(2H,d,J=11.2Hz),7.65(2H,t,J=12.6Hz),7.42(4H,t,J=8.3Hz) ,7.31(4H,d,J=7.8Hz),7.22(2H,t,J=7.8Hz),6.49(7H,t,J=11.7Hz),6.27(2H,t,J=11.7Hz)

[0103] [Compound 4 (ICG-C9)] To a stirred solution of compound 2 (0.5 g, 1.45 mmol) in acetic anhydride (10 mL) and compound 3 (215 mg, 0.69 mmol) at room temperature, sodium acetate (200 mg, 2.42 mmol) was added. Acetic acid (4 mL) was added dropwise under an inert atmosphere. The mixture was stirred at 120 °C for 1 h and cooled to room temperature. The reaction mixture was transferred to 30 mL of diethyl ether, and the resulting precipitate was dissolved in 2-propanol:water (4:1, 50 mL), diluted with additional 2-propanol (300 mL), and stored at 4 °C overnight for recrystallization. The precipitate was collected by centrifugation, and the resulting residue was further purified by column chromatography using chloroform / methanol (8:2) as the eluent. Compound 4, ICG-C9, was thus obtained as a brown solid (270 mg, 23% yield). The mass spectrometry (MS) and nuclear magnetic resonance (NMR) analysis results for compound 4 are shown below. High-resolution mass spectrometry (m / z): Molecular formula C 45 H 49 N2O6S2 [M] + against Calculated value: 777.3026 Actual measurement: 777.3005 1 H NMR (DMSO-d6,500MHz): δ=8.22(2H,d,J=8.3Hz),7.89-8.05(6H,m),7.76-7.80(1H,m),7.74(2H,d,J=9.3Hz),7.59-7.66(3H,m),7.42-7.50(3 H,m),6.59(2H,t,J=12.7Hz),6.42-6.51(2H,m),4.18(4H,t,J=6.3Hz) ,1.94-2.02(4H,m),1.90(12H,s),1.71-1.85(8H,m),1.02-1.06(2H,m)

[0104] [Cytotoxicity test of compound 4] HeLa cells were plated in a 96-well plate at 6 × 10 cells per well. 3The cells were seeded in individual wells and cultured overnight. Aqueous solutions of compound 4 at concentrations of 0.1 nM, 1 nM, 10 nM, and 100 nM were prepared as test drugs. Each test drug concentration was added to the wells, and HeLa cells were cultured for an additional 6, 24, or 48 hours. After each incubation period, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reagent was added to the wells, and the cells were cultured for an additional 2 hours.

[0105] After 2 hours of incubation, the solubilization solution was added to the wells, and the formazan produced in the live cells was dissolved by pipetting. The 96-well plate was sealed and placed in a 37°C incubator overnight to completely solubilize the formazan. Measurements were taken at 570 nm (formazan dye) and 650 nm (background) using a plate reader, and the cell viability of wells containing each concentration of test drug was calculated, with the cell viability of wells without test drug being set at 100%. The results are shown in Figure 1. These test results demonstrate that compound 4, at concentrations of 1 to 100 nM, exhibits no cytotoxicity over 6, 24, and 48 hours of incubation.

[0106] Example 2: Synthesis of ICG-C11 ICG-C11, a compound of the present invention, was synthesized according to the synthesis scheme shown below.

[0107] [ka]

[0108] The synthesis scheme of compound 8 is shown below.

[0109] [ka]

[0110] [Compound 5] Triethylphosphonoacetic acid (5.2 g, 22.1 mmol) was added dropwise to a stirred solution of sodium hydride (60% in mineral oil, 833 mg, 20.8 mmol) in tetrahydrofuran at 0 °C under an inert atmosphere. The mixture was stirred at 0 °C for 30 minutes, and then 2,4-hexadienal 1 (2 g, 20.8 mmol) was added dropwise. The reaction was warmed to room temperature, stirred for approximately 2 hours, quenched with ammonium chloride, and extracted with ethyl acetate. The organic phase was dried over sodium sulfate, filtered, and concentrated in vacuo to give a residue. This was purified by column chromatography using hexane / ethyl acetate (9.5:0.5) as the eluent. Compound 5 was thus obtained as a white solid (3.25 g, 94% yield). Mass spectrometry (MS) and nuclear magnetic resonance (NMR) analysis of compound 5 are shown below. High-resolution mass spectrometry (m / z): Molecular formula C 10 H 14 O2 [M+H] + against Calculated value: 167.1066 Actual value: 167.1068 1 H NMR(CDCl3,500MHz):δ=7.31(1H,dd,J=11.3,4.4Hz),6.53(1H,dd,J=10.7,4.4Hz),6.14-6.24(2H,m) ,5.90-5.98(1H,m),5.86(1H,d,J=15.1Hz),4.21(2H,m),1.34(3H,d,J=6.4Hz),1.30(3H,t,J=7.5Hz)

[0111] [Compound 6] Diisobutylaluminum hydride (1.0 M in hexane, 45 mL, 45 mmol) was added dropwise to a stirred dichloromethane solution of compound 5 (2.5 g, 15 mmol) at -60 °C under an inert atmosphere. The reaction was stirred at -60 °C for 30 minutes and then at 0 °C for 1 hour. Water (10 mL) was slowly added, followed by sodium hydroxide (10 mL of a 15% aqueous solution) to quench the reaction, yielding a white precipitate. The mixture was vigorously stirred at room temperature for 1 hour, magnesium sulfate was added, and the mixture was filtered through Celite containing dichloromethane. The organic phase was concentrated in vacuo to give the crude product, which was purified by column chromatography using hexane / ethyl acetate (8:2) as the eluent. Compound 6 was thus obtained as a white solid (1.65 g, 89% yield). Mass spectrometry (MS) and nuclear magnetic resonance (NMR) analysis of compound 6 are shown below. High resolution mass spectrometry (m / z): Molecular formula C8H 10 O [M+H-HO] + against Calculated value: 107.0855 Actual measurement: 107.0860 1 H NMR(CDCl3,500MHz):δ=6.20-6.28(2H,m),6.06-6.14(2H,m),5.71-5.83(2H,m),4.20(2H,d,J=5.8Hz),1.79(2H,d,J=6.8Hz),1.40(1H,s)

[0112] [Compound 7] Activated manganese oxide (7.4 g, 84.7 mmol) was added to a solution of compound 6 (1.5 g, 12.1 mmol) in dichloromethane (30 mL) at room temperature under an inert atmosphere, and the reaction mixture was stirred overnight. The reaction mixture was filtered, and the filtrate was concentrated to obtain the crude product, which was purified by column chromatography using hexane / ethyl acetate (9:1) as an eluent. Compound 7 was thus obtained as a yellow solid (1.08 g, 73% yield). The mass spectrometry (MS) and nuclear magnetic resonance (NMR) analysis results for compound 7 are shown below. High resolution mass spectrometry (m / z): Molecular formula C8H 10 O[M+H] + against Calculated value: 123.0804 Actual measurement: 123.0804 1 H NMR(CDCl3,500MHz):δ=9.58(1H,d,J=9.8Hz),7.12(1H,dd,J=11.3,3.9Hz),6.65(1H,dd,J=10.3,4.4Hz), 6.34(1H,dd,J=10.7,4.0Hz),6.21(1H,m),6.14(1H,dd,J=7.8,7.3Hz),6.05(1H,m),1.87(3H,d,J=7.8Hz)

[0113] [Compound 8] Phosphoryl chloride (2.5 g, 16.4 mmol) was added dropwise to a solution of dimethylformamide (3.2 mL, 42.5 mmol) and methanol (0.4 mL) at 0 °C under an inert atmosphere with stirring. The reaction temperature was raised to 40 °C, and a solution of compound 7 (1 g, 8.2 mmol) in dimethylformamide (0.5 mL) was added dropwise. The reaction mixture was stirred at 40 °C for approximately 4 hours. The reaction mixture was cooled to room temperature (RT), added to aqueous sodium perchlorate (1 g in 20 mL), and extracted with dichloromethane (25 mL). The organic phase was filtered through a layer of sodium sulfate. Aniline (1.5 g, 16.4 mmol) was added dropwise to the stirred dichloromethane phase at room temperature and stirred overnight. The resulting precipitate was collected by filtration and washed several times with dichloromethane. Compound 8 was thus obtained as a green solid (1.25 g, 45% yield). The results of mass spectrometry (MS) and nuclear magnetic resonance (NMR) of compound 8 are shown below. High-resolution mass spectrometry (m / z): Molecular formula C 21 H 20 N2 [M+H] + against Calculated value: 301.1699 Actual measurement: 301.1699 1H NMR(CD3OD,500MHz):δ=8.21(2H,d,J=11.2Hz),7.52(2H,t,J=13.2Hz),7.4(4H,t,J=8.3Hz),7.367(1H,d ,J=12.7Hz),7.27(4H,d,J=7.8Hz),7.18(2H,t,J=7.3Hz),6.47(2H,t,J=13.2Hz),6.26(2H,t,J=11.7Hz)

[0114] [Compound 9] To a solution of compound 2 (250 mg, 0.72 mmol) and sodium acetate (178 mg, 2.17 mmol) in ethanol (10 mL) at room temperature, compound 8 (244 mg, 0.72 mmol) was added while stirring the solution. Acetic anhydride (0.4 mL, 4.34 mmol) was then added dropwise under an inert atmosphere. The reaction was continued for 30 minutes, and the solvent was removed in vacuo. The residue was purified by column chromatography using chloroform / methanol (9:1) as an eluent. Compound 9 was thus obtained as a golden-brown solid (274 mg, 64% yield). The mass spectrometry (MS) and nuclear magnetic resonance (NMR) analysis results for compound 9 are shown below. High-resolution mass spectrometry (m / z): Molecular formula C 36 H 38 N2O4S [M+H] + against Calculated value: 595.2625 Actual value: 595.2617 1H NMR(CD3OD,500MHz):δ=8.35(1H,d,J=7.3Hz),8.20-8.25(2H,m),8.11(1H,d,J=8.3Hz),7.86-7.94(2H,m),7. 76(1H,t,J=7.3Hz),7.65(1H,t,J=7.8Hz),7.53-7.61(3H,m),7.28-7.36(3H,m),7.12(1H,d,J=14.6Hz),6.86 (2H,q,J=14.1Hz),6.69(1H,t,J=11.2Hz),6.49(1H,t,J=13.6Hz),6.20(1H,t,J=11.7Hz),5.23(2H,t,J=13.7 Hz),4.59(2H,t,J=7.3Hz),2.9(2H,t,J=7.3Hz),2.14(2H,quintet,J=7.8Hz),2.00(6H,s),1.89-1.98(2H,m)

[0115] [Compound 10 (ICG-C11)] To a solution of compound 2 (29 mg, 0.084 mmol) and sodium acetate (12 mg, 0.139 mmol) in ethanol (3 mL) at room temperature, acetic anhydride (9 μL, 0.092 mmol) was added while stirring the solution. Compound 9 (50 mg, 0.084 mmol) dissolved in ethanol (3 mL) was added to the reaction mixture under an inert atmosphere. The mixture was stirred at 60 °C for 1 hour. The solvent was removed in vacuo, and the residue was purified by column chromatography using chloroform / methanol (7.5:2.5). Compound 10, ICG-C11, was thus obtained as a brown solid (33 mg, 48% yield). The mass spectrometry (MS) and nuclear magnetic resonance (NMR) analysis results of compound 10 are shown below. High-resolution mass spectrometry (m / z): Molecular formula C 47 H 51 N2O6S2 [M] + against Calculated value: 803.3183 Actual measurement: 803.3221 1H NMR (DMSO-d6,500MHz): δ=8.21(2H,d,J=8.3Hz),8.01(4H,d,J=7.3Hz),7.81( 2H,t,J=13.1Hz),7.71(2H,t,J=8.8Hz),7.61(2H,t,J=6.8Hz),7.47(2H,t,J= 7.3Hz),7.34(2H,t,J=12.7Hz),7.01(1H,t,J=12.7Hz),6.48-6.57(4H,m),6. 44(2H,d,J=13.6Hz),4.17(4H,t,J=6.8Hz),1.88(12H,s),1.75-1.81(12H,m)

[0116] [Cytotoxicity test of compound 10] HeLa cells were cultured and cell viability was measured in the same manner as in the cytotoxicity test for compound 4, except that compound 10 was used instead of compound 4 to prepare the test drug. The results are shown in Figure 2. The test results revealed that compound 10, like compound 4, did not exhibit cytotoxicity at concentrations of 1 to 100 nM under culture conditions of 6, 24, and 48 hours.

[0117] Example 3: Synthesis of ICG-C9-NHS ICG-C9-NHS, a compound of the present invention, was synthesized according to the synthesis scheme shown below.

[0118] [ka]

[0119] [Compound 11] To a mixture of compound 2 (1 g, 2.89 mmol), sodium acetate (712 mg, 8.68 mmol), and ethanol (25 mL) at room temperature, compound 3 (900 mg) was added while stirring. Acetic anhydride (1.7 mL, 17.37 mmol) was then added dropwise under an inert atmosphere. The reaction was continued for 30 minutes, the solvent was removed in vacuo, and the residue was purified by column chromatography using chloroform / methanol (9:1) as an eluent. Compound 11 was thus obtained as a golden-brown solid (1.12 g, 68% yield). The mass spectrometry (MS) and nuclear magnetic resonance (NMR) analysis results for compound 11 are shown below. High-resolution mass spectrometry (m / z): Molecular formula C 34 H 36 N2OS's [M+H] + against Calculated value: 569.2468 Actual value: 569.2481 1 H NMR(CD3OD,500MHz):δ=8.35(1H,d,J=8.3Hz),8.22(1H,dd,J=11.25,3.4Hz),8.18(1H,d,J=8.8Hz),8.11(1H,d,J=8.3Hz ),8.00-8.04(1H,m),7.93(1H,d,J=8.7Hz),7.76(1H,t,J=7.3Hz),7.56-7.67(4H,m),7.31-7.36(3H,m),7.07(1H,d,J=1 5.1Hz),6.95(1H,dd,J=11.3Hz,3.0Hz),6.75(1H,dd,J=11.3,3.0Hz),6.33(1H,dd,J=11.7,2.5Hz),5.30(1H,dd,J=11.7 Hz,2.0Hz),4.58(2H,t,J=7.8Hz),2.90(2H,t,J=7.3Hz),2.10-2.16(2H,m),2.00(6H,s),1.98(3H,s),1.95-1.97(2H,m)

[0120] [Compound 12] The synthesis scheme of compound 12 is shown below.

[0121] [ka]

[0122] A solution of 1,1,2-trimethyl-1H-benzo[e]indole (compound 1) (2.5 g, 11.9 mmol) and 6-bromohexanoic acid (7 g, 35.8 mmol) in acetonitrile (100 mL) was stirred at 90 °C under an inert atmosphere for 2 days. The solvent was removed in vacuo, and the residue was dissolved in dichloromethane (25 mL). The product was precipitated by dilution with diethyl ether, filtered, and washed with diethyl ether. Compound 12 was thus obtained as a pale green solid (3.8 g, 79% yield). Mass spectrometry (MS) and nuclear magnetic resonance (NMR) analysis of compound 12 are shown below. High-resolution mass spectrometry (m / z): Molecular formula C 21 H 26 NO2 [M] + against Calculated value: 324.1958 Actual measurement: 324.1949 1 H NMR (DMSO-d6,500MHz): δ=8.38(1H,d,J=8.8Hz),8.29(1H,d,J=8.8Hz),8.22(1H,d,J=7.9Hz),8.15(1H,d,J=8.8Hz),7.78(1H,t,J=7.4Hz),7.7 3(1H,t,J=6.9Hz),4.57(2H,t,J=7.8Hz),2.93(3H,s),2.23(2H,t,J=7. 3),1.87-1.93(2H,m),1.75(6H,s),1.54-1.60(2H,m),1.43-1.49(2H,m)

[0123] [Compound 13] To a mixture of compound 12 (164 mg, 0.405 mmol), sodium acetate (55 mg, 0.673 mmol), and ethanol (8 mL) at room temperature, acetic anhydride (43 μL, 0.446 mmol) was added while stirring. Compound 11 (230 mg, 0.405 mmol) dissolved in ethanol (10 mL) was added to the reaction mixture under an inert atmosphere. The mixture was stirred at 60 °C for 1 hour. The solvent was removed in vacuo, and the residue was purified by column chromatography using chloroform / methanol (8:2) as an eluent. Compound 13 was thus obtained as a dark green solid (234 mg, 76% yield). The mass spectrometry (MS) and nuclear magnetic resonance (NMR) analysis results for compound 13 are shown below. High-resolution mass spectrometry (m / z): Molecular formula C 47 H 52 N2O5S [M+H] + against Calculated value: 757.3669 Actual value: 757.3666 1 H NMR(CD3OD,500MHz):δ=8.20(2H,t,J=7.8Hz),7.85-8.00(7H,m),7.58-7.65(3H,m),7.52(1H,d,J=9.3H z),7.34-7.48(4H,m),6.50-6.64(3H,m),6.42(1H,d,J=13.6Hz),6.25(1H,d,J=13.1Hz),4.24(2H,t,J= 6.3Hz),4.16(2H,t,J=6.8Hz),3.61(1H,q,t,J=6.8Hz),2.92(2H,t,J=6.8Hz),2.29(2H,t,J=7.3Hz),1. 99-2.07(4H,m),1.86(2H,quintet,J=7.3Hz),1.71(2H,quintet,J=7.3Hz),1.53(2H,quintet,J=7.3Hz)

[0124] [Compound 14(ICG-C9-NHS)] Under an inert atmosphere, a solution of N,N'-dicyclohexylcarbodiimide (136 mg, 0.660 mmol) in chloroform (1 mL) was added dropwise to a stirred solution of compound 13 (200 mg, 0.264 mmol) and N-hydroxysuccinimide (76 mg, 0.660 mmol) in chloroform (1 mL) under stirring. The reaction mixture was stirred at room temperature for 4 hours. The solvent was removed in vacuo, and the residue was purified by column chromatography using chloroform / methanol (9:1) as an eluent. Compound 14, ICG-C9-NHS, was thus obtained as a dark green solid (125 mg, 55% yield). Mass spectrometry (MS) and nuclear magnetic resonance (NMR) analysis of compound 14 are shown below. High-resolution mass spectrometry (m / z): Molecular formula C 51 H 55 N3O7S [M+H] + against Calculated value: 854.3833 Actual value: 854.3828 1 H NMR(CD3OD,500MHz):δ=8.21(2H,t,J=7.3Hz),7.96(6H,m),7.62(3H,m),7.53(1H,d,J=8. 8Hz),7.34-7.53(4H,m),6.50-6.65(3H,m),6.42(1H,d,J=13.6Hz),6.27(1H,d,J=13.1Hz ),4.24(2H,t,J=6.3Hz),1.46(2H,t,J=7.3Hz),2.92(2H,t,J=7.3Hz),2.65-2.70(2H,m), 2.00-2.07(4H,m),1.97(12H,s),1.82-1.92(6H,m),1.61-1.73(2H,m),1.39-1.45(2H,m)

[0125] Example 4: Synthesis of ICG-C11-NHS ICG-C11-NHS, a compound of the present invention, was synthesized according to the synthesis scheme shown below.

[0126] [ka]

[0127] [Compound 15] To a solution of compound 12 (250 mg, 0.618 mmol) and sodium acetate (101 mg, 1.236 mmol) in ethanol (15 mL) at room temperature, compound 8 (208 mg, 0.618 mmol) was added while stirring. Acetic anhydride (175 μL, 1.855 mmol) was then added dropwise under an inert atmosphere. The reaction was continued for 30 minutes, and the solvent was removed in vacuo. The residue was purified by column chromatography using chloroform / methanol (9:1) as an eluent. Compound 15 was thus obtained as a golden-brown solid (310 mg, 77% yield). Mass spectrometry (MS) and nuclear magnetic resonance (NMR) analysis of compound 15 are shown below. High-resolution mass spectrometry (m / z): Molecular formula C 38 H 41 N2O3 [M] + against Calculated value: 573.3111 Actual value: 573.3108 1 H NMR(CD3OD,500MHz):δ=8.36(1H,d,J=8.3Hz),8.25(1H,dd,J=11.2,4.0Hz),8.19(1H,d,J=8.8Hz),8.12(1H,d,J=8.3Hz),7.89(2H,m),7.77(1H ,t,J=7.3Hz),7.67(1H,t,J=7.3Hz),7.60(2H,j,J=7.4Hz),7.54(1H,t,J=7.8Hz),7.36(1H,dd,J=11.8,2.4Hz),7.30(2H,d,J=7.4Hz),7.04(1H, d,J=15.1Hz),6.79-6.90(2H,m),6.71(1H,dd,J=11.3,3.4Hz),6.50(1H,dd,J=11.3,3.0Hz),6.21(1H,dd,J=11.3,3.4Hz),5.23(1H,dd,J=11.3 ,3.0Hz),4.56(2H,t,J=7.3Hz),2.29(2H,t,J=7.3Hz)2.01(6H,s),1.92-1.97(5H,m),1.69(2H,quintet,J=6.8Hz),1.54(2H,quintet,J=7.3Hz)

[0128] [Compound 16] To a solution of compound 15 (110 mg, 0.168 mmol) and N-hydroxysuccinimide (48 mg, 0.42 mmol) in chloroform (10 mL) was added dropwise a solution of N,N'-dicyclohexylcarbodiimide (86 mg, 0.42 mmol) in chloroform (1 mL) under an inert atmosphere at 0 °C while stirring. The reaction mixture was stirred at room temperature for 2 hours. The solvent was removed in vacuo, and the residue was purified by column chromatography using chloroform / methanol (9.5:0.5) as an eluent. Compound 16 was thus obtained as a brown solid (88 mg, 70% yield). The mass spectrometry (MS) and nuclear magnetic resonance (NMR) analysis results for compound 16 are shown below. High-resolution mass spectrometry (m / z): Molecular formula C 42 H 44 N3O5 [M] + against Calculated value: 670.3275 Actual value: 670.3272 1 H NMR(CD3OD,500MHz):δ=8.36(1H,d,J=8.3Hz),8.24(1H,t,J=11.7Hz),8.18(1H,d,J=9.3Hz),8.12(1H,d,J=7.8Hz),7.87- 7.89(2H,m),7.77(1H,t,J=7.3Hz),7.67(1H,t,J=7.8Hz),7.54-7.61(4H,m),7.29-7.38(3H,m),7.04(1H,d,J=14.6Hz),6 .78-6.90(2H,m),6.72(1H,t,J=13.6Hz),6.50(H,t,J=11.2Hz),6.21(1H,t,J=14.1Hz),5.24(1H,t,J=11.7Hz),4.57(2H, t,J=6.3Hz),2.76-2.84(4H,m),2.01(6H,s),1.89-1.97(4H,m),1.85(2H,quintet,J=6.8Hz),1.64(2H,quintet,J=5.0Hz)

[0129] [Compound 17(ICG-C11-NHS)] To a solution of compound 2 (55 mg, 0.160 mmol) and sodium acetate (13 mg, 0.160 mmol) in ethanol (3 mL) at room temperature, acetic anhydride (15 μL, 0.160 mmol) was added while stirring the solution. Compound 16 (80 mg, 0.106 mmol) dissolved in ethanol (5 mL) was added to the reaction mixture under an inert atmosphere. The mixture was stirred at 60 °C for 1 hour. The solvent was removed in vacuo, and the residue was purified by column chromatography using chloroform / methanol (9:1) as an eluent. Compound 17, ICG-C11-NHS, was thus obtained as a dark green solid (42 mg, 45% yield). The mass spectrometry (MS) and nuclear magnetic resonance (NMR) analysis results of compound 17 are shown below. High-resolution mass spectrometry (m / z): Molecular formula C 53 H 57 N3O7S [M+H] + against Calculated value: 880.3990 Actual value: 880.3985 1 H NMR (DMSO-d6,500MHz): δ=8.25(1H,d,J=8.3Hz),8.18(1H,d,J=8.8Hz),8.06(2H,t,J=8.8Hz),7.99(2H,d,J=8.8Hz),7.92(1H,t,J=12.7Hz),7 .80(1H,d,J=8.8Hz),7.71(1H,t,J=12.7Hz),7.65(1H,t,J=7.3Hz),7.60(2H,t,J=8.8Hz),7.51(1H,t,J=7.8Hz),7.37-7.44(2H,m),7.27(1H, t,J=12.7Hz),7.01(1H,t,J=13.1Hz),6.45-6.64(5H,m),6.22(1H,d,J=13.7Hz),4.25(2H,t,J=6.8Hz),4.09(2H,t,J=7.3Hz),2.80(4H,s),2. 68(2H,t,J=6.8Hz),1.90(6H,s),1.88(6H,s),1.82-1.86(3H,m),1.68-1.79(7H,m),1.50(2H,quintet,J=7.3Hz),1.40(2H,quintet,J=6.8Hz)

[0130] [Other synthesis methods for ICG-C11-NHS] ICG-C11-NHS can also be synthesized according to the synthesis scheme shown below. Other synthesis methods for ICG-C11-NHS will be specifically described below.

[0131] [ka]

[0132] [Compound 18] Acetic anhydride (53 μL, 0.555 mmol) was added to a solution of compound 2 (175 mg, 0.505 mmol) and sodium acetate (21 mg, 0.838 mmol) in ethanol (10 mL) at room temperature. Compound 15 (330 mg, 0.505 mmol) dissolved in ethanol (10 mL) was then added to the reaction mixture under an inert atmosphere, and the mixture was stirred at 60 °C for approximately 1.5 hours. The solvent was removed in vacuo, and the residue was purified by column chromatography using chloroform / methanol (8:2) as an eluent. Compound 18 was thus obtained as a dark brown solid (127 mg, 64%). Mass spectrometry (MS) and nuclear magnetic resonance (NMR) analysis of compound 18 are shown below. High-resolution mass spectrometry (m / z): Molecular formula C 49 H 54 N2O5S [M+H] + against Calculated value: 783.3826 Actual value: 783.3817 1H NMR (DMSO-d6,500MHz): δ=8.25(1H,d,J=8.8Hz),8.19(1H,d,J=8.8Hz),8.06(2H,t,J=8.3Hz),8.00(2H,d,J=8.8Hz),7.92(1H,t,J=12.7Hz ),7.80(1H,d,J=8.8Hz),7.71(1H,t,J=12.7Hz),7.65(1H,t,J=7.3Hz),7.60(2H,t,J=8.8Hz),7.51(1H,t,J=7.3Hz),7.37-7.44(2H,m),7.2 7(1H,t,J=12.7Hz),7.01(1H,t,J=12.7Hz),6.45-6.63(5H,m),6.22(1H,d,J=13.1Hz),4.24(2H,t,J=6.3Hz),4.09(2H,t,J=6.8Hz),2.14( 2H,t,J=6.8Hz),1.90(6H,s),1.88(6H,s),1.82-1.84(2H,m),1.68-1.79(4H,m),1.55(2H,quintet,J=7.3Hz),1.40(2H,quintet,J=6.8Hz)

[0133] [Compound 17 (ICG-C11-NHS)] To a stirred solution of compound 18 (53 mg, 0.0677 mmol) and N-hydroxysuccinimide (20 mg, 0.169 mmol) in chloroform (8 mL) at 0 °C under an inert atmosphere, a solution of N,N'-dicyclohexylcarbodiimide (35 mg, 0.169 mmol) in chloroform (1 mL) was added dropwise, and the reaction mixture was stirred at room temperature for 4 h. The solvent was removed in vacuo, and the residue was purified by column chromatography using chloroform / methanol (9:1) as an eluent. ICG-C11-NHS (compound 17) was thus obtained as a dark brown solid (31 mg, 52%). Mass spectrometry (MS) and nuclear magnetic resonance (NMR) analysis of the resulting compound 17 were as previously described.

[0134] Example 5: Synthesis of ICG-C9-maleimide ICG-C9-maleimide, a compound of the present invention, was synthesized according to the synthesis scheme shown below.

[0135] [ka]

[0136] [Compound 19] To a stirred solution of compound 13 (60 mg, 0.079 mmol) in dichloromethane (10 mL) at room temperature under an inert atmosphere, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) (75 mg, 0.198 mmol), N,N-diisopropylethylamine (DIPEA) (31 mg, 0.237 mmol), and 1-(2-aminoethyl)maleimide hydrochloride (21 mg, 0.119 mmol) were added. The reaction mixture was stirred at room temperature for approximately 4–5 h. The solvent was removed in vacuo, and the residue was purified by column chromatography using chloroform / methanol (9:1) to give compound 19 (45 mg, 64%) as a dark brown solid. Mass spectrometry (MS) and nuclear magnetic resonance (NMR) analyses of compound 19 are shown below. High resolution mass spectrometry (m / s): Molecular formula C 53 H 58 N4O6S [M+H] + against Calculated value: 879.4149 Actual value: 879.4121 1H-NMR (DMSO-d6,500MHz):δ=8.56(1H,d,J=3.4Hz),8.36(1H,d,J=8.3Hz),8.22(2H,dd,J=17.5,8.3Hz),7.99-8.06(4H,m),7.85-7.88(2H,m), 7.78(1H,d,J=8.8Hz),7.59-7.65(3H,m),7.43-7.51(3H,m),7.34-7.37(1H,m),6.46-6.64(4H,m),6.31(1H,d,J=13.2Hz),4.22(2H,t,J =6.3Hz),4.12(2H,t,J=6.8Hz),3.58(1H,s),3.38(2H,t,J=5.8Hz),3.15(2H,t,J=5.3Hz),1.98(2H,t,J=4.9Hz),1.90(6H,s),1.89(6H, s),1.84(2H,q,J=7.3Hz),1.77(2H,q,J=6.8Hz),1.69(2H,quintet,J=7.3Hz),1.51(2H,quintet,J=7.3Hz),1.34(2H,quintet,J=7.3Hz)

[0137] Example 6: Synthesis of ICG-C9-alkyne ICG-C9-alkyne, a compound of the present invention, was synthesized according to the synthesis scheme shown below.

[0138] [ka]

[0139] [Compound 20] To a stirred solution of compound 13 (60 mg, 0.079 mmol) in CHCl (10 mL) at room temperature under an inert atmosphere, HATU (75 mg, 0.198 mmol), DIPEA (31 mg, 0.238 mmol), and propargylamine (9 mg, 0.159 mmol) were added. The reaction mixture was stirred at room temperature for approximately 4–5 h. The solvent was removed in vacuo, and the residue was purified by column chromatography using chloroform / methanol (9:1) to give compound 20 (43 mg, 68%) as a dark brown solid. Mass spectrometry (MS) and nuclear magnetic resonance (NMR) analyses of compound 20 are shown below. High resolution mass spectrometry (m / s): Molecular formula C 53 H 58 N4O6S [M+H] + against Calculated value: 793.3907 Actual value: 793.3790 1 H NMR (DMSO-d6,500MHz): δ=8.68(1H,d,J=4.3Hz),8.48(1H,d,J=8.3Hz),8.19-8.24(3H,m),8.00-8.06(4H,m),7.88(1H, t,J=13.2),7.78(1H,d,J=8.8Hz),7.60-7.65(3H,m),7.44-7.51(3H,m),6.50-6.64(3H,m),6.31(1H,d,J=13.6Hz),4.22 (2H,t,J=6.3Hz),4.13(2H,t,J=6.8Hz),3.80-3.82(2H,m),3.06(1H,s),2.08(2H,t,J=7.3Hz),1.90(6H,s),1.89(6H,s) ,1.84(2H,q,J=6.8Hz),1.68-1.79(4H,m),1.56(2H,quintet,J=7.3Hz),1.38(2H,quintet,J=6.8Hz),1.22-1.25(2H,m)

[0140] Example 7: Synthesis of ICG-C11-maleimide ICG-C11-maleimide, a compound of the present invention, was synthesized according to the synthesis scheme shown below.

[0141] [ka]

[0142] [Compound 21] To a stirred solution of compound 18 (50 mg, 0.064 mmol) in dichloromethane (10 mL) at room temperature under an inert atmosphere, HATU (60 mg, 0.160 mmol), DIPEA (25 mg, 0.192 mmol), and 1-(2-aminoethyl)maleimide hydrochloride (17 mg, 0.10 mmol) were added. The reaction mixture was stirred at room temperature overnight. The solvent was removed in vacuo, and the residue was purified by column chromatography using chloroform / methanol (9:1) to give compound 21 (32 mg, 55%) as a dark green solid. The mass spectrometry (MS) and nuclear magnetic resonance (NMR) analysis results of compound 21 are shown below. High resolution mass spectrometry (m / s): Molecular formula C 53 H 58 N4O6S [M+H] + against Calculated value: 905.4306 Actual value: 905.4295 1H NMR (DMSO-d6,500MHz): δ=8.94(2H,s),8.24(1H,d,J=8.3Hz),8.18(1H,d,J=8.8Hz),8.05(2H,t,J=8.3Hz),7.97-8.00(2H,m),7.87-7.90(2H, m),7.79(1H,d,J=8.8Hz),7.57-7.75(5H,m),7.50(1H,t,J=7.3Hz),7.3 6-7.44(2H,m),7.28(1H,t,J=12.7Hz),6.98(2H,s),6.46-6.62(4H,m), 6.25(1H,d,J=13.7Hz),4.23(2H,t,J=7.3Hz),4.09(2H,t,J=5.3Hz),3.38(2H,t,J=5.8Hz),3.11-3.15(2H,m),1.97(2H,t,J=7.3Hz),1.89(6H, s),1.87(6H,s),1.81-1.86(2H,m),1.76(2H,quintet,J=6.8Hz),1.68(2H,quintet,J=6.8Hz),1.50(2H,quintet,J=7.3Hz),1.32-1.37(2H,m)

[0143] Example 8: Synthesis of ICG-C11-alkyne ICG-C11-alkyne, a compound of the present invention, was synthesized according to the synthesis scheme shown below.

[0144] [ka]

[0145] [Compound 22] To a stirred solution of compound 18 (50 mg, 0.064 mmol) in dichloromethane (10 mL) at room temperature under an inert atmosphere, HATU (61 mg, 0.160 mmol), DIPEA (25 mg, 0.192 mmol), and propargylamine (7 mg, 0.128 mmol) were added. The reaction mixture was stirred at room temperature overnight. The solvent was removed in vacuo, and the residue was purified by column chromatography using chloroform / methanol (9:1) to give compound 22 (30 mg, 58%) as a dark green solid. The mass spectrometry (MS) and nuclear magnetic resonance (NMR) analysis results of compound 22 are shown below. High resolution mass spectrometry (m / s): Molecular formula C 53 H 58 N4O6S [M+H] + against Calculated value: 920.4142 Actual measurement: 920.4130 1 H NMR (DMSO-d6,500MHz):δ=8.17-8.24(3H,m),8.05(2H,t,J=7.8Hz),8.00(2H,d,J=8.3Hz),7.90(1H,t,J=13.2Hz),7.70-7.79(2H,m) ,7.58-7.66(3H,m),7.50(1H,t,J=7.3Hz),7.43(1H,t,J=7.3Hz),7.28(1H,t,J=13.2Hz),7.01(1H,t,J=12.7),6.46-6.62(4H,m),6.2 4(1H,d,J=13.2Hz),4.23(2H,t,J=6.8Hz),4.09(2H,t,J=5.3Hz),3.80-3.82(2H,m),3.06(1H,s),2.91(2H,s),2.08(2H,t,J=7.3Hz), 1.89(6H,s),1.87(6H,s),1.81-1.84(2H,m),1.76(2H,quintet,J=7.3Hz),1.70(2H,quintet,J=6.8Hz),1.37(2H,quintet,J=6.8Hz)

[0146] [Examples 9 and 10] [Preparation of short-wave infrared fluorescent agents 1 and 2] 1 mg of compound 4 (ICG-C9) was dissolved in 1 mL of dimethyl sulfoxide (Fujifilm Wako Pure Chemical Industries, Ltd.). Next, 0.1 mL of this solution was added to 0.9 mL of 1% by weight bovine serum albumin (Sigma-Aldrich). 1 mL of this solution was dialyzed (spectrum dialysis, MWCO: 50 kDa) against distilled water. This yielded an aqueous solution of a fluorescent probe (1 mg / mL, 1% bovine serum albumin) for imaging blood vessels and lymph nodes. This aqueous solution is designated short-wave infrared fluorescent agent 1.

[0147] Further, short-wave infrared fluorescent agent 2 was prepared in the same manner as short-wave infrared fluorescent agent 1, except that compound 10 (ICG-C11) was used instead of compound 4.

[0148] [Imaging of blood vessels and lymph nodes using short-wave infrared fluorescent agents 1 and 2] <Imaging of blood vessels in the lower limbs and abdomen of mice> A hairless mouse (male, 5 weeks old, Hos:HR-1, Japan SLC Co., Ltd.) was anesthetized with isoflurane (Fujifilm Wako Pure Chemical Industries, Ltd.) and 0.2 mL of short-wave infrared fluorescent agent 1 was injected into its tail vein. Short-wave infrared fluorescent imaging of blood vessels in the lower limbs and abdomen of the hairless mouse was then performed.

[0149] Shortwave infrared fluorescence imaging of blood vessels in the lower limbs and abdomen of hairless mice was also performed in the same manner as for shortwave infrared fluorescence imaging with shortwave infrared fluorescent agent 1, except that shortwave infrared fluorescent agent 2 was used instead of shortwave infrared fluorescent agent 1. Fluorescence images were taken using a cooled InGaAs shortwave infrared camera (C10633-34, Peltier-cooled + water-cooled -70°C, dark current 132 electrons / pixel / s) manufactured by Hamamatsu Photonics K.K.

[0150] In the short-wave infrared fluorescence imaging using short-wave infrared fluorescent agent 1, the excitation wavelength was 905 nm and the fluorescence detection wavelength was 1000 nm. In the short-wave infrared fluorescence imaging using short-wave infrared fluorescent agent 2, the excitation wavelength was 975 nm and the fluorescence detection wavelength was 1100 nm. In both short-wave infrared fluorescence imaging, the excitation light intensity was 20 to 40 mW / cm. 2The exposure time was 5 to 10 seconds.

[0151] Figure 3 shows bright-field images of the lower limbs and abdomen of a hairless mouse, as well as photographs showing short-wave infrared fluorescence images obtained with short-wave infrared fluorescent agent 1. Figure 4 shows bright-field images of the lower limbs and abdomen of a hairless mouse, as well as photographs showing short-wave infrared fluorescence images obtained with short-wave infrared fluorescent agent 2. As shown in Figures 3 and 4, both short-wave infrared fluorescent agent 1 and short-wave infrared fluorescent agent 2 can form clear short-wave infrared images of blood vessels in the limbs and abdomen. More specifically, the short-wave infrared fluorescence images obtained with short-wave infrared fluorescent agent 1 and short-wave infrared fluorescent agent 2 are both clear, with strong fluorescent signal intensity and little image blurring. Thus, short-wave infrared fluorescent agent 1 and short-wave infrared fluorescent agent 2 enable not only practical but also practically excellent short-wave infrared fluorescence imaging of living organisms.

[0152] <Imaging of cerebral blood vessels in mice> Hairless mice (male, 5 weeks old, Hos:HR-1, Japan SLC Co., Ltd.) were anesthetized with isoflurane (Fujifilm Wako Pure Chemical Industries, Ltd.) and 0.2 mL of shortwave infrared fluorescent agent 1 was injected into their tail vein. Shortwave infrared fluorescence imaging of the cerebral blood vessels of the hairless mice was then performed. Fluorescence images were taken using a cooled InGaAs shortwave infrared camera (C10633-34, Peltier-cooled + water-cooled, -70°C, dark current 132 electrons / pixel / s, manufactured by Hamamatsu Photonics K.K.).

[0153] In addition, short-wave infrared fluorescence imaging of the cerebral blood vessels of hairless mice was performed in the same manner as short-wave infrared fluorescence imaging using short-wave infrared fluorescent agent 1, except that short-wave infrared fluorescent agent 2 was used instead of short-wave infrared fluorescent agent 1.

[0154] Furthermore, for comparison, shortwave infrared fluorescence imaging of the cerebral blood vessels of hairless mice was performed in the same manner as the shortwave infrared fluorescence imaging using shortwave infrared fluorescent agent 1, except that shortwave infrared fluorescent agent C1 was used instead of shortwave infrared fluorescent agent 1. Shortwave infrared fluorescent agent C1 is a fluorescent agent prepared in the same manner as shortwave infrared fluorescent agent 1, except that ICG was used instead of compound 4.

[0155] In the short-wave infrared fluorescence imaging using short-wave infrared fluorescent agent 1, the excitation wavelength was 905 nm and the fluorescence detection wavelength was 1000 nm. The excitation light intensity was 10 mW / cm. 2 and the exposure time was 1 second.

[0156] For short-wave infrared fluorescence imaging using short-wave infrared fluorescent agent 2, the excitation wavelength was 975 nm and the fluorescence detection wavelength was 1100 nm. The excitation light intensity was 20–40 mW / cm. 2 The exposure time was 1 to 2.5 seconds.

[0157] For short-wave infrared fluorescence imaging using the short-wave infrared fluorescent agent C1, the excitation wavelength was 758 nm and the fluorescence detection wavelength was 900 nm. The excitation light intensity was 5 mW / cm. 2 and the exposure time was 1 second.

[0158] Figure 5 shows photographs representing bright-field images of the top of the head of a hairless mouse and short-wave infrared fluorescence images using short-wave infrared fluorescent agents C1, 1, and 2. As shown in Figure 5, short-wave infrared fluorescent agents 1 and 2 are able to form clearer, more detailed images of the short-wave infrared light of the cerebral blood vessels of a hairless mouse that can be observed externally, compared to short-wave infrared fluorescent agent C1 containing ICG.

[0159] Examples 11 and 12 [Preparation of short-wave infrared fluorescent agents 3 and 4] Shortwave infrared fluorescent agents 3 and 4 were prepared. In shortwave infrared fluorescent agent 3, compound 14 (ICG-C9-NHS) is bound to bovine serum albumin (BSA) via an amide group containing a carbonyl group, which is the residue of a reactive crosslinking group. In shortwave infrared fluorescent agent 4, compound 17 (ICG-C11-NHS) is bound to BSA via the same amide group. The preparation schemes for shortwave infrared fluorescent agents 3 and 4 are shown below.

[0160] [ka]

[0161] Five mg of bovine serum albumin (BSA) was dissolved in 1 mL of 10 mM sodium carbonate solution and reacted with 0.1 mL of a dimethyl sulfoxide solution of compound 14 (1 mg / mL). This yielded compound 14 (ICG-C9-BSA) conjugated with BSA. Purification was performed using a gel filtration column (PD10, GE Healthcare) with physiological phosphate buffer (PBS) as the eluent. ICG-C9-BSA was dissolved in PBS to a concentration of 1 mg / mL. The resulting aqueous solution was designated short-wave infrared fluorescent agent 3.

[0162] Further, short-wave infrared fluorescent agent 4 was prepared in the same manner as short-wave infrared fluorescent agent 3, except that compound 17 was used instead of compound 14.

[0163] [Imaging of blood vessels and lymph nodes using short-wave infrared fluorescent agents 3 and 4] <Imaging of blood vessels in the lower limbs and abdomen of mice> Short-wave infrared fluorescent imaging of blood vessels in the lower limbs and abdomen of hairless mice was performed in the same manner as in Example 5, except that short-wave infrared fluorescent agent 3 or short-wave infrared fluorescent agent 4 was used instead of short-wave infrared fluorescent agent 1.

[0164] In the short-wave infrared fluorescence imaging using the short-wave infrared fluorescent agent 3, the excitation wavelength was 905 nm and the fluorescence detection wavelength was 1000 nm. The excitation light intensity was 10 mW / cm. 2The exposure time was 5 seconds. Photographs showing bright-field images of the lower limbs and abdomen of the hairless mouse and short-wave infrared fluorescent images using short-wave infrared fluorescent agent 3 are shown in FIG.

[0165] For short-wave infrared fluorescence imaging using short-wave infrared fluorescent agent 4, the excitation wavelength was 975 nm and the fluorescence detection wavelength was 1100 nm. The excitation light intensity was 20–40 mW / cm. 2 The exposure time was 5 to 15 seconds. Photographs showing bright-field images of the lower limbs and abdomen of the hairless mouse and short-wave infrared fluorescent images using short-wave infrared fluorescent agent 4 are shown in FIG.

[0166] As shown in Figures 6 and 7, shortwave infrared fluorescent agent 3 and shortwave infrared fluorescent agent 4, like shortwave infrared fluorescent agent 1 and shortwave infrared fluorescent agent 2, can form clear shortwave infrared images of blood vessels in the lower limbs and abdomen.

[0167] <Imaging of cerebral blood vessels in mice> Short-wave infrared fluorescent imaging of cerebral blood vessels in hairless mice was performed in the same manner as in Example 5, except that short-wave infrared fluorescent agent 3 or short-wave infrared fluorescent agent 4 was used instead of short-wave infrared fluorescent agent 1.

[0168] Furthermore, for comparison, shortwave infrared fluorescence imaging of the cerebral blood vessels of hairless mice was performed in the same manner as in Example 5, except that shortwave infrared fluorescent agent C2 was used instead of shortwave infrared fluorescent agent 1. Shortwave infrared fluorescent agent C2 is a fluorescent agent prepared in the same manner as shortwave infrared fluorescent agent 1, except that ICG-NHS was used instead of compound 14. ICG-NHS can be purchased from Goryo Chemical Co., Ltd.

[0169] In the short-wave infrared fluorescence imaging using the short-wave infrared fluorescent agent 3, the excitation wavelength was 905 nm and the fluorescence detection wavelength was 1000 nm. The excitation light intensity was 10 mW / cm. 2 and the exposure time was 7.5 seconds.

[0170] For short-wave infrared fluorescence imaging using short-wave infrared fluorescent agent 4, the excitation wavelength was 975 nm and the fluorescence detection wavelength was 1100 nm. The excitation light intensity was 20–40 mW / cm. 2 and the exposure time was 1 second.

[0171] For short-wave infrared fluorescence imaging using the short-wave infrared fluorescent agent C2, the excitation wavelength was 758 nm and the fluorescence detection wavelength was 900 nm. The excitation light intensity was 5 mW / cm. 2 and the exposure time was 20 seconds.

[0172] Figure 8 shows photographs representing bright-field images of the top of the head of a hairless mouse and shortwave infrared fluorescence images obtained with shortwave infrared fluorescent agents C2, 3, and 4. As shown in Figure 8, shortwave infrared fluorescent agents 3 and 4, like shortwave infrared fluorescent agents 1 and 2, are able to produce clear, detailed images of the cerebral blood vessels of a hairless mouse that can be observed externally with shortwave infrared light. In contrast, it was difficult to observe the cerebral blood vessels of a hairless mouse externally using the shortwave infrared image obtained with shortwave infrared fluorescent agent C2.

[0173] Examples 13 to 15 [Preparation of Complexes 1 to 3] Conjugates 1 and 2 were prepared. In conjugate 1, compound 14 (ICG-C9-NHS) is conjugated to an antibody (Erbitux) via an amide group containing a carbonyl group, which is the residue of a reactive crosslinking group. In conjugate 2, compound 17 (ICG-C11-NHS) is conjugated to an antibody via the same amide group. The preparation schemes for conjugates 1 and 2 are shown below.

[0174] [ka]

[0175] 2 mg of anti-human EGFR monoclonal antibody (Erbitux, Merck Serono Co., Ltd.) was dissolved in 1 mL of 10 mM sodium carbonate solution, and 50 μL of a dimethyl sulfoxide solution of compound 14 (1 mg / mL) was added to the solution to obtain conjugate 1 (ICG-C9-Erbitux), in which compound 14 was modified with Erbitux. Purification was performed using a gel filtration column.

[0176] In addition, Conjugate 2 (ICG-C11-Erbitux) in which Compound 17 was modified with Erbitux was prepared in the same manner as Conjugate 1, except that Compound 17 was used instead of Compound 14.

[0177] [Fluorescence properties] The fluorescence spectra of aqueous solutions prepared by dissolving each of complexes 1 and 2 in PBS were measured. The concentrations of the solutions measured were 1 mg / mL. The excitation wavelengths were 785 nm for complex 1 and 900 nm for complex 2. The fluorescence spectra of complexes 1 and 2 are shown in Figure 9. As shown in Figure 9, the fluorescence emission peak of complex 1 was approximately 950 nm, and the fluorescence emission peak of complex 2 was approximately 1100 nm.

[0178] Furthermore, Conjugate 3 (ICG-C11-Kadcyla) was prepared in the same manner as Conjugate 2, except that an anti-HER2 antibody-tubulin polymerization inhibitor conjugate (Kadcyla (registered trademark of F. Hoffmann-La Roche, Chugai Pharmaceutical Co., Ltd.), an antibody-drug conjugate (ADC), was used instead of the anti-human EGFR monoclonal antibody. Similar to Conjugate 2, in Conjugate 3, compound 17 (ICG-C11-NHS) is linked to the antibody in the antibody-drug conjugate via the amide group containing a carbonyl group. The excitation wavelength and fluorescence peak of Conjugate 3 are substantially the same as those of Conjugate 2.

[0179] Examples 16 to 18 [Preparation of short-wave infrared fluorescent agents 5-7] Complex 1 was diluted with PBS to obtain an aqueous solution of Complex 1 at a concentration of 1 mg / mL. This aqueous solution was designated as short-wave infrared fluorescent agent 5.

[0180] Shortwave infrared fluorescent agent 6 was prepared in the same manner as shortwave infrared fluorescent agent 5, except that complex 2 was used instead of complex 1. Also, shortwave infrared fluorescent agent 7 was prepared in the same manner as shortwave infrared fluorescent agent 5, except that complex 3 was used instead of complex 1.

[0181] [Fluorescence imaging of breast cancer tumors using short-wave infrared fluorescent agents 5 and 6] <Creation of breast cancer model mice> Five-week-old nude mice (BALB / c Slc-nu / n, Japan SLC) were inoculated with human breast cancer cells (MDA-MB-468 (ATCC), approximately 1.5 × 10 7 The tumors were transplanted into the second breast of the mouse's lower leg. This produced tumor-bearing mice as breast cancer model mice. The mice were then grown for 2-3 weeks, allowing the tumors to grow to a few millimeters in size.

[0182] <Fluorescence imaging of breast cancer tumors in breast cancer model mice> The tumor-bearing mice were anesthetized with isoflurane (Fujifilm Wako Pure Chemical Industries, Ltd.) and 0.2 mL of short-wave infrared fluorescent agent 5 was injected into their tail vein. Then, in vivo short-wave infrared fluorescence imaging of the breast cancer tumors in the tumor-bearing mice was performed. The tumor-bearing mice were then dissected, and the breast cancer tumors and organs (heart, kidney, spleen, and liver) were removed. Then, ex vivo short-wave infrared fluorescence imaging of the removed breast cancer tumors and each organ was performed.

[0183] In the short-wave infrared fluorescence imaging using the short-wave infrared fluorescent agent 5, the excitation wavelength was 905 nm and the fluorescence detection wavelength was 1000 nm. The intensity of the excitation light was 10 mW / cm. 2The exposure time was 15 seconds. Photographs showing bright-field images of breast cancer tumors in nude mice in vivo and short-wave infrared fluorescence images obtained with short-wave infrared fluorescent agent 5 are shown in FIG. 10. Photographs showing bright-field images of breast cancer tumors, heart, kidney, spleen, and liver in nude mice ex vivo and short-wave infrared fluorescence images obtained with short-wave infrared fluorescent agent 5 are shown in FIG. 11.

[0184] In addition, short-wave infrared fluorescence imaging was performed in vivo and ex vivo in the same manner as the short-wave infrared fluorescence imaging using short-wave infrared fluorescent agent 5, except that short-wave infrared fluorescent agent 6 was used instead of short-wave infrared fluorescent agent 5.

[0185] In the short-wave infrared fluorescence imaging using the short-wave infrared fluorescent agent 6, the excitation wavelength was 975 nm and the fluorescence detection wavelength was 1100 nm. The excitation light intensity was 20 to 40 mW / cm. 2 The exposure time was 15 to 30 seconds. Photographs showing bright-field images of breast cancer tumors in nude mice in vivo and short-wave infrared fluorescence images using short-wave infrared fluorescent agent 6 are shown in Figure 12. Photographs showing bright-field images of breast cancer tumors, heart, kidney, spleen, and liver in nude mice ex vivo and short-wave infrared fluorescence images using short-wave infrared fluorescent agent 6 are shown in Figure 13.

[0186] 10, 11, 12, and 13, breast cancer tumors emit short-wave infrared fluorescence due to the accumulation of Complex 1 or Complex 2. As such, it is clear that both short-wave infrared fluorescent agent 5 and short-wave infrared fluorescent agent 6 specifically bind to breast cancer tumors and can form images of breast cancer tumors by detecting short-wave infrared light.

[0187] Furthermore, as is clear from Figures 10, 11, 12, and 13, short-wave infrared fluorescence from Complex 1 or Complex 2 was also observed in the heart, kidney, spleen, and liver. The fluorescence from the heart and kidney was weaker than that from the spleen and liver. The reason for the weak fluorescence from the heart is thought to be that blood does not stagnate in the heart but simply passes through it. The reason for the weak fluorescence from the kidney is thought to be that the complex that reaches the kidney is excreted from the body in urine, and therefore the amount of complex stored there is smaller than in the spleen and liver.

[0188] [Fluorescence imaging of breast cancer tumors using short-wave infrared fluorescent agent 7] Human breast cancer cells were treated with KPL-4 cells (provided by Kawasaki Medical School, approximately 1.5 × 10 7 Tumor-bearing mice were generated in the same manner as described above for breast cancer model mice, except that shortwave infrared fluorescent agent 7 was used instead of shortwave infrared fluorescent agent 6. Tumor-bearing mice were then grown to a size of several millimeters. Fluorescence imaging of breast cancer tumors in breast cancer model mice was then performed in vivo and ex vivo in the same manner, except that shortwave infrared fluorescent agent 7 was used instead of shortwave infrared fluorescent agent 6. Bright-field images of breast cancer tumors in nude mice in vivo and shortwave infrared fluorescent images using shortwave infrared fluorescent agent 7 are shown in Figure 14. Bright-field images of breast cancer tumors, heart, kidney, spleen, and liver in nude mice ex vivo and shortwave infrared fluorescent images using shortwave infrared fluorescent agent 7 are shown in Figure 15.

[0189] As is clear from Figures 14 and 15, shortwave infrared fluorescent agent 7, like shortwave infrared fluorescent agent 6, specifically binds to breast cancer tumors and can form images of breast cancer tumors by detecting shortwave infrared light.

[0190] [Examples 19 and 20] [Preparation of Complexes 4 and 5] Conjugates 4 and 5 were prepared. In conjugate 4, compound 19 (ICG-C9-maleimide) is conjugated to an antibody (Herceptin) via a succinimide group, which is a residue of a reactive crosslinking group. In conjugate 5, compound 21 (ICG-C11-maleimide) is conjugated to the antibody via the succinimide group. The preparation schemes for conjugates 4 and 5 are shown below.

[0191] [ka]

[0192] Two mg of anti-human HER2 monoclonal antibody (Herceptin, Chugai Pharmaceutical Co., Ltd.) was dissolved in 1 mL of 10 mM sodium carbonate solution, and 20 μL of NHS-Fluorescein (Thermo Scientific) in dimethyl sulfoxide (1 mg / mL) was added and incubated at room temperature for 1 hour. Next, 2 mg of dithiothreitol (Tokyo Chemical Industry Co., Ltd.) was added and the mixture was left for 30 minutes. Excess dithiothreitol was removed by gel filtration using PBS as the eluent. 50 μL of a dimethyl sulfoxide solution (1 mg / mL) of compound 19 (ICG-C9-maleimide) or compound 21 (ICG-C11-maleimide) was added to the solution and incubated at room temperature for 2 hours. Purification was performed using a gel filtration column to obtain conjugate 4 (ICG-C9-maleimide-Herceptin) and conjugate 5 (ICG-C11-maleimide-Herceptin).

[0193] [Examples 21 and 22] [Preparation of Complexes 6 and 7] Conjugates 6 and 7 were prepared. In conjugate 6, compound 20 (ICG-C9-alkyne) is bound to an antibody (Herceptin) via a triazole group, which is the residue of a reactive crosslinking group. In conjugate 7, compound 22 (ICG-C11-alkyne) is bound to the antibody via the triazole group. The preparation schemes for conjugates 6 and 7 are shown below.

[0194] [ka]

[0195] Two mg of anti-human HER2 monoclonal antibody (Herceptin, Chugai Pharmaceutical Co., Ltd.) was dissolved in 1 mL of 10 mM sodium carbonate solution, to which 20 μL of Azido-PEG4-NHSester (Tokyo Chemical Industry Co., Ltd.) dimethyl sulfoxide solution (1 mg / mL) was added and allowed to react at room temperature for 1 hour. Unreacted Azido-PEG4-NHSester was removed by gel filtration using PBS as the eluent, and an Azido-PEG4-modified anti-human EGFR monoclonal antibody was prepared. Next, 10 μL of tris(3-hydroxypropyltriazolylmethyl)amine (3 mg / mL aqueous solution, Sigma-Aldrich), 10 μL of copper sulfate (0.5 mg / mL, CuSO 5H O, Nacalai Tesque), and 50 μL of compound 20 (ICG-C9-alkyne) or compound 22 (ICG-C11-alkyne) in dimethyl sulfoxide (1 mg / mL) were added to 0.5 mL of PBS solution. Then, 10 μL of sodium ascorbate (100 mg / mL, Nacalai Tesque) was added and the mixture was left for 1 minute. To this solution, 1 mL of Azido-PEG4-modified anti-human EGFR monoclonal antibody (2 mg / mL) was added and allowed to react for 12 hours at room temperature. Purification was performed by centrifugation and gel filtration (PD10 column, GE Healthcare) to obtain conjugate 6 (ICG-C9-alkyne-Herceptin) and conjugate 7 (ICG-C11-alkyne-Herceptin).

[0196] [Examples 23 and 24] [Preparation of short-wave infrared fluorescent agents 8 and 9] Complex 4 was diluted with PBS to obtain an aqueous solution of Complex 4 at a concentration of 1 mg / mL. This aqueous solution is designated as short-wave infrared fluorescent agent 8. Complex 5 was also diluted with PBS to obtain an aqueous solution of Complex 5 at a concentration of 1 mg / mL. This aqueous solution is designated as short-wave infrared fluorescent agent 9.

[0197] [Breast cancer imaging using short-wave infrared fluorescent agents 8 (complex 4) and 9 (complex 5)] Nude mice implanted with HER2-overexpressing breast cancer cells (KPL-4) were injected with 0.2 mL of short-wave infrared fluorescent agent 8 (1 mg / mL of complex 4) and 0.2 mL of short-wave infrared fluorescent agent 9 (1 mg / mL of complex 5) via the tail vein. Three days later, in vivo short-wave infrared fluorescence imaging of breast cancer tumors in the tumor-bearing mice was performed. The imaging conditions for fluorescence imaging were an excitation wavelength of 905 nm and a fluorescence detection wavelength of 1000 nm or more. The excitation light intensity was 20 mW / cm. 2 The exposure time was 5 seconds. Figure 16 shows bright-field and short-wave infrared fluorescence images of breast cancer tumors in nude mice in vivo using short-wave infrared fluorescent agent 8 and short-wave infrared fluorescent agent 9. These results demonstrate that breast cancer tumors can also be clearly detected using short-wave infrared fluorescent agents 8 and 9.

[0198] [Examples 25 and 26] [Preparation of short-wave infrared fluorescent agents 10 and 11] Complex 6 was diluted with PBS to obtain an aqueous solution of Complex 6 at a concentration of 1 mg / mL. This aqueous solution is designated as short-wave infrared fluorescent agent 10. Complex 7 was also diluted with PBS to obtain an aqueous solution of Complex 7 at a concentration of 1 mg / mL. This aqueous solution is designated as short-wave infrared fluorescent agent 11.

[0199] [Breast cancer imaging using short-wave infrared fluorescent agents 10 (complex 6) and 11 (complex 7)] Nude mice implanted with HER2-overexpressing breast cancer cells (KPL-4) were injected with 0.2 mL of short-wave infrared fluorescent agent 10 (1 mg / mL of complex 6) and 0.2 mL of short-wave infrared fluorescent agent 11 (1 mg / mL of complex 7) via the tail vein. Three days later, in vivo short-wave infrared fluorescence imaging of breast cancer tumors in tumor-bearing mice was performed. The imaging conditions for fluorescence imaging were an excitation wavelength of 905 nm and a fluorescence detection wavelength of 1000 nm or more. The excitation light intensity was 20 mW / cm. 2The exposure times were 10 seconds (shortwave infrared fluorescent agent 10) and 30 seconds (shortwave infrared fluorescent agent 11). Photographs showing bright-field and shortwave infrared fluorescent images of breast cancer tumors in nude mice in vivo using shortwave infrared fluorescent agent 10 and shortwave infrared fluorescent agent 11 are shown in Figure 17. These results demonstrate that breast cancer tumors can also be clearly detected using shortwave infrared fluorescent agents 10 and 11.

[0200] [Examples 27 and 28] [Preparation of Complexes 8 and 9] The recombinant annexin V protein was synthesized according to the method published in our paper (Setsuko Tsuboi and Takashi Jin, ChemBioChem 18, 2231-2235, 2017). 50 μL of a dimethyl sulfoxide solution (1 mg / mL) of compound 14 (ICG-C9-NHS) or compound 17 (ICG-C11-NHS) was added to 1 mL of a PBS solution (1 mg / mL) of annexin V and incubated for 1 hour. Purification was performed using a gel filtration column to obtain conjugates 8 (ICG-C9-annexin V) and 9 (ICG-C11-annexin V).

[0201] [Examples 29 and 30] [Preparation of short-wave infrared fluorescent agents 12 and 13] Complex 8 was diluted with PBS to obtain an aqueous solution of complex 8 at a concentration of 0.5 mg / mL. This aqueous solution is designated as short-wave infrared fluorescent agent 12. Complex 9 was also diluted with PBS to obtain an aqueous solution of complex 9 at a concentration of 0.5 mg / mL. This aqueous solution is designated as short-wave infrared fluorescent agent 13.

[0202] [Imaging apoptosis (cell death) in breast cancer cells using short-wave infrared fluorescent agents 12 (complex 8) and 13 (complex 9)] Nude mice implanted with HER2-overexpressing breast cancer cells (KPL-4) were injected with 0.1 mL of Kadcyla (2 mg / mL) via the tail vein. Three days later, the tumor area was injected with short-wave infrared fluorescent agent 12 (0.5 mg / mL of complex 8) or short-wave infrared fluorescent agent 13 (0.5 mg / mL of complex 9). The next day, in vivo short-wave infrared fluorescence imaging of breast cancer tumors in tumor-bearing mice was performed. The imaging conditions for fluorescence imaging were an excitation wavelength of 905 nm and a fluorescence detection wavelength of 1000 nm or higher. The excitation light intensity was 20 mW / cm. 2 The exposure times were 5 seconds (short-wave infrared fluorescent agent 12) and 10 seconds (short-wave infrared fluorescent agent 13). Figure 18 shows bright-field and short-wave infrared fluorescent images of in vivo breast cancer tumors in nude mice injected with Kadcyla (left) and nude mice not injected with Kadcyla (right) for short-wave infrared fluorescent agent 12 and short-wave infrared fluorescent agent 13, respectively.

[0203] Fluorescence images show that Kadcyla induces apoptosis (cell death) in tumor cells. The control image (right) shows a nude mouse breast cancer tumor that was not injected with Kadcyla, and no significant accumulation of short-wave infrared fluorescent dyes 12 and 13 was observed.

[0204] [Time-lapse imaging of apoptosis (cell death) in breast cancer cells using short-wave infrared fluorescent agent 13 (complex 9)] Nude mice implanted with HER2-overexpressing breast cancer cells (KPL-4) were injected with 0.1 mL of Kadcyla (2 mg / mL) via the tail vein, and three days later, the tumor was injected with short-wave infrared fluorescent agent 13 (complex 9 at a concentration of 0.5 mg / mL). Three, five, and 11 days later, in vivo short-wave infrared fluorescence imaging of breast cancer tumors in tumor-bearing mice was performed. The imaging conditions for fluorescence imaging were an excitation wavelength of 905 nm and a fluorescence detection wavelength of 1000 nm or higher. The excitation light intensity was 20 mW / cm. 2The exposure time was 10 seconds. Figure 19 shows bright-field and short-wave infrared fluorescent images of in vivo breast cancer tumors in nude mice injected with Kadcyla (short-wave infrared fluorescent agent 13) at specific time points.

[0205] Fluorescence images confirmed that Kadcyla treatment induced apoptosis (cell death) in tumor cells, resulting in a reduction in tumor size. These results demonstrate that this short-wave infrared fluorescent agent is effective in confirming the efficacy of antibody-drug conjugates (ADCs) in animal experiments. [Industrial Applicability]

[0206] The present invention can be used in short-wave infrared fluorescence imaging technology, and is expected to enable visualization of deep inside living organisms with a resolution far superior to that of conventional technology.

Claims

1. A compound represented by the following formula (1): 【Chemical 1】 (In formula (1), n ​​represents an integer of 3 to 5, and X represents a salt of a sulfonic acid group or a crosslinking group reactive to a molecular recognition agent.)

2. 2. The compound of claim 1, wherein the reactive crosslinking group comprises one or more organic groups selected from the group consisting of an N-hydroxysuccinimide ester group, an alkynyl group, a maleimide group, and an azide group.

3. The compound according to claim 1 or 2, wherein n is 3 or 4.

4. The compound according to any one of claims 1 to 3, represented by any one of the following formulas (2) to (9): 【Chemistry 2】

5. A complex in which a molecular recognition agent and the compound according to any one of claims 1 to 4 are bound to each other via a residue of the reactive crosslinking group in the compound.

6. The complex according to claim 5 , wherein the molecular recognition agent is an antibody or a fragment of the antibody having antigen-binding ability.

7. A short-wave infrared fluorescent agent containing either or both of the compound according to any one of claims 1 to 4 and the complex according to claim 5 or 6.

8. A first step of synthesizing a first compound represented by the following formula (1a): a second step of replacing the anilino group in the formula (1a) with a structure represented by the following formula (1b); a third step of replacing the phenylimino group in the formula (1a) with a structure represented by the following formula (1c); A method for producing a compound represented by the following formula (1): 【Chemistry 3】 (In the above formula, n represents an integer of 3 to 5, and X represents a salt of a sulfonic acid group or a crosslinking group reactive with a molecular recognition agent.)

9. 9. The method for producing a compound according to claim 8, wherein n is 3, X is a salt of a sulfonic acid group, and the second step and the third step are carried out at the same time.

10. The method for producing a compound according to claim 8, wherein n is 4, X is a salt of a sulfonic acid group, and the third step is carried out after the second step.

11. The method for producing a compound according to claim 8 , wherein n is 3, X is the reactive crosslinking group, and the third step is carried out after the second step.

12. The method for producing a compound according to claim 8 , wherein n is 4, X is the reactive crosslinking group, and the third step is carried out before the second step.

Images