Squaric acid compound, pharmaceutically acceptable salt or deuterated compound thereof, preparation method therefor and use thereof

Abstract

The present application relates to a squaric acid compound or a pharmaceutically acceptable salt or deuterated compound thereof, a preparation method therefor and a use thereof. The structural general formula of the squaric acid compound is shown as formula I, II, III, IV, V or VI.

Description

Squaric acid compounds, pharmaceutically acceptable salts or deuterated compounds thereof, and preparation methods and applications thereof

[0001] Related applications

[0002] This application claims priority to Chinese patent application No. 2023117686258, filed on December 20, 2023, entitled “Squaric acid compounds or pharmaceutically acceptable salts thereof, or deuterated compounds and preparation methods and applications thereof,” the entire text of which is hereby incorporated by reference. Technical Field

[0003] The present application relates to the field of organic fluorescent probes, and in particular to a squaric acid compound or a pharmaceutically acceptable salt or deuterated compound thereof, and a preparation method and application thereof. Background Art

[0004] Optical imaging, especially fluorescence imaging, which has developed rapidly and become widely used in recent years, utilizes specific fluorescent molecular probes to label specific molecules or cells. Its spatial resolution can reach the millimeter level. It is well known to life scientists and has been widely used in in vitro imaging. It is very popular among scientists. It has many advantages, such as high sensitivity, rapidity and simplicity, low cost, and relatively high throughput. Key issues in optical imaging include autofluorescence, quenching, photobleaching, and low tissue penetration depth.

[0005] Compared to fluorescence imaging in the visible region (400-700 nm), fluorescence imaging in the near-infrared (NIR) window (700-1700 nm) offers significant advantages in terms of reduced photon scattering, lower absorption, and minimal autofluorescence interference (Chem. Soc. Rev., 2018, 47, 4258). NIR imaging offers high resolution and a high signal-to-noise ratio, and holds great potential for molecular diagnostics and therapeutic applications. Over the past decade, fluorescence imaging in the first near-infrared region (NIR-I, 700–900 nm) has been widely used in basic research, preclinical research, and clinical diagnostics, including the FDA-approved indocyanine green (ICG) and methyl blue (MB). However, compared to NIR-I fluorescence imaging, NIR-II offers greater penetration depth, superior imaging quality, signal-to-noise ratio, and sensitivity due to reduced tissue autofluorescence, reduced photon scattering, and low photon absorption. At present, biological NIR-II fluorescence imaging reagents mainly include carbon nanotubes, quantum dots, rare earth-doped nanoparticles, organic small molecules and conjugated polymers.

[0006] Compared with inorganic nanomaterials, organic small molecule dyes have gradually attracted the interest of researchers due to their advantages such as clear structure, small molecular weight, easy metabolism, and safety. Researchers have attempted to push the emission wavelength of small molecule dyes to the near-infrared region by optimizing structural design and synthesis routes. These diverse NIR dye structures have enriched the NIR fluorescent probe library by rationally designing the main chain and substituent groups of the dye structure. For example, cyanines and DAD types have shown good water solubility, quantum yield and molar absorptivity in biological systems and tissues, and have been widely developed for use as NIR-II fluorescent probes. However, cyanine dyes are unstable to light, have poor chemical stability, low photothermal conversion efficiency, small Stokes shift, and most of the developed dyes are located in NIR-I. DAD-type dyes have disadvantages such as large molecular weight, small molar absorptivity, and slow metabolism in the body. Summary of the Invention

[0007] Based on this, the purpose of the present application is to provide an aromatic acid compound or a pharmaceutically acceptable salt or deuterated compound thereof that emits near-infrared light, is photostable, has a small molecular weight, a large Stokes shift and a large molar absorption coefficient, which can be used as a fluorescent probe and a near-infrared second zone contrast agent.

[0008] In one aspect, a squarylium compound or a pharmaceutically acceptable salt or deuterated compound thereof is provided, wherein the structure of the squarylium compound is shown in Formula I, II, III, IV, V or VI:

[0009] Among them, X m- 、X n- and X p- are independently anions;

[0010] m, n and p are independently selected from any integer from 1 to 10;

[0011] Each R1 is independently selected from H, C1-C 12 Alkyl, C1-C 12 Alkoxy, C1-C8 alkylsilyl, hydroxy C1-C8 alkyl, amino C1-C8 alkyl, aldehyde C1-C8 alkyl, mercapto C1-C8 alkyl, halogenated C1-C8 alkyl, acyloxy C1-C8 alkyl, amino, halogen, nitro, carboxyl C1-C6 alkyl, substituted or unsubstituted C6-C 10 At least one of aryl or 5-10 membered heteroaryl, -(CH2)n1-COOCH2CH2Si(CH3)3, -(CH2)n2-(CH2CH2O)n3-R and -(CH2)n2-(OCH2CH2)n3-R;

[0012] R2 is selected from hydroxyl, C1-C12 At least one of an alkoxy group, a C1-C8 alkylsilyl group, a hydroxy C1-C8 alkyl group, a carboxyl group, an amino group, -NR5R6, a mercapto group, -SR7, and a malononitrile group;

[0013] R3, R4, R5, R6 and R7 are independently selected from H, C1-C8 alkyl, hydroxyl, amino, carboxyl, C1-C 12 Alkyl, C1-C 12 Alkoxy, C1-C8 alkylsilyl, hydroxy C1-C8 alkyl, amino C1-C8 alkyl, aldehyde C1-C8 alkyl, mercapto C1-C8 alkyl, halogenated C1-C8 alkyl, acyloxy C1-C8 alkyl, amino, halogen, sulfonic acid, carboxyl C1-C6 alkyl, substituted or unsubstituted C6-C 10 At least one of aryl or 5-10 membered heteroaryl, -(CH2)n1-COOCH2CH2Si(CH3)3, -(CH2)n2-(CH2CH2O)n3-R and -(CH2)n2-(OCH2CH2)n3-R;

[0014] n1 is any integer from 0 to 10, n2 is any integer from 0 to 10, and n3 is any integer from 1 to 500;

[0015] R is selected from H, C1-C8 alkyl, hydroxyl, amino, carboxyl, sulfonic acid, halogen, mercapto, and At least one of;

[0016] Indicates the attachment site.

[0017] In one embodiment, the general structural formula of the squaric acid compound is shown in Formula I-1, II-1, III-1, IV-1, VI-1 or VI-1:

[0018] In one embodiment, the C6-C 10 The substituents in the aryl group or the 5-10 membered heteroaryl group are selected from at least one of a C1-C8 alkyl group, a C1-C8 alkoxy group, a C1-C8 alkylsilyl group, a hydroxy C1-C8 alkyl group, an amino C1-C8 alkyl group, a mercapto C1-C8 alkyl group, a halogenated C1-C8 alkyl group and a carboxyl C1-C6 alkyl group.

[0019] In one embodiment, m, n and p are independently selected from any integer from 1 to 4.

[0020] In one embodiment, X - Each independently selected from I - Br - 、BF4 -or ClO4 - .

[0021] In one embodiment, the square acid compound has any of the following structures:

[0022] The present application also provides a method for preparing the above-mentioned square acid compound, and the technical solution is as follows:

[0023] (1) A method for preparing a square acid compound as described in Formula I, comprising the following steps:

[0024] Compound 1 undergoes a nucleophilic substitution reaction with compound a-1 to obtain compound 2-1, wherein compound a-1 is halogenated R3; compound 1 undergoes a nucleophilic substitution reaction with compound a-2 to obtain compound 2-2, wherein compound a-2 is halogenated R4;

[0025] Compound 2-1 undergoes a Grignard reaction with compound b to obtain compound 3-1, and compound 2-2 undergoes a Grignard reaction with compound b to obtain compound 3-2, wherein compound b is a Grignard reagent;

[0026] Compound 3-1, compound 3-2 and compound c undergo condensation reaction to obtain compound 4 (ie, compound IV), wherein compound c is a squaric acid;

[0027] Compound 4 reacts with compound d-1 to obtain the compound shown in I, wherein compound d-1 contains R2;

[0028] wherein R1, R2, R3 and R4 are as defined above.

[0029] In one embodiment, the compound a-1 is Br-R3.

[0030] In one embodiment, the molar ratio of the compound 1 to the compound a-1 is 1:(1-1.5).

[0031] In one embodiment, the compound a-2 is Br-R4.

[0032] In one embodiment, the molar ratio of the compound 1 to the compound a-2 is 1:(1-1.5).

[0033] In one embodiment, the compound b is methylmagnesium chloride.

[0034] In one embodiment, the molar ratio of the compound 2-1 to the compound b is 1:(1-1.5).

[0035] In one embodiment, the molar ratio of the compound 2-2 to the compound b is 1:(1-1.5).

[0036] In one embodiment, the molar ratio of the compound 3-1 to the compound c is (1-3):1.

[0037] In one embodiment, the molar ratio of the compound 3-2 to the compound c is (1-3):1.

[0038] In one embodiment, the molar ratio of the compound 4 to the compound d-1 is 1:(1-1.5).

[0039] (2) A method for preparing a square acid compound as described in Formula II above, comprising the following steps:

[0040] Compound 5 undergoes a condensation reaction with compound c to obtain compound 6 (i.e., compound V), wherein compound c is a squaric acid;

[0041] Compound 6 reacts with compound d-2 to obtain the compound shown in II, wherein compound d-2 contains R2;

[0042] wherein R1, R2, R3 and R4 are as defined above.

[0043] In one embodiment, the molar ratio of the compound 5 to the compound c is (2-3):1.

[0044] In one embodiment, the molar ratio of the compound 6 to the compound d-2 is 1:(1-1.5).

[0045] (3) A method for preparing a square acid compound as described in Formula III above, comprising the following steps:

[0046] Compound 3-1 is substituted with compound e to generate compound 7, wherein compound e is diethyl squarate;

[0047] Compound 7 undergoes a hydrolysis reaction with compound f to generate compound 8, wherein compound f is a base;

[0048] Compound 8 and compound 5 undergo condensation reaction to generate compound 9 (i.e., compound VI);

[0049] Compound 9 reacts with compound d-3 to obtain the compound shown in III, wherein compound d-3 contains R2;

[0050] wherein R1, R2, R3 and R4 are as defined above.

[0051] In one embodiment, the molar ratio of the compound 3-1 to the compound e is (1-1.5):1;

[0052] In one embodiment, the molar ratio of the compound 8 to the compound 5 is 1:(1-1.5);

[0053] In one embodiment, the molar ratio of the compound 9 to the compound d-3 is 1:(1-1.5).

[0054] In another aspect, a fluorescent probe is provided, comprising the above-mentioned squaraine compound or a pharmaceutically acceptable salt or a deuterated form thereof.

[0055] In another aspect, a near-infrared zone II contrast agent is provided, comprising the above-mentioned squaraine compound or a pharmaceutically acceptable salt or a deuterated form thereof.

[0056] In yet another aspect, a method for monitoring early-stage liver damage is provided, comprising imaging a subject in need thereof using the squaraine compound described above, or a pharmaceutically acceptable salt or deuterated compound thereof, the fluorescent probe described above, or the near-infrared zone II contrast agent described above.

[0057] This application has at least the following beneficial effects:

[0058] The squaraine compounds provided in this application use squaraine as the central ring. By changing the donor and acceptor, a class of donor-π-acceptor (D-π-A) type fluorescent molecules that emit near-infrared light are developed. They are photostable, have a small molecular weight, a large Stokes shift and a large molar absorption coefficient, and have a certain photothermal effect. They are very suitable for use as fluorescent probes, especially near-infrared fluorescent probes, and can further be used to prepare near-infrared zone II contrast agents for in vivo imaging applications such as in vivo metabolism research in small animals and lymphangiography.

[0059] The preparation process of the squaric acid compound of the present application is simple, the raw materials are readily available, the cost is low, it is very suitable for scale-up production, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the conventional technology, the following briefly introduces the drawings required for use in the embodiments or the conventional technology descriptions. Obviously, the drawings described below are merely embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on the disclosed drawings without any creative work.

[0061] Figure 1 is the UV absorption spectrum of compound Ia.

[0062] Figure 2 is the fluorescence emission spectrum of compound Ia.

[0063] Figure 3 is a diagram of the metabolism of compound Ia in normal mice.

[0064] FIG4 is a diagram showing the metabolism of compound Ia in normal mice and mice with carbon tetrachloride liver injury model.

[0065] FIG5 is an in vivo fluorescence image obtained 30 minutes after injection of compound Ia into normal mice and mice with carbon tetrachloride liver injury model.

[0066] FIG6 is a qPCR graph of primary hepatocytes extracted from mice with carbon tetrachloride liver injury model.

[0067] Figure 7 is a metabolic diagram of compound Ia in Mate 1 transporter inhibitor model mice.

[0068] FIG8 is a graph showing the photostability test of compound Ia and indocyanine green (ICG) currently used in clinical practice. DETAILED DESCRIPTION

[0069] The following will be combined with the drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0070] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application.

[0071] Where “including,” “having,” and “comprising” are used herein, it is intended to cover a non-exclusive inclusion, and another component may also be added unless a clear limiting term such as “only,” “consisting of,” etc. is used.

[0072] In the present application, “further”, “further”, “particularly”, etc. are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of the present application.

[0073] In this application, "at least one" means more than one, such as one, two, or more than two. "Multiple" or "several" means at least two, such as two, three, etc., and "multi-layer" means at least two layers, such as two, three, etc., unless otherwise specifically defined. In the description of this application, "several" means at least one, such as one, two, etc., unless otherwise specifically defined.

[0074] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein are understood to include any and all subranges subsumed therein.

[0075] Unless otherwise specified, all steps of the present application may be performed sequentially or randomly. For example, the method includes steps (a) and (b), which means that the method may include steps (a) and (b) performed sequentially, or may include steps (b) and (a) performed sequentially. For example, the method may further include step (c), which means that step (c) may be added to the method in any order. For example, the method may include steps (a), (b) and (c), or may include steps (a), (c) and (b), or may include steps (c), (a) and (b), etc.

[0076] Unless mentioned otherwise, terms in the singular may include plural forms and should not be construed as having one number.

[0077] In this application, “above” or “below” includes the number itself. For example, “1 below” includes 1.

[0078] Unless otherwise specified, the temperature parameters in this application allow for both constant temperature treatment and temperature fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows for temperature fluctuations within the accuracy range of instrument control. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are permitted.

[0079] In this application, the number of atoms described in the numerical range includes both integer endpoints of the numerical range and each integer between the two endpoints. For example, "C1-C10 alkyl" means an alkyl group having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.

[0080] In this application, When R is selected from a single bond, express It indicates that the connection point of the substituent R to the benzene ring is not limited.

[0081] In this application, Indicates the attachment site.

[0082] As used herein, "halogen" or "halo" refers to -F, -Cl, -Br or -I.

[0083] In this application, the term "alkyl" refers to a monovalent residue formed by the loss of one hydrogen atom from a saturated hydrocarbon containing a primary (normal) carbon atom, a secondary carbon atom, a tertiary carbon atom, a quaternary carbon atom, or a combination thereof. Phrases containing this term, for example, "C1-C10 alkyl" refers to an alkyl group containing 1 to 10 carbon atoms, and each occurrence can independently be C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, or C10 alkyl. Suitable examples include, but are not limited to, methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, -CH(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, i-butyl, -CH2CH(CH3)2), 2-butyl (s-Bu, s-butyl, -CH(C H3)CH2CH3), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH3)3), 1-pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-1-butyl (- CH2CH2CH(CH3)2), 2-methyl-1-butyl (-CH2CH(CH3)CH2CH3), 1-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH( )2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3 and octyl (-(CH2)7CH3).

[0084] As used herein, "haloalkyl" refers to an alkyl group substituted with one or more halogen (chlorine, fluorine, bromine, or iodine) atoms. Polyhaloalkyl groups have the same or mixed types of halogen atoms. "Perhaloalkyl" refers to an alkyl group in which every hydrogen atom is replaced by a halogen atom. A haloalkyl group that is "fully halogenated" at a particular carbon atom means that all hydrogen atoms attached to that carbon are replaced by halogen atoms. Representative mono-, di-, and trihaloalkyl groups include: chloromethyl, chloroethyl, bromomethyl, bromoethyl, iodomethyl, iodoethyl, chloropropyl, bromopropyl, iodopropyl, 1,1-dichloromethyl, 1,1-dibromomethyl, 1,1-dichloropropyl, 1,2-dibromopropyl, 2,3-dibromopropyl, 1-chloro-2-bromoethyl, 2-chloro-3-bromopropyl, trifluoromethyl, trichloromethyl, and the like.

[0085] In this application, "cycloalkyl" refers to a non-aromatic hydrocarbon containing ring carbon atoms, which can be a monocyclic alkyl, a spirocyclic alkyl, or a bridged cycloalkyl. Phrases containing this term, for example, "C3-C10 cycloalkyl" refers to a cycloalkyl containing 3 to 10 carbon atoms, each occurrence of which can be independently C3 cycloalkyl, C4 cycloalkyl, C5 cycloalkyl, C6 cycloalkyl, C7 cycloalkyl, C8 cycloalkyl, C9 cycloalkyl, or C10 cycloalkyl. Suitable examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. In addition, "cycloalkyl" may also contain one or more double bonds. Representative examples of cycloalkyl containing double bonds include cyclopentenyl, cyclohexenyl, cyclohexadienyl, and cyclobutadienyl.

[0086] In this application, the "number of ring atoms" refers to the number of atoms among the atoms that constitute the ring itself of a structural compound (e.g., a monocyclic compound, a condensed ring compound, a cross-linked compound, a carbocyclic compound, a heterocyclic compound) formed by atoms bonded together to form a ring. When the ring is substituted with a substituent, the atoms contained in the substituent are not included in the ring atoms. The same applies to the "number of ring atoms" described below unless otherwise specified. For example, the number of ring atoms of a benzene ring is 6, the number of ring atoms of a naphthalene ring is 10, and the number of ring atoms of a biphenyl ring is 12.

[0087] In this application, the term "aryl, aromatic group or aromatic group" refers to a hydrocarbon group containing at least one aromatic ring, such as benzene, naphthalene, anthracene, fluoranthene, phenanthrene, triphenylene, acenaphthene, fluorene, biphenyl, terphenyl and derivatives of the above aromatic groups.

[0088] In this application, the term "arylene" refers to an aromatic hydrocarbon group derived from an aromatic ring compound by removing two hydrogen atoms. It can be a monocyclic arylene, a condensed-ring arylene, or a polycyclic arylene. For polycyclic rings, at least one is an aromatic ring system. For example, "C6-C10 arylene" refers to an arylene group containing 6 to 10 carbon atoms, and each occurrence can independently be C6 arylene, C7 arylene, C8 arylene, C9 arylene, or C10 arylene. Suitable examples include, but are not limited to, phenylene, biphenylene, naphthalene, anthracene, phenanthrene, perylene, triphenylene, and their derivatives.

[0089] In this application, the term "cycloalkylene" refers to a hydrocarbon group having two monovalent radical centers derived from a cycloalkyl group by removing two hydrogen atoms. It can be a monocycloalkylene, a spirocycloalkylene, or a bridged cycloalkylene. For example, "C3-C10 cycloalkylene" refers to a cycloalkylene group containing 3 to 9 carbon atoms, and each occurrence can be independently C3 cycloalkylene, C4 cycloalkylene, C5 cycloalkylene, C6 cycloalkylene, C7 cycloalkylene, C8 cycloalkylene, or C9 cycloalkylene. Suitable examples include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, and cycloheptylene. In addition, "cycloalkylene" may also contain one or more double bonds. Representative examples of cycloalkylene groups containing double bonds include cyclopentenylene, cyclohexenylene, cyclohexadienylene, and cyclobutadienylene.

[0090] In the present application, "A and B are independently selected from x, y or z" means that A and B are independent events, and event A does not affect the occurrence of event B. Therefore, when A is selected from x, B can be selected from any one of x, y or z; when A is selected from y, B can be selected from any one of x, y or z; when A is selected from z, B can be selected from any one of x, y or z.

[0091] In the present application, "substituted" means that a hydrogen atom in a substituted group is replaced by a substituent.

[0092] In the present application, "substituted or unsubstituted" means that the defined group may be substituted or unsubstituted. When the defined groups are substituted, they are understood to be optionally substituted with groups acceptable in the art, including but not limited to: straight-chain alkyl groups having 1-20 carbon atoms, branched or cycloalkyl groups having 3-20 carbon atoms, heterocyclyl groups having 3-20 ring atoms, aryl groups having 5-20 ring atoms, heteroaryl groups having 5-20 ring atoms, silanyl groups, carbonyl groups, alkoxycarbonyl groups, aryloxycarbonyl groups, carbamoyl groups, haloformyl groups, formyl groups, -NRR', cyano groups, isocyano groups, isocyanate groups, thiocyanate groups, isothiocyanate groups, hydroxyl groups, trifluoromethyl groups, nitro groups or halogen groups, and the above groups may also be further substituted with substituents acceptable in the art, selected substituents including but not limited to: straight-chain alkyl groups having 1-20 carbon atoms, branched or cycloalkyl groups having 3-20 carbon atoms, heterocyclyl groups having 3-20 ring atoms, aryl groups having 5-10 ring atoms, heteroaryl groups having 5-10 ring atoms, -N RR′, cyano, hydroxy, trifluoromethyl, nitro or halogen; it is understood that R and R′ in -NRR′ are each independently substituted by an acceptable group in the art, including but not limited to H, a straight-chain alkyl group having 1-6 carbon atoms, a branched or cycloalkyl group having 3-8 carbon atoms, a heterocyclyl group having 3-8 ring atoms, an aryl group containing 5-10 ring atoms or a heteroaryl group containing 5-10 ring atoms; wherein, the straight-chain alkyl group having 1-6 carbon atoms, the branched or cycloalkyl group having 3-8 carbon atoms, the heterocyclyl group having 3-8 ring atoms, the aryl group having 5-10 ring atoms or the heteroaryl group having 5-10 ring atoms are optionally further substituted, including but not limited to the following substituents: a straight-chain alkyl group having 1-6 carbon atoms, a branched or cycloalkyl group having 3-8 carbon atoms, an aryl group having 5-10 ring atoms (in one embodiment, a phenyl or naphthyl group) or a heteroaryl group having 5-10 ring atoms.

[0093] Inorganic nanomaterials have attracted significant interest due to their excellent optical properties. However, inorganic materials are slowly metabolized in the body and retained in the liver and spleen for extended periods, limiting their long-term biosafety. Compared to inorganic nanomaterials, organic small-molecule dyes have gradually attracted research interest due to their well-defined structures, small molecular weight, ease of metabolism, and safety. Researchers have attempted to push the emission wavelengths of small-molecule dyes into the near-infrared region by optimizing structural design and synthetic routes. These diverse NIR dye structures have enriched the NIR fluorescent probe library through the rational design of the backbone and substituent groups in the dye structure. Many organic dyes, such as cyanines, DADs, BODIPYs, and porphyrins, have demonstrated excellent water solubility, quantum yield, and molar absorptivity in biological systems and tissues. However, currently, only cyanines and DADs have been widely developed as NIR-II fluorescent probes. Since cyanine dyes are unstable to light, have poor chemical stability, low photothermal conversion efficiency, small Stokes shift, and most of the developed dyes are located in NIR-I, DAD-type dyes have disadvantages such as large molecular weight, small molar absorption coefficient, and slow metabolism in the body.

[0094] The liver has extremely complex and important physiological functions. As the body's primary metabolic organ, it is closely linked to numerous complex biological processes, including drug metabolism, excretion, bile secretion, phagocytosis, and immunity. Liver diseases have a high morbidity and mortality rate worldwide. Acute and chronic liver injury, as well as drug- and alcohol-induced liver injury, pose a serious threat to human health and life. With the continuous introduction of new drugs, drug-induced hepatotoxicity will become a significant clinical issue. However, commonly used biomarkers, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), are insensitive to early liver damage, and manifestations of muscle and kidney damage can also lead to false-positive diagnoses. Furthermore, with the secretion and accumulation of ALT and AST, when these two indicators rise to a detectable level, it indicates that liver disease has reached a very serious stage. At this point, the opportunity for timely treatment is lost, and the disease may progress and become life-threatening. Even in the terminal stages of the disease, ALT levels may decline. Therefore, ALT cannot be considered a true predictor. Histopathological examination, or liver biopsy, is currently considered the gold standard for diagnosing, staging, and grading liver disease. However, this technique is invasive and patient compliance is poor. Furthermore, complications can occur at any time during liver tissue collection, and the partial liver tissue sample collected often does not represent the true condition of the entire liver, potentially leading to misjudgment of the course of liver injury. Early detection of liver damage allows for timely liver-protective interventions. Therefore, the development of a reliable liver injury monitoring method would greatly help ensure drug safety and improve treatment efficiency. Although currently reported probes offer key advantages such as high selectivity and real-time feedback, relatively few near-infrared (NIR) II small molecule fluorescent probes are available for early monitoring of drug-induced liver injury. The FDA-approved fluorescent probe ICG, used clinically, is primarily used for liver resection surgical navigation and preoperative and postoperative liver function assessment. Its use in monitoring acute liver injury has been less studied, and ICG's inherent photophysical instability limits its application in bioimaging.

[0095] At present, some squaric acid dyes have been reported. Squaric acid dyes have high molar absorption coefficients (>10 5 M -1 cm -1 ), excellent photostability, easy absorption and wavelength tunability, etc. However, the emission spectra of most of the reported squaric acid dyes are located in the near-infrared region, with limited penetration depth and high tissue background, which affects the reliability of the experimental results; at the same time, the reported dyes are basically fat-soluble, with poor water solubility and low bioavailability.

[0096] The present application provides an aromatic acid compound or a pharmaceutically acceptable salt or deuterated compound thereof. These aromatic acid compounds or pharmaceutically acceptable salt or deuterated compounds have the characteristics of emitting near-infrared light, being photostable, having a small molecular weight, a large Stokes shift, and a large molar absorptivity. Furthermore, they are water-soluble and have high bioavailability. Therefore, they can be used as fluorescent probes and in the preparation of contrast agents, particularly near-infrared region II contrast agents.

[0097] The technical solution is as follows:

[0098] A squaric acid compound or a pharmaceutically acceptable salt or deuterated compound thereof, having the general structural formula I, II, III, IV, V or VI:

[0099] Among them, X m- 、X n- and X p- are independently anions;

[0100] m, n and p are independently selected from any integer from 1 to 10;

[0101] R1 and R 1’ are independently selected from H, C1-C 12 Alkyl, C1-C 12 Alkoxy, C1-C8 alkylsilyl, hydroxy C1-C8 alkyl, amino C1-C8 alkyl, aldehyde C1-C8 alkyl, mercapto C1-C8 alkyl, halogenated C1-C8 alkyl, acyloxy C1-C8 alkyl, amino, halogen, nitro, carboxyl C1-C6 alkyl, substituted or unsubstituted C6-C 10 At least one of aryl or 5-10 membered heteroaryl, -(CH2)n1-COOCH2CH2Si(CH3)3, -(CH2)n2-(CH2CH2O)n3-R and -(CH2)n2-(OCH2CH2)n3-R;

[0102] R2 is selected from hydroxyl, C1-C 12 At least one of an alkoxy group, a C1-C8 alkylsilyl group, a hydroxy C1-C8 alkyl group, a carboxyl group, an amino group, -NR5R6, a mercapto group, -SR7, and a malononitrile group;

[0103] R3, R4, R5, R6 and R7 are independently selected from H, C1-C8 alkyl, hydroxyl, amino, carboxyl, C1-C 12 Alkyl, C1-C 12Alkoxy, C1-C8 alkylsilyl, hydroxy C1-C8 alkyl, amino C1-C8 alkyl, aldehyde C1-C8 alkyl, mercapto C1-C8 alkyl, halogenated C1-C8 alkyl, acyloxy C1-C8 alkyl, amino, halogen, sulfonic acid, carboxyl C1-C6 alkyl, substituted or unsubstituted C6-C 10 At least one of aryl or 5-10 membered heteroaryl, -(CH2)n1-COOCH2CH2Si(CH3)3, -(CH2)n2-(CH2CH2O)n3-R and -(CH2)n2-(OCH2CH2)n3-R;

[0104] n1 is any integer from 0 to 10, n2 is any integer from 0 to 10, and n3 is any integer from 1 to 500;

[0105] R is selected from H, C1-C8 alkyl, hydroxyl, amino, carboxyl, sulfonic acid, halogen, mercapto, and At least one of;

[0106] Indicates the attachment site.

[0107] In one embodiment, the general structural formula of the squaric acid compound is shown in Formula I-1, II-1, III-1, IV-1, VI-1 or VI-1:

[0108] In one embodiment, each R1 is independently selected from hydrogen, nitro, methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, -CH(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, i-butyl, -CH2CH(CH3)2), 2-butyl (s-Bu, s -butyl, -CH(CH3)CH2CH3), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH3)3), 1-pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl- 1-butyl (-CH2CH2CH(CH3)2), 2-methyl-1-butyl (-CH2CH(CH3)CH2CH3), 1-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3) CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3 and octyl (-(CH2)7CH3).

[0109] In one embodiment, each R1 is independently selected from hydroxy C1-C8 alkyl, and in one embodiment is hydroxy C1-C4 alkyl.

[0110] In one embodiment, each R1 is independently selected from amino C1-C8 alkyl, and in one embodiment is amino C1-C4 alkyl.

[0111] In one embodiment, each R1 replaces the C6-C 10The substituents in the aryl group or the 5-10 membered heteroaryl group are selected from at least one of a C1-C8 alkyl group, a C1-C8 alkoxy group, a C1-C8 alkylsilyl group, a hydroxy C1-C8 alkyl group, an amino C1-C8 alkyl group, a mercapto C1-C8 alkyl group, a halogenated C1-C8 alkyl group and a carboxyl C1-C6 alkyl group.

[0112] In one embodiment, R3 and R4 are independently selected from hydrogen, methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, -CH(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, i-butyl, -CH2CH(CH3)2), 2-butyl (s-Bu, s -butyl, -CH(CH3)CH2CH3), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH3)3), 1-pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl- 1-butyl (-CH2CH2CH(CH3)2), 2-methyl-1-butyl (-CH2CH(CH3)CH2CH3), 1-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3) CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3 and octyl (-(CH2)7CH3).

[0113] In one embodiment, R3 and R4 are independently selected from hydroxy C1-C8 alkyl, and in one embodiment, hydroxy C1-C4 alkyl.

[0114] In one embodiment, R3 and R4 are independently selected from amino C1-C8 alkyl, and in one embodiment, amino C1-C4 alkyl.

[0115] In one embodiment, R3 and R4 are substituted with the C6-C 10 The substituents in the aryl group or the 5-10 membered heteroaryl group are selected from at least one of a C1-C8 alkyl group, a C1-C8 alkoxy group, a C1-C8 alkylsilyl group, a hydroxy C1-C8 alkyl group, an amino C1-C8 alkyl group, a mercapto C1-C8 alkyl group, a halogenated C1-C8 alkyl group and a carboxyl C1-C6 alkyl group.

[0116] In the present application, R2 is selected from hydroxyl, O - 、C1-C 12 At least one of an alkoxy group, a C1-C8 alkylsilyl group, a hydroxy C1-C8 alkyl group, a carboxyl group, an amino group, -NR6R7, a mercapto group, -SR8 and a malononitrile group.

[0117] In one embodiment, R5, R6 and R7 are independently selected from hydrogen, methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, -CH(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, i-butyl, -CH2CH(CH3)2), 2-butyl (s-Bu, s-butyl, -CH(CH3)CH2C H3), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH3)3), 1-pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-1-butyl (-CH2CH2CH(CH3)2), 2-methyl-1-butyl (-CH 2CH(CH3)CH2CH3), 1-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl 3), octyl (-(CH2)7CH3), -(CH2)n1-COOCH2CH2Si(CH3)3, -(CH2)n2-(CH2CH2O)n3-R and -(CH2)n2-(OCH2CH2)n3-R.

[0118] In one embodiment, R5, R6 and R7 are independently selected from hydroxy C1-C8 alkyl, and in one embodiment, hydroxy C1-C4 alkyl.

[0119] In one embodiment, R5, R6 and R7 are independently selected from amino C1-C8 alkyl, and in one embodiment, amino C1-C4 alkyl.

[0120] In one embodiment, R5, R6 and R7 replace the C6-C 10 The substituents in the aryl group or the 5-10 membered heteroaryl group are selected from at least one of a C1-C8 alkyl group, a C1-C8 alkoxy group, a C1-C8 alkylsilyl group, a hydroxy C1-C8 alkyl group, an amino C1-C8 alkyl group, a mercapto C1-C8 alkyl group, a halogenated C1-C8 alkyl group and a carboxyl C1-C6 alkyl group.

[0121] It is understandable that in this application, X m- 、X n- and X p- are all anions, m, n and p are independently selected from any integer from 1 to 10, i.e., m, n and p are independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, m, n and p are independently selected from any integer from 1 to 4, i.e., 1, 2, 3 or 4. In another embodiment, m, n and p are independently selected from 1 or 2. In other embodiments, m, n and p are all 1, X - Each independently selected from I - Br - 、BF4 - or ClO4 - .

[0122] It is understood that in the present application, n1 is any integer from 0 to 10, that is, n1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, n1 is any integer from 0 to 6.

[0123] It is understood that in the present application, n2 is any integer from 0 to 10, that is, n2 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, n2 is any integer from 0 to 6.

[0124] It is understood that in the present application, n3 is any integer from 1 to 500, that is, n3 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 or 500. In one embodiment, n3 is any integer from 1 to 300. In another embodiment, n3 is any integer from 1 to 150.

[0125] In one embodiment, the square acid compound has any of the following structures:

[0126] The present application also provides a method for preparing the above-mentioned square acid compound, and the technical solution is as follows:

[0127] (1) A method for preparing a square acid compound as described in formula (I) above, comprising the following steps:

[0128] Compound 1 undergoes a nucleophilic substitution reaction with compound a-1 to obtain compound 2-1, wherein compound a-1 is halogenated R3; compound 1 undergoes a nucleophilic substitution reaction with compound a-2 to obtain compound 2-2, wherein compound a-2 is halogenated R4;

[0129] Compound 2-1 undergoes a Grignard reaction with compound b to obtain compound 3-1, and compound 2-2 undergoes a Grignard reaction with compound b to obtain compound 3-2, wherein compound b is a Grignard reagent;

[0130] Compound 3-1, compound 3-2 and compound c undergo condensation reaction to obtain compound 4 (ie, compound IV), wherein compound c is a squaric acid;

[0131] Compound 4 reacts with compound d-1 to obtain the compound shown in I, wherein compound d-1 contains R2;

[0132] wherein R1, R2, R3 and R4 are as defined above.

[0133] In one embodiment, the compound a-1 is Br-R3.

[0134] In one embodiment, the molar ratio of the compound 1 to the compound a-1 is 1:(1-1.5).

[0135] In one embodiment, the compound a-2 is Br-R4.

[0136] In one embodiment, the molar ratio of the compound 1 to the compound a-2 is 1:(1-1.5).

[0137] In one embodiment, the compound b is methylmagnesium chloride.

[0138] In one embodiment, the molar ratio of the compound 2-1 to the compound b is 1:(1-1.5).

[0139] In one embodiment, the molar ratio of the compound 2-2 to the compound b is 1:(1-1.5).

[0140] In one embodiment, the molar ratio of the compound 3-1 to the compound c is (1-3):1.

[0141] In one embodiment, the molar ratio of the compound 3-2 to the compound c is (1-3):1.

[0142] In one embodiment, the molar ratio of the compound 4 to the compound d-1 is 1:(1-1.5).

[0143] In one embodiment, a method for preparing a square acid compound as described in formula (I) above comprises the following steps:

[0144] Compound 1 (5.91 mmol) was dissolved in DMF. Sodium hydride (7.09 mmol) was added portionwise in an ice bath at 0°C, and the mixture was stirred for 10 minutes. Methyl iodide (0.56 mL, 7.09 mmol) was then added in an ice bath. The mixture was then allowed to react overnight at room temperature under nitrogen. After the reaction, the mixture was extracted with saturated sodium chloride solution and ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was then purified with silica gel and column chromatography to yield compound 2-1.

[0145] Take compound 2-1 (5.07mmol) in a two-necked flask, dissolve it in anhydrous tetrahydrofuran, and replace nitrogen. Add methylmagnesium chloride (2.03mL, 6.08mmol) under heating conditions at 60°C, and heat under reflux to react overnight. After the reaction is completed, pour the reaction solution into a 2M hydrochloric acid solution under an ice bath to quench the reaction. Then add a saturated aqueous solution of sodium fluoroborate and stir for 30 minutes. Solids will appear in the solution. The green solid is centrifuged, washed with a small amount of water, and dried to obtain compound 3-1;

[0146] Squaric acid (0.87 mmol) and compound 3-1 (1.75 mmol) were added to a flask. 10 mL of n-butanol and 10 mL of toluene were added as reaction solvents. Toluene was added to the water separator until the outlet was reached. The atmosphere was purged with nitrogen and the reaction solution was stirred at 130°C for 3-4 hours. After the reaction, the toluene was removed by rotary evaporation under reduced pressure. The resulting solution was filtered, and the solid was washed with anhydrous ether until the ether was colorless. Compound 4-1 was obtained after the solid was passed through a silica gel column.

[0147] Compound 4-1 (0.3 mmol) was added to a two-necked flask, the atmosphere was purged with nitrogen, and anhydrous dichloromethane was added to dissolve the mixture. The atmosphere was purged with nitrogen again, and methyl trifluoromethanesulfonate (0.36 mmol) was added via syringe. The mixture was allowed to react overnight at room temperature. After completion of the reaction, the mixture was quenched with a 5 wt% aqueous sodium bicarbonate solution, extracted with water and dichloromethane, and the organic phase was dried over anhydrous sodium sulfate. The filtrate was then purified by silica gel column chromatography to yield Compound I.

[0148] (2) A method for preparing a square acid compound as described in formula (II) above, comprising the following steps:

[0149] Compound 5 undergoes a condensation reaction with compound c to obtain compound 6 (i.e., compound V), wherein compound c is a squaric acid;

[0150] Compound 6 reacts with compound d-2 to obtain the compound shown in II, wherein compound d-2 contains R2;

[0151] wherein R1, R2, R3 and R4 are as defined above.

[0152] In one embodiment, the molar ratio of the compound 5 to the compound c is (2-3):1.

[0153] In one embodiment, the molar ratio of the compound 6 to the compound d-2 is 1:(1-1.5).

[0154] In one embodiment, a method for preparing a square acid compound as described in formula (II) above comprises the following steps:

[0155] Squaric acid (0.87 mmol) and compound 5-1 (1.75 mmol) were added to a flask. 10 mL of n-butanol and 10 mL of toluene were added as reaction solvents. Toluene was added to the water separator until the outlet was reached. The nitrogen atmosphere was replaced and the reaction solution was stirred at 130°C for 3-4 hours. After the reaction was completed, the toluene was removed by rotary evaporation under reduced pressure. The resulting solution was filtered and the solid was washed with anhydrous ether until the ether was colorless. Compound 6-1 was obtained by column chromatography on silica gel.

[0156] Compound 6-1 (0.3 mmol) was added to a two-necked flask, the atmosphere was purged with nitrogen, and anhydrous dichloromethane was added to dissolve the mixture. The atmosphere was purged with nitrogen again, and methyl trifluoromethanesulfonate (0.36 mmol) was added via syringe. The mixture was allowed to react overnight at room temperature. After completion of the reaction, the mixture was quenched with a 5 wt% aqueous sodium bicarbonate solution, extracted with water and dichloromethane, and the organic phase was dried over anhydrous sodium sulfate. The filtrate was then sampled with silica gel and passed through a column to yield Compound II.

[0157] (3) A method for preparing a square acid compound as described in formula (III) above, comprising the following steps:

[0158] Compound 3-1 is substituted with compound e to generate compound 7, wherein compound e is diethyl squarate;

[0159] Compound 7 undergoes a hydrolysis reaction with compound f to generate compound 8, wherein compound f is a base;

[0160] Compound 8 and compound 5 undergo condensation reaction to generate compound 9 (i.e., compound VI);

[0161] Compound 9 reacts with compound d-3 to obtain the compound shown in III, wherein compound d-3 contains R2;

[0162] wherein R1, R2, R3 and R4 are as defined above.

[0163] In one embodiment, the molar ratio of the compound 3-1 to the compound e is (1-1.5):1;

[0164] In one embodiment, the molar ratio of the compound 8 to the compound 5 is 1:(1-1.5);

[0165] In one embodiment, the molar ratio of the compound 9 to the compound d-3 is 1:(1-1.5).

[0166] In one embodiment, a method for preparing a square acid compound as described in formula (III) above comprises the following steps:

[0167] Compound 3-1 (3.98 mmol), diethyl squarate (3.32 mmol) and triethylamine (8.96 mmol) were added to the bottle, and ethanol was added. The atmosphere was replaced with nitrogen and the mixture was heated under reflux at 90°C overnight. The reaction solution was cooled to room temperature, the solvent was removed, the sample was stirred, and the mixture was passed through a column to obtain compound 7.

[0168] Compound 7-1 (1.20 mmol) was added to a bottle and dissolved in ethanol. A 40% aqueous sodium hydroxide solution was added under reflux and reacted for 2-3 hours. After the reaction was completed, the mixture was cooled to room temperature and 2M HCl solution was added to adjust the pH to neutral. The reaction solution was concentrated and 5 mL of ice ethanol solution was added to produce a solid, which was filtered to obtain compound 8-1.

[0169] Compound 8-1 (1.2 mmol) and compound 5 (1.75 mmol) were added to a flask, along with 10 mL of n-butanol and 10 mL of toluene as the reaction solvents. Toluene was added to the water separator until the outlet was reached, and nitrogen was purged. The reaction solution was stirred at 130°C for 3-4 hours. After the reaction, the toluene was removed by rotary evaporation under reduced pressure, the resulting solution was filtered, and the solid was washed with anhydrous ether until the ether was colorless. Compound 9-1 was obtained after the solid was passed through a silica gel column.

[0170] Compound 9-1 (0.3 mmol) was added to a two-necked flask, the atmosphere purged with nitrogen, and anhydrous dichloromethane was added to dissolve the mixture. The atmosphere was purged with nitrogen again, and methyl trifluoromethanesulfonate (0.36 mmol) was added via syringe. The mixture was allowed to react overnight at room temperature. After completion of the reaction, the mixture was quenched with 5% aqueous sodium bicarbonate solution, extracted with water and dichloromethane, and the organic phase was dried over anhydrous sodium sulfate. The filtrate was then purified by silica gel column chromatography to yield Compound III.

[0171] The present application also provides the application of the above-mentioned square acid compound, and the technical solution is as follows:

[0172] A fluorescent probe comprises the above-mentioned squaraine compound or a pharmaceutically acceptable salt or deuterated compound thereof.

[0173] A near-infrared zone II contrast agent comprises the above-mentioned squaraine compound or a pharmaceutically acceptable salt or deuterated compound thereof.

[0174] A method for monitoring early-stage liver injury comprises imaging a subject in need thereof using the aforementioned squaraine compound, or a pharmaceutically acceptable salt or deuterated compound thereof, the aforementioned fluorescent probe, or the aforementioned near-infrared zone II contrast agent. Also provided is the use of the aforementioned squaraine compound, or a pharmaceutically acceptable salt or deuterated compound thereof, the aforementioned fluorescent probe, or the aforementioned near-infrared zone II contrast agent for monitoring early-stage liver injury in a subject in need thereof.

[0175] Specific embodiments are listed below to illustrate the present application.

[0176] Table 1 Structures of compounds synthesized in Example

[0177] Example 1: Synthesis of Compound Ia

[0178] Compound 1a (3 g, 17.73 mmol) and potassium tert-butoxide (1.99 g, 17.73 mmol) were weighed into a three-necked flask and dissolved in ultra-dry THF. The reaction mixture was refluxed and stirred at 70°C for 10 minutes. 1b (2.41 g, 17.73 mmol, 1.81 mL) was then added via syringe and allowed to react for 2 hours. A white precipitate was produced during the reaction. The reaction progress was monitored by high-performance liquid chromatography (HPLC). Once reactant 1a had reacted completely, no further treatment was required and the reaction could proceed directly to the next step.

[0179] After compound 1a reacts completely to form 1c, heating is stopped, 18-crown ether-6 (4.69 g, 17.73 mmol) is added, and the mixture is stirred for 10 minutes. After nitrogen protection is replaced, methylmagnesium chloride (4.64 g, 62.1 mmol, 20.70 mL) is slowly added dropwise using a syringe. The reaction is continued for 2-3 hours, and the reaction progress is monitored by HPLC. After the reaction is complete, the reaction solution is cooled to room temperature. In an ice bath, the reaction solution is poured into 3M HCl (35.5 mL) and stirred for 20 minutes. 30 mL of ethanol is added and stirring is continued for 10 minutes. The resulting suspension is centrifuged, and the solid is washed with acetonitrile, filtered, and dried to obtain a green solid 1d (4.1 g, yield 75.9%).

[0180] 1H NMR(400MHz,D2O)δ8.48(d,J=7.3Hz,1H),8.39(d,J=8.1Hz,1H),8.07(dd,J=9.6,7.9Hz,2H),7.84(t,J=7.7Hz,1H) ,7.73(t,J=7.8Hz,1H),4.52(t,J=7.6Hz,2H),2.89(t,J=7.5Hz,2H),2.06(p,J=7.7Hz,2H),1.83(p,J=7.6Hz,2H).

[0181] 13 C NMR (126MHz, D2O) δ171.2,138.4,137.8,134.1,130.6,130.1,128.9,128.4,127.9,121.8,120.1,49.6,45.9,27.5,21.1.

[0182] Compound 1e (0.2 g, 1.75 mmol) and compound 1d (1.12 g, 3.51 mmol) were added to a flask, along with 10 mL each of n-butanol and toluene as solvents. Toluene was added to the trap, and the atmosphere was purged with nitrogen. The reaction mixture was stirred at 130°C for 3-4 hours. After the reaction, the toluene was removed by rotary evaporation under reduced pressure. The resulting solution was filtered, and the solid was washed with anhydrous ether until the ether was colorless. The resulting solid was dissolved in water and passed through a reverse-phase silica gel column to obtain compound Ia as a green solid.

[0183] 1 H NMR (400MHz, DMSO-d6) δ9.00(d,J=7.4Hz,2H),8.11(d,J=8.0Hz,2H),7.88(t,J=7.7Hz,2H),7.68(d,J=8.2Hz,2H),7.60(t,J=7.7Hz,2 H),7.49(d,J=7.3Hz,2H),6.27(s,2H),4.32(t,J=7.3Hz,4H),2.57(t,J=7.5Hz,5H),1.89(p,J=7.4Hz,5H),1.75(h,J=7.2,6.2Hz,5H).

[0184] 13 C NMR (126MHz, DMSO) δ181.6,175.8,149.5,141.1,130.7,129.8,129.6,129.4,129.2,129.1,124.4,121.6,108.7,91.8,50.9,43.2,27.6,22.5.

[0185] ESI-LR:

[0186] Ⅰa:expected MW:about685.Found:about 685

[0187] Example 2: Synthesis of Compound Ib

[0188] Sodium hydride (0.28 g) was added portionwise to a DMF solution of 1a (1 g) at 0°C. The mixture was stirred for 15 min, and iodoethane (567 μL) was added. The reaction was allowed to react at room temperature for 12 h. Completion of the reaction was monitored by TLC. The reaction solution was poured into saturated brine and extracted three times with ethyl acetate. The organic layers were combined, dried, and filtered through a silica gel column to afford compound 2b (0.8 g, 68% yield).

[0189] 1 H NMR(500MHz,Chloroform-d)δ7.93–7.87(m,1H),7.81(dd,J=7.5,1.6Hz,1H),7.67(t,J=7.5Hz ,1H),7.64–7.56(m,2H),6.99(dd,J=6.5,2.5Hz,1H),4.11(q,J=8.0Hz,2H),1.31–1.24(m,3H).

[0190] To a solution of 2b (0.5 g) in acetic acid was added nitric acid at 0°C, and the reaction mixture was incubated at 50°C for 12 h. Completion of the reaction was monitored by TLC. The reaction mixture was extracted with water and ethyl acetate, and the organic layer was dried and passed through a silica gel column to obtain compound 2c (0.46 g, 75% yield).

[0191] 1 H NMR(500MHz,Chloroform-d)δ9.01(dd,J=7.6,1.5Hz,1H),8.21(d,J=7.5Hz,1H),7.86(dd,J=7.5,1. 5Hz,1H),7.75(t,J=7.4Hz,1H),7.25(d,J=7.5Hz,1H),4.12(q,J=8.0Hz,2H),1.28(t,J=8.0Hz,3H).

[0192] Compound 2c (0.5 g) was dissolved in THF, and the atmosphere was purged with nitrogen. Methylmagnesium chloride (2.5 mL) was then added, and the reaction was continued at 70°C for 12 h. Completion of the reaction was monitored by HPLC. The mixture was cooled to room temperature, and the reaction solution was poured into ice-water containing HCl (2.5 mL). KI (1 g) was then added, and the mixture was filtered to obtain compound 2d (0.4 g, 76% yield).

[0193] 1H NMR(500MHz,Chloroform-d)δ9.02(dd,J=7.6,1.6Hz,1H),8.37(d,J=7.5Hz,1H),7.83(t,J=7.5Hz,1H),7. 67(dd,J=7.5,1.6Hz,1H),7.53(d,J=7.5Hz,1H),4.80(q,J=8.0Hz,2H),2.88(s,2H),1.53(t,J=8.0Hz,3H).

[0194] Compound 1e (100 mg) and compound 2d (710 mg) were weighed, 10 mL each of n-butanol and toluene were added, nitrogen was replaced, and the reaction was carried out at 130°C for 12 h. The completion of the reaction was monitored by TLC. Compound 2e (0.2 g, 40% yield) was obtained by column chromatography.

[0195] 1 H NMR(500MHz,Chloroform-d)δ9.01(dd,J=7.6,1.6Hz,1H),8.43(d,J=7.5Hz,1H),8.26–8.18(m,3H),7.75–7.66(m,3H),7.65–7.58(m,2H) ,7.54(t,J=7.5Hz,1H),7.33(d,J=7.5Hz,1H),4.83(q,J=8.0Hz,2H),4.09(q,J=8.0Hz,2H),1.50(t,J=8.0Hz,3H),1.35(t,J=8.0Hz,3H).

[0196] Compound 2e (10 mg) was dissolved in THF, and methyl trifluoromethanesulfonate (100 μL) was added. The mixture was stirred at room temperature for 5 h. The reaction progress was monitored by HPLC. After completion, the solvent was removed by rotary evaporation. The dried compound was dissolved in ultra-dry DMSO and NH2-PEG5K (3 mg) was added. The reaction was allowed to react at room temperature for 12 h. After completion of the reaction, the product was dialyzed using a dialysis bag and purified on a C18 reverse-phase column. The final product was purified by MALDI-TOF-MS.

[0197] MALDI-TOF-MS:

[0198] Ⅰb:expected MW:about5558.Found:about 5550

[0199] Example 3: Synthesis of Compound Ic

[0200] Compound Ia (10 mg) was dissolved in THF, and methyl trifluoromethanesulfonate (100 μL) was added. The reaction was stirred at room temperature for 5 h. The reaction progress was monitored by HPLC. After completion, the solvent was removed by rotary evaporation. The dried compound was dissolved in ultra-dry DMSO and 3-amino-N-tert-butyloxycarbonylalanine (3 mg) was added. The reaction was allowed to proceed to the next step. After completion of the reaction, the product was dialyzed using a dialysis bag and purified on a C18 reverse-phase column. The final product was verified by MALDI-TOF-MS.

[0201] 1 H NMR(500MHz,Chloroform-d)δ8.49(s,1H),8.45–8.37(m,2H),8.24(s,1H),8.05–7.97(m,3H),7.80(dtd,J=7.5,4.6,4. 1,2.3Hz,3H),7.71(dt,J=7.5,1.5Hz,1H),7.69–7.62(m,3H),7.55(dt,J=7.5,1.8Hz,2H),7.50(td,J=7.4,5.0Hz,2H),7 .38(t,J=7.5Hz,1H),7.15(dd,J=7.5,1.7Hz,1H),4.32–4.23(m,1H),4.22–4.04(m,3H),3.96–3.83(m,2H),3.82–3.72(m ,1H),3.03–2.91(m,2H),2.94–2.81(m,2H),2.09–2.02(m,1H),2.05–1.98(m,2H),2.02–1.93(m,1H),1.90–1.73(m,4H).

[0202] MALDI-TOF-MS:

[0203] Ⅰc:expected MW:about771.Found:about 771

[0204] Example 4: Synthesis of Compound Id

[0205] Compound 1e (0.1 g, 1.75 mmol) and compound 3a (0.675 g, 3.51 mmol) were added to a flask, along with 10 mL each of n-butanol and toluene as solvents. Toluene was added to the separatory opening, and the atmosphere was purged with nitrogen. The reaction mixture was stirred at 130°C for 3-4 hours. After the reaction, the toluene was removed by rotary evaporation under reduced pressure. The resulting solution was filtered, and the solid was washed with anhydrous ether until colorless. The resulting solid was dissolved in water and passed through a silica gel column to afford compound Id (0.2 g, 38.75% yield).

[0206] 1 H NMR(500MHz,Chloroform-d)δ7.80(dd,J=7.5,2.3Hz,1H),7.60(dt,J=2.2,0.9Hz,1H),7.41(d,J=7 .5Hz,1H),7.26(dd,J=7.6,1.6Hz,1H),6.92–6.88(m,1H),6.71(dd,J=11.0,1.4Hz,2H),6.51(dd,J= 7.5,1.5Hz,1H),3.49(dq,J=10.1,8.0Hz,8H),3.17(t,J=7.1Hz,2H),2.91–2.83(m,2H),2.87–2.76 (m,2H),2.75(t,J=7.1Hz,2H),2.01(p,J=7.1Hz,2H),1.64(p,J=7.1Hz,2H),1.17(t,J=8.0Hz,12H).

[0207] Example 5: Synthesis of Compound Ie

[0208] Compound 4b (0.5 g) was dissolved in ethanol (3 mL) and heated to reflux. Compound 4a (0.3 mL) and triethylamine (0.6 mL) were dissolved in ethanol (0.3 mL) and added to the reaction solution. The reaction was allowed to react for 12 h. After the reaction, the reaction solution was cooled to room temperature, the solvent was removed under reduced pressure, and the solid was passed through a silica gel column to obtain compound 4c (0.35 g, 52% yield).

[0209] 1 H NMR(500MHz,Chloroform-d)δ7.84–7.78(m,1H),7.81(s,3H),7.67(dt,J=7.9,1.7Hz,2H),7.51–7.41(m,6H),6.94 (dd,J=7.5,1.5Hz,2H),4.19(q,J=8.1Hz,4H),4.09(q,J=8.0Hz,4H),1.47(t,J=8.0Hz,6H),1.32(t,J=8.0Hz,6H).

[0210] Compound 4c (0.3 g) was dissolved in ethanol, heated to reflux, and 40% aqueous sodium hydroxide solution (0.2 mL) was added. The reaction was continued for 2 h. The solvent was removed and the product was purified by column chromatography to obtain compound 4d (0.21 g, 49% yield).

[0211] 1H NMR(500MHz,Chloroform-d)δ7.84–7.75(m,2H),7.65–7.60(m,2H),7.51–7.41( m,3H),6.90(dd,J=7.5,1.6Hz,1H),4.10(q,J=8.0Hz,2H),1.32(t,J=8.0Hz,3H).

[0212] Compound 4d (0.1 g) and compound 3a (0.15 g) were added to a flask, and 10 mL each of n-butanol and toluene were added. The atmosphere was replaced with nitrogen, and the mixture was reacted at 130°C for 12 h. The solvent was removed and the mixture was purified by column chromatography to obtain compound Ie (90 mg, 49% yield).

[0213] 1 H NMR(500MHz,Chloroform-d)δ7.79(ddd,J=10.6,7.5,2.0Hz,2H),7.68–7.62(m,3H),7.60(d t,J=2.4,0.9Hz,1H),7.49(q,J=7.5Hz,2H),6.97(dd,J=7.5,1.5Hz,1H),6.80(dd,J=7.6,1. 6Hz,1H),6.65–6.61(m,1H),4.09(q,J=8.0Hz,2H),3.48(q,J=8.0Hz,4H),2.91–2.76(m,2H) ,2.72(t,J=7.1Hz,2H),2.01(p,J=7.1Hz,2H),1.32(t,J=8.0Hz,3H),1.17(t,J=8.0Hz,6H).

[0214] Example 6 Synthesis of Compound IF

[0215] Compound Ia (10 mg) was dissolved in THF, methyl trifluoromethanesulfonate (100 μL) was added, and the reaction was stirred at room temperature for 5 h. The reaction progress was monitored by HPLC. After the reaction was completed, the solvent was removed by rotary evaporation. The next step was to dissolve the spin-dried compound in ultra-dry DMSO. The peptide cRGDyk (15 mg) was added and reacted at room temperature for 12 h. After the reaction was completed, the product was dialyzed using a dialysis bag and purified by a C18 reverse phase column. The final product was verified by MALDI-TOF-MS. MALDI-TOF-MS. If: expected MW: about 1286.46. Found: about 1286.5

[0216] Example 7: Synthesis of Compound Ig

[0217] Compound 4a (100 mg) was dissolved in ethanol, and then 5b (77 mg) and triethylamine (76 mg) were added thereto. The mixture was stirred at room temperature for 12 h. The solvent was removed by rotary evaporation, and the sample was passed through a column to obtain compound 5c.

[0218] 1 H NMR (600MHz, Chloroform-d) δ4.74 (q, J = 7.1Hz, 2H), 1.48 (s, 9H), 1.44 (t, J = 7.1Hz, 3H).

[0219] Compounds 5c and 1d were added to a vial, followed by toluene and n-butanol (10 ml each). The atmosphere was purged with nitrogen and the reaction was continued at 130°C for 7-8 hours. The reaction was stopped, cooled to room temperature, and filtered to obtain compound 5d. Compound 5d was then placed in a vial and deprotected with TFA. The reaction was continued for 5-6 hours, and the solvent was removed and passed through a reverse-phase column to obtain compound Ig.

[0220] 1 H NMR (400MHz, DMSO-d6) δ10.00(t,J=6.2Hz,1H),8.83(d,J=7.5Hz,1H),8.32(d,J=8.1Hz,1H) ,8.25(d,J=8.1Hz,1H),8.18(dd,J=7.6,4.6Hz,1H),8.00(q,J=7.8Hz,2H),7.89(d,J=8.0Hz ,1H),7.84–7.61(m,5H),6.79(s,1H),6.43(s,1H),5.75(s,1H),4.71(d,J=6.1Hz,2H),4.39 (dt,J=26.7,7.7Hz,4H),2.62(dt,J=19.7,7.2Hz,4H),2.07–1.97(m,2H),1.96–1.71(m,6H).

[0221] 13 C NMR (126MHz, DMSO) δ174.3,171.1,169.7,158.7,157.6,152.9,151.5,141.1,140.8,132.6,132.3,131.3,130.2,129.8,129.6,129.4,129.3, 124.6,124.4,124.2,122.9,110.1,93.8,93.1,51.1,50.6,46.0,44.2, 44.0,40.5,40.3,40.2,40.0,39.8,39.7,39.5,27.9,27.6,22.9,22.9.

[0222] Example 8: Synthesis of Compound Ih

[0223] Compound 4a (100 mg) was dissolved in ethanol, and then 6b (60 mg) and triethylamine (76 mg) were added thereto. The mixture was stirred at room temperature for 12 h. The solvent was removed by rotary evaporation, and the sample was mixed and passed through a column to obtain compound 6c.

[0224] 1 H NMR (600MHz, Chloroform-d) δ4.75(q,J=7.1Hz,2H),4.68(s,1H),3.53(q,J=6.6Hz,2H),3.39(t,J=6.4Hz,2H),1.89(q,J=6.6Hz,2H),1.44(t,J=7.1Hz,3H).

[0225] Compounds 6c and 1d were added to the bottle, followed by toluene and n-butanol (10 ml each), and nitrogen was replaced. The reaction was carried out at 130°C for 7-8 hours, and the reaction was stopped. The mixture was cooled to room temperature, filtered, and filtered to obtain compound Ih.

[0226] 1 H NMR(500MHz,Chloroform-d)δ7.82(t,J=7.6Hz,1H),7.74–7.65(m,2H),7.60(dt,J=7.5,1.6Hz,1H),7.58–7.53(m,3H), 7.50(dd,J=7.5,1.5Hz,1H),7.50–7.45(m,2H),7.47–7.37(m,1H),7.16(dd,J=7.3,1.6Hz,1H),6.74(s,1H),6.62(s,1H ),6.44(s,2H),6.10(t,J=4.3Hz,1H),4.63(t,J=7.0Hz,2H),4.16–4.08(m,2H),3.24(td,J=7.1,4.3Hz,2H),3.15(t,J= 7.1Hz,2H),2.84(t,J=7.1Hz,2H),2.81–2.75(m,2H),2.09(pd,J=6.9,0.7Hz,2H),2.03–1.86(m,4H),1.89–1.77(m,4H).

[0227] Example 9: UV absorption spectrum of compound Ia

[0228] Compound Ia was dissolved in deionized water and methanol, and 1.5 mL of each solution was added to a 1 cm thick quartz cuvette. The absorption spectra of the different probes were measured on a UV2600 UV-visible spectrophotometer, with the background subtracted and zeroed using the solvent used to prepare the solution. The wavelength range was 300-1400 nm. The absorption peaks of compound Ia in water and methanol were 738 nm and 858 nm, respectively. These results indicate that compound Ia exhibits strong near-infrared absorption. Furthermore, the absorption spectra of compound Ia in different solvents differ, possibly due to its aggregation state in the solvents.

[0229] Example 10: Fluorescence emission spectrum of compound Ia

[0230] Compound Ia was dissolved in deionized water and methanol, and 200 μL of each aliquot was pipetted into a 2-cm-thick quartz cuvette. Excitation was performed using an 808-nm laser, and the emission wavelength was recorded using an IHR320 fluorescence spectrometer over a wavelength range of 825-1400 nm. Compound Ia exhibited an emission peak at 925 nm, falling within the near-infrared region II. These results demonstrate that compound Ia emits near-infrared region II light, making it suitable for use as a near-infrared region fluorescent contrast agent.

[0231] Example 11: Biodistribution of Compound Ia in Normal Mice

[0232] The mice used in the experiment were purchased with the approval of the Shanghai Laboratory Animal Center, and the animal experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences. The experiments used normal 6-week-old Balb / c female mice. Compound Ia was dissolved in PBS and injected into the mice through the tail vein. In vivo imaging was studied using a near-infrared second-zone camera, excited by an 808nm laser, and a 1000nm long-pass filter. The results are shown in Figure 3. Figure 3 shows the biodistribution of compound Ia in mice. The fluorescence intensity is concentrated in the liver and intestine, indicating that the probe is metabolized by the liver and intestine. Compound Ia is rapidly metabolized in the body, has no obvious toxicity, and has good biocompatibility.

[0233] Example 12: Experimental study of compound Ia in carbon tetrachloride liver injury model mice

[0234] C57BL / 6J male mice, 6-8 weeks old and weighing 18-22 grams, were selected from the same batch. Mice in the liver injury group were intraperitoneally injected with 2 mg / kg of 10% carbon tetrachloride (CCl4) in olive oil. Mice in the normal control group were intraperitoneally injected with the same dose of 0.9% saline. Twenty-four hours after CCl4 treatment, blood samples were collected retroorbitally. Serum alanine aminotransferase (ALT) activity, a common marker of liver function, was measured. ALT concentrations increased dramatically in the liver injury group, indicating successful establishment of the liver injury model. Eighteen hours after CCl4 injection, compound Ia dissolved in phosphate-buffered saline (PBS) was injected via the tail vein into mice in the liver injury and control groups. In vivo imaging was performed using a near-infrared zone II camera, excited by an 808 nm laser and using an 1100 nm long-pass filter. The results are shown in Figures 4 and 5. Compared with normal mice, the metabolism of compound Ia in the carbon tetrachloride liver injury model group mice was significantly slower, indicating that compound Ia can be used for faster monitoring of early liver damage.

[0235] Example 13: Mechanism Experiment of Carbon Tetrachloride Liver Injury Model Mouse

[0236] According to the steps in Example 12, carbon tetrachloride liver injury mouse model was performed, and the mice were then anesthetized. Mouse primary liver cells were extracted, and the genes of related efflux transporters in the liver tissue were subsequently tested by qPCR experiments. The results are shown in Figure 6. It can be seen that the expression levels of slc47a1, slc47a2, abcc3, abcc6, and abcg2 genes were all reduced, and the expression level of slc47a1 gene decreased the most significantly. The experimental results show that when the liver is damaged, the efflux transporter function of the liver is affected, which may lead to a decrease in the clearance ability of metabolites and drugs, thereby slowing down the metabolic process of compound Ia in the liver. In addition, by extracting primary mouse hepatocytes and constructing a damaged cell model at the cellular level, qPCR confirmed that slc47a1 may be a potential target of compound Ia.

[0237] Example 14: Compound Ia in a mouse model of the slc47a1-related Mate 1 transporter inhibitor. Six- to eight-week-old C57BL / 6j male mice weighing 18-22 g were selected from the same batch. For the efflux Mate 1 transporter inhibitor imatinib group, imatinib was weighed on an electronic balance at a dose of 25 mg / kg or 50 mg / kg. Imatinib was then solubilized with a small amount of DMSO and then added to a 1:1 volume ratio of polyethylene glycol 300 and 1x PBS to a final volume of 200 μL. After a 30-minute wait, 200 μL of the pre-prepared Compound Ia solution was injected. Fluorescence images of the abdomen of each mouse were collected at 1, 3, 5, 8, 10, 15, 25, 30, 45, 60, 90, and 120 minutes after Compound Ia injection. Acquisition conditions were: 808 nm wavelength, 1100 nm long-pass filter for receiving and transmitting wavelengths, and 200 ms exposure time. The results are shown in Figure 7. Compared to the normal control group, the metabolism of compound Ia in the liver of mice treated with imatinib was significantly inhibited and slowed at 10, 15, 30, and 45 minutes. These results suggest that imatinib affects the metabolism of compound Ia by inhibiting the Mate1 transporter associated with slc47a1.

[0238] Example 15: Photostability test of compound Ia

[0239] The photostability of compound Ia SQ 905 was determined using indocyanine green (ICG), a near-infrared dye approved by the FDA and widely used in clinical practice, as a control. ICG was dissolved in water and compound Ia SQ 905 was dissolved in methanol. Over a period of 25 minutes, a 108 mW / cm 2 An 808 nm laser was continuously irradiated to a methanol solution of compound Ia and an aqueous solution of ICG, during which the fluorescence intensity was recorded using a near-infrared zone II camera (MARS, Artemis Intelligent Imaging, Shanghai, China). Fluorescence intensity was recorded every minute for the first 15 minutes, and then every 5 minutes thereafter. The fluorescence intensity of the region of interest (ROI) in the captured image was calculated using ImageJ software and compared with the initial fluorescence intensity to obtain the ratio of the fluorescence intensity at each time point to the initial fluorescence intensity. A curve was drawn using GraphPad Prism 8.0.2 software, which reflects the photostability of compound Ia.

[0240] Photostability is an important indicator for evaluating probes. If the probe has poor photostability, photobleaching and quenching will occur during the experiment, which will affect the fluorescence quantitative data during the experiment and make the experimental results unreliable. Photostability greatly affects the clinical transformation potential and application scenarios of subsequent probes. Therefore, the photostability of compound Ia was evaluated in this example. A methanol solution of compound Ia and an aqueous solution of ICG were taken for photostability investigation. As shown in Figure 8, the fluorescence intensity of the methanol solution of compound Ia did not show significant attenuation after continuous irradiation with an 808nm laser for 25 minutes; however, the fluorescence intensity of the aqueous solution of ICG as a control was attenuated by 83% after continuous irradiation with an 808nm laser for 20 minutes. The data show that compound Ia has better photostability than ICG, and it can be used as a near-infrared zone II probe for subsequent biological applications.

[0241] Compounds Ib, Ic, Id, Ie, If, Ig, and Ih were tested using the methods described in Examples 9 through 15. The results demonstrated that these compounds exhibit strong near-infrared light absorption and emit light in the near-infrared region II, making them suitable for use as fluorescent contrast agents in the near-infrared region II. These compounds also exhibit rapid in vivo metabolism, lack significant toxicity, exhibit good biocompatibility, and possess excellent photostability, making them suitable for rapid monitoring of early-stage liver damage.

[0242] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0243] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that a person of ordinary skill in the art may make various modifications and improvements without departing from the spirit of the present application, and these modifications and improvements fall within the scope of protection of the present application. Therefore, the scope of protection of the present patent application shall be determined by the appended claims.

Claims

1. A squaric acid compound or a pharmaceutically acceptable salt or deuterated compound thereof, characterized in that: The general structural formula of the square acid compound is shown in Formula I, II, III, IV, V or VI: Among them, X m- , X n- and X p- are independently anions; m, n and p are independently selected from any integer from 1 to 10; Each R1 is independently selected from H, C1-C 12 Alkyl, C1-C 12 Alkoxy, C1-C8 alkylsilyl, hydroxy C1-C8 alkyl, amino C1-C8 alkyl, aldehyde C1-C8 alkyl, mercapto C1-C8 alkyl, halogenated C1-C8 alkyl, acyloxy C1-C8 alkyl, amino, halogen, nitro, carboxyl C1-C6 alkyl, substituted or unsubstituted C6-C 10 At least one of aryl or 5-10 membered heteroaryl, -(CH2)n1-COOCH2CH2Si(CH3)3, -(CH2)n2-(CH2CH2O)n3-R and -(CH2)n2-(OCH2CH2)n3-R; R2 is selected from hydroxyl, O - 、C1-C 12 At least one of an alkoxy group, a C1-C8 alkylsilyl group, a hydroxy C1-C8 alkyl group, a carboxyl group, an amino group, -NR5R6, a mercapto group, -SR7, and a malononitrile group; R3, R4, R5, R6 and R7 are independently selected from H, C1-C8 alkyl, hydroxyl, amino, carboxyl, C1-C 12 Alkyl, C1-C 12 Alkoxy, C1-C8 alkylsilyl, hydroxy C1-C8 alkyl, amino C1-C8 alkyl, aldehyde C1-C8 alkyl, mercapto C1-C8 alkyl, halogenated C1-C8 alkyl, acyloxy C1-C8 alkyl, amino, halogen, sulfonic acid, carboxyl C1-C6 alkyl, substituted or unsubstituted C6-C 10 At least one of aryl or 5-10 membered heteroaryl, -(CH2)n1-COOCH2CH2Si(CH3)3, -(CH2)n2-(CH2CH2O)n3-R and -(CH2)n2-(OCH2CH2)n3-R; n1 is any integer from 0 to 10, n2 is any integer from 0 to 10, and n3 is any integer from 1 to 500; R is selected from H, C1-C8 alkyl, hydroxyl, amino, carboxyl, sulfonic acid, halogen, mercapto, and At least one of; Indicates the connection site.

2. The squaric acid compound or the pharmaceutically acceptable salt or deuterated compound thereof according to claim 1, characterized in that: The general structural formula of the square acid compound is shown in Formula I-1, II-1, III-1, IV-1, V-1 or VI-1:

3. The squaric acid compound or the pharmaceutically acceptable salt or deuterated compound thereof according to claim 1 or 2, characterized in that: Replace the C6-C 10 The substituents in the aryl or 5-10 membered heteroaryl are selected from at least one of C1-C8 alkyl, C1-C8 alkoxy, C1-C8 alkylsilyl, hydroxy C1-C8 alkyl, amino C1-C8 alkyl, mercapto C1-C8 alkyl, halogenated C1-C8 alkyl and carboxyl C1-C6 alkyl.

4. The squaric acid compound or a pharmaceutically acceptable salt or deuterated compound thereof according to any one of claims 1 to 3, characterized in that: m, n and p are independently selected from any integer from 1 to 4.

5. The squaric acid compound or a pharmaceutically acceptable salt or deuterated compound thereof according to any one of claims 1 to 4, characterized in that: m, n and p are all 1, X - Each independently selected from I - Br - 、BF4 - or ClO4 - .

6. The squaric acid compound or a pharmaceutically acceptable salt or deuterated compound thereof according to any one of claims 1 to 5, characterized in that: The square acid compound has any of the following structures:

7. A method for preparing the squaric acid compound or the pharmaceutically acceptable salt or deuterated compound thereof according to any one of claims 1 to 6, characterized in that: The steps include: (1) Compound 1 undergoes a nucleophilic substitution reaction with compound a-1 to obtain compound 2-1, wherein compound a-1 is halogenated R3; compound 1 undergoes a nucleophilic substitution reaction with compound a-2 to obtain compound 2-2, wherein compound a-2 is halogenated R4; Compound 2-1 undergoes a Grignard reaction with compound b to obtain compound 3-1, and compound 2-2 undergoes a Grignard reaction with compound b to obtain compound 3-2, wherein compound b is a Grignard reagent; The compound 3-1, the compound 3-2 and the compound c undergo a condensation reaction to obtain the compound 4, wherein the compound c is a squaric acid; Compound 4 reacts with compound d-1 to obtain the compound shown in I, wherein compound d-1 contains R2; (2) Compound 5 undergoes a condensation reaction with compound c to obtain compound 6, wherein compound c is a squaric acid; Compound 6 reacts with compound d-2 to obtain the compound shown in II, wherein compound d-2 contains R2; (3) Compound 3-1 is substituted with compound e to generate compound 7, wherein compound e is diethyl squarate; Compound 7 undergoes a hydrolysis reaction with compound f to generate compound 8, wherein compound f is a base; Compound 8 and compound 5 undergo condensation reaction to generate compound 9; Compound 9 reacts with compound d-3 to obtain the compound shown in III, wherein the compound d-3 contains R2; Wherein, R1, R2, R3 and R4 are defined as in any one of claims 1 to 6.

8. The method for preparing a squaric acid compound or a pharmaceutically acceptable salt or deuterated compound thereof according to claim 7, characterized in that: Satisfy at least one of the following (1) to (9): (1) The molar ratio of the compound 1 to the compound a-1 is 1:(1-1.5), and the molar ratio of the compound 1 to the compound a-2 is 1:(1-1.5); (2) The molar ratio of the compound 2-1 to the compound b is 1:(1-1.5), and the molar ratio of the compound 2-2 to the compound b is 1:(1-1.5); (3) The molar ratio of the compound 3-1 to the compound c is (1-3):1, and the molar ratio of the compound 3-2 to the compound c is (1-3):1; (4) The molar ratio of the compound 4 to the compound d-1 is 1:(1-1.5); (5) The molar ratio of the compound 5 to the compound c is (2-3):1; (6) The molar ratio of the compound 6 to the compound d-2 is 1:(1-1.5); (7) The molar ratio of the compound 3-1 to the compound e is (1 to 1.5):1; (8) The molar ratio of the compound 8 to the compound 5 is 1:(1-1.5); (9) The molar ratio of the compound 9 to the compound d-3 is 1:(1-1.5).

9. A fluorescent probe, characterized in that The invention comprises the square acid compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt or deuterated compound thereof.

10. A near-infrared zone II contrast agent, characterized in that: The invention comprises the square acid compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt or deuterated compound thereof.

11. A method for monitoring early liver damage, comprising imaging a subject in need thereof using the squaric acid compound or a pharmaceutically acceptable salt or deuterated compound thereof according to any one of claims 1 to 6, the fluorescent probe according to claim 9, or the near-infrared zone II contrast agent according to claim 10.

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