Test reagents and fractional quantitative methods for polyamines

Abstract

To provide a test drug and a fractional determination method for polyamines.SOLUTION: The invention provides a compound represented by the general formula in the figure, or an isomer thereof. [In the formula, L1 and L2 are divalent polyether chains; L3 is a divalent group cleavable at a terminal or inside; L4 is absent or a linking group; n5 is an integer from 0 to 4; and R5 at each occurrence is independently a substituent, or two of the R5 groups may form a ring with carbon atoms to which they are respectively linked.]SELECTED DRAWING: None

Description

[Technical Field]

[0001] This disclosure relates to a diagnostic reagent and a method for the fractional quantification of polyamines. This disclosure also relates to a diagnostic reagent and a method for the fractional quantification of polyamines in vivo that can be applied to the diagnosis of cancer, Parkinson's disease, and other conditions, and that can be performed simply and rapidly. [Background technology]

[0002] In vivo polyamines (primarily referring to four types in humans: stresin, cadaverine, spermidine, and spermine, but not limited to these) influence the processes of protein synthesis and nucleic acid synthesis, are essential factors for cell proliferation, and have also been linked to cancer and Parkinson's disease. However, measuring their concentrations usually requires complicated procedures and expensive equipment, and there is a need for the development of effective and simple quantitative methods.

[0003] Compounds for measuring polyamines have been developed conventionally (Patent Documents 1-2). [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Patent No. 5044779 [Patent Document 2] International Publication No. 2019 / 151381 [Overview of the project] [Means for solving the problem]

[0005] For example, this disclosure provides the following items: (Item 1) Compounds represented by the following general formula I: [ka] Equation I [In the formula, L 1is a divalent polyether chain, L 2 is a divalent polyether chain, L 3 is a divalent group cleavable at the terminal or internally, L 4 either does not exist or is a linking group, n 5 is an integer from 0 to 4, R 5 is, in each occurrence, independently, a substituent or two of R 5 may together with the carbon atoms to which they are respectively attached form a substituted or unsubstituted ring, Ar 1 and Ar 2 are independently the same or different aromatic groups. Or an isomer thereof. (Item 2) Ar 1 and Ar 2 are both the same or different benzene ring structures, L 4 is a linking group, the compound according to Item 1 or an isomer thereof. (Item 3) L 1 is -CH2-(OCH2CH2) n11 -OCH2- [where n11 is an integer from 1 to 10.] is the compound according to Item 1 or 2 or an isomer thereof. (Item 4) L 2 is -CH2-(OCH2CH2) n11 -OCH2- [where n22 is an integer from 1 to 10.] is the compound according to any one of Items 1 to 3 or an isomer thereof. (Item 5) L 3 is -CO2-, -SO3-, or -CONH- is the compound according to any one of Items 1 to 4 or an isomer thereof. (Item 6) L 4 is -O-, -CH2-O-, -O-CH2-, -O-(CH2) nA compound or isomer of any one of items 1 to 5, which is -O-, -CH2-O-CH2-, or -OC(=O)-O-, where n is an integer from 1 to 10. (Item 7) R 5 The compound or its isomer described in any one of items 1 to 6, which, in each occurrence, is independently selected from the group consisting of an unsubstituted or halogenated linear or branched alkyl group, an unsubstituted or halogenated linear or branched alkyloxy group, a halogen atom, a nitro group, a carboxylic acid group or its derivative, a sulfonic acid group or its derivative, and an amino group or its derivative. (Item 8) Ar 1 and Ar 2 The compounds or isomers described in any one of items 1 to 7, which independently have a benzene ring structure, a naphthalene ring structure, an anthracene ring structure, or a phenanthrene ring structure in each occurrence. (Item 9) Ar 1 -OH and L 1 A portion formed by, or Ar 2 -OH and L 2 The parts formed by this are independent in each occurrence, [ka] Selected from the group consisting of, where * is L 4 Or in formula I above, Ar 1 Ar 2 and L 3 A compound or its isomer described in any one of items 1 to 8, representing a bond site that can bond to the carbon atom to which the compound is bonded. (Item 10) The compound according to any one of items 1 to 9, wherein the isomer is a tautomer, a ketone isomer, or a protonated ketone isomer. (Item 11) The aforementioned compound, [ka] The compound described in item 1. (Item 12) The aforementioned compound, [ka] The compound described in item 1. (Item 13) The aforementioned compound, [ka] The compound described in item 1. (Item 14) The aforementioned compound, [ka] The compound described in item 1. (Item 15) L 3 However, it is -CO2-, and L 4 A compound listed in any of items 1 to 10, which is -O-. (Item 16) The aforementioned compound, [ka] The compound described in item 15. (Item 17) The aforementioned compound, [ka] The compound described in item 16. (Section 17A) n 5 The compounds listed in item 17, wherein the value is 0. (Section 17B) L 3 However, it is -CO2-, and L 4 is -CH2-O-, -O-CH2-, -O-(CH2) nA compound described in any of items 1 to 10, which is -O-, -CH2-O-CH2-, or -OC(=O)-O-, where n is an integer from 1 to 10. (Section 17C) The aforementioned compound, [ka] The compound described in item 17B. (Item 18) [ka] It is a compound. (Section 18A) [ka] It is a compound. (Section 18B) [ka] It is a compound. (Item 19) A diagnostic reagent for detecting endogenous polyamines containing any of the compounds listed in items 1-18B. (Item 20) The diagnostic reagent described in item 19, further comprising another compound having different affinity for spermine and spermidine from the aforementioned compound. (Item 21) A method for the fractional determination of spermine and spermidine, comprising the steps of contacting a sample with at least two compounds having different affinities to spermine and spermidine, and fractional determination of spermine and spermidine based on the difference in the affinities of the at least two compounds to spermine and spermidine. (Item 22) The fractional quantitative method according to item 21, wherein the at least two compounds include at least one compound from any of items 1 to 17C. (Item 23) A method for the fractional determination of spermine and spermidine, comprising contacting a sample with one of the compounds from items 1 to 17C and measuring the color change or fluorescence using a spectrophotometer. (Item 24) (i) Contact one of the compounds from items 1 to 17C with the sample and measure the color change or fluorescence using a spectrophotometer. (ii) Contact a compound from item 1 to 17C, different from the compound used in (i) above, with the sample and measure the color change or fluorescence using a spectrophotometer, and (iii) Calculate the amounts of spermine and spermidine based on the measurement results of steps (i) and (ii) above. A method for the fractional quantitative determination of spermine and spermidine, characterized by the following. (Item 25) A test or diagnostic agent for identifying diseases based on endogenous polyamines, containing any of the compounds listed in items 1-17C. (Item 26) The tests or diagnostic agents described in item 25, wherein the disease includes cancer, Parkinson's disease, or Alzheimer's disease. (Item 27) In the subjects, polyamines shall be measured by the method described in any one of items 21 to 24, and Based on the measured polyamine information, an indicator is presented to show whether the subject is suspected of having a disease based on their in vivo polyamine levels. including A method for identifying endogenous polyamine-based diseases in subjects. (Item 28) The method according to item 27, wherein the disease includes cancer, Parkinson's disease, or Alzheimer's disease. [Effects of the Invention]

[0006] A diagnostic reagent and a technology for the fractional quantification of polyamines are provided with unprecedented sensitivity, speed, and ease of use. A diagnostic reagent and fractional quantification method are provided that enable simple and rapid fractional quantitative measurement of polyamines in vivo, which can be applied to the diagnosis of cancer, Parkinson's disease, and other conditions.

[0007] We can provide diagnostic reagents that exhibit different color and fluorescence reactions to spermidine and spermine, respectively. Furthermore, by using multiple diagnostic reagents that react differently to spermidine and spermine, the concentrations of spermidine and spermine can be easily, quickly, and quantitatively determined from the color and fluorescence reaction results of each reagent.

[0008] Compared to conventional methods that involve introducing UV or fluorescently active sites such as pyrene into polyamines and quantifying them from their UV or fluorescence intensity, or using ELISA, the method disclosed herein has the advantage of eliminating the need for pretreatment and large-scale equipment, and makes it possible to easily quantify individual concentrations, which was not possible with conventional methods.

[0009] This disclosure provides an effective, simple, and rapid quantitative method for polyamines. Unlike reagents that exhibit selectivity only for spermidine and spermine among polyamines and can quantify the sum of both (Patent Documents 1-2, Non-Patent Document 1), this method allows for the separate quantification of spermidine and spermine, enabling applications in the early diagnosis of cancer and Parkinson's disease.

[0010] Furthermore, the compounds disclosed herein exhibit high sensitivity, enabling the measurement of polyamines in vivo. This allows for the selective quantification of individual concentrations of spermidine and spermine in vivo, such as in bacteria and animals. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 shows a comparison of the activity of compound 4 against spermine and spermidine. [Figure 2] Figure 2 shows the colorimetric recognition of compounds 1-3 with spermidine (4). Conditions: [Compounds 1-3] = [Spermidine (4)] = 1.0 × 10⁻⁴ M, methanol, 25.0°C. UV measurement path length = 1 cm. Inset: Diagram of color change at spermidine binding. [Figure 3]Figure 3 shows the color change between compound 3 and the amine. Conditions: [Compound 3] = 5.0 × 10⁻⁶ M, [Amine] = 5.0 × 10⁻⁶ M, methanol, 25.0°C. UV measurement path length = 1 cm. Inset: Diagram of color change. [Figure 4] Figure 4 shows the visual detection of spermidine (4) and spermine (5) among the biogenic amines (9-14) using compound 3. (a) UV-vis spectra of compound 3 and amines (9-14). (b) Photographs of the color change in compound 3 due to interaction with amines (9-14). Conditions: [Compound 3] = 5.0 × 10⁻⁶ M, [9-14] = 5.0 × 10⁻⁶ M, [8] = 1.0 × 10⁻⁴ M, 25°C, methanol. Path length of UV measurement = 1 cm. (c) Color change between compound 3 and three types of biogenic amines. Conditions: [Compound 3] = 1.0 × 10⁻⁵ M, [Amine] = 6.0 × 10⁻³ M, [8] = 1.0 × 10⁻⁴ M, 25°C, methanol. Path length of UV measurement = 1 cm. [Figure 5] Figure 5 shows a comparison of spermidine concentrations determined by colorimetric and HPLC methods. [Figure 6] Figure 6(a) shows the change in the absorption spectrum of compound 104 upon addition of spermidine. Figure 6(b) shows the difference spectrum of (a). Conditions: [Compound 104] = 1.0 × 10⁻⁶ M, [Spermidine] = 1.0 × 10⁻⁴ M, in MeOH, 25°C. [Figure 7] Figure 7(a) shows the change in the absorption spectrum of compound 104 upon addition of spermine. Figure 7(b) shows the difference spectrum of (a). Conditions: [Compound 104] = 2.5 × 10⁻⁶ M, [Spermine] = 1.0 × 10⁻⁴ M, in MeOH, 25°C. [Figure 8] Figure 8(a) shows the approximate curve for compound 104 and spermine. Figure 8(b) shows the approximate curve for compound 104 and spermidine. [Figure 9] Figure 9 shows the UV-vis spectra of compound 104 and NaOH. Conditions: [Compound 104] = 1.0 × 10⁻⁵ M, NaOH = 1.0 × 10⁻² M, in MeOH, 25°C. [Figure 10] Figure 10 shows a plot of actual and calculated concentrations. [Figure 11] Figure 11 shows the titration curves of the colorimetric reagent compound 222 and endogenous polyamines. [Figure 12] Figure 12 shows the titration curves of the colorimetric reagent compound 223 and endogenous polyamines. [Figure 13] Figure 13 shows the absorption spectrum of compound 278 as the pH changes. [Figure 14] Figure 14 shows the fluorescence spectrum of compound 278 as the pH changes. [Figure 15] Figure 15 shows the fluorescence spectra when spermidine or spermine is added to compound 290. [Modes for carrying out the invention]

[0012] The following provides further details about this disclosure. Throughout this specification, singular expressions should be understood to include the concept of their plural form unless otherwise specified. Accordingly, singular articles (for example, "a," "an," and "the" in English) should be understood to include the concept of their plural form unless otherwise specified. Furthermore, terms used herein should be understood to have the meaning commonly used in the art unless otherwise specified. Accordingly, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains. In case of any conflict, this specification (including definitions) shall prevail.

[0013] (term)

[0014] In this specification, "about" and "approximately" refer to ±10% of the number they modify, unless otherwise specified.

[0015] In this specification, "polyether chain" means a hydrocarbon chain in which one or more oxygen atoms are inserted, which may have one or more substituents. In this specification, if it is cyclic, it may be referred to as a crown ether (chain).

[0016] As used herein, the "divalent group cleavable at the terminal or inside" refers to a divalent group containing a cleavable bond such as an ester or an amide. Examples include -CO2-, -SO3-, or -CONH-.

[0017] As used herein, the "linking group" means a divalent group that links two different or identical aromatic groups (e.g., Ar 1 and Ar 2 ). Examples include -O-, -CH2-O-, -O-CH2-, -O-(CH2) n -O-, -CH2-O-CH2-, or -O-C(=O)-O-, where n is an integer from 1 to 10.

[0018] As used herein, the "halogen atom" includes fluorine atom, chlorine atom, bromine atom, and iodine atom.

[0019] As used herein, the "aromatic group" means a cyclic group having aromaticity, and may have the structure of benzene ring, naphthalene ring, anthracene ring, or phenanthrene ring.

[0020] As used herein, the "alkyl group" means a linear or branched alkyl group. For example, it may have 1 to 12 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms, but the number of carbon atoms is not limited thereto. Examples include methyl, ethyl, propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, isobutyl, sec-butyl, tert-butyl), etc.

[0021] As used herein, the "isomer" refers to any compound having a structure generated by the movement of a hydrogen atom in addition to the structural formula explicitly shown for the compound of the present disclosure (e.g., the compound of formula I) and the structure shown interchangeably by the movement of electrons, including a keto isomer, a protonated keto isomer, etc.

[0022] In this specification, "ketone isomer" means a different or identical aromatic group of the compound of this disclosure (e.g., the compound of formula I) Ar 1 and Ar 2 This refers to an isomer in which the OH group on the base has been converted into a ketone by the movement of a hydrogen atom. In this specification, "protonated ketone isomer" means a ketone isomer that has been protonated, and is considered to have a structure in which the oxygen of the ketone is protonated.

[0023] In this specification, "alkyloxy group" is also called an alkoxy group, and refers to a structure in which an alkyl group is bonded to an oxygen atom (RO-, where R is the alkyl group).

[0024] In this specification, "nitro group" refers to a group having the structure -NO2.

[0025] In this specification, "carboxylic acid group" refers to a group represented by CO2H, and "carboxylic acid group derivative" refers to any derivative of a carboxylic acid group, for example, CO2R c1 CONH2, CONHR c2 or CONR c3 R c4 R c1 , R c2 , R c3 , R c4 Each of these can independently be a linear or branched alkyl group (for example, having 1 to 4 carbon atoms, but not limited to these).

[0026] In this specification, "sulfonic acid group" refers to a group represented as SO3H, and a derivative of a sulfonic acid group refers to any group of a sulfonic acid group, for example, SO3R s1 SO2NH2, SO2NHR s2 or SO2NR s3 R s4 It is represented as R s1 , R s2 , R s3 , R s4 Each of these is independently a linear or branched alkyl group (for example, having 1 to 4 carbon atoms, but not limited to these).

[0027] In this specification, "amino group" refers to NH2, and "amino group derivative" refers to NHR 11 , NR 21 R 22 , and N + R 31 R 32 R 33 (R 11 , R 21 , R 22 , R 31 , R 32 , R 33 Each of these is an independent linear or branched alkyl group (for example, having 1 to 4 carbon atoms, but not limited to these).

[0028] (Preferred embodiment) Preferred embodiments of the Disclosure are described below. The embodiments provided below are provided for a better understanding of the Disclosure, and it will be understood that the scope of the Disclosure should not be limited to the descriptions below. Accordingly, it will be obvious that those skilled in the art can make appropriate modifications within the scope of the Disclosure, taking into consideration the descriptions herein. It will also be understood that the embodiments of the Disclosure below can be used individually or in combination.

[0029] The following provides further details regarding this disclosure.

[0030] This disclosure may provide a diagnostic reagent and a differential quantification method that can be used for the diagnosis of cancer and Parkinson's disease to easily and quickly perform differential quantitative measurement of polyamines in vivo.

[0031] Conventional diagnostic reagents have enabled the detection of polyamines in vivo through color reactions, but while the total amount can be measured, it has not been possible to differentiate and quantify spermidine and spermine. This disclosure provides diagnostic reagents that exhibit different color or fluorescent responses to spermidine and spermine, respectively. Furthermore, it provides a differential quantification method that uses multiple diagnostic reagents having different reactions to spermidine and spermine to quantitatively determine the concentrations of spermidine and spermine, respectively, from the color or fluorescent response results of each diagnostic reagent.

[0032] According to this disclosure, the concentrations of spermidine and spermine in vivo can be easily and rapidly quantified using the color or fluorescence response of a diagnostic reagent, enabling early detection of cancer and Parkinson's disease, and rapid diagnosis during cancer surgery. (Novel polyether-based substances)

[0033] In one aspect, the present disclosure provides a compound represented by the following general formula I (the whole or a sub-concept thereof also referred to as the compound of the present disclosure): [ka] Equation I [In the formula, L 1 It is a divalent polyether chain, L 2 It is a divalent polyether chain, L 3 It is a divalent group that can be cleaved at its terminal or internally, L 4 It either does not exist or is a linking group. n 5 is an integer from 0 to 4, R 5 In each occurrence, independently, it is either a substituent or R 5 Two of these may, together with the carbon atoms to which they are linked, form a substituted or unsubstituted ring. Ar 1 and Ar 2are each independently the same or different aromatic groups. Or an isomer thereof is provided.

[0034] In one embodiment, Ar 1 and Ar 2 are both the same or different benzene ring structures, L 4 is a linking group.

[0035] In one embodiment, L 1 is -CH2-(OCH2CH2) n11 -OCH2-[where n11 is an integer from 1 to 10.].

[0036] In one embodiment, L 2 is -CH2-(OCH2CH2) n11 -OCH2-[where n22 is an integer from 1 to 10.].

[0037] In one embodiment, L 3 is -CO2-, -SO3-, or -CONH-.

[0038] In one embodiment, L 4 is -O-, -CH2-O-, -O-CH2-, -O-(CH2) n -O-, -CH2-O-CH2-, or -O-C(=O)-O-, and n is an integer from 1 to 10. Without wishing to be bound by theory, when L 4 is present (when it is a linking group), the relative configuration of the crown ether ring is fixed, so the measurement using the compound of the present disclosure is relatively stable, which is desirable.

[0039] In one embodiment, R 5is independently selected, at each occurrence, from the group consisting of a linear or branched alkyl group which is unsubstituted or substituted with a halogen atom, a linear or branched alkyloxy group which is unsubstituted or substituted with a halogen atom, a halogen atom, a nitro group, a carboxylic acid group or a derivative thereof, a sulfonic acid group or a derivative thereof, and an amino group or a derivative thereof, and preferably, R 5 is independently selected, at each occurrence, from the group consisting of a linear or branched alkyl group having 1 to 4 carbon atoms which is unsubstituted or substituted with a halogen atom, a linear or branched alkyloxy group having 1 to 4 carbon atoms which is unsubstituted or substituted with a halogen atom, a halogen atom, a nitro group (NO2), a carboxylic acid group (CO2H) or a derivative thereof (CO2R c1 , CONH2, CONHR c2 or CONR c3 R c4 is exemplified, and R c1 , R c2 , R c3 , R c4 can each independently be a linear or branched alkyl group (for example, having 1 to 4 carbon atoms), a sulfonic acid group (SO3H) or a derivative thereof (SO3R s1 , SO2NHʹ, SO2NHR s2 or SO2NR s3 R s4 is represented by, and R s1 , R s2 , R s3 , R s4 can each independently be a linear or branched alkyl group (for example, having 1 to 4 carbon atoms), an amino group or a derivative thereof (NHR 11 , NR 21 R 22 , and N + R 31 R 32 R 33 (R 11 , R 21 , R 22 , R 31 , R 32 , R 33 are each independently selected (for example, having 1 to 4 carbon atoms, but not limited thereto) from a linear or branched alkyl group).

[0040] In one embodiment, Ar 1 and Ar 2 Each occurrence may independently have the same or different benzene ring structure, naphthalene ring structure, anthracene ring structure, or phenanthrene ring structure, etc. In a preferred embodiment, in one compound, Ar 1 and Ar 2 These may be different or identical. This is because it becomes easier to set the distance between the two crown ether rings to be different, thereby making it easier to distinguish between the affinity for spermine and spermidine.

[0041] In one embodiment, Ar 1 -OH and L 1 A portion formed by, or Ar 2 -OH and L 2 The parts formed by this are independent in each occurrence, [ka] Selected from the group consisting of, where * is L 4 Or in formula I above, Ar 1 Ar 2 and L 3 This represents a bond site that can bond to the carbon atom to which it is bonded.

[0042] In one embodiment, the isomer is a tautomer, a ketone isomer, or a protonated ketone isomer.

[0043] In one embodiment, the compound is [ka] That is the case.

[0044] A-3 is a compound that significantly improves sensitivity. [ka]

[0045] Compound A-3 exhibits similar selectivity for spermidine and spermine, allowing for the quantification of their sum, although measuring individual concentrations is not always desirable. While we do not wish to be constrained by theory, this phenomenon is thought to be due to the longer molecular length of spermine binding to the binding site in the cross-linking color complex shown on the right of the figure above. However, in this disclosure, compounds like compound A-3, which exhibit similar selectivity for spermidine and spermine, can be used to distinguish and measure spermidine and spermine relatively by combining them with other compounds that exhibit different selectivity for spermidine and spermine. Furthermore, this disclosure provides other such compounds.

[0046] In preferred embodiments of this disclosure, a compound that can quantify spermidine separately from spermine is desirable, and one example of this is the synthesis of a compound with a longer distance between binding sites, creating a situation where spermidine cannot reach.

[0047] To create such a situation, the distance between the oxygen atoms of the phenolic hydroxyl groups present in the two crown ether rings of the compound of this disclosure may be a distance that is not reachable in spermidine but is reachable in spermine, and can be set to, for example, about 11 angstroms or more, but is not limited to this.

[0048] An example of a situation that can be generated is when the aromatic Ar in the substance of formula I... 1 and Ar 2 The fact that at least one of them has a structure different from a benzene ring can create a situation where the distance between the bonding sites is longer. If one is phenol and the other is naphthol, it can be approximately 10.8 angstroms. Therefore, Ar 1 and Ar 2 If both are not phenols, the distance between hydroxyl groups can generally exceed 11 angstroms, which may distinguish spermidine from spermine.

[0049] Specific examples of its structure include the following: Ar 1 However, it has a benzene ring structure, Ar 2 It can be a naphthalene ring structure, anthracene ring structure, or phenanthrene ring structure. Alternatively, Ar 2 However, it has a benzene ring structure, Ar 1 This can be a naphthalene ring structure, an anthracene ring structure, or a phenanthrene ring structure.

[0050] Ar 1 However, it has a naphthalene ring structure, Ar 2 It can be a naphthalene ring structure, anthracene ring structure, or phenanthrene ring structure. Alternatively, Ar 2 However, it has a naphthalene ring structure, Ar 1 This can be a naphthalene ring structure, an anthracene ring structure, or a phenanthrene ring structure.

[0051] Ar 1 However, it has an anthracene ring structure, Ar 2 It can be an anthracene ring structure or a phenanthrene ring structure. Alternatively, Ar 2 However, it has an anthracene ring structure, Ar 1 This can be an anthracene ring structure or a phenanthrene ring structure.

[0052] Ar 1 However, it has a phenanthrene ring structure, Ar 2 This can be a phenanthrene ring structure.

[0053] In one embodiment, the compound is [ka] That is the case.

[0054] In one embodiment, the compound is [ka] That is the case.

[0055] In one embodiment, the compound is [ka] That is the case.

[0056] In one embodiment, L 3 However, it is -CO2-, and L 4 However, it is -O-.

[0057] In one embodiment, the compound is [ka] That is the case.

[0058] In one embodiment, the compound is [ka] That is the case.

[0059] In one embodiment, n 5 It is 0.

[0060] In one embodiment, L 3 However, it is -CO2-, and L 4 is -CH2-O-, -O-CH2-, -O-(CH2) n The vectors are -O-, -CH2-O-CH2-, or -OC(=O)-O-, where n is an integer from 1 to 10.

[0061] In one embodiment, the compound is [ka] That is the case.

[0062] In one embodiment, [ka] That is the case.

[0063] In one embodiment, [ka] That is the case.

[0064] In one embodiment, [ka] That is the case.

[0065] In one embodiment, L 1 is -CH2-(OCH2CH2) n11 -OCH2-[In the given expression, n11 is an integer from 1 to 10.] and L 2 is -CH2-(OCH2CH2) n11 -OCH2-[In this expression, n2² is an integer between 1 and 10.] L 1 and L 2 They may be the same or different.

[0066] In one embodiment, L 1 is -CH2-(OCH2CH2) n11 -OCH2-[In the given expression, n11 is an integer from 1 to 10.] and L 2 is -CH2-(OCH2CH2) n11 -OCH2-[In the given expression, n2² is an integer from 1 to 10.] and Ar 1 and Ar 2 Each instance independently has the same or different benzene ring structure, naphthalene ring structure, anthracene ring structure, or phenanthrene ring structure. 1 and L 1 The structure formed by and 2 and L 2 The resulting structures may be the same or different.

[0067] In one embodiment, L 1 is -CH2-(OCH2CH2) n11 -OCH2-[In the given expression, n11 is an integer from 1 to 10.] and L 2 is -CH2-(OCH2CH2) n11 -OCH2-[In the given expression, n2² is an integer from 1 to 10.] and Ar 1 and Ar 2 Each instance independently has the same or different benzene ring structure, naphthalene ring structure, anthracene ring structure, or phenanthrene ring structure, L 4 It does not exist, or L 4 is -O-, -CH2-O-, -O-CH2-, -O-(CH2) n The vectors are -O-, -CH2-O-CH2-, or -OC(=O)-O-, where n is an integer from 1 to 10.

[0068] In one embodiment, L 1 is -CH2-(OCH2CH2) n11 -OCH2-[In the given expression, n11 is an integer from 1 to 10.] and L 2 is -CH2-(OCH2CH2) n11 -OCH2-[In the given expression, n2² is an integer from 1 to 10.] and Ar 1 and Ar 2 Each instance independently has the same or different benzene ring structure, naphthalene ring structure, anthracene ring structure, or phenanthrene ring structure, L 4 It does not exist, or L 4 is -O-, -CH2-O-, -O-CH2-, -O-(CH2) n -O-, -CH2-O-CH2-, or -OC(=O)-O-, where n is an integer from 1 to 10, L 3 These are -CO2-, -SO3-, or -CONH-.

[0069] In one embodiment, L 1 is -CH2-(OCH2CH2) n11-OCH2-[In the given expression, n11 is an integer from 1 to 10.] and L 2 is -CH2-(OCH2CH2) n11 -OCH2-[In the given expression, n2² is an integer from 1 to 10.] and Ar 1 and Ar 2 Each instance independently has the same or different benzene ring structure, naphthalene ring structure, anthracene ring structure, or phenanthrene ring structure, L 4 It does not exist, or L 4 is -O-, -CH2-O-, -O-CH2-, -O-(CH2) n -O-, -CH2-O-CH2-, or -OC(=O)-O-, where n is an integer from 1 to 10, L 3 is -CO2-, -SO3-, or -CONH-, and R 5 In each appearance, independently selected from the group consisting of unsubstituted or halogen-substituted linear or branched alkyl groups, unsubstituted or halogen-substituted linear or branched alkyloxy groups, halogen atoms, nitro groups, carboxylic acid groups or their derivatives, sulfonic acid groups or their derivatives, and amino groups or their derivatives, preferably R 5 In each occurrence, independently, these are: unsubstituted or halogenated linear or branched alkyl groups having 1 to 4 carbon atoms, unsubstituted or halogenated linear or branched alkyloxy groups having 1 to 4 carbon atoms, halogen atoms, nitro groups (NO2), carboxylic acid groups (CO2H) or their derivatives (CO2R c1 CONH2, CONHR c2 or CONR c3 R c4 R c1 , R c2 , R c3 , R c4 Each of these can independently be a linear or branched alkyl group (for example, having 1 to 4 carbon atoms), a sulfonic acid group (SO3H) or a derivative thereof (SO3R s1 SO2NH2, SO2NHR s2 or SO2NR s3 R s4 It is represented as R s1 , Rs2 , R s3 , R s4 Each is independently a linear or branched alkyl group (for example, having 1 to 4 carbon atoms), and an amino group or its derivative (NHR 11 , NR 21 R 22 , and N + R 31 R 32 R 33 (R 11 , R 21 , R 22 , R 31 , R 32 , R 33 Each of these is independently selected from the group consisting of linear or branched alkyl groups (for example, having 1 to 4 carbon atoms, but not limited to these).

[0070] In one embodiment, L 3 is -CO2-, -SO3-, or -CONH-, and R 5 In each appearance, independently selected from the group consisting of unsubstituted or halogen-substituted linear or branched alkyl groups, unsubstituted or halogen-substituted linear or branched alkyloxy groups, halogen atoms, nitro groups, carboxylic acid groups or their derivatives, sulfonic acid groups or their derivatives, and amino groups or their derivatives, preferably R 5 In each occurrence, independently, these are: unsubstituted or halogenated linear or branched alkyl groups having 1 to 4 carbon atoms, unsubstituted or halogenated linear or branched alkyloxy groups having 1 to 4 carbon atoms, halogen atoms, nitro groups (NO2), carboxylic acid groups (CO2H) or their derivatives (CO2R c1 CONH2, CONHR c2 or CONR c3 R c4 R c1 , R c2 , R c3 , R c4 Each of these can independently be a linear or branched alkyl group (for example, having 1 to 4 carbon atoms), a sulfonic acid group (SO3H) or a derivative thereof (SO3R s1SO2NH2, SO2NHR s2 or SO2NR s3 R s4 It is represented as R s1 , R s2 , R s3 , R s4 Each is independently a linear or branched alkyl group (for example, having 1 to 4 carbon atoms), and an amino group or its derivative (NHR 11 , NR 21 R 22 , and N + R 31 R 32 R 33 (R 11 , R 21 , R 22 , R 31 , R 32 , R 33 Each of these is independently selected from the group consisting of linear or branched alkyl groups (for example, having 1 to 4 carbon atoms, but not limited to these).

[0071] In one embodiment, L 3 is -CO2-, -SO3-, or -CONH-, L 4 It does not exist, or L 4 is -O-, -CH2-O-, -O-CH2-, -O-(CH2) n -O-, -CH2-O-CH2-, or -OC(=O)-O-, where n is an integer from 1 to 10, R 5 In each appearance, independently selected from the group consisting of unsubstituted or halogen-substituted linear or branched alkyl groups, unsubstituted or halogen-substituted linear or branched alkyloxy groups, halogen atoms, nitro groups, carboxylic acid groups or their derivatives, sulfonic acid groups or their derivatives, and amino groups or their derivatives, preferably R 5 In each occurrence, independently, these are: unsubstituted or halogenated linear or branched alkyl groups having 1 to 4 carbon atoms, unsubstituted or halogenated linear or branched alkyloxy groups having 1 to 4 carbon atoms, halogen atoms, nitro groups (NO2), carboxylic acid groups (CO2H) or their derivatives (CO2R c1CONH2, CONHR c2 or CONR c3 R c4 R c1 , R c2 , R c3 , R c4 Each of these can independently be a linear or branched alkyl group (for example, having 1 to 4 carbon atoms), a sulfonic acid group (SO3H) or a derivative thereof (SO3R s1 SO2NH2, SO2NHR s2 or SO2NR s3 R s4 It is represented as R s1 , R s2 , R s3 , R s4 Each is independently a linear or branched alkyl group (for example, having 1 to 4 carbon atoms), and an amino group or its derivative (NHR 11 , NR 21 R 22 , and N + R 31 R 32 R 33 (R 11 , R 21 , R 22 , R 31 , R 32 , R 33 Each of these is independently selected from the group consisting of linear or branched alkyl groups (for example, having 1 to 4 carbon atoms, but not limited to these).

[0072] In one embodiment, Ar 1 and Ar 2 Each instance independently has the same or different benzene ring structure, naphthalene ring structure, anthracene ring structure, or phenanthrene ring structure, L 3 is -CO2-, -SO3-, or -CONH-, L 4 It does not exist, or L 4 is -O-, -CH2-O-, -O-CH2-, -O-(CH2) n -O-, -CH2-O-CH2-, or -OC(=O)-O-, where n is an integer from 1 to 10, L 3is -CO2-, -SO3-, or -CONH-, and R 5 In each appearance, independently selected from the group consisting of unsubstituted or halogen-substituted linear or branched alkyl groups, unsubstituted or halogen-substituted linear or branched alkyloxy groups, halogen atoms, nitro groups, carboxylic acid groups or their derivatives, sulfonic acid groups or their derivatives, and amino groups or their derivatives, preferably R 5 In each occurrence, independently, these are: unsubstituted or halogenated linear or branched alkyl groups having 1 to 4 carbon atoms, unsubstituted or halogenated linear or branched alkyloxy groups having 1 to 4 carbon atoms, halogen atoms, nitro groups (NO2), carboxylic acid groups (CO2H) or their derivatives (CO2R c1 CONH2, CONHR c2 or CONR c3 R c4 R c1 , R c2 , R c3 , R c4 Each of these can independently be a linear or branched alkyl group (for example, having 1 to 4 carbon atoms), a sulfonic acid group (SO3H) or a derivative thereof (SO3R s1 SO2NH2, SO2NHR s2 or SO2NR s3 R s4 It is represented as R s1 , R s2 , R s3 , R s4 Each is independently a linear or branched alkyl group (for example, having 1 to 4 carbon atoms), and an amino group or its derivative (NHR 11 , NR 21 R 22 , and N + R 31 R 32 R 33 (R 11 , R 21 , R 22 , R 31 , R 32 , R 33Each of these is independently selected from the group consisting of linear or branched alkyl groups (for example, having 1 to 4 carbon atoms, but not limited to these). (Test reagents, method for differentiating and quantifying spermine and spermidine)

[0073] This disclosure provides a diagnostic reagent for detecting endogenous polyamines, including the compounds of this disclosure.

[0074] In one embodiment, the diagnostic reagent of the present disclosure further comprises another compound having a different affinity for spermine and spermidine than the aforementioned compound.

[0075] This disclosure provides a method for the fractional determination of spermine and spermidine, comprising the steps of contacting a sample with at least two compounds having different affinities to spermine and spermidine, and fractional determination of spermine and spermidine based on the differences in the affinities of the at least two compounds to spermine and spermidine.

[0076] In one embodiment, the at least two types of compounds include at least one of the above-mentioned compounds.

[0077] The testing and differential quantification methods disclosed herein can utilize compounds with different distances between binding sites to create molecules that allow for the differentiation and quantification of target polyamines. These can be used as novel diagnostic reagents for polyamines that have not been previously provided.

[0078] In one embodiment, the sum of spermidine and spermine can be determined using a suitable compound such as compound A-3, the concentration of spermine alone can be determined from the novel compound, and the concentration of spermidine can be determined from the difference between the two. In another embodiment, even if the selectivity for spermidine and spermine is not extremely high, it is possible to determine the individual concentrations from the degree of color development using multiple color-developing molecules. The principle is as follows.

[0079] Let X and Y be color-producing molecules. The association constants of X with spermidine and spermine are KXspd and KXspm, respectively, and their molar extinction coefficients are εXspd and εXspm. The association constants of X with respect to spermidine and spermine are KYspd, KYspm, and εYspd, εYspm, respectively. These values ​​can be measured using pure spermidine and spermine solutions alone.

[0080] Next, given a mixed solution of spermidine and spermine at unknown concentrations, if an excess amount of color-developing molecules is used relative to spermidine and spermine, and most of the spermidine and spermine (e.g., 95% or more) is incorporated into the color-developing molecules, then if the absorbances of these molecules are measured using color-developing molecules X and Y, and the absorbance when using X is denoted as AbsX and the absorbance when using Y is denoted as AbsY, then they can be expressed by the following equations. AbsX=εXspd[spermidine]+εXspm[spermine] AbsY=εYspd[spermidine]+εYspm[spermine] In other words, it is possible to determine the respective concentrations by solving a system of equations containing two unknowns measured under appropriate conditions.

[0081] Furthermore, by using Z in addition to X and Y as the color-developing molecule, it is possible to determine the concentrations of each combination of X and Y, X and Z, and Y and Z. By statistically analyzing these combinations using methods such as Student's t-test, it is possible to determine the concentrations more accurately.

[0082] In one embodiment, for example, the following compounds of this disclosure may be used as molecules corresponding to X, Y, Z, etc. above.

[0083] [ka] (The above compounds include alcoholic forms, ketone forms, and protonated ketone forms.)

[0084] By combining variations in substituents on the benzene ring, it is possible to synthesize many color-reactive molecules, enabling more accurate quantification of spermidine and spermine. Furthermore, compounds in which two phenol crown rings are cross-linked with oxygen exhibit fluorescence, allowing for more sensitive quantification using fluorescence response in addition to color response.

[0085] This disclosure provides a method for the fractional determination of spermine and spermidine, comprising contacting the compound of this disclosure with a sample and measuring the color or fluorescence using a spectrophotometer.

[0086] In this disclosure, (i) the compound is brought into contact with a sample and the color or fluorescence is measured with a spectrophotometer, (ii) Contact the sample with a compound different from the compound used in (i) above, and measure the color change or fluorescence with a spectrophotometer, and (iii) Calculate the amounts of spermine and spermidine based on the measurement results of steps (i) and (ii) above. This invention provides a method for the fractional quantitative determination of spermine and spermidine, characterized by the following features.

[0087] This disclosure provides a test or diagnostic agent for identifying diseases based on endogenous polyamines, or a test or diagnostic method that includes quantifying the amount of polyamines using the compounds of this disclosure. Identifying based on endogenous polyamines means any disease based on endogenous polyamines, such as cancer, Parkinson's disease, or Alzheimer's disease.

[0088] In one embodiment of the present disclosure, the present disclosure provides a method for identifying endogenous polyamine-based diseases in a subject, comprising measuring polyamines in the subject using the method of the present disclosure, and, based on the measured polyamine information, presenting an index of whether the subject is suspected of having an endogenous polyamine-based disease. Diseases to be identified may include cancer, Parkinson's disease, or Alzheimer's disease. For early diagnosis of Parkinson's disease, see, for example, Saiki, S et al. Ann Neurol. 2019 Aug; 86(2): 251-263. That is, in patients with Parkinson's disease... Diacetylspermidine has been identified as correlating with disease severity, and the conversion of spermidine to spermine is consistently reduced in Parkinson's disease patients regardless of age. It has been found that polyamines and their metabolites in the blood are biomarkers for early diagnosis and severity assessment of Parkinson's disease. It is understood that seven polyamines (spermine) and their metabolites (diacetylspermine, N1-acetylspermine, N8-acetylspermine, diacetylspermine, spermidine, N-1-acetylspermine, and spermine) in the serum of Parkinson's disease patients can serve as biomarkers for diagnosis and severity assessment of Parkinson's disease. The technology of this disclosure allows for the appropriate fractionation and quantification of these seven types.

[0089] However, in Parkinson's disease patients, spermine production, which is thought to have a longevity effect due to its autophagy-inducing action, is consistently reduced regardless of age. In the Parkinson's disease patient group, diacetylspermidine, N1-acetylspermidine, N8-acetylspermidine, diacetylspermine, and spermidine were significantly increased, while spermine was decreased. Of these metabolites, diacetylspermidine was found to be elevated in correlation with the severity of Parkinson's disease. Furthermore, it has been found that Parkinson's disease can be diagnosed with high probability based on the concentration of each of the seven polyamines, suggesting that they can be used as biomarkers. It has been found that not only dopaminergic neurons in the substantia nigra but also other nerve axonal networks are damaged in Parkinson's disease patients. It has also been found that the higher the diacetylspermidine level, the more severe the axonal damage in the brain, and the technology disclosed herein can be applied. The spermine / spermidine ratio is significantly lower in Parkinson's disease patients, and while the spermine / spermidine ratio decreases with age in healthy individuals, it remains low regardless of age in the Parkinson's disease patient group, so the method disclosed herein can be used for diagnosis. Among the seven polyamine compounds, spermine has the highest autophthongic effect in nervous system cells. It exhibits azithromycin-inducing ability, which can also be utilized.

[0090] In recent years, the spermine / spermidine ratio has been reported to be a biomarker for the early diagnosis of Parkinson's disease. Parkinson's disease is a progressive neurodegenerative disease that causes motor impairments such as tremors in the limbs and difficulty walking. With a prevalence of 140 people per 100,000, it is the second most common neurodegenerative disease in Japan and is designated as an intractable disease. Furthermore, it has been shown that spermidine levels in the body are significantly elevated in Parkinson's disease patients, while the levels of its metabolite, spermine, are decreased. For this reason, the ratio of the two can be used as a marker for the intractable disease Parkinson's disease (Shinji Saiki et al., ANN NEUROL, 2019, 86, 251-263). [Table 1-1]

[0091] Thus, using the technology disclosed herein, early diagnosis and assessment of disease severity of Parkinson's disease are possible by measuring blood polyamines and related metabolites. The spermine / spermidine ratio can be used for pre-symptomatic diagnosis in the very early preclinical and prodromal stages.

[0092] (Method for producing the compound disclosed herein) The compounds of general formula I described herein can be produced by the following methods.

[0093] In compounds of general formula I, L 4 If the bond is not directly ether-bonded, it can be produced by the following process.

[0094] [ka]

[0095] In the formula, Y is an oxygen protecting group, such as an allyl group. X is a halogen group, for example, a butyl group. RLi is an alkyllithium, for example, sec-butyllithium. L 1 , L 2 , L 3 , n 5 , R 5 Ar 1 and Ar 2 These are as specified independently in the specification.

[0096] RLi (alkyllithium) is added dropwise to a solution containing the compound of formula 15A, and then formula 16A is added to obtain the compound of formula 17A. The oxygen protecting group Y of formula 17A is deprotected to obtain the compound of compound 3A.

[0097] In compounds of general formula I, L 4 If the linking group is an ether crosslink, it can be manufactured by the following process. [ka]

[0098] In the formula, Y is an oxygen protecting group, such as an allyl group. RLi is an alkyllithium, for example, sec-butyllithium. X is a halogen group, R x1 , R x2 , and R x3 This is a leaving group, for example, a halogen (F, Cl, Br, I), L 1 , L 2 , L 3 , L 4 , n 5 , R 5 Ar 1 and Ar 2 This is as specified in the specification.

[0099] In compounds of general formula I, L 4 If it is a linking group, it can be manufactured by the following process. [ka]

[0100] In the formula, Y is an oxygen protecting group, such as an allyl group. RLi is an alkyllithium, for example, sec-butyllithium. X is a halogen group, R x1 , R x2 , and R x3 This is a leaving group, for example, a halogen (F, Cl, Br, I), L 1 , L 2 , L 3 , L 4 , n 5 , R 5 Ar 1 and Ar 2 This is as specified in the specification.

[0101] In compounds of general formula I, L 4 If it is an ether crosslink, it can be manufactured by the following process. [ka]

[0102] In the formula, L 1 , L 2 , L 3 , L 4 , n 5 , R 5 Ar 1 and Ar 2 These are as specified independently in the specification.

[0103] The compound of formula 15B is reacted with the compound of formula 16B and RLi (alkyllithium) to obtain the compound of formula 17X. The compound of formula 17X and R x L 3 The reaction is carried out to deprotect the oxygen protecting group Y and obtain the target compound 3B.

[0104] References such as scientific literature, patents, and patent applications cited herein are incorporated herein by reference to the same extent as they are specifically described herein.

[0105] The present disclosure has been described above with reference to preferred embodiments for ease of understanding. The present disclosure will be described in more detail below with reference examples, examples and test examples, which are provided for illustrative purposes only and the present disclosure is not limited thereto. The compound names shown in the following reference examples and examples do not necessarily follow IUPAC nomenclature. Abbreviations may be used for simplification, but these abbreviations are synonymous with those described above. The scope of the present disclosure is not limited to the embodiments or examples specifically described herein, but is limited only by the claims. [Examples]

[0106] (Example 1: Identifiable Compounds) We synthesized and evaluated prototype 4, in which the distance between binding sites was extended (Figure 1). In Figure 1b, only spermine shows a fainter coloration; this is because the coloring complex absorbs in the near-infrared region and is invisible to the naked eye. As can be seen from Figure 1c, we succeeded in creating approximately a three-fold difference in color intensity between spermidine and spermine, proving that our research approach was correct.

[0107] As shown in Figures 1b and 1c, compound 4 has the ability to distinguish between spermidine and spermine, but its selectivity is approximately 3 times greater in terms of absorbance, and its association constant with spermine is also 10. 6 It's been ordered and quoted.

[0108] [ka]

[0109] (Experimental Item 1: Determination of the composition ratio of the complex) We will confirm whether compound 4, whose synthesis has been successfully achieved, forms a 1:1 complex with spermine. This confirmation is directly related to whether spermine forms the expected bridging complex. When molecules with multiple binding sites associate, they may form unexpected complexes, which can be a major obstacle to subsequent improvement research. We will confirm the 1:1 complex using the mole ratio method, job plot method, and NMR analysis.

[0110] The composition ratio of compound 4, spermine, and the complex with spermine was confirmed to be 1:1 as expected by job plotting and DOSY NMR measurements.

[0111] (Experimental Item 2: Measurement of Association Constant) The association constants of compound 4 with spermine and spermidine will be determined through titration experiments. Since this study involves biological samples, the presence of water in the recognition site is unavoidable. Furthermore, this molecular recognition system is driven by strong ion pairs and hydrogen bonds following acid-base reactions between the phenol and amine moieties; however, the effectiveness of these is diminished in the presence of water. The association constants will be measured in various solvents to identify the solvent system in which compound 4 exhibits maximum function, and this information will be used for future research.

[0112] The association constant (Ka) and molar extinction coefficient (e) of compound 4, spermine, and spermidine were determined through titration experiments. The association constant (Ka) and molar extinction coefficient (e) in methanol at 25 degrees Celsius are as follows. Spermine: Ka = 1.3 x 10⁻⁶ 6 e = 6.5 x 10 4 Spermidine: Ka = 1.2 x 10⁻⁶ 5 e = 1.8 x 10 4 In other words, it was revealed that compound 4 exhibits an affinity for spermine that is approximately 10 times greater in terms of association constant and approximately 3.6 times greater in terms of molar extinction coefficient.

[0113] (Experimental Item 3: Synthesis of Candidate Compounds) The synthesis of compounds 5 through 10 will be carried out sequentially. The synthesis method for xanthones, in which benzene rings are extended in various directions to form the basic skeleton, has already been established. From previous synthetic studies, it has been found that constructing the two crown ether moieties is a challenge, but the synthesis will be achieved using the template method and the high-dilution method. In order to advance the synthetic studies more efficiently, separation and purification by column chromatography will be performed day and night using two preparative fraction collectors.

[0114] We semi-automated the separation and purification process using two preparative fraction collectors to synthesize the compounds. Although not the compounds originally planned, we synthesized compounds 223, 222, and 14 according to a new idea. We also succeeded in synthesizing key intermediate 236, which is directly linked to a compound in which two crown rings are bridged with ether. [ka]

[0115] (Experimental Item 4: Functional Evaluation) In Experiment Item 3, the function of the synthesized compound will be evaluated. This will involve utilizing the findings from Experiment Item 2 to measure the association constant and identify a compound that meets the aforementioned numerical target.

[0116] The function of the synthesized compounds was evaluated. The association constants of the evaluated compounds are shown above. The association constant is approximately 10. 6 The above large values ​​confirm high sensitivity. Compound 14 is a novel compound, consisting of a benzene ring on one side and a naphthalene ring on the other.

[0117] (Example 2: Detection of polyamines in vivo using phenolsulfonphthalein) Amidst the need for a quantitative method for polyamines that does not require complicated procedures, compound 101 developed by the present inventors forms a color-reactive complex by interacting with spermine and spermidine at three sites. This makes it possible to quantify the sum of spermine and spermidine. [ka] A color-reactive complex of compound 101 and spermidine.

[0118] Polyamines in the body are present at a concentration of 10 μM in the red blood cells of cancer patients, and in order to quantify polyamines using actual biological samples, a detection sensitivity of at least 10 μM is required. -5 A level of M or higher is required. However, the detection sensitivity of compound 101 did not reach that level. Therefore, by using phenolsulfonphthalein as the basic skeleton of the host molecule, the detection sensitivity was greatly improved, and 10 -7 We developed compound 102 with sensitivity M. The detection sensitivity of compound 102 is at a level suitable for practical use. [ka] A color complex of compound 102 and spermidine.

[0119] As shown above, compound 102 also forms a color-reactive complex similar to compound 101. In this case, it takes the form of a sulfonate ion, which is the conjugate base of a strong acid, and interacts more strongly with the amine moiety, resulting in improved detection sensitivity.

[0120] The development of compound 102 improved detection sensitivity, making it possible to quantify the sum of spermine and spermidine at a practical level. Therefore, the inventors aimed to further functionalize the compound and achieve selectivity for either spermine or spermidine. Focusing on the distance between substrate recognition sites of the host molecule and the length of the polyamine, the inventors succeeded in achieving selectivity for spermine by extending the basic skeleton to form compound 103, which is naphthol sulfonphthalein. [ka] The distance between the substrate recognition site and the length of the polyamine.

[0121] The association constants and molar extinction coefficients of compounds 101, 102, and 103 and their respective polyamines are shown below. [Table 1A] The association constant of compound 103 and spermine is 1.31 × 10⁻⁶. 6 M -1 This is because the association constant of spermidine is 1.19 × 10⁻⁶. 5 M -1 It showed a value approximately 10 times larger compared to [another compound]. This indicates that compound 103 has selectivity for spermine.

[0122] Focusing on the distance between substrate recognition sites in compound 102 and the length of the polyamine, we synthesized compound 103 with naphthol sulfonphthalein as its basic skeleton and successfully achieved selective recognition of spermine. However, compound 103 may adopt the structure shown below due to rotation of the C7-C2' bond, and a shorter distance between substrate recognition sites may result in a slight color response to spermidine as well. [ka] Distance between the conformation of compound 103 and the substrate recognition site.

[0123] Given this background, we hypothesized that it would be possible to quantify spermine and spermidine by synthesizing multiple host molecules and solving a system of equations, as shown in Equation 2-1 below, for each host molecule. Abs host = ε spd ×[spermidine] + ε spm ×[sprmine] (Equation 2-1)

[0124] The spermine / spermidine ratio has been reported to be a biomarker for the early diagnosis of Parkinson's disease, and this research was initiated with the goal of determining the spermine / spermidine ratio using the method described above.

[0125] (Development of length-recognizing molecules based on phenolsulfonphthalein) (Synthesis of novel phenolsulfonphthalein derivatives) Previous studies have demonstrated the quantitative determination of the sum of spermine and spermidine using phenolsulfonphthalein as a host molecule. However, selective quantitative determination of spermine and spermidine proved difficult. Therefore, we synthesized a novel phenolsulfonphthalein derivative compound 104, which has a longer distance between substrate recognition sites than phenolsulfonphthalein. We aimed to determine the relative abundance of spermine and spermidine by computation by using this compound in combination with the previously synthesized phenolsulfonphthalein. [ka] Structure of compound 104

[0126] First, compound 113, a component of compound 104, was synthesized using the synthetic route shown in Scheme 3-1. After brominating the 4th and 7th positions of compound 105 to synthesize the dibromo compound 106, compound 107 was obtained by reducing the bromine at the 4th position with tin. Next, compound 108 was synthesized by methyl esterification of the carboxylic acid, and compound 109 was obtained by subsequent formylation, which introduced a formyl group at the 4th position. Subsequently, compound 110 was synthesized by protecting the hydroxyl group with an allyl group. Further reduction with lithium aluminum hydride yielded compound 111, and chlorination yielded compound 112. Finally, compound 113 was obtained by reacting compound 112 with tetraethylene glycol. [ka] Scheme3-1. Synthetic route of compound 113

[0127] The synthesis route of compound 104 is shown in Scheme 3-2. First, the synthesized compounds 113 and 114 were reacted with s-butyllithium to obtain lithiated compounds by halogen metal exchange, and then reacted with o-sulfobenzoic acid cyclic anhydride 115, followed by dehydration with acid to obtain compound 117. Next, compound 117 was reacted with potassium carbonate as a base and Pd(PPh3)4 as a catalyst to deprotect the allyl group, thereby achieving the synthesis of the target compound 104. [ka] Scheme 3-2. Synthetic route of compound 104

[0128] (Functional evaluation of host-guest complexes for spermine and spermidine) (Evaluation of association constant and molar extinction coefficient) The host-guest complex of compound 104 with respect to spermine and spermidine was evaluated. First, titration was performed based on ultraviolet-visible absorption spectroscopy. A methanol solution of the guest molecule was added to a methanol solution of the host molecule, and the absorption spectrum was measured at each titration volume, tracking its changes. Subsequently, the association constant and molar extinction coefficient were calculated using the nonlinear least squares method. In performing the analysis, it was assumed that a fast equilibrium exists between the colored and colorless complexes, and the obtained values ​​were treated as reflecting the weighted average of the two types of complexes. Furthermore, all titration results could be analyzed using a 1:1 complex formation model. From this, it was considered that the host-guest complex is a 1:1 bridging complex.

[0129] Figure 6(a) shows the absorption spectrum obtained when compound 104 was titrated with spermidine as a guest molecule. Figure 6(b) also shows the spectrum showing the difference between the host molecule and the complex. In the spectrum shown in (a), the absorption at 424 nm originating from compound 104 gradually decreases, accompanied by an isosbestic point, and a new absorption appears at 574.5 nm. This can be interpreted as compound 104 forming a complex with spermidine, expanding the resonance system and showing absorption on the longer wavelength side.

[0130] Next, Figure 7(a) shows the absorption spectrum obtained when compound 104 was titrated with spermine as a guest molecule. Figure 7(b) also shows the spectrum showing the difference between the host molecule and the complex. In the spectrum shown in (a), the absorption at 424 nm originating from compound 104 gradually decreases, accompanied by an isosbestic point, and a new absorption appears at 588.5 nm. This can be interpreted as compound 104 forming a complex with spermine, expanding the resonance system and showing absorption on the longer wavelength side. Furthermore, a wavelength increase of 14 nm was observed compared to the case of the spermidine complex. This is thought to be due to the destabilization of the ground state of compound 104 during complex formation with spermine, resulting in a decrease in the HOMO-LUMO energy gap.

[0131] The change in absorbance at the maximum absorption wavelength of the complex was plotted on the vertical axis, and the volume of host solution added on the horizontal axis. The association constant (K) and molar extinction coefficient (ε) of compound 104, spermine, and spermidine were calculated using a nonlinear least squares method. Figure 8 shows the change curves for each guest compound.

[0132] The association constants and molar extinction coefficients for spermine and spermidine calculated below are shown. For comparison, data for compounds 102 and 103 are also included. [Table 1B]

[0133] The association constant between compound 104 and spermidine was on the order of approximately 100,000, and the association constant with spermine was on the order of approximately 1,000,000. This indicates that compound 104 selectively associates with spermine, and we believe that this selectivity is due to an extended distance between the substrate recognition sites compared to phenolsulfonphthalein. Furthermore, the molar extinction coefficient was smaller compared to compounds 102 and 103.

[0134] Next, we compared the maximum absorption wavelength with that of a base that does not form a crosslinking complex. The absorption spectra of the host molecule, the host molecule after base addition, and the host molecule after spermine addition are shown below (Figure 9). When coloration was achieved with sodium hydroxide without structural constraints, absorption appeared 18.5 nm shorter than in the case of spermine. This suggests that the two aromatic rings are significantly twisted when the crosslinking complex is formed. Furthermore, the absorbance when spermine was added was smaller than when sodium hydroxide was added, confirming that the molar extinction coefficient was indeed smaller.

[0135] Based on these results, we hypothesize that structural distortion occurs during cross-linking complex formation, and because the π electrons do not spread easily in a structure that is significantly more unstable than the most stable structure, the molar extinction coefficient becomes smaller.

[0136] (Quantitative determination of spermine and spermidine using simultaneous equations) We attempted to quantify spermine and spermidine using multiple host molecules. In the following, spermidine will be referred to as A and spermine as B.

[0137] First, the absorbance when a host molecule solution is added to a mixed solution of A and B to form a complex can be expressed as follows (Equation 3-1) using the molar extinction coefficients of each complex determined experimentally. Abs = ε A [Complex A] + ε B [Complex B] (Formula 3-1) (ε A : Molar extinction coefficient of complex A, ε B (Molar extinction coefficient of complex B)

[0138] Here, if there is an excess amount of host molecules and the complex formation rate with guest molecules is 99% or higher, we can assume that all guest molecules exist as complexes. Therefore, [Complex A] = [A], [Complex B] = [B] By approximating this, (Equation 3-1) can be replaced with (Equation 3-2) below. Abs = ε A [A] + ε B [B] (Formula 3-2)

[0139] Abs and ε A , ε B Since this can be measured, we thought that we could find the unknowns [A] and [B] by setting up (Equation 3-2) for multiple host molecules and solving it as a system of equations.

[0140] A mixed solution of spermine and spermidine was added to the host molecule solution, and ultraviolet-visible absorption spectroscopy was performed. The concentrations in the solution and the measured absorbance values ​​under each condition are shown below. [Table 1C]

[0141] The difference between the absorbance measured under each concentration condition and the absorbance of the host molecule alone was calculated and is shown below. This value represents the absorbance derived from the complex. [Table 1D]

[0142] The molar extinction coefficient was calculated by setting up the following system of equations using the obtained absorbance values ​​(Equations 3-3, 3-4). The solution to the system of equations was found for all combinations, and the average value of these solutions was taken as the molar extinction coefficient. Abs1 = ε spm × [spm]1+ ε spd × [spd]1 (Equation 3-3) Abs² = ε spm × [spm]² + ε spd × [spd]2 (Equation 3-4) [Table 1E]

[0143] The measured absorbance and calculated molar extinction coefficient values ​​were substituted into Equation 3-2 to solve the system of equations, and the calculated concentrations of spermine and spermidine are shown below. Furthermore, the actual and calculated concentrations were plotted with spermidine concentration on the vertical axis and spermine concentration on the horizontal axis (Figure 10). [Table 1F]

[0144] As described above, the actual concentrations and calculated values ​​are close, demonstrating that the inventors have successfully quantified the concentrations of spermine and spermidine using multiple host molecules. Furthermore, it is believed that the measurement error can be further reduced by increasing the number of host molecules.

[0145] (Development of carbon-crosslinked phenolsulfonphthalein derivatives) (Synthesis of substrates with introduced carbon crosslinks)

[0146] The goal was to control the steric properties of phenolsulfonphthalein by introducing carbon bridges and to investigate the behavior observed in complex formation and color response. We planned the synthesis of compound 126. The synthetic route is shown below (Scheme 4-1). [ka] Scheme 4-1. Synthetic route of compound 126

[0147] The synthesis route of compound 129 is shown in Scheme 4-2. First, the carboxylic acid of commercially available compound 130 was esterified to obtain compound 131, and then compound 118 was synthesized by hydroxymethylation. Next, bromine was introduced using bromine to obtain compound 119. Subsequently, regioselective MOM protection of the hydroxyl group was performed to obtain compound 132, and then the other hydroxyl group was protected with an allyl group to synthesize compound 133. Further reduction with lithium borohydride followed by chlorination of the hydroxyl group yielded compound 135. Finally, tetraethylene glycol and a base were reacted to form a crown ether ring, and the synthesis of the target compound 129 was achieved in a total yield of 20%. [ka] Scheme 4-2. Synthetic route of compound 129

[0148] We aimed to synthesize a phenolsulfonphthalein derivative with a carbon bridge by performing a coupling reaction using the synthesized compound 129. The synthesis route of compound 139 is shown in Scheme 4-3. First, the synthesized compound 129 was reacted with s-butyllithium to exchange bromine and lithium, and then coupled by nucleophilic attack with compound 115 to obtain reaction intermediate 136. Next, compound 137 was obtained by dehydration cyclization with acid. The phenol hydroxyl group of compound 137 was carbon bridged to obtain compound 138. Subsequently, the allyl group was deprotected to achieve the synthesis of the target compound 139. [ka] Scheme 4-3. Synthetic route of compound 139

[0149] (Experimental section) General operations Tetrahydrofuran (THF) was dried over metallic sodium and distilled immediately before use with benzophenone ketyl as an indicator. All reactions were performed using thin-layer chromatography (TLC; Silica gel 60 F). 254 The samples were tracked using one or a combination of the following methods on a 0.25 mm Merck (0.25 mm) scale: (1) UV irradiation observation, (2) immersion in phosphomolybdic acid aqueous solution, and (3) heating on a hot plate. A SilicaFlash F60 (SiliCycle) was used for normal-phase column chromatography. For gel permeation chromatography, two columns, Shodex GPC H-2001L and Shodex GPC H-2002L, were linked together, and chloroform (flow rate 3.5 mL / min) was used as the eluent.

[0150] Physical properties and spectral measurements The melting point was measured using Yanagimoto Seisakusho Micro Melting Point Apparatus MP-J3 and is uncorrected. Nuclear magnetic resonance (NMR) spectra were obtained using JEOL JNM-ECZS 400. 1 H; 400 MHz, 13 The measurement was performed at C (100 MHz). The standard substance was deuterated chloroform when used as the solvent. 1 In H, tetramethylsilane was added at 0.00 ppm. 13 In C, when CDCl3 was set to 77.0 ppm and deuterium methanol was used as the solvent, 1 In H, CD3OD was 3.30 ppm. 13The concentration at C was set to 49.00 ppm. The chemical shift (δ) of the physical properties is expressed in ppm, and the coupling constant (J) is expressed in Hz. Peak multiplicity is abbreviated as follows: s; singlet, d; doublet, t; triplet, q; quartet, br; broadened, m; multiplet. Infrared absorption (IR) spectra were measured using a JASCO FT / IR-4600 spectrometer, and the values ​​were measured in cm. -1 The results were displayed as follows: High-resolution mass spectrometry (HRMS) was performed using SolariX FT-ICR-MS (Bruker). Ultraviolet-visible absorption (UV) spectroscopy was performed using a JASCO V-650 spectrometer. Fluorescence (FL) spectroscopy was performed using a JASCO FP-8600 spectrometer.

[0151] Instruments and solvents used in the instrumental analysis Deuterated chloroform (CDCl3) was used at 99.8% atm% D (Cambridge Isotope Laboratories, Inc., +0.05% v / v% TMS). Deuterated methanol was used at 99.8% atm% D (Cambridge Isotope Laboratories, Inc., +0.05% v / v% TMS). For ultraviolet-visible absorption (UV) spectroscopy measurements, a spectroscopic solvent purchased from Wako Pure Chemical Industries was used.

[0152] (Synthesis of compound 109) [ka] Compound 108 (6.75 g, 24.01 mmol) was dissolved in dichloromethane (100 mL), then titanium tetrachloride (5.27 mL, 48.02 mmol) and dichloromethyl methyl ether (6.39 mL, 72.03 mmol) were added. The mixture was then heated until the dichloromethane refluxed and stirred for 16 hours. The reaction solution was then cooled to room temperature, and water was added to quench the reaction. The mixture was extracted with chloroform and washed with water and saturated brine. The extract was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the solvent was removed by distillation under reduced pressure. Compound 109 (7.00 g, 94%) was obtained as a yellow solid.

[0153] compound 109: yellow powder; mp = 192-193°C; IR(KBr)3072, 2956, 2889, 1670, 1576, 1500 cm -1 ; 1 H-NMR (400 MHz, CDCl3) δ 11.83 (s, 1H), 10.92 (s, 1H), 9.09 (d, J = 8.8 Hz, 1H), 8.61 (s, 1H), 7.99 (d, J = 2.0 Hz, 1H), 7.76 (dd, J =9.2, 2.4 Hz, 1H), 4.08 (s, 3H); 13 C-NMR(100 MHz, CDCl3) δ 191.3, 169.2, 163.9, 139.1, 135.6, 133.2, 131.8, 127.8, 126.1, 119.0, 115.1, 53.4; HRMS (ESI) calcd. for C 13 H9 79 BrO4Na ([M + Na] + ) 330.9576. found 330.9577, calcd. for C 13 H9 81 BrO4Na ([M + Na] + ): 332.9556, found 332.9557.

[0154] (Synthesis of compound 110) [ka]

[0155] Compound 109 (617.3 mg, 1.997 mmol) was dissolved in N,N-dimethylformamide (10 mL), and potassium carbonate (690.0 mg, 4.993 mmol), sodium iodide (29.9 mg, 0.1997 mmol), and allyl bromide (259 μl, 2.996 mmol) were added in sequence. The mixture was stirred at room temperature for 14 hours. Then, 1 M HCl aq. was added to stop the reaction. The mixture was extracted with ethyl acetate and washed with water and saturated brine. After drying the extract over anhydrous sodium sulfate, the drying agent was filtered off, and the solvent was removed by distillation under reduced pressure. Compound 110 (613.3 mg, 88%) was obtained as a white solid.

[0156] compound 110: white powder; mp = 118-119°C; IR (KBr) 3421, 3070, 2947, 2904, 2362, 1709, 1684, 1581 cm -1 ; 1 H-NMR (400 MHz, CDCl3) δ 10.79 (s, 1H), 9.11 (d, J = 9.6 Hz, 1H), 8.53 (s, 1H), 8.04 (d, J = 2.0 Hz, 1H), 7.77 (dd, J = 9.4, 2.0 Hz, 1H), 6.13 (m, 1H), 5.42 (ddd, J = 17.0, 2.8, 1.6 Hz, 1H), 5.34 (ddd, J = 10.4, 2.2, 1.2 Hz, 1H), 4.68 (ddd, J = 6.0, 2.0, 1.2 Hz, 2H), 4.00 (s, 3H); 13 C-NMR (100 MHz, CDCl3) δ 192.3, 1652, 162.4, 138.9, 134.6, 132.1, 131.2, 131.1, 130.8, 127.1, 125.3, 124.2, 120.8, 119.8, 79.2, 52.8; HRMS (ESI) calcd. for C 16 H 1379 BrO4Na ([M + Na] + ) 370.9889. found: 370.9889. calcd. for C 16 H 13 81 BrO4Na ([M + Na] + ): 372.9869, found 372.9869.

[0157] (Synthesis of compound 111) [ka] Compound 110 (100 mg, 0.286 mmol) was dissolved in dry tetrahydrofuran (5 mL) and cooled to 0°C. Lithium aluminum hydride (32.4 mg, 0.858 mmol) was slowly added in small amounts and stirred for 5 hours. The reaction was stopped by adding 1 M HCl aq. The solution was extracted with ethyl acetate and washed with 1 M HCl aq., water, and saturated brine. The extract was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the solvent was removed by vacuum distillation. Compound 111 (81.2 mg, 88%) was obtained as a white solid.

[0158] compound 111: white powder; mp = 154-156°C; IR (KBr) 3305, 2978, 2872, 2359, 1591 cm -1 ; 1 H-NMR (400 MHz, CDCl3) δ 8.11 (d, J = 9.2 Hz, 1H), 8.02 (d,J = 2.0 Hz, 1H), 7.87 (s, 1H), 7.59 (dd, J = 9.2 Hz, 2.0 Hz, 1H), 6.17 (m, 1H), 5.50 (ddd, J = 17.2 Hz, 1.6 Hz, 1.6 Hz, 1H), 5.29 (ddd, J = 10.4, 1.6, 1.2 Hz, 1H), 5.08 (s, 2H), 4.82 (d, J = 0.4 Hz, 2H), 4.53 (ddd, J = 5.6, 1.6, 1.2 Hz, 2H);13 C-NMR (100 MHz, CDCl3) δ 155.1, 137.3, 135.1, 133.8, 132.8, 131.1, 130.3, 128.8, 128.0, 127.6, 119.8, 117.7, 77.8, 60.7, 55.9; HRMS (ESI) calcd. for C 15 H 15 79 BrO3Na ([M + Na] + ): 345.0096, found 345.0096. calcd. for C 15 H 15 81 BrO3Na ([M + Na] + ) 347.0076, found 374.0076.

[0159] (Synthesis of Compound 112) [ka] Compound 112 (2.94 g, 9.09 mmol) was dissolved in chloroform (50 mL). Thionyl chloride (1.98 mL, 27.27 mmol) and N,N-dimethylformamide (0.5 mL) were added, and the mixture was stirred at room temperature for 24 hours. Water was added to stop the reaction. The mixture was extracted with chloroform and washed with water and saturated brine. The extract was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the solvent was removed by vacuum distillation. The residue was purified by silica gel column chromatography (chloroform:methanol = 50:1) to obtain compound 112 (2.81 g, 86%) as a white solid.

[0160] compound 112: white powder; mp = 125-126°C; IR (KBr) 2987, 2870, 2360, 1649, 1587 cm -1 ; 1H-NMR (400 MHz, CDCl3) δ 7.99 (d, J = 2.0 Hz, 1H), 7.96 (d, J = 9.2 Hz, 1H), 7.86 (s, 1H), 7.67 (dd, J = 8.8 Hz, 2.0 Hz, 1H), 6.25-6.15 (m, 1H) 5.57 (ddd, J = 17.2 Hz, 2.8 Hz, 1.2 Hz, 1H), 5.38 (dd, J =10.4 Hz, 1.2 Hz, 1H), 5.09 (s, 2H), 4.79 (s, 2H), 4.69 (d, J = 5.2 Hz, 2H) 13 C-NMR (100 MHz, CDCl3) 154.1, 132.9, 131.1, 131.0, 130.9, 130.6, 125.5, 125.3, 119.8, 118.3, 76.6, 41.3, 37.3; HRMS (ESI) calcd. for C 15 H 13 79 Br 35 Cl2ONa ([M + Na] + ) 380.9419, found 380.9417,calcd. for C 15 H 13 81 Br 35 Cl2ONa ([M + Na] + ) or C 15 H 13 79 Br 35 Cl 37 ClONa ([M + Na] + ) 382.9397, found 382.9397, calcd. for 81 Br 35 Cl 37 ClONa ([M + Na] + ) or 79 Br 37 Cl2ONa ([M + Na] + ) 384.9369, found 382.9397, 81 Br 37 Cl2ONa ([M + Na] +) 368.9369, found 368.9368.

[0161] (Synthesis of compound 113) [ka] Compound 113 (4.55 g, 12.64 mmol) and tetraethylene glycol (2.18 mL, 12.64 mmol) were dissolved in 50 mL of dry tetrahydrofuran and placed in an isobaric dropping funnel. Sodium hydride (1.51 g, 37.92 mmol) and potassium iodide (3.15 g, 18.96 mmol) were dissolved in 100 mL of dry tetrahydrofuran and placed in a round-bottom flask. The temperature was raised until the tetrahydrofuran refluxed, and the apparatus was assembled to allow for gradual dropwise addition from the dropping funnel into the flask. After the addition was complete, the mixture was stirred for 27 hours. The temperature was lowered to room temperature, and then quenched with 1 M HClaq. Extraction was performed with ethyl acetate, and the mixture was washed with 1 M HClaq, water, and saturated brine. The extract was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the solvent was removed by vacuum distillation. The residue was purified by silica gel column chromatography (hexane:ethyl acetate = 2:1) to obtain compound 113 (3.21 g, 52%) as a white solid.

[0162] compound 113: white powder; mp = 124-126°C; IR (KBr) 2875, 1589, 1113 cm -1 ; 1H-NMR (400 MHz, CDCl3) 7.96 (d, J = 8.8 Hz, 1H), 7.92 (d, J = 2.0 Hz, 1H), 7.65 (s, 1H), 7.55 (dd, J = 9.2 Hz, 2.4 Hz, 1H), 6.23 (m, 1H), 5.58 (dddd, J = 17.2, 2.0, 2.0, 2.0, 1H), 5.27 (dddd, J = 10.4, 2.0, 2.0, 2.0, 1H), 5.12 (d, J = 10.8 Hz, 1H), 5.06 (dddd, J = 13.6, 4.8, 1.6, 1.6 Hz, 1H), 5.00 (d, J = 10.0 Hz, 1H), 4.93 (d, J = 10.0 Hz, 1H), 4.83 (dddd, J = 13.6, 5.2, 1.6,1.6 Hz, 1H), 4.30 (d, J = 10.8 Hz, 1H) 3.86-3.34 (m, 16H); 13 C-NMR (100 MHz, CDCl3) δ 156.5, 135.9, 132.7, 132.5, 131.4, 130.5, 130.0, 129.7, 126.4, 125.8, 118.5, 115.2, 77.5, 70.6, 70.4, 70.3, 70.2, 70.1, 69.9, 69.8, 68.4, 63.6; HRMS (ESI) calcd. for C 23 H 29 79 BrO6Na ([M + Na] + ) 503.1039, found 503.1035, calcd. for C 23 H 29 81 BrO6Na ([M + Na] + ) 505.1020, found 505.1015.

[0163] (Synthesis of compound 117)

change

[0164] Compound 117: white powder; 1 H-NMR (400 MHz, CDCl3) 8.12-8.07 (m, 1H), 7.87 (d, J = 6.9 Hz, 1H), 7.79 (d, J = 6.4 Hz, 1H), 7.71 (s, 1H), 7.64 (m, 2H), 7.53 (t, J = 8.2 Hz, 1H), 7.47 (s, 1H), 7.23 (m, 1H), 7.18 (d, J = 5.0 Hz, 1H), 6.27-6.14 (m, 2H), 5.54 (dd, J = 28.8, 17.4 Hz, 2H), 5.24 (dd, J = 19.2, 10.5 Hz, 2H), 5.15-5.05 (m, 2H), 5.00-4.94 (m, 4H), 4.87-4.82 (m, 3H), 4.27-4.23 (m, 1H), 4.08 (t, J = 4.8 Hz, 2H), 3.82-3.36 (m, 34H)

[0165] (Synthesis of compound 104) [ka] Compound 117 (30.0 mg, 0.0325 mmol) was dissolved in methanol (1.0 mL), and Pd (PPh3)4 (0.8 mg, 0.00065 mmol) and potassium carbonate (13.5 mg, 0.0977 mmol) were added. After stirring at room temperature for 3 hours, Amberlyst 15 ion-exchange resin (60 mg) was added, and the mixture was stirred for 1.5 hours. After filtering out the Amberlyst 15 ion-exchange resin, the solvent was removed by distillation under reduced pressure. Compound 104 (20.2 mg, 74%) was obtained as an orange solid.

[0166] Compound 104: orange powder; 1 H-NMR (400 MHz, CDCl3) δ 8.75 (s, 1H), 8.36 (d, J = 8.2 Hz, 1H), 7.93 (d, J = 9.1 Hz, 1H), 7.85 (d, J = 1.8 Hz, 1H), 7.66 (s, 1H), 7.64 (d, J = 2.7 Hz, 1H), 7.56 (dd, J = 8.7, 1.8 Hz, 1H), 7.51-7.47 (m, 1H), 7.34-7.31 (m, 1H), 6.93 (d, J = 6.9 Hz, 1H), 5.15 (dd, J = 15.3, 11.2 Hz, 2H), 4.93 (d, J = 9.6 Hz, 1H), 4.80 (q, J = 11.0 Hz, 2H), 4.71 (d, J = 9.1 Hz, 1H), 3.76-3.41 (m, 36H)

[0167] (Synthesis of Compound 140) [ka] Compound 113 (1 g, 2.08 mmol) was dissolved in dry THF (20 mL) and cooled to -78°C. Then, n-BuLi (1.4 mL, 2.16 mmol) was slowly added dropwise over 5 minutes, and the mixture was stirred for 25 minutes. Separately from the reaction solution, o-sulfobenzoic acid cyclic anhydride (150 mg, 0.830 mmol) was dissolved in dry THF (20 mL), and this prepared THF solution was slowly added dropwise to the reaction solution using a syringe over 15 minutes, and the mixture was stirred for 2.75 hours. The mixture was allowed to return to room temperature, extracted with ethyl acetate, washed with water and saturated aqueous solution, dried over anhydrous sodium sulfate, filtered off the drying agent, and the solvent was removed by vacuum distillation. Impurities were removed from the residue by silica gel column chromatography (chloroform:methanol = 30:1 to 15:1), and several highly polar components were obtained by replacing the developing solvent with methanol. The obtained highly polar components were dissolved in (tetrahydrofuran / 1 M HCl aq. = 1:1) and stirred at room temperature for 16 hours. Extraction was performed with ethyl acetate and washed with 1 M HCl aq. and saturated brine. After drying the extract over anhydrous sodium sulfate, the drying agent was filtered off and the solvent was removed by distillation under reduced pressure. The residue was purified by silica gel chromatography (chloroform:methanol = 40:1), and after drying, the resulting pale yellow amorphous material was washed with a small amount of methanol to obtain compound 140 (301 mg, 37%) as a white solid.

[0168] Compound 140: white powder; mp = 176-178°C; IR (KBr) 3428, 2869, 1631, 1604, 1504, 1452, 1349, 1132, 1110 cm -1 ; 1H-NMR (400 MHz, CDCl3) δ 8.14 (d, J = 9.0 Hz, 2H), 7.92-7.90 (m, 1H), 7.76-7.69 (m, 3H), 7.68-7.64 (m, 1H), 7.64 (d, J = 1.8Hz, 2H), 7.50 (dd, J = 9.0, 2.4 Hz, 2H), 7.47-7.42 (m, 1H), 6.28-6.21 (m, 2H), 5.62-5.56 (m, 2H), 5.29-5.25 (m, 2H), 5.16-5.13 (m, 2H), 5.11-5.06 (m, 2H), 5.04-5.00 (m, 2H), 4.98-4.94 (m, 2H), 4.90-4.83 (m, 2H), 4.28-4.24 (m, 2H), 3.87-3.36 (m, 32H), 13 C-NMR (100 MHz, CDCl3) δ 157.9, 157.5, 141.4, 141.3, 141.2, 136.2, 136.0, 136.0, 135.9, 135.6, 134.3, 134.2, 134.2, 134.1, 133.5, 133.5, 133.4, 132.5, 132.5, 132.4, 132.4, 132.4, 132.3, 132.3, 129.5, 129.4, 129.3, 129.2, 127.9, 127.7, 127.4, 127.3, 126.5, 126.4, 126.3, 126.3, 126.2, 126.2, 126.2, 125.9, 125.6, 125.5, 125.1, 125.1, 124.9, 124..8, 122.2, 122.2, 122.1, 115.3, 115.3, 96.9, 96.9, 6.8, 77.7, 70.8, 70.07, 70.7, 70.7, 70.4, 70.3, 70.3, 70.3, 70.3, 70.2, 70.2, 70.2, 70.2, 70.1, 70.0, 70.0, 69.9, 69.8, 69.8, 68.3, 68.3, 68.2, 68.1, 63.7, 63.7, 63.6; HRMS (ESI) calcd. for C 53 H 62 O15 SNa ([M + Na] + ) 993.3701, found 993.3686.

[0169] (Synthesis of compound 103) [ka]

[0170] Compound 140 was dissolved in methanol (1 ml), and Pd(PPh3)4 (0.7 mg, 0.000618 mmol) and potassium carbonate (12.8 mg, 0.0926 mmol) were added. The mixture was stirred at room temperature for 4 hours, and then Amberlyst 15 ion-exchange resin (60 mg) was added and the mixture was stirred for 1 hour. After filtering out the Amberlyst 15 ion-exchange resin, the solvent was removed by distillation under reduced pressure. Compound 103 (17.2 mg, 63%) was obtained as a yellow solid.

[0171] Compound 103: dark yellow powder; 232°C (decomp.); IR (KBr) 3423, 2904, 2867, 1614, 1417, 1351, 1299, 1253, 1184 cm -1 ; 1 H-NMR (400 Hz, CD3OD) δ 8.07 (d, J = 7.6 Hz, 1H), 7.65 (dd, J = 2.0 Hz, 2H), 7.56-7.51 (m, 3H), 7.50 (s, 2H), 7.47 (ddd, J = 9.2, 9.2, 1.6 Hz, 1H), 7.18 (dd, J = 9.6, 2.0 Hz, 2H), 7.05 (d, J = 6.4 Hz, 1H), 4.74 (s, 4H), 4.39 (s, 4H), 3.69-3.49 (m, 32H); 13C-NMR (100 MHz, CD3OD) δ 146.2, 142.8, 140.3, 139.5, 137.1, 134.7, 133.0, 132.6, 132.2, 130.7, 130.5, 129.4, 126.6, 122.4, 121.4, 71.9, 71.3, 71.2, 71.0, 70.9, 70.8, 70.7, 70.5, 70.1, 64.8; 47 H 54 O 15 S ([M] - ), 889.3099, found 889.3099.

[0172] (Synthesis of Compound 141) [ka]

[0173] Compound 114 (292.0 mg, 0.678 mmol) was dissolved in dry THF (9 mL) and cooled to -78°C. Then, s-BuLi (722 μL, 0.7588 mmol) was slowly added dropwise, and the mixture was stirred for 15 minutes. Separately from the reaction solution, o-sulfobenzoic acid cyclic anhydride (50 mg, 0.271 mmol) was dissolved in dry THF (1.5 mL), and this prepared THF solution was slowly added dropwise to the reaction solution using a syringe, and the mixture was stirred at room temperature for 42 hours. 1 M HCl aq. (1.0 ml) was added, and the mixture was stirred for a further 9 hours. The solvent was removed by distillation under reduced pressure, methanol (2 ml) was added to the residue, and the precipitated solid was purified by filtration. The filtrate was further purified by silica gel column chromatography (chloroform:methanol = 5:1) to obtain compound 141 (87.6 mg, 37%) as a white solid.

[0174] Compound 141: white powder; 1H-NMR (400 MHz,CDCl3) δ 7.85 (d, J = 7.8 Hz, 1H), 7.71 (td, J = 7.5, 1.1 Hz, 1H), 7.66-7.61 (m, 1H), 7.41 (d, J = 7.8 Hz, 1H), 7.31 (d, J = 2.7 Hz, 2H), 7.17 (d, J = 2.7 Hz, 2H), 6.23-6.13 (m, 2H), 5.50 (dq, J = 17.3, 1.8 Hz, 2H), 5.22 (dd, J = 10.5, 2.3 Hz, 2H), 4.99-4.91 (m, 4H), 4.86 (td, J = 10.1, 5.5 Hz, 4H), 4.17-4.04 (m, 4H), 3.70-3.41 (m, 36H)

[0175] (Synthesis of compound 119) [ka] Compound 118 (6.72 g, 33.91 mmol) was dissolved in diethyl ether (130 ml), and Br2 (1.75 ml, 33.91 mmol) was added at 0°C. The mixture was stirred for 10 minutes, and the reaction was stopped by adding saturated sodium sulfite aqueous solution. The solution was extracted with ethyl acetate and washed with water and saturated brine. After drying the extract over anhydrous sodium sulfate, the drying agent was filtered off, and the solvent was removed by distillation under reduced pressure. The resulting solid was washed with chloroform to obtain compound 119 (9.06 g, 96%) as a white solid.

[0176] Compound 119: white powder; 1 H-NMR (400 MHz, CDCl3) δ 11.22 (s, 1H), 8.17 (s, 1H), 7.96 (s, 1H), 5.01 (s, 2H), 3.93 (s, 3H); 13 C-NMR (100 MHz, CDCl3) δ 169.8, 159.8, 157.8, 132.8, 113.5, 106.5, 101.2, 57.7, 52.6

[0177] (Synthesis of Compound 132) [ka] Compound 119 (2.00 g, 7.218 mmol) was dissolved in acetone (100 ml), and potassium carbonate (997.6 mg, 7.218 mmol) and chloromethyl methyl ether (548 μL, 7.218 mmol) were added. The mixture was then stirred at room temperature for 16.5 hours. Water was added to stop the reaction. Ethyl acetate was added for extraction, the mixture was washed with water and saturated brine, and dried over anhydrous sodium sulfate. After filtering off the drying agent, the solvent was removed by reduced pressure. The residue was purified by silica gel column chromatography (hexane:ethyl acetate = 3:1) to obtain compound 132 (1.61 g, 70%) as a white solid.

[0178] Compound 132: white powder; 1 H-NMR (400 MHz, CDCl3) δ 11.24 (s, 1H), 8.04 (s, 1H), 5.16 (s, 2H), 4.77 (d, J = 7.3 Hz, 2H), 3.96 (s, 3H), 3.68 (s, 3H), 3.00 (t, J = 7.1Hz, 1H)

[0179] (Synthesis of Compound 133) [ka]

[0180] Compound 132 (100 mg, 0.311 mmol) was dissolved in DMF (2 ml), and potassium carbonate (47.2 mg, 0.342 mmol) and allyl bromide (29.6 μL, 0.342 mmol) were added. The mixture was then stirred at room temperature for 15.5 hours. Water was added to stop the reaction, ethyl acetate was added for extraction, the mixture was washed with water and saturated brine, and dried over anhydrous sodium sulfate. After filtering off the drying agent, the solvent was removed by distillation under reduced pressure. The residue was purified by silica gel column chromatography (hexane:ethyl acetate = 5:1) to obtain compound 133 (78.0 mg, 69%) as a white solid.

[0181] Compound 133: white powder; 1 H-NMR (400 MHz, CDCl3) δ 8.07 (s, 1H), 6.14 (m, 1H), 5.43 (dd, J = 16.9, 1.4 Hz, 1H), 5.29 (d, J = 10.5 Hz, 1H), 5.17 (s, 2H), 4.71 (d, J = 6.9 Hz, 2H), 4.59 (d, J = 5.9 Hz, 2H), 3.89 (d, J = 9.6 Hz, 3H), 3.63 (d, J = 31.6 Hz, 3H), 3.29 (t, J = 7.3 Hz, 1H)

[0182] (Synthesis of compound 134) [ka] Compound 133 (100 mg, 0.277 mmol) was dissolved in THF (1 mL), and lithium borohydride (6.0 mg, 0.277 mmol) was added at 0°C. The temperature was then raised to 80°C and the mixture was stirred under reflux for 15 hours. Water was added to stop the reaction, ethyl acetate was added for extraction, the mixture was washed with water and saturated brine, and dried over anhydrous sodium sulfate. After filtering off the drying agent, the solvent was removed under reduced pressure to obtain compound 134 (82.8 mg, 90%) as a white solid.

[0183] Compound 134: white powder; 1 H-NMR (400 MHz, CDCl3) δ 7.59 (s, 1H), 6.11 (dd, J = 16.9, 10.5 Hz, 1H), 5.45 (dd, J = 17.2, 1.6 Hz, 1H), 5.32-5.29 (m, 1H), 5.14 (d, J = 11.0 Hz, 2H), 4.67 (d, J = 7.3 Hz, 4H), 4.57 (td, J = 3.4, 1.8 Hz, 2H), 3.68-3.66 (m, 3H), 3.53(t, J = 7.1 Hz, 1H)

[0184] (Synthesis of Compound 135) [ka] Compound 134 (50.0 mg, 0.138 mmol) was dissolved in dichloromethane (1 mL), and DMF and thionyl chloride (12.1 μL, 0.166 mmol) were added. The mixture was stirred at room temperature for 7 minutes, and the reaction was stopped by adding water. Ethyl acetate was added for extraction, the mixture was washed with water and saturated brine, and dried over anhydrous sodium sulfate. After filtering off the drying agent, the solvent was removed under reduced pressure to obtain compound 135 (51.9 mg, 99%) as a white solid.

[0185] Compound 135: white powder; 1 H-NMR (400 MHz, CDCl3) δ7.65 (s, 1H), 6.18-6.10 (m, 1H), 5.52 (dq, J = 17.0, 1.5 Hz, 1H), 5.35 (dt, J = 10.5, 1.4 Hz, 1H), 5.22 (d, J = 5.0 Hz, 2H), 4.78-4.76 (m, 2H), 4.62 (td, J = 3.5, 1.5 Hz, 2H), 4.58 (s, 2H), 3.70 (s, 3H)

[0186] (Synthesis of Compound 129) [ka]

[0187] Compound 135 (530.0 mg, 1.432 mmol) and tetraethylene glycol (247.2 μL, 1.432 mmol) were dissolved in dry tetrahydrofuran (5 mL) and placed in an isobaric dropping funnel. Sodium hydride (171.8 mg, 4.296 mmol) and potassium iodide (356.5 mg, 2.148 mmol) were dissolved in dry tetrahydrofuran (5 mL) and placed in a round-bottom flask. The temperature was raised until the tetrahydrofuran refluxed, and the mixture was gradually added dropwise from the dropping funnel into the flask. After the addition was complete, the mixture was stirred for 3.5 hours. The temperature was lowered to room temperature, and water was added to stop the reaction. The mixture was extracted with ethyl acetate and washed with water and saturated brine. The extract was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the solvent was removed by reduced pressure distillation. The residue was purified by silica gel chromatography (hexane:ethyl acetate = 1:1) to obtain compound 129 (450.0 mg, 64%) as a white solid.

[0188] Compound 129: white powder; 1 H-NMR (400 MHz, CDCl3) δ 7.46 (s, 1H), 6.23-6.13 (m, 1H), 5.52 (dd, J = 17.2, 1.6 Hz, 1H), 5.23 (dd, J = 10.5, 1.4 Hz, 1H), 5.13 (dd, J = 25.4, 5.3 Hz, 2H), 4.94 (t, J = 5.0 Hz, 2H), 4.86 (d, J = 10.5 Hz, 1H), 4.65 (dd, J = 13.3, 9.6 Hz, 2H), 4.09 (d, J = 10.5 Hz, 1H), 3.69-3.41 (m, 19H).

[0189] Synthesized compound 129 is reacted with s-butyllithium (2.8 equivalents), and after the exchange of bromine and lithium, it is coupled to compound 115 (1.0 equivalent) by nucleophilic attack in distilled THF at -78°C to room temperature to obtain reaction intermediate 136. Next, compound 137 is obtained by dehydration cyclization with acid (1M aqueous HCl solution). The phenolic hydroxyl group of compound 137 is cross-linked with K2CO3 (9.0 equivalents) and BrCH2Cl (6.0 equivalents) to obtain compound 138. Subsequently, K2CO3 (3.0 equivalents) and Pd(PPh3)4 (0.02 equivalents) are reacted in MeOH at room temperature to deprotect the allyl group and synthesize the target compound 139.

[0190] (Example 3: Substitutive compound (2)) Compound 3, shown in Scheme 1 below, was synthesized. Bromocrown ether 15 (synthesized according to the method of Tsubaki et al. (J. Org. Chem. 2005, 70, 4609)) was treated with s-BuLi at -78°C, and then reacted with commercially available sulfobenzoic anhydride (16) to obtain compound 17. Due to the low solubility of compound 17 in methanol, pure 17 was obtained in 75% yield as a pale yellow powder by pulverizing the reaction residue with methanol. The two allyl groups of compound 17 were removed under Pd(PPh3)4 and K2CO3 conditions to obtain compound 3 in 85% yield as an orange powder. Regarding the structure of compound 3, it is assumed to exist as a tautomer between a ring-opened sulfonic acid-quinone form and a cyclic sulfonate-phenol form. The NMR of compound 3 in MeOH-d4 showed a broad signal attributable to its complex tautomerism. By adding excess trifluoroacetic acid, the complex signal shifted to a single dominant signal, allowing us to determine the structure of compound 3. [ka] Scheme 1. Synthetic route to compound 3

[0191] To test the function of compound 3, the color development of compounds 1-3 against spermidine bonds was compared. Compounds 1-3 (1 × 10⁻⁶ -4After adding spermidine (1 equivalent) to the solution (in M methanol), the absorption at 560–570 nm increased. Significant differences in color intensity were observed among the three hosts, with compound 3 showing overwhelmingly higher sensitivity to spermidine compared to compounds 1 and 2 (Figure 2). [ka]

[0192] Next, a color change was induced in compound 3 through interaction with a series of amines, including primary amines, e.g., n-hexylamine (6), secondary amines, e.g., dipropylamine (7), and tertiary amines, e.g., N-ethylpiperidine (8) (Figure 3). The addition of 10 equivalents of (7) or (8) to a methanol solution of compound 3 did not produce a meaningful color change detectable by UV. When n-hexylamine (6) was added to compound 3, a slight increase in absorption was detected by UV, but the color change was not visible to the naked eye. In contrast, when compound 3 interacted with spermidine (4), the color of the solvent changed distinctly from clear to purple. These results indicate that bulkiness around the amino group plays a crucial role in color development. Specifically, the crown ether moiety prevented the bulky secondary and tertiary amino groups from approaching the phenolic hydroxyl group of compound 3. Furthermore, for primary monoamines, the formation of colored ternary complexes (one molecule of compound 3 and two molecules of compound 6) was suppressed by a large entropy cost (Figure 3).

[0193] Next, the selectivity of compound 3 for biogenic amines (4, 5, and 9-14) was investigated (Figure 4). The UV-vis spectrum was obtained in the presence of 20 equivalents of N-ethylpiperidine (8) in methanol, and compound 3 (5 × 10⁻¹⁰) was analyzed. -6 M) and guest amine (5 × 10 -6Measurements were taken using M). Since some biogenic amines are supplied as HCl salts, the addition of 20 equivalents of an excess of 8 generates free amines. On the other hand, as described above, no color development was observed between compound 3 and amine 8 (Figure 3). As shown in Figure 4a, of the guest substances tested, compound 3 showed a weak color response when interacting with cadaverine (10), as well as a clear color change response when interacting with spermidine (4) and spermine (5). These three color responses were visible to the naked eye (Figure 4b). In previous studies, it was found that compound 2 could clearly distinguish spermidine (4) and spermine (5) from the shorter cadaverine (10). Compared to compound 2, compound 3 showed higher sensitivity to 4 and 5. The detection limit of biogenic amines, as determined by the calibration curve, is approximately 10. -7 It is M and meets the requirements for clinical use, therefore it is valuable.

[0194] Figure 4c shows the response of compound 3 to spermidine (4) in the presence of dopamine (11) and tryptamine (14). Specifically, 11 (approximately 2 equivalents) was added to a solution of compound 3 in increments of 0.2 equivalents. Then, 14 (approximately 2 equivalents) was added to the solution, and finally, 4 was added in excess, and the absorption at 571.5 nm was monitored. When 11 and 14 were assayed individually, a negligible increase in absorption was observed. In contrast, compound 3 changed color in response to the addition of 4. This response plateaued after the addition of 1 equivalent of 4. These results indicate that compound 3 was able to recognize 4 with high affinity, even in the presence of primary amines.

[0195] Job plots showing the interaction between compound 3 and guest amines 4 and 5 all have peaks at 0.5 mole, indicating that the stoichiometry of the colored complex is consistent with host / guest = 1 / 1. For the proposed complex between compound 3 and spermidine (4), the terminal amino group of 4 bridges the two phenol crown rings of the host, and the protonated inner amino group of 4 captures the host sulfonate, similar to (b) above.

[0196] The association constant (Ka) and molecular absorption coefficient (ε) of compound 3, which crosslinks guests 4, 5, or 10, were determined by UV-vis titration and analyzed by nonlinear least squares. The results are summarized in Table 1, including comparisons with similar measurements obtained for compounds 1 and 2. A very large association constant (10) was observed. 7 M -1 ) was observed in the binding of compound 3 and guest 4. In contrast, the corresponding values ​​for compound 1 or 2 binding to guest 4 were approximately 10, respectively. 3 and 10 4 Similarly, the molar absorption coefficients (ε=82,000) of compound 3 and guest 4 were approximately 16 times and 2 times higher than those of compound 1 and compound 2, respectively, for guest 4. The molar absorption coefficient obtained for compound 3 bound to guest 4 was comparable to that of the parent phenolsulfonphthalein. These results indicate that the dramatic improvement in sensitivity was caused by the high affinity of compound 3 for spermidine (4). Table 1. Association constants (Ka) and molar absorption coefficients (ε) of spermidine (4), spermine (5), and compound 3.

[0197] [Table 1] Ka and ε were determined by nonlinear least squares method using the software program SPANA. Condition: (b) [Compound 3] 0 = 1 × 10 -6 M [4]0 = 1 × 10 -5 M, (c) [Compound 3]0=1×10 -6 M, [5] 0 = 1 × 10 -5 M, (d) [Compound 3]0=1×10 -5 M, 25℃, methanol.

[0198] We attempted to determine the endogenous spermidine concentration of E. coli using highly sensitive compound 3. Since E. coli lacks the synthetase that converts spermidine to spermine, there is no background signal due to the interaction between compound 3 and endogenous spermine. Therefore, E. coli (MG1655) was cultured and cell-free extracts were prepared. The extracts were divided into two aliquots. One was treated with phthalaldehyde and mercaptoethanol to produce UV-detectable spermidine derivatives, which were measured by HPLC. The other portion was added directly to a solution of compound 3, and the spermidine concentration was estimated by measuring the color change. The data are shown in Table 2.

[0199] [Table 2] Table 2. Measurement of endogenous spermidine concentration by colorimetric assay or HPLC.

[0200] A comparison of concentrations determined by the two methods revealed that the colorimetric assay (compound 3) yielded a value approximately 20 times higher than that obtained by HPLC. This result suggests that an unknown non-spermidine component in the cell-free extract bound to compound 3. This interference is due to compound 3 containing two 18-phenol crown-6-ether substructures, and therefore K + It was thought that this could originate from ions. The calibration curve between K+ and compound 3 has a slope of approximately 160, while the corresponding slope between spermidine and compound 3 is approximately 59,000, therefore K + The interference didn't seem to be much of a problem.

[0201] However, K in E. coli + The reported concentration is 180-200 mM. Therefore, even if the interaction gradient with compound 3 is shallow, K in the molecular recognition system + The impact could not be ignored. K + To prevent the effects of K +Spermidine measurements were performed in the presence of [2.2.2]cryptand, which has a particularly high affinity for [2.2.2]. + The effect could be masked by the cryptand, and it was expected that the tertiary amino group in the cryptand would not affect the phenol crown of compound 3. Therefore, compound 3 could selectively interact with spermidine (4).

[0202] The spermidine concentrations in 20 different preparations of E. coli cell-free extracts were determined by both HPLC and colorimetric methods (Table 3 and Figure 5). Similar results were obtained using both approaches. When the data obtained by each method were plotted against each other (Figure 6), the slope of the graph was 1, indicating a 97% correlation coefficient. The spermidine concentration in E. coli could be determined using an optimized colorimetric method based on detecting unbound interactions between the host and guest in a protic solvent.

[0203] Table 3. Determination of spermidine concentration using colorimetric and HPLC methods (2). [Table 3]

[0204] (Compound 17) A solution of n-BuLi (1.66 M hexane solution, 3.04 ml, 5.06 mmol) was added dropwise to a solution of 15 (2.00 g, 4.60 mmol) in dry THF (60 ml) under nitrogen at -78°C. After stirring for 35 minutes, 16 (338 mg, 1.84 mmol) in THF solution (15 ml) was added dropwise. The resulting solution was stirred at -78°C, then warmed to room temperature and stirred for 45 hours. Next, hydrochloric acid (1 M) was added to the reaction mixture. After stirring for 6 hours, all solvent was evaporated under reduced pressure. The residue was treated with 1 M hydrochloric acid to obtain a white powder. The powder was collected and washed with a small amount of methanol to obtain 17 (478 mg, 30% yield). An additional amount of 17 (83 mg, 5% yield) was recovered from the mother liquor. Compound 17: white powder; R f=0.72(CHCl3-MeOH,4:1),mp=142-147℃;IR(KBr)3465,2873,1637,1350,1103cm -1 .

[0205] (Compound 3) NaBH4 (26 mg, 0.69 mmol) was added under nitrogen to a solution of 17 (150 mg, 0.17 mmol) and Pd(PPh3)4 (4 mg, 5.2 μmol) in MeOH (20 ml). After stirring at room temperature for 17 hours, Amberlust 15 ion exchange resin (15 mg) was added. The reaction mixture was stirred until the solvent changed color from clear to orange, and then the resin was filtered. The solvent was concentrated under reduced pressure to obtain the residue. The residue (148 mg) was purified by recirculating preparative HPLC using continuously connected Shodex GPC H-2001L (20 × 600 mm) and GPC H-2002L (20 × 600 mm) columns to obtain compound 3 (81 mg, 60%) as an orange solid. Compound 3: Orange solid; R f =0.72(H2O-MeOH,1:2),mp=149-154℃;IR(KBr)3435,2873,2360,1630,1600,1352,1090cm -1 ; 1 H-NMR (270.05MHz, solvent CD3OD) δ7.91(dd,J=7.3,1.4Hz,1H),7.76(ddd,J=7.3,7.3,1.4Hz,1H),7.70(ddd,J=7.3,7.3,1.4H HRMS(FAB + )C 39 H 51 O 15 S (M + H + Calculated value for ): 791.2948. Measured value: 791.2922.

[0206] (Example 4: Development of a color-responsive reagent with added substituents) Chromogenic reagent compound 203, which has phenolsulfonphthalein as its core, was a highly sensitive detection reagent for spermine and spermidine. However, as the practical application of chromogenic reagent compound 203 progressed, false positives due to cadaverine were identified as a problem. The association constant of 203 with cadaverine is 3.2 × 10⁻⁶. 5 M -1 On the other hand, the association constant for spermidine is 1.2 × 10⁻⁶. 7 M -1 Compound 203 has a 38-fold difference in association constants between cadaverine and spermidine, but because the amount of polyamines in the body varies greatly from person to person, there was concern about false positives for cadaverine in humans who have abnormally high levels of cadaverine. [ka] Structure of polyamines in living organisms

[0207] Therefore, the inventors worked on developing a novel color reagent compound 222, which is compound 203 with a methoxy group, an electron-donating group, introduced into it, and compound 223, which is compound 203 with a nitro group, an electron-withdrawing group, introduced into it. [ka] Structures of novel colorimetric reagent compounds 222 and 223 (Synthesis of chromatic responsive reagents with added substituents) First, in order to synthesize compound 222, compound 227 was synthesized by introducing a methoxy group (Hernandez, Vincent S. PCT Int. Appl. 2011, 219, 011060199). Commercially available 3-methoxytoluene (224) was sulfonated with sulfuric acid to synthesize compound 225. Next, compound 226 was synthesized by oxidation with potassium permanganate, and compound 227 was synthesized by dehydration cyclization with thionyl chloride (Scheme 2-1). [ka] Scheme 2-1. Synthesis of Compound 227

[0208] Next, compound 205 was lithiated with s-butyllithium and then reacted with compound 227 to synthesize compound 228. Finally, the allyl group was deprotected (Vutukuri, DR; Bharathi, P.; Yu, Z.; Rajasekaran, K.; Tran, M.; Thayumanavan. SJ Org. Chem. 2003, 68, 1146-1149), and a novel chromogenic reagent compound 222 was synthesized by introducing a methoxy group (Scheme 2-2). [ka] Scheme 2-2. Synthesis of colorimetric reagent compound 222

[0209] Next, in order to synthesize compound 223, compound 230 was synthesized by introducing a nitro group. Compound 229 was nitrated using sulfuric acid and nitric acid to obtain compound 230 (Scheme 2-3). [ka] Scheme 2-3. Synthesis of Compound 230

[0210] Next, compound 223 was synthesized. Compound 205 was lithiated with s-butyllithium, then reacted with compound 230 to synthesize compound 231. Finally, the allyl group was deprotected. 18 A novel colorimetric reagent compound, 223, was synthesized by introducing a nitro group (Scheme 2-4). [ka] Scheme 2-4. Synthesis of Compound 223

[0211] (Evaluation of host-guest complexes for three types of endogenous polyamines) We successfully synthesized novel colorimetric reagent compounds 222 and 223 by introducing various substituents. Subsequently, we evaluated the host-guest complexes with the endogenous polyamines spermine, spermidine, and cadaverine. Titration was performed based on ultraviolet-visible absorption spectroscopy, and the association constant (K) and molar extinction coefficient (ε) were calculated using the nonlinear least squares method.

[0212] First, the association constants of compound 222 with respect to three types of polyamines were measured (Figure 11). [Table 4]

[0213] A methanol solution of the colorimetric reagent compound 222 was orange, and when a methanol solution of polyamine was added dropwise, it changed to purple. The association constant of compound 222 with spermidine is 4.6 × 10⁻⁶. 6 M -1 This value was 0.38 times that of compound 203 to spermidine. Furthermore, the association constant of compound 222 to spermine was 8.9 × 10⁻⁶. 6 M -1 This value was 0.56 times that of compound 203 with respect to spermine. Compound 222 showed decreased sensitivity as a colorimetric reagent for spermidine and spermine. This is thought to be because the sulfonate was destabilized by the extrusion of electrons from the methoxy group, causing the equilibrium to shift towards the ring-closed colorless form and thus reducing sensitivity. On the other hand, the association constant of compound 222 with respect to cadaverine was 2.0 × 10⁻⁶. 5 M -1 The association constant for compound 222 with cadaverine was 0.66 times that of compound 203, and the decrease in sensitivity was similar to that of spermine and spermidine. From these results, it was found that compound 222 does not show improved selectivity with cadaverine compared to compound 203.

[0214] Next, the association constants of compound 223 with respect to three types of polyamines were measured (Figure 12). [Table 5]

[0215] A methanol solution of the colorimetric reagent compound 223 was orange, and when a methanol solution of polyamine was added dropwise, it changed to purple. The association constant of compound 223 with spermidine is 1.0 × 10⁻⁶. 7 M -1 The association constant of compound 203 with spermidine was 0.83 times. The association constant of compound 223 with spermine was 1.8 × 10⁻⁶. 7 M -1 The association constant of compound 203 with spermine was 1.13 times higher. The association constant of compound 223 with cadaverine was 1.6 × 10⁻⁶. 5 M -1 The association constant for compound 223 with cadaverine was 0.5 times that of compound 203, indicating lower sensitivity compared to spermine and spermidine. From these results, it was found that compound 223 showed improved selectivity for cadaverine compared to compound 203.

[0216] General operations The reaction solvents used—tetrahydrofuran (THF), dimethylformamide (DMF), 1,2-dichloroethane (DCE), toluene, and dichloromethane—were dried using activated molecular sieves. Moisture-sensitive reactions were carried out in dry flasks under a nitrogen atmosphere using rubber septums. All reactions were performed using thin-layer chromatography (TLC; Silica gel 60°F). 254 The samples were tracked using a 0.25 mm (Merck) probe, and the reaction was detected by one or a combination of the following methods: (1) UV (254 nm, 365 nm) irradiation observation, (2) immersion in phosphomolybdic acid aqueous solution, and (3) heating on a hot plate. A WT-100-M (Honda Giken Co., Ltd.) ultrasonic generator was used.

[0217] Purification of compounds For normal-phase column chromatography, SiliaFlash F60 (Silicycle) was used. For preparative TLC, Silica gel 60 F 254 A 0.5 mm Merck column was used. For gel permeation chromatography (GPC), an LC-2000plus system (JASCO, pump: PU-2086; UV detector: UV-2075; RI detector: RI-2031) was used, with two columns, GPC H-2001 (20 × 500 mm, Showa Denko) and GPC H-2002 (20 × 500 mm, Showa Denko), linked together, and chloroform (flow rate 3.5 mL / min.) used as the eluent.

[0218] Physical properties and spectral measurements The melting point was measured using a Yanako Micro Melting Point Apparatus M-J3 and is uncorrected. Nuclear magnetic resonance (NMR) spectra were obtained using a Bruker 400 Ultra Shield. 1 H, 400 MHz); JNM-ECZS 400 ( 13 The measurement was taken at C, 100 MHz. The chemical shift value (d) is: 1 For H, deuterated chloroform (7.26 ppm) and deuterated methanol (3.31 ppm) were used as internal standards, and the coupling constant (J) was expressed in Hz. 13 For C, deuterated chloroform (77.00 ppm) and deuterated methanol (49.00 ppm) were used as internal standards, respectively. Peak multiplicity was abbreviated as follows: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), brm (broad multiplet). Infrared absorption (IR) spectra were measured using a Horiba FT-IR 720 spectrometer, and cm -1The results were expressed as follows. High-resolution mass spectrometry (HRMS) was measured using SolariX FT-ICR-MS (Bruker). Ultraviolet-visible absorption spectra were measured using a JASCO V-650 spectrophotometer (JASCO). Fluorescence emission spectra were measured using a JASCO FP-8600 spectrophotometer (JASCO).

[0219] Reagents used in instrumental analysis Deuterated chloroform (CDCl3) was prepared using Cambridge Isotope Laboratories, Inc.'s Chloroform-D, (D, 99.8%) + 0.05% v / v% TMS. Deuterated methanol (CD3OD) was prepared using Cambridge Isotope Laboratories, Inc.'s Methanol-D4, (D, 99.9%). Deuterated DMSO was prepared using Cambridge Isotope Laboratories, Inc.'s (D, 99.9%) + 0.05% v / v% TMS.

[0220] (Synthesis of compound 231) [ka] Under a nitrogen atmosphere, compound 205 (2.82 g, 6.55 mmol) was dissolved in anhydrous THF (30 mL), cooled to -78°C, and s-butyllithium (1.05 M cyclohexane-n-hexane solution, 7.0 mL, 7.34 mmol) was added dropwise. After stirring for 10 minutes, an anhydrous THF solution of compound 230 (599.5 mg, 2.62 mmol) (8 mL) was added dropwise. The mixture was allowed to rise naturally to room temperature, and then 1 M hydrochloric acid was added to stop the reaction. Hexane was added, and the solvent was removed by vacuum distillation. The residue was purified by column chromatography (chloroform:acetone = 1:1) to obtain compound 231 (128.9 mg, 5%) as a white solid.

[0221] 231: white powder. mp. IR. 1H NMR (400 MHz, CDCl3) δ 8.45 (d, J = 8.2 Hz, 1H), 7.94 (t, J = 8.0 Hz, 1H), 7.80 (d, J = 7.8 Hz, 1H) , 7.29 (d, J = 2.7 Hz, 2 also), 7.16 (d, J = 2.7 Hz, 2H), 6.17 (qd, J = 11.0, 5.1 Hz, 2H), 5.51 (dd, J = 17.4, 1.4 Hz, 2H), 5.22 (dd, J = 10.5, 1.4 Hz, 2H), 4.96 (d, J = 4.6 Hz, 4H), 4.83-4.88 (m, 4H), 4.08-4.14 (m, 4H), 3.42-3.63 (m, 32H). 13 C NMR (100 MHz , CDCl3) δ . HRMS (ESI) Calcd for C 45 H 57 NO 17 S ([M + Na] + ) 938.3239, Found 938.3222.

[0222] (Synthesis of compound 223) [ka] At room temperature, methanol (1 mL) was added to compound 231 (48.2 mg, 0.05 mmol) and the mixture was stirred. Then, tetrakistriphenylphosphine palladium (1.4 mg, 1.21 × 10) was added. -3 The mixture was mixed with potassium carbonate (21.4 mg, 0.15 mmol) and stirred for 4 hours. Upon adding Amberlyst 15 (144.5 mg), the reaction solution changed from purple to a clear orange color. After filtering off Amberlyst 15, the solvent was removed by distillation under reduced pressure, and compound 223 (33.5 mg, 73%) was obtained as an orange solid.

[0223] 223: orange powder. mp ℃. IR 1H NMR (400 MHz, TFA : CDCl3= 7 : 1) δ 8.43 (d, J = 7.8 Hz, 1H), 7.91 (t, J = 7.8 Hz, 1H), 7.63 (d, J = 8.2 Hz, 1H), 7.26 (s, 4H), 7.05 (s, 4H), 4.65 (s, 8H), 3.79 (d, J = 6.9 Hz, 32H). HRMS (ESI) Calcd for C 39 H 48 NO 17 S ([M] + ) 834.2648, Found 834.2623. (Example 5: Crosslinked compound)

[0224] Next, a cross-linked compound is prepared to reduce rotation of crown ether moieties such as the naphthol crown moiety.

[0225] This suppresses the phenomenon of the phenolic hydroxyl group at the center of the binding site wobbling, fixing it in the optimal conformation for spermine incorporation, and improving the association constant and discriminative ability.

[0226] Therefore, in this embodiment, two naphthol crown rings are linked and immobilized to synthesize a compound with an optimal length for spermine. Based on this idea, compounds 5 to 10 are created. These are a group of compounds in which a phenol crown ring and a naphthol crown ring are combined, and the orientation of the naphthol crown ring is immobilized by oxygen crosslinking. Although the structural formulas below show cyclic sulfonic acid esters, cyclic esters can also be synthesized. [ka]

[0227] Starting with a xanthone, compound 232 was synthesized by converting the benzyl position on the lower side of the xanthone skeleton to a leaving group, and then compound 233 was obtained by substituting it with tetraethylene glycol. Subsequently, a functional group was also introduced on the upper side of the xanthone skeleton to obtain compound 252. Since it was found that the terminal end of the tetraethylene glycol was the reaction site, the compound was first protected with a MOM group, and then the morpholinomethyl group was chlorinated. After that, the MOM group was deprotected, and compound 236 was synthesized by cyclizing the two crown ethers in one step (Scheme 5-1). [ka] Scheme 5-1. Synthesis of Compound 236

[0228] (Synthesis of compound 239) [ka] At room temperature, compound 238 (151.1 mg, 0.35 mmol) was dissolved in DMF (0.5 mL) and THF (1.5 mL). Potassium carbonate (103.0 mg, 0.75 mmol), allyl bromide (103 μL, 0.75 mmol), and sodium iodide (4.8 mg, 0.03 mmol) were added at 0°C, and the temperature was raised to 40°C. After stirring for 9 hours, saturated aqueous ammonium chloride was added to stop the reaction. Chloroform was added to separate the two layers, and the organic layer was washed with water and saturated brine, then dried over anhydrous sodium sulfate. After filtering off the drying agent, the solvent was removed by reduced pressure. The residue was purified by column chromatography (chloroform:methanol = 20:1) to obtain compound 239 (84.6 mg, quant) as a white solid.

[0229] 239: white powder. mp 171 -191.3℃. IR (KBr) 3435, 2958, 2852, 2806, 1651, 1599, 1454, 1425, 1354, 1333, 1273, 1213, 1115, 1092, 991, 866, 779. 1H NMR (400 MHz, CDCl3) d 8.27 (d, J = 9.2 Hz, 2H), 6.97 (d, J = 9.2 Hz, 2H), 6.12-6.03 (m, 2H), 5.52-5.47 (m, 2H), 5.35-5.33 (m, 2H), 4.71-4.70 (m, 4H), 3.96 (s, 4H), 3.67 (t, J = 4.6 Hz, 8H), 2.61 (t, J = 4.2 Hz, 8H). 13 C NMR (100 MHz , CDCl3) d 176.3, 162.2, 159.1, 132.3, 127.5, 117.9, 115.7, 113.3, 108.8, 69.4, 67.1, 53.5, 49.9. HRMS (ESI) Calcd for C 29 H 34 N2O6([M + H] + ) 507.2490, Found 507.2480.

[0230] (Synthesis of compound 232) [ka] At room temperature, compound 239 (1.26 g, 2.49 mmol) was dissolved in DCE (20 mL), ethyl chloroformate (1.2 mL, 12.43 mmol) was added, and the mixture was heated under reflux for 2.5 hours. The solvent was removed by distillation under reduced pressure, the precipitated solid was filtered, and after washing with ethyl acetate, compound 232 (963.7 mg, 96%) was obtained as a white solid.

[0231] 232: white powder. mp 200 - 203 ℃. IR (KBr) 3433, 1651, 1601, 1433, 1286, 1219, 1192, 1115, 1088, 793. 1H NMR (400 MHz, CDCl3) d 8.29 (d, J = 8.8 Hz, 2H), 6.98 (d, J = 8.8 Hz, 2H), 6.14-6.05 (m, 2H), 5.52-5.47 (m, 2H), 5.39-5.35 (m, 2H), 5.08 (s, 4H), 4.79-4.77 (m, 4H). 13 C NMR (100 MHz , CDCl3) d 175.4, 161.2, 155.2, 131.9, 128.8, 118.4, 115.7, 113.8, 109.0, 69.7, 34.4. HRMS (ESI) Calcd for C 21 H 18 Cl2O4([M + Na] + ) 427.0474, Found 427.0478.

[0232] (Synthesis of compound 233) [ka] At room temperature, compound 232 (30.0 mg, 0.07 mmol) was dissolved in dry THF (2 mL) and dry DMF (1 mL), and TBAI (2.9 mg, 0.007 mmol) and tetraethylene glycol (380 μL, 2.22 mmol) were added, and the mixture was heated to 40°C. Sodium hydride (45.6 mg, 1.11 mmol) was then added and the mixture was stirred for 39 minutes, after which water was added to stop the reaction. Ethyl acetate was added to separate the two layers, and the organic layer was washed with water and saturated brine, and then dried over anhydrous sodium sulfate. After filtering off the drying agent, the solvent was removed by reduced pressure. The residue was purified by column chromatography (chloroform:methanol = 25:1) to obtain compound 233 (27.0 mg, 51%) as a clear oil and compound 240 (18.8 mg, 35%) as a white solid.

[0233] 233: colorless oil. IR (neat) 3465, 2918, 2871, 1651, 1599, 1429, 1352, 1277, 1215, 1111, 1036, 991, 933, 887, 839, 789, 723, 546. 1 H NMR (400 MHz, CDCl3) d 8.28 (d, J = 9.2 Hz, 2H), 6.96 (d, J = 8.8 Hz, 2H), 6.12-6.04 (m, 2H), 5.48-5.44 (m, 2H), 5.35-5.32 (m, 2H), 4.97 (s, 4H), 4.74-4.73 (m, 4H), 3.81-3.55 (m, 32H). 13 C NMR (100 MHz , CDCl3) d 162.1, 159.1, 132.4, 128.4, 118.0, 115.6, 113.7, 108.9, 72.6, 70.5, 70.2, 69.9, 69.5, 61.6, 61.1. HRMS (ESI) Calcd for C 37 H 52 O 14 ([M + Na] + ) 743.3249, Found 743.3250.240: white powder. mp 110.7 - 125.0℃. IR (KBr) 3433, 2924, 2970, 1655, 1601, 1431, 1352, 1277, 1219, 1111, 1093, 987, 928, 841, 791, 729, 669, 546. 1 H NMR (400 MHz, CDCl3) d 8.27 (d, J = 8.8 Hz, 2H), 6.95 (d, J = 9.2 Hz, 2H), 6.11-6.02 (m, 2H), 5.48-5.43 (m, 2H), 5.33-5.31 (m, 2H), 4.99 (s, 4H), 4.72-4.71 (m, 4H), 3.76-3.46 (m, 16H). 13C NMR (100 MHz, CDCl3) d 176.0, 162.3, 161.1, 156.0, 132.3, 131.9, 128.8, 128.3, 118.4, 118.0, 115.7, 115.0, 113.9, 113.8, 109.0, 108.9, 70.7, 70.6, 70.2, 70.0, 69.7, 69.5, 61.1, 34.4. HRMS (ESI) Calcd for C 28 H 32 O9([M + Na] + ) 549.2095, Found 549.2101.

[0234] (Synthesis of compound 249) [ka] At room temperature, dissolve compound 233 (25.1 mg, 0.03 mmol) in methanol (5 mL) and tetrakistriphenylphosphine palladium (0.8 mg, 6.96 × 10⁻¹⁶) in methanol. -4 The mixture was mixed with potassium carbonate (14.9 mg, 0.10 mmol) and stirred for 1 hour. Upon adding Amberlyst 15 (67.2 mg), the reaction solution changed from clear to yellow. After filtering off Amberlyst 15, the solvent was removed by distillation under reduced pressure to obtain compound 249 (20.9 mg, 94%) as orange oil.

[0235] 249: orange powder. mp 192.3 - 211.7 ℃. IR (KBr) 3396, 2891, 1649, 1583, 1539, 1493, 1429, 1329, 1277, 1201, 1095, 1001, 951, 837, 694. 1 H NMR (400 MHz, CD3OD) d 8.02 (d, J = 8.8 Hz, 2H), 6.86 (d, J = 8.8 Hz, 2H), 4.95 (s, 4H), 3.80-3.31 (m, 32H). 13C NMR (100 MHz , CD3OD) d 159.4, 127.8, 120.0, 112.0, 73.5, 71.2, 70.9, 70.7, 70.0, 63.6, 61.6. HRMS (ESI) Calcd for C 31 H 44 O 14 ([M + Na] + ) 663.2623, Found 663.2633.

[0236] (Synthesis of compound 234) [ka] At 0°C, 100 μL, 1.16 mmol of morpholine was mixed with 470 μL, 5.80 mmol of 37% formalin aqueous solution and 1 mL of acetic acid, and the mixture was heated to room temperature. After stirring for 1 hour, toluene was added and the solvent was removed under reduced pressure to obtain a mixture of morpholine (250) and iminium 251 (366.5 mg). The resulting mixture was dissolved in 4 mL of dry toluene, compound 249 (22.8 mg, 0.04 mmol) was added, and the mixture was heated to 70°C. After stirring for 1.5 hours, water was added to stop the reaction. Chloroform was added to separate the two layers, and the organic layer was washed with water and saturated brine, then dried over anhydrous sodium sulfate. After filtering off the drying agent, the solvent was removed under reduced pressure. The residue was purified by column chromatography (chloroform:methanol = 10:1) to obtain compound 234 (20.8 mg, 741%) as a white solid.

[0237] 234: white powder. IR (neat) 3462, 3005, 2866, 1649, 1618, 1489, 1452, 1423, 1350, 1319, 1271, 1182, 1117, 1005, 908, 864, 798, 754, 665. 1H NMR (400 MHz, CDCl3) d 7.96 (s, 2H), 4.92 (s, 4H), 3.84 (s, 4H), 3.77-3.56 (m, 40H), 2.62 (s, 8H). 13 C NMR (100 MHz , CDCl3) d 162.9, 126.7, 118.1, 113.9, 72.6, 70.5, 70.3, 69.8, 66.6, 61.6, 61.3, 52.7. HRMS (ESI) Calcd for C 41 H 62 O 16 ([M + Na] + ) 861.3992, Found 861.3984.

[0238] (Synthesis of Compound 252) [ka] At room temperature, compound 234 (283.7 mg, 0.39 mmol) was dissolved in DMF (3 mL), and cesium carbonate (271.6 mg, 0.83 mmol), allyl bromide (65 μL, 0.68 mmol), and sodium iodide (5.0 mg, 0.04 mmol) were added at 0°C. The temperature was raised to 30°C. After stirring for 3 hours, saturated aqueous ammonium chloride was added to stop the reaction. Ethyl acetate was added to separate the two layers, and the organic layer was washed with water and saturated brine, then dried over anhydrous sodium sulfate. After filtering off the drying agent, the solvent was removed under reduced pressure, and compound 252 (147.0 mg, 47%) was obtained as a white solid. Chloroform was added to the aqueous layer obtained by liquid-liquid separation to separate the two layers, and the organic layer was washed with water and saturated brine, then dried over anhydrous sodium sulfate. After filtering off the drying agent, the solvent was removed under reduced pressure. The residue was purified by column chromatography (chloroform:methanol = 20:1) to obtain compound 252 (62.2 mg, 20%) as a white solid.

[0239] 252: white powder. 1H NMR (400 MHz, CDCl3) δ 8.29 (s, 2H), 6.16 (qd, J = 10.9, 5.6 Hz, 2H), 5.49 (d, J = 16.9 Hz, 2H), 5.31 (d, J = 10.5 Hz, 2H), 4.91 (s, 4H), 4.73 (d, J = 5.0 Hz, 4H), 3.56-3.89 (m, 44H), 2.51 (s, 8H). 13 C NMR (100 MHz, CDCl3) δ 195.33, 176.32, 163.07, 155.50, 133.64, 129.25, 128.38, 120.31, 117.78, 117.43, 77.20, 72.51, 70.59, 70.52, 70.35, 70.27, 67.29, 66.99, 62.32, 61.64, 57.51, 53.52, 29.68. HRMS (ESI) Calcd for C 47 H 70 N2O 16 ([M + Na] + ) 941.4618, Found 941.4636.

[0240] (Synthesis of compound 260) [ka] At room temperature, compound 252 (13.2 mg, 0.01 mmol) was dissolved in dry THF (1.5 mL), and chloromethyl methyl ether (2.2 μL, 0.03 mmol) and sodium hydride (1.1 mg, 0.33 mmol) were added, and the temperature was raised to 30°C. After stirring for 6.5 hours, water was added to stop the reaction. Ethyl acetate was added to separate the two layers, and the organic layer was washed with water and saturated brine, then dried over anhydrous sodium sulfate. After filtering off the drying agent, the solvent was removed by reduced pressure. The residue was purified by column chromatography (chloroform:methanol = 20:1) to obtain compound 260 (11.7 mg, 81%) as a white solid.

[0241] 260: White powder.1 H NMR (400 MHz, CDCl3) δ 8.28 (s, 2H), 6.15 (qd, J = 11.0, 5.5 Hz, 2H), 5.49 (d, J = 16.9 Hz, 2H), 5.31 (d, J = 10.5 Hz, 2H), 4.89 (s, 4H), 4.73 (d, J = 5.0 Hz, 4H), 4.64 (s, 4H), 3.82 (t, J = 4.6 Hz, 4H), 3.59-3.68 (m, 40H), 3.35 (d, J = 1.4 Hz, 6H), 2.51 (s, 8H). HRMS (ESI) Calcd for C 51 H 78 N2O 18 ([M + Na] + ) 1029.5142, Found 1029.5161.

[0242] (Synthesis of Compound 262) [ka] At room temperature, compound 260 (11.8 mg, 0.01 mmol) was dissolved in DCE (1 mL), ethyl chloroformate (5.6 μL, 0.06 mmol) was added, and the mixture was heated under reflux for 6 hours. The solvent was removed by distillation under reduced pressure, and the residue was purified by column chromatography (hexane:ethyl acetate = 5:1) to obtain compound 262 (9.0 mg, 85%) as a white solid.

[0243] 262: white powder. 1H NMR (400 MHz, CDCl3) δ 8.40 (s, 2H), 6.18 (qd, J = 11.0, 5.5 Hz, 2H), 5.53 (dd, J = 17.2, 1.6 Hz, 2H), 5.35 (dd, J = 10.3, 1.1 Hz, 2H), 4.90 (d, J = 14.6 Hz, 4H), 4.74 (t, J = 4.8 Hz, 8H), 4.63 (d, J = 5.5 Hz, 4H), 3.55-3.83 (m, 32H), 3.35 (s, 6H).

[0244] (Synthesis of compound 235) [ka] At room temperature, compound 262 (40.6 mg, 0.04 mmol) was mixed with 1 mL of 4 M dioxane hydrochloride and stirred for 1 hour. Water was then added to stop the reaction. Ethyl acetate was added to separate the two layers. The organic layer was washed with water and saturated brine, and then dried over anhydrous sodium sulfate. After filtering off the drying agent, the solvent was removed by reduced pressure. The residue was purified by column chromatography (ethyl acetate) to obtain compound 235 (30.4 mg, 83%) as a white solid.

[0245] 235: White powder. 1 H NMR (400 MHz, CDCl3) δ 8.40 (s, 2H), 6.18 (td, J = 11.2, 5.5 Hz, 2H), 5.54 (dd, J = 17.2, 1.6 Hz, 2H), 5.35 (dd, J = 10.3, 1.1 Hz, 2H), 4.90 (s, 4H), 4.73-4.75 (m, 8H), 3.55-3.85 (m, 32H).

[0246] (Synthesis of Compound 236) [ka] At room temperature, compound 235 (42.1 mg, 0.05 mmol) was dissolved in dry DMF (1 mL), potassium iodide (28.9 mg, 0.17 mmol) and sodium hydride (7.7 mg, 0.19 mmol) were added, and the mixture was stirred for 5 hours. Water was then added to stop the reaction. Ethyl acetate was added to separate the two layers, and the organic layer was washed with water and saturated brine, and then dried over anhydrous sodium sulfate. After filtering off the drying agent, the solvent was removed by reduced pressure. The residue was purified by column chromatography (ethyl acetate : methanol = 20 : 1) to obtain compound 236 (8.7 mg, 22%) as a white solid.

[0247] 236: white powder. 1 H NMR (400 MHz, CDCl3) δ 8.25 (d, J = 4.1 Hz, 2H), 6.21-6.29 (m, 2H), 5.58 (d, J = 17.4 Hz, 2H), 5.29 (d, J = 10.5 Hz, 2H), 5.12-5.18 (m, 2H), 5.00-5.06 (m, 6H), 4.86 (dd, J = 10.1, 6.4 Hz, 2H), 4.28 (dd, J = 11.0, 2.7 Hz, 2H), 3.41-3.79 (m, 32H). 13 C NMR (100 MHz, CDCl3) δ 164.91, 155.99, 155.87, 135.75, 129.75, 129.70, 129.18, 129.07, 121.32, 117.01, 116.93, 115.71, HRMS (ESI) Calcd for C 39 H 52 O 14 ([M + K] + ) 783.2989, Found 783.2983.

[0248] (Synthesis of Compound 271) [ka] Under a nitrogen atmosphere, compound 270 (96.7 mg, 0.42 mmol) was dissolved in anhydrous THF (1 mL), cooled to -78°C, and n-butyllithium (1.58 M n-hexane solution, 240 μL, 0.38 mmol) was added dropwise. After stirring for 30 minutes, anhydrous THF solution of compound 264 (100.1 mg, 0.32 mmol) (2 mL) was added dropwise. The mixture was allowed to rise naturally to room temperature, and then 1N hydrochloric acid solution (0.3 mL) was added to stop the reaction. Ethyl acetate was added to separate the two layers, and the organic layer was washed with water and saturated brine, and then dried over anhydrous sodium sulfate. After filtering off the drying agent, the solvent was removed by reduced pressure. The residue was purified by column chromatography (hexane:ethyl acetate = 15:1) to obtain compound 271 (54.8 mg, 39%) as a yellow solid.

[0249] 271: Yellow powder. 1 H NMR (400 MHz, CDCl3) δ 7.91 (d, J = 7.8 Hz, 1H), 7.68 (dd, J = 7.5, 1.6 Hz, 1H), 7.40 (dtd, J = 20.6, 7.4, 1.4 Hz, 2H), 7.01 (d, J = 8.7 Hz, 2H), 6.72 (d, J = 2.3 Hz, 2H), 6.64 (dd, J = 8.7, 2.7 Hz, 2H), 6.03-6.12 (m, 2H), 5.68 (s, 1H), 5.44 (dq, J = 17.4, 1.5 Hz, 2H), 5.32 (dq, J = 10.5, 1.4 Hz, 2H), 4.57 (td, J = 3.3, 1.8 Hz, 4H), 3.91-3.95 (m, 2H), 3.67-3.70 (m, 2H), 2.90 (s, 1H).

[0250] (Synthesis of compound 274) [ka] Under a nitrogen atmosphere, compound 270 (54.2 mg, 0.24 mmol) was dissolved in anhydrous THF (1 mL), cooled to -78°C, and n-butyllithium (1.58 M n-hexane solution, 160 μL, 0.24 mmol) was added dropwise. After stirring for 10 minutes, anhydrous THF solution of compound 264 (50.3 mg, 0.16 mmol) (2 mL) was added dropwise. The mixture was allowed to rise naturally to room temperature, then 1N hydrochloric acid solution (2 mL) was added and the temperature was raised to 50°C. After stirring for 2 hours, ethyl acetate was added to separate the two layers. The organic layer was washed with water and saturated brine, and then dried over anhydrous sodium sulfate. After filtering off the drying agent, the solvent was removed by reduced pressure. The residue was purified by column chromatography (hexane:ethyl acetate = 15:1) to obtain compound 274 (37.2 mg, 55%) as a yellow solid.

[0251] 274: Yellow powder. 1 H NMR (400 MHz, CDCl3) δ 7.54 (d, J = 7.8 Hz, 1H), 7.42 (t, J = 7.5 Hz, 1H), 7.35 (t, J = 7.3 Hz, 1H), 7.11 (d, J = 8.7 Hz, 1H), 6.89 (d, J = 7.8 Hz, 1H), 6.77 (d, J = 8.7 Hz, 1H), 6.72 (t, J = 2.5 Hz, 3H), 6.63 (ddd, J = 13.4, 8.8, 2.4 Hz, 2H), 6.05 (dq, J = 22.6, 5.3 Hz, 2H), 5.42 (dd, J = 17.4, 1.4 Hz, 2H), 5.30 (d, J = 10.5 Hz, 2H), 4.55 (d, J = 5.0 Hz, 4H), 3.23 (d, J = 5.5 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 159.27, 159.23, 151.61, 151.26, 146.52, 138.38, 132.85, 132.82, 130.96, 130.32, 129.47, 128.52, 123.46, 122.51, 117.95, 116.65, 116.17, 111.75, 101.23, 101.15, 99.88, 83.77, 77.21, 68.94.

[0252] (Synthesis of compound 265) [ka] At room temperature, compound 274 (15.1 mg, 0.04 mmol) was added to dry toluene (1 mL), and the dichlorocymenruthenium dimer (1.2 mg, 0.33 × 10) was dissolved. -3 Adding N,N'-diisopropylcarbodiimide (5.6 μL, 0.04 mmol) and stirring for 2.5 hours, water was added to stop the reaction. Ethyl acetate was added to separate the two layers, and the organic layer was washed with water and saturated brine, then dried over anhydrous sodium sulfate. After filtering off the drying agent, the solvent was removed by reduced pressure. The residue was purified by column chromatography (hexane:ethyl acetate = 10:1) to obtain compound 265 (9.4 mg, 63%) and compound 275 (4.8 mg, 32%).

[0253] 265: Yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.02 (d, J = 7.3 Hz, 1H), 7.64 (td, J = 14.4, 7.2 Hz, 2H), 7.16 (d, J = 7.3 Hz, 1H), 6.78 (d, J = 2.3 Hz, 2H), 6.68 (d, J = 8.7 Hz, 2H), 6.62 (dd, J = 8.7, 2.3 Hz, 2H), 6.00-6.08 (m, 2H), 5.43 (d, J = 16.9 Hz, 2H), 5.32 (d, J = 10.5 Hz, 2H), 4.57 (d, J = 5.0Hz, 4H). 275: Yellow oil. 1 H NMR (400 MHz, CDCl3) δ 8.02 (d, J = 7.8 Hz, 1H), 7.60-7.69 (m, 2H), 7.16 (d, J = 7.8 Hz, 1H), 6.75 (dd, J = 16.5, 1.8 Hz, 2H), 6.60-6.68 (m, 3H), 6.52 (dd, J = 8.2, 1.8 Hz, 1H), 6.03 (q, J = 5.3 Hz, 1H), 5.30-5.45 (m, 2H), 4.56 (dd, J = 5.3, 1.1 Hz, 2H).

[0254] (Synthesis of compound 290 using compound 265) Using compound 265, compound 290 is synthesized in the same manner as the synthesis of compound 236. [ka]

[0255] (Synthesis of compound 281) [ka] Compound 280 is dissolved in DCE at room temperature, ethyl chloroformate is added, and the mixture is heated under reflux. The solvent is removed by distillation under reduced pressure, the precipitated solid is filtered, and after washing with ethyl acetate, compound 281 is obtained.

[0256] (Synthesis of compound 282) [ka] At room temperature, compound 281 is dissolved in dry THF and dry DMF, TBAI and tetraethylene glycol are added, and the temperature is raised to 40°C. Then sodium hydride is added and the mixture is stirred, and water is added to stop the reaction. Ethyl acetate is added to separate the two layers, the organic layer is washed with water and saturated brine, and then dried over anhydrous sodium sulfate. After filtering off the drying agent, the solvent is removed by reduced pressure. The residue is purified by column chromatography (chloroform:methanol = 25:1) to obtain compound 282.

[0257] (Synthesis of compound 283) [ka] At room temperature, compound 282 is dissolved in methanol, and tetrakistriphenylphosphine palladium and potassium carbonate are added and the mixture is stirred. After adding Amberlyst 15, Amberlyst 15 is filtered off, and the solvent is removed by distillation under reduced pressure to obtain compound 283.

[0258] (Synthesis of compound 284) [ka] At 0°C, morpholine was mixed with 37% formalin aqueous solution and acetic acid, and the mixture was heated to room temperature. After stirring, toluene was added and the solvent was removed under reduced pressure to obtain a mixture of morpholine and iminium-51. The resulting mixture was dissolved in dry toluene, compound 281 was added, and the mixture was heated to 70°C. After stirring, water was added to stop the reaction. Chloroform was added to separate the two layers, and the organic layer was washed with water and saturated brine, then dried over anhydrous sodium sulfate. After filtering off the drying agent, the solvent was removed by reduced pressure. The residue was purified by column chromatography (chloroform:methanol = 10:1) to obtain compound 284.

[0259] (Synthesis of compound 285) [ka] At room temperature, compound 284 is dissolved in DMF, and cesium carbonate, allyl bromide, and sodium iodide are added at 0°C, and the temperature is raised to 30°C. After stirring, saturated aqueous ammonium chloride is added to stop the reaction. Ethyl acetate is added to separate the two layers, the organic layer is washed with water and saturated brine, and then dried over anhydrous sodium sulfate. After filtering off the drying agent, the solvent is removed under reduced pressure to obtain compound 282a. Chloroform is added to the aqueous layer obtained by liquid-liquid separation to separate the two layers, the organic layer is washed with water and saturated brine, and then dried over anhydrous sodium sulfate. After filtering off the drying agent, the solvent is removed under reduced pressure. The residue is purified by column chromatography (chloroform:methanol = 20:1) to obtain compound 285.

[0260] (Synthesis of Compound 286) [ka] At room temperature, compound 285 is dissolved in dry THF, chloromethyl methyl ether and sodium hydride are added, and the temperature is increased. After stirring, water is added to stop the reaction. Ethyl acetate is added to separate the two layers, the organic layer is washed with water and saturated brine, and then dried over anhydrous sodium sulfate. After filtering off the drying agent, the solvent is removed by reduced pressure. The residue is purified by column chromatography (chloroform:methanol = 20:1) to obtain compound 286.

[0261] (Synthesis of compound 287) [ka] Compound 286 is dissolved in DCE at room temperature, ethyl chloroformate is added, and the mixture is heated under reflux. The solvent is removed by distillation under reduced pressure, and the residue is purified by column chromatography (hexane:ethyl acetate = 5:1) to obtain compound 287.

[0262] (Synthesis of compound 288) [ka] At room temperature, 4M dioxane hydrochloride is added to compound 287 and stirred, then water is added to stop the reaction. Ethyl acetate is added to separate the two layers, the organic layer is washed with water and saturated brine, and then dried over anhydrous sodium sulfate. After filtering off the drying agent, the solvent is removed by reduced pressure. The residue is purified by column chromatography (ethyl acetate) to obtain compound 288.

[0263] (Synthesis of compound 289) [ka] At room temperature, compound 288 is dissolved in dry DMF, potassium iodide and sodium hydride are added, and the mixture is stirred. Water is then added to stop the reaction. Ethyl acetate is added to separate the two layers, and the organic layer is washed with water and saturated brine, then dried over anhydrous sodium sulfate. After filtering off the drying agent, the solvent is removed by reduced pressure. The residue is purified by column chromatography (ethyl acetate : methanol = 20 : 1) to obtain compound 289.

[0264] Compound 290 is synthesized by deprotecting the allyl group of compound 289. [ka]

[0265] (Synthesis of compound 290 using compound 236) Compound 290 was synthesized using compound 236 as the starting material, via compound 289. [ka] (Synthesis of compound 289) [ka] Under a nitrogen atmosphere, 2-iodobenzoic acid (225.5 mg, 0.91 mmol) was dissolved in anhydrous THF (0.8 mL), and isopropylmagnesium chloride-lithium chloride complex (1.3 M THF solution, 1.4 mL, 1.82 mmol) and compound 236 (67.6 mg, 0.09 mmol) were added at 0°C. The mixture was allowed to rise naturally and then returned to room temperature, after which 1 M hydrochloric acid was added to stop the reaction. Ethyl acetate was added to separate the two layers, and the organic layer was washed with water and saturated brine, and then dried over anhydrous sodium sulfate. After filtering off the drying agent, the solvent was removed by reduced pressure distillation. The residue was purified by column chromatography (hexane:ethyl acetate = 1:1) and (ethyl acetate:methanol = 30:1) to obtain compound 289 (26.3 mg, 34%) as yellow oil.

[0266] 289: Yellow oil. 1 H NMR (400 MHz, CDCl3) δ 8.01 (d, J = 7.3 Hz, 1H), 7.75-7.57 (m, 2H), 7.32 (d, J = 7.8 Hz, 1H), 6.66 (t, J = 14.9 Hz, 1H), 6.51 (d, J = 11.4 Hz, 1H), 6.21 (m, J = 11.3, 5.7 Hz, 2H), 5.53 (dd, J = 16.9, 6.4 Hz, 1H), 5.25-5.18 (m, 1H), 5.06-4.94 (m, 6H), 4.90-4.71 (m, 2H), 4.52-4.39 (m, 2H), 3.92-3.78 (m, 4H), 3.73-3.19 (m, 32H)

[0267] (Synthesis of compound 290) [ka] At room temperature, dissolve compound 289 (23.1 mg, 0.03 mmol) in methanol (1 mL) and tetrakistriphenylphosphine palladium (0.6 mg, 5.44 × 10⁻¹⁴). -4( mmol) and potassium carbonate (11.7 mg, 0.08 mmol) were added. After stirring for two and a half hours, the reaction was stopped by adding 1 M hydrochloric acid solution. Ethyl acetate was added to separate the two layers, which were washed with water and saturated brine, and then dried over anhydrous sodium sulfate. After filtering off the drying agent, the solvent was removed by distillation under reduced pressure. The residue was purified by column chromatography (ethyl acetate:methanol = 10:1) to obtain compound 290 (11.3 mg, 54%) as a yellow solid.

[0268] 290: Yellow powder. 1 H NMR (400 MHz, CDCl3) δ 8.53-8.87 (2H), 7.99 (d, J = 7.3 Hz, 1H), 7.67-7.58 (m, 2H), 7.13 (d, J = 7.3 Hz, 1H), 6.49 (s, 2H), 5.01 (s, 4H), 4.44 (dd, J = 32.2, 10.7 Hz, 4H), 3.82-3.63 (m, 32H)

[0269] Figure 15 shows the fluorescence spectra when spermidine or spermine is added to compound 290.

[0270] (Synthesis of compound 291 using compound 123) Compound 291 is synthesized using compound 123. [ka]

[0271] (Synthesis of compound 291) [ka] Under a nitrogen atmosphere, compound 123 (41.1 mg, 0.08 mmol) was dissolved in anhydrous THF (0.3 mL), and s-butyllithium (1.2 M hexane solution, 0.08 mL, 0.10 mmol) was added dropwise at -78°C. Anhydrous THF solution of phthalic anhydride (5.0 mg, 0.03 mmol) (0.1 mL) was added, and the temperature was gradually raised to room temperature. 1 M hydrochloric acid solution (0.1 mL) was added, and the mixture was stirred for two and a half hours. Ethyl acetate was added, and the mixture separated into two layers. The organic layer was washed with water and saturated brine, and dried over anhydrous sodium sulfate. After filtering off the drying agent, the solvent was removed by reduced pressure. The residue was purified by column chromatography (hexane:ethyl acetate = 1:1) to obtain compound 291 (11.8 mg, 37%) as a yellow solid. 1H-NMR (400 MHz, CHLOROFORM-D) δ 7.85 (d, J = 7.8 Hz, 1H), 7.62-7.56 (dd, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.45 (s, 2H), 7.35-7.29 (dd, 1H), 6.22-6.08 (m, 4H), 5.51 (d, J = 16.9 Hz, 2H), 5.45-5.40 (m, 2H), 5.28-5.21 (m, 4H), 4.98-4.89 (m, 4H), 4.86 (d, J = 10.5 Hz, 2H), 4.62 (d, J = 9.6 Hz, 4H), 4.56 (qd, J = 5.9, 1.1 Hz, 2H), 4.46 (qd, J = 5.9, 1.1 Hz, 2H), 4.10-4.07 (m, 2H), 3.70-3.41 (m, 32H).

[0272] (Synthesis of compound 294 using compound 129) Compound 294 is synthesized using compound 129. [ka]

[0273] (Synthesis of Compound 293) [ka] Under a nitrogen atmosphere, compound 129 (41.5 mg, 0.08 mmol) was dissolved in anhydrous THF (0.3 mL), and s-butyllithium (1.2 M hexane solution, 0.08 mL, 0.10 mmol) was added dropwise at -78°C. Anhydrous THF solution of phthalic anhydride (5.0 mg, 0.03 mmol) (0.1 mL) was added, and the temperature was gradually raised to room temperature. 1 M hydrochloric acid solution (0.1 mL) was added, and the mixture was stirred for two and a half hours. Ethyl acetate was added to separate the mixture into two layers. The organic layer was washed with water and saturated brine, and dried over anhydrous sodium sulfate. After filtering off the drying agent, the solvent was removed by reduced pressure. The residue was purified by column chromatography (hexane:ethyl acetate = 1:1) to obtain compound 293 (1.7 mg, 5%) as a white solid.

[0274] (Synthesis of Compound 294) [ka] Compound 293 (1.7 mg) is dissolved in methanol (0.5 mL), and tetrakistriphenylphosphine palladium (0.1 mg, 3.8 × 10⁻⁶) is added. -5 mmol), potassium carbonate (0.8 mg, 5.8 × 10⁻⁶). -3 mmol) was added. The temperature was raised to 50°C, and the reaction was stopped by adding 1M hydrochloric acid (0.5 mL). Ethyl acetate was added to separate the two layers, and the organic layer was washed with water and saturated brine. Anhydrous sodium sulfate was added and dried, and after filtering off the drying agent, the solvent was removed by vacuum distillation to obtain compound 294 (1.7 mg). ESI-MS 837.21(M+K) + A peak corresponding to the molecular weight of the designed compound was observed.

[0275] (Example 6: Development of amide-type fluorescent dyes) During the investigation of the synthesis of fluorescently responsive reagents, compound 268 containing an amide was obtained. To confirm whether compound 268 fluoresces, the allyl group was deprotected and compound 278 was synthesized (Scheme 6-1).

[0276] [ka] Scheme 6-1. Synthesis of Compound 278

[0277] The behavior of compound 278 in buffer solutions with pH 1–13 was investigated. First, the UV-vis spectra are shown in Figure 13. In the strongly acidic spectrum, the maximum absorption wavelength was 447 nm, and the absorption intensity was high at pH 1. No significant absorption was observed at pH 3–5, suggesting that it is a neutral species. At pH 6–8, absorption was observed at 500 nm, and the intensity increased as the solution became more basic. Similarly, absorption was observed at 500 nm at pH 9–13. The absorption intensity was highest at pH 8 and decreased as the solution became more basic.

[0278] [ka] compound 278

[0279] Next, fluorescence measurements were performed on compound 278 at pH 1–13 (Figure 14). At pH 13, fluorescence was emitted at 520 nm, and in the pH range of 12–9, fluorescence was emitted at 528 nm. In the pH range of 8–6, fluorescence was emitted at 530 nm. At the acidic pH of 2–1, fluorescence was emitted at 523 nm. From the UV-vis spectrum, it was found that cationic, neutral, and monoanionic species were generated with changes in pH.

[0280] It is understood that the amide-type compounds of the present invention, like compound 278, can be used as fluorescent dyes.

[0281] (Example 7: Diagnosis of Parkinson's disease) The spermine / spermidine ratio has been reported to be a biomarker for the early diagnosis of Parkinson's disease. It has been shown that spermidine levels in the body are significantly elevated in Parkinson's disease patients, while the levels of its metabolite, spermine, are decreased. Therefore, the ratio of the two can be used as a marker for the intractable disease Parkinson's disease.

[0282] As described above, by using multiple host molecules and solving the simultaneous equations shown in Equation 2-1 for each host molecule, spermine and spermidine can be quantified. Abs host = ε spd ×[spermidine] + ε spm ×[sprmine] (Equation 2-1) For example, by using a host molecule such as the one shown in Example 2 above, the spermine / spermidine ratio can be determined and used as a biomarker for early diagnosis.

[0283] (Note) As described above, the Disclosure has been illustrated using preferred embodiments thereof, but it is understood that the scope of the Disclosure should be interpreted solely by the claims. This Application claims priority over Japanese Patent Application No. 2021-21230 (filed February 12, 2021), the entire contents of which are incorporated herein by reference. It is understood that the patents, patent applications and documents cited herein should be incorporated herein by reference as if their contents were specifically described herein. [Industrial applicability]

[0284] This technology holds potential for adding polyamine concentration as an item in health checkups, and could be used for the early detection of cancer and Parkinson's disease. Furthermore, by adding fluorescence responsiveness and developing a spray application method, it could be applied to rapid diagnosis during cancer surgery (determining how much of the cancer should be removed).

Claims

1. Compounds represented by the following general formula I: 【Chemistry 1】 Equation I [In the formula, L ４ It either does not exist or is a linking group. n ５ is an integer from 0 to 4, R ５ In each occurrence, independently, is either a substituent or R ５ Two of these may, together with the carbon atoms to which they are linked, form a substituted or unsubstituted ring. Ar １ -OH and L １ The portion formed by and Ar ２ -OH and L ２ The parts formed by this are independent in each occurrence, 【Chemistry 2】 selected from the group consisting of, * is L ４ or in the formula I, Ar １ Ar ２ and L ３ represents a bonding point that can be bonded to the carbon to which they are bonded L ３ is, -CO ２ -, -SO ３ -, or -CONH-, and -CO ２ - and -CONH- carbon atoms, -SO ３ - The sulfur atoms are each R ５ Bonded to a benzene ring which may be substituted, L ３ ga-CO ２ - and Ar １ and Ar ２ If both have the same or different benzene ring structures, L ４ is a linking group, L ３ is, -SO ３ - When n ５ is an integer from 1 to 4, and R5 is a nitro group, and / or Ar １ and Ar ２ At least one of them independently has a naphthalene ring structure, an anthracene ring structure, or a phenanthrene ring structure in each appearance. The aforementioned linking groups are -O-, -CH ２ -O-, -O-CH ２ -, -O-(CH ２ ) ｎ -O-, -CH ２ -O-CH ２ A compound or its isomer, wherein n is an integer from 1 to 10, or -O-C(=O)-O-, and n is an integer from 1 to 10.

2. Ar １ and Ar ２ If both have the same or different benzene ring structures, L ４ The compound or its isomer according to claim 1, wherein the linking group is...

3. L ４ is -O-, -CH ２ -O-, -O-CH ２ -, -O-(CH ２ ) ｎ -O-, -CH ２ -O-CH ２ The compound or its isomer according to claim 1 or 2, wherein n is an integer from 1 to 10, or -O-C(=O)-O-.

4. R ５ The compound or isomer of any one of claims 1 to 3, wherein each occurrence is independently selected from the group consisting of an unsubstituted or halogenated linear or branched alkyl group, an unsubstituted or halogenated linear or branched alkyloxy group, a halogen atom, a nitro group, a carboxylic acid group or its derivative, a sulfonic acid group or its derivative, and an amino group or its derivative.

5. The aforementioned compound, 【Chemistry 4】 The compound according to claim 1.

6. The aforementioned compound, 【Transformation 5】 The compound according to claim 1. 【Request Item 7】 【Transformation 6】 It is a compound.

8. L ３ However, -CO ２ - and L ４ The compound according to any one of claims 1 to 4, wherein the compound is -O-.

9. The aforementioned compound, 【Transformation 7】 The compound according to claim 8.

10. The aforementioned compound, 【Transformation 8】 The compound according to claim 9.

11. n ５ The compound according to claim 10, wherein is 0.

12. L ３ However, -CO ２ - and L ４ However, -CH ２ -O-, -O-CH ２ -, -O-(CH ２ ) ｎ -O-, -CH ２ -O-CH ２ The compound according to any one of claims 1 to 4, wherein it is - or -O-C(=O)-O-, and n is an integer from 1 to 10.

13. The aforementioned compound, 【Chemical Engineering 8A】 The compound according to claim 12. 【Request Item 14】 【Chemistry 9】 It is a compound. 【Request Item 15】 【Chemistry 9A】 It is a compound. 【Request Item 16】 【Chemistry 9B】 It is a compound.

17. A diagnostic reagent for detecting polyamines in vivo, comprising any compound from claims 1 to 16.

18. The diagnostic reagent according to claim 17, further comprising another compound having different affinity for spermine and spermidine from the aforementioned compound.

19. The method includes the steps of contacting a sample with at least two compounds having different affinities to spermine and spermidine, and separating and quantifying spermine and spermidine based on the difference in affinities of the at least two compounds to spermine and spermidine, wherein the at least two compounds include at least one compound from any of claims 1 to 13. Hmm, a method for the differential determination of spermine and spermidine.

20. A method for the fractional determination of spermine and spermidine, comprising contacting a sample with any compound from claims 1 to 13 and measuring the color change or fluorescence with a spectrophotometer.

21. (i) Contact any compound from claims 1 to 13 with a sample and measure the color or fluorescence using a spectrophotometer. (ii) Contacting a compound from any of claims 1 to 13, different from the compound used in (i) above, with the sample and measuring the color change or fluorescence with a spectrophotometer, and (iii) Calculate the amounts of spermine and spermidine based on the measurement results of steps (i) and (ii) above. A method for the fractional quantitative determination of spermine and spermidine, characterized by the following.

22. A test or diagnostic agent for identifying diseases based on in vivo polyamines, comprising any compound from claims 1 to 13.

23. The test or diagnostic agent according to claim 22, wherein the disease includes cancer, Parkinson's disease, or Alzheimer's disease.

24. A method for providing information on polyamines in a subject as an indicator of whether the subject is suspected of having a disease based on polyamines in the subject, In a sample derived from the subject, the polyamine is measured using the compound described in any one of claims 1 to 16 or the diagnostic reagent described in claim 17 or 18. Methods including (excluding diagnostic acts by physicians).

25. The method according to claim 24, wherein the disease includes cancer, Parkinson's disease, or Alzheimer's disease.

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